diff --git a/.gitattributes b/.gitattributes index f54812e46d96b85fdb8714735ca13a781700be5f..3cddeb53cc3eef7b8e5d367f205b4214a40f1221 100644 --- a/.gitattributes +++ b/.gitattributes @@ -136,3 +136,4 @@ pdf-files/vestibular-evoked-myogenic-potential-vemp-triggere-nxbdfin.pdf filter= pdf-files/workflow-for-beta-range-forest-plots-bootstrap-rid-czcix2ue.pdf filter=lfs diff=lfs merge=lfs -text TXT.jsonl filter=lfs diff=lfs merge=lfs -text Base64.jsonl filter=lfs diff=lfs merge=lfs -text +Caduceus_Data.jsonl filter=lfs diff=lfs merge=lfs -text diff --git a/Caduceus_Data.jsonl b/Caduceus_Data.jsonl new file mode 100644 index 0000000000000000000000000000000000000000..4b6b174157fe90c9692850d524e647fc4fec7d27 --- /dev/null +++ b/Caduceus_Data.jsonl @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:67ede707c95a611f239e95e27c19ca7be5e2868a6c00d8f2999e8255a94dc19d +size 285639134 diff --git a/markdown-output/0-1m-edta-0-2m-mgcl2-0-2m-ascorbate-buffer-c2yyfv.md b/markdown-output/0-1m-edta-0-2m-mgcl2-0-2m-ascorbate-buffer-c2yyfv.md new file mode 100644 index 0000000000000000000000000000000000000000..dcb9a86f71a5929a3976565e961bcb640b8c309e --- /dev/null +++ b/markdown-output/0-1m-edta-0-2m-mgcl2-0-2m-ascorbate-buffer-c2yyfv.md @@ -0,0 +1,97 @@ +```markdown +# Goal/Experiment: +Preparation of iron chloride resuspension buffer using disodium EDTA dihydrate and magnesium chloride in Tris buffer. + +# 0.1M EDTA-0.2M MgCl2-0.2M Ascorbate Buffer + +## Abstract +Preparation of iron chloride resuspension buffer using disodium EDTA dihydrate and magnesium chloride in Tris buffer. + +Citation: Seth John, Bonnie Poulos, Christine Schirmer 0.1M EDTA-0.2M MgCl2-0.2M Ascorbate Buffer. protocols.io dx.doi.org/10.17504/protocols.io.c2yyfv + +Published: 20 Jul 2015 + +## Guidelines + +### Recipe as developed by Seth: +| Reagent | Formula Weight | Amount | Final Concentration | +|----------------------------|----------------|---------|---------------------| +| Tris-base | FW=121.14 | 1.51g | 0.125M | +| Na₂-EDTA dihydrate | FW= 372.24 | 3.72g | 0.1M | +| MgCl₂ hexahydrate | FW=203.3 | 4.07g | 0.2M | +| Ascorbic Acid | FW=176.12 | 3.52g | 0.2M | +| 5N NaOH | | ~4.0ml | to pH 6.5 final | +| MilliQ H₂O | | to 100ml| | + +1. **Tris-base**: A common buffering agent. +2. **Na₂-EDTA dihydrate**: Chelating agent to bind divalent cations. +3. **MgCl₂ hexahydrate**: Source of Mg²⁺ ions. +4. **Ascorbic Acid**: Reductant to improve virus infectivity. +5. **5N NaOH**: Used to adjust pH. + +*Oxalic acid can be substituted for ascorbic acid to improve virus infectivity. Use oxalic acid dihydrate (FW=126.07) at 2.52g/100ml for 0.2M.* + +### 2X Ascorbic Acid Buffer: +Keep the amount of Tris-base, water, and NaOH the same, but double the amount of EDTA, Mg and ascorbate. Check the pH and add NaOH or HCl to get final pH to 6.5. If increasing 2x, use 1 ml for every 2 mg Fe. + +### Notes: +- The new formulation uses EDTA and MgCl₂. +- Ensure EDTA has a pH above 8.0 to dissolve. +- Ascorbic acid may come out of solution if the pH is very high (above 5.0). + +### Recipe tested with diluted amounts of key reagents: +| Reagent | ½ Na₂-EDTA (1.86g/100ml) | ½ MgCl₂ (2.04g/100ml) | ½ Oxalic Acid-2H₂O (1.46g/100ml) | +|---------------------------|--------------------------|------------------------|-----------------------------------| +| Tris-base 1.51g/100ml | clear; pH 10.79 | clear; pH 10.82 | clear; pH 10.78 | +| Na₂-EDTA 3.72g/100ml | clear | clear | clear | +| MgCl₂·6H₂O 4.07g/100ml | clear; pH 7.68 | clear; pH 4.89 | clear; pH 4.59 | +| 5N NaOH | none; pH 7.68 | 1.25ml; pH 7.23 | 1.5ml; pH 7.51 | +| Oxalic acid·2H₂O 2.52g/100ml | white; pH 1.68 | cloudy; pH 3.02 | white; pH 3.30 | + +- Final results showed best results with ½ MgCl₂, intermediate results with ½ Oxalic acid, and worst results with ½ Na₂-EDTA. + +![Photo of solutions after final pH](path_to_image) + +## Protocol + +### 1x Buffer + +#### Step 1. +Dissolve 1.51g Tris-base in 80ml Milli Q water. + +#### Step 2. +Dissolve 3.72g Na₂-EDTA dihydrate into solution. +> **Note:** pH will be ~10.0 + +#### Step 3. +Once EDTA is in solution, dissolve 4.07g MgCl₂. +> **Note:** pH will drop to ~8.0 + +#### Step 4. +Add 3ml of NaOH. +> **Note:** This will drop the pH to ~4.5 and the solution will become cloudy indicating that the EDTA is coming out of solution. + +#### Step 5. +Dissolve the reductant (3.52g of ascorbic acid or 2.52g of oxalic acid). +> **Note:** The pH will increase to ~8.3 and the solution will clear up. + +#### Step 6. +Once the reductant is in solution, add the last 1ml of NaOH. + +#### Step 7. +Check the pH using pH paper (the buffer should be at pH 6.0 - 6.5). +> **Note:** The solution may need some minor adjusting with NaOH or HCl to achieve a pH of 6.0, which is ideal for good recovery of viruses. + +#### Step 8. +Check the volume and add MilliQ water for a total volume of 100ml. + +#### Step 9. +Store the buffer in the dark (bottle wrapped in foil) and visually inspect prior to use. It should be clear without precipitates. +> **Note:** The buffer will start to change color after about 24 hours but it is okay to use if slightly discolored. Do not use after about 36 hours. + +## Warnings +- EDTA needs a pH above 8.0 to dissolve and will come out of solution at pH below 5.0. +- Ascorbic acid may come out of solution if the pH is very high. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/16s-and-gyrb-bacterial-amplification-c6ywzfxe.md b/markdown-output/16s-and-gyrb-bacterial-amplification-c6ywzfxe.md new file mode 100644 index 0000000000000000000000000000000000000000..ee811ae8fb5808f79890e0acac94cfdbd12f32d3 --- /dev/null +++ b/markdown-output/16s-and-gyrb-bacterial-amplification-c6ywzfxe.md @@ -0,0 +1,204 @@ +```markdown +## Goal/Experiment: +The goal of this experiment is to amplify the 16S and gyrB bacterial genes using PCR for subsequent sequencing with PacBio Sequel II and Illumina MiSeq platforms. + +# 16S and GyrB Bacterial Amplification + +**DOI:** [dx.doi.org/10.17504/protocols.io.36wgg31nylk5/v1](dx.doi.org/10.17504/protocols.io.36wgg31nylk5/v1) + +**Author:** Robert Nichols +**Institution:** Pennsylvania State University + +**Protocol Status:** Working +We use this protocol and it's working. + +**Created:** January 03, 2024 +**Last Modified:** July 12, 2024 +**Protocol Integer ID:** 92918 +**Keywords:** PCR, gyrB, PacBio, MiSeq + +--- + +## Abstract +This protocol is used for the amplification of the bacterial _gyrB_ gene and the _16S_ gene for both PacBio Sequel II and Illumina MiSeq sequencing. This protocol is used in the paper titled *Long-read Sequencing Increases the Accuracy and Specificity of the gyrB Phylogenetic Marker Gene*. + +--- + +## Materials + +- **Isolated bacterial DNA** +- **Nuclease-free water** (VWR Cat # 103307-278) +- **Invitrogen Platinum SuperFi PCR Master Mix** (ThermoFisher Scientific, Cat # 12368250) +- **1× TAE** (Tris base [Millipore Sigma, Cat # 648311], acetic acid [Millipore Sigma, Cat # 695092], and EDTA [Millipore Sigma, Cat # E9884]) buffer +- **OmniPur agarose** (VWR, Cat # EM-2070) +- **GelRed dye** (VWR, Cat # 10098-684) +- **6x Gel loading dye** (no SDS) (Biolabs, Cat# B7025S) +- **100-bp DNA ladder** (VWR, Cat# PAG2101) +- **Ice bath** +- **NanoDrop UV-Vis Spectrophotometer Lite** (Thermo-Scientific) +- **Sterile 0.2-ml thin-wall PCR Tubes**, strips of 8 tubes (Denville) +- **Sterile 0.5- to 10-µl pipettes** (Denville) +- **Sterile 10- to 200-µl pipettes** (Denville) +- **Sterile 1000-µl pipettes** (Denville) +- **T100 Thermal cycler** (BioRad) +- **Gel electrophoresis box** (Labnet) +- **ChemiDoc XRS+** (BioRad) + +--- + +## Primer Information + +| Primer Name | Primer Description | Primer Sequence | Platform | +|------------------|--------------------------------------------------|-----------------------------------------------|-----------| +| **V4_16S_F** | Forward primer for V4 16S sequencing | TCGTCGGCAGCGTCAGATGTGTATAAGA GACAGTGYCAGCMGCCGCGGTAA | MiSeq | +| **V4_16S_R** | Reverse primer for V4 16S sequencing | GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGGGTATTACNVGGGTWTTCAAT | MiSeq | +| **FL_16S_F** | Forward primer for full-length 16S sequencing | /5AmMc6/GCAGTCGAACATGTAGCTGACTCAGGTCACAGRGTYTGAATMGGCTCA | PacBio | +| **FL_16S_R** | Reverse primer for V4 full-length sequencing | /5AmMc6/TGCGACGTCTTGGCACAGATCACTCGAAATGRETCAGGCTTAGR | PacBio | +| **SR_GyrB_Bac_F**| Forward primer to amplify the _gyrB_ gene from Bacteroidaceae | TCGTCGGCAGCGTCAGATGTGTATAAGA GACAGGGGTAAARTTCGAYAAAGG | MiSeq | +| **SR_GyrB_Bac_R**| Reverse primer to amplify the _gyrB_ gene from Bacteroidaceae | GTCTCGTGGGCTCGGAGATGTGTATAAGAGACRT TTYYTCTTCRCGCGCGTAACG | MiSeq | +| **SR_GyrB_Bif_F**| Forward primer to amplify the _gyrB_ gene from Bifidobacteriaceae | TCGTCGGCAGCGTCAGATGTGTATAAGA GACAGGACCRACGGNCGNGCG | MiSeq | +| **SR_GyrB_Bif_R**| Reverse primer to amplify the _gyrB_ gene from Bifidobacteriaceae | GTCTCGTGGGCTCGGAGATGTGTATAAGAGACCT CCGCTTTGNAACGWAATGC | MiSeq | +| **SR_GyrB_Lac_F**| Forward primer to amplify the _gyrB_ gene from Lachnospiraceae | TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCGGGGWCG GayCGAATTAG | MiSeq | +| **SR_GyrB_Lac_R**| Reverse primer to amplify the _gyrB_ gene from Lachnospiraceae | GTCTCGTGGGCTCGGAGATGTGTATAAGAGACGGA TRGGTCTGGACCTGRCRTTTCCG | MiSeq | +| **LR_GyrB_Bac_F**| Forward primer to amplify the _gyrB_ gene from Bacteroidaceae | /5AmMc6/GCAGTCGAACATGTAGCTGACTCAGGTCACATACGAYGTT | PacBio | +| **LR_GyrB_Bac_R**| Reverse primer to amplify the _gyrB_ gene from Bacteroidaceae | /5AmMc6/TGCGACGTCTTGGCACAGATCACTCGAAAGATTYGCTGTCART | PacBio | +| **LR_GyrB_Bif_F**| Forward primer to amplify _gyrB_ gene from Bifidobacteriaceae | /5AmMc6/GCAGTCGAACATGTAGCTGACTCAGGTCAGACCGCGWCTCGA | PacBio | +| **LR_GyrB_Bif_R**| Reverse primer to amplify the _gyrB_ gene from Bifidobacteriaceae | TCGARGTSAGCATTCTGCCC | PacBio | +| **LR_GyrB_Lac_F**| Forward primer to amplify the _gyrB_ gene from Lachnospiraceae | /5AmMc6/GCAGTCGAACATGTAGCTGACTCAGGTCAGCTTYACNCGNWNTA | PacBio | +| **LR_GyrB_Lac_R**| Reverse primer to amplify the _gyrB_ gene from Lachnospiraceae | /5AmMc6/TGCGACGTCTTGGCACAGATCACTCGAAAGATRCCWGWTNNTT | PacBio | + +--- + +## Before Start +Before starting, ensure you have isolated bacterial DNA and selected primers for amplification. + +--- + +## Prepare DNA for Amplification + +1. **Thaw the isolated DNA** +2. **Measure DNA concentration on the Nanodrop** + This requires only 1 μL of isolated DNA. Concentration values typically range from 100 ng/μL to 400 ng/μL. The NanoDrop provides an estimate of total DNA concentration. For more accurate results, submit samples for quantification on a Bioanalyzer. +3. **Create a 100 μL aliquot at 10 ng/μL concentration**. + 3.1. **Calculate required DNA volume** + - Divide 1000 by the average DNA concentration. + Example: If average DNA concentration is 254 ng/μL, then 1000/254 = 3.94. Therefore, use 3.94 μL of original DNA. + 3.2. **Calculate water volume** + - Subtract the calculated volume from 100. Example: 100 - 3.94 = 96.06 μL of nuclease-free water. + - Result: 3.94 μL original DNA + 96.06 μL nuclease-free water = 10 ng/μL DNA solution. + +--- + +## 16S and _gyrB_ PacBio and Illumina Amplicon PCR Protocol + +### PCR Mix Preparation + +4. **Prepare the PCR mix.** + +| Reagent | Concentration | Volume to make 20 µL of product | +|------------------------|--------------|-----------------------| +| Forward primer | 10 µM | 0.4 µL | +| Reverse primer | 10 µM | 0.4 µL | +| Platinum SuperFi Master Mix | N/A | 10 µL | +| Nuclease-Free water | N/A | 8.2 µL | + +Adjust volumes based on the number of samples plus one or two extra to ensure sufficient master mix availability. For example, for 15 sample PCR, multiply each volume by 17 (15 samples + 2 extra). + +5. **Fill PCR wells** + Fill adequate number of wells with 19 μL of master mix per well. +6. **Add DNA to wells** + Add 1 μL of 10 ng/μL DNA directly into the master mix of each well. +7. **Mix and spin** + Ensure reagents are mixed by gently flicking and quickly spinning in a mini centrifuge. +8. **Run PCR** + +### PCR Settings + +8.1 **16S samples for MiSeq** + +| Cycle Number | Time | Temperature | Description | +|--------------|------------|-------------|---------------------| +| 1 cycle | 2 minutes | 98°C | Initial denaturation | +| 25 cycles | 10 seconds | 98°C | Denaturation | +| | 20 seconds | 56.6°C | Annealing | +| | 15 seconds | 72°C | Extension | +| 1 cycle | 5 minutes | 72°C | Final extension | + +> Optimize PCR cycles for specific requirements to reduce chimeric sequences. + +8.2 **16S samples for PacBio** + +| Cycle Number | Time | Temperature | Description | +|--------------|------------|-------------|---------------------| +| 1 cycle | 30 seconds | 95°C | Initial denaturation | +| 25 cycles | 30 seconds | 95°C | Denaturation | +| | 30 seconds | 57°C | Annealing | +| | 1 minute | 72°C | Extension | +| 1 cycle | 5 minutes | 72°C | Final extension | + +> Optimize PCR cycles to reduce chimeric sequences. + +8.3 **GyrB samples for MiSeq** + +| Cycle Number | Time | Temperature | Description | +|--------------|------------|-------------|---------------------| +| 1 cycle | 2 minutes | 98°C | Initial denaturation | +| 30 cycles | 10 seconds | 98°C | Denaturation | +| | 20 seconds | 56.6°C | Annealing | +| | 15 seconds | 72°C | Extension | +| 1 cycle | 5 minutes | 72°C | Final extension | + +> Optimize PCR cycles to reduce chimeric sequences. + +8.4 **GyrB samples for PacBio** + +| Cycle Number | Time | Temperature | Description | +|--------------|------------|-------------|---------------------| +| 1 cycle | 30 seconds | 95°C | Initial denaturation | +| 30 cycles | 30 seconds | 95°C | Denaturation | +| | 30 seconds | 57°C | Annealing | +| | 1 minute | 72°C | Extension | +| 1 cycle | 5 minutes | 72°C | Final extension | + +> Optimize PCR cycles to reduce chimeric sequences. + +--- + +## Check for Amplification + +9. **Create a 1x agarose gel** + - Combine 1 g of agarose and 100 mL of 1x TAE. Microwave for 1 minute and 45 seconds. + - Pour into a mold with appropriate comb. Add 10 μL of Gel Red dye (10,000x). Let cool for 45 minutes to 1 hour. + +10. **Prep amplicons for electrophoresis** + +| Reagent | Concentration | Volume | +|------------------|---------------|--------| +| Gel loading dye | 6x | 8 µL | +| Nuclease-free water | NA | 16 µL | + + - Multiply volumes by the number of samples. + - Add 20 µL of amplified product to wells with 24 µL of dye-water mix. + +11. **Run electrophoresis** + - Run gel at 80 volts for 1 hour. + - Check gel in a gel doc to see amplified bands. + +--- + +## Clean Amplicon Samples with Gel Clean-up Kit + +12. **Cut out bands from gel** + - Use specialized pipette tips to punch out bands under UV light (wear proper PPE). + +13. **Clean-up with QIAquick Gel Extraction Kit** + - Dissolve gel punch-outs in provided buffer at 50°C for 10 minutes. Add dissolved punch-out mixture to columns. Wash twice with provided buffers and elute with nuclease-free water or elution buffer. + +14. **Submit for sequencing** + - MiSeq: 250x250 Illumina MiSeq + - PacBio: PacBio Sequel II + +--- + +**endofoutput** +``` \ No newline at end of file diff --git a/markdown-output/2-agarose-gel-cac7sazn.md b/markdown-output/2-agarose-gel-cac7sazn.md new file mode 100644 index 0000000000000000000000000000000000000000..fb2c5221d2cc67e2701da2345f8cd5395a72e8e1 --- /dev/null +++ b/markdown-output/2-agarose-gel-cac7sazn.md @@ -0,0 +1,115 @@ +```markdown +# Goal/Experiment: +To prepare and use a 2% agarose gel for the separation and analysis of DNA fragments through gel electrophoresis. + +# 2% Agarose Gel + +**Author:** George Testo +**Affiliation:** The Pathogen & Microbiome Institute +**Date:** May 31, 2022 +**Protocol Shared via:** [protocols.io link](https://protocols.io/view/2-agarose-gel-cac7szan) +**Date of Protocol:** Jun 03, 2022 + +**Disclaimer:** +TO THE EXTENT ALLOWED BY LAW, LIFE TECHNOLOGIES AND/OR ITS AFFILIATE(S) WILL NOT BE LIABLE FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING YOUR USE OF IT. + +## Introduction + +Agarose gel electrophoresis is a method used in biochemistry, molecular biology, genetics, and clinical chemistry to separate a mixed population of macromolecules such as DNA or proteins in a matrix of agarose, one of the two main components of agar. The molecules are separated by applying an electric field to move the charged molecules through an agarose matrix, with separation primarily based on size. + +Routinely, agarose gels are suitable for separating DNA of various size ranges, can be stained and visualized under UV light, and DNA fragments can be extracted from the gel easily. + +## Reagents + +- 1 x DI Water +- 1 x bottle of TAE (or TBE) +- 1 x bottle of Agarose Powder +- 1 x bottle of SYBR Safe +- 1 x bottle of 6x Loading Dye + +## Supplies + +- Green temporary seal(s) +- Foil seal(s) + +## Equipment + +- 10µL pipette, tip box, & tips +- 20µL pipette, tip box, & tips +- 200uL pipette, tip box, & tips +- 1000uL pipette, tip box, & tips +- Serological pipette & 10mL tip + +### Note on SYBR™ Safe Dye: +SYBR™ Safe DNA gel stain showed no or very low mutagenic activity in tests and is not classified as hazardous waste under U.S. Federal regulations. However, standard care should be taken when handling and disposing of this reagent in compliance with all local regulations. + +#### Storage: +SYBR™ Safe DNA gel stain can be stored between 2°C to 25°C. SYBR™ Safe in DMSO freezes at low temperatures; it must be completely thawed and mixed before use. + +## Procedure + +### Pouring a Standard 2% Agarose Gel (21 min) + +1. **Measure 1g of Agarose.** + +2. **Prepare Agarose Solution:** + - Mix agarose powder with **49mL of DI water and 1mL of 50x TAE** in a microwavable flask. + +3. **Dissolve Agarose:** + - Microwave for **1-3 minutes** until the agarose is completely dissolved. + > **Note:** Do not overboil to avoid buffer evaporation which alters gel percentage. Prefer pulse microwaving to avoid boiling. + +4. **Cool Agarose Solution:** + - Let the solution cool down to **about 50°C for about 5 minutes**. + +5. **(Optional) Stain Agarose:** + - Add **5uL of SYBR Safe** to the flask. + > **Note:** SYBR Safe binds to DNA, allowing visualization under UV light. If using EtBr, handle it with caution as it's a mutagen. + +6. **Pour Gel:** + - Pour the agarose into a gel tray with well comb in place. + +7. **Solidify Gel:** + - Place newly poured gel at **4°C for 15 minutes** or let it sit at room temperature for 30 minutes until completely solidified. + > **Note:** Pour slowly to avoid disrupting bubbles. Use a pipette tip to push the bubbles away if they form. + +### Loading Samples and Running an Agarose Gel (21 min) + +8. **Add Loading Buffer to Samples:** + - Prepare ladder dilution: **Molecular Grade Water + 10uL of 1kb Ladder**. + - Mix at least **10uL of ladder or sample with 2uL of 6x Loading Dye** for a total volume of 12uL. + > For lower DNA concentrations, mix **15uL of ladder or sample with 2.5uL of 6x Loading Dye.** + +9. **Place Gel in Box:** + - Once solidified, place the agarose gel into the gel box (electrophoresis unit). + +10. **Add Running Buffer:** + - Fill the gel box with **1x TAE** (or TBE) until the gel is covered. + +11. **Load Ladder:** + - Carefully load a molecular weight ladder into the first lane of the gel. + +12. **Load Samples:** + - Carefully load your samples into the additional wells of the gel. + +13. **Run Gel:** + - Run the gel at **80-150V** until the dye line is approximately **75-80% of the way down the gel**. + > Typical run time is about 1-1.5 hours, depending on gel concentration and voltage. + > **Note:** Always run to RED (positive electrode). + +### Analyzing Your Gel (21 min) + +14. **Stop Run:** + - Turn OFF power, disconnect electrodes, and carefully remove the gel from the box. + > If EtBR was used, place the gel into running buffer and destain. + +15. **Visualize Gel:** + - Visualize DNA fragments using a UV light device. + > **Note:** For longer storage or less damage, use long-wavelength UV and minimize exposure time. + +**Caution:** When using UV light, protect your hands and eyes by wearing appropriate PPE (Personal Protective Equipment). + +--- + +**End of Output** +``` diff --git a/markdown-output/2023-genometrakr-proficiency-testing-exercise-puls-cs8uwhww.md b/markdown-output/2023-genometrakr-proficiency-testing-exercise-puls-cs8uwhww.md new file mode 100644 index 0000000000000000000000000000000000000000..23ce0c96ba6e5f0f91e2897b3d1973143bc40602 --- /dev/null +++ b/markdown-output/2023-genometrakr-proficiency-testing-exercise-puls-cs8uwhww.md @@ -0,0 +1,192 @@ +```markdown +# 2023 GenomeTrakr Proficiency Testing Exercise (PulseNet Harmonized) V.7 + +## Goal/Experiment: +The goal of this proficiency testing exercise is to provide standardized guidelines for GenomeTrakr (GT) laboratories to process isolates, generate sequencing data, and report results for the 2023 GenomeTrakr Proficiency Testing exercise. The exercise aims to ensure consistent and accurate detection of foodborne pathogens using Whole Genome Sequencing (WGS). + +### Authors: +Maria Balkey¹, Ruth Timme¹, Julie Haendiges¹, Tina.Pfefer¹ +¹ U.S. Food and Drug Administration + +## Abstract: +This Standard Operating Procedure (SOP) outlines guidelines for processing the isolates for the 2023 GenomeTrakr (GT) Proficiency Testing exercise. It is applicable to all GenomeTrakr labs participating in the 2023 GenomeTrakr Proficiency Testing exercise (PulseNet Harmonized). + +### Version Updates: +- **v5:** Added links to GalaxyTrakr_PT_exercise_report_2022.xlsx +- **v6:** added 2023 PT strains; edited Steps 1, 2, 4, and 5 to include references to growth of Listeria on BHIA plates; Updated Tables in Steps 7.1 and 8.1 with sample information for 2023 PT strains. +- **v7:** Section 9 and Section 11: deleted file GalaxyTrakr_PT_exercise_report_2023.xlsx; uploaded new file version GalaxyTrakr_PT_exercise_report_2023-v2.xlsx + +### Proficiency Testing Isolates: +The FDA GenomeTrakr program will ship the following proficiency testing isolates in April 2023: + +| Strain ID | Organism | +| ------------- | ----------------------- | +| ESP23-5286 | Escherichia/Shigella | +| ESP23-9201 | Escherichia/Shigella | +| SAP23-3307 | Salmonella | +| LMP23-6714 | Listeria | + +Completion of the entire proficiency test entails the following: +- GT participating laboratory generates sequencing data (fastq files) using the PT strains provided by CDC through FDA-CFSAN-WGS Program-GT in accordance with GT and/or PulseNet SOPs. +- Populate sample sheet according to 2023 GT Proficiency Testing exercise (PulseNet Harmonized) SOP. +- Submission of sequencing records to the appropriate project on BaseSpace or Isilon according to GT SOPs. +- By participating in the 2023 GT Proficiency Testing exercise (PulseNet Harmonized), GT labs provide consent to use the PT exercise data in subsequent analysis and manuscript publications. Participants will be acknowledged for their contribution on any publication that might require processing data from the 2023 GT PT exercise. + +## Materials +### Materials Needed: +- Sterile sturdy forceps +- 1 ml pipetman +- 1 ml sterile pipet tips +- 1 µl and/or 10 µl sterile inoculating loop + +### Reagents Needed: +- Trypticase Soy + 5% Sheep Blood Agar plates (BAP) or equivalent media +- BHIA plates +- Sterile reagent grade water or Phosphate Buffered Saline (0.01M PBS; pH 7.4) +- BHI broth +- 70% isopropyl alcohol + +## Safety Warnings +- **Biological Safety Warning:** _Escherichia/Shigella_, _Salmonella_, and _Listeria_ strains are considered Level 2 biological agents by the U.S. Department of Health and Human Services. Use appropriate precautions when handling the vial or culture. Carry out laboratory work in a biological safety cabinet when applicable to ensure aseptic conditions and personal safety. + +## Before Start Instructions: +There are four sections in this protocol: +1. Culture preparation of lyophilized isolates. +2. Sequencing +3. Data Transfer + +## Culture Preparation +### 1. Salmonella, Escherichia/Shigella, and Listeria Lyophilized cultures: +#### Day 1: +1. Document the isolate number(s) and the lyophilized date(s) for your records. Wipe the aluminum cover and outside of the vial with isopropyl alcohol. Using sturdy forceps, aseptically remove the aluminum cover and rubber stopper from the vial containing the lyophilized culture. Wipe the outside of the rubber stopper and neck of the vial with isopropyl alcohol before removing the stopper. +2. Re-suspend the lyophilized cells with 1 ml of sterile reagent grade water. Allow to stand for a few minutes and/or mix gently to produce a uniform suspension. Plate 10 µl of this suspension onto a blood agar plate (BAP) (or BHIA plate for Listeria) and incubate at 37°C overnight in aerobic conditions. It is recommended to plate in duplicate to ensure adequate growth. +3. Add the rest of the suspension to 5 mL of BHI broth and incubate the culture overnight at 37°C in aerobic conditions. + +#### Day 2 and 3: +4. Check the BAPs and BHIA plates; if the culture appears pure, pick an isolated colony, and streak it on a fresh plate; incubate at 37°C overnight in aerobic conditions. Use the growth from this plate to make DNA templates of the PT strains. Transfer culture to fresh medium and incubate at 37°C overnight; this will ensure that the same culture can be retested, if necessary. + +#### Optional: +5. If plates don't show bacterial growth, prepare a new plate by taking a loop from the BHI overnight culture (prepared at step 3), streak it on a BAP or BHIA plate as appropriate and incubate at 37°C overnight in aerobic conditions. On the next day check BAP or BHIA plate and proceed as step 4. + +## Sequencing +### 6. Perform DNA extraction, library preparation and sequencing according to lab's normal workflow described on SOPs posted at GenomeTrakr protocols.io: + - [Manual DNA Extraction Using Qiagen DNeasy Blood & Tissue Kit](https://www.protocols.io/view/manual-dna-extraction-using-qiagen-dneasy-blood-an-81wgqb391qvk/v1) + - [Procedure for Operation and Maintenance of the Illumina MiSeq](https://www.protocols.io/view/procedure-for-operation-and-maintenance-of-the-ill-rm7vz8z52wx1/v1) + - [Illumina DNA Prep (M)-Tagmentation Library Preparation](https://www.protocols.io/view/illumina-dna-prep-m-tagmentation-library-preparati-x54v9m7ezg3e/v2) + - [DNA Quantification Using the Qubit Fluorometer](https://www.protocols.io/view/dna-quantification-using-the-qubit-fluorometer-81wgbp3x3vpk/v1) + +### PulseNet Labs: +- Will process isolates according to PulseNet Guidelines. +- Isolates must be processed exactly as any routine isolate would be processed in the laboratory. + +### 7. Sequencing sample sheets must be filled out according to Table 1: +#### 7.1 Sample_ID: +Include in this field the values from the Sample_ID column of Table 1,*do not modify these IDs*. You will also find this identifier in the vial of the lyophilized culture. + +| Sample_ID | Sample_Name | Project | Description | +| ------------ | ----------------------| -----------------------------------------| ------------| +| ESP23-5286 | ESP23-5286-M0XXXX-20230424 | PR0403_2023_Proficiency_Testing_Exercise | Escherichia/Shigella | +| ESP23-9201 | ESP23-9201-M0XXXX-20230424 | PR0403_2023_Proficiency_Testing_Exercise | Escherichia/Shigella | +| SAP23-3307 | SAP23-3307-M0XXXX-20230424 | PR0403_2023_Proficiency_Testing_Exercise | Salmonella | +| LMP23-6714 | LMP23-6714-M0XXXX-20230424 | PR0403_2023_Proficiency_Testing_Exercise | Listeria | + +#### Non PulseNet Labs: +NextSeq 1000/2000 and MiSeq systems running Windows 10 (MCS v4 and up) have only Sample_ID available in the sequencing sample sheet, populate the Sample_ID column with the values in the Sample_ID column of Table 1. + +### 7.2 Sample_name: +#### Non PulseNet Labs: +- Fill out this field according to example provided in column Sample_Name of table 1. Include the isolate identifier, instrument ID (M0XXXX where "XXXX" corresponds to the instrument identifier) and run start date. (e.g. ESP23-5286_M01001-20230424). + +#### PulseNet Labs: +- Include the isolate identifier, lab ID, Instrument ID (M0XXXX where "XXXX" corresponds to the instrument identifier) and run start date. (e.g. ESP23-5286-GA-M01001-20230424). + +### 7.3 Project: +- Please fill out the project field with the project identifier PR0403_2023_Proficiency_Testing_Exercise. + +### 7.4 Description: +- For your use only, we do not track this field. Organism names might be included in this field. + +## Replicates +### 8. Are you running more than one set of PT isolates in a run? +- **YES:** Proceed to Step 8.1 +- **NO:** Proceed to Step 9. + +### 8.1 Modify Isolate identifiers (IDs at vial of lyophilized culture) to create unique identifiers for PT replicated isolates by adding suffixes such as: "_2" or "_3" to isolate identifiers. + +#### Non-PulseNet Laboratories: +If you choose to run replicates, the sample sheet must contain unaltered isolate identifiers for each PT strain in the Sample_name field. Identifiers in the sample_ID field must include the isolate identifier, replicate, instrument Id and start run date. + +| Sample_ID | Sample_Name | Project | Description | +| ----------------------- | ----------- | ----------------------------------- |-----------------------| +| ESP23-5286-1-M0XXXX-20230424 | ESP23-5286 | PR0403_2023_Proficiency_Testing_Exercise | Escherichia/Shigella | +| ESP23-9201-1-M0XXXX-20230424 | ESP23-9201 | PR0403_2023_Proficiency_Testing_Exercise | Escherichia/Shigella | +| SAP23-3307-1-M0XXXX-20230424 | SAP23-3307 | PR0403_2023_Proficiency_Testing_Exercise | Salmonella | +| LMP23-6714-1-M0XXXX-20230424 | LMP23-6714 | PR0403_2023_Proficiency_Testing_Exercise | Listeria | +... + +#### PulseNet Laboratories: +- Identifiers in the sample_ID and sample_Name fields must include the isolate identifier, replicate, lab ID, instrument Id and start run date. + +| Sample_ID | Sample_Name | Project | Description | +| --------------------------------- | ------------------- | ----------------------------------- |-----------------------| +| ESP23-5286_1_GA_M0XXXX_20230424 | ESP23-5286_1_GA_M0XXXX_20230424 | PR0403_2023_Proficiency_Testing_Exercise | Escherichia/Shigella | +| ESP23-9201_1_GA_M0XXXX_20230424 | ESP23-9201_1_GA_M0XXXX_20230424 | PR0403_2023_Proficiency_Testing_Exercise | Escherichia/Shigella | +| SAP23-3307_1_GA_M0XXXX_20230424 | SAP23-3307_1_GA_M0XXXX_20230424 | PR0403_2023_Proficiency_Testing_Exercise | Salmonella | +| LMP23-6714_1_GA_M0XXXX_20230424 | LMP23-6714_1_GA_M0XXXX_20230424 | PR0403_2023_Proficiency_Testing_Exercise | Listeria | +... + +### 8.2 Project: +- Please fill out the project field with the project identifier PR0403_2023_Proficiency_Testing_Exercise. + +### 8.3 Description: +- For your use only, we do not track this field. + +## Quality of sequencing run +### 9. Data analysis results must be reported to GenomeTrakr in the spreadsheet included in the attachment. +- Check the quality of your sequencing records by following the SOP [Assessing sequence quality in GalaxyTrakr](https://). Note that Shigella isolates will be predicted as Escherichia coli with PubMLST scanning of contigs. The result is acceptable. Report your findings in the spreadsheet GalaxyTrakr_PT_exercise_report_2023-v2.xlsx, sheet MicroRunQC. +- Run the tool [ShigaTyper](https://) for Shigella genoserotyping. Report your findings in the spreadsheet GalaxyTrakr_PT_exercise_report_2023, sheet ShigaTyper. +- Run the tool [SeqSero2](https://) for Salmonella serotyping. Report your findings in the spreadsheet GalaxyTrakr_PT_exercise_report_2023, sheet SeqSero. + +## Data Transfer +### 10. Data Transfer +After checking the quality of your records, transfer the data and associated QC results to GenomeTrakr. + +#### 10.1 Sharing a run in BaseSapce +- Click the Runs tab in the Illumina BaseSpace website. +- Select the run that you would like to share with the GenomeTrakr team. +- Go to the summary tab and click at the share button. +- Enter the email address for the FDA team (gnometrakr@fda.hhs.gov), and then click Add Collaborator. +- Click Save Settings. Your run will be automatically shared with the GenomeTrakr team. + +#### 10.2 Sharing a project in BaseSapce +- Click the Projects tab in the Illumina BaseSpace website. +- Select the project (PR0403_2023_Proficiency_Testing_Exercise) that you would like to share with the GenomeTrakr team. +- Click the share project button. +- Enter the email address for the FDA team (gnometrakr@fda.hhs.gov), and then click Add Collaborator. +- Click Save Settings. Your project will be automatically shared with the GenomeTrakr team. + +#### 10.3 Labs inside the FDA network +- Must share the sequencing files by transferring the sequencing run folder to the isilon storage drive. + +### 11. PT Exercise Completion Notification +- Notify GenomeTrakr of your completion of the PT exercise by sending an email to: gnometrakr@fda.hhs.gov. The subject line should include “2023 WGS Proficiency Testing_YourLabName”, attach sequencing sample sheet and include the following information in the body of the email: + - Run name: + - Sequenced by: + - Results submitted by: + - MiSeq ID: + - Flow cell ID: + - SOP Protocol: (Select one of the options) + - PulseNet SOP + - GenomeTrakr SOP + +#### Non-PulseNet Laboratories, please include +- GalaxyTrakr_PT_exercise_report_2023-v2.xlsx in the attachments of the notification email. + +#### PulseNet Laboratories +- Must send the 2023 Proficiency Testing exercise data and results to PulseNet. + +### 12. +00:00:00 + +endofoutput +``` diff --git a/markdown-output/3-39-tagseq-library-preparation-protocol-cgcbtssn.md b/markdown-output/3-39-tagseq-library-preparation-protocol-cgcbtssn.md new file mode 100644 index 0000000000000000000000000000000000000000..d13c41412c06c0a8c39efbb7f84728de82aa7295 --- /dev/null +++ b/markdown-output/3-39-tagseq-library-preparation-protocol-cgcbtssn.md @@ -0,0 +1,257 @@ +```markdown +# Goal/Experiment: +3' TagSeq Library Preparation Protocol + +## Abstract + +Purpose: This protocol is designed for constructing libraries targeting the 3' ends of mRNA for gene expression profiling and offers an alternative to standard RNA-Seq. + +**DOI:** +[dx.doi.org/10.17504/protocols.io.q26g7yrr3gwz/v1](dx.doi.org/10.17504/protocols.io.q26g7yrr3gwz/v1) + +**Protocol Citation:** +anni.wang 2022. 3' TagSeq Library Preparation Protocol. protocols.io [URL](https://protocols.io/view/3-39-tagseq-library-preparation-protocol-cgcbtssn) + +**License:** +This is an open access protocol distributed under the terms of the [Creative Commons Attribution License](https://creativecommons.org/licenses/by/4.0/). + +**Created:** +Sep 08, 2022 +**Last Modified:** +Sep 09, 2022 + +## Materials + +| Reagent | Vendor/Catalog # | Storage | +| --------------------------------------- | --------------------------- | ----------------- | +| 10mM dNTPs | NEB/N0447L | Freezer | +| 0.1M DTT (aliquots) | ThermoFisher/707265ML | Freezer | +| 5X First Strand (FS) buffer | Takara(Clontech)/639537 | Freezer | +| SMARTScribe Reverse Transcriptase | Takara(Clontech)/639537 | Freezer | +| Klentaq DNA polymerase | DNA Polymerase tech/SKU: 100 | Freezer | +| 10X Klentaq 1 buffer | DNA Polymerase tech/SKU: 100 | Freezer | +| 3iLL-30TV primer | IDT | Freezer | +| 5iLL primer | IDT | Freezer | +| Template Switching Oligos (TSO) 4 oligos | IDT | -80 | +| Agencourt AMPure Beads XP | Beckman Coulter A63881 | Fridge | +| 80% EtOH | Prepared fresh | Flammable cabinet | +| Eppendorf twin-tec 96-well PCR plate | Eppendorf (VWR:95041-440) | Shelf | +| Adhesive PCR plate foil seal | Fisher | Shelf | +| 96-well plate magnet | ThermoFisher AM10027 | Shelf | +| Bioanalyzer kits (HS and Pico) | Aligent: HS DNA: 5067-4626; RNA 6000 Pico: 5067-1513 | Fridge | +| Index Primers | IDT | Freezer | +| Pippin Prep Cassettes/Reagents | BDF1510 | Shelf and Fridge | +| MinElute Column | Qiagen #28006 | Fridge | +| PB Binding Buffer | Qiagen #28006 | Shelf | +| PE Wash Buffer | Qiagen #28006 | Shelf | + +### Template Switching Oligos (4) Specifications + +1. Order on IDT [IDT URL](https://idtdna.com) + - Go to "DNA&RNA" -> "Custom RNA oligos" + - Order RNA oligos by the tube and customize each oligo in their template. + +**General Specifications:** +- Product: 100nmole RNA Oligo +- Guarantee: 0.3 nmol +- Purification: RNase-Free HPLC +- Additional Services: Level II Setup Fee + +### Index Primer 96 Well Adapter Plate + +**General Specifications:** +- Guarantee: 1.38 nmol +- Final Concentration: 10 µM +- Quantity: 1.38 nmol +- Buffer: IDTE Buffer pH 8.0 (10mM Tris-HCl/0.1 mM EDTA) +- Purification: Standard Desalting +- Plate Product: Tru-Seq - Compatible Indexing Primer, 16 rxn +- Plate Type: Matrix- Screw Top + +## RNA Fragmentation and RT Primer Annealing + +### 1. RNA Fragmentation and RT Primer Annealing + +*Input material should be at least 25 µl of high-quality DNAse-treated RNA in water normalized to 50 ng/µl (acceptable range: 5ng/µl to 100ng/µl).* + +1.1 Clean bench, pipettes, ice bucket, and all equipment with RNaseZAP and 70% EtOH. Thaw RNA and reagents on ice. + +1.2. Preheat the thermocycler to 95°C for 10-15 min. + +1.3. Label a 96-well plate with appropriate information (plate#, date, initials, and SOP step). + +1.4. Pipette RNA and nuclease-free H2O to a total of 10 µl (50ng-1000ng) into each well. This can be done using a liquidator or a 20 µl multichannel pipette. + +1.5. Prepare the RNA fragmentation/RT master mix: + +| Reagent | Volume per Sample | Volume in Master Mix for 40 Samples +10% (44 total) | +| ---------------- | ----------------- | -------------------------------------------------- | +| dNTPs (10mM) | 1 µl | 44 µl | +| 0.1M DTT | 2 µl | 88 µl | +| 5x FS buffer | 4 µl | 176 µl | +| 3iLL-30TV (10uM) | 1 µl | 44 µl | +| Total RNA (H2O if needed) | 10 µl | 440 µl | +| Total volume | 18 µl | 352 µl | + +1.6. Mix the master mix by gentle inversion or pipetting and briefly spin in a mini-centrifuge. + +1.7. Aliquot 8 µl of master mix into each RNA sample using a multi-channel pipette, pipette 3-5 times to mix. + +1.8. Cover the plate with foil seal, briefly spin, and start the fragmentation program as specified in an RT thermocycler. + +1.9. Remove the plate and incubate on ice for 2 minutes. + +1.10. **Optional:** Perform fragment analysis on a subset of samples if necessary. Replace 1.5 µl of 1x FS buffer into each well. + +### 2. First-Strand cDNA Synthesis + +2.1. Briefly centrifuge and remove foil seal from the RNA plate, return the plate to ice. + +2.2. Prepare the master mix: + +| Reagent | Volume per Sample | Volume in Master Mix for 40 Samples +10% (44 total) | +| ------------------- | ----------------- | -------------------------------------------------- | +| Template switch oligo pool (10 µM) | 0.1 µl | 4.4 µl | +| SMARTScribe RT | 1 µl | 44 µl | + +2.3. Add 2 µl of the oligo/RT master mix to each sample. Pipet 4-5 times to mix. + +2.4. Start the first strand cDNA synthesis program in a thermocycler: + + - 42°C for 1 hr + - 65°C for 15 min + +### 3. AMPure Bead Purification + +3.1. Vortex AMPure beads well, dispense enough (45 µl/sample) to a 50 mL reservoir. Prepare a second reservoir with nuclease-free H2O. + +3.2. Briefly centrifuge the first-strand cDNA plate in a mini plate spinner. Add 30 µl of H2O to each sample, bringing the total volume to 50 µl. + +3.3. Perform a 0.9x AMPure bead purification: adding 45 µl AMPure beads, mixing well, incubating 15 minutes at RT. + +3.4. Prepare 80% EtOH during incubation (200 µl EtOH/sample). + +3.5. Place the plate on the magnet until beads are collected (approx. 5 min). + +3.6. Wash beads with 100 µl 80% EtOH, incubate 30 sec, discard wash. Repeat. + +3.7. Remove remaining EtOH and let samples air dry for 3-5 min. Avoid over-drying. + +3.8. Resuspend beads in 15 µl H2O, mix well, and collect on magnet. + +3.9. Transfer 10 µl of cleaned cDNA to new 96-well plate using Liquidator or multi-channel. Label the plate accordingly. + +3.10. This is a safe stopping point. Cover plate with foil seal and store in freezer. + +### 4. cDNA Amplification + +4.1. Prepare the master mix for cDNA amplification: + +| Reagent | Volume per sample | Volume in Master Mix for x10 Samples | +| -------------------------- | ----------------- | ------------------------------------ | +| RNA/DNA-free H2O | 6 µl | 60 µl | +| dNTPs (10mM) | 0.5 µl | 5 µl | +| 10x Klentaq 1 buffer | 2 µl | 20 µl | +| 10 µM 5iLL oligo | 0.5 µl | 5 µl | +| 10 µM 3iLL-30TV oligo | 0.5 µl | 5 µl | +| Klentaq | 0.5 µl | 5 µl | +| Purified cDNA | 10 µl | 100 µl | +| Total Volume | 20 µl | 200 µl | + +4.2. Mix by inversion, briefly spin. + +4.3. Aliquot 10 µl of master mix into each cDNA sample. Pipet, cover with foil seal, spin, and start thermocycler program: + +| RNA Input | PCR Cycle # | +| -------------------- | ------------- | +| <150 ng | 18 cycles | +| 150-400 ng | 14 cycles | +| 400-1000 ng | 10 cycles | + + - Initial Denaturation: 94°C, 5 min + - Denaturation: 94°C, 1 min + - Annealing: 63°C, 2 min + - Extension: 72°C, 2 min + - Hold: 4°C, infinite + +### 5. AMPure Cleanup + +5.1. Vortex AMPure beads, dispense 45 µl/sample to reservoir. Prepare a second reservoir with H2O. + +5.2. Centrifuge cDNA plate, add 30 µl H2O, bringing volume to 50 µl. + +5.3. Add 45 µl AMPure beads, mix well, incubate 15 min at RT, wash twice with 100 µl 80% EtOH. + +5.4. Remove any remaining ethanol and air-dry for 3-5 min. + +5.5. Resuspend samples in 22 µl water, mix well, collect beads on magnet for 5 min. + +5.6. Transfer 10 µl amplified cDNA into two new 96-well plates; one for Index PCR, the other for storage (freezer). + +### 6. Index Addition via PCR + +6.1. Document the index primer correlates. Add 3 µl of index (3.9µM) to each sample. + +6.2. Prepare the following master mix: + +| Reagent | Volume per sample | Volume in Master Mix for x10 Samples | +| -------------------- | ----------------- | ------------------------------------ | +| RNA/Nuclease-free H2O| 12.65 µl | 126.5 µl | +| dNTPs | 0.75 µl | 7.5 µl | +| 10x PCR buffer | 3 µl | 30 µl | +| Klentaq | 0.6 µl | 6 µl | +| Amplified cDNA | 10 µl | 100 µl | +| Total Volume | 30 µl | 300 µl | + +6.3. Mix by inversion, briefly spin. + +6.4. Add master mix to samples depending on index plate used. Run thermocycler program: + + - Initial Denaturation: 95°C, 5 min + - Denaturation: 95°C, 40 sec (4 cycles) + - Annealing: 63°C, 2 min + - Extension: 72°C, 2 min + - Hold: 4°C, infinite + +### 7. AMPure Cleanup + +7.1. Vortex AMPure beads, dispense 27 µl/sample to reservoir. Prepare a second reservoir with H2O. + +7.2. Perform 0.9X AMPure bead purification: + + - Add 27 µl AMPure beads + - Mix 10 times + - Incubate 15 min at RT + - Wash with 100 µl 80% EtOH (twice) + - Remove ethanol, air dry for 3-5 min + - Resuspend samples in 28 µl water and mix well + +7.3. Remove 25 µl to a new 96-well plate, label accordingly. + +### 8. PicoGreen and Library QC + +*Supplement with tapestation protocol if desired.* + +8.1. PicoGreen the plate using standards starting at 50ng/µl. Run and save values. + +8.2. Verify library concentration using Qubit if PicoGreen shows low levels. + +8.3. Troubleshoot as necessary, repeat library prep if needed. + +### 9. Pooling + +9.1. Designate pools of 24-32 samples, aiming for 50ng per sample. + +9.2. Enter the ng amount for sample pooling, adjust as necessary. + +9.3. Centrifuge final library plate, label and store tubes. + +### 10. Size Selection + +10.1. Adjust pool volume to 60 µl, use Speedvac for drying or H2O for adjustment. + +10.2. Run 2% dye-free gel on Blue Pippin system to size-select 350-550bp fragments. + +10.3. Label tubes as final libraries, confirm size selection, and store. + +**endofoutput** \ No newline at end of file diff --git a/markdown-output/3-level-sci-rna-seq-with-facs-buxdnxi6.md b/markdown-output/3-level-sci-rna-seq-with-facs-buxdnxi6.md new file mode 100644 index 0000000000000000000000000000000000000000..f3b7d525b2fe914fe9d1839f33d11acb6d383a48 --- /dev/null +++ b/markdown-output/3-level-sci-rna-seq-with-facs-buxdnxi6.md @@ -0,0 +1,238 @@ +```markdown +# Goal/Experiment: +The goal of this experiment is to perform a 3-level sci RNA-Seq (single-cell combinatorial indexing RNA sequencing) with the addition of Fluorescence Activated Cell Sorting (FACS) to decrease background noise during the library preparation stage. + +## 3-level sci RNA-Seq with FACS + +**David Fraser Read¹, Cole Trapnell¹** +¹University of Washington, Dept. of Genome Sciences + +**DOI:** [dx.doi.org/10.17504/protocols.io.buxdnxi6](https://dx.doi.org/10.17504/protocols.io.buxdnxi6) + +### Abstract +This protocol is a variant of "3 level sci RNA-Seq" which includes a FACS sorting step before the PCR stage. This notable addition helps to decrease background in the library preparation. + +### Guidelines +This protocol is an adaptation and includes: +- Addition of FACS sorting to reduce background noise. +- Omission of the USER enzyme reaction step. +- Modified reverse transcription temperature ramp to enhance the number of unique molecular identifiers (UMIs) recovered per nucleus. + +### Materials + +#### Supplies +- **Nuclease-free water** (Ambion, AM 9937) +- **Snap Cap FACS Tube** (Corning, 08-771-23) +- **SUPERase In RNase Inhibitor 20 U/μL** (Thermo Fisher Scientific, AM2696) +- **BSA 20 mg/mL** (NEB, B9000S) +- **1M Tris-HCl (pH 7.4)** (Thermo Fisher Scientific, AM9759) +- **5M NaCl** (Thermo Fisher Scientific, AM9759) +- **1M MgCl2** (Thermo Fisher Scientific, AM9530G) +- **Triton X-100 for molecular biology** (Sigma Aldrich, 93443-100ML) +- **10mM dNTP** (Thermo Fisher Scientific, R0192) +- Indexed oligo-dT primers (100uM, 5'/5Phos/CAGACGNNNNNNNNNNT10bp barcode) +- **Superscript IV RNase Inhibitor** (Invitrogen, 10777019) +- **Quick ligation kit** (NEB, M2200L) +- **Elution buffer** (Qiagen, 19086) +- **NEBNext Ultra II Non-Directional RNA Second Strand Synthesis Module** (NEB, E7550S) +- **DNA binding buffer** (Zymo Research, D4004-1-L) +- **AMPure XP beads** (Beckman Coulter, A63882) +- **Ethanol** (Sigma Aldrich, 459844-4L) +- **Qubit dsDNA HS kit** (Invitrogen, Q32854) +- **Qubit tubes** (Invitrogen, Q32856) +- **Nextera 96 plate** (Illumina) +- Various falcon tubes and tips +- **BrightLine™ Hemacytometer** (Sigma Aldrich) +- **LoBind clear 1.5 mL PCR clean** (Eppendorf, 03-395-565; 22343102) + +#### Equipment +- **FACS Aria II Sorter** with 96 well plate holder +- **Ice buckets** +- **Refrigerated centrifuge** with 15 mL tube holders +- **ScreenTape** (Agilent) +- **Qubit** (Thermo) + +#### N7-loaded Tn5 +The original use of the protocol involved custom Tn5 loaded with Nextera N7 adapters (commercial equivalent example: Illumina FC-121-1030). Alternatively, unloaded Tn5 can be purchased and adapters loaded per the method described [here](https://www.biorxiv.org/content/10.1101/2019.12.17.879304v1.full.pdf). + +## Protocol + +### Buffer Preparation + +1. **Nuclei Buffer:** + - Combine: + - 10 mM Tris-HCl (pH 7.4) + - 10 mM NaCl + - 3 mM MgCl2 + - Store at 4°C. + +2. **Nuclei Suspension Buffer (NSB):** + - 1 mL Nuclei Buffer + - 10 μL BSA + - 10 μL SUPERaseIn + - Chill on ice. + +3. **Nuclei Buffer with BSA (NBB):** + - 1 mL Nuclei Buffer + - 10 μL BSA + - Chill on ice. + +4. **10% Triton X-100 Stock:** + - 1 mL Triton X-100 + - 9 mL Nuclease-free water. + - Store at 4°C. + +5. **Permeabilization Buffer:** + - 500 μL per sample: + - 12.5 μL of 10% Triton X-100 + - 487.5 μL NSB + - Pre-chill on ice. + +### Permeabilization + +6. **Thaw** + - Thaw frozen aliquots at 37°C in water bath. + +7. **Buffer Addition** + - Add 400 μL of Permeabilization Buffer. Mix gently. + +8. **Incubate** + - Incubate for 3 minutes on ice. + +9. **Pellet and Resuspend** + - Pellet at 500g for 5 min (4°C), discard supernatant and resuspend. + +10. **Recentrifuge** + - Pellet at 500g for 5 min (4°C), discard supernatant. + +11. **Resuspend** + - Resuspend in 300 μL NSB and count nuclei with hemocytometer. + +### Reverse Transcription + +12. **Setup RT reaction** + - 30,000 nuclei in 22 μL Nuclei buffer + - 2 μL 10mM dNTP + - 2 μL indexed oligo-dT primer (100uM) + - Incubate at 55°C for 5 min, then cool on ice. + +13. **Prepare RT Mix** + - 8 μL SuperScript IV First-Strand Buffer + - 2 μL 100mM DTT + - 2 μL SuperScript IV reverse transcriptase + - 2 μL RNaseOUT RNase Inhibitor + +14. **Distribute RT mix and Incubate** + - Distribute 14 μL to each well. Incubate at following steps: + - 4°C for 2 mins + - 10°C for 2 mins + - 20°C for 2 mins + - 30°C for 2 mins + - 40°C for 2 mins + - 50°C for 2 mins + - 53°C for 15 mins + - 55°C for 10 mins + - Add 60 μL ice-cold NBB post reaction. + +15. **Pool Nuclei** + - Pool Nuclei, pellet at 600 RCF for 10 min (4°C). + +### Ligation + +16. **Resuspend Nuclei** + - Resuspend nuclei in 1 mL NSB. + +17. **Distribute and Add Indexing Oligos** + - Distribute 10 μL to each well, add 8 μL indexing oligos (100uM). + +18. **Prepare Ligation Mix** + - Combine: + - 2 μL Quick Ligase + - 20 μL Quick Ligase buffer + - Distribute 22 μL to each well. + +19. **Mix and Ligate** + - Mix by pipetting, then incubate at 25°C for 10 min. + - Add 60 μL NBB, pool all wells. + +20. **Spin and Resuspend** + - Add 10 mL NBB, spin at 600 RCF, 10 min (4°C), supernatant discarded. + - Resuspend in 1 mL Elution Buffer. + +21. **Add DAPI and Filter** + - Add 10 μL of 300 μM DAPI, mix gently. + - Filter through a 35 μM FACS tube. + +### FACS Sorting + +22. **Sort Nuclei** + - Add 4 μL Elution Buffer to each well, sort based on DAPI. + +### Second Strand Synthesis + +23. **Volume Check** + - Ensure volume in well is ~12 μL. Adjust input volumes if necessary. + +24. **Prepare and Add Second Strand Mix** + - For each well, prepare: + - 1.33 μL second strand buffer + - 0.67 μL enzyme mix + - Add 2 μL per well. + +25. **Incubate** + - Incubate at 16°C for 3 hours. + +### Tagmentation + +26. **Make TD Buffer** + - 1.2 mL tagmentation salt buffer + - 300 μL dimethylformamide + +27. **Prepare Mix** + - 12.5 μL 2x TD buffer, + - 12.5 μL second tagmentation mix + - (optional: 0.02 μL N7 loaded Tn5) + - Incubate 5 min at 55°C. + +### Ampure Bead Purification + +28. **Add 50 μL of Ampure Beads** + - Incubate 5 min, transfer to magnet, incubate 3 min more. + +29. **Wash beads** + - Twice with ~150 μL 80% ethanol. + +30. **Resuspend Beads** + - Add 17 μL EB. + +### Post-Bead Cleanup + +31. **Bead Cleanup** + - With .7 volumes bead volume, wash with 80% ethanol. + +### PCR + +32. **Setup PCR** + - 2 μL indexed P5 PCR primer (10uM) + - 2 μL P7 primer (10uM) + - 20 μL NEBNext master mix + - PCR setting: + - 72°C 5 min + - 98°C 30 secs + - 17 cycles: 98°C 10 secs, 66°C 30 secs, 72°C 30 secs + - 72°C 5 min + +### Quantify and Sequence + +33. **Quantify & Sequence Sample** + - Quantified using Qubit and Agilent ScreenTape. + - Sequenced on Illumina Nextseq 2000, 100 bp kit. + + - Read settings: + - Read 1: 34 bases + - Read 2: 66 bases + - Index 1: 10 bases + - Index 2: 10 bases + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/605ceft-resting-medium-no-selection-c4mqyu5w.md b/markdown-output/605ceft-resting-medium-no-selection-c4mqyu5w.md new file mode 100644 index 0000000000000000000000000000000000000000..9b24db12521782c35a2dae1b81d8192630decfbb --- /dev/null +++ b/markdown-output/605ceft-resting-medium-no-selection-c4mqyu5w.md @@ -0,0 +1,136 @@ +```markdown +# 605CefT - Resting Medium (no selection) + +## Author +[leiboffs](https://protocols.io/researchers/70532) +Oregon State University, College of Agricultural Sciences, Department of Botany and Plant Pathology + +## Disclaimer +**DISCLAIMER – FOR INFORMATIONAL PURPOSES ONLY; USE AT YOUR OWN RISK** + +The protocol content here is for informational purposes only and does not constitute legal, medical, clinical, or safety advice, or otherwise; content added to [protocols.io](https://protocols.io) is not peer reviewed and may not have undergone a formal approval of any kind. Information presented in this protocol should not substitute for independent professional judgment, advice, diagnosis, or treatment. Any action you take or refrain from taking using or relying upon the information presented here is strictly at your own risk. + +## License +This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. + +## Protocol Status +Working +We use this protocol and it's working + +## Created +Nov 06, 2023 + +## Last Modified +Nov 07, 2023 + +## Protocol Integer ID +90512 + +## Funding Acknowledgement +NSF +Grant ID: IOS-2211435 + +## Goal/Experiment +This protocol provides guidelines for preparing 605CefT medium, used in the transformation protocol for somatic embryogenesis of B104 immature maize embryos. The focus is on preventing Agrobacterium contamination and encouraging rapid plant growth. + +## Abstract +This part of the Leiboff Lab maize transformation protocol aims to transfer embryos from Co-cultivation Medium 562v-MSM to Resting Medium 605CefT to suppress Agrobacterium contamination. This ensures growth and preparation of embryos for further experimental procedures. + +Embryos will be transferred scutellum side up after 3 days of infection, using 605CefT for 7 days before moving to 605CefTB. 605CefT includes synthetic auxin (2,4-D) and uses Cefotaxime and Timentin for suppressing Agrobacterium contamination. + +### Professional/Scientific Terms and Reagents +- **2,4-D (2,4-Dichlorophenoxyacetic acid):** A synthetic auxin used for promoting callus formation and shoot growth. +- **Cefotaxime and Timentin:** Antibiotics used to control Agrobacterium contamination. +- **Sucrose and D-Glucose:** Sugars that support rapid plant growth. +- **Casein Hydrolysate:** A mixture of amino acids and peptides used as a nitrogen source. +- **Phytagar:** A gelling agent used to solidify the medium. + +### Equipment and Vendors +- **pH Meter:** Hanna Instruments +- **Autoclave:** Cord 3112 and 4112 +- **Beakers, Stir Bars, and Graduated Cylinders:** Standard lab suppliers + +### Alternative Methods +If specific reagents are difficult to source: +- Casein Hydrolysate can be replaced with a similar complex nitrogen source. +- If synthetic auxin (2,4-D) is unavailable, consider using another auxin-like Indole-3-acetic acid (IAA). + +## Planning +1. Estimate the volume of 605CefT needed: + + \[ + \text{Volume} = 30 \text{mL} \times \text{Number Plates} + \] + +2. Prepare mixing ingredients: + - **Retrieve Heat-Stable Ingredients:** + - 605 Medium + - Casein Hydrolysate + - 2,4-D (5 mg/mL) + - Sucrose + - D-Glucose + - Agar, Phyto + +3. Equipment set-up: + - Graduated cylinder + - Beaker with a stir bar + +## Procedure + +### Mixing Heat-Stable Ingredients + +1. **Clean equipment:** + - Rinse stir bar, beaker, and graduated cylinder with MQ H2O. + +2. **Prepare solution in beaker:** + - Add approximately 90% of final volume using MQ H2O to the beaker. + - Place on a magnetic stir plate. + +3. **Add ingredients:** + - Use a dry spatula/pipette for each ingredient. + - Use the table for specific quantities. + + | Ingredient | 100 mL | 200 mL | 300 mL | 600 mL | + |------------|--------|--------|--------|--------| + | 605 Medium | 1.1 g | 2.2 g | 3.3 g | 6.6 g | + | Casein Hydrolysate | 0.03 g | 0.06 g | 0.09 g | 0.18 g | + | 2,4-D | 11.5 µL | 23 µL | 34.5 µL | 69 µL | + | Sucrose | 2.0 g | 4.0 g | 3.0 g | 6.0 g | + | D-Glucose | 0.06 g | 0.12 g | 0.18 g | 0.36 g | + +4. **Adjust pH:** + - Set pH to 5.7 using 0.1 M KOH, stir solution. + +### Bring to Target Volume and Autoclave + +1. Bring solution to target volume: + - Add water, transfer to a graduated cylinder. + +2. Add Phytoagar: + - According to required mass. + +3. Autoclave: + - Loosely cap the bottle, autoclave using 'Liquid' setting for 20-25 min. + +### Adding Heat-sensitive Ingredients +1. Retrieve solution from autoclave, cool to 55°C. +2. Add heat-sensitive ingredients: + - Use table below for quantities. + + | Ingredient | 100 mL | 200 mL | 300 mL | 600 mL | + |------------|--------|--------|--------|--------| + | Dicamba | 120 µL | 240 µL | 360 µL | 720 µL | + | Silver nitrate | 340 µL | 680 µL | 1020 µL | 2040 µL | + | Cef | 100 µL | 200 µL | 300 µL | 600 µL | + | Tim | 33 µL | 67 µL | 100 µL | 200 µL | + +### Final Steps + +1. Pour media into clean sterile plates (~30 mL/plate). +2. Close plates to solidify in a laminar flow hood. + +## Storage +- Store plates upside-down at 4°C for up to 1 week. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/605ceftb-resting-medium-basta-selection-c4utywwn.md b/markdown-output/605ceftb-resting-medium-basta-selection-c4utywwn.md new file mode 100644 index 0000000000000000000000000000000000000000..6722ac1d08e36b53a1d58b3a343ead190b1a9208 --- /dev/null +++ b/markdown-output/605ceftb-resting-medium-basta-selection-c4utywwn.md @@ -0,0 +1,207 @@ +```markdown +# Goal/Experiment: +### Maize Transformation Protocol for Somatic Embryogenesis of B104 Immature Embryos Using Resting Medium (basta selection) + +## Title: 605CefTB - Resting Medium (basta selection) + +### Author: +leiboffs1 + +1 Oregon State University, College of Agricultural Sciences, Department of Botany and Plant Pathology + +### DISCLAIMER +> DISCLAIMER – FOR INFORMATIONAL PURPOSES ONLY; USE AT YOUR OWN RISK +> +> The protocol content here is for informational purposes only and does not constitute legal, medical, clinical, or safety advice, or otherwise; content added to [protocols.io](https://protocols.io) is not peer reviewed and may not have undergone a formal approval of any kind. Information presented in this protocol should not substitute for independent professional judgment, advice, diagnosis, or treatment. Any action you take or refrain from taking using or relying upon the information presented here is strictly at your own risk. You agree that neither the Company nor any of the authors, contributors, administrators, or anyone else associated with [protocols.io](https://protocols.io), can be held responsible for your use of the information contained in or linked to the protocol or any of our Sites/Apps and Services. + +## ABSTRACT +This protocol is a part of the Leiboff Lab maize transformation protocol for somatic embryogenesis of B104 immature embryos. This protocol is a combination of methods from Chen et al. 2022 and Kang et al. 2022 with modifications for material availability. It is intended for the GRF-GIF/BBM somatic embryogenesis transformation strategy with the LBA4404 Met- auxotrophic Agrobacterium strain. + +Embryos will be transferred (scutellum side up) from Resting Medium 605CefT to Resting Medium 605CefTB, 10 days after infection (DAI). 605CefT should be used for 7 days, before moving embryos to Shoot Maturation Medium 13329A. Resting Medium contains added synthetic auxin (2,4-D) to encourage callus and shoot growth. 605CefTB is high in sucrose and uses a small amount of glucose to encourage rapid plant growth. 605CefTB contains 5 mg/L of bialaphos (preferred for basta selection in maize over glufosinate) as a plant selective agent, and uses both Cefotaxime and Timentin to control Agrobacterium contamination. Concentrations here are sufficient to control the LBA4404 Met- strain, but not wild-type LBA4404 in prior trials. + +605CefTB solid media should be prepared in 15x100 (standard) petri plates, planning for ~20 embryos per plate. Material on 605CefTB will be sealed with micropore tape and incubated at 28°C in the dark. Embryos are ready to move off 605CefTB after 1 week. Noticeable growth should occur on the scutellum side, indicating somatic embryo establishment. + +## Planning +1. Estimate the volume of 605CefTB needed: + \[ + \text{Volume} = 30mL \times \text{NumberPlates} + \] + + Round up the volume. Check the table below to plan your media needs. + +## Mixing Heat-Stable Ingredients +2. Retrieve the following heat-stable ingredients: + - 605 Medium - Stored in Main Lab, 4C Refrigerator, Top Shelf + - Casien Hydrolysate - Stored in Main Lab, Chemical shelf 'C', use Megraw stock + - 2,4-D (5 mg/mL) - Stored in Main Lab, -20C Freezer, Bottom drawer 'Tissue Culture 1' + - Sucrose - Stored in Main Lab, Chemical shelf 'S', use Fowler refillable container + - D-Glucose - Stored in Main Lab, Chemical shelf 'G' + - Agar, Phyto - Stored in Main Lab, Chemical shelf 'A' + +3. Retrieve a graduated cylinder for measuring your final solution. + - Place a stir bar in a beaker 1.5x the volume of your solution. + - Rinse stir bar and beaker with MQ H2O, discard rinse water in sink. + +## Preparation of Medium +4. Add approximately 90% of final media volume in MQ H2O to the beaker. + - Place beaker on a magnetic stir plate. + - Turn stir plate on to generate a vigorous stir. + +5. Using fresh weigh paper and dry spatula/scoopula/pipette tip for each ingredient, add the following to your beaker: + +| Volume (mL) | 605 Medium (g) | Casien Hydrolysate (g) | 2,4-D (μL) | Sucrose (g) | D-Glucose (g) | +|---------------|-----------------|------------------------|------------|-------------|---------------| +| 100 | 1.1 | 0.03 | 11.5 | 2.0 | 0.06 | +| 200 | 2.2 | 0.06 | 23 | 4.0 | 0.12 | +| 300 | 3.3 | 0.09 | 34.5 | 3.0 | 0.18 | +| 600 | 6.6 | 0.18 | 69 | 6.0 | 0.36 | + +6. Thoroughly rinse all used tools with running water. + - Place clean tools in drying rack. + - Return chemical reagents to their original storage location. + +## Adjust Solution pH to 5.7 with 0.1 M KOH +7. Turn on the Hanna Instruments pH meter. + - Unscrew and remove the small green pH probe exchange cover and set cap aside. + - Gently remove probe from storage tube and set storage tube aside. + - Using squeeze bottle, rinse the glass probe with H2O, catch rinse water in a waste beaker. + - Gently blot probe with laboratory tissue paper to dry. + +8. Using adjustable arm, lower the pH probe into the beaker with stir plate on. + - Ensure stir bar does not strike the probe. + - Electrode at the base of the probe must be fully submerged. + +9. Using a plastic transfer pipette, add 0.1M KOH to solution until pH 5.7 is measured. + - Note: KOH can be added rapidly until pH 5.4, then one drop at a time to reach pH 5.7. pH between 5.6 - 5.8 is acceptable. + +10. Using the adjustable arm, remove pH probe from beaker. + - Using squeeze bottle, rinse the glass probe with H2O, catch rinse water in a waste beaker. + - Gently blot probe with laboratory tissue paper to dry. + - Return probe to storage tube – ensure the electrode bulb is fully submerged in storage solution. + - Return and secure the small probe exchange cover. + - Turn off the pH meter. + +## Bring Solution to Target Volume, Add Phytoagar, and Autoclave +11. Turn off the stir plate and remove your beaker. + - Hold a large stir bar in your hand to stabilize the one in your beaker. + - Pour solution into the graduated cylinder– do not include the stir bar. + - Add a small amount (50-100 mL) of water to your beaker. + - Carefully add water from the beaker to the graduated cylinder until solution reaches the target volume– do not include the stir bar. + +12. Retrieve a clean dry bottle and matching cap. + - Using fresh weigh paper and dry spatula/scoopula: + + | Volume (mL) | Phytoagar (g) | + |-------------|---------------| + | 100 | 0.6 | + | 200 | 1.2 | + | 300 | 1.8 | + | 600 | 3.6 | + + - Add phytoagar to dry bottle. + - Note: Adding phytoagar to dry bottle avoids clumping which is undesirable for final media. + +13. Loosely place cap on bottle. + - Add a small piece of autoclave tape on cap and bottle. + - Place bottle in an autoclave-safe bin. + - Autoclave 20-25 min using the 'Liquid' setting. + - Note: Recommended autoclaves are in Cord 3112 and 4112. Complete cycle will take ~1 hr. + +14. Rinse all used tools and glassware in running water. + - Place clean items on drying rack. + - Return chemical reagents to original storage location. + +## Adding Heat-sensitive Ingredients +15. Return to autoclave to pick up your solution– Be prompt, sucrose can degrade if left too long. + - Using autoclave gauntlets, gently seal cap of bottle. + - Swirl autoclaved solution to evenly mix phytoagar. + +16. Carefully return to lab with autoclave bin and sealed bottle. + - Place sealed solution into large 55°C water bath in main lab. + - Discard any liquid remaining in autoclave bin and return to bin storage. + - Note: Solution needs to reach ~55°C before adding heat-sensitive ingredients. + +17. Retrieve the following heat-sensitive ingredients: + - Dicamba (1 mg/mL) - Stored in Main Lab, -20C Freezer, Bottom drawer 'Tissue Culture 2' + - Silver nitrate (1 mg/mL) - Stored in Main Lab, -20C Freezer, Bottom drawer 'Tissue Culture 2' + - Cefotaxime (100 mg/mL), 'Cef' - Stored in Main Lab, -20C Freezer, 'Antibiotics 2' + - Timentin (300 mg/mL), 'Tim' - Stored in Main Lab, -20C Freezer, 'Antibiotics 2' + - Bialaphos (1 mg/mL) - Stored in Main Lab, -20C Freezer, 'Tissue Culture 3' + +18. Turn on laminar flow hood, airflow, and lamp. + - Using 70% EtOH spray bottle and paper towels, sterilize working area inside laminar flow hood. + - Retrieve sterile petri plates. + - Using fine-tipped sharpie, write '605CefT' and date along bottom rim of plate. + +19. When solution reads 55°C with digital thermometer gun: + - Transfer sealed bottle to laminar flow hood. + - Bottle should be warm, but safe to handle. + - Sterilize outside of bottle and gloved hands with 70% ethanol spray. + +20. Using fresh filter tip for each ingredient, add the following to your bottle: + +| Volume (mL) | Dicamba (μL) | Silver nitrate (μL) | Cef (μL) | Tim (μL) | Bialaphos (μL) | +|-------------|---------------|---------------------|----------|----------|----------------| +| 100 | 120 | 340 | 100 | 33 | 500 | +| 200 | 240 | 680 | 200 | 67 | 1000 | +| 300 | 360 | 1020 | 300 | 100 | 1500 | +| 600 | 720 | 2040 | 600 | 200 | 3000 | + + Used tips may be disposed of in regular lab waste – no contact with rDNA or modified cells is anticipated. + +21. Gently swirl media bottle to mix thoroughly, but avoid introducing bubbles. + - Pour media into plates, ~30 mL per plate. + - Note: Each plate should be more than half-full with media. + - Close plates to solidify in laminar flow hood. + +22. Using paper towels, clean any spilled media and discard in regular lab waste. + - When plates are poured, rinse media bottle in lab sink and hang on bottle rack to dry. + - Return reagents to original storage location. + - Using 70% EtOH spray bottle and paper towels, sterilize working area inside laminar flow hood for next worker. + +23. Leave closed plates to solidify in laminar flow hood with the fan on, 3 hrs - overnight. + - Note: Keep plates ~10 cm (4 in) away from back of flow hood to avoid drying out. + +When plates are solid, wrap in a clean plate bag or individually seal with parafilm and store upside-down at 4°C, up to 1 week. + +--- + +## Terms and Reagents: +1. **605 Medium:** Basal medium for plant tissue culture. +2. **Casien Hydrolysate:** Protein hydrolysate used as a complex supplement. +3. **2,4-D (2,4-Dichlorophenoxyacetic acid):** Synthetic auxin to promote plant growth and callus induction. +4. **Sucrose:** Disaccharide sugar critical for energy and carbon source. +5. **D-Glucose:** Simple sugar used for metabolic energy. +6. **Phytoagar:** Solidifying agent for culture media. +7. **Dicamba:** Synthetic auxin used as a growth regulator. +8. **Silver Nitrate:** Used for its antimicrobial properties. +9. **Cefotaxime:** Antibiotic to control bacterial contamination. +10. **Timentin:** Combination antibiotic for broader antibacterial spectrum. +11. **Bialaphos:** Phosphinothricin-based herbicide for plant selection. +12. **KOH (Potassium hydroxide):** Used to adjust pH. + +## Equipment: +- Magnetic Stir Plate +- Graduated Cylinder +- Beaker +- Stir Bar +- Laminar Flow Hood +- Autoclave (`Cord 3112` and `Cord 4112`) +- Digital Thermometer Gun +- pH Meter (`Hanna Instruments`) +- Petri Plates (15x100 mm) + +## Vendors: +- Chemical shelf locations in Main Lab + +## Alternatives: +- 2,4-D can be substituted with other auxins based on availability. +- Phytoagar can be replaced with other high-quality agars, though consistency should be tested. + +## Funders: +- NSF, Grant ID: IOS-2211435 + +--- + +**endofoutput** +``` \ No newline at end of file diff --git a/markdown-output/a-cellprofiler-computational-pipeline-to-quantify-dhja34ie.md b/markdown-output/a-cellprofiler-computational-pipeline-to-quantify-dhja34ie.md new file mode 100644 index 0000000000000000000000000000000000000000..2efe9b59d812a974de1659780c2774798d799846 --- /dev/null +++ b/markdown-output/a-cellprofiler-computational-pipeline-to-quantify-dhja34ie.md @@ -0,0 +1,125 @@ +```markdown +# Goal/Experiment: +To quantify the density of mouse striatal dopaminergic processes using a CellProfiler computational pipeline. + +# A CellProfiler Computational Pipeline to Quantify the Density of Mouse Striatal Dopaminergic Processes + +### DOI +[dx.doi.org/10.17504/protocols.io.x54v92km4l3e/v1](https://dx.doi.org/10.17504/protocols.io.x54v92km4l3e/v1) + +### Authors +- **Ebsy Jaimon** +- **Sreeja V Nair** +- **Suzanne R Pfeffer** + +**Department of Biochemistry, Stanford University School of Medicine and Aligning Science Across Parkinson's** + +### Protocol Citation +_Ebsy Jaimon, Sreeja V Nair, Suzanne R Pfeffer 2024. A CellProfiler computational pipeline to quantify the density of mouse striatal dopaminergic processes. **protocols.io**_ + +### License +This is an open access protocol distributed under the terms of the [Creative Commons Attribution License](https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. + +### Protocol Status +Working + +### Created +July 19, 2024 + +### Last Modified +July 19, 2024 + +### Protocol Integer ID +103746 + +### Keywords +- ASAPCRN +- LRRK2 +- Primary cilia +- Dorsal striatum + +### Funders Acknowledgement +Aligning Science Across Parkinson's - Grant ID: ASAP-000463 + +## Abstract +Here, we present a CellProfiler software pipeline to quantify the density and intensity of dopaminergic processes in the mouse striatum. The dopaminergic processes in the striatum are stained using an anti-tyrosine hydroxylase antibody. The same sections are stained using an antibody that recognizes total neuronal NeuN (neuronal nuclear antigen biomarker) for staining normalization. For the examples shown herein, images were acquired using a Zeiss LSM 900 laser scanning confocal microscope with a 63X 1.4 oil immersion objective. + +*References:* +1. Stirling DR, Swain-Bowden MJ, Lucas AM, Carpenter AE, Cimini BA, Goodman A (2021). CellProfiler 4: improvements in speed, utility, and usability. BMC Bioinformatics, 22 (1), 433. PMID: 34507520 PMCID: PMC8431850. + +## Materials +1. `.czi` files from Zeiss Laser Scanning Microscope +2. FIJI/ImageJ +3. CellProfiler software 4.0+ + +## Methods + +### Batch Process Images +1. Use the FIJI macro as described in [dx.doi.org/10.17504/protocols.io.3byl4bpo8vo5/v1](https://dx.doi.org/10.17504/protocols.io.3byl4bpo8vo5/v1) to Z-project the `.czi` images from the Zeiss LSM microscope. +2. Open the images that need to be processed, choose the output folder, run the code for maximum intensity Z projection, and save the file as .TIFF. + +### Import Files and Extract Metadata +1. Open CellProfiler. Go to the Images module, drag and drop the maximum intensity projected .TIFF files as indicated. Select "no filtering" in the filter images option. +2. Go to the Metadata module: + - Set _Extract Metadata?_ to Yes. + - Set _Metadata extraction method_ to Extract from image file headers. + - Set _Extract metadata from_ to All images. + - Click _Extract metadata_. + - Click on Add another extraction method. + - Set _Metadata extraction method_ to Extract from file/folder names. + - Set _Metadata source_ to File name. + - Set Regular expression to extract from file name: + ``` + ^.*br(?P[0-9]{1,2}).*#(?P[0-9]{2}) + ``` + Note these steps: + - In Regex, ^ indicates the beginning of the file name. + - The program recognizes and extracts brain and image numbers. + - Click _update_ to populate the metadata field. + - Set _Metadata data type_ to Text. +3. Go to the NamesAndTypes module: + - Assign a name to images matching rules. + - Process as 3D: No + - Match "All" of the following rules. + - Select the rule criteria: Metadata/Does/Have C matching/0 + - Name to assign these images: TyrosineHydroxylase + - Set the image type: Grayscale image + - Click on Add another image and set similar parameters for NeuN staining. + +### Density Measurement +1. To binarize the images: + - Select the input image: TyrosineHydroxylase + - Name the output image: Thresholded_TyrosineHydroxylase + - Threshold strategy: Global + - Thresholding method: Minimum Cross-Entropy + - Set threshold scales and corrections as required. + +Note: Use test settings to ensure the best results. + +2. Add `MeasureImageAreaOccupied` module: + - Measure the area occupied by: Binary image. + - Select binary images to measure: Thresholded_TyrosineHydroxylase. + +### Intensity Measurement +1. To segment Tyrosine Hydroxylase and NeuN: + - Add IdentifyPrimaryObjects module. + - Use advanced settings: Yes. + - Segment objects according to parameters. + - Adjust modules and test mode settings. + +2. Segment NeuN: + - Add Smooth module, then IdentifyPrimaryObjects for segmentation. + - Similar advanced settings as previous steps. + +3. Add modules MeasureObjectIntensity and MeasureObjectSizeShape to measure the integrated intensity and area of the objects (THoverjects and NeuNobject). + +### Export Data +- Add ExportToSpreadsheet module: + - Select measurements to export under specific categories and criteria. + - Save the pipeline and run the analysis. + +## Protocol References +1. Stirling DR, Swain-Bowden MJ, Lucas AM, Carpenter AE, Cimini BA, Goodman A (2021). CellProfiler 4: improvements in speed, utility and usability. BMC Bioinformatics, 22 (1), 433. PMID: 34507520 PMCID: PMC8431850. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/a-high-throughput-assay-for-quantifying-phenotypic-bxptpmnn.md b/markdown-output/a-high-throughput-assay-for-quantifying-phenotypic-bxptpmnn.md new file mode 100644 index 0000000000000000000000000000000000000000..77963fe35dc61533d94479ecf013aac144687833 --- /dev/null +++ b/markdown-output/a-high-throughput-assay-for-quantifying-phenotypic-bxptpmnn.md @@ -0,0 +1,105 @@ +```markdown +# Goal/Experiment: +This experiment aims to measure 10 phenotypic traits of centric diatoms using high-throughput methodologies. This versatile assay provides detailed insights into the various characteristics of microalgae, particularly focusing on growth rates, reactive oxygen species production, photophysiological traits, and other key indicators of cellular health and activity. + +## A High-Throughput Assay for Quantifying Phenotypic Traits of Microalgae + +### Authors +Phoebe Argyle1,2, Jana Hinners3, Nathan G. Walworth4, Sinéad Collins5, Naomi M. Levine4, Martina A. Doblin1,6 + +1. Climate Change Cluster, University of Technology Sydney, Sydney, NSW, 2007, Australia +2. Ministry of Marine Resources, Cook Islands +3. Institute of Coastal Ocean Dynamics, Helmholtz-Zentrum Hereon, 21502, Geesthacht, Germany +4. Department of Biological Sciences, University of Southern California, Los Angeles, CA, 90089-0371, USA +5. Institute of Evolutionary Biology, University of Edinburgh, Edinburgh, EH9 3JF, UK +6. Sydney Institute of Marine Science, Mosman, NSW, 2088, Australia + +### Abstract +This outlines a workflow for measuring 10 phenotypic traits of centric diatoms using a variety of methodologies. + +### Citation +- Argyle, P. A., Hinners, J., Walworth, N. G., Collins, S., Levine, N. M., & Doblin, M. A. (2021). A high-throughput assay for quantifying phenotypic traits of microalgae. *Frontiers in microbiology*, 12, 706235. + +## Protocol Steps + +### Set up Experimental Cultures + +1. **Initial Setup (Approx. 5 minutes)**: + - Grow experimental cultures in 12-well tissue culture plates. + - Use triplicate cultures per treatment. + - Initial cell concentration: 2000 cells/mL, adjustable based on growth expectations. + +2. **Stock Preparation**: + - Add 400 µL of stock culture (at 11000 cells/mL) to 4 mL of growth media per well, achieving 4.4 mL total volume with 2000 cells/mL. + - Measure concentration of initial stock using flow cytometry: + ```markdown + **Protocol:** Measuring Growth Rates of Diatom Cells in Culture + **Created By:** Phoebe Argyle + ``` + +3. **Concentration Adjustment**: + - Use centrifugation (1000 x g, 20°C, 5 minutes) to adjust concentration if required. + +4. **Sealing Plates**: + - Seal plates with breathable seal (Breathe-Easy® sealing membrane from Sigma-Aldrich, SKU: Z380059-1PAK). + +### Track Growth + +3. **Initial Fluorescence Measurement**: + - Take in vivo fluorescence measurement post-inoculation with a microplate reader: + ```markdown + **Protocol:** Measuring Growth Rates of Diatom Cells in Culture + **Created By:** Phoebe Argyle + ``` + +4. **Daily Monitoring**: + - Measure in vivo fluorescence daily at least 1 hour post-photoperiod onset (e.g., 9 am post 6 am light onset on a 12:12 light cycle). + - Monitor the growth phase and note exponential growth for trait measurement. + +### Trait Measurements + +5. **Mid-Exponential Phase**: + - Once culture reaches mid-exponential phase, begin trait measurements. + - Ensure staggered harvesting due to varying readiness of culture wells. + +### Detailed Workflow (Quantitative Phenotyping Assay - QPA) + +#### At Mid-Exponential Phase + +6. **Split Culture (Flow Cytometry Preparation)**: + - 200 µL culture + PFA fixative for flow cytometry. + +7. **Reactive Oxygen Species (ROS) Measurement**: + - 2 x 500 µL culture (1 stained H2DCFDA + 1 blank control). + - 2-hour dark incubation at growth temperature, followed by fluorescence reading. + +8. **Photophysiological Traits**: + - Daily in vivo fluorescence tracking. + - Measure using Water-PAM for Rapid Light Curves. + +9. **Silicification**: + - 1 mL culture + 1 mL artificial seawater into a quartz cuvette. + - Analyze post-Water-PAM measurements. + +10. **Additional Flow Cytometry**: + - Use BODIPY 10-minute incubation, followed by flow cytometry. + - Traits measured: Cell Size, Granularity, Chlorophyll a, Neutral Lipids, Silicification. + +### Statistical Analysis + +11. **Data Analysis**: + - Perform Principal Component Analysis (PCA) to visualize multivariate trait data. + - Identify differences between strains/species and relationships between traits. + +```markdown +### References +- Argyle, P., et al. (2021). High-throughput assay for quantifying phenotypic traits of microalgae. Frontiers in microbiology, 12, 706235. +``` + +```markdown +- Licenses: This protocol follows the Creative Commons Attribution License. +- Protocol Status: Working, created on Aug 25, 2021. +``` + +## endofoutput +``` \ No newline at end of file diff --git a/markdown-output/a-high-throughput-cost-efficient-library-preparati-rt8d6rw.md b/markdown-output/a-high-throughput-cost-efficient-library-preparati-rt8d6rw.md new file mode 100644 index 0000000000000000000000000000000000000000..f082681c889ae3a9cab0f19cad7a70cd735c6974 --- /dev/null +++ b/markdown-output/a-high-throughput-cost-efficient-library-preparati-rt8d6rw.md @@ -0,0 +1,219 @@ +```markdown +Goal/Experiment: +To develop a high throughput, cost-efficient library preparation protocol for large-scale next generation sequencing using a Tn5 transposase-based library construction procedure. + +# A High Throughput, Cost-efficient Library Preparation Protocol for Large Scale Next Generation Sequencing (Version 2) + +yunjun Zan, Örjan Carlborg + +## Abstract + +Previously, Picelli et al. (Picelli et al., 2014) reported a Tn5 transposase-based library construction procedure for Illumina sequencing. Here, we describe an optimized procedure for high throughput library preparation to facilitate large-scale sequencing that does not rely on advanced lab equipment. The Tn5 transposase used can be purified using a publicly available construct (Picelli et al., 2014). Reaction buffers and primers can be prepared using standard chemicals available from common suppliers. + +**Citation:** yanjun Zan, Örjan Carlborg A high throughput, cost-efficient library preparation protocol for large scale next generation sequencing. *protocols.io* dx.doi.org/10.17504/protocols.io.rt8d6rw +**Published:** 23 Jul 2018 + +## Guidelines + +### 1. Introduction + +Previously, Picelli et al. (Picelli et al., 2014) reported a Tn5 transposase-based library construction procedure for Illumina sequencing. Here, we describe an optimized procedure for high throughput library preparation to facilitate large-scale sequencing that does not rely on advanced lab equipment. The Tn5 transposase used can be purified using a publicly available construct (Picelli et al., 2014). Reaction buffers and primers can be prepared using standard chemicals available from common suppliers. + +### 2. DNA Input Recommendations + +#### 2.1. Sensitivity to DNA Preparation Protocol + +We have tested this protocol with DNA prepared using several different methods (QIAGEN Maxi Blood kits, QIAGEN DNeasy Blood & Tissue Kits, QIAGEN Gentra Puregene Blood Kit) with DNA eluded in water and TE. We did not find the protocol to be sensitive to these factors. + +#### 2.2. DNA Quality and Quantity + +The quality of the input DNA is not a major concern for preparation of sequencing libraries using this protocol. We have prepared libraries using DNA stored in -20°C for up to ~20 years from chicken and foxes with good results. + +This protocol is optimized for DNA input of 10 ng. Lower DNA input, 1-5 ng, will yield sufficient amount of library for sequencing by increasing the number of PCR cycles. If possible, we would recommend using 10 ng to reduce the amount of PCR duplicates. + +## Protocol + +### 3. Enzyme Purification + +**Step 1:** +The enzyme was produced from a plasmid constructed by Picelli et al. (Picelli et al., 2014), which has been deposited to AddGene ([http://www.addgene.org/](http://www.addgene.org/), pTXB1-Tn5; plasmid # 60240). A protocol describing enzyme purification from this is available in Picelli et al. (Picelli et al., 2014). + +### 4. Buffer Preparation + +**Step 2:** + +#### 4.1. 2xTn5 Dialysis Buffer (DF) + +| Component | Final Concentration | 1L (H₂O added to vol.) | +|--------------------|-----------------------|------------------------------| +| 100 mM Hepes, pH 7.2 | 100 mL 1M or 23.83 g | +| 200 mM NaCl | 11.69 g NaCl | +| 0.2 mM EDTA | 400 ul 500 mM | +| 2 mM DTT | 2 ml 1M | +| 0.2% Triton X-100 | 2 ml Triton X-100 | +| 20% Glycerol | 252 g 100% Glycerol | + +#### 4.2. 5X TAPS-MgCl₂ + +- 50 mM TAPS-NaOH at pH 8.5, 25 mM MgCl₂ + +All chemicals are ordered from Sigma ([https://www.sigmaaldrich.com/](https://www.sigmaaldrich.com/)). + +### 5. Workflow + +**Step 3:** +The workflow includes the following steps (numbers in parentheses refer to sections below): Primer annealing (6) - indexing primer preparation (7) - Assemble transposon (8) - Tagmentation and deactivating the Tn5 (9) - PCR amplification (10) - Double size selection (11) - Quantification and Pooling (12). + +### 6. Primer Annealing + +**Step 4:** +Three oligos, Tn5-rev, Tn5-A and Tn5 B were ordered from IDT ([eu.idtdna.com](https://eu.idtdna.com/)) with standard desalting. Their sequences are: + +- **Tn5-rev:** + `5′- [phos]CTGTCTCTTATACACATCT-3′` +- **Tn5-A (Illumina FC-121-1030):** + `5′-TCGTGGCAGCGTCAGATGTGTATAAGAGACAG-3′` +- **Tn5-B (Illumina FC-121-1031):** + `5′-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-3′` + +Two double-stranded primers, Primer A and Primer B are obtained by mixing equal molar of the corresponding oligos. + +- **Primer A:** mix equal molar Tn5-rev with Tn5-A +- **Primer B:** mix equal molar Tn5-rev with Tn5-B + +Prepare the two primers (A & B) separately using two 1.5 ml Eppendorf tubes. First denature them by incubating at 70°C for 1 min and then anneal them by chilling on ice afterwards. If performed in a PCR tube, the incubation time should be reduced to 30s. + +### 7. Indexing Primer Preparation + +**Step 5:** +PCR indexing primers can be synthesized using, for example, the Illumina adapter sequences described on page 14 in the Illumina Nextera XT Index Kit v2 (Index 2 i5/i7 adapters; [https://support.illumina.com/content/dam/illumina-support/documents/documentation/chemistry_documentation/experiment-design/illumina-adapter-sequences-1000000002694-06.pdf](https://support.illumina.com/content/dam/illumina-support/documents/documentation/chemistry_documentation/experiment-design/illumina-adapter-sequences-1000000002694-06.pdf)). We ordered the indexing primers from IDT with standard desalting. The primers were diluted to 10 uM and arranged in a 96 well plate. As 24 and 16 primers are available at the i5/i7 sides, 384 unique indexes are available. We recommend users to be careful in this step to avoid contamination. + +**Figure 1. Layout of the indexing primer plate.** In this case, well A1 will contain index primer N702 and S502 with concentration both at 10 uM. + +### 8. Transposon Assembly in Solution and Assay of Transposase Activity + +**Step 6:** + +#### 8a. Transposon Assembly + +The transposon needs to be assembled every time before tagmentation as the assembled solution cannot be stored in -20°C. We assume that the Tn5 is stored in 1X DF buffer as described in Picelli et al. (Picelli et al., 2014). + +##### 8a.1. Reaction (sufficient for 280 DNA samples): + +| Reagent | Volume | +|---------------------------|----------------| +| Tn5 (100 μl | 340 μg)) | 100 μl | +| Pre-annealed primer A (100uM) | 64 μl | +| Pre-annealed primer B (100uM) | 64 μl | +| 2X DF buffer | 128 μl | +| **Total** | 356 μl | + +Incubate at room temperature for at least 60 min, we have extended it to several hours without problems. +*Note:* This reaction uses 100 ul Tn5, assuming the concentration of Tn5 is 3.4 μg/μl. The volume of Tn5 should be adjusted according to the actual concentration of the prepared enzyme to ensure 340 μg Tn5 is added. + +#### 8b. Assay of Transposase Activity + +Every time a new batch of enzyme is purified, an assay of its activity should be performed. This can be skipped if an old batch with known activity is used (proceed to step 9). + +##### 8b.1. Reaction + +| Reagent | Volume | +|----------------------------------|--------| +| H₂O | 13.5 μl| +| 5X TAPS-MgCl₂-PEG 8000 | 4 μl | +| Target DNA at 50 ng/μl | 1 μl | +| Tn5 | 1.5 μl | +| **Total** | 20 μl | + +Incubate the reaction for 10 min at 55°C. Then add 2.5 ul 0.2% SDS and incubate another 7 min at 55°C. Load sample on an agar gel, where a successfully assembled transposase should produce a smear ranging in size from 100-1000 bp. + +### 9. Tagmentation + +**Step 7:** + +#### 9.1. Reaction + +| Reagent | Volume | +|----------------------------------|--------| +| 5X TAPS buffer | 2 μl | +| 40% PEG | 2 μl | +| Tn5 | 1.2 μl | +| DNA(10 ng/μl) | 1μl | +| Water | 3.8 μl | +| **Total** | 10 μl | + +Incubate reaction at 55°C for 10 min. Add 2.5 ul 0.2% SDS and incubate at 55°C for another 7 min to deactivate the Tn5. + +### 10. PCR Enrichment + +**Step 8:** + +#### 10.1. Reaction + +| Reagent | Volume | +|----------------------------------------|--------| +| Tagmentation product from from above (step 9) | 12.5 μl| +| 5X PCR buffer | 5 μl | +| HiFi PCR Enzyme | 0.2 μl | +| dNTP (10mM) | 0.3 μl | +| Index1 (10 uM) | 2.5 μl | +| Index2 (10 uM) | 2.5 μl | +| Water | 2 μl | +| **Total** | 25 μl | + +#### 10.2. PCR Program + +- 72°C 3min (Gap filling) +- 10 cycles of: + - 98°C 30s + - 98°C 30s + - 63°C 30s + - 72°C 3min + +### 11. Double Size Selection Using AMPure Beads + +**Step 9:** + +Check if there is evaporation, especially for wells located around the border of the 96 well plate. If so, fill them to the correct volume with water before bead purification is initiated. + +We use Ambion Magnetic Stand-96, (P/N: AM10027). However, any magnetic stand for 96-well plate will work equally well. Double-size selection is performed to cut under-tormented fragments and remove primer/primer dimers. The resulting insert size should be around 350 bp. + +#### 11.1 Procedure + +1. Warm up AMPure beads to room temperature (30 min in room temperature - RT). +2. Add 7.5 ul beads to each sample, mix it evenly using a vortex and then incubate it for 10 min at RT. Leave it on the magnetic stand until the solution is clear to remove large fragments (> 1kb). +3. Pipette the supernatant into a new tube and add 4.8 ul beads, incubate for 10 min at RT and put it on the magnetic stands until the solution is clear (5min) to remove small fragments (<200bp). +4. Add 70 ul 80% ethanol, quickly flip the plates (with the magnets stand on) and pour out the liquid. Wipe the plate clean using a paper towel. +5. Repeat step 4 one more time and dry the samples by leaving them at RT for 5 min. +6. Add 20 ul water to the sample and suspend the beads by vortex. Incubate for 5-10 min at RT and then put the plate on magnetic stands until the solution is clear (5min). +7. The sequencing library is obtained by carefully pipetting off the supernatant to a new tube. We recommend discarding the last 3-4 ul to avoid contamination. + +### 12. Quality Control and Pooling + +**Step 10:** +The concentration of the generated library can be measured using Qubit™ dsDNA HS Assay Kit. For example, Qubit for individual samples or a TECAN Infinite® 200 PRO for high-through measurements. + +To save time and costs, checking of insert sizes are performed after pooling of the libraries. Equal amount of DNA (20 ng) from each sample are mixed in a pool. Insert sizes for the libraries in the pooled sample are then checked using a TapeStation (Agilent High Sensitivity D1000 ScreenTape). If primer contamination is observed, additional bead selection can be performed (adding 0.75X beads to the pool and repeat 11.1 step 4 to 7 as described above). + +### 13. Sequencing + +**Step 11:** +We have obtained high-quality sequences from libraries prepared using this protocol of Illumina HiSeq 4000 and HiSeq X10. In principle, however, the libraries should be useful for sequencing on any Illumina sequencing platform supporting the Nextera protocol. + +### 14. Troubleshooting + +**Step 12:** +For troubleshooting, we refer the reader to the full publication describing the development of the original protocol (Picelli et al., 2014). + +### 15. Acknowledgements + +**Step 13:** +We thank Simone Picelli for helpful advice with the initial library preparation and the Protein Science Facility (PSF) at the Karolinska Institute, Stockholm for help with production of the Tn5 enzyme. pTXB1-Tn5 was a gift from Rickard Sandberg (Addgene plasmid # 60240). + +### 16. References + +**Step 14:** +Picelli, S., Björklund, A. K., Reinius, B., Sagasser, S., Winberg, G., & Sandberg, R. (2014). Tn5 transposase and tagmentation procedures for massively scaled sequencing projects. *Genome Research,* 24(12), 2033-2040. [http://doi.org/10.1101/gr.177881.114](http://doi.org/10.1101/gr.177881.114) + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/a-membrane-enriched-preparation-of-culture-samples-bdapi2dn.md b/markdown-output/a-membrane-enriched-preparation-of-culture-samples-bdapi2dn.md new file mode 100644 index 0000000000000000000000000000000000000000..c247bc091cc4dce537f45c8e513454347b292025 --- /dev/null +++ b/markdown-output/a-membrane-enriched-preparation-of-culture-samples-bdapi2dn.md @@ -0,0 +1,168 @@ +```markdown +# Goal/Experiment: +Preparation of culture samples for mass spectrometry-based proteomics + +# A membrane-enriched preparation of culture samples for mass spectrometry-based proteomics + +*Gwendolyn Gallagher¹* +¹Gwendolyn Gallagher [University of Chicago], Jacob Waldbauer [University of Chicago] + +Coleman Lab +Gwendolyn Gallagher + +## Abstract + +### Purpose: +Preparation of culture samples for mass spectrometry-based proteomics + +### Principle: +Utilizing a membrane-enrichment method of lysing cells and preparing peptides has yielded higher representation of membrane proteins in our mass spectrometry-based proteomic results. Traditional methods do not adequately extract or digest hydrophobic, transmembrane proteins. Particularly, we can now see full expression patterns of proteorhodopsin, something we could not detect using traditional mass spec proteomics prep. This protocol builds on the work of Molloy (2008) Methods Mol Biol (doi:10.1007/978-1-60327-064-9_30), Erde et al. (2014) J. Proteome Res. (doi:10.1021/pr4010019), and Waldbauer, et al. (2017) Anal. Chem. (doi:10.1021/acs.analchem.7b02752). + +### Summary: +Pure culture samples were spun down and flash frozen for proteomics. A carbonate extraction protocol was used for membrane enrichment before eFASP. The membrane fraction was enzymatically digested with both chymotrypsin and trypsin and the cytosolic fraction was digested with just trypsin. These samples were then ready to be processed further by in vitro isotopic peptide labeling (diDO-IPTL). + +## Materials Text + +### Equipment +- QSonica high power sonicator +- Optima MAX-XP Beckman Coulter centrifuge +- Regular benchtop centrifuge for Eppendorf tubes +- Labconco CentriVap Cold Trap +- Sonicator bath +- Dry Block +- Incubator (37°C) +- Vortex + Eppendorf tube attachment +- 10, 20, 200, and 1000 μL pipettes +- Tube racks + +### Materials +- 10, 20, 200, and 1000 μL tips +- Wash solution +- Carbonate extraction solution +- Polypropylene microfuge tube (Beckman Coulter: 357448) +- Exchange buffer +- 1x LDS buffer +- Dithiothreitol (DTT) +- Iodoacetamide +- Digestion Buffer +- Peptide Recovery Buffer +- Protein LoBind Tube (Eppendorf: 022431081) +- Filtrate tubes and Vivacon 500 (30,000 MWCO HY) concentrator (Sartorius) +- Parafilm +- Ethyl acetate +- Trifluoroacetic acid (TFA) + +### Reagents and Solutions +- **Wash Solution:** 50 mM Tris-HCl, pH 7.5 +- **Carbonate extraction solution:** 100 mM sodium carbonate +- **Exchange buffer:** 8 M urea, 0.2% (w/v) deoxycholate, 1 M ammonium bicarbonate +- **1x LDS buffer:** 0.666 g Tris HCl, 0.682 g Tris Base, 0.800 g LDS, 0.006 g EDTA, 4 g glycerol in 20 mL milliQ +- **Digestion Buffer:** 50 mM ammonium bicarbonate with 0.2% (w/v) deoxycholate +- **Peptide Recovery Buffer:** 50 mM ammonium bicarbonate + +## Cell Lysis Protocol (3h) + +1. **Lysis:** + - Cell pellets resuspended in 333 μL wash solution and lysed with QSonica high power sonication (15 min, 1 sec pulse, Ampl 85%) + - All samples were previously derived from 4.5 mL pure cultures spun down and flash frozen + +2. **Centrifugation:** + - After sonication, the tubes were centrifuged (2500×g, 8 min) to pellet unlysed debris + +3. **Carbonate Addition:** + - Supernatant was drawn off and added to 830 μL carbonate extraction solution in a polypropylene microfuge tube + - It is very important to check that tubes are compatible with ultracentrifuge + +4. **Shaking:** + - Shake samples in polypropylene tubes in 4°C for 1 hour + +5. **Membrane Pellet:** + - After balancing tubes with additional carbonate extraction solution, membrane pellets were spun down in an ultracentrifuge (115,000×g, 1 hr) + +6. **Fraction Separation:** + - Draw off supernatant and preserve as “cytosolic” fraction and save pellet as “membrane” fraction. + +## Cytosolic Fraction Prep (1h) + +7. **Dilution:** + - Dilute cytosolic fraction samples in 1:1 in exchange + 20 mM DTT. Additional Eppendorf tubes may be necessary. + +8. **Cysteine Alkylation:** + - Alkylate cysteine thiols with 60 nM iodoacetamide and incubate at room temperature for an hour in the dark. + +## Membrane Fraction Prep (3h) + +9. **Sonication:** + - Disturb membrane pellets with QSonica high power sonication (10 min, 1 sec pulse, Ampl 85%) in 500 μL LDS buffer + 20 mM DTT. + +10. **Heating:** + - Incubate samples at 95°C for 20 minutes + +11. **Cooling:** + - Incubate samples at 37°C for 30 minutes + +12. **Cysteine Alkylation:** + - Alkylate cysteine thiols with 60 nM iodoacetamide and incubate at room temperature for an hour in the dark. + +## Enhanced Filter Aided Sample Preparation (eFASP) (3d) + +13. **Filter Loading:** + - Mix 50 μL lysate (membrane or cytosolic fraction) with 400 μL exchange buffer on filter unit. + +14. **Centrifugation:** + - Spin at 14,000×g for 10 minutes and discard filtrate + +15. **Repeat Steps:** + - Repeat steps 13-14 until all lysate is concentrated on filter + +16. **Washing:** + - Wash filter unit 3 times with 200 μL exchange buffer by spinning at 14,000×g for 10 minutes. Discard filtrate each time. + +17. **Digestion Buffer:** + - Wash filter 2 times with 200 μL digestion buffer (spin down at 14,000×g for 10 min) + +18. **Transfer:** + - Transfer filter unit to passivated collection tube. + +19. **Peptide Digestion Incubation:** + + **For MEMBRANE fraction:** + - Add 100 μL digestion buffer and 2 μg chymotrypsin on filter. Incubate overnight at room temperature (seal tubes with parafilm). After overnight incubation, add 2 μg Trypsin and incubate again overnight at room temperature. + + **For CYTOSOLIC fraction:** + - Add 100 μL digestion buffer and 2 μg trypsin on filter. Incubate overnight at room temperature (seal tubes with parafilm). + +20. **Centrifugation:** + - Centrifuge (14,000×g for 10 minutes). + - **Keep filtrate** + +21. **Peptide Recovery:** + - Add 50 μL peptide recovery buffer and centrifuge for 10 minutes at 14,000×g. + - **Keep filtrate** + +22. **Recovery Replication:** + - Repeat step 21 + +23. **Ethyl Acetate:** + - Add 200 μL ethyl acetate to the filtrate and transfer to LoBind tube. + +24. **TFA Addition:** + - Add 2.5 μL TFA and vortex gently. + +25. **Sonication:** + - Nearly fill each tube with ethyl acetate, sonicate for 10 s (note: not high power), centrifuge at 14,000×g for 10 minutes, then discard upper organic layer. + +26. **Replication:** + - Repeat step 25 two more times. + +27. **Dry Block:** + - Place sample tubes (uncovered/caps off) in Dry Block set to 60°C for 5 minutes. + +28. **Freezing:** + - Freeze sample (-80°C), then centrivap to dryness. + +29. **Usage:** + - Dried samples can now be used for IPTL labeling or can be loaded on mass spec in 2% Acetonitrile, 0.1% formic acid as an unlabeled sample. + +endofoutput +``` diff --git a/markdown-output/a-novel-laboratory-method-to-simulate-climatic-str-b927r8hn.md b/markdown-output/a-novel-laboratory-method-to-simulate-climatic-str-b927r8hn.md new file mode 100644 index 0000000000000000000000000000000000000000..b7693347b47bb127e9fc7931a58a5317e766a256 --- /dev/null +++ b/markdown-output/a-novel-laboratory-method-to-simulate-climatic-str-b927r8hn.md @@ -0,0 +1,105 @@ +```markdown +# Goal/Experiment: +To isolate individual ticks and control their environment in order to examine how different humidity levels affect the survival and host-seeking behavior of three medically important tick species. + +# A Novel Laboratory Method to Simulate Climatic Stress with Successful Application to Experiments with Medically Relevant Ticks V.2 + +**Authors:** +Sang Hyo Kim¹, Caleb Nielebeck¹, Lauren Dedmon¹, Mark Pangilinan¹, Jahred Quan¹, William Ota¹, Javier D. Monzón¹ +¹Pepperdine University + +**DOI:** +[dx.doi.org/10.17504/protocols.io.rm7vzyo8rlx1/v2](dx.doi.org/10.17504/protocols.io.rm7vzyo8rlx1/v2) + +**Citation:** +Sang Hyo Kim, Caleb Nielebeck, Lauren Dedmon, Mark Pangilinan, Jahred Quan, William Ota, Javier D. Monzón. 2022. A Novel Laboratory Method to Simulate Climatic Stress with Successful Application to Experiments with Medically Relevant Ticks. *protocols.io*. DOI:10.17504/protocols.io.rm7vzyo8rlx1/v2 + +## Introduction + +This protocol details a novel method to isolate individual ticks and manipulate their environment. We successfully used this method to investigate how humidity affects survival and host-seeking (questing) behavior of three species of ticks: the lone star tick (Amblyomma americanum), American dog tick (Dermacentor variabilis), and black-legged tick (Ixodes scapularis). We placed 72 adult females of each species into individual plastic tubes and separated them into three experimental relative humidity (RH) treatments representing distinct climates: 32% RH, 58% RH, and 84% RH. For 30 days, we assessed the survival and questing behavior of each tick. + +## Materials + +### Required Ticks +- 72 adult female *Amblyomma americanum* +- 72 adult female *Dermacentor variabilis* +- 72 adult female *Ixodes scapularis* + +### Equipment +- 1 Climate Chamber (e.g. Percival I-41VL) +- 216 - 20 cm x 2.5 cm Clear PETG plastic tubes +- 216 - 20 cm Wooden skewers +- 36 - 2 L Airtight containers +- 12 - 32% RH Boveda Two-Way Humidity Control Packs +- 12 - 58% RH Boveda Two-Way Humidity Control Packs +- 12 - 84% RH Boveda Two-Way Humidity Control Packs +- 1 Temperature/Relative Humidity Data Logger (e.g. ONSET UX100-003) +- 70% Ethanol +- Colored dot stickers +- Sharpie + +### Other Tools +- Entomology forceps +- 30 cm ruler +- White surface (e.g. lab bench diaper) + +> **Note:** Always handle ticks with blunt entomology forceps, as regular forceps can injure them. Always handle ticks over a white surface so that they can easily be spotted in case they are dropped. + +### Alternative Methods +For hard-to-find supplies such as specific humidity control packs, these can be made using saturated salt solutions to maintain different humidity levels: +- 32% RH: Saturated solution of magnesium chloride +- 58% RH: Saturated solution of sodium bromide +- 84% RH: Saturated solution of potassium chloride + +## Experimental Setup + +### Preparation +1. **Incubator Setup:** + - Program the climate chamber to cycle between 20°C and 30°C with specific temperature increments. + - Ensure a 12:12 light:dark photoperiod. + +2. **Setup Time: 2h** + - Place a single tick with one wooden skewer in each tube and seal with a cap, labeling each tube with an individual identifier. + - Place six tubes in each airtight container along with a humidity pack, labeling each container. + - Confirm the humidity in one container of each RH level with the data logger. + - Program the climate chamber. + +### Program the Climate Chamber +To cycle between 20°C to 30°C, the temperature increments should be as follows: + - 3:00 - 25°C + - 6:00 - 27.5°C + - 9:00 - 30°C + - 12:00 - 27.5°C + - 15:00 - 25°C + - 18:00 - 22.5°C + - 21:00 - 20°C + - 24:00 - 22.5°C + +### Data Collection: 5w 5d +3. Place all the airtight containers, filled with ticks and humidity packs, into the climate chamber and start the program. + +4. Each day thereafter, during the 9:00 to 12:00 or 30°C increment, assess each tick for survivorship and questing behavior. Only take one container out of the chamber at a time. Collect a binary outcome for survivorship and questing, and measure the tick's height (to the nearest 0.5 cm) in the tube if it is found questing. + +5. Periodically move the data logger to a new bin to confirm that no unexpected changes to the climate inside the containers have occurred. + +6. Repeat steps 4 and 5 for 30 days or until all ticks have died. + +### Questing Qualifications +- The individual is not walking but is still with its front legs extended. + +![](https://example.com/amblyomma_questing.jpg) +*Example of an **Amblyomma** tick questing.* + +![](https://example.com/dermacentor_questing.jpg) +*Example of a **Dermacentor** tick questing.* + +### Death Qualifications +- If any tick appears dead, lightly blow on it since ticks respond to carbon dioxide exhaled by potential hosts. +- If the tick does not move at all in 2 minutes, it should be counted as dead and placed in 70% ethanol. + +## Results +This method allows for in-depth analysis of tick behavior under controlled humidity conditions, providing valuable insights into their survivorship and questing behavior. + +--- +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/a-protocol-for-agrobacterium-mediated-transformati-8vghw3w.md b/markdown-output/a-protocol-for-agrobacterium-mediated-transformati-8vghw3w.md new file mode 100644 index 0000000000000000000000000000000000000000..1ed8f162cbd75adbb4d302bee0f03e9e77b3a960 --- /dev/null +++ b/markdown-output/a-protocol-for-agrobacterium-mediated-transformati-8vghw3w.md @@ -0,0 +1,195 @@ +```markdown +# Goal/Experiment: +A protocol for **Agrobacterium** mediated transformation of **Mimulus guttatus** from leaf petiole explants. + +## Authors +**Srinidhi Hollalu1, Benjamin Blackman2** +1Department of Plant & Microbial Biology, UC Berkeley, 2University of California, Berkeley + +## Aim: +This protocol aims to conduct Agrobacterium-mediated transformation of Mimulus guttatus from leaf petiole explants including the following procedures: +1. Surface sterilization of seeds +2. Agrobacterium culture preparation +3. Agrobacterium infection and co-cultivation +4. Callus induction and shoot induction +5. Rooting of shoots + +--- + +## Guidelines: +1. **Check Active Ingredients:** Verify active ingredient in herbicide formula (Basta or Phosphinothricin) before use to modify the selection protocol accordingly. +2. **Pilot Kill-Curve Test:** May be necessary to determine optimum herbicide concentration for each M. guttatus population. +3. **Sterilization of Antibiotics/Hormones:** Antibiotics and hormones must be filter-sterilized and added to medium post-autoclaving. +4. **Prepare Fresh Medium:** Cool solid medium overnight at room temperature post-pouring in petri-dish to avoid condensation. +5. **Herbicide Resistance:** This protocol was standardized for herbicide resistance selection. + +--- + +## Materials + +| Name | Catalog # | Vendor | +|-------------------------------------------|------------|-----------------------| +| Timentin (Ticarcillin-clavulanate) | T-104-2 | Gold Biotechnology | +| Cefotaxime | C-104-25 | Gold Biotechnology | +| Phosphinothricin | P-165-250 | Gold Biotechnology | +| 4-CPPU | C279 | Phytotech Labs | +| Meta-toplin | T841 | Phytotech Labs | +| Murashige & Skoog basal salts with vitamins | M404 | Phytotech Labs | +| Acetosyringone | 2478-38-8 | Sigma Aldrich | + +--- + +### Growth Medium Composition + +**Murashige & Skoog Basal Salt Medium (MS salts)** + +| Components | Concentration | +|------------------------------------------|----------------| +| Murashige & Skoog basal salt medium | 4 g/L | +| Sucrose | 20 g/L | +| Calcium gluconate | 1.3 g/L | +| MES (2-(N-Morpholino) ethanesulfonic acid hydrate) | 0.25 g/L | +| Gelrite | 0.25% | +| Adjust pH to 5.6 with KOH before adding Gelrite | + +**Agrobacterium Virulence Induction Medium** + +| Components | Concentration | +|------------------------------------------|----------------| +| Murashige & Skoog basal salt medium | 2 g/L | +| Sucrose | 10 g/L | +| MES | 0.5 g/L | +| 2-(N-Morpholino) ethanesulfonic acid hydrate | 0.25 g/L | +| Acetosyringone (Dissolved in DMSO & added before use) | 200 µM | +| Adjust pH to 5.5 with KOH & autoclave | + +**Co-Cultivation Medium** + +| Components | Concentration | +|------------------------------------------|----------------| +| Murashige & Skoog basal salt medium | 4 g/L | +| Sucrose | 20 g/L | +| Calcium gluconate | 1.3 g/L | +| MES | 0.25 g/L | +| 2-(N-Morpholino) ethanesulfonic acid hydrate | 0.25 g/L | +| Gelrite | 0.25% | +| CPPU | 1 mg/L | +| Acetosyringone* | 100 µM | +| * Filter sterilize before adding to the medium | + +**Callus Induction Medium** + +| Components | Concentration | +|------------------------------------------|----------------| +| Murashige & Skoog basal salt medium | 4 g/L | +| Sucrose | 20 g/L | +| Calcium gluconate | 1.3 g/L | +| MES | 0.25 g/L | +| 2-(N-Morpholino) ethanesulfonic acid hydrate | 0.25 g/L | +| Gelrite | 0.25% | +| CPPU | 1 mg/L | +| Timentin (Ticarcillin-clavulanate)* | 200 mg/L | +| Cefotaxime* | 50 mg/L | +| Phosphinothricin* | 6 mg/L | +| * Filter sterilize before adding to the medium | + +**Shoot Induction Medium** + +| Components | Concentration | +|------------------------------------------|----------------| +| Murashige & Skoog basal salt medium | 4 g/L | +| Sucrose | 20 g/L | +| Calcium gluconate | 1.3 g/L | +| MES | 0.25 g/L | +| 2-(N-Morpholino) ethanesulfonic acid hydrate | 0.25 g/L | +| Gelrite | 0.25% | +| Meta-toplin* | 0.1 mg/L | +| Timentin (Ticarcillin-clavulanate)* | 200 mg/L | +| Cefotaxime* | 50 mg/L | +| Phosphinothricin* | 6 mg/L | +| * Filter sterilize before adding to the medium | + +**Root Induction Medium** + +| Components | Concentration | +|------------------------------------------|----------------| +| Murashige & Skoog basal salt medium | 2 g/L | +| Sucrose | 10 g/L | +| Calcium gluconate | 1.3 g/L | +| MES | 0.25 g/L | +| 2-(N-Morpholino) ethanesulfonic acid hydrate | 0.25 g/L | +| Gelrite | 0.25% | +| Naphthalene Acetic Acid (NAA)* | 0.1 mg/L | +| Timentin (Ticarcillin-clavulanate)* | 200 mg/L | +| Cefotaxime* | 50 mg/L | +| Phosphinothricin* | 6 mg/L | +| * Filter sterilize before adding to the medium | + +**Safety Warnings** +For safety information and warnings, please refer to the SDS (Safety Data Sheet). + +--- + +## Procedure + +### Surface Sterilization of Seeds (2.5-3 months) + +1. **Collect Mature Seeds** + - From plants grown in greenhouse/growth chamber to reduce contamination risk. +2. **Sterilize Seeds** + - Place seeds in **1.5 ml** Eppendorf tube. + - Fill tube with surface sterilization solution (**15% laundry bleach and a drop of hand soap**). + - Shake vigorously for **8-10 min**. + - Discard sterilizing solution; **rinse seeds with sterile water** by shaking for **30 sec**. Repeat 5-6 times. + - Add **1 ml** sterilized water to later plate seeds. +3. **Store Seeds** + - **4 ºC** for at least **2-3 weeks** to cold stratify seeds. +4. **Transfer & Grow Seeds** + - Spread sterilized seeds on growth medium. + - Incubate jars at **20-21 ºC** under cool fluorescent lamps. +5. **Harvest Shoots** + - Subculture onto new jars as needed to avoid senescence. + +### Agrobacterium Culture Preparation (4 days) + +1. **Streak Agrobacterium EH105** + - Harboring binary plasmid on LB/YEP agar plate; incubate at **28 ºC** for **two days**. +2. **Inoculate Culture** + - Single colony into **10 ml** YEP liquid medium with rifampicin (40 µg/mL) and appropriate antibiotic. + - Incubate at **28 ºC** for **36-48 hrs** at **200 rpm**. +3. **Centrifuge Cultures** + - Pellet at **4000 rpm**; resuspend in **5 ml** virulence induction medium. + - Incubate for **3-4 hrs** with gentle shaking **(50-80 rpm)** in darkness at **Room temperature**, adjusting pH to **5.5**. +4. **Adjust Agrobacterium OD** + - To **0.2** using liquid half-strength MS medium. + +### Agrobacterium Infection and Co-cultivation (3-4 days) + +1. **Infect Petioles** + - Pull shoots onto sterile petri dish; infect each petiole with Agrobacterium on co-cultivation medium. + - Incubate in darkness/low light at **Room temperature** for **2-3 days**. +2. **Day 3**: + - Wash explants in sterile timentin (100 mg/L) + cefotaxime (50 mg/L) solution and transfer to callus induction medium with phosphinothricin. + - Incubate at **21 ºC** under cool fluorescent lamps. + +### Callus Induction and Shoot Induction (4-5 months) + +1. **Subculture Explants after 21-24 days** + - Retain partial callus and culture on callus induction medium. Repeat subculturing every **21-24 days**. + - Transfer mature callus to Meta-toplin medium with acclimation. +2. **Signatures of Differentiation** + - Transfer compact callus to shoot induction medium. + +### Rooting of Shoots (3 weeks) + +1. **When Shoots Emerge** + - Separate and plate individual shoots on rooting medium. +2. **Subculture** + - Half-strength rooting medium for further rooting/hardening. +3. **Move Shoots to Greenhouse** + - Move rooted shoots to potting medium and leave for at least two weeks to harden. + +--- + +`endofoutput` +``` \ No newline at end of file diff --git a/markdown-output/a-sars-cov-2-surveillance-sequencing-protocol-opti-butbnwin.md b/markdown-output/a-sars-cov-2-surveillance-sequencing-protocol-opti-butbnwin.md new file mode 100644 index 0000000000000000000000000000000000000000..f59860e5855b2682d187f586a3b355ba34783ac0 --- /dev/null +++ b/markdown-output/a-sars-cov-2-surveillance-sequencing-protocol-opti-butbnwin.md @@ -0,0 +1,235 @@ +```markdown +# Goal/Experiment: +To identify and monitor SARS-CoV-2 variant evolution, using a surveillance sequencing protocol optimized for Oxford Nanopore PromethION. + +--- + +## A SARS-CoV-2 Surveillance Sequencing Protocol Optimized for Oxford Nanopore PromethION + +**Authors**: +Jannatul Ferdous, Torri Weathers, Visva Bharati Barua, Erin Stiers, Adam France, Kevin C Lambirth, Cynthia Gibas, Jessica A Schlueter +*UNC Charlotte* + +**Published**: October 15, 2021 +**DOI**: [dx.doi.org/10.17504/protocols.io.butbnwin](https://dx.doi.org/10.17504/protocols.io.butbnwin) + +--- + +### Introduction +To identify and monitor SARS-CoV-2 variant evolution, UNC Charlotte has introduced a surveillance sequencing program. This protocol, optimized for the Oxford Nanopore PromethION, prepares SARS-CoV-2 viral genome libraries for next-generation sequencing. The protocol is designed to work in a 96-well format, producing sufficient sequence data for genome assembly to meet GISAID and NCBI submission standards. It addresses isolate sequencing failures due to low viral titers and provides guidelines for cost-effective sequencing of high Cq value clinical samples. + +--- + +### Safety and Preparations +- **Wear PPE** at all times. +- **Clean workbench** and pipettes with 70% ethanol before use. +- **Handle samples carefully** during SPRI clean-up, barcoding, and adapter ligation to avoid significant loss of beads. + +--- + +### Reagents and Equipment +#### cDNA Synthesis +- **ABI HC cDNA kit** (Fisher cat #4368813) +- **10x RT Buffer** (ABI HC cDNA kit, Fisher cat #4368813) +- **10x RT Random primers** (ABI HC cDNA kit, Fisher cat #4368813) +- **25x dNTPs Mix**, 100 mM +- **Multiscribe RT**, 50 U/µL (ABI HC cDNA kit, Fisher cat #4368813) +- **MgCl2**, 50 mM + +#### ARTIC PCR Amplification +- **Q5 Reaction Buffer** (New England Biolabs, cat #M0491L) +- **Q5 High Fidelity Polymerase** (New England Biolabs, cat #M0491L) +- **ARTIC V3 primer pool 1** (IDT DNA, diluted to 10 µM) +- **ARTIC V3 primer pool 2** (IDT DNA, diluted to 10 µM) +- **2.5 mM dNTP Mix** + +#### SPRI Clean-up +- **AMPure XP beads** (Beckman Coulter cat #A63881) +- **80% Ethanol**, fresh +- **Omega EB** (Omega BioTek, PD089) + +#### Qubit Quantification +- **Invitrogen Qubit 1x dsDNA HS Assay Kit** (Fisher cat #Q33231) + +#### Library End-Repair +- **Ultra II End-Prep Reaction Buffer** +- **Ultra II End-Prep Reaction Enzyme** + +#### Sample Barcoding +- **Native Barcoding Expansion 96** (Oxford Nanopore, EXP-NBD196) +- **Ultra II Ligation Master Mix** (New England Biolabs cat #E7546L) +- **Ultra II Ligation Enhancer** (New England Biolabs cat #E7546L) +- **Short Fragment Buffer** (Oxford Nanopore, EXP-SFB001) +- **Elution Buffer** (EB) + +#### Adapter Ligation +- **Adapter Mix II** (Oxford Nanopore, EXP-MRT001 or part of EXP-NBD196) +- **Quick T4 DNA Ligase** (New England Biolabs cat #E6056L) +- **Short Fragment Buffer** (Oxford Nanopore, EXP-SFB001) +- **Elution Buffer** (EB; Oxford Nanopore EXP-AUX001) + +--- + +### Protocol + +#### cDNA Synthesis + +1. **Reagents:** + - 10x RT Buffer + - 50mM MgCl2 + - 10x RT Random Primer + - 25x dNTPs Mix + - Multiscribe RT + +2. **Prepare Master Mix 1 (MM1) in a 1.5 mL Lobind tube on ice:** + - *For 96 samples* + Nuclease Free Water: 424 µL + 10x Random Primer: 212 µL + 25x dNTPs Mix: 84.8 µL + +3. **Array RNA into Plate and Mix:** + - *For Regular Samples:* + Array 10 µL RNA into a 96-well plate on an ice block. Add 6.8 µL of MM1 to each well. + + - *For High Cq Samples:* + Array 20 µL RNA and add 13.6 µL MM1 to each well. + + - *For Reduced Cost Samples:* + Array 5 µL RNA and add 3.4 µL MM1 to each well. + +4. **Incubate Reaction:** + - Seal plate, mix by quick spin. Incubate at 65°C for 5 minutes. + +5. **Prepare Master Mix 2 (MM2) in a 1.5 mL Lobind tube on ice:** + - *For 96 samples* + 10x RT Buffer: 212 µL + Multiscribe RT: 106 µL + MgCl2: 21.2 µL + +6. **Add MM2 to Each Well:** + - *For Regular Samples:* Add 3.2 µL MM2 to the RNA plate. + - *For High Cq Samples:* Add 6.4 µL MM2 per well. + - *For Reduced Cost Samples:* Add 1.6 µL MM2 per well. + +7. **Incubate Further:** + - Seal and mix by quick spin. + 25°C for 10 minutes + 37°C for 2 minutes + 85°C for 5 minutes + Hold at 4°C + +8. **Store Plate:** + - For long term: -20°C + - Short term: 4°C + +#### ARTIC PCR Amplification + +9. **Reagents:** + - 5x Q5 Reaction Buffer + - Q5 High Fidelity Polymerase + - V3 ARTIC primer pool 1 (diluted to 10 µM) + - V3 ARTIC primer pool 2 (diluted to 10 µM) + +10. **PCR Plate Setup:** + - **ARTIC Pool 1:** + Master Mix: + Nuclease-free water: 927.5 µL + Reaction Buffer: 530 µL + V3 Pool 1: 424 µL + 2.5mM dNTP mix: 212 µL + Q5 High fidelity polymerase: 26.5 µL + + - **ARTIC Pool 2:** + Master Mix: + Nuclease-free water: 927.5 µL + Reaction Buffer: 530 µL + V3 Pool 2: 424 µL + 2.5mM dNTP mix: 212 µL + Q5 High fidelity polymerase: 26.5 µL + +11. **Distribute PCR Components for ARTIC Pools 1 & 2:** + - Mix 20 µL MM1 into the ARTIC Pool 1 plate and add 5 µL of cDNA. + - Mix 20 µL MM2 into the ARTIC Pool 2 plate and add 5 µL of cDNA. + +12. **Set PCR Cycling Program:** + - 1 Cycle: 98°C for 30 seconds + - 20 Cycles: 94°C for 16 seconds → 65°C for 5 minutes → 94°C for 16 seconds → 65°C for 5 minutes + - Hold at 4°C. + + **Store at** + - Long term: -20°C + - Short term: 4°C + +#### SPRI Clean-Up + +13. **Reagents:** + - AMPure XP Beads + - 80% Ethanol Fresh + - Omega EB + +14. **Prepare Fresh 80% Ethanol:** + - Mix 40 mL 100% Ethanol with 10 mL H2O in a 50 mL tube. + +15. **Clean-up Procedure:** + - Spin PCR Product Plates. + - Transfer and mix AMPure Beads. + - Incubate 15 minutes. + - Wash with 80% ethanol. + - Elute DNA with 33 µL Omega EB. + +#### Qubit Quantification + +16. **Reagents:** + - Qubit Quantification Kit (dsDNA HS Assay) + - Standards (Included in Kit) + +17. **Qubit reagent preparation:** + - dsDNA HS Buffer: 24278 µL + - Qubit Dye: 122 µL + - Mix well, vortex and spin. + +18. **Run Qubit Assay Using Standard Protocols:** + +#### Library End-Repair + +19. **Reagents:** + - NEB Ultra II End-Prep Reaction Buffer & Enzyme + +20. **Prepare Master Mix for 96 Samples:** + - Reaction Buffer: 213.59 µL + - Enzyme: 91.425 µL + - Mix and distribute into the 96-well plate. + +21. **Standardize DNA:** + - Normalization using qubit data and spreadsheet. + - Incubate, perform end-repair. + +#### Sample Barcoding + +22. **Reagents:** + - Native Barcoding Expansion (EXP NBD 196) + - NEB Ultra II Ligation Master Mix and Enhancer + +23. **Prepare Master Mix:** + - Nuclease-Free Water: 604.2 µL + - Ligation Master Mix: 1060 µL + - Ligation Enhancer: 31.8 µL + - Mix thoroughly. + +#### Adapter Ligation & Final Steps + +24. **Reagents:** + - Adapter Mix II + - NEB Quick Ligase and Buffer + - Elution Buffer + +25. **Complete Ligation Steps:** + - Mix Polishes, Spin, Elute, and Store. + +--- + +**End of Document** +--- + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/a-scoping-review-of-remote-rehabilitation-interven-bh9aj92e.md b/markdown-output/a-scoping-review-of-remote-rehabilitation-interven-bh9aj92e.md new file mode 100644 index 0000000000000000000000000000000000000000..186f177019f74d10b2d34468b187d3edd3c9a0ac --- /dev/null +++ b/markdown-output/a-scoping-review-of-remote-rehabilitation-interven-bh9aj92e.md @@ -0,0 +1,124 @@ +```markdown +Goal/Experiment: Conduct a scoping review of “remote” rehabilitation interventions to address COVID-19 sequalae. + +# A Scoping Review of “Remote” Rehabilitation Interventions to Address COVID-19 Sequelae + +**Authors:** +Julie Whitney1, Lindsay Bearne1, Patrick White1, Arietta Spinou1, Emma Godfrey1, Matthew O’Connell1, Julia Fox-Rushby2, Graham Fisher2, Katie Sheehan1 + +1King’s College London, University of London +2Patient representative + +DOI: [dx.doi.org/10.17504/protocols.io.bh9aj92e](https://dx.doi.org/10.17504/protocols.io.bh9aj92e) + +--- + +### Abstract + +**Plain English Summary** + +Many people who have been unwell with COVID-19 are suffering long-term problems with their fitness and ability to participate in usual activities of daily life. Symptoms include muscle weakness, breathlessness, changes in sensation, pain and fatigue. There are also effects on psychological and mental health. Rehabilitation could help with these issues. However, there are challenges in providing this rehabilitation because so many patients need to access these services and because face to face treatment is not always possible due to social distancing. + +There has been increasing interest in remote rehabilitation interventions from researchers and app developers in recent years. Remote interventions use technology such as video, smartphone applications, and interactive conferencing (e.g., Zoom) to deliver rehabilitation programmes. + +We will search for evidence about remote rehabilitation interventions and application stores to identify existing remote interventions that might meet the needs of COVID-19 survivors. This information will allow rehabilitation teams to direct patients to suitable remote interventions. This may be needed to replace or to enhance their rehabilitation programme. + +--- + +## Attachments + +[scoping review protocol version 4.pdf](#) + +### Protocol Citation + +Julie Whitney, Lindsay Bearne, Patrick White, Arietta Spinou, Emma Godfrey, Matthew O’Connell, Julia Fox-Rushby, Graham Fisher, Katie Sheehan 2020. A scoping review of “remote” rehabilitation interventions to address COVID-19 sequelae. *protocols.io*. [dx.doi.org/10.17504/protocols.io.bh9aj92e](https://dx.doi.org/10.17504/protocols.io.bh9aj92e) + +### License + +This is an open access protocol distributed under the terms of the [Creative Commons Attribution License](https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. + +### Protocol Details + +**Created:** Jul 06, 2020 +**Last Modified:** Jul 08, 2020 +**Protocol Integer ID:** 38914 + +--- + +## 1. Protocol Registration + +The protocol is registered on protocols.io. + +## 2. Inclusion Criteria + +Papers/applications that describe a rehabilitation intervention: + +- Aimed at long-term symptoms known to be associated with COVID-19 (e.g., fatigue, breathlessness, weakness). +- Aimed at the community-based or self-management level of acuity (excluding hospital or inpatient rehabilitation). +- Delivered remotely. + +The intervention type, dose, and delivery should be clearly described/replicable. The intervention should be currently accessible as an application/web-based tool to the public (although may be behind a paywall). + +Programs should be available in English language and based in the UK. + +Types of evidence sources: meta-analysis, systematic review, randomized controlled trial, non-randomized or before/after study design as well as selected commentaries. + +## 3. Search Strategy + +The review will use a five-step search strategy: + +1. An initial search of two databases (MEDLINE and CINAHL) with selected papers analysed for potential additional keywords. +2. A full search across all databases (Embase, Cochrane trials register) using any newly identified keywords as search terms. +3. A search of reference lists of selected papers. +4. A search of grey literature including conference proceedings from tech in health conferences; selected commentaries. +5. App stores search (Apple, Google Play, and NHS Apps library) and in the website [www.fnd.io](https://fnd.io). + +## 4. Data Extraction + +Data from papers: + +- **Source details** + - Author / date + - Conditions/populations for which the intervention is designed (including acuity) + - Media type + +- **Type of rehabilitation** + - Type of exercise/activity + - Dose (intensity/frequency/duration) of intervention + +- **Delivery of the intervention** + - Qualifications/profession of developer/instructor + - Motivational tools incorporated into the intervention (use template to quantify) + - Theories to support delivery + - Opportunity for social interaction/peer support incorporated into the intervention + - Follow up/support provided within the tool/intervention + - Costs of the interventions (simple i.e. whether it requires a fee to use) + +Data from applications: + +- **Source information** + - App name, platform, version, developer, size, star rating, number of installs. + - Privacy policy statement and medical product status. + - Conditions/populations for which the intervention is designed (and acuity) + +- **Type of rehabilitation** + - Type of exercise/activity + - Dose (intensity/frequency/duration) of intervention + +- **Delivery of the intervention** + - Qualifications/profession of developer/instructor + - Motivational tools incorporated into the intervention (use template to quantify) + - Theories to support delivery + - Opportunity for social interaction/peer support incorporated into the intervention + - Follow up/support provided within the tool/intervention + - Costs of the interventions (simple i.e. whether it requires a fee to use) + +## 5. Analysis + +Interventions will be summarised narratively according to target condition and demographic, media type and rehabilitation type (dose—frequency, intensity, duration—and support provided) using tables and/or charts. Interventions relevant to different COVID-19 rehabilitation needs (addressing different symptoms) will be illustrated using tables and/or charts. + +**Consultation:** +Findings will be reviewed by a stakeholder group of rehabilitation clinicians and academics that have assembled for a related project. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/a-versatile-nuclei-extraction-protocol-for-single-crw4v7gw.md b/markdown-output/a-versatile-nuclei-extraction-protocol-for-single-crw4v7gw.md new file mode 100644 index 0000000000000000000000000000000000000000..e60110d2256f96da71f1ef051d18adeea09c7f72 --- /dev/null +++ b/markdown-output/a-versatile-nuclei-extraction-protocol-for-single-crw4v7gw.md @@ -0,0 +1,129 @@ +```markdown +# Goal/Experiment: +Develop a versatile nuclei extraction protocol for single-cell multimodal ATAC and gene expression analysis in non-model species to ensure high-quality nuclei isolation for subsequent molecular biology applications. + +# A Versatile Nuclei Extraction Protocol for Single Cell Multimodal ATAC and Gene Expression in Non-Model Species + +**Authors:** +Rose Ruiz Daniels1, Richard S Taylor1, Ioannis Konstantinidis2, Sarah Salisbury1, Diego Perojil Morata1, Jorge Manuel de Oliveira Fernandes2, Emily Clark1, Dan Macqueen1, Diego Robledo1 +**Affiliations:** +1. The Roslin Institute and Royal (Dick) School of Veterinary Studies, The University of Edinburgh, Edinburgh EH25 9RG, UK. +2. Nord University, Bodø, Hovedbygning 2060, Norway. + +**DOI:** [10.17504/protocols.io.bp2l69wnrlqe/v1](https://doi.org/10.17504/protocols.io.bp2l69wnrlqe/v1) + +## Abstract + +This protocol presents a modified version of an approach to extract nuclei from various tissue types for single-cell sequencing and aims to extract high-quality nuclei for single-cell multimodal ATAC and gene expression analysis using the 10x Chromium system. Key modifications include varied RNase inhibitor concentrations, nuclear isolation buffer quantities, and specific steps for multimodal analysis. + +For bulk ATAC-seq, use protease inhibitor cocktail PIC instead of RNase inhibitor in the snRNA-seq version of this protocol. + +### Goal +Develop a high-quality nuclei extraction protocol from diverse tissue types for single-cell multi-omic applications ensuring optimal nuclei integrity and yield. + +## Guidelines + +**Trial Preparation:** +Conduct a trial run before library preparation, especially with new tissue types, to adjust parameters (e.g., mincing times, filter size) and optimize nuclei quality. Adjusting these parameters helps prevent RNAse introduction and ensures the production of nuclei with intact membranes. + +## Materials + +### Equipment and Supplies +- **Noyes Spring Scissors - Tungsten Carbide** (Fine Science Tools Catalog #15514-12) +- **Tungsten Carbide Straight Scissors 11.5 cm** (Fine Science Tools Catalog #14558-11) +- **40 µm Falcon™ Cell Strainers** (Fisher Scientific Catalog #08-771-2) +- **Corning™ Falcon™ Test Tube with 35 µm Cell Strainer Snap Cap** (Corning Catalog #352235) +- **pluriStrainer Mini 20 µm (Cell Strainer)** (pluriSelect Catalog #43-10020-50) +- X500 Eppendorf DNA LoBind Tubes, 1.5ml, PCR clean +- Cryotube +- 6-well tissue culture plate (Stem Cell Technologies) +- Falcon tubes 15 ml (Corning) +- **INCYTO C-Chip™ Disposable Hemacytometers** (VWR International Catalog #82030-468) + +## Sampling and Storage for Nuclear Isolation + +### Sampling +- **Euthanize and Process:** Euthanize animals humanely and immediately process the tissues. +- **Sample Preparation:** Freeze ~60 mg of sammond tissue in a cryotube and flash freeze in liquid nitrogen. If liquid nitrogen is unavailable, use dry ice. The tissue preservation step is critical. +- **Storage:** Store tissue samples at -80°C for up to one year. Older samples may still yield viable nuclei but require testing. + +### Critical Steps +- **Immediate Freezing:** Preserve tissue samples as quickly as possible to maintain nuclear integrity. + +## Reagents + +Chill all reagents on ice before use. + +**2X Stock of Salt-Tris Solution (10 mL)** + +| Component | Catalog Number | Stock Solution | Volume | Final Concentration | +|-----------------------|--------------------------------------------|----------------|--------|---------------------| +| NaCl (5 M) RNase-free | Thermo Fisher Scientific Catalog #AM9759 | 292 µL | | 146 mM | +| Tris-HCl pH 7.5 | Thermo Fisher Catalog #15567027 | 100 µL | | 10 mM | +| CaCl2 1 M Solution | VWR International Catalog #97062-820 | 10 µL | | 1 mM | +| MgCl2 1.00 M Solution | MilliporeSigma (Sigma-Aldrich) Catalog M1028 | 210 µL | | 21 mM | +| Nuclease-free Water | VWR International Catalog #97062-794 | 9388 µL | | | + +### RNase Inhibitor-Containing Buffers + +**Protector RNase Inhibitor** (Merck MilliporeSigma Catalog #3335399001) + +**1X ST Buffer Solution** +- Dilute 2X ST with equal volumes of nuclease-free water (1:1) to create the 1X solution. +- 250 µL RNase inhibitor (200 U/mL) per 1X ST solution (10 mL). + +**Working Solutions:** +1X ST with additional components: + +| Component | Catalog Number | Volume | Final Concentration | +|------------------------|---------------------------------|---------|---------------------| +| 2X ST Buffer | | 2 mL | | +| 1% Tween-20 | Sigma-Aldrich Catalog #P-7949 | 120 µL | | +| 2% BSA | NEB Biolabs Catalog #B9000S | 20 µL | | +| Nuclease-free water | | 1810 µL | | +| RNase inhibitor | | 50 µL | 1000 U/mL | + +Prepare and chill solutions immediately before use. + +## Nucleus Isolation Workflow for ST-based Buffers + +### Step 1: Initial Sample Preparation (30m) +- Keep samples frozen on dry ice until nuclei isolation. +- Pre-chill the centrifuge to 4°C. +- Place frozen tissue into a 6-well plate with 1 mL TST buffer. +- If tissue sticks to the cryotube, transfer using tweezers on dry ice. + +### Step 2: Mincing (10m) +- Mince tissue with Tungsten Carbide scissors for 30 seconds, then switch to Noyes Spring Scissors for total of 10 minutes for hard tissues. +- For soft tissues, use spring scissors only for 10 minutes. + +#### Step 2.1: Dissociation +- Pipette tissue gently using a P1000 pipette with a low-retention tip. +- Critical Step: Assess timing by examining nuclei under a microscope. + - **Head Kidney Nuclei:** Insufficient dissociation time - not ideal. + - **Blood Nuclei:** Perfect dissociation - ideal. + - **Liver Nuclei:** Over-dissociation - not ideal. + +### Step 3: Lysate Processing +- Pass lysate through a 40 µm cell strainer. + - Rinse with 1 mL TST and add 3 mL freshly prepared ST buffer. + - Collect 5 mL of lysate in a Falcon tube on ice. + +### Step 4: Centrifugation (5m) +- Centrifuge at 500 x g for 5 minutes at 4°C in a swinging bucket centrifuge. + +### Step 5: Pellet Resuspension +- Remove excess liquid with a P200 pipette carefully without disturbing the pellet. +- Gently resuspend the pellet with a P1000 pipette aiming to recover 6000 nuclei using a diluted nuclei buffer containing 1U/µL RNase inhibitor. + +### Step 6: Nuclei Counting +- Use a C-chip disposable haemocytometer to count nuclei. +- Confirm counts and assess debris and nuclear membrane integrity. + +### Step 7: Confirmation +- Confirm nuclei counts with a Bio-Rad TC20 for viable cells. Ideal live cell proportion: 1-4%. + +--- + +## End of Output +``` \ No newline at end of file diff --git a/markdown-output/aav-injection-in-the-nodose-ganglia-in-mouse-cfpgtmjw.md b/markdown-output/aav-injection-in-the-nodose-ganglia-in-mouse-cfpgtmjw.md new file mode 100644 index 0000000000000000000000000000000000000000..c7f27901fd0eb987b18c087ef58bc0e387a76555 --- /dev/null +++ b/markdown-output/aav-injection-in-the-nodose-ganglia-in-mouse-cfpgtmjw.md @@ -0,0 +1,105 @@ +```markdown +# Goal/Experiment: +The goal of this experiment is to inject Adeno-Associated Virus (AAV) into the nodose ganglia in mice to study the role of specific cell populations in the bidirectional communication between the brain and body via the vagus nerve. This protocol aims to describe a practical surgical approach to access the vagal trunk and the jugular-nodose ganglion (JNG) complex in mice. + +# AAV Injection in the Nodose Ganglia in Mouse +### Santiago Unda¹, Michael G. Kaplitt¹ +¹Weill Cornell Medicine + +#### ASAP Collaborative Research Network + +#### Kaplitt Protocols + +#### Eileen Ruth Torres +#### Weill Cornell Medicine + +#### DOI: +[dx.doi.org/10.17504/protocols.io.5qpvobjmdl4o/v1](https://dx.doi.org/10.17504/protocols.io.5qpvobjmdl4o/v1) + +#### Protocol Status: +Working - We use this protocol and it is working + +### Abstract +The gut-brain axis links the visceral organs to the medulla oblongata via the vagus nerve. Accessing the afferent vagal pathway is crucial to dissect the role of cell populations in the communication between the brain and body. The jugular-nodose ganglion (JNG) complex has varied neural subpopulations responsible for sensing physiological conditions of the thoracic and abdominal organs. Studying these ganglia is challenging in small animals due to their size and location. Herein, we describe a practical surgical approach to the vagal trunk and the JNG complex in mice. + +## Materials + +| Equipment | Type | Brand | SKU | Link | +| --------- | ---- | ----- | --- | ---- | +| 10μl, Neuros Syringe, Model 1701 RN, 33 gauge | Syringe | Hamilton | 65460-06 | | +| Sub-Microliter Injection System | Injection System | World Precision Instruments | N/A | [wpiinc.com](https://www.wpiinc.com/var-3167-sub-microliter-injection-system) | +| NanoFil Application Kits | Application Kit | World Precision Instruments | IO-KIT | [wpiinc.com](https://www.wpiinc.com/var-3327-nanofil-application-kits) | +| 36-gauge Beveled NanoFil needle | Needle | World Precision Instruments | NF36BV-2 | | +| Micromanipulator | Manipulator | Miller Design | P#10 | | + +## Before Start Instructions + +1. Disinfect the surgical work surface with 70% ethanol and prepare sterile instruments (e.g., fine scissors, forceps, retractor). +2. Use gauzes, staples, and swabs sterilized by autoclaving. +3. For multiple surgeries, clean and re-sterilize instruments with 70% ethanol or a dry bead sterilizer between mice. +4. A surgical mask, clean lab coat, hair bonnet, and sterile gloves should be worn. +5. These ganglia are approximately 1mm wide and located deep in the cervical carotid triangle. A surgical microscope will be needed for the entire procedure. + +## Preparation of the Surgical Setup + +1. **Turn on the heating pad to 37°C.** +2. **Position the surgical microscope.** +3. **Prepare the AAV aliquot:** + - Thaw the aliquot, mix well, and keep on ice. + - For intraneural injections (vagus trunk), use the mosaic AAVrg/rh10 for better efficiency and mainly transduce afferent neurons with minimal impact on the efferent vagal pathway. Titer: 1-3×10¹²vg/ml; Volume: 4-6μl. + - For intraganglionic injections, use AAV9. Titer: 1-3×10¹²vg/ml; Volume: 2-3μl. +4. **Withdraw the AAV with a 10μl 33G syringe.** +5. **Remove locking cap and gasket from the 10μl syringe.** +6. **Connect the NanoFil sub-microliter injection system.** +7. **Attach the SilFlex tubing to the 10μl syringe and the other end to the Neuros Syringe.** +8. **Secure the 36G beveled NanoFil needle to the injection holder.** +9. **De-gas the NanoFil system by slowly pushing the plunger.** + +## Surgery + +1. **Anesthetize mice** using a mixture of ketamine (110mg/kg) and xylazine (8mg/kg) or isoflurane. +2. **Shave** the whole anterior cervical area with an electric razor or shaving cream. +3. **Lay the mouse flat** on supine position on a heating pad. +4. **Apply ophthalmic ointment** to the mouse’s eyes. +5. **Sterilize the surgical area** with 70% alcohol, complex iodine, and 70% alcohol again. Place a surgical gauze in the sterile area. +6. **Make a small incision** in the skin with straight thin scissors or a scalpel. +7. **Retract the submandibular glands** laterally to expose the cervical musculature. +8. **Dissect the sternocleidomastoid muscle** laterally and the omohyoid muscle medially. +9. **Identify the carotid bifurcation** and gently dissect the connective tissue surrounding the area. +10. **Remove the connective tissue** surrounding the vagus trunk above the carotid bifurcation (Proceed if intra neural injections are required). +11. **Identify the temporal bone** underneath the posterior belly of the digastric muscle. +12. **Dissect the muscle fibers**, next to the mastoid notch of the temporal bone to reveal the JNG swelling. + +## Stepwise Procedure + +1. **Make a small incision** in the middle of the neck. +2. **Retract the submandibular glands** laterally. +3. **Dissect the sternocleidomastoid muscle** laterally and the omohyoid muscle medially. +4. **Identify the carotid bifurcation** and gently dissect the connective tissue surrounding the vagus trunk above it. +5. **Remove the connective tissue** around the vagus trunk above the carotid bifurcation. (Intra neural injections proceed). +6. **Identify the temporal bone** and dissect the muscle fibers to reveal JNG swelling. +7. **Visualize JNG**, proceed with intraganglionic injection. +8. **Open the muscle fibers** behind the posterior belly of the digastric muscle for clear view if needed. +9. **Injection volume and method**: Use a micromanipulator or free-handed; inject using 10μl syringe at 2nl/s infusion rate with a total volume of 500nl. + +## Post-Surgery Procedures + +1. **Close the skin** with sterile suture, apply povidone-iodine. +2. **Apply antibiotic ointment** and inject 5mg/kg Carprofen for analgesia. +3. **Maintain the mouse under a heat lamp** until fully awake; return to the cage when fully recovered. + +## Visual Guide +![Visual Guide](surgical_steps_image.jpg) +1. Midline incision +2. Submandibular glands +3. Muscle dissection +4. Carotid bifurcation +5. Left vagus trunk +6. Intra neural injection site + +End of Protocol. + +--- +End of Output. +``` +endofoutput \ No newline at end of file diff --git a/markdown-output/activation-and-intracellular-staining-of-whole-blo-hukb6uw.md b/markdown-output/activation-and-intracellular-staining-of-whole-blo-hukb6uw.md new file mode 100644 index 0000000000000000000000000000000000000000..8d8e0f7efb37725c73f536e3826c801c033a010d --- /dev/null +++ b/markdown-output/activation-and-intracellular-staining-of-whole-blo-hukb6uw.md @@ -0,0 +1,127 @@ +```markdown +# Goal/Experiment: +**Activation and Intracellular Staining of Whole Blood: For the Detection of Intracellular Cytokines and Other Intracellular Targets** + +## BioLegend, Inc. + +### Abstract +This is part of BioLegend's, "Intracellular Flow Cytometry Staining Protocol: For the Detection of Intracellular Cytokines and Other Intracellular Targets." + +### Guidelines + +#### Application Notes + +1. **Activated Cell Preparation:** + - Activated cell populations can be prepared from in vivo-stimulated tissues or vitro-stimulated cultures (e.g., antigen-specific activation or mitogen-induced). + - For cytokine and chemokine detection, include a protein transport inhibitor such as brefeldin A (BioLegend Cat. No. 420601) or monensin (BioLegend Cat. No. 420701) in the last 4-6 hours of cell culture activation. + - The cells can be suspended and distributed to 12 x 75 mm plastic tubes or microwell plates for immunofluorescent staining. + +2. **Optimization of Stimulation Conditions:** + - Different cytokines/chemokines have different production peaks. Stimulation conditions for each stimulant should be optimized. + +3. **Surface Marker Antibodies:** + - Some antibodies recognizing native cell surface markers may not bind to fixed/denatured antigens. + - It is recommended that staining of cell surface antigens be done with live, unfixed cells PRIOR to fixation/permeabilization and staining of intracellular targets. + +#### Note +To confirm specific anti-cytokine staining, a blocking experiment is recommended: +- Cells fixed/permeabilized then preincubated with an excess amount of unlabeled anti-cytokine antibody and/or the recombinant cytokine of interest is preincubated with fluorophore-conjugated anti-cytokine antibody before its addition to the cells. + +### Related Information + +1. Assenmacher, M., et al. 1994. Eur. J. Immunol. 24:1097. +2. Elson, L.H., et al. 1995. J. Immunol. 1995. 154:4294. +3. Jung T, et al. 1993. J. Immunol. Methods 159:197. +4. Prussin C., et al. 1995. J. Immunol. Methods 188:117. +5. Vikingsson A., et al. 1994. J. Immunol. Methods 173:219. + +### Reagent List + +1. **Cell Staining Buffer** (BioLegend Cat. No. 420201) +2. **Monensin** (BioLegend Cat. No. 420701) +3. **RBC Lysis Buffer** (BioLegend Cat. No. 420301) +4. **Brefeldin A** (BioLegend Cat. No. 420601) +5. **Fixation Buffer** (BioLegend Cat. No. 420801) +6. **Intracellular Staining Perm Wash Buffer** (BioLegend Cat. No. 421002) +7. **Cyto-Last™ Buffer** (BioLegend Cat. No. 422501) + +### Protocol + +#### Activation + +**Step 1.** +- Dilute heparinized whole blood 1:1 with sterile appropriate tissue culture medium. + +**Step 2.** +- Perform in vitro cellular stimulation by either antigen or mitogen. +- If staining intracellular cytokines or chemokines (e.g., IFN-γ or IL-4), add a protein transport inhibitor such as brefeldin A (BioLegend Cat. No. 420601) or monensin (BioLegend Cat. No. 420701). + +**Step 3.** +- Aliquot 200 µl of the whole blood cell suspension into 12 x 75 mm plastic tubes. +- Incubate for 4-6 hours in 5% CO2 at 37°C. + +**Step 4.** +- Add 2 ml of 1X Red Blood Cell Lysis Buffer (BioLegend Cat. No. 420301) and incubate for 5-10 minutes at room temperature. + +**Step 5.** +- Centrifuge at 350 x g for 5 minutes and discard the supernatant. + +**Step 6.** +- Wash cells 1X with Cell Staining Buffer. + +**Step 7.** +- If staining intracellular antigens (e.g., IFN-γ or IL-4), first perform cell surface antigen staining as described in BioLegend’s [Cell Surface Immunofluorescence Staining Protocol](https://biolegend.com) then fix cells in 0.5 ml/tube Fixation Buffer (BioLegend Cat. No. 420801) in the dark for 20 minutes at room temperature. + +**Step 8.** +- Centrifuge at 350 x g for 5 minutes and discard the supernatant. + +**Step 9.** +- To put the experiment "on hold" for future staining and analysis: + - Wash cells 1x with Cell Staining Buffer (BioLegend Cat. No. 420201). + - Resuspend cells in Cell Staining Buffer and store cells at 4°C (short term) or in 90% FCS/10% DMSO for storage at -80°C (long term, for fixed cells without surface antigen staining). + + _Note: Alternatively, cells can be kept in Cyto-Last™ Buffer (BioLegend Cat. No. 422501) for the storage of cytokine-producing cells for up to two weeks._ + +#### Permeabilization + +**Step 10.** +- Dilute 10X Intracellular Staining Perm Wash Buffer (BioLegend Cat. No. 421002) to 1X in DI water. + +**Step 11.** +- Resuspend fixed cells in Intracellular Staining Perm Wash Buffer and centrifuge at 350 x g for 5-10 minutes. (1/3) + +**Step 12.** +- Resuspend fixed cells in Intracellular Staining Perm Wash Buffer and centrifuge at 350 x g for 5-10 minutes. (2/3) + +**Step 13.** +- Resuspend fixed cells in Intracellular Staining Perm Wash Buffer and centrifuge at 350 x g for 5-10 minutes. (3/3) + +#### Intracellular Staining + +**Step 14.** +- Resuspend fixed/permeabilized cells in residual Intracellular Staining Perm Wash Buffer and add a predetermined optimum concentration of fluorophore-conjugated antibody of interest (e.g., PE anti-IFN-γ) or an appropriate negative control for 20 minutes in the dark at room temperature. + +**Step 15.** +- Wash with 2 ml of Intracellular Staining Perm Wash Buffer and centrifuge at 350 x g for 5 minutes. (1/2) + +**Step 16.** +- Wash with 2 ml of Intracellular Staining Perm Wash Buffer and centrifuge at 350 x g for 5 minutes. (2/2) + +**Step 17.** +- If primary intracellular antibody is biotinylated, perform fluorophore-conjugated Streptavidin incubations and subsequent washes in Intracellular Staining Perm Wash Buffer. + +**Step 18.** +- Resuspend fixed and intracellularly labeled cells in 0.5 ml Cell Staining Buffer and analyze with appropriate controls. + +#### Flow Cytometric Analysis + +**Step 19.** +- Set PMT voltage and compensation using cell surface staining controls. +- Set quadrant markers based on blocking controls, isotype controls, or unstained cells. + + _Note: For proper flow cytometric analysis, cells stained by this method should be inspected by light microscopy and/or flow light scatter patterns to confirm that they are well dispersed._ + + _Note: Bivariate dot plots or probability contour plots can be generated upon data analysis to display the frequencies of and patterns by which individual cells coexpress certain levels of cell surface antigen and intracellular cytokine proteins._ + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/adding-solid-fertilisers-to-soil-in-pot-experiment-4engtde.md b/markdown-output/adding-solid-fertilisers-to-soil-in-pot-experiment-4engtde.md new file mode 100644 index 0000000000000000000000000000000000000000..acebc8330f686181f0ca085a546f289f87a8453c --- /dev/null +++ b/markdown-output/adding-solid-fertilisers-to-soil-in-pot-experiment-4engtde.md @@ -0,0 +1,96 @@ +```markdown +# Goal/Experiment: +To determine the appropriate methods for calculating and adding granular/dry fertilizers to soil in pot experiments before planting. + +## Adding Solid Fertilisers to Soil in Pot Experiments V.2 +**Matema L.E. Imakumbili** +Sokoine University of Agriculture +[dx.doi.org/10.17504/protocols.io.4engtde](http://dx.doi.org/10.17504/protocols.io.4engtde) + +--- + +### Abstract +This protocol describes how rates of granular/dry fertilizer can be calculated and incorporated into soil for pot experiments before planting. + +### Guidelines +It is important to clean all apparatus (shovels, pots, brushes, etc.) before use. This can be done by washing the equipment with tap water and soap. Thoroughly rinse off the soap with clean tap water. All equipment must be properly dried before use. + +### Materials + +- Soil +- Fertilizer (e.g., urea, TSP, muriate of potash) +- Large plastic sheet (2 m × 5 m) +- Weighing scale (5-20 kg capacity) +- Empty pots +- Analytical balance +- Small plastic bags (2 cm × 15 cm) +- Marker pen +- Mechanical kitchen/hanging scale (20 kg capacity) +- A brush or small broom to sweep soil with +- A dustpan + +### 1. Determining the Amount of Fertilizer to Add to Soil in Pots +Field-based fertilizer rates (in kg/ha) are often used and converted to pot-based rates (in g/kg or mg/kg of soil) in pot experiments. This conversion requires some calculations. + +#### Fertilizer Calculation Example: +In this example, we will add potassium to the soil at a rate of 80 kg/ha. The fertilizer used is muriate of potash (MOP), also known as potassium chloride (KCl), which contains 60% potassium oxide (K2O). + +##### Calculation: +1. Amount of potassium to be added to each kilogram of soil: + +\[ +\text{mass of } K \text{ (kg)} = \frac{80 \text{ kg}}{2,000,000 \text{ kg of soil}} = 0.04 \text{ g K/kg of soil} +\] + +2. Potassium to be added to 5 kg of oven-dry soil: + +\[ +\text{mass of } K \text{ to be added to 5 kg of soil} = 0.04 \text{ g K/kg } \times 5 \text{ kg} = 0.2 \text{ g K} +\] + +3. Calculate the equivalent K2O needed: + +\[ +\text{Mass of one mole of } K_2O = 2 \times \text{K} + \text{O} = 78.1966 \text{ g K/kg} +\] + +4. Using the mass ratio: + +\[ +\frac{0.2 \text{ g K}}{78.1966 \text{ g K}} = 0.241 \text{ g K}_2O +\] + +5. Adjust for the 60% purity of MOP: + +\[ +\text{Amount of } MOP = \frac{0.241 \text{ g K}_2O \times 100 \text{ g MOP}}{60 \text{ g K}_2O} = 0.402 \text{ g MOP} +\] + +### 2. Weighing the Fertilizer to Be Added to Pots +Using an analytical balance with an accuracy of ± 0.0001 g, weigh out the required amount of muriate of potash to be added to each pot. Ensure correct labeling and separate packing of the weighed packets of fertilizer for easy identification. + +### 3. Mixing the Fertilizer into the Pot + +#### Procedure: +1. Use a large plastic sheet for mixing the soil and fertilizer. +2. Divide the soil for each treatment into roughly equal portions. +3. Mix the fertilizer into a small amount of soil first and then into a larger amount. +4. Combine all parts thoroughly to ensure uniform mixing. + +**Figures:** +- **Fig 1.** Fertilizer packed in small plastic bags for pot experiments. +![Fig 1](image_url) + +- **Fig 2.** Bulked pile of soil on a plastic sheet. +![Fig 2](image_url) + +### Notes: + +1. **Accuracy:** Precision is crucial for each step of the process. +2. **Hygroscopic Nature:** Fertilizers tend to absorb moisture; pack tightly. +3. **Contamination:** Regularly clean equipment to avoid cross-contamination. + +--- + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/addition-of-rna-sequins-to-sample-for-rna-sequenci-x8cfrsw.md b/markdown-output/addition-of-rna-sequins-to-sample-for-rna-sequenci-x8cfrsw.md new file mode 100644 index 0000000000000000000000000000000000000000..339e3a6e709469e170b569b6a69770dc9bdb2574 --- /dev/null +++ b/markdown-output/addition-of-rna-sequins-to-sample-for-rna-sequenci-x8cfrsw.md @@ -0,0 +1,85 @@ +```markdown +Goal/Experiment: +The goal of the experiment is to assess the impact of technical bias and sample complexity in RNA sequencing by using RNA sequins as internal controls. RNA sequins act as synthetic genes to normalize and control variations in RNA sequencing experiments. + +# Addition of RNA Sequins to Samples for RNA Sequencing + +**Version 1** +**Tim Mercer** +**Garvan Institute of Medical Research** +[doi.org/10.17504/protocols.io.x8cfrsw](dx.doi.org/10.17504/protocols.io.x8cfrsw) + +## Abstract +RNA sequencing can measure both gene or isoform expression and reconstruct novel and complex spliced isoforms. However, the sheer size and complexity of the transcriptome, as well as technical bias, can confound analysis with RNA-seq. To assess the impact of these variables, we developed a set of RNA sequins that represent synthetic genes that act as internal controls during RNA sequencing. + +Each RNA sequin represents an individual isoform, with multiple isoforms forming artificial gene loci that are encoded within the *in silico* chromosome (chrIS). By modulating the relative abundance of individual sequin isoforms, we can emulate alternative splicing while modulating the abundance of multiple isoforms to emulate gene expression. Accordingly, RNA sequins are mixed at different concentrations to emulate differences in gene expression and alternative splicing. + +By sequentially diluting sequins, we can establish a reference ladder across a range of gene expressions. We formulate multiple alternative mixtures that differ in the concentration of individual sequins. By comparing mixtures, we can emulate differential gene expression and alternative splicing between samples. By contrast, RNA sequins with invariant concentrations between mixtures provide static scaling factors that enable quantitative normalization between multiple RNAseq libraries. + +The RNA sequin mixture is added to a user’s RNA sample at a fractional concentration prior to library preparation. The combined sample and sequins then undergo sequencing. The sequins can then be distinguished in the output library by their synthetic sequence, and analyzed as internal controls. + +For further detailed background on the design, validation, and use of sequins, we refer users to "Spliced synthetic genes as internal controls in RNA sequencing experiments" by Hardwick et al., (2016) Nature Methods. + +**External Link:** +[www.sequinstandards.com](www.sequinstandards.com) + +**This protocol accompanies the following publication:** +**Hardwick et. al., Spliced synthetic genes as internal controls in RNA sequencing experiments. (2016) Nature Methods.** + +## Protocol Status +**Working** +We use this protocol in our group, and it is working. + +## Materials + +| Name | Catalog # | Vendor | +|---------------------|-----------|---------| +| RNA sequins standards | [View](https://www.sequinstandards.com) | Sequins | + +## Step Materials + +| Name | Catalog # | Vendor | +|---------------------|-----------|---------| +| RNA sequins standards | [View](https://www.sequinstandards.com) | Sequins | + +### Re-suspension and Storage of Sequins + +1. **RNA Sequins Standards** + ![RNA Sequins Standards](https://www.sequinstandards.com) + + Upon receipt of RNA sequins, first check to ensure they have not thawed during shipment and immediately transfer the RNA sequins to frozen storage at -80°C (sequins should not be stored in a -20°C frost-free freezer). + + ![RNA Sequin Mixture Traces](https://www.sequinstandards.com) + + *Figure 1. Example traces of RNA sequins using an Agilent 2100 BioAnalyzer with the RNA Nano Kit (Agilent Technologies) for (left upper) neat Sequin Mixture A and (left lower) neat Sequins Mixture B. Also shown are example traces for (right upper) K562 with Sequin Mixture A and (right lower) GM12878 with Sequins Mixture B.* + +2. Each tube contains RNA sequins provided in solution in 10 µL nuclease-free water at a concentration of 15 ng/µL. On first thaw, spin the tube down to collect the contents at the bottom of the tube, and prepare smaller single-use aliquots to minimize subsequent freeze-thaw cycles. The exact amount of RNA sequins required for a single-use aliquot depends on the sample input required for your preferred library preparation method. + + **Table 1. Guidelines for diluting RNA sequins according to sample RNA amounts (recommended 1% spike-in).** + + | Sample RNA | Sequin Mass | Sequin Volume (dilution from 15 ng/µL stock)| + |------------|-------------|---------------------------------------------| + | 20 ng | 0.2 ng | 1 µL (1:75) | + | 50 ng | 0.5 ng | 1 µL (1:30) | + | 100 ng | 1.0 ng | 1 µL (1:15) | + | 500 ng | 5.0 ng | 1 µL (1:3) | + | 1000 ng | 10.0 ng | 1 µL (2:3) | + +#### Addition of Sequins to Sample, Library Preparation, and Sequencing + +3. The diluted RNA sequins should then be added directly to the sample RNA prior to any subsequent processing steps (such as poly-A enrichment or rRNA depletion). While this enables an assessment of these processing steps, the amount and dilution of RNA sequins added may need to be modified accordingly. + + > **COMMENT:** RNA sequins are provided in two alternative mixture formulations: Mix A and B. Each contains the same sequin isoforms, however they have been formulated at molar ratios. This emulates fold-change differences in gene expression and alternative splicing between the two mixtures. If a gene-profiling RNAseq experiment is being performed to identify differences in gene expression and splicing between two conditions, it is suggested that Mixture A and B be added alternately to separate samples from each condition being compared (ensure that both mixtures are not added to a single sample). This enables the use of RNA sequins to assess the detection of fold-change differences between samples. + +4. Use the combined sample and sequins as input according to the protocol of your preferred library preparation kit. + + > **COMMENT:** The downstream library preparation workflow may require the user to concentrate the sample RNA after the addition of the RNA sequins. RNA samples can be concentrated using either ethanol precipitation, SPRI® bead purification (e.g. RNAClean® XP, Beckman Coulter), column-based methods (e.g. RNA Clean & Concentrator™ Kit, Zymo Research), or using vacuum centrifugation. + + ![Successful Sequenced Libraries](https://www.sequinstandards.com) + + *Figure 2. Successful sequin-containing (total) RNA Libraries. A, K562 with Sequins Mix A. B, GM12878 with Sequins Mix B. Samples analyzed by Agilent 2100 BioAnalyzer trace (size distributions sequenced on an Illumina HiSeq 2500 Instrument).* + +5. The library that is generated from the combined RNA sample and sequins is then sequenced per manufacturer’s instructions. In this example, Illumina HiSeq 2500 was used. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/adrenal-chromaffin-cell-cultures-bpkzmkx6.md b/markdown-output/adrenal-chromaffin-cell-cultures-bpkzmkx6.md new file mode 100644 index 0000000000000000000000000000000000000000..291c86e8f1d5975bcc0b932611ffc239f0792518 --- /dev/null +++ b/markdown-output/adrenal-chromaffin-cell-cultures-bpkzmkx6.md @@ -0,0 +1,130 @@ +```markdown +# Goal/Experiment: +To culture adrenal chromaffin cells derived from Sprague Dawley rats aged 7 to 12 days. + +# Adrenal Chromaffin Cell Cultures + +**Authors:** +Ellen Kantar1, David Sulzer1 +1Columbia University + +**Date:** +May 18, 2021 + +**DOI:** +[dx.doi.org/10.17504/protocols.io.bpkzmkx6](https://dx.doi.org/10.17504/protocols.io.bpkzmkx6) + +## Abstract +This protocol details the culturing of adrenal chromaffin cells from rats. Adrenal glands from 7- to 12-day-old Sprague Dawley rats are dissected in ice-cold Hanks Balanced Salt Solution (HBSS). + +### Keywords +rat-derived cultures, cell culture, adrenal, chromaffin, rat + +### License +This protocol is distributed under the terms of the [Creative Commons Attribution License](https://creativecommons.org/licenses/by/4.0/), permitting unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. + +## Guidelines +### Suggestions for Plating Density for Rat and Mouse CC Cultures: +- 3 × 10-day-old rat pups – 8 dishes +- 5 × 10-day-old rat pups – 16 dishes +- 2 adult mice – 12 dishes +- 10 × 10-day-old mouse pups – 12 dishes + +## Materials + +### Reagents: +- **Ketamine (Anaesthetic) if decapitation is not an approved protocol: Ketase®** + *Vendor*: FORT DODGE + *Catalog #: NDC-0856-2013-01* + +- **L-Glutamine solution, 200 mM** + *Vendor*: Sigma + *Catalog #: G2150* + +- **Penicillin-Streptomycin** + *Vendor*: Sigma + *Catalog #: P0781* + +- **Fetal Bovine Serum, qualified, heat-inactivated, United States** + *Vendor*: Thermo Fisher + *Catalog #: 16140063* + +- **DMEM - Low Glucose** + *Vendor*: Sigma + *Catalog #: D5546* + +- **HBSS, no calcium, no magnesium, no phenol red** + *Vendor*: Thermo Fisher + *Catalog #: 14175095* + +- **Collagenase Type I** + *Vendor*: Worthington Biochemical Corporation + *Catalog #: LS004197* + +- **Deoxyribonuclease I** + *Vendor*: Worthington Biochemical Corporation + *Catalog #: LS002006* + +### Preparation: + +#### CC Media (200 mL): +- 2 mL L-Glutamine +- 240 µL Pen-Strep +- 20 mL Fetal Bovine Serum, heat-inactivated +- 180 mL DMEM + +#### Trituration Solution (110 mL): +- 10 mL HBSS +- 30 µL DNase stock (final concentration 0.02%) +- 100 µL Fetal Bovine Serum, heat-inactivated + +#### Preparation of DNase I Stock Solution: +- Reconstitute with HBSS to a concentration of 2000 U/mL (e.g., a vial with 20 mg and 3364 U/mg is reconstituted with 33.64 mL HBSS). +- Store in 500 µL aliquots at -80 °C. + +### Safety Warnings: +For hazard information and safety warnings, please refer to the SDS (Safety Data Sheet). + +## Protocol Steps: + +1. **Animal Preparation:** + - Animals are decapitated (anaesthetize the animals with Ketase® KETAMINE, FORT DODGE® NDC-0856-2013-01 if decapitation is not an approved protocol). + +2. **Body disinfection and pinning:** + - Decapitate and pin the body belly-down. Spray with 70% ethanol. + +3. **Back skin opening:** + - Cut the skin along the spinal column (easier starting from the neck) and pull it out to both sides, using scissors to separate the skin from underlying tissue. The back of the body is now open. + +4. **Removal of adrenal glands:** + - Grab the vertical column with forceps and pull it up while cutting along both sides through the ribs. Alternate cuts on each side as you work back. Once the diaphragm is reached, open the scissors and place onto the diaphragm approximately 1/3 of the way up from the bottom. Pull the spine up while holding the diaphragm down. Expose the abdominal cavity showing two kidneys with adrenal glands on top. + +5. **Isolation of adrenal glands:** + - Remove the glands with fine forceps (preferably curved), pinch off the tissue under the glands, and pull up. Place into ice-cold HBSS (Ca²⁺/Mg²⁺-free). + +6. **Removal of capsule:** + - Adrenal glands are encased by adipose tissue and a capsule. Remove using two fine forceps, pull the capsule open like a bag, and use the other to roll off the gland. Cut the adrenal glands in half or thirds depending on their size. + +7. **Cleaning and digestion:** + - After several washes with HBSS (using a sterile plastic transfer pipette), digest the tissue with Collagenase I in 10 mL HBSS, Ca²⁺/Mg²⁺-free, (250-350 U/mL, Worthington) for about 30:00 at 37 °C with stirring. Stop the digestion once the solution becomes cloudy. + +8. **Rinse and triturate:** + - Rinse the digested tissue with HBSS and triturate gently in a solution containing 1% heat-inactivated fetal bovine serum and 0.02% DNase I. Use large bore tech-tips for trituration, medium bore if needed. + +9. **Centrifuge and resuspend cells:** + - Centrifuge dissociated cells at 1000 x g, 00:03:00 to form a pellet. Resuspend in culture medium with DMEM, 10% fetal bovine serum, 50 U/mL penicillin, 50 µg/mL streptomycin, and 2 mM Glutamine. + +10. **Plating cells:** + - Plate cell suspension on poly-D-lysine and laminin-coated glass wells in 50 mm dishes (cells from 5 rat 10-day-old pups onto 16 dishes), and after 2:00:00, flood dishes with culture medium (3 mL per dish). + +11. **Incubation:** + - Maintain cells in a 5% CO₂ incubator at 37 °C. Conduct all measurements between 1 and 7 post-plating days. + +--- + +Please refer to the "Ventral Midbrain Cultures" protocol for instructions on how to prepare and coat dishes. + +--- + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/agarose-gel-electrophoresis-1-2-with-ethidum-bromi-ds76hm.md b/markdown-output/agarose-gel-electrophoresis-1-2-with-ethidum-bromi-ds76hm.md new file mode 100644 index 0000000000000000000000000000000000000000..b31a4351850023bdd7ccff9514c4c73149c956a8 --- /dev/null +++ b/markdown-output/agarose-gel-electrophoresis-1-2-with-ethidum-bromi-ds76hm.md @@ -0,0 +1,133 @@ +```markdown +# Agarose Gel Electrophoresis, 1.2% with Ethidium Bromide + +### Goal/Experiment: +The goal of this experiment is to separate DNA molecules based on size using agarose gel electrophoresis. This protocol is suitable for checking DNA after a restriction digest and can resolve DNA fragments ranging from 400bp to 7kb. + +### Abstract +This protocol separates molecules based on size and is great for checking DNA after a restriction digest. The protocol utilizes a 1.2% agarose gel, sufficient for resolving DNA from 400bp to 7kb. The electrophoresis system has a 7.5cm x 10cm gel bed area with an approximately 15cm electrode distance. The minimum volume for a 7.5cm x 10cm x 0.5cm gel is 37.5mL, and the maximum volume for a 50mL gel is 0.67cm in height. + +**Citation**: Harold Bien Agarose gel electrophoresis, 1.2% with Ethidium bromide. protocols.io dx.doi.org/10.17504/protocols.io.ds76hm +**Published**: 11 Sep 2015 + +--- + +### Before Start +- Ensure you have a DNA sample ready, either from PCR or a recently performed restriction digest. +- Dilute the 50X TAE Buffer to 1X using sterile filtered water. +- For the dual-sided 1.0mm gel comb MHC-10-0816 the maximum volume is 35/13μL. +- For the dual-sided 1.5mm gel comb MHC-15-1014, the maximum sample volume is 38/25μL. + +--- + +### Materials + +| Reagent/Equipment | Specification | Vendor | +|-------------------------------------------------------|---------------------------------|--------------------------------------------------| +| 2-Log DNA Ladder | 100-200 gel lanes | N3200S by New England Biolabs | +| Gel Loading Dye, Purple (6X), no SDS | 4.0 mL | B7025S by New England Biolabs | +| GeneMate LE Quick Dissolve Agarose | 500g | E-3119-500 by Bioexpress | +| Ethidium bromide | 10mg/mL, 10mL | X328-10ML by Amresco | +| TAE (Tris-Acetate-EDTA) Buffer, 50X | | K915 by Amresco | +| Horizontal mini-gel kit | | MHU-202 by C.b.s Scientific | + +--- + +### Protocol + +#### Prep Work + +##### Step 1 +Weigh out 0.6 g (1.2% w/v of 50mL) agarose and add it to the Erlenmeyer flask. + +- **Amount:** 1 g +- **Reagents:** GeneMate LE Quick Dissolve Agarose, 500g [E-3119-500 by Bioexpress] + +##### Step 2 +Add 50mL of 1x TAE buffer. + +- **Amount:** 50 mL +- **Reagents:** TAE (Tris-Acetate-EDTA) Buffer, 1X + +##### Step 3 +Place Erlenmeyer flask in microwave. Set to wait 30 seconds, then full power (P10, 1250W) for 20 seconds followed by low power (P1, 125W) for 30 seconds or until the solution is clear and agarose is completely dissolved. + +**Duration:** 50 seconds + +**Notes:** +- Ensure the agarose is fully dissolved in the buffer solution. + +##### Step 4 +Remove Erlenmeyer flask from microwave and let it sit on the lab bench to cool just until you can comfortably pick it up. + +**Duration:** 3 minutes + +##### Step 5 +Add 1μL concentrated ethidium bromide (10mg/mL) into the flask and swirl to mix, taking care not to introduce bubbles. + +- **Amount:** 1 μL +- **Reagents:** Ethidium bromide, 10mg/mL, 10mL [X328-10ML by Amresco] +- **Notes:** The final concentration of ethidium bromide will be 10μg/50mL or 0.2μg/mL (Carcinogen, handle with care). + +##### Step 6 +Place gel tray on clamp and clamp securely. Add well plates where you want wells and use a level to ensure it is balanced. + +**Notes:** +- Good well balance is crucial for even sample loading. + +--- + +### Running the Agarose Gel + +#### Running the Gel + +##### Step 7 +Pour contents of the Erlenmeyer Flask into the gel tray and let it sit for 30 minutes, or until a blue tint appears. + +- **Duration:** 30 minutes + +##### Step 8 +Remove the well plates carefully to avoid tearing the gel and remove the tray from the clamp, ensuring the gel remains in the tray. + +##### Step 9 +Place gel tray into the gel electrophoresis apparatus with the wells closer to the negative/black end. + +##### Step 10 +Pour additional TAE Buffer to fill each side of the apparatus and to create a thin layer of buffer covering the top of the gel. + +##### Step 11 +Prepare DNA ladder and samples by adding 6x blue dye. + +- **Reagents:** Gel Loading Dye Blue (6X) - 4.0 ml [B7021S by New England Biolabs] +- **Notes:** + - Dilute the DNA ladder 1:10 in sterile filtered water when using the 1.5mm thick 14-well lane. + +##### Step 12 +Pipette your samples into each well. + +- **Notes:** + - For the 1.5mm gel comb MHC-15-1014, recommended DNA mass is 200-500ng for 14-well. + +##### Step 13 +Place the lid on the apparatus and plug cables into the high voltage power supply. Run at 100V (6.6V/cm) for 45-60 minutes or until the loading dye has sufficiently migrated down the gel. + +- **Duration:** 45-60 minutes +- **Notes:** + - Ensure that the negative terminal (typically black) is plugged into the negative/black terminal on the power supply and the wells are at the negative side. + +--- + +#### Visualizing DNA Bands + +##### Step 14 +Gel can be imaged on UV transilluminator through the UV-transparent gel tray or removed and wrapped in plastic wrap for storage at 4°C for later use. + +--- + +### Warnings +Ethidium Bromide potentially acts as a mutagen or carcinogen. Handle with proper safety measures. + +--- + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/aktiv-formulations-keto-bhb-100-safe-and-effective-b96qr9dw.md b/markdown-output/aktiv-formulations-keto-bhb-100-safe-and-effective-b96qr9dw.md new file mode 100644 index 0000000000000000000000000000000000000000..371f54ae626ce6236368e6346f7c7994eccdb6d0 --- /dev/null +++ b/markdown-output/aktiv-formulations-keto-bhb-100-safe-and-effective-b96qr9dw.md @@ -0,0 +1,77 @@ +```markdown +# Goal/Experiment: +Evaluate the claims and effectiveness of Aktiv Formulations Keto BHB as a safe and effective weight-loss supplement. + +## Aktiv Formulations Keto BHB 100% Safe and Effective Formulation! (Legit Or Scam?) + +### Product Information +- **Product Name:** [Aktiv Formulations Keto BHB](https://aktivformulationsketobhb.com) +- **Composition:** Natural +- **Side Effects:** NIL +- **Price:** [Check Online/Offline](https://aktivformulationsketobhb.com) +- **Rating:** (User ratings not provided) +- **Official Sponsor:** [aktivformulationsketobhb.com](https://aktivformulationsketobhb.com) + +### Overview +Aktiv Formulations Keto BHB is a weight reduction dietary supplement designed to help users reduce excess fat effectively. The supplement claims to harness natural ingredients to boost the body's fat-burning process, primarily through ketosis. + +--- + +## What Exactly Is Aktiv Formulations Keto BHB Diet Program? + +Aktiv Formulations Keto BHB is advertised as a weight-loss nutritional supplement aimed at reducing extra fat and maintaining a lean body. It does this by leveraging the processes associated with ketosis, where the body burns fats instead of carbohydrates for energy. + +### Key Functions: +1. **Promotes Weight Loss:** Helps reduce extra fat and maintain a lean body. +2. **Natural Ingredients:** Contains BHB ketones, Rice-flour, Silicon Dioxide, and Green Coffee-bean. +3. **Burns Fat Efficiently:** Targets body portions like buttocks, back, thighs, and wrists. +4. **Inhibits Fat Accumulation:** Prevents the storage of new fat in the body. + +### Ingredients and Their Functions: +1. **BHB Ketones:** A combination of BHB magnesium and BHB sodium, critical in inducing ketosis and providing energy by burning fat. +2. **Rice-flour:** Provides low carbohydrates and aids in burning fats naturally. Acts as an anti-oxidant to promote fat reduction. +3. **Silicon Dioxide:** Enhances the body's ability to lose weight by encouraging fat reduction naturally. +4. **Green Coffee-bean:** Contains chlorogenic acid that helps in weight reduction and boosts metabolism. Also contains caffeine which acts as a natural stimulant. + +### Additional Useful Compounds: +1. **Hoodia Gordonii:** Known for suppressing appetite, essential for those facing challenges with cravings and excess weight. +2. **Magnesium:** Assists in further fat reduction, supports overall body health, and aids muscle fortification. + +--- + +## Mechanism of Action +### Aktiv Formulations Keto BHB's Working Principle: +- **Induces Ketosis:** By leveraging BHB ketones, the supplement aims to switch the body's fuel source from carbohydrates to fats, promoting efficient weight loss. +- **Enhances Fat Burning:** Burns off saved fats while inhibiting carbohydrate conversion into fats. + +**Ketosis Details:** Ketosis occurs when the body burns fat instead of carbs due to lower carbohydrate intake. This is facilitated by the BHB ketones present in the supplement. + +### Proper Usage: +- The supplement is provided in capsule form. +- Recommended dosage: Two capsules daily. +- Drink plenty of water and engage in regular physical activities to enhance the supplement's effectiveness. + +### Suitable For: +- Individuals seeking substantial weight loss. +- People ready to supplement their diet with a ketogenic regimen. +- Adults (not recommended for individuals under 20, pregnant women, or those with high blood pressure). + +### Potential Side Effects: +- Reported as minimal to zero due to the natural composition. +- Users are advised to consult with physicians if they have underlying medical conditions. + +### Purchase Information: +Aktiv Formulations Keto BHB can be purchased online through the [official website](https://aktivformulationsketobhb.com). + +--- + +## Conclusion +Aktiv Formulations Keto BHB appears to be a credible natural weight loss supplement focused on leveraging the ketogenic process. Its efficacy is enhanced when combined with regular exercise and a balanced diet. + +### Recommended Actions: +- Ensure adherence to a ketogenic diet. +- Engage in regular physical exercises. +- Monitor health regularly and consult a healthcare provider as needed. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/algorithm-for-gestational-age-assessment-at-birth-bawbifan.md b/markdown-output/algorithm-for-gestational-age-assessment-at-birth-bawbifan.md new file mode 100644 index 0000000000000000000000000000000000000000..86b07c9be16c52e133cea111515e20d64e71c9db --- /dev/null +++ b/markdown-output/algorithm-for-gestational-age-assessment-at-birth-bawbifan.md @@ -0,0 +1,107 @@ +```markdown +# Goal/Experiment: +The goal of this protocol is to develop a reliable algorithm for assessing the gestational age of a newborn at birth. This is achieved through the use of either the mother's last menstrual period (LMP) or an obstetric ultrasound conducted early in the pregnancy. The aim is to standardize and automate the process to provide the most accurate estimate of gestational age at birth. + +# Algorithm for Gestational Age Assessment at Birth + +**Authors**: Zilma Reis, Juliano de Souza Gaspar, Sara Oliveira Elias, Regina Amelia Lopes Pessoa De Aguiar +**Institution**: Universidade Federal de Minas Gerais +**Published**: Jan 16, 2020 +**DOI**: [10.17504/protocols.io.bawbifan](http://dx.doi.org/10.17504/protocols.io.bawbifan) + +## Abstract +This protocol presents an algorithm for gestational age assessment when a reliable last menstrual period or an obstetric ultrasound is available in the birth scenario. A software was developed to automatically process data entries into the best estimate of the gestational age at birth. + +It shall be used by the multicenter team of researchers, duly trained in accordance with the Good Clinical Practice Protocol, during the enrollment of newborns. This protocol is complementary documentation for scientific publications related to the clinical trials. + +Relevant Clinical Trials: +- "Prematurity detection evaluating interaction between the skin of the newborn and light: protocol for the preemie-test multicenter clinical trial in Brazilian hospitals to validate a new medical device." Register number: [RBR-3f5bm5](http://ensaiosclinicos.gov.br/rg/RBR-3f5bm5). +- "Premature or small for gestational age? International multicenter trial protocol for classification of the low birth weight newborn through the optical properties of the skin." Register number: [RBR33rnjf](http://ensaiosclinicos.gov.br/rg/RBR33rnjf). + +## Guidelines +The scientific references for the best recommendation to assist gestational age calculation at birth were: +1. Committee on Obstetric Practice, the Society for Maternal-Fetal Medicine. Committee Opinion No 700: Methods for estimating the due date. Obstet Gynecol. 2017;129(5):e150-4. PMID: 28426621. DOI: [10.1097/AOG.0000000000002046](http://dx.doi.org/10.1097/AOG.0000000000002046). +2. Papageorghiou AT, Kennedy SH, Salomon LJ, et al. International standards for early fetal size and pregnancy dating based on ultrasound measurement of crown-rump length in the first trimester of pregnancy. Ultrasound Obstet Gynecol 2014;44:641-8. DOI: [10.1002/uog.13448](http://dx.doi.org/10.1002/uog.13448). +3. Nguyen TH, Larsen T, Engholm G, et al. Increased adverse pregnancy outcomes with unreliable last menstruation. Obstet Gynecol 2000;95(6 Pt 1):867-73. DOI: [10.1016/s0029-7844(99)00639-0](http://dx.doi.org/10.1016/s0029-7844(99)00639-0). + +## Materials +- **Reagents and Equipment:** + - Standardized Clinical Trial Data Collection Form + - Tablet for data collection + +## Safety Warnings +- Ensure that patient consent forms are completed. +- Obtain necessary ethical approvals for conducting the research. + +## Before Starting +Researchers should identify and prepare to interview the woman if she is conscious and agrees to participate in the study. Sources of data to assist in determining gestational age should include antenatal obstetric ultrasound reports with their images and medical records from prenatal care. + +## Methodology + +### 1. Menstrual Cycle Reference + +#### 1.1 The Last Menstrual Period (LMP) Reference +The interview aims to qualify the information about the last menstrual period, in terms of its reliability. +- **Documentary Analysis**: + - When did your last menstrual period occur? Please confirm if it was the first day of menstruation. + - Are you sure about this date? + - Are your menstrual cycles regular? Clarify if the cycle duration did not change for more than one week. + - Two months before your last menstrual period, did you use any contraceptive method (pills, injections, Mirena IUD, skin implant, vaginal ring)? + - Two months before your last menstrual period, had you had an abortion or delivered a child? + +- **Further Verification**: + - Take a picture of the personal prenatal care book where the LMP is reported to verify the accuracy. + +### 2. Ultrasound Reference + +#### 2.1 The First Obstetric Ultrasound Reference +Assess the best antenatal ultrasound record to determine gestational age. The assessment should occur between 7 weeks 0 days and 13 weeks 6 days of pregnancy, and the crown-rump-length (CRL) of the embryo is the primary reference. + +- Chronologically organize the antenatal obstetric ultrasound reports. +- Select the first gestational assessment with an available CRL measurement between 7w+0d and 13w+6d. +- Take note of the assessment date and reported gestational age. +- Verify the CRL measurement, using the International Fetal Size standard for early pregnancy to adjust the gestational age accordingly. + +### 3. Gestational Age Assessment, According to an Automated Algorithm +- Use software to process pregnancy dating at birth, following best practices for gestational age calculation. + +The algorithm automates methods as follows: +- **Adjustment of CRL to the Intergrowth-21st Reference**: Based on international standards (Papageorghiou AT et al., 2014). +- **Reliable Last Menstrual Period Concept**: Addressing increased adverse pregnancy outcomes with unreliable LMP (Nguyen TH et al., 2000). +- **Gestational Age Assisted by CRL or LMP**: According to recommendations by the Committee on Obstetric Practice (2017). + +#### 3.1 Data Collection +Insert the following details into the information system: + +| Variable | Data | Details | +|-----------------|------------------------------------------------|-----------------| +| LMP_Date | Date of the last menstrual period | Date | +| LMP_1 | Question 1 | Yes/No | +| LMP_2 | Question 2 | Yes/No | +| LMP_3 | Question 3 | Yes/No | +| LMP_4 | Question 4 | Yes/No | +| LMP_5 | Question 5 | Yes/No | +| LMP_GA_Days | Number of days between the LMP_Date and Childbirth_Date | Date | +| US_CRL | Crown-rump-length (mm) reported in the ultrasound | Value | +| GA_CRL | Gestational age reported in the ultrasound (days) | Value | +| Childbirth_Date | Date of the childbirth | Date | +| US_Date | Date of the reference obstetric ultrasound | Date | +| US_GA | Gestational age reported in the ultrasound (days) | Value | +| DIFF | Day difference by LMP and US days of pregnancy | Value | +| DB_Intergrowth | Database with Intergrowth's CRL standard curves | Reference values| + +#### 3.2 Decision-tree Details +Refer to the algorithm's decision tree for handling different data points regarding LMP and ultrasound measurements. + +### 4. Experts Checking and Adjustments +Researchers are required to recheck data, referring to sources such as photos of ultrasound reports, digital images of the crown-rump length measurement, and copies of personal prenatal care records. Gestational age data should be validated against registered information. + +Once validation is complete, the algorithm will process the optimal dating for gestational age at birth. + +## Conclusion +This protocol provides a systematic process to determine the gestational age of a newborn at birth reliably. The accuracy of the assessment is enhanced by integrating data from two primary sources: the last menstrual period and early obstetric ultrasound measurements. + +--- + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/alm-window-surgery-bqstmwen.md b/markdown-output/alm-window-surgery-bqstmwen.md new file mode 100644 index 0000000000000000000000000000000000000000..912161a30c2e28ca30ee8e847c03b4ba9fc4889a --- /dev/null +++ b/markdown-output/alm-window-surgery-bqstmwen.md @@ -0,0 +1,88 @@ +```markdown +**Goal/Experiment:** +The goal of this protocol is to provide a detailed, step-by-step method for performing ALM (anterior lateral motor) window surgery in mice. This includes preparation, craniotomy, window installation, recovery, and post-operative care. This method is designed for researchers conducting in vivo imaging and other experimental studies on cortical structures. + +# ALM Window Surgery + +**Authors:** +Kayvon Daie¹, Tim Wang¹, Amrita Singh¹, Arseny Finkelstein¹, Marton Rozsa¹, JJ Kim², Karel Svoboda¹ + +¹Janelia Research Campus; ²HHMI Janelia Research Campus, Johns Hopkins University School of Medicine + +**Publication Date:** Dec 15, 2020 +**Last Modified:** Oct 23, 2023 +**Protocol ID:** 45619 + +## Abstract +Protocol for Head Post and Cranial Window Surgery developed at Janelia Research Campus in the Svoboda Lab. + +Developed and improved by: +- Karel Svoboda for Trachtenberg et al 2002 +- Anthony Holtmaat for Holtmaat et al 2006 and Holtmaat et al 2009 +- Daniel Huber for Huber et al 2012 + +Compiled and modified by various researchers over the years, the latest version was compiled by JJ Kim in 2019. + +**Note:** This outline provides practical step-by-step advice. Surgeons must adhere to the official animal protocol and current procedures posted on the Svoboda lab wiki. + +## Guidelines +### ALM Craniotomy Considerations +- **Coordinates**: 1.5 mm lateral and 2.5 mm anterior to Bregma. The medial edge of the circle should fall over the midline. +- **Head post placement**: Just posterior to the outer edge of the glass window. +- **Skull preparation**: Rotate the skull when drilling and measuring. +- **Blood vessel management**: Handle major blood vessels carefully to minimize bleeding. +- **Triple glass window**: Use 2.5 mm/2.5 mm/3 mm glasses. +- **Thinning the skull**: The skull varies in thickness; lateral edges are particularly thick. +- **Avoid dural thickening**: Thin down the skull adequately. +- **Bleeding management**: Blood coagulates in ~90 seconds. + +## Protocol + +### Animal Preparation +1. **Weigh Mouse**: Transfer mouse to a new cage if needed. Note ear tags or mark first mouse. +2. **Prepare Cage**: Allow mouse to adjust to a new cage several hours before surgery. +3. **Food Preparation**: Remove wirebar, provide food pellets, and add dietgel to the cage. + +### Drug Preparation +4. **Buprenex dilution**: Dose 0.1 mg/kg, draw up using an insulin syringe. Administer dexamethasone (optional), marcaine (50 µL), ketoprofen (5 mg/kg). + +### Preparation and Mounting +5. **Surgical Prep**: + - **Gloves and Gown**: Wear protective clothing. + - **Prepare Viruses**: If needed. + - **Surgery Area**: Sterilize with Virkon-s and set up the necessary equipment. + +6. **Induction**: + - **Isoflurane**: Turn it to 3%, place the mouse in the induction chamber, and monitor. +7. **Securing the mouse**: + - **Nose Cone**: Gently secure the mouse ensuring comfort. +8. **Prepare for Surgery**: + - Cover eyes with artificial tears and inject marcaine subcutaneously. + - Apply betadine and perform a clean incision to remove scalp and expose skull. + +### Craniotomy and Window Installation +18. **Drill Window**: + - *Mark and drill*: Carefully thin the skull without penetrating fully. + - *Install Window*: Place and secure the triple glass window. + +### Recovery +38. **Post-Surgery**: + - Lower isoflurane and clean the surgical site. + - Secure mouse on a heating pad until recovery reflexes are observed. + - Administer ketoprofen and buprenex, record the end time and details. + +### Virus Injections (If Applicable) +44. **Virus Preparation**: + - Backfill pipette with mineral oil, prepare injection apparatus, and spin down the virus if needed. +48. **Performing Injections**: + - Use stereotactic equipment to inject virus precisely. + +### Post-Operation Care +41. **Noting Care**: Ensure post-op care instructions are clear for follow-up. + +## End Notes +- **References**: Include scholarly articles for detailed readings. +- **Attachments**: Detailed diagrams and supplementary PDF for visualization. + +**endofoutput** +``` \ No newline at end of file diff --git a/markdown-output/amplicon-multiplex-pcr-sequencing-of-rift-valley-f-ckb2usqe.md b/markdown-output/amplicon-multiplex-pcr-sequencing-of-rift-valley-f-ckb2usqe.md new file mode 100644 index 0000000000000000000000000000000000000000..75eda1003a71b044b55a3790aa0f912de722c82e --- /dev/null +++ b/markdown-output/amplicon-multiplex-pcr-sequencing-of-rift-valley-f-ckb2usqe.md @@ -0,0 +1,277 @@ +```markdown +# Goal/Experiment: +Amplicon multiplex PCR sequencing of Rift Valley fever virus (RVFV) on Illumina MiSeq + +## Abstract +Amplicon sequencing protocol for Rift Valley fever virus (RVFV). + +## RNA Extraction + +1. Extract viral RNA from serum or cell-culture supernatants using QIAamp Viral RNA kit (QIAGEN, Hilden, Germany), according to the manufacturer’s instructions. Begin with a volume of 140 µL. + +## RT-qPCR + +2. Determine cycle threshold (Ct) values on RNA samples using probe-based reverse transcription quantitative real-time PCR against the highly conserved domain on the L-segment of the virus (using 5' Fam reporter dye and 3' BHQ1 quencher dye). + +| RVFV segment | Primer name | Sequence 5’-3’ | +|--------------|--------------------|--------------------------------------| +| L | RVFL-2912FwdGG | TGAAAATTCCCTGAGACACATGG | +| L | RVFL-2981revAC | ACTTCCTTGCATCACTGATG | +| L | RVFL-probe-2950 | CAATGTAAGGGGCCTGTTGTGGCAGCTGTG | + +Table 1. Primers and probe set used for RT-qPCR assay (Bird et al., 2007). + +### Mix the following components in PCR strip-tubes/plate + +| Component | Volume (µL) | +|-----------------------------------|-------------| +| KiCqStart™ One-Step Probe RT-qPCR ReadyMix™ | 7.5 | +| Nuclease-free water | 4.75 | +| RVFV Oligos (2912FwdGG, 2981revAC, probe-2950) | 0.75 | +| RNA | 2.0 | +| Total | 15 | + +Note: Set up the reaction on ice. Incubate the reaction on an Applied Biosystems machine as follows: + +- 50 °C for 10:00 +- 95 °C for 2:00 +- 95 °C for 0:03, 40 cycles +- 60 °C for 0:30 + +## cDNA Synthesis + +3. Prepare RNA samples and include a negative control (nuclease-free water) per library. If previously frozen, mix by vortexing briefly and quick spin to collect the liquid. At all times, keep the samples on ice. Mix the following components in PCR strip-tubes/plate. Gently mix by pipetting and performing quick spin to collect the liquid. + +| Component | Volume (µL) | +|-----------------|-------------| +| LunaScript RT Supermix (5X) | 2 | +| Template RNA | 8 | +| Total | 10 | + +Note: To prevent pre-PCR contamination the mastermix should be added to the PCR strip-tubes/plate in the mastermix cabinet, which should be cleaned with decontamination wipes and UV sterilized before and after use. RNA samples should be added in the extraction/sample addition cabinet which should be cleaned with decontamination wipes and UV sterilized before and after use. + +3. Incubate the reaction as follows: + - 25 °C for 2:00 + - 55 °C for 10:00 + - 95 °C for 1:00 + - Hold at 4 °C + +## Primer Pool Preparation + +4. If making up primer pools from individual oligos, fully resuspend lyophilized oligos in 1xTE to a concentration of 100 micromolar (µM), vortex thoroughly, and spin down. + +### Preparing the primer pools: + +4.1 Sort all odd regions primers into one or more tube racks. Add 5 µL of each odd region primer to a 1.5 mL Eppendorf tube labeled "Pool 1 (100 µM)". Repeat the process for all even region primers for Pool 2. These are your 100 µM stocks of each primer pool. + +Note: Primers should be diluted and pooled in the mastermix cabinet which should be cleaned with decontamination wipes and UV sterilized before and after use. + +4.2 Dilute 100 µM pools 1:10 in molecular grade water, to generate 10 µM primer stocks. + +Note: Primers are used at a final concentration of 15 nanomolar (nM) per primer. In this case, V1 pools have 38 primers in pool 1 and 36 primers in pool 2, so the requirements are approx. 1.4 µL primer pool (100 µM) per 25 µL reaction. + +Note: Make up several 100 µL aliquots of 10 µM primer dilutions and freeze them in case of degradation and/or contamination. + +## Multiplex PCR + +5. Set up the two PCR reactions per sample as follows in strip-tubes or plates. Gently mix by pipetting and pulse spin the tube to collect liquid at the bottom of the tube. + +| Component | Reaction 1 (µL) | Reaction 2 (µL) | +|-------------------------------|-------------------------|-------------------------| +| Q5 Hotstart Mastermix Buffer (5X) | 12.5 | 12.5 | +| V1 Primer Pool 1 | 1.425 | 0 | +| V1 Primer Pool 2 | 0 | 1.35 | +| Nuclease-free water | 6.575 | 6.65 | +| Mastermix Volume | 20.5 | 20.5 | +| (cDNA) | 4.5 | 4.5 | +| **Total reaction Volume** | **25** | **25** | + +Note: To prevent pre-PCR contamination the mastermix for each pool should be made up in the mastermix cabinet, which should be cleaned with decontamination wipes and UV sterilized before and after use and aliquoted into PCR strip-tubes/plate. + +### 5.2 Add 4.5 µL cDNA to each of the PCR reactions, gently mix by pipetting and pulse spin the tube to collect liquid at the bottom of the tube. + +Note: cDNA should be added in the extraction and sample addition cabinet which should be cleaned with decontamination wipes and UV sterilized before and after use. + +### Set up the following program on the thermal cycler: + +| Step | Temperature | Time | Cycles | +|----------------|-------------|------------------|---------| +| Heat activation| 98 °C | 0:30 | 1 | +| Denaturation | 95 °C | 0:15 | 35 | +| Annealing | 63 °C | 5:00 | 35 | +| Hold | 4 °C | Indefinite | 1 | + +## Amplicon Clean-up + +6. Combine the two pools of amplicons. Add 12.5 µL of each primer pool (Pool 1 and Pool 2, total of 25 µL) in new PCR strip-tubes/plate. Perform NEBNext Sample Purification Beads/AMPure XP bead cleanup as follows: + +### 6.1 Add 20 µL (0.8X) of AMPure XP beads (thoroughly vortexed and at Room temperature). Cover the plate with seal, gently mix on a plate mixer and pulse spin to bring down the components at the bottom of the tube. Incubate at Room temperature for 5 minutes. + +### 6.2 Place the tube/plate on a magnetic stand for 5 minutes or until the beads have pelleted and the supernatant is completely clear. + +### 6.3 Remove and discard the liquid from each well with a multichannel pipette, being careful not to touch the bead pellet. + +### Note: Caution: do not discard the beads. + +### 6.4 Add 200 µL of freshly prepared, Room temperature 80% ethanol to each well/tube, incubate for 30 seconds at Room temperature and then carefully remove and discard the supernatant. + +### Note: Be careful not to disturb the beads that contain DNA targets. + +### 6.5 Repeat ethanol wash (step 6.3 and 6.4). Be sure to remove all visible liquid after the second wash. If necessary, briefly spin the tube/plate, place back on the magnet and remove traces of ethanol with a p10 pipette tip. + +### 6.6 Air dry the beads for up to 5 minutes while the tube/plate is on the magnetic stand with the lid open. + +### Note: Caution: Do not over-dry the beads. This may result in lower recovery of DNA. Elute the samples when the beads are still dark brown and glossy looking, but when all visible liquid has evaporated. When the beads turn lighter brown and start to crack, they are too dry. + +### 6.7 Remove the tube/plate from the magnetic stand. Elute the DNA target from the beads by adding 28 µL 0.1X TE or Elution Buffer (EB). + +### 6.8 Mix well by pipetting up and down 10 times, or on a vortex mixer. Incubate for at least 2 minutes at room temperature. If necessary, quickly spin the sample to collect the liquid from the sides of the tube or plate wells before placing back on the magnetic stand. + +### 6.9 Place the tube/plate on the magnetic stand. After 5 minutes (or when the solution is clear). + +### 6.10 Transfer 25 µL to a new PCR tube, ensuring no beads are transferred. + +## Gel Electrophoresis or Tapestation + +7. Use remaining volumes from Pool 1 and Pool 2 to confirm amplification (step 5.3). + +### 7.1 Make 1% agarose gels with enough wells for all samples. + +### 7.2 Load 2 µL of the 100 bp ladder into gel on either side of each row of wells. + +### 7.3 Dispense 2 µL of 6X loading dye into each sample with a multichannel pipette, mix and load 2 µL of this mix into the gel. + +### 7.4 Run at 240V for 20 minutes. Visualize PCR products, confirm bands of approximately 400bp. + +### Run pooled cDNA amplicons on a TapeStation® without cleanup. To run on a TapeStation, dilute an aliquot of the pooled amplicons 10-fold with 0.1X TE Buffer and run 2 µL on a DNA High Sensitivity ScreenTape. + +## Amplicon Quantification + +8. Quantify amplicons using Qubit dsDNA High Sensitivity kit and plate reader according to directions. + +## Library Preparation + +9. Prepare sequencing libraries with NEBNext Ultra II RNA Library Prep kit at half volume, as follows. + +### 9.1 End-Prep + +Add the following components to a sterile nuclease-free tube: + +| Component | Volume (µL) | +|-----------------------------------|-------------| +| NEBNext Ultra II End Prep Enzyme Mix | 1.5 | +| NEBNext Ultra II Reaction Buffer | 3.5 | +| Targeted cDNA amplicon | 25 | +| **Total volume** | **30** | + +Set a 100 µL or 200 µL pipette to 25 µL and then pipette the entire volume up and down at least 10 times to mix thoroughly. Perform a quick spin to collect all liquid from the sides of the tube. + +In a thermal cycler with lid heated to 75 °C, run the following program: + +| Temperature | Time | +|-------------|----------| +| 20 °C | 30:00 | +| 65 °C | | +| 4 °C | Indefinite| + +### 9.2 Adaptor-ligation + +Add the following components directly to the End Prep Reaction Mixture: + +| Component | Volume (µL) | +|------------------------------|-------------| +| End Prep Reaction Mixture (step 9.1) | 30 | +| NEBNext Adaptor for Illumina | 1.25 | +| NEBNext Ultra II Ligation Master Mix | 15 | +| **Total volume** | **46.25** | + +Note: Mix the NEBNext Ultra II Ligation Master Mix by pipetting up and down several times prior to adding to the reaction. The NEBNext adaptor is provided in NEBNext Oligo kits. NEB has several oligo options which are supplied separately from the library prep kit. Please see www.neb.com/oligos for additional information. + +### Do not premix adaptor with the Ligation Master Mix. + +### 9.3 Set a 100 µL or 200 µL pipette to 40 µL and then pipette the entire volume up and down at least 10 times to mix thoroughly. Perform a quick spin to collect all liquid from the sides of the tube. + +Note: Caution: The NEBNext Ultra II Ligation Master Mix is very viscous. Care should be taken to ensure adequate mixing of the ligation reaction, as incomplete mixing will result in reduced ligation efficiency. The presence of a small amount of bubbles will not interfere with performance. + +### 9.4 Incubate at 20 °C for 15 minutes in a thermal cycler with the heated lid off. + +### 9.5 Add 1.5 µL of USER® Enzyme to the ligation mixture from Step 9.4. + +Note: Steps 9.5 and 9.6. are only required for use with NEBNext Adaptors. USER enzyme can be found in the NEBNext Multiplex Oligos (www.neb.com/oligos). + +### 9.6 Mix well and incubate at 37 °C for 15 minutes with the heated lid set to ≥ 47 °C. + +Note: Samples can be stored overnight at –20°C. Note: Only a portion of the ligation reaction (7.5 µl) will move forward to PCR enrichment. + +## PCR Enrichment of Adaptor-ligated DNA + +10. Follow Section 10.1 if you are using the following oligos: Use option A for any NEBNext Oligo kit where index primers are supplied in tubes. These kits have the forward and reverse primers supplied in separate tubes. Primers are supplied at 10 micromolar (µM). + + Follow Section 10.2. if you are using the following oligos: Use Option B for any NEBNext Oligo kit where index primers are supplied in a 96-well plate format. These kits have the forward and reverse (i7 and i5) primers combined. Primers are supplied at 10 micromolar (µM). + +### 10.1 Add the following components to a sterile strip tube: +Separate Forward and Reverse Primers + +| Component | Volume (µL) | +|---------------------------------------|-------------| +| Adaptor Ligated DNA Fragments (step 9.4 or 9.6) | 7.5 | +| NEBNext Library PCR Master Mix | 12.5 | +| Universal PCR Primer/i5 Primer | 2.5 | +| Index (X) /i7 Primer | 2.5 | +| **Total volume** | **25** | + +### 10.2 Add the following components to a sterile strip tube: +Premixed Forward and Reverse Primers + +| Component | Volume (µL) | +|---------------------------------------|-------------| +| Adaptor Ligated DNA Fragments (step 9.4 or 9.6) | 7.5 | +| Adaptor Ligated DNA Fragments (step 9.4 or 9.6) | 12.5 | +| Index Primer Mix | 5 | +| **Total volume** | **25** | + +### 10.3 Set a 100 µL pipette to 20 µL and then pipette the entire volume up and down at least 10 times to mix thoroughly. Perform a quick spin to collect all liquid from the sides of the tube. + +### 10.4 Run the PCR program to amplify the libraries: + +| Step | Temperature | Time | Cycles | +|-----------------------|-------------|----------------|--------| +| Initial Denaturation | 98 °C | 0:30 | 1 | +| Denaturation | 98 °C | 0:10 | 7 | +| Annealing | 65 °C | 1:15 | 7 | +| Extension | 65 °C | 5:00 | 1 | +| Hold | 4 °C | Indefinite | 1 | + +## Library Clean-up + +11. Clean Up Libraries: +Repeat the same clean up process as step 6 using 20 µl of AMPure XP beads or NEBNext Sample Purification Beads and 28 µL of Elution Buffer (EB)/ 0.1X TE. + +## Library Quantification and Normalization + +12. + +### 12.1 Analyze 2 µL library using a Qubit dsDNA HS Assay kit. + +### 12.2 Calculate the molarity value using the following formula. Use the band size from gel electrophoresis or Tapestation readings (step 7). + +Library concentration (µg/µL) / (660 g/mol * average library size (bp)) * 10^6. + +### 12.3 Normalize each library by dilution with nuclease free water. + +### 12.4 Pool equal volume (e.g., 5 µL) from each of the normalized libraries into a single 1.5 mL Eppendorf tube. + +## Sequencing + +13. Denature and load pooled libraries as follows: + +### 13.1 Denature the pooled libraries by mixing 5 µL of pooled libraries and 5 µL of 0.2N NaOH solution. + +### 13.2 Incubate for 5 minutes. + +### 13.3 Add 990 µL of HT1 buffer and mix well with denatured pooled library by pipetting up and down 10 times with P1000. + +### 13.4 Load 600 µL of the denatured, diluted pooled library into the loading position of the Illumina reagent cartridge (V2, 300 cycle kit). Load reagent cartridge, flow cell, and PR2 buffer into MiSeq instrument, confirm the metrics and start the run. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/an-axenic-plant-culture-system-for-sporobolus-alte-cu5ewy3e.md b/markdown-output/an-axenic-plant-culture-system-for-sporobolus-alte-cu5ewy3e.md new file mode 100644 index 0000000000000000000000000000000000000000..28f349b57ed75ebd8baee658ec1d39b7e51e9cee --- /dev/null +++ b/markdown-output/an-axenic-plant-culture-system-for-sporobolus-alte-cu5ewy3e.md @@ -0,0 +1,164 @@ +```markdown +# An axenic plant culture system for Sporobolus alterniflorus + +### Goal/Experiment: +Develop a reliable protocol for generating axenic Sporobolus alterniflorus plants derived from seeds for in vitro culture purposes. + +## Abstract + +* **Sporobolus alterniflorus** is a native grass crucial to the U.S. East and Gulf coasts, particularly for salt marsh ecosystems. It tolerates various abiotic stresses, including high salinity, anoxia, and toxic sulfide concentrations. This protocol focuses on generating axenic plants from seeds, offering a simplified and efficient alternative to existing methods. + +## Guidelines + +This protocol involves the following steps: +1. Collection of seeds in the field. +2. Seed germination. +3. In vitro establishment of cultures. + +A modified MS (Murashige and Skoog) medium is selected to favor root adventitious regeneration and lateral shoot development without altering plant morphology. + +## Materials + +### General Supplies +- 1 L plastic containers +- Aluminum foil +- Tweezers +- Surgical blades +- 50 mL conical tubes +- Petri dishes +- Pyrex 1L bottles +- ~1 L glass culture vessels + +### Reagents +- Soil +- Bleach (commercial, typically around 5-6%) +- Autoclaved distilled water +- Modified Murashige and Skoog medium +- Indole-3-acetic acid (IAA) +- Kinetin +- Agar +- Sucrose + +### Equipment +- Growing lights +- pH meter +- Autoclave +- Tissue culture hood + +### Safety Warnings +**Caution**: Bleach is harmful. Hazards include skin and eye burns and severe damage. + +### Ethics Statement +No animals or humans were involved in this protocol. + +## Before Start Instructions +Consult your institution about procedures related to soil processing and disposal. + +## Procedure + +### 1. Seed Storage and Germination + +#### 1.1 Seed Storage (30m) +1. Collect mature seeds from flower stalks in the field. +2. Transfer to a clean ziplock bag. +3. Keep seeds wet and store at 4°C in darkness. +4. Properly stored seeds are viable for 6-12 months. + +#### 1.2 Preparation of Containers for Seed Germination (3d) +1. Fill autoclave containers 1/4 full with soil and saturate completely with water. +2. Cover openings with aluminum foil and autoclave for 1h. +3. After 48h, repeat autoclaving and let cool to room temperature before sowing seeds. + +#### 1.3 Seed Preparation and Germination (Surface Sterilization) (2w) +1. Place seeds in a 50 mL conical tube. +2. Add 40 mL 20% commercial bleach solution. +3. After 20m, remove bleach and rinse twice with autoclaved distilled water (5m each). +4. Sow seeds in autoclaved soil containers under 100 μE light at 20°C, 16h light/8h dark. +5. Germination in 1-2 weeks. + +**Note**: High seed numbers per container ensure a continuous seedling supply. Glumes removal is labor-intensive and negligible for this experiment. + +![Figure 1](img1.png) +*Fig.1 - Mature seeds preparation.* + +### 2. Removal of Root System + +#### 2.1 Remove Seedlings from Soil (10m) +1. Pull seedlings gently from the soil. +2. Clean by placing in autoclaved water. +3. Transfer to a clean work area. + +#### 2.2 Remove Radicle from Seedling (10m) +1. Use tweezers and clean blades to remove root tissue and external seed covers. +2. Maintain secondary roots from the crown area. + +![Figure 2](img2.png) +*Fig.2 - Smooth cordgrass seedlings.* + +![Figure 3](img3.png) +*Fig.3 - Removal of root tissues.* + +### 3. Surface Sterilization of Trimmed Seedlings + +#### 3.1 Surface Sterilization (25m) +1. Place 10-15 seedlings in a 50 mL tube with 40 mL 20% bleach solution. +2. Incubate at room temperature for 20 minutes. + +#### 3.2 Rinsing (20m) +1. Rinse seedlings in an autoclave hood. +2. Follow with 3 rinses in autoclaved distilled water. +3. Incubate 5m between each rinse. + +### 4. Inducing New Roots in Surface Sterilized Trimmed Seedlings + +#### 4.1 Root Induction Medium (Prepare in Advance) (2h) +1. **Modified MS Medium**: + - 4.43g MS powder in 1L autoclave bottle. + - 30g/L sucrose. + - Adjust pH to 5.8. + - Add 8g/L agar, autoclave for 40m. + - Pour into sterile Petri dishes under hood (~25 mL/dish). + +**Note**: Modified MS includes specific macro-and micronutrients and vitamin concentrations enhancing root formation. + +#### 4.2 Transfer Seedlings to Root Induction Medium (1h) +1. Place seedlings on the agar surface, ensuring crown area contact. +2. Seal Petri dishes with parafilm. + +#### 4.3 Root Induction (1w) +1. Illuminate seedlings (~100 μE light, 16h light/8h dark). +2. Roots will emerge within ~3 days. + +![Figure 4](img4.png) +*Fig.4 - Root Induction.* + +### 5. Transfer Re-Rooted Seedlings into Culture Vessels + +#### 5.1 Prepare the Plant Growing Medium (2h) +1. Prepare medium as in step 4.1. +2. Autoclave 150 mL for 15 minutes in culturing vessels. + +#### 5.2 Transfer the Rooted Seedlings to Culture Vessels (1h) +1. Under the hood, place seedlings into the 1 L vessels with media. +2. Sterilize media by autoclaving for 30 minutes. +3. Grow under ~200 μE light, 16h light/8h dark. + +![Figure 5](img5.png) +*Fig.5 - Re-rooted seedlings in culture chamber.* + +### 6. In Vitro Propagation of *Sporobolus alterniflorus* + +#### 6.1 Lateral Shoot and Rhizomes Development (1h) +1. Separate new shoots from mother plants to initiate individual cultures. +2. Develop new vitroplants within 4-6 weeks. + +**Note**: Transfer plants to hormone-free MS medium once roots are established for continued growth. + +## License +This protocol is distributed under the Creative Commons Attribution License, permitting unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. + +## Citation +Elena L. Peredo, Suzanne M Thomas, Zoe Cardon. (2023). An axenic plant culture system for Sporobolus alterniflorus. DOI: [10.17504/protocols.io.x54v9d94qg3e/v1](https://dx.doi.org/10.17504/protocols.io.x54v9d94qg3e/v1). + +**End of Output** +``` \ No newline at end of file diff --git a/markdown-output/an-improved-deep-learning-method-for-predicting-dn-2rdgd26.md b/markdown-output/an-improved-deep-learning-method-for-predicting-dn-2rdgd26.md new file mode 100644 index 0000000000000000000000000000000000000000..787819390fbe1f75e513f19296417a5b887e0517 --- /dev/null +++ b/markdown-output/an-improved-deep-learning-method-for-predicting-dn-2rdgd26.md @@ -0,0 +1,79 @@ +```markdown +# Goal/Experiment: +Develop a deep learning method for predicting DNA-binding proteins based on contextual features in amino acid sequences. + +# An improved deep learning method for predicting DNA-binding proteins based on contextual features in amino acid sequences + +*PLOS One* +*Ruixiong Ma* +*USTB* + +[DOI Link http://dx.doi.org/10.17504/protocols.io.2rdgd26](http://dx.doi.org/10.17504/protocols.io.2rdgd26) + +## Abstract +With the explosively increased amount of newly discovered proteins, predicting the function of these proteins from amino acid sequences is becoming one of the main challenges in functional annotation of genomes. + +Nowadays a number of computational approaches have been developed to predict DNA-binding proteins effectively and accurately from amino acid sequences, such as SVM, DNABP, and CNN-RNN. However, these methods do not consider the context in amino acid sequences, which makes it difficult for them to capture sequence features adequately. + +In this paper, we propose CNN-BiLSTM, a new method for predicting DNA-binding proteins, elaborately reconciling convolutional neural network and bi-directional long short-term memory recurrent neural network. CNN-BiLSTM can explore the potential contextual relationships of amino acid sequences to obtain more features than traditional models. + +The experimental results show that the prediction accuracy of the proposed CNN-BiLSTM method on the test set is 96.5%, which is 7.8% higher than that of SVM, 9.6% higher than that of DNABP and 3.7% higher than that of CNN-RNN respectively. + +Being tested on 20,000 independent samples provided by UniProt that weren't involved in model training, the accuracy of CNN-BiLSTM is 94.5%, which is 12% higher than that of SVM, 4.9% higher than that of DNABP and 4% higher than that of CNN-RNN respectively. + +The model training process is visualized and compared with that of CNN-RNN, and it is found that the training process of CNN-BiLSTM support better generalization from the training data set, which shows that CNN-BiLSTM has a wider range of adaptations to protein sequences. + +On the independent samples set, CNN-BiLSTM presents better credibility, for its predicted scores are closer to the labels of the samples than those of CNN-RNN. Therefore, the proposed CNN-BiLSTM is a more powerful method for identifying DNA-binding proteins. + +## External Link +[https://doi.org/10.1371/journal.pone.0225317](https://doi.org/10.1371/journal.pone.0225317) + +## Guidelines +This is a method of recognizing DNA binding proteins by deep learning. + +## Materials Text +[Various encrypted data, not transcribed here for brevity] + +## Safety Warnings +Pay attention to the temperature of the computer. + +## Before Starting +### What you need to prepare: + +- Python 3 +- TensorFlow +- Keras + +## Step-by-Step Procedure +### Step 1: Prepare the dataset +- **Data Extraction**: In the process of extracting data from UniProt, we removed those sequences with length less than 50 or greater than 1,280 amino acids, resulting in 17,651 DNA-binding protein sequences selected as positive samples. At the same time, we got 50,500 non-DNA-binding protein sequences as negative samples. + +**UniProt**: UniProt is a comprehensive, high-quality and freely accessible resource of protein sequence and functional information. It provides the scientific community with a vivid, integrated and richly annotated view of protein knowledge. + +- **Independent Samples**: Taking sequences from both positive and negative samples as independent test samples, we selected 500 sequences each. + +- **Training and Test Sets**: For the remaining 17,151 positive and 50,000 reverse samples, we randomly selected 85% of them as training sets and the remaining 15% as test sets for model training. + +### Step 2: Build Model +The deep learning model is composed of four parts: + +1. **Coding Layer**: Each amino acid is represented as a particular number. +2. **Embedding Layer**: Translates amino acid sequences into continuous vectors. +3. **Convolution Layer**: Consists of two convolutions and two maximal pooling operations. +4. **BiLSTM Layer**: Grasp the context features of amino acid sequences. + +We use the Keras platform to build this model. + +### Step 3: Model Training +- The data is trained in the built model using GPU. +- At the end of this process, we get a DNA binding protein predictor. + +## References +This is an open access protocol distributed under the terms of the Creative Commons Attribution License [License URL](https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. + +--- + +*Note*: This compiled information assists in understanding and applying the described process, ensuring adequate preparation of materials and systematic implementation of the steps. + +`endofoutput` +``` \ No newline at end of file diff --git a/markdown-output/analysis-of-islet-function-by-insulin-enzyme-linke-bz7bp9in.md b/markdown-output/analysis-of-islet-function-by-insulin-enzyme-linke-bz7bp9in.md new file mode 100644 index 0000000000000000000000000000000000000000..e1c99c9c334cdde6c625ec0a8dd74dd79a3f62ee --- /dev/null +++ b/markdown-output/analysis-of-islet-function-by-insulin-enzyme-linke-bz7bp9in.md @@ -0,0 +1,139 @@ +```markdown +# Goal/Experiment: +The goal of this experiment is to quantify the insulin secretion and content from human pancreatic islets after glucose stimulation to assess islet function. This protocol provides a detailed methodology for performing Insulin Enzyme-linked Immunosorbent Assay (ELISA) to measure insulin concentrations. + +# Analysis of Islet Function by Insulin Enzyme-linked Immunosorbent Assay (ELISA) + +**Date of Publication:** December 02, 2021 +**DOI:** [dx.doi.org/10.17504/protocols.io.bz7bp9in](https://dx.doi.org/10.17504/protocols.io.bz7bp9in) +**Cited by:** IIDP-HIPP + +This Standard Operating Procedure (SOP) is based on the Vanderbilt University Medical Center Human Islet Phenotyping Program (HIPP) Islet Functional Analysis. This SOP provides the HIPP procedure for measuring islet insulin content and secretion to assess islet function. It defines the assay method used by the Human Islet Phenotyping Program (HIPP) for the qualitative determination of Purified Human Pancreatic Islet product, post-shipment, for use in National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)-sponsored research in the Integrated Islet Distribution Program (IIDP). + +**SOP #: HIPP-09-v01** + +## Organizations & Key Terms + +### Integrated Islet Distribution Program (IIDP) +The IIDP is a grant-funded program commissioned and funded by the NIDDK to provide quality human islets to the diabetes research community to advance scientific discoveries and translational medicine. + +### IIDP Coordinating Center (CC) +The IIDP CC integrates an interactive group of academic laboratories including the subcontracted IIDP centers. + +### Human Islet Phenotyping Program (HIPP) +The HIPP is a subcontracted entity of the IIDP through the COH and Vanderbilt University. + +### Islet Equivalent (IEQ) +An islet with a diameter of 150 μm determined mathematically by compensating for islet shape. + +### Islet Perfusion Assay +A functional assay that acquires dynamic hormone secretory profiles simultaneously from islet cell types. + +### Enzyme-linked immunosorbent assay (ELISA) +A sensitive in vitro assay technique used to measure concentrations of antigens by making use of an enzyme conjugated to an antibody recognizing an antigen of interest. + +## Equipment + +1. The following equipment is necessary to assess human islet function by Insulin ELISA: + - **1.1 Micropipettes:** (10-100 µL, 20-200 µL, and 100-1000 µL ranges) + - **1.2 Multi-channel micropipette:** (20-200 µL range) + - **1.3 Computer:** with Excel (Microsoft) and Prism (Graphpad) software + - **1.4 epMotion Liquid Handling Workstation:** (Eppendorf 5075) + - **1.5 Benchmark Orbi shaker:** (BT1502) + - **1.6 Fisherbrand accuWash microplate washer:** (5165100) or similar plate washer (14-377-577 or 14-377-578) + - **1.7 BMG Labtech CLARIOstar microplate reader:** (Plus Model) + - **1.7.1 CLARIOstar software** + - **1.7.2 MARS data analysis software** + - **1.8 Vortex mixer** + +## Supplies and Materials + +2. The following supplies and materials are necessary to assess human islet function by Insulin ELISA: + - **2.1 Human Insulin ELISA Kit, Mercodia** + - **Catalog #10-1113-10** + - **2.1.1 Coated 96-well plates:** (20-2622) + - **2.1.2 Calibrators 0, 1, 2, 3, 4, 5:** Insulin concentrations are known values provided by manufacturer (20-2615, 20-2616, 20-2617, 20-2618, 20-2619, 20-2620) + - **2.1.3 Enzyme Conjugate 11X:** (20-2631) + - **2.1.4 Enzyme Conjugate Buffer:** (20-2630) + - **2.1.5 Washer Buffer 21X:** (20-3194) + - **2.1.6 Substrate TMB:** (20-3136) + - **2.1.7 Stop Solution:** (20-2694) + - **2.2 Buffer, Mercodia** + - **Catalog #10-1195-01** + - **2.3 Human Diabetes Antigen Controls** + - **Catalog #10-1134-01** + - **2.4 50 µL epMotion pipette tip, Eppendorf** + - **Catalog #30014421** + - **2.5 10 mL (Fisher Scientific 13-678-11E) and 25 mL (Fisher Scientific 13-678-11) serological pipets** + - **2.6 2 mL microcentrifuge tube, Sarstedt** + - **Catalog #72.695.500** + - **2.7 200 µL (ART P-200) and 1000 µL (ART P-1250) pipette tips** + +## Procedures + +### 1. Preparation of Samples, Standards, and Internal Quality Controls +1.1 Thaw archived samples intended for analysis in room temperature water. Once thawed, invert capped samples ten times to thoroughly mix. + +1.2 Retrieve the islet hormone extracts and keep on ice. + +1.3 Prepare serial dilutions of hormone extract (1:100, 1:1000, 1:2000, 1:5000, and 1:10000) in 2 mL tubes using the Calibrator 0 media from the ELISA Kit or Diabetes Sample Buffer (See Figure 1). Vortex each tube to mix contents before generating subsequent dilutions. + +1.4 Generate 1:3 dilutions for perfusion fractions #23, #24, #25, and #43 by adding 40 µL sample to 80 µL of Calibrator 0 or Diabetes Sample Buffer in 2 mL tubes. + +1.5 Transfer all Calibrators and Antigen Controls from original bottles to 2 mL tubes. + +### 2. Preparation of Enzyme Conjugate and Wash Buffer Solutions + +2.1 Prepare Enzyme Conjugate 1X solution by diluting Enzyme Conjugate 11X in Enzyme Conjugate Buffer. Mix gently. Prepare a volume sufficient to add 100 µL to each well (see step 3.2). + +2.2 Prepare Wash Buffer 1X solution by diluting Wash Buffer 21X in redistilled water. Mix thoroughly. Prepare a volume sufficient to add 4.2 mL to each well (see step 3.4). + +### 3. Performing Insulin Assay + +3.1 By using epMotion 5075 or hand-pipetting, pipette 25 µL each of Calibrators and Antigen Controls (in duplicate), samples, extract dilutions, and sample dilutions into appropriate wells of ELISA 96-well plate. + +3.2 Add 100 µL of enzyme conjugate 1X solution to each well. + +3.3 Incubate ELISA 96-well plate on a microplate shaker (900 rpm, orbital movement) for 1 hour at room temperature (18-25°C). + +3.4 Using the plate washer, wash 6 times with 700 µL wash buffer 1X solution. After final wash, invert and tap the plate firmly against absorbent paper. Do not include soak step in washing procedure. + +3.5 Add 200 µL Substrate TMB into each well. + +3.6 Incubate on the bench for 15 minutes at room temperature (18-25°C). + +3.7 Add 50 µL Stop Solution to each well. Mix thoroughly for 5 seconds by tapping gently on all sides of the plate without dispersing liquid in wells. + +3.8 Using the microplate reader, determine the optical density and insulin concentration of each well within 30 minutes of adding stop solution. Set to 450 nm. + +### 4. Data Analysis + +4.1 Values for all standards must be within ±15% of their expected values and replicate values of each standard must have a Coefficient of Variation (CV) ≤20%. If standards vary beyond these limits, the assay must be repeated. + +4.2 Values for quality control samples, corresponding to lower and upper assay detection ranges, must be within their known ranges. If QCs vary beyond these limits, the assay must be repeated. + +4.3 Calculate the average of the insulin concentrations from the 4 extract dilutions to determine insulin content, expressed as ng/mL. + +4.4 Normalize secreted insulin concentrations per islet volume (IEQs), expressed as ng/100 IEQs/min and islet insulin content, expressed as % content/min. + +4.5 Use Prism software to create graphs and to calculate stimulation index (SI) and area under curve (AUC) values. + - 4.5.1 **Stimulation index (SI):** A ratio calculated as maximum response to a given stimulus relative to baseline. + - 4.5.2 **Area under curve (AUC):** Calculated by integrating islet secretory response to a given stimulus over time. + +## Data Storage and Reporting + +### 5. Data Storage and Reporting + +5.1 To facilitate data management and ensure data security, the VUMC HIPP uses an institutional server-based platform for data storage and analysis. + +5.2 Upon analysis completion, the VUMC HIPP uploads raw data, including hormone levels, data analysis, and graphical representations of each human islet perfusion into the IIDP HIPP database. Example of human islet perfusion results performed in HIPP is shown in **Figure 1**. + +5.3 Functional data on islet insulin and glucagon secretion will be uploaded within 3 business days to the HIPP database built by IIDP programming team and immediately available to IIDP-affiliated investigators and islet isolation centers. + +## Deviations and Resolutions + +### 6. Deviations and Resolutions +Document any deviations that occurred during this protocol that affect the final results and report with the analysis of the assay. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/analysis-of-the-time-evolution-of-auditory-steady-wejfbcn.md b/markdown-output/analysis-of-the-time-evolution-of-auditory-steady-wejfbcn.md new file mode 100644 index 0000000000000000000000000000000000000000..0e9427ba656ca32aa6d113d9ea790a9b846394ac --- /dev/null +++ b/markdown-output/analysis-of-the-time-evolution-of-auditory-steady-wejfbcn.md @@ -0,0 +1,132 @@ +```markdown +# Goal/Experiment: +Analysis of the time evolution of auditory steady-state responses (ASSR) recorded in rats. + +## Analysis of the time evolution of auditory steady-state responses (ASSR) recorded in rats + +#### Version 2 + +**Authors:** +1. Pavel Prado - Advanced Center for Electrical and Electronic Engineering (AC3E), Universidad Técnica Federico Santa María, Chile +2. Eduardo Martínez-Montes - Cuban Neuroscience Center +3. Matías Zañartu - Department of Electronic Engineering, Universidad Técnica Federico Santa María, Valparaíso, Chile + +dx.doi.org/10.17504/protocols.io.wejfbcn + +## Abstract + +Auditory steady-state responses (ASSRs) are brain oscillations locked to the periodic properties of acoustic stimuli. Audiological tests based on the acquisition of ASSR are useful for estimating hearing sensitivity, mainly because multiple hearing frequencies can be simultaneously assessed, and the auditory response can be objectively detected using statistical tests. + +Typically, the extraction of the auditory response from the measured signal relies on averaging epochs of the EEG, time-locked to the stimulus. This assumes that the auditory response is steady over time and that averaging increases the signal-to-noise ratio of the measurement. + +Since time-domain averaging of epochs within a recording does not allow distinction between methodological and physiological related variations in the amplitude of the ASSR, we designed a protocol for analyzing the dynamics of the auditory response during the acquisition procedure. The protocol allows us to compute the ASSR amplitude at a given time window without being compromised by the segments of the preceding EEG. + +## Guidelines + +The study must be performed under the approval of the local Animal Research and Ethics Committee. Specifically, this study followed the guidelines of the Cuban Neuroscience Center and the National Center for Animal Breeding of Cuba. + +## Safety Warnings + +Handle animals following standard safety procedures. Standard safety procedures should also be adhered to when handling disposable needles and syringes. National and local electrical safety regulations must be followed. + +## Before Starting + +Care, feeding, breeding, and maintenance of animals should follow standard local guidelines. Animals should be housed in a standard bio-clean animal room under a 12-hour light/dark cycle at 22-24°C, with free access to food and tap water. + +## Preparation + +1. **Anesthesia:** + - Administer ketamine (75.0 mg/kg, intraperitoneal) and diazepam (5.0 mg/kg, intraperitoneal). + +2. **Supplemental anesthesia:** + - Maintain the animal in an areflexic state with supplemental doses during the experiment. + +3. **Atropine sulfate:** + - Administer 0.06 mg/kg intramuscularly to decrease mucosal secretions. + +4. **Temperature control:** + - Maintain body temperature at 37.0±0.1°C using a body temperature control system. + +5. **Post-experiment care:** + - Return animals to the colony after recovery from anesthesia—animal sacrifice is not required. + +## Acoustic Stimulation and EEG Recording + +6. **Presentation:** + - Acoustic stimuli are presented monaurally via an ER-3A Etymotic Research Insert Earphone. + +7. **Custom ear molds:** + - Use custom-fitted ear molds to replace the original foam for coupling the earphone to the rat’s ear. + +8. **Stimulation system calibration:** + - Refer acoustic levels to a Brüel & Kjær artificial ear (type 4152). Calibrate with a Brüel & Kjær 2250 sound level meter and type 4144 microphone. + +9. **Acoustic stimuli generation:** + - Generate stimuli using standard hardware/software. Example: continuous tones of 8 kHz sinusoidally-modulated in amplitude at 115 Hz are generated using the ASSR software module of the AUDIX system (Havana, Cuba). Stimulus intensity is fixed at 50 dB SPL. + +10. **Electrophysiological responses:** + - Record responses differentially using stainless-steel needle electrodes inserted subdermally (vertex positive; neck negative; thorax reference). + - Amplify recordings with a gain of 1.2x10^4 and band-pass filter frequencies from 10 to 300 Hz. + +12. **Digitization:** + - Digitize the output at 16-bit resolution and sample at 920 Hz. + +13. **Artifact rejection:** + - Reject segments with electrical oscillations exceeding 50 mV online. + +14. **Data acquisition:** + - Complete 60 artifact-free epochs of 4.45 s duration each (4096 time-points each). Allow 10 minutes between consecutive recordings. Thirty recordings are acquired from each animal. + +## Data Processing + +15. **Software Processing:** + - Perform data processing using in-house MATLAB codes (MathWorks, USA). + +16. **Data Matrix Arrangement:** + - Rearrange the 60 sequential epochs of the 30 recordings offline into a data matrix of 30 rows and 60 columns (one matrix per animal). + +17. **Noise Influence Reduction (Optional):** + - Modify the dataset to reduce noise influence on auditory response computation. + +18. **Column-wise Averaging:** + - Average the 30 epochs column-wise for each time window to reduce EEG background noise and detect the ASSR amplitude. + +19. **ASSR Amplitude Computation:** + - Compute the amplitude for each group of epochs using Fast Fourier Transform (FFT). Use an FFT length of 4096 time-points, aligning with the length of an epoch (4.45 s). With sampling at 920 Hz, the FFT resolution is 0.22 Hz. Windowing technique is not implemented. + +20. **Spectral Amplitude:** + - Define amplitude as the spectral amplitude at 115 Hz. Vector average the amplitude of 30 spectral components to calculate residual noise level (RNL). + +21. **Statistical Comparison:** + - Compare ASSR amplitudes with corresponding RNL using Hotelling’s T2 multivariate test in the AUDIX system, considering both amplitude and phase oscillations. + +22. **Time Evolution:** + - Plot ASSR amplitudes as a function of time. Fit to time courses using negative exponential functions if R^2>0.85 and p<0.05. + +23. **Statistical Tests:** + - Apply statistical tests, such as One-way ANOVAs (p<0.05) and post-hoc analyses (Tukey test, p<0.05), as needed to analyze the stability of the ASSR amplitude and RNL. + +### Calculation of Adaptive Behavior Index: + +When an adaptive behavior is detected, the adaptation index (P_adapt) of the response is calculated using the equation: + +\[ P_{\text{adapt}} = 100 \left( \frac{A_{\text{mp}\max} - A_{\text{mpadapt}}}{A_{\text{mp}\max}} \right) \] + +Where: +- \( A_{\text{mp}\max} \) represents the maximum amplitude of the fitted curve. +- \( A_{\text{mpadapt}} \) represents its asymptotic value (defined as the amplitude estimated when the recording length was three times the time constant of the fitted exponential function). + +## Summary + +A summary of the protocol: +1. Presenting acoustic stimuli modulated in amplitude at 115 Hz. +2. Organizing the dataset (matrix with 60 columns and 30 rows). +3. Spectral analysis of the averaged measurement. +4. Graphical representation of ASSR dynamics. + +![Protocol Diagram](https://example.com/path/to/protocol-diagram.png) + +This protocol is distributed under the terms of the Creative Commons Attribution License. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/antioxidant-rescue-of-c-elegans-behaviour-on-keio-cgehttb6.md b/markdown-output/antioxidant-rescue-of-c-elegans-behaviour-on-keio-cgehttb6.md new file mode 100644 index 0000000000000000000000000000000000000000..e66976dcfa407cccc7fe97ea600a56afa4af33ce --- /dev/null +++ b/markdown-output/antioxidant-rescue-of-c-elegans-behaviour-on-keio-cgehttb6.md @@ -0,0 +1,92 @@ +```markdown +# Goal/Experiment: +The goal of this experiment is to screen candidate behaviour-modifying E. coli/BW25113 single-gene deletion mutants from the 'Keio Collection’ to investigate their effects on *Caenorhabditis elegans* behaviour in the presence of antioxidants. + +# Antioxidant Rescue of *C. elegans* Behaviour on Keio E. coli Mutants (6-Well Plates) + +DOI: [dx.doi.org/10.17504/protocols.io.j8nlkw5kdl5r/v1](https://dx.doi.org/10.17504/protocols.io.j8nlkw5kdl5r/v1) + +Author: Saul Moore +Imperial College London, MRC London Institute of Medical Sciences (LMS) + +## Abstract +Protocol for screening candidate behaviour-modifying *E. coli* BW25113 single-gene deletion mutants from the 'Keio Collection' to investigate their effects on *Caenorhabditis elegans* behaviour in the presence of antioxidants. + +## Disclaimer +**DISCLAIMER – FOR INFORMATIONAL PURPOSES ONLY; USE AT YOUR OWN RISK** + +## License +This is an open access protocol distributed under the terms of the Creative Commons Attribution License. + +## Materials +- 6-well flat bottom plates ('imaging plates') +- 60mm Petri plates ('maintenance plates') +- 90mm Petri plates ('nursery plates') +- 50mL Erlenmeyer flasks + +## Reagents +- 500mL LB broth +- 1L NGM agar (for ingredients, see protocol for making NGM agar) + +## Preparing NGM Agar + Pouring Plates +1. Prior to screening, prepare the materials needed for screening *C. elegans* on selected Keio *E. coli* mutants: + - 6-well plates (imaging plates) + - 15 mL Falcon tubes + - 50 mL Erlenmeyer flasks + - 90 mm Petri plates (maintenance plates) + - 150 mm Petri plates (nursery plates) +2. Make 1L normal Nematode Growth Media (NGM) agar, following the protocol: [Making normal NGM for imaging plates (Cabreiro Lab)](dx.doi.org/10.17504/protocols.io.6bhhaj6) +3. Pour 15 mL NGM agar into each 60 mm maintenance plate, and 35 mL NGM agar into each 90 mm nursery plate, following the protocol for Plate pouring [here](dx.doi.org/10.17504/protocols.io.6bhhaj6). Keep the remaining agar warm in a water bath set to 65°C, for pouring into 6-well imaging plates afterwards. +4. Using the Integra ViaFill, dispense 4 mL NGM agar into each well of the 6-well plates, following the protocol: [Dispensing agar into multiwell plates](dx.doi.org/10.17504/protocols.io.8g2ww7e) +5. Leave the plates on the lab bench (with lids on) until the agar has cooled and solidified (approximately 1 hour, timing depends on humidity). + +## Preparing Worms +9. Inoculate 10 mL LB broth media with *E. coli* BW25113 (Keio background wild-type strain, used as negative control and for raising worms, no Kanamycin) in an Erlenmeyer flask for overnight culture following the protocol: [Inoculating a Liquid Bacterial Culture](dx.doi.org/10.17504/protocols.io.dd8i2h6) +10. Place the inoculation in a shaking incubator at 37°C at 200 rpm and leave to grow overnight. +11. Remove the BW culture from the shaking incubator and place in 4°C fridge until seeding. +12. Remove the plates from storage and the BW culture from the fridge, and leave on the bench for approximately 30 minutes to acclimate to room temperature. +13. Using aseptic technique, seed the 60 mm maintenance plates each with approximately 250 µL of BW25113 culture. +14. Leave under hood until dry (with lids on, timing depends on humidity). +15. Using a platinum pick, gently pick 30 adult N2 Bristol *C. elegans* onto each maintenance plate, and store in an incubator at 20°C. +16. After 24 hours, remove the adult worms, leaving the eggs behind to hatch into L1 larvae. +17. Inoculate a further 10 mL LB broth with BW25113 bacteria for overnight culture (no Kanamycin), following the protocol [here](dx.doi.org/10.17504/protocols.io.5dbp2i6) and place in a shaking incubator at 37°C, 200 rpm. +18. After 24 hours, remove the culture from the incubator, and the 90 mm nursery plates from storage, and leave to acclimate on bench top for 30 minutes. +19. Seed the nursery plates each with approximately 1 mL of fresh BW25113 culture. Leave under hood until dry. +20. Wash the worms off the BW-seeded maintenance plates, into two 15 mL Falcon tubes. +21. Perform an egg prep on worms in the Falcon tubes, following the protocol: [Egg Prep for Bleach Synchronization (Cabreiro Lab)](dx.doi.org/10.17504/protocols.io.4h4gp3w) + +## Preparing Bacteria +23. Fill 2 separate Erlenmeyer flasks with 25 mL LB. Add 50 µg/mL Kanamycin to one flask, and leave the other flask without Kanamycin for the BW25113 control. +24. Remove the required Keio frozen stock plates from -80°C containing the strains for antioxidant testing. Gently remove the aluminium film and leave to partially thaw for a minute or so. + > **Safety Information**: To avoid damaging the bacterial stocks through repeated freeze-thawing, do not let the wells completely defrost. Just enough to be able to pick up some cells with the replicator. +25. Inoculate the Erlenmeyer flasks with the desired strains for antioxidant testing from Keio frozen stock plates, following the protocol: [Inoculating a Liquid Bacterial Culture](dx.doi.org/10.17504/protocols.io.dd8i2h6) +26. Incubate the cultures overnight at 37°C in a shaking incubator at 200 rpm. +27. Remove the overnight cultures from the incubator. Inoculate 2 more Erlenmeyer flasks for a second round of overnight cultures from the first, this time without Kanamycin (to avoid exposing the worms to the antibiotics), and incubate overnight at 37°C at 200 rpm. +28. After 24 hours, remove the cultures from the incubator and store at 4°C until used for experiments. + +## Seeding Imaging Plates (6-Well) +29. Remove the imaging plates from 4°C storage. +30. Ensure that imaging plates have lost approximately 3-5% of their original weight (so that they are not too wet for imaging when seeded). Place under a hood or drying cabinet until they have. +31. Remove overnight cultures of Keio strains from 4°C storage. Using a pipette, seed 30 µL of bacterial culture into the wells of each 6-well imaging plate. +32. Place the seeded plates under a laminar flow hood to dry for 20 minutes, then place in an incubator at 25°C (no shaking) for 7 hours 40 minutes (total lawn growth time: 8 hours). +33. After 8 hours, remove the plates from the incubator and store at 4°C. + +## Adding Antioxidants (6-Well) +34. On the day of tracking, remove the seeded imaging plates from 4°C, and dry for 30 minutes under a laminar flow hood. +35. Remove the antioxidants from 4°C. Prepare 100 mM NAC or Vitamin C (in H2O). +36. Using a pipette, dispense 40 µL of antioxidant solution into each desired well of the 6-well imaging plates (for a final concentration of 1 mM in 4 mL agar). +37. Leave the plates to dry under a hood for a further 30 minutes. Record the weight of the plates after drying (as weight at imaging). + +## Picking Worms + Hydra Tracking (6-Well) +38. Prior to tracking, ensure that the imaging cave air conditioning is turned on (and there has not been a power-cut) and also empty the dehumidifier waste water tray (see pre-imaging checklist). +39. Remove the nursery plates from the incubator. +40. Using a platinum worm pick, carefully pick 10 Day1 worms onto the edge of the lawns in each well of the 6-well imaging plates, then place in incubator at 20°C until tracking (at +4 hours on food). +41. 30 minutes prior to tracking with the Hydra rig (each run is performed every 20-30 minutes), remove 5 imaging plates from the 20°C incubator and leave to acclimate in the imaging cave. +42. Record worm behaviour on the bacterial food for 15 minutes at the 4-hour timepoint (25 fps, exposure: 25000 msec, blue-light stimulation). + +## Post-Tracking +43. After tracking, discard the plates in a biological waste bin. +44. Check tracking checklist to ensure that all videos have been saved correctly: `'/Volumes/behavgenom$/Documentation/Protocols/analysis/tracking-checklist-20210210.docx'` + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/application-of-phyto-pam-ii-compact-version-for-ru-cjgtujwn.md b/markdown-output/application-of-phyto-pam-ii-compact-version-for-ru-cjgtujwn.md new file mode 100644 index 0000000000000000000000000000000000000000..76a35a8465a79e61ac4ec482eaf64e8dc34b52c6 --- /dev/null +++ b/markdown-output/application-of-phyto-pam-ii-compact-version-for-ru-cjgtujwn.md @@ -0,0 +1,109 @@ +```markdown +# Goal/Experiment: +To acquire photosynthetic efficiency (Fv/Fm) and quantum yield of photosystem II (Y(II)) of Microcystis aeruginosa cultures using far-red acclimation with the PHYO-PAM-II Compact Version. + +# Application of PHYTO-PAM-II (Compact Version) For Running Rapid Light Curves on Cyanobacterial Samples + +**Forked from**: [Application of PHYTO-PAM-II (Compact Version) on Aureococcus anophagefferens cultures for photosynthetic efficiency and quantum yield of PSII](https://dx.doi.org/10.17504/protocols.io.eq2ly7jqelx9/v1) + +### Citation: +Gwen Stark, Emily E. Chase, Steven W. Wilhelm 2022. Application of PHYTO-PAM-II (Compact Version) For Running Rapid light curves on Cyanobacterial samples. protocols.io https://dx.doi.org/10.17504/protocols.io.eq2ly7jqelx9/v1 + +### Contributors: +Gwen Stark1, Emily E. Chase2, Steven W. Wilhelm1 +1. The University of Tennessee, Knoxville +2. University of Tennessee, Knoxville + +--- + +## Abstract +A protocol to acquire photosynthetic efficiency (Fv/Fm) and quantum yield of photosystem II (Y(II)) of Microcystis aeruginosa cultures using far-red acclimation. + +## Keywords +- Photosynthetic efficiency +- Quantum yield +- Fv/Fm +- HAB +- Chlorophyll fluorescence +- Cyanobacteria + +## License +This is an open access protocol distributed under the terms of the [Creative Commons Attribution License](https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. + +--- + +## Guidelines +The collection of quantum yield and photosynthetic efficiency is highly sensitive to modifications in sampling protocol. + +## Materials +- PHYTO-PAM-II Compact Version and components +- Laptop with a USB port, and PhytoWin_3 installed +- Measurement cuvettes +- At least 3 mL of culture material for each sample +- 70% Ethanol +- KimWipes +- Necessary materials and setup for both dark adapting cultures, and a no light or low light environment for sampling + +## Before Starting +Familiarize yourself with the PHYTO-PAM-II equipment, manufacturer’s provided manual, and the basics of chlorophyll fluorescence parameters for full use of data collected and accuracy of results. + +--- + +## Equipment Preparation +1. **Setting Up the PHYTO-PAM-II Compact Version:** + - Toggle the power and connect to a laptop via USB. + - Open the PhytoWin_3 program and select "Phyto Compact Unit." + - Ensure the program is in "MEASURE" mode, and that the "ML" light is green (indicating it is ready). + - Ensure that "AL" and "FR" lights are not selected. + +![Equipment Setup](image1.png) + +2. **Adjusting Settings:** + - Modify Phyto-Pam settings based on the species being tested. + - Use the "View Pulse" check box to observe the saturation pulse kinetics, ensuring a distinct plateau is seen. + +--- + +## Sample Preparation +3. **Sample Transfer:** + - Transfer 3 mL of culture to a quartz cuvette and minimize exposure time. + - Place the cuvette in the optical port and cover with the darkening hood. + +4. **Auto-Adjust Gain:** + - Perform a zero-offset function using Zoff-determination to blank the PHYTO-PAM-II before runs. + +5. **Far-Red Acclimation:** + - Apply far-red light acclimation to induce photosystem I and oxidize the PQ pool. + - Dark acclimation is not recommended for cyanobacteria. + + > **Note:** Evaluate the necessity between no acclimation and weak far-red acclimation for consistent results. + +--- + +## Measurement Acquisition +6. **Experimental Measurements:** + - Measurements can now be collected. + +7. **Running Far-Red Light Acclimation:** + - Transfer 3 mL of culture into the cuvette, auto-adjust gain, and run acclimation. + - Turn on the "ML" button and wait for the light to turn green. + +8. **Taking Measurements:** + - Choose Fv/Fm measurements or run a rapid light curve. + - Adjust PAR and exposure times appropriately. + - Aim for the ETR curve to hit a maximum peak and level off. + +9. **Recording Data:** + - Data will be recorded automatically. + - Use 70% ethanol to clean the cuvette between different treatments. + +10. **Data Storage:** + - Save and export data as a .csv file. + - Unplug the equipment and charge fully for long-term storage. + +> **Recommendation:** Regularly charge the equipment if stored unused for extended periods. + +--- + +### endofoutput +``` \ No newline at end of file diff --git a/markdown-output/around-the-horn-pcr-and-cloning-rf2d3qe.md b/markdown-output/around-the-horn-pcr-and-cloning-rf2d3qe.md new file mode 100644 index 0000000000000000000000000000000000000000..131f82fbd620968c5471c3a6f4664951d1644b51 --- /dev/null +++ b/markdown-output/around-the-horn-pcr-and-cloning-rf2d3qe.md @@ -0,0 +1,142 @@ +```markdown +# Goal/Experiment: +The goal of this experiment is to perform 'around-the-horn' or 'divergent' PCR, where primers extend in opposite directions on a plasmid to generate a linear product. + +# Around-the-horn PCR and Cloning +*Stephen Floor* + +## Abstract +This protocol is designed for 'around-the-horn' or 'divergent' PCR, where primers go around most or all of a plasmid but are pointed away from each other so they generate a linear product. Note that this protocol is written for Q5 polymerase but works fine with other polymerases. To switch polymerases, just change the PCR reaction setup. + +**Citation:** Stephen Floor Around-the-horn PCR and cloning. [protocols.io](https://dx.doi.org/10.17504/protocols.io.rf2d3qe) doi:10.17504/protocols.io.rf2d3qe +**Published:** 03 Jul 2018 + +## Before start +Program the thermocycler with the PCR program in the main protocol. + +## Materials +- **Q5 Hot Start High-Fidelity DNA Polymerase** - 500 units (M0493L) by New England Biolabs +- **Phusion Hot Start Flex DNA Polymerase** - 100 units (M0535S) by New England Biolabs +- **HotStart ReadyMix (KAPA HiFi PCR kit)** (KK2601) by Kapa Biosystems +- **dNTP** (639125) by Takara +- **Forward primer (25 µM)** by Contributed by users +- **Reverse primer (25 µM)** by Contributed by users +- **Template** (5 ng/µl) by Contributed by users + +## Protocol + +### Design Primers +The basic idea of this protocol is that primers head 'away' from each other on a plasmid backbone, which gives a lot of flexibility in what can be done. Downsides are that it generates a linear product instead of a circular one, so it must be phosphorylated and ligated, and the PCR process takes a while. + +Insertion (region in blue are inserted): +![Insertion Diagram](img_insertion) + +Deletion (region in red is deleted): +![Deletion Diagram](img_deletion) + +Note that deletions and insertions can be combined. This strategy can be helpful when designing products for Gibson cloning. + +### Set up the PCR +**Step 1.** +Mix the following on ice: + +| Reagent | Volume for 1 reaction | +|---------------------------|-----------------------| +| Q5 buffer | 10 µl | +| dNTPs (10 mM) | 1 µl | +| Forward primer (25 µM) | 1 µl | +| Reverse primer (25 µM) | 1 µl | +| Template (5 ng/µl) | 5 µl | +| Q5 polymerase | 0.5 µl | +| ddH₂O | 31.5 µl | + +### Run the PCR +**Step 2.** +Run this PCR: + +- 95°C for 2 minutes +- 95°C for 15 seconds +- 65°C for 15 seconds +- 72°C for 10 minutes + +Repeat the above 30 times + +- 72°C for 15 minutes + +Note that the extension times at 72°C can be adjusted to the plasmid. In general, allow 1 minute for each kb of plasmid. + +### Remove the Template DNA +**Step 3.** +This strategy will have high background unless you remove the template DNA. DNA from most *E. coli* strains is methylated and can be degraded by the relatively nonspecific restriction enzyme Dpn1. + +Add 1 µl Dpn1 to each PCR tube and incubate for 30 minutes to overnight at 37°C. + +### Purify the PCR Product +**Step 4.** +In general, it is recommended to gel purify PCR products from these reactions both to further get rid of template DNA and to avoid cloning any truncated PCR products. + +**Step 5.** +Pour a 1% agarose gel in 0.5X TBE. + +- 75 ml 0.5X TBE +- 750 mg agarose + +Mix and microwave until boiling and clear - about 90 seconds +Mix and check that the agarose is dissolved. +Add 7.5 µl SYBR safe + +**Step 6.** +Casting gel +Assemble the gel cassette with combs. Use combs that are big enough to accommodate the entire PCR. Typically, these have four or five lanes per gel. Can use two combs per gel. +Pour hot agarose into cassette and let cool to RT. + +**Step 7.** +Running gel +Put RT gel into a tank with 0.5X TBE +Ensure gel is submerged in TBE. + +Load ladder into one well. Typically, 10 µl ladder is sufficient, even in large lanes. +Load samples into remaining lanes. +Run gel at 120V for 30 minutes + +**Step 8.** +Cut PCR product bands +Image gel on the blue light imager +Prepare one 1.5 ml tube for each successful band +Cut band out with a clean razor blade and transfer to tube. + +**Step 9.** +Gel purify the PCR product according to a mini-spin protocol, eluting in 15 µl. Quantify the product using a nanodrop. Good yields are 50 ng/µl; often yields are 10 ng/µl (which can still work). + +### Phosphorylate, Ligate, and Transform +**Step 10.** +Ligation of unphosphorylated DNA can be accomplished by simultaneous phosphorylation using T4 PNK and ligation with T4 DNA ligase. The reaction setup is simple but ensure you use T4 DNA ligase buffer and **not** T4 PNK buffer, since PNK buffer has no ATP. + +**Step 11.** +**Phosphorylation** +Mix the following in a tube: +- 1 µl 10X T4 ligase buffer +- 1 µl PNK +- 50 ng of gel purified PCR product +- Water to 9 µl + +Incubate for 30 minutes at 37°C + +**Step 12.** +**Ligation** +Move to RT, add 1 µl T4 DNA ligase +Incubate for 1 or more hours at RT (1 hour typically sufficient) + +**Step 13.** +**Transformation** +Thaw competent cells from -80°C on ice +Add 4 µl reaction to 33 µl competent cells in a microfuge tube +Incubate on ice for 25 minutes +Heat shock at 42°C for 1 minute +Incubate on ice for 2 minutes +Add 180 µl LB or SOC media +Shake at 37°C for 1 hour +Plate 75 µl on a plate with proper antibiotic + +**endofoutput** +``` \ No newline at end of file diff --git a/markdown-output/assay-for-determination-of-functional-concentratio-cstjwekn.md b/markdown-output/assay-for-determination-of-functional-concentratio-cstjwekn.md new file mode 100644 index 0000000000000000000000000000000000000000..43a761202b5866d466bd1a81687a315eb049062a --- /dev/null +++ b/markdown-output/assay-for-determination-of-functional-concentratio-cstjwekn.md @@ -0,0 +1,182 @@ +```markdown +# Goal/Experiment: +Assay for determination of functional concentration of Tn5 transposase + +## Assay for determination of functional concentration of Tn5 transposase +**Adrian Mcnairn** +*Cornell University* + +## Abstract +Tn5 is used by multiple labs worldwide for its ability to introduce DNA oligos and barcode sequences into libraries and for genomic assays. In many instances, labs produce their own Tn5 enzyme in-house rather than from commercial sources. Our method provides a means of determining the functional concentration of Tn5 in preparations by qPCR to standardize amounts of Tn5 used in assays and identify batch/lot variability. The current standard for assaying in-house produced Tn5 is a plasmid smear. Purified Tn5 is loaded with oligonucleotides and mixed with a plasmid substrate. The plasmid is then run on an agarose gel and checked for smearing, indicating the DNA was cut by the Tn5. The amount of Tn5 is standardized by measuring protein concentration using a protein assay or absorbance at 280nm which provides an approximation based on total protein present, not the functional concentration. This method is modified from Rykalina et al. to evaluate and empirically determine the functional concentration of Tn5 transposomes in homemade enzyme preps by qPCR. + +The principle is based on decreased Cp (or Cq) values correlating with increased fragmentation caused by the transposome. The change in Cp values functions as a measurement of the activity of the Tn5 at that concentration of oligonucleotide (the greater the number of cycles required to produce a product correlates with the increased cleavage of the plasmid substrate by Tn5) as plasmids with transposomes insertions (cleaved regions) will not amplify. The change in Cp values is then plotted against the oligonucleotide concentration in a line graph, and a plateau will appear at the concentration at which the Tn5 is saturated by oligo. The first point in the plateau is the functional concentration. + +This method may also be applied to testing the efficiency of changing DNA sequences in oligos used to assemble transposomes as the activity of the transposase is dependent upon the binding sequences contained within the oligo as well as secondary structure formed by the oligo. This property enables testing of oligo variations and barcode efficiencies in our assay. For instance, oligos containing different lengths or different barcodes sequences can be assembled in transposomes and tested in comparison to standard oligos. + +## Materials + +| Reagent | Vendor | Catalog Number | +|-------------------------------------|-----------------|------------------| +| HEPES, pH 7.2 (1M) | FisherScientific| AAJ16924K2 | +| NaCl (5M) | Invitrogen | AM9760G | +| EDTA (0.5M) | Invitrogen | AM9260G | +| Triton X-100 (10%) | VWR | 97063-864 | +| DTT (1M) | Krackeler | 45-43816-50ML | +| Glycerol (100%) | VWR | MK509202 | +| Nuclease-free water | Invitrogen | AM9932 | +| Tn5 or TDE1 | homemade or Illumina | 20034197 | +| SDS (10%) | Invitrogen | 15553027 | +| pUC19 | NEB | N3041S | +| EcoRI | NEB | R3101S | +| Zymo Research Clean and Concentrate-5| Zymo | D4014 | +| LUNA 2x qPCR master mix | NEB | M3003S | +| Qubit 1X dsDNA HS Assay Kit | Invitrogen | Q33231 | + +### Equipment: +- Eppendorf Thermomixer +- qPCR thermocycler +- Qubit + +### Primers: + +**Transposome Primers** + +| A | B | +|-------------------------------------|------------------------------------------------| +| Tn5ME_Rev | /5Phos/CTGTCTCTTATACACATCT | +| Tn5ME-A (Illumina FC-121-1030) | TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG | +| Tn5ME-B (Illumina FC-121-1031) | GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG | + +**qPCR Primers** + +| A | B | C | +|----------------------|----------------------------------|-----------------| +| Name | Sequence | pUC19 location(bp) | +| 591bp_pUC19_Tn5_F | GCTCACTCAAAGGCGTAAT | 748-1319 | +| 591bp_pUC19_Tn5_R | CTTCAGCAGAGCGAGATAC | | +| 602bp_pUC19_Tn5_F | CTTTCCACAGCGTTTTGGG | 2396-248 | +| 602bp_pUC19_Tn5_R | GCTGCGTAATAGCGAAGAG | | +| 610bp_pUC19_Tn5_F | CCTATCTCAGCGATCTGTTATTTC | 1651-2241 | +| 610bp_pUC19_Tn5_R | GCGCGGTATTATCCCGTATT | | + +## Transposome preparation and assembly + +1. **Prepare ME A/Rev or ME-B/rev oligos by annealing equal concentrations of primers.** Typically target 40uM stocks. + Example: 40uL 100uM Tn5-ME-A + 40uL 100uM Tn5-ME-B + 20uL nuclease-free water or 0.1x TE (can be scaled as needed). + Thermocycler program: 95°C for 2min, slow cool to 25deg at 0.1°C/sec, total time ~22 minutes. + **Duration:** 22 min + +2. **Prepare working stocks of annealed oligos by serial dilution (1:1):** + - Concentrations: 1.25uM, 2.5uM, 5uM, 10uM, 20uM, 40uM + +3. **Prepare Tn5 transposomes by combining 9uL of the Tn5 to be tested with 1uL annealed oligos** + - Final concentrations: 0.125uM, 0.25uM, 0.5uM, 1uM, 2uM, 4uM + +| Oligo Concentration (uM)| 1.25 | 2.5 | 5 | 10 | 20 | 40 | +|-------------------------|-----|-----|---|----|----|----| +| Oligo (uL) | 1 | 1 | 1 | 1 | 1 | 1 | +| Tn5 (uL) | 9 | 9 | 9 | 9 | 9 | 9 | +| Final vol (uL) | 10 | 10 | 10| 10 | 10 | 10 | +| Final Conc. (uM) | 0.125 | 0.25 | 0.5 | 1 | 2 | 4 | + +4. **Incubate for 21 hours at 25°C with shaking (600rpm) on Eppendorf Thermomixer** + **Note:** Shorter incubation (1 hour) is possible but longer incubation provides more consistent results. + **Duration:** Overnight 21 h + +## Template preparation + +1. **Digest pUC19 with EcoRI to generate a linear template:** + a. Column purify the plasmid DNA and adjust concentration to 25ng/uL + b. Digest reaction may be scaled to provide a stock of linearized plasmid for future use + **Duration:** 1 h + +## Tagmentation + +1. **In duplicate, prepare tagmentation master mix and test each concentration of transposome on 25ng linearized pUC19** + a. Include a no Tn5 control for reference (0uM) + b. For no Tn5 control, increase water to 11.5uL/reaction + c. 10X Tango restriction enzyme buffer (ThermoFisher) or 10X CutSmart buffer (NEB) are used to provide the magnesium Tn5 requires for tagmentation. + +| Stock | Vol per 25uL rxn (uL) | +|--------------------------------|-----------------------| +| 10X Tango RE Buffer | 2.5 | +| Nuclease-free water | 18 | +| DMF | 2.5 | +| Linearized pUC19 (25ng/uL) | 1 | +| Tn5 | 1 | + +**Tagmentation Master Mix:** + +2X Tagmentation Buffer + +| Stock | Vol for 10mL | Final Conc. | Supplier | +|--------------------------------|--------------|-------------|--------------------------| +| 1M Tris-HCl pH7-8 | 200uL | 20mM | Invitrogen #AM9850G | +| 1M MgCl2 | 100uL | 10mM | Invitrogen #AM5930G | +| Nuclease-free water | 9.7mL | | Invitrogen AM9932 | + +2. **Incubate at 37°C for 1 hour at 600rpm** + +3. **Stop the reaction by adding 1uL of 2% SDS to each reaction (final conc. 0.08%)** + a. 55°C for 7 minutes to remove transposomes + **Duration:** 7 min + +4. **Quench SDS by adding 3uL 10% Triton X-100 to reactions** + +5. **Final volume should be 29uL** + +6. **Qubit or nanodrop DNA for normalization (~0.86ng/uL)** + a. Quantifying the DNA first enables the assay to be more quantitative + +## qPCR + +1. **Dilute transposed DNA 1:10 in nuclease-free water for qPCR:** + a. Using too much DNA may lead to difficulty in determining Cp and higher data variability + b. Controls: linearized pUC19 without Tn5 and an NTC control + +2. **Setup qPCR reactions using primer pairs (recommend running at least 2 pairs):** + a. Pairs 591, 602, and 610 may be run on the same program + +3. **Run qPCR using the following settings:** + a. 95°C for 1 min, followed by 35 (minimum) to 45 cycles of 95°C for 15 sec, 60°C for 20 sec, 72°C for 30 sec, then a melt curve + **Note:** Two-step protocols may also be used alternating 95°C for 15 sec, 60°C for 20 sec + +4. **Analyze and graph delta Cp versus concentration:** + a. The no Tn5 control provides the reference value as the plasmid should be intact (i.e., Subtract the Geomean of the no Tn5 replicates from the Cp of the test samples). + b. The higher the delta Cp, the more cleavage of the plasmid, indicating higher Tn5 tagmentation activity + c. The concentration at which the graph plateaus or peaks at is the functional concentration of the Tn5 prep + +## Data Analysis Example + +| Primer pair: 610 | | | | +|------------------|------------------|------------------|---------| +| Sample | Cp | Ave Cp | delta Cp| +| 0.5 | 8.89 | 8.78 | 8.88 | 0.74 | +| 0.5 | 9.12 | 8.81 | 8.97 | 0.83 | +| 1 | 9.74 | 9.80 | 9.74 | 1.62 | +| 1 | 9.67 | 9.99 | 9.79 | 1.68 | +| 2.2 | 11.36 | 11.34 | 11.49 | 3.26 | +| 2.2 | 11.37 | 11.67 | 11.57 | 3.40 | +| 3.5 | 12.52 | 12.63 | 12.63 | 4.49 | +| 3.5 | 12.70 | 12.74 | 12.86 | 4.63 | +| 5 | 9.83 | 9.94 | 9.92 | 1.76 | +| 5 | 9.79 | 10.05 | 9.97 | 1.80 | +| noTn5 | 8.20 | 8.02 | 8.18 | -0.01| +| noTn5 | 8.25 | 7.97 | 8.21 | 0.00 | +| NTC | 24.04 | 23.92 | 23.96 | 15.81| +| NTC | 24.27 | 24.25 | 24.26 | 16.12| + +Example results from qPCR testing of a Tn5 prep +| Primer pair: 610 | | | | +|------------------|------------------|------------------|----------| +| Conc. (uM) | ave delta CP | stdev | +| 0.5uM | 0.79 | 0.06 | +| 1uM | 1.65 | 0.04 | +| 2.2uM | 3.33 | 0.10 | +| 3.5uM | 4.56 | 0.10 | +| 5uM | 1.78 | 0.03 | + +![Graph](data:image/png;base64,...) + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/assessing-coastal-risk-and-the-economics-of-climat-miyc4fw.md b/markdown-output/assessing-coastal-risk-and-the-economics-of-climat-miyc4fw.md new file mode 100644 index 0000000000000000000000000000000000000000..ba75eff9a22a862a3549557d5edc1907761ff27d --- /dev/null +++ b/markdown-output/assessing-coastal-risk-and-the-economics-of-climat-miyc4fw.md @@ -0,0 +1,92 @@ +```markdown +Goal/Experiment: + +The goal of this experiment is to assess coastal flood risk and the economics of climate adaptation. This involves identifying areas at risk, quantifying losses and damages under various scenarios, and comparing potential adaptation solutions using cost-benefit analysis. + +# Assessing Coastal Risk and the Economics of Climate Adaptation + +**Authors:** Borja G. Reguero, David N. Bresch + +**Citation:** Borja G. Reguero, David N. Bresch Assessing Coastal Risk and the Economics of Climate Adaptation. protocols.io dx.doi.org/10.17504/protocols.io.miyc4fw + +**Published:** 10 Jan 2018 + +## Abstract + +As climate change progresses, so does the risk from hurricanes, flooding, and other natural disasters. As sea levels rise, tropical cyclones will pose a greater risk of extreme flooding and are likely to inflict the greatest damages on highly populated shorelines. This protocol describes a quantitative risk assessment framework to evaluate the cost-effectiveness of different adaptation measures. These options may include natural solutions (e.g., oyster reef restoration), structural solutions (e.g., seawalls), and policy measures (e.g., home elevation or coastal development policies). + +## Guidelines + +The protocol is part of the ‘Economics of Climate Adaptation’ framework and is implemented in climada, particularly the ‘coastal module’. + +For more information, consult: [www.swissre.com/eca/](http://www.swissre.com/eca/) + +## Before Start + +Risk occurs at the intersection of economic assets and the hazard of coastal flooding. Adaptation can impact each risk component. There are three parts to assess the cost-effectiveness of adaptation measures: + +### 1. Assessing Current Risk + +Risk is quantified as a probable loss. The total loss from a natural hazard (e.g., floods) is a combination of three factors: + +- **Hazard (or 'peril')**: Defined by the location, frequency, and intensity of events. +- **Assets exposed**: Defined by the location and value of buildings and assets. +- **Damages to assets**: The relationship between the extent of damage and event intensity, determined by damage (or vulnerability) curves. + +### 2. Assessing Future Risk + +Future risk arises from climate and economic changes. Factors include land subsidence, sea level rise, and changes in storm patterns. Coastal exposure changes, due to development intensification, also contribute to future risk. + +### 3. Assessing the Economics of Adaptation Measures + +The cost and benefits (losses averted) of adaptation measures are assessed by: + +1. Defining adaptation strategies. +2. Estimating the benefits of each measure in protecting a percentage of property. +3. Calculating the cost of each measure. +4. Calculating the Net Present Value (NPV) of costs and benefits, calculated as follows: + - Calculate both baseline risk (current) and future risk (e.g., 2050). + - Discount benefits and costs to present terms. +5. Calculating the benefit-to-cost ratio. + +Cost estimates for each adaptation measure can be derived from the review of past projects or local estimates. + +## Protocol + +### Assessment of Coastal Flooding + +**Step 1.** +- Historical and synthetic storms are simulated with climada. +- Total water levels are calculated based on surges, tides, sea level rise, and wave runup. + +**Software Package (Matlab):** [CLIMADA - COASTAL, 1.0](https://github.com/borjagreguero/climada_coastal_hazards_module) +**Dataset:** [Sea Level Rise NOAA measurements](#) + +### Assessment of Coastal Exposure + +**Step 2.** +- In GIS, data on building value is calculated for each ground height. +- Flood maps are created using a bathtub approach for each ground height. + +**Dataset:** [Elevation model](#) + +### Calculation of Damages + +**Step 3.** +- Damages are calculated in climada, considering asset distribution for each ground height. + +**Software Package (Matlab):** [CLIMADA - COASTAL, 1.0](https://github.com/borjagreguero/climada_coastal_hazards_module) + +### Calculation of Cost and Benefits + +**Step 4.** +a) Estimate adaptation strategies for hazard attenuation, location, cost, and protection percentage. + +b) Calculate net present value over the period the adaptation measure is designed for. + +**Software Package (Matlab):** [CLIMADA - COASTAL, 1.0](https://github.com/borjagreguero/climada_coastal_hazards_module) + +--- + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/assessment-of-prepulse-inhibition-ppi-of-the-acous-cr4cv8sw.md b/markdown-output/assessment-of-prepulse-inhibition-ppi-of-the-acous-cr4cv8sw.md new file mode 100644 index 0000000000000000000000000000000000000000..08a991195dbc64863ab1a4db402c4a5c6def9a65 --- /dev/null +++ b/markdown-output/assessment-of-prepulse-inhibition-ppi-of-the-acous-cr4cv8sw.md @@ -0,0 +1,103 @@ +```markdown +# Goal/Experiment: +**Goal:** Assessment of Prepulse Inhibition (PPI) of the Acoustic Startle Reflex in Rodents + +## Assessment of Prepulse Inhibition (PPI) of the Acoustic Startle Reflex in Rodents + +**Authors:** Franciele Kich Giongo1, Matheus Gallas-Lopes1, Ana P Herrmann1 +**Institution:** Universidade Federal do Rio Grande do Sul + +1 Universidade Federal do Rio Grande do Sul + +### Abstract +The acoustic startle reflex is an automatic and involuntary response that occurs in humans and other animals when they are exposed to sudden and intense auditory stimuli, such as loud noises. This response can be influenced by several factors, including the intensity and timing of the stimulus, the individual's state of alertness, and contextual factors. It can also be modulated by prior exposure to a weak prepulse shortly before the startling stimulus, a phenomenon known as prepulse inhibition (PPI). + +PPI reflects the brain's ability to filter out irrelevant or non-threatening sensory information and is an important indicator of sensorimotor gating. Here we describe a protocol to measure PPI in mice and rats using a startle chamber from Insight® (SP, Brazil). + +## Materials + +### Software +| Name | OS | Developer | +|---------------------------------|---------|--------------------------| +| Monitor de Sobressalto para Ratos e Camundongos | Windows | Insight Equipamentos Ltda | + +### Equipment +| Name | Brand | SKU | Link | +|--------------------------|------------------------|-----|------------------------------------------------------| +| EP-175 Startle Sobressalto | Insight Equipamentos Ltda | N/A | [EP-175 Startle Sobressalto](https://www.insightltda.com.br/produto/ep-175-startle-sobressalto/) | + +## Before Start Instructions +This is a step-by-step guide developed at PsychoLab to set up the software used to assess prepulse inhibition (PPI) of the acoustic startle reflex in mice or rats using the equipment acquired from Insight Equipamentos Ltda. + +- Ensure the speakers and ventilation of the apparatus are turned on. +- Avoid using any light source inside the box. +- Read the entire protocol before starting. + +## Setting up the Protocol + +1. **To create a new protocol:** Click on the logo in the upper left corner and select "Edit" and then "Procedure". + +2. **To create a new prepulse protocol:** Select "Create a new procedure file starting with a standard PPI block" and name the protocol. + +3. **If you already have a saved protocol file:** Select "Open an existent procedure file" and then select the file (e.g., example.sbs). + +4. **To calibrate the background noise:** + - Place the decibel meter inside the equipment, just above the accelerometer. + - Ensure the decibel meter’s battery is charged or new. + - Position the decibel meter on the accelerometer and adjust the detection range as necessary (range 30-80 dB for background noise; 80-130 dB for other measurements). + - To see the readings of the decibel meter, remove the lid of the equipment and close the door. + +5. **Select the speaker:** The button to select the speaker is pointed by the red arrow. Speaker identification will be shown in the box "Confirmação de Ips" for selection. + +6. **Adjust the intensity:** Adjust the intensity of the sound manually in the percentage bar and press play. Check the decibel meter to see if the desired range is shown. For other stimuli, repeat the process outlined in step 2. + +7. **Note percentages:** Take notes of the percentages shown in each stimulus for easier stimulus calibration. + +8. **Configure stimulus settings:** Set the duration of each period in the first line according to your protocol. Each stimulus will have its own configuration regarding background noise, white noise, pure tone, shock, and light. + +9. **Save stimulus settings:** Once you have entered the stimulus settings for each block, click the checked box icon below the table, then save. + +10. **To create new stimuli blocks:** Click on the "add PPI" icon. Set the desired percentage for pulse and prepulse. Adjust the decibel meter accordingly. + +11. **Scale calibration:** + - First, remove the decibel meter from the scale. + - Click on the option "No, scale calibration presents considerable errors. Repeat scale calibration process now". + - Check whether the scale displays the weight of 50g correctly. + - Calibrate the scale at the beginning of the tests, if measuring is accurate, no need to recalibrate. + - Click on "Yes, current scale calibration is satisfactory. Execute the next step". + +12. **Session configuration:** Create stimuli presentation order. + - Check the box for habituation with background noise if needed. + - Transfer blocks from procedure blocks to execution session. + - Transfer blocks in the same order, and randomly as needed. + - Fill in the session parameters (habituation period, inter-trial interval, etc.). + - Click on "Execute procedure file now". + +13. **Adjust scale gain:** + - For mice, set scale gain to 1. + - For rats up to 200g, set scale gain to 2. + - For rats over 200g, set scale gain to 3. + +## Running the Protocol + +1. **To start the test:** Place the subject in the apparatus, close the lid, and click the play icon. The test will start automatically after selecting where to save the summary results. + - If there is a habituation period, a countdown will show. + - The session will end and a notification will pop up. + +## Retrieving the Data + +1. **Full reports:** Click on "Reports" on the main screen to export full reports. Use parameters set in step 8 as filters. Export data in `.xls`, `.doc`, or `.txt`. + +## Data Analysis + +1. **Reactivity Values:** + - Use the `mean value` of each stimulus for pulse and prepulse reactivity. + +2. **Prepulse Inhibition Values:** + - Use the formula `(pulse alone - prepulse + pulse) / pulse alone * 100` for each combination. + +3. **Mean PPI:** + - Use the mean value of all %PPI calculated for each combination of stimulus. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/assessment-of-tuberculosis-transmission-probabilit-dc622zge.md b/markdown-output/assessment-of-tuberculosis-transmission-probabilit-dc622zge.md new file mode 100644 index 0000000000000000000000000000000000000000..5a0783e0e2c1c1165bc38afd7f869f898ecc3d56 --- /dev/null +++ b/markdown-output/assessment-of-tuberculosis-transmission-probabilit-dc622zge.md @@ -0,0 +1,111 @@ +```markdown +## Goal/Experiment: +Assess and compare the probability of tuberculosis transmission in three Thai prisons based on five dynamic models. + +## Assessment of Tuberculosis Transmission Probability in Three Thai Prisons Based on Five Dynamic Models + +**DOI:** [dx.doi.org/10.17504/protocols.io.6qpvr868zlmk/v1](dx.doi.org/10.17504/protocols.io.6qpvr868zlmk/v1) + +**Authors:** +- Nithinan Mahawan, Thanapoom Rattananupong, Puchong Sri-Uam, Wiroj Jiamjarasrangsi + 1Faculty of Medicine, Chulalongkorn University + 2Center for Safety, Health and Environment of Chulalongkorn University + +**Corresponding Author:** +- Nithinan Mahawan, Faculty of Medicine, Chulalongkorn University + +**Protocol Status:** Working + +**Created:** May 05, 2024 +**Last Modified:** June 25, 2024 +**Protocol Integer ID:** 99258 + +**Keywords:** Probability of tuberculosis transmission, dynamic models, model parameters, prisons + +--- + +## Abstract +This study aimed to assess and compare the probability of tuberculosis (TB) transmission based on five dynamic models: the Wells–Riley equation, two Rudnick & Milton-proposed models based on air changes per hour (ACH) and liters per second per person (L/s/p), the model proposed by Issarow et al., and the Applied Susceptible-Exposed-Infected-Recovered (SEIR) TB transmission model. The study aimed to determine the impact of model parameters on such probabilities in three Thai prisons. The results revealed that the median (Quartiles 1 and 3) of TB transmission probability among these cells was 0.052 (0.017, 0.180). Compared to the pioneering Wells–Riley model, the remaining models projected a discrepant TB transmission probability from less to more commensurate to the degree of model modification. The ventilation rate and the number of infectious TB patients were the greatest impact factors on the estimated TB transmission probability. All stakeholders must urgently address these influential parameters to reduce TB transmission in prisons. + +--- + +## Materials and Methods + +### Materials +1. **Absolute Ventilation Rate Measurement:** + - Using the Kimo HQ210 with SCOH 112 probe (Sauermann Industries, ZA Bernard Mouliend, Montpon, France) for measuring CO₂ concentrations. + +2. **ACH (Air Changes per Hour):** + - Classical metric assessing infection control risk; wind speed in cells using a hot wire thermo-anemometer (Model SDL350, Extech Instruments, Waltham, MA). + +### Before Start +1. **Literature Review Goals:** + - Incidence and prevalence of TB in prisons globally and in Thailand. + - Evidence of prisons as TB reservoirs. + - Humanitarian issues and health problems. + - Factors affecting TB transmission in prisons. + - Comparison of dynamic models of TB transmission. + +2. **Contact Department of Corrections:** + - Walkthrough to assess suitability in June 2019. + +--- + +## Research Ethics +1. Ethical approval from Chulalongkorn University Faculty of Medicine (Ref: 610/63). +2. Permission from the Department of Corrections, Ministry of Justice needed. +3. Inform and gain consent from relevant stakeholders, but inmate personal information not required for the study. + +--- + +## Data Collection +1. Train research team on protocols. +2. CO₂ collection inside and outside cells using Kimo HQ210; measured by trained inmates and researchers. +3. Measure wind speeds with thermo-anemometer (Model SDL350). +4. Collection of infection data by health volunteers. +5. Survey of cell architecture and characteristics. +6. Literature review for model parameters. + +--- + +## Statistical Analysis +1. **Estimation of TB Transmission Probability:** + - Calculation based on dynamic models. + - Assess agreement using Spearman’s rank correlation. + - Bland–Altman analysis for agreement pattern. + - Model parameter influence using Wilcoxon rank-sum (Mann-Whitney test) and linear regression. + +2. **Parameters Analysed:** + - Ventilation rates. + - Number of infectious inmates. + +--- + +## Results and Conclusion +1. **Variant Model Projections:** + - Different transmission probabilities across models. + - Wells–Riley model as reference showed low to high probability variance. + +2. **Risk Factors:** + - Key factors: low ventilation rates, high TB inmate numbers. + - Models: Similar patterns of TB transmission probability with varying high and low extremes. + - Urged addressing ventilation and inmate number issues. + +3. **Recommendations:** + - Validation and further studies required for more accurate TB incidence prediction in prison settings. + +--- + +## Protocol References +1. World Health Organization. Global tuberculosis report 2023. Geneva: WHO; 2023. +2. United States Agency International Development. Tuberculosis in prisons: a growing public health challenge [Internet]. USAID; 2014 [Cited 2021 August 8]. +3. World Health Organization [Internet]. Tuberculosis: Key facts. [Cited 2021 August 8]. +4. World Prison Brief, Institute for Crime & Justice Policy Research, Birkbeck University of London. +5. Walter KS, Martinez L, et al. Lancet. 2021;397(10284):1591-6. +6. Mabud TS, de Lourdes Delgado Alves M, et al. PLoS Med. 2019;16(1):e1002737. +... [Additional 30 references] ... + +--- + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/aureococcus-anophagefferens-population-count-and-r-cgzftx3n.md b/markdown-output/aureococcus-anophagefferens-population-count-and-r-cgzftx3n.md new file mode 100644 index 0000000000000000000000000000000000000000..16614fb1a784e39e80c84a97643668e58a948b9b --- /dev/null +++ b/markdown-output/aureococcus-anophagefferens-population-count-and-r-cgzftx3n.md @@ -0,0 +1,93 @@ +```markdown +# Goal/Experiment: +To identify and count the population and relative size of the brown tide alga *Aureococcus anophagefferens* using flow cytometry techniques, specifically utilizing the Violet Side Scatter (SSC) channel on a Beckman Coulter CytoFLEX S Flow Cytometer. + +# Aureococcus anophagefferens Population Count, and Relative Size (Violet SSC) by Flow Cytometry (CytoFLEX S Flow Cytometer Beckman Coulter) + +### Authors +- Emily E. Chase +- Alex Truchon +- Steven W Wilhelm + +*[The University of Tennessee, Knoxville](https://www.utk.edu)* + +Published Dec 01, 2022 + +### DOI +[https://dx.doi.org/10.17504/protocols.io.q26g7yby9gwz/v1](https://dx.doi.org/10.17504/protocols.io.q26g7yby9gwz/v1) + +### Keywords +- cytoflex +- flow cytometry +- violet side scatter +- cell size +- cell counts +- Aureococcus anophagefferens + +### Abstract +A method for obtaining relative cell size, population density, etc., of the brown tide algae *Aureococcus anophagefferens* by a Violet Side Scatter (SSC) configuration on a Beckman Coulter CytoFLEX S Flow Cytometry System (CytExpert software). + +### License +This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. + +--- + +## Guidelines +To achieve the intended results of this protocol, a configuration using a violet laser must be set up for a CytoFLEX S Flow Cytometer. This protocol is based on a CytoFLEX with Violet SSC on the violet laser at the 405/10 position. Refer to the "Setting Up Violet Side Scatter (VSSC) Channel" section of the CytoFLEX manual. Visualization of *A. anophagefferens* can be achieved without a violet laser, but it will not be possible to determine relative cell size. + +## Materials +- **CytoFLEX S Flow Cytometer**: A flow cytometry device used to count and analyze particles suspended in a fluid. +- **CytExpert Software + desktop computer with appropriate specs**: Software used to control the CytoFLEX system and analyze data. +- **96 well CoStar Assay Plates [REF#3795]**: Plates used for sample preparation and analysis. +- **1000mL pipette and tips**: For transferring samples. + + **Note**: Sheath fluid + cleaning fluid + Milli-Q water (or alternative) as required for cytometer maintenance only. + +## Before Starting +Boot up the CytExpert Software and follow the start up protocol. Familiarity with the machine and its settings will permit necessary adjustments for your experiments and help ensure accurate results. + +## Protocol + +### CytoFLEX Experiment Setup + +1. **Create a new experiment with sample acquisition settings as follows:** + + - FSC 200 + - SSC 40 + - VioSSC 75 + - FITC 200 + - PerCP 150 + - PB450 1 (placeholder; this could be set for a different dye than Pacific Blue shown here, this will not change the results) + - Threshold: (manual) PerCP 5000 + + Set the flow rate to medium (30 µL/minute), the sampling to 60 seconds (not by events), and the display to 100,000 events. + + **Note:** + - Flow rate may need to be adjusted for a high abort percentage (>10%), both adjusting the rate (custom or otherwise) and diluting dense culture samples will solve this problem. Users should aim for a certain number of events per second, typically between 300–2000. + - Settings can be adjusted in real time by selecting "run" on samples and changing the acquisition settings. + +2. **Optional**: Create a combination of density plots and histograms best suited for your analyses. An example set up is provided in the image below (Figure 2), where **A. anophagefferens** populations are clearly achieved. + + - It is recommended to set up a histogram with time on the x-axis (final histogram) to account for the machine's measuring consistency as sampling will often start out slower and then stabilize. + + ![Figure 1. Detector configuration setup for the CytoFLEX Flow Cytometer used to establish this protocol.](image_url_1) + +3. **Sampling Process**: + - 250 µL of each sample to be measured (e.g., *A. anophagefferens* culture) are pipetted into a 96 well CoStar [REF#3795] Assay Plate round bottom plate (can be substituted) and loaded into the CytoFLEX. + - After opening the "Plate" window and clicking "Add Plate", samples can then be labelled. + + **Note:** + - Volumes can be reduced after taking into account the flow rate and sample timing. + +4. **Data Acquisition**: + - Labelled samples can then be run sequentially using the "Auto Record". + - Upon completion, data can be either exported (most conveniently as a .csv) or reviewed using the "Statistics" window according to all events and events within user defined gated populations. + - Population counts are achieved by number of events within the *A. anophagefferens* gates, and subsequent relative size of events (cells) can be achieved through Violet Side Scatter channel results. + + **Note:** + - Relative size by Violet SSC cannot be achieved for every cell type (normally "larger" microalgae cannot), a priori comparisons with other cell size calculation methods (e.g., FlowCam) must be conducted. + +![Figure 2. CytoFLEX S Flow Cytometer discovery of Aureococcus anophagefferens cell populations. Lassoed (i.e., gated) populations represent A. anophagefferens classified events.](image_url_2) + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/automated-bar-seq-library-preparation-and-pooling-dhu936z6.md b/markdown-output/automated-bar-seq-library-preparation-and-pooling-dhu936z6.md new file mode 100644 index 0000000000000000000000000000000000000000..4ea8d2fc061919ce044caa563f906fb3dce4e869 --- /dev/null +++ b/markdown-output/automated-bar-seq-library-preparation-and-pooling-dhu936z6.md @@ -0,0 +1,136 @@ +```markdown +# Goal/Experiment: +The goal of this protocol is to prepare and pool barcoded DNA libraries for multiplexed Illumina sequencing using automated steps. This involves several rounds of PCR, cleanup processes, and pooling samples to ensure balanced representation in sequencing. + +## Automated Bar-Seq Library Preparation and Pooling V.2 + +### DOI +[dx.doi.org/10.17504/protocols.io.3byl49qdjgo5/v2](https://dx.doi.org/10.17504/protocols.io.3byl49qdjgo5/v2) + +### Authors +- David Ross, Nina Alperovich + +### Affiliation +- NIST + +### Collaborating Initiative +- Open Datasets Initiative + +### License +This protocol is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. + +### Protocol Status +- Working + +### Creation Date +- May 24, 2024 + +### Last Modified +- July 25, 2024 + +### Protocol Integer ID +- 104065 + +--- + +## Abstract + +**Protocol for automated Bar-Seq Library preparation** + +This protocol prepares 96 DNA samples, representing 24 samples from 4 different timepoints, for multiplexed Illumina sequencing. The process starts with two rounds of PCR, each followed by a bead-based cleanup. The first round of PCR attaches primers that serve as tags to identify the timepoint and sample. The second round of PCR attaches flow-cell adapters required for Illumina sequencing. Following the PCR and cleanup steps, the protocol outlines procedures for pooling samples together to ensure balanced representation during sequencing. The process includes quantifying the DNA concentration and diluting/ pooling samples from the same timepoint. + +#### NOTES: +- Prepare the magnetic bead suspension before implementing this protocol, following the **Preparation of Sera-mag SpeedBeads protocol**. +- This protocol should be implemented after the **Automation Protocol for Plasmid DNA Extraction from E. coli protocol**. +- A fragment analyzer can also be used instead of a gel to determine if the PCR cleanup process was successful. + +--- + +## Materials + +### Starting Samples +- 96 DNA samples resulting from the Automation Protocol for Plasmid DNA Extraction from *E. coli* protocol + +### Reagents +- Nuclease-free water (ThermoFisher Scientific 4387936) +- 80% Absolute Ethanol (Fisher Bioreagents BP2818500) +- Elution Buffer (Qiagen 19086) +- Phusion Flash PCR Mastermix (ThermoFisher Scientific F548L) +- Multiplexing Primers +- Universal Illumina Primers + +### Labware +- Three 96-well DeepWell reagent plates (Abgene AB-0765) - one used as a reagent plate and two used as midi plates +- Two 96-well PCR plates (Bio-Rad HSP9635 or HSP9645) +- Three PCR plate lids (Agilent 202497-100) +- 96-well output plate (Eppendorf 30603303) + +### Primers + +| Primer Name | Description | Sequence | +|-------------|-------------|----------| +| BarSeq_1_F1 | forward barSeq PCR 1 primer, with sample multiplex tag: CG | ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNNNNGTGATTGGCCTAGACGTGTGATAgactcagtc | +| BarSeq_1_F2 | forward barSeq PCR 1 primer, with sample multiplex tag: AT | ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNNNNTTACTTAGCAGCCTCAGACGTGTGATAgactcagtc | +| BarSeq_1_F3 | forward barSeq PCR 1 primer, with sample multiplex tag: TC | ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNNNNTCTCTAGGCCCTAGACGTGTGATAgactcagtc | +| BarSeq_1_F4 | forward barSeq PCR 1 primer, with sample multiplex tag: GA | ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNNNNCCGATAGGCCTAGACGTGTGATAgactcagtc | +| BarSeq_1_F5 | forward barSeq PCR 1 primer, with sample multiplex tag: GG | ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNNNNGATTGTGGCACTCTTCCAGACGTGTGATAgactcagtc | +| BarSeq_1_F6 | forward barSeq PCR 1 primer, with sample multiplex tag: CC | ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNNNGGGTAGGACGACTAGACGTGTGATAgactcagtc | +| BarSeq_1_F7 | forward barSeq PCR 1 primer, with sample multiplex tag: GA | ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNNNGTAGAAGGCACTGAACGACTCTGC | +| BarSeq_1_F8 | forward barSeq PCR 1 primer, with sample multiplex tag: TC | ACACTCTTTCCCTACACGACGCTCTTCCGATCTCTAGAGGATGACTCGACTCTGAAG | +| BarSeq_2_F | forward barSeq PCR 2 primer | AATGATACGGCGACCACCGAGATCTACACTC | +| BarSeq_2_R | reverse barSeq PCR 2 primer| CAAGCAGAAGACGGCATACGATCTCGTATGCCGTCTTCTGCTTG | + +[Table continues similarly for other primers...] + +## Methods + +### Transfer Plasmid DNA to PCR Plate and Dilute with DI Water + +1. Pre-heat the on-deck thermocycler (ODTC) for 1st PCR step. +2. Remove lids from PCR-Plate 1, Sample-Plate, and Reagent Plate. +3. Pipette 10 µL nuclease-free water to each well in PCR plate 1. + - Mix 6x after dispensing. + - Dispense 1 mm above the bottom of the well. +4. Transfer 35 µL of extracted plasmid DNA to each well of PCR Plate 1. + - This step is done 24 times (one sample at a time). +5. Remove plasmid sample input Sample-Plate. + +### Run First PCR -Using Primers to Identify Samples from Each Timepoint + +6. Add 28.125 µL Master Mix with reverse primer for the appropriate timepoint and sample to each well of PCR plate 1. + - Dispense 0.5 mm below liquid surface, with liquid following On. +7. Add 28.125 µL Master Mix with forward primer for the appropriate timepoint and sample to each well of PCR plate 1. + - Mix 10x after dispensing. + - Dispense 0.5 mm below liquid surface, with liquid following On. +8. Place a PCR plate lid on the PCR plate 1. +9. Move PCR-Plate 1 to ODTC. +10. Run the First PCR using the following conditions (101.25 µL volume): + - 98°C for 60 s + - 3 cycles at: + - 98°C for 10 s + - 58°C for 20 s + - 72°C for 20 s + - 72°C for 60 s + - 23°C for 10 s + +### First PCR Cleanup Part 1: Bind Template Plasmid DNA to Beads and Keep the Supernatant + +11. Pipette 54 µL magnetic bead suspension into each well of Midi Plate 1. + - Bead ratio: 0.6x +12. Move PCR plate 1 from ODTC; take lid off PCR plate 1. +13. Shake Midi plate 1 for 10 s at 1800 RPM. +14. Transfer 90 µL of each sample from PCR plate 1 to Midi Plate 1. +15. Incubate for 7 minutes at room temperature. +16. Move Midi Plate 1 to the magnet base and wait for 4 minutes. +17. Remove the supernatant (209.4 µL). + +[Continue similarly for all steps] + +... + +### Protocol References + +This protocol is based on a similar protocol described by Tack et al., Mol Syst Biol (2021), [https://doi.org/10.15252/msb.202010179](https://doi.org/10.15252/msb.202010179). + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/automated-procedure-for-estimation-of-methylation-b3ptqmnn.md b/markdown-output/automated-procedure-for-estimation-of-methylation-b3ptqmnn.md new file mode 100644 index 0000000000000000000000000000000000000000..a4c2765f6b84db67154b8956fce2d4733408151c --- /dev/null +++ b/markdown-output/automated-procedure-for-estimation-of-methylation-b3ptqmnn.md @@ -0,0 +1,124 @@ +```markdown +# Goal/Experiment: +This protocol outlines an automated procedure for estimating methylation levels using Methylation-Sensitive High-Resolution Melting (MS-HRM) analysis. The goal is to facilitate the detection and quantification of disease-related DNA methylation changes which can provide clinically relevant information in personalized patient care. + +# Automated Procedure for Estimation of Methylation Levels in MS-HRM Analysis + +**Authors:** +- Sally Samsø Mathiasen +- Jan Bińkowski +- Tina Kjeldsen +- Tomasz K Wojdacz +- Lise Lotte Hansen + +**Affiliations:** +- ¹Department of Biomedicine, Aarhus University, Aarhus DK-8000, Denmark +- ²Independent Clinical Epigenetics Laboratory, Pomeranian Medical University, Szczecin, Poland +- ³Department of Biomedicine, Aarhus University, Aarhus DK-8000, Denmark + +## Abstract +Testing for disease-related DNA methylation changes provides clinically relevant information in personalized patient care. Methylation-Sensitive High-Resolution Melting (MS-HRM) is a method used for measuring methylation changes and has already been employed in diagnostic settings. This method uses one set of primers that initiate the amplification of both methylated and non-methylated templates. Quantification of methylation levels using MS-HRM is hampered by PCR bias, leading to inaccurate calculations. This protocol utilizes the Area Under the Curve (AUC), a derivative of the HRM curves, and least square approximation (LSA) to improve accuracy. Limitations of the technique have been comprehensively evaluated, leading to a procedure that allows methylation level inference with specific measurement limitations. + +## Protocol Citation +``` +Sally Samsø Mathiasen, Jan Bińkowski, Tina Kjeldsen, Tomasz K Wojdacz, Lise Lotte Hansen. Automated procedure for estimation of methylation levels in MS-HRM analysis. protocols.io. https://protocols.io/view/automated-procedure-for-estimation-of-methylation-b3ptqmn +``` + +## License +This protocol is made available under the terms of the [Creative Commons Attribution License](https://creativecommons.org/licenses/by/4.0/), permitting unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. + +## Experimental Procedure + +### Data Import +1. **MS-HRM Data Preparation for the Analyses (when using Light Cycler system – other PCR systems may require adjusting the data format):** + 1.1 Normalize the HRM curves using the Gene Scanning software (we recommend default settings for normalization). + 1.2 Generate difference plots for each normalized melting curve with the 100% methylation melting curve as the baseline/reference. + 1.3 If data for any samples contain obvious outliers, consider removing them (note the name of the outlier). + 1.4 Export the difference plot as a text file (example layout in supplementary materials S8-S11). + + > **IMPORTANT:** Calculations using the Methylation Level Calculator (MLC) require layout as described in "Plate set up" section of the MLC template. Modify columns and rows accordingly for other layouts. + +### Calculation of Experiment Specific Standard Curve + +2. **Methylation Levels Estimation Procedure:** + 2.1 Open the Methylation Levels Calculator (MLC). + 2.2 Open the exported text file from LC480 instrument (e.g., S10-S11 MGMT assay text without outliers). + 2.3 Copy all data and paste into the "imported data" sheet starting in cell B3. Ensure names in rows 2 and 3 match. + + > **IMPORTANT:** Make sure the digital separator in the file exported from LC480 and Excel are the same. Modify the MLC for different sample layouts accordingly. + + 2.4 MLC will calculate and display AUC for each control and sample in row 1 of "Imported data" sheet. + 2.5 Check if AUC for each replicate in row 1 is within the acceptable range. Replace outliers with 0 if necessary. + > **IMPORTANT:** If MLC does not perform calculations automatically, change Excel settings to Automatic (`Formulas > Calculation options > Automatic`). + + 2.6 Go to sheet "0 variable": + - Panel 1: AUC for each control replicate is calculated. + - Panel 2: Equation 1 calculates theoretical AUC for each methylation level. + - Panel 3: Theoretical and obtained AUC values for controls are plotted. + + 2.7 Go to sheet "1 variable": + - Panel 1: AUC for each control replicate is calculated. + - Panel 2: Equation 2 calculates theoretical AUC with M value set to 1. + - Panel 3: Theoretical and obtained AUC values for controls are plotted. + + 2.8 Go to sheet "1 variable after LSA": + - Solver Add-in is used. + + 2.9 Go to `Data > Solver > Solve`. Recalculate M value by LSA and recalculate standard curve. + + 2.10 Go to the sheet "2 variables": + - Panel 1: AUC for each control replicate is calculated. + - Panel 2: Equation 3 calculates theoretical AUC with N value set to 1. + - Panel 3: Theoretical and obtained AUC values for controls are plotted. + + 2.11 Go to sheet "2 variables after LSA": + - Solver Add-in is used. + + 2.12 Go to `Data > Solver > Solve`. Recalculate M and N values by LSA and recalculate standard curve. + +### Estimation of Methylation Level in Unknown Samples + +3. **Estimation of Methylation Level in Unknown Samples:** + - MLC uses polynomial trend function for calculation. + + 3.1 Go to sheet "PTF": + - Panel 1: Transform standard curve to describe methylation level as a function of AUC. + - Panel 2: Polynomial trend function describes the standard curve. + + 3.2 Go to sheet "USC": + - Panel 1.1: Sample name. + - Panel 1.2: AUC for each replicate. + - Panel 1.3: Methylation level calculated using equation 3 with M and N variables. + +### Calculation of Experiment Specific Detection Window + +4. **Calculation of Experiment Specific Detection Window:** + 4.1 Go to sheet "Cut off (CO)": + - Panel 1.1-1.2: Calculate AUC for each control replicate. + - Panel 1.3-1.4: Calculate standard deviation and mean for each control replicate. + - Panel 2: Plot normal distribution for each control. + + 4.2 Go to sheet "Detection window": + - Panel 1.1-1.2: Calculate AUC for each control replicate. + - Panel 1.3-1.4: Calculate standard deviation and mean for each control replicate. + - Panel 2: Calculate overlap between consecutive controls. + - Panel 3: Fill lower (10%) and upper limits (50%-60%) of detection window in cells P7 and Q7. + +### Calculation of Methylation Levels in the Assay Specific Detection Window + +5. **Calculation of Methylation Levels in the Assay Specific Detection Window:** + 5.1 Go to sheet "2 variables within DW": + - Solver Add-in is used. If calculations are not automatic, change Excel settings to Automatic. + + 5.2 Go to `Data > Solver > Solve`. Recalculate M and N values by LSA and standard curve. + + 5.3 Go to sheet "PTF within DW": + - The same procedure is applied within the detection window. + + 5.4 Go to sheet "USC within DW": + - Panel 1.1: Sample name. + - Panel 1.2: AUC for each sample replicate. + - Panel 1.3: Calculate methylation level with M and N variables within detection window. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/behavioural-phenotyping-of-c-elegans-on-uv-killed-b2dhqa36.md b/markdown-output/behavioural-phenotyping-of-c-elegans-on-uv-killed-b2dhqa36.md new file mode 100644 index 0000000000000000000000000000000000000000..9d825d4b228526233ed7b7ab9a5e4661b5e030b1 --- /dev/null +++ b/markdown-output/behavioural-phenotyping-of-c-elegans-on-uv-killed-b2dhqa36.md @@ -0,0 +1,196 @@ +```markdown +# Goal/Experiment: +Behavioural phenotyping of *C. elegans* on UV-killed *E. coli* mutants + +## Introduction + +**Saul Moore** +Imperial College London +[DOI: 10.17504/protocols.io.b2dhqa36](https://dx.doi.org/10.17504/protocols.io.b2dhqa36) +*Behavioral Genomics* + +**Abstract:** +Protocol for screening candidate behaviour-modifying *E. coli* BW25113 single-gene deletion mutants from the 'Keio Collection', to investigate their effects on *Caenorhabditis elegans* behaviour when killed by ultraviolet (UV) light. + +## Materials + +- 12 x Whatman Square Well Flat Bottom UNIPLATE, 7701-1651 +- 25 x ThermoFisher Scientific Nunc™ 96-Well Polystyrene Round Bottom Microwell Plates, Non-Treated, [268200](https://www.thermofisher.com/order/catalog/product/268200) +- 100 x 60mm Petri plates +- 3 x 90mm Petri plates +- 3 x 150mm Petri plates +- 2 x 50mL Erlenmeyer flask +- 1 x 96-pin replicator +- 500mL LB broth media +- 1L NGM agar (for ingredients, see protocol for making NGM agar) +- 110 x 15mL Falcon tubes + +## Preparing NGM Agar and Pouring Plates + +1. Prior to screening, prepare the materials needed for screening *C. elegans* on selected *Keio* *E. coli* mutants (9 candidate mutants + wild-type BW control). For a single experiment replicate (10 biological replicates of each mutant, screened in 2 runs with the laboratory's 'Hydra' imaging rig): + +- 12 Whatman 96-square-well flat-bottom plates ('imaging plates') +- 25 Nunc™ 96-round-well round-bottom microwell plates ('culture' plates) +- 3 x 150mm Petri plates ('nursery' plates) +- 3 x 90mm Petri plates ('maintenance' plates) +- 100 x 60mm Petri plates ('uv-killing plates') +- 110 x 15mL Falcon tubes +- 2 x 50mL Erlenmeyer flasks + +2. Make 1L normal Nematode Growth Media (NGM) agar, following the protocol: [Making normal NGM for imaging plates (Cabreiro Lab)](https://dx.doi.org/10.17504/protocols.io.b2dhqa36). + +3. Pour 20ml NGM agar into each maintenance plate, and 50ml NGM agar into each nursery plate, following the protocol for plate pouring: [Plate pouring protocol]( https://dx.doi.org/10.17504/protocols.io.6bhhaj6). + +![Alert](https://dx.doi.org/10.17504/protocols.io.6bhhaj6) **Keep the remaining agar warm in a water bath set to 65°C, for dispensing into 96-well imaging plates afterwards.** + +4. Using the Integra ViaFill, dispense 200μL of NGM agar into each well of the 10 imaging plates, following the protocol: [Dispensing agar into multiwell plates](https://dx.doi.org/10.17504/protocols.io.b2dhqa36). + +5. Leave the plates on the lab bench (with lids on) until the agar has cooled and solidified (approximately 1 hour, timing depends on humidity). + +6. Measure the weight of 3 imaging plates (with lids on) and record average plate weight on day of pouring. + +7. Dry the imaging plates under a hood (or drying cabinet) until the plates lose between 3-5% of their original plate weight (with lids on). + +8. Store the imaging plates upside-down at 4°C until used for experiments. + +## Seeding Petri Plates and Worm Maintenance + +9. Inoculate 10ml LB broth media with *E. coli* BW25113 (Keio background wild-type strain, used as negative control and for raising worms, no Kanamycin) in an Erlenmeyer flask for overnight culture following the protocol: [Inoculating a Liquid Bacterial Culture](https://dx.doi.org/10.17504/protocols.io.b2dhqa36). + +10. Place the inoculation in a shaking incubator at 37°C at 200 rpm and leave to grow overnight. + +11. Remove the BW culture from the shaking incubator and place in 4°C fridge until seeding. + +12. Remove the plates from storage and the BW culture from the fridge, and leave on the bench for approximately 30 minutes to acclimate to room temperature. + +13. Using aseptic technique, seed the maintenance plates each with 400μL of BW25113 culture. + +14. Leave under hood until dry (with lids on, timing depends on humidity). + +15. Using a platinum pick, gently pick 30 adult N2 Bristol *C. elegans* onto each maintenance plate, and store in an incubator at 20°C (Monday). + +16. After 24 hours, remove the adult worms, leaving the eggs behind to hatch into L1 larvae (Tuesday). + +17. Inoculate a further 10ml LB broth media with BW25113 bacteria for overnight culture, following the protocol in step #9 and place in a shaking incubator at 37°C, 200 rpm (Wednesday). + +18. After 24 hours, remove the culture from the incubator, and the nursery plates from storage, and leave to acclimate on bench top for 30 minutes (Thursday). + +19. Seed the nursery plates each with 1mL of fresh BW25113 culture. Leave under hood until dry. + +20. Wash the worms off the BW-seeded maintenance plates, into two 15mL Falcon tubes (Friday). + +21. Perform an egg prep on worms in the Falcon tubes, following the protocol: [Egg Prep for Bleach Synchronization](https://dx.doi.org/10.17504/protocols.io.b2dhqa36). + +22. At around noon the next day, wash L1 larvae off the empty plate and re-feed onto the BW-seeded nursery plates using a glass Pasteur pipette. Aim to dispense around 3000 worms per plate. Incubate at 20°C (Saturday). + +## Inoculating from Frozen Stocks (96-well) + +23. Remove the required stock plates from -80°C containing the selected candidate strains. Gently remove the aluminium film and leave to partially thaw for a minute or so. + +![Alert](https://dx.doi.org/10.17504/protocols.io.b2dhqa36) **To avoid damaging the bacterial stocks through repeated freeze-thawing, do not let the wells completely defrost. Just enough to be able to pick up some cells with the replicator.** + +24. Inoculate individual vials containing 4mL LB broth and 50μg/mL Kanamycin from the selected wells of the Keio frozen stock plates, following the protocol: [Inoculating a Liquid Bacterial Culture](https://dx.doi.org/10.17504/protocols.io.b2dhqa36). + +25. Wet some tissue with MilliQ water, wrap the culture plates in the tissue, and incubate overnight at 37°C (no shaking). + +![Alert](https://dx.doi.org/10.17504/protocols.io.b2dhqa36) **The tissue provides humidity that aids growth, while the presence of Kanamycin should prevent contamination.** + +26. Also inoculate 10mL LB broth media in an Erlenmeyer flask with BW control, and place in a shaking incubator overnight at 37°C, 200 rpm. + +27. Using a multi-pipette, fill half of wells (those designated for live bacterial cultures) of 10 x 96-well culture plates with 200μL LB broth (as per the desired plate layout). Fill those wells with 50μg/mL Kanamycin, except for the wells that are reserved for BW control. + +28. Remove the overnight cultures from the incubator. Using a sterile pipette tip (or inoculation loop), inoculate the wells of the culture plates with strains from the overnight culture vials designated for live culture. Inoculate the wells without Kanamycin with the BW control. + +Optional: Make a template stock plate for -80°C storage (live strain layout only): mix 200μL culture with 15% glycerol in each well. + +29. Fill another round of individual 15mL Falcon tubes each with 4mL fresh LB broth, for overnight culture of strains destined for UV-treatment. Add 50μg/mL Kanamycin to all tubes except those reserved for the UV-treated BW control. + +30. For strains designated for UV-treatment, inoculate the new Falcon tubes from the previous overnight culture, following the above protocol in step #24. + +31. Incubate both the live cultures in 96-well format (no shaking) and the cultures for UV-treatment in vials (shaking) overnight at 37°C. + +32. Remove the overnight cultures from the incubator. Again, fill half of the wells of 10 culture plates (designated for live bacteria) with 200μL LB broth. This time do not add Kanamycin. (Thursday). + +33. Inoculate the second round of overnight cultures from the first in 96-well format (for live bacteria), using a 96-pin replicator, following the protocol: [Growing overnight bacterial culture in 96WP](https://dx.doi.org/10.17504/protocols.io.b2dhqa36). + +34. Fill another round of 15mL Falcon tubes with 4mL fresh LB broth for the second round of inoculations of the overnight cultures in vials for UV-treatment (no Kanamycin). + +35. Inoculate the new vials from the previous overnight cultures, by following the above protocol for Inoculating a Liquid Bacterial Culture in step #24. + +36. Place the 96-well culture plates (no shaking) and the vials (shaking) in an incubator for overnight culture at 37°C. + +## UV-Killing Bacteria + +37. Clean the CL-1000 Ultraviolet crosslinker machine by wiping down with distilled MilliQ water and 70% ethanol. Turn on the UV light and leave for 5 minutes to decontaminate (Friday). + +38. Remove the overnight cultures in vials from the incubator, add 4mL fresh LB broth to each culture vial (total 8mL) and pour into empty 60mm plates for UV-killing (10 replicates for each strain tested; 10 strains = 100 plates). + +39. Place the plates inside the machine, and remove their lids. + +40. Expose the bacterial cultures to UV light (365nm wavelength) for 10 minutes. + +41. Remove the plates from the machine, replace the lids, and leave to stand for 5 minutes. + +42. Repeat steps #40 to #42 six more times, to ensure that the bacteria are dead. + +![Alert](https://dx.doi.org/10.17504/protocols.io.b2dhqa36) **This process may need to be repeated (in batches of up to 25 plates) due to the maximum capacity of the UV machine.** + +43. Transfer the bacterial cultures to separate 15mL Falcon tubes, and top up to 15mL with LB broth. + +44. Centrifuge the bacteria for 10 minutes at 4,000 rpm to pellet the bacteria at the bottom of the tubes. + +45. Remove the supernatant using a plastic Pasteur pipette, and store at 4°C. + +## Seeding Imaging Plates (96-well) + +46. Remove the imaging plates from 4°C storage and record the average weight of 3 randomly selected plates (Friday). + +47. Ensure that imaging plates have lost approximately 3-5% of their original weight. Place under a hood or drying cabinet until they have. + +48. Remove overnight cultures of live Keio strains and the pelleted dead Keio strains from 4°C storage. + +49. Re-suspend the bacteria by adding 3mL LB broth and vortexing. + +50. Add 200μL of re-suspended dead bacterial culture to the empty wells of the overnight culture plate with live bacteria, to complete the experimental plate layout, with an equal proportion of wells with live bacterial cultures and wells with dead bacterial cultures. + +51. Using the Integra ViaFlo, seed 10μL of bacterial culture from the wells of each live overnight culture plate into the corresponding wells of each imaging plate. + +![Alert](https://dx.doi.org/10.17504/protocols.io.b2dhqa36) **Ensure correct plate orientation under the Integra ViaFlo, with well A1 in the top left corner.** + +52. Place seeded plates under a hood to dry for 20 minutes, then place in an incubator at 25°C (no shaking) for 7 hours 40 minutes (total lawn growth time: 8 hours). + +53. After 8 hours, remove the plates from the incubator and store at 4°C. + +## COPAS Worm-Sorting and Hydra Tracking (96-well) + +54. Prior to tracking, ensure that the imaging cave air conditioning is turned on (and there has not been a power-cut) and also empty the dehumidifier waste water tray (see pre-imaging checklist) (Tuesday). + +![Alert](https://dx.doi.org/10.17504/protocols.io.b2dhqa36) **Normal temperature range: 19 - 21°C, Humidity: 35 - 45%** + +55. Remove the nursery plates from the incubator. Wash the worms off the plates into two 15ml Falcon tubes using approximately 10mL sterile PBS 'A' buffer. + +56. Fill up the tubes to 15ml with PBS 'A' and centrifuge at 1000rpm for 2 minutes. + +57. Remove the supernatant using a Pasteur pipette. + +58. Repeat steps #57 to #58 four more times to thoroughly rinse off any remaining control BW25113 bacteria. + +59. Re-suspend the worms and divide them equally into two 50ml Falcon tubes (for the COPAS), and fill them both up to approximately 40ml with PBS 'A'. + +60. Use the COPAS to dispense three Day1 adult worms into each well of the 10 imaging plates, following the protocol: [COPAS wormsorter v.2](https://dx.doi.org/10.17504/protocols.io.b2dhqa36). + +61. Leave the plates to dry under a hood for 30 minutes to 1 hour (until dry, timing depends on humidity), then place in incubator at 20°C until tracking (at +4 hours on food). + +![Alert](https://dx.doi.org/10.17504/protocols.io.b2dhqa36) **Check that worms are crawling (not swimming) on plates, and lawns appear matt in colour (not wet).** + +62. 30 minutes prior to tracking with the Hydra rig (every 20 minutes, 2 runs in total), remove 5 imaging plates from the 20°C incubator and leave to acclimate in the imaging cave. + +63. Record worm behaviour on the bacterial food for 15 minutes at the 4-hour timepoint (25 fps, exposure: 25000 msec, blue-light stimulation). + +64. After tracking, discard the plates in a biological waste bin. + +65. Check tracking checklist to ensure that all videos have been saved correctly: +`'/Volumes/behavgenom$/Documentation/Protocols/analysis/tracking-checklist-20210210.docx'` + +_endofoutput_ +``` \ No newline at end of file diff --git a/markdown-output/bgiseq-500-wgs-library-construction-ps5dng6.md b/markdown-output/bgiseq-500-wgs-library-construction-ps5dng6.md new file mode 100644 index 0000000000000000000000000000000000000000..b00e2d0fc4056540c5e6a010f70a9566b58432cf --- /dev/null +++ b/markdown-output/bgiseq-500-wgs-library-construction-ps5dng6.md @@ -0,0 +1,249 @@ +```markdown +# Goal/Experiment: +Construct a Whole Genome Sequencing (WGS) Library using BGI's BGISEQ-500. + +# BGISEQ-500 WGS Library Construction + +**Authors:** Jie Huang, Xinming Liang, Yuankai Xuan, Chunyv Geng, Yuxiang Li, Haorong Lu, Shoufang Qu, Xianglin Mei, Hongbo Chen, Ting Yu, Nan Sun, Junhua Rao, Jiahao Wang, Wenwei Zhang, Ying Chen, Sha Liao, Hui Jiang, Xin Liu, Zhaopeng Yang, Feng Mu, Shangxian Gao + +## Abstract +BGISEQ-500 is a desktop sequencer developed by BGI. Using DNA nanoball and combinational probe anchor synthesis developed from Complete Genomics™ sequencing technologies, it generates short reads at a large scale. Library construction on the platform includes fragmentation, size selection, end repair and A-tailing, adaptor ligation, PCR amplification, and splint circularization. + +## Materials +- Fresh 80% ethanol by [XILONG SCIENTIFIC](https://www.xilong.cn/) +- ERAT Buffer by Contributed by users +- ERAT Enzyme by Contributed by users +- Adapter Mix by Contributed by users +- Ligation Buffer by Contributed by users +- Ligation Enzyme by Contributed by users +- TE buffer by [Ambion](https://www.thermofisher.com/) +- PCR Enzyme Mix by Contributed by users +- Splint Buffer by Contributed by users +- Digestion Buffer by Contributed by users +- Digestion Enzyme by Contributed by users + +## Protocol + +### Overview +Step 1. + +![Flow Chart](images/flow_chart.png) + +### DNA Fragmentation +**Step 2.** + +#### 1) Input Genomic DNA Sample + +**Genomic DNA Sample Recommendation** + +| Nucleic Acid | High-quality genomic DNA | +|--------------|---------------------------| +| Molecular Weight | >23k bp | +| Amount | 1µg | +| Concentration | ≥12.5ng/µL | +| Purity | OD260/OD280=1.82.0 | + +> High-quality genomic DNA should be free of salt or organics. It could run as an intact band with DNA length >23kb during 1% agarose gel electrophoresis. + +#### 2) Fragmentation +Use the Covaris Focused-ultrasonicator for genomic DNA fragmentation following the instructions of the instrument. Optimization should be performed on DNA prior to the experiment and analyzed with agarose electrophoresis or an Agilent 2100 BioAnalyzer. + +**Sequencing with Input Amount Reaction Volume Derived Fragments** + +| | PE 100 | PE 50 | PE 150 | +|------------------------|--------|--------|--------| +| Input Amount | 1µg | 1µg | 1µg | +| Reaction Volume | 80µL | 80µL | 80µL | +| Derived Fragments | 100-700 bp (main band≈200-300 bp) | 100-500 bp (main band≈200 bp) | 100-700 bp (main band≈400 bp) | + +#### 3) Bead-based Cleanup +1. Place AMPure XP magnetic beads at room temperature (RT) for 30 min, fully thaw before use. +2. **PE100:** Pipette 48µL AMPure XP magnetic beads to 80µL shearing product, and mix well by gently pipetting 10 times, incubate for 5 min at room temperature *(PE50: Pipette 40µL AMPure XP magnetic beads to 80µL shearing product, and mix well by gently pipetting 10 times, incubate for 5 min at room temperature. PE150: Pipette 44µL AMPure XP magnetic beads to 80µL shearing product, and mix well by gently pipetting 10 times, incubate for 5 min at room temperature.)” +3. After brief centrifugation, place the non-stick tube on the magnet for 2 min until the liquid clears, carefully transfer the supernatant to a new non-stick tube with a pipette. +4. **PE100:** Pipette 16µL AMPure XP magnetic beads to 128µL supernatant, mix well by gently pipetting 10 times, and incubate at room temperature for 5 min. *(PE50: Pipette 40µL AMPure XP magnetic beads to 160µL supernatant, mix well by gently pipetting 10 times, and incubate at room temperature for 5 min. PE150: Pipette 12µL AMPure XP magnetic beads to 124µL supernatant, mix well by gently pipetting 10 times, and incubate at room temperature for 5 min.)* +5. After brief centrifugation, place the non-stick tube on the magnet for 2 min until the liquid clears, remove and discard the supernatant with a pipette. +6. Add 500µL of fresh 80% ethanol, while the tube remains on the magnet, then, rotate the tubes in the rack by half turns 4 times to wash the beads then carefully remove and discard the supernatant after 1 min. +7. Repeat step 6 once, and remove all liquid from the tube without disrupting the beads. + +#### 4) Homogenization +1. Use a double-strand DNA quantification kit such as Qubit® dsDNA HS Assay Kit or Quant-iT™ PicoGreen® dsDNA Assay Kit, and quantify the sample as per the instructions of the quantification kit. +2. Remove 50ng of sample (calculated based on its concentration) to a new 0.2mL PCR tube, and add NF water to the final volume of 40µL. + +### End Repair and Tailing +**Step 3.** + +1. Prepare the mixture as follows in PCR tube (do not vortex enzymes): + + **Components** + + | DNA | Volume | + |----------------|--------------| + | DNA | 40µL | + | ERAT Buffer | 7.1µL | + | ERAT Enzyme | 2.9µL | + | **Total** | **50µL** | + +2. Mix well by gently pipetting (Do not mix by vortexing), concentrate the reaction liquid to tube bottom by brief centrifugation. +3. Place the PCR tube containing the reaction mixture of the above step in a Thermal Cycler, and initiate the reaction as per the following conditions: + + | Temperature | Time | | + |-------------|------|--| + | Heated lid | On | | + | 37°C | 30 min | | + | 65°C | 15 min | | + | 4°C | Hold | | + +### Ligate Adapters +**Step 4.** + +1. Add 5µL of Adapter Mix to above PCR tube, and mix well by pipetting. Now 16 Adapter Mix are available, 8 libraries in one lane strategy, every sample with 4 different barcodes. +2. Prepare the following reaction mixture (Note: Ligation Buffer II is viscous, pipette slowly): + + **Components** + + | | Volume | + |-----------------|--| + | Ligation Buffer | 23.4µL | + | Ligation Enzyme | 1.6 µL | + | **Total** | **25µL** | + +3. Add 25µL of the above reaction mixture to the reaction solution containing adapters from above step. +4. Place the tube in a Thermal Cycler, then initiate reaction as per following condition: + + | Temperature | Time | | + |-------------|------|----------------------| + | Heated lid | On | | + | 23°C | 30 min | | + | 4°C | Hold | | + +5. After ligation, add 20µL TE to the final volume of 100µL, then transfer the entire volume to a non-stick tube containing 50µL of room temperature AMPure beads and mix by slow pipetting 10 times to avoid bubble formation. + +### Purify Ligated DNA +**Step 5.** + +1. Incubate at room temperature for 5 min. +2. After brief centrifugation, place the non-stick tube on the magnet for 2 min until the liquid clears, remove and discard the supernatant with a pipette. +3. Add 500µL of fresh 80% ethanol, while the tube remains on the magnet, then, rotate the tubes in the rack by half turns 4 times to wash the beads. Carefully remove and discard the supernatant after 1 min. +4. Repeat step 4 once, remove all liquid from tube without disrupting the beads. +5. Open the cap of non-stick tube, while the tube remains on the magnet, and dry at room temperature for 3 min. +6. Remove the non-stick tube from the magnet, add 46µL of TE for DNA elution, mix well by pipetting, and incubate at room temperature for 5 min. +7. After brief centrifugation, place the non-stick tube on the magnet for 2 min until the liquid clears, transfer all 44µL of supernatant to a new 0.2mL PCR tube ready for PCR in the next step or store at -20°C. + +### PCR +**Step 6.** + +1. Prepare the PCR reaction mixture as follows: + + **Components** + + | | Volume | + |----------------|----| + | DNA | 44µL | + | PCR Enzyme Mix | 50µL | + | PCR Primer Mix | 6µL | + | **Total** | 100µL | + +2. Place the above PCR tube in a Thermal Cycler and initiate the reaction as per the following conditions: + + | Temperature | Time | Cycles | + |-------------|------|--------| + | Heated lid | On | | + | 95°C | 3 min | | + | 98°C | 20 sec | | + | 60°C | 15 sec | 8 | + | 72°C | 30 sec | | + | 72°C | 10 min | | + | 4°C | Hold | | + +### Purify PCR Product +**Step 7.** + +1. Place AMPure XP magnetic beads at room temperature 30 min in advance, mix well by vortexing before use. +2. Add 100µL of AMPure XP magnetic beads to 100µL of PCR product, mix well by gently pipetting 10 times, and incubate at room temperature for 5 min. +3. After brief centrifugation, place the non-stick tube on the magnet for 2 min until the liquid clears, remove and discard the supernatant with a pipette. +4. Add 500µL of fresh 80% ethanol, while the tube remains on the magnet, then rotate the tubes in the rack by half-turns 4 times to wash the beads. Carefully remove and discard the supernatant after 1 min. +5. Repeat step 4 once, try to suck up all liquid from the tube's bottom. +6. Open the cap of the non-stick tube, while the tube remains on the magnet, and dry at room temperature for 3 min. +7. Remove the non-stick tube from the magnet, add 32µL of TE water for DNA elution, mix well by pipetting, and incubate at room temperature for 5 min. +8. After brief centrifugation, place the non-stick tube on the magnet for 2 min until the liquid clears, transfer the supernatant to a new non-stick tube. Proceed to the next step of the reaction or store at -20°C. + +### Homogenization +**Step 8.** + +1. Use double strrand DNA quantification kit such as Qubit® dsDNA HS Assay Kit or Quant-iT™ PicoGreen® dsDNA Assay Kit, and quantify the sample as per the instructions of the quantification kit. +2. It is recommended to mix samples of different Barcodes here. +3. Add mixed sample (calculated based on its concentration) to a PCR tube, and add NF water to the final volume of 48µL. + +### Circularization +**Step 9.** + +1. Denature the homogenized PCR product on a Thermal Cycler at 95°C for 3 min, then immediately transfer to an ice bath. +2. Prepare reaction mixture on ice as per the following system: + + **Components** + + | | Volume | + |-----------------|--| + | Splint Buffer | 11.6µL | + | Ligation Enzyme | 0.2 µL | + | **Total** | 11.8µL | + +3. Add 11.8µL of the above reaction mixture to 48µL of denatured DNA. +4. Place the above PCR tube in a Thermal Cycler and initiate the reaction as per the following conditions: + + | Temperature | Time | | + |-------------|------|--| + | Heated lid | On | | + | 37°C | 30 min | | + | 4°C | Hold | | + +### Digestion +**Step 10.** + +1. Prepare digestion reaction solution on ice as per the following system: + + **Components** + + | | Volume | + |----------------|----| + | Digestion Buffer | 1.4µL | + | Digestion Enzyme | 2.6 µL | + | **Total** | 4µL | + +2. After the circularization reaction is finished, directly add 4µL of digestion reaction solution into the circularized DNA solution, mix well and briefly centrifuge, then place the PCR tube in a Thermal Cycler, and initiate the reaction as per the following conditions: + + | Temperature | Time | | + |-------------|------|--| + | Heated lid | on | | + | 37°C | 30 min | | + | 4°C | Hold | | + +3. Add 7.5µL of Digestion Stop Buffer to each reaction, mix well to terminate the reaction. +4. Transfer all the reaction solution to a new non-stick tube, ready for purification. + +### Purify Digestion Product +**Step 11.** + +1. Place AMPure XP magnetic beads and place at room temperature for 30 min in advance. Mix well by vortexing before use. +2. Pipette 168µL AMPure XP magnetic beads to the digestion product, mix well by pipetting 10 times, and incubate at room temperature for 10 min. +3. After transient centrifugation, place the non-stick tube on the magnet for 2 min until the liquid clears, remove and discard the supernatant with a pipette. +4. Add 500µL of fresh 80% ethanol, while the tube remains on the magnet, then, rotate the tubes in the rack by half turns 4 times to wash the beads. Carefully remove and discard the supernatant after 1 min. +5. Repeat step 4 once, and try to suck up all liquid from the tube bottom. +6. Open the cap of a non-stick tube, while the tube remains on the magnet, and dry at room temperature for 3 min. +7. Remove the non-stick tube from the magnet, add 32µL of TE for DNA elution, mix well by pipetting and incubate at room temperature for 10 min. +8. After brief centrifugation, place the non-stick tube on the magnet for 2 min until the liquid clears, transfer the supernatant to a new non-stick tube. Store at -20°C, ready for preparing DNB. + +## Reagents and Their Functions: +- **ERAT Buffer**: Used for end repair and A-tailing of fragmented DNA. +- **ERAT Enzyme**: Enzyme used along with ERAT buffer for end repair and A-tailing. +- **Adapter Mix**: Mix of different adapters used for ligation. +- **Ligation Buffer/Enzyme**: Reagents used for ligating the adapters to the DNA fragments. +- **TE Buffer**: Tris-EDTA buffer used for elution and storage of DNA. +- **PCR Enzyme Mix**: Enzymes for PCR amplification of the ligated DNA. +- **Splint Buffer and Digestion Buffer/Enzyme**: Reagents used for the circularization and digestion steps to create DNA nanoballs. +- **AMPure XP Beads**: Magnetic beads used for purification steps. + +--- + +**endofoutput** +``' diff --git a/markdown-output/big-redesign-protocol-version-2-chddt226.md b/markdown-output/big-redesign-protocol-version-2-chddt226.md new file mode 100644 index 0000000000000000000000000000000000000000..4e652eb6ffee1f8f7e2a36c39dca7e295bf3a1d7 --- /dev/null +++ b/markdown-output/big-redesign-protocol-version-2-chddt226.md @@ -0,0 +1,236 @@ +```markdown +# Goal/Experiment: +The goal of this experiment is to provide a comprehensively structured and detailed big redesign protocol. + +# Big Redesign Protocol Version-2 V.1 +*Authors: Maria Gul¹, Katarina¹* \ +*Affiliation: 1qa* \ +*Version 1, Published on Oct 04, 2022* + +--- + +## Disclaimer +``` +Lorem ipsum dolor sit amet, consectetur adipiscing elit. Integer in sapien. Praesent id justo in neque elementum ultrices. Cras elementum. Sed ac dolor sit amet purus malesuada congue. Nunc auctor. Quis autem vel eum iure reprehenderit qui in ea voluptate velit esse quam nihil molestiae consequatur, vel illum qui dolorem eum fugiat quo voluptas nulla pariatur? In convallis. +``` + +## Abstract +``` +Lorem ipsum dolor sit amet, consectetur adipiscing elit. Integer in sapien. Praesent id justo in neque elementum ultrices. Cras elementum. Sed ac dolor sit amet purus malesuada congue. Nunc auctor. Quis autem vel eum iure reprehenderit qui in ea voluptate velit esse quam nihil molestiae consequatur, vel illum qui dolorem eum fugiat quo voluptas nulla pariatur? In convallis. + +Lorem ipsum dolor sit amet, consectetur adipiscing elit. Integer in sapien. Praesent id justo in neque elementum ultrices. Cras elementum. Sed ac dolor sit amet purus malesuada congue. Nunc auctor. Quis autem vel eum iure reprehenderit qui in ea voluptate velit esse quam nihil molestiae consequatur, vel illum qui dolorem eum fugiat quo voluptas nulla pariatur? In convallis. Etiam quis quam. Aliquam ornare wisi eu metus. Curabitur bibendum justo non orci. Pellentesque sapien. Nullam at arcu a est sollicitudin euismod. Phasellus rhoncus. Maecenas libero. Praesent vitae arcu tempor neque lacinia pretium. Duis aute irure dolor in reprehenderit in voluptate velit esse cillum dolore eu fugiat nulla pariatur. Phasellus faucibus molestie nisl. Vivamus ac leo pretium faucibus. + +Etiam neque. Lorem ipsum dolor sit amet, consectetur adipiscing elit. Nulla est. Etiam ligula pede, sagittis quis, interdum ultricies, scelerisque eu. Nulla quis diam. Curabitur ligula sapien, pulvinar a vestibulum quis, facilisis vel sapien. Sed convallis magna eu sem. Nulla accumsan, elit sit amet varius semper, nulla mauris mollis quam, tempor suscipit diam nulla vel leo. Etiam ligula pede, sagittis quis, interdum ultricies, scelerisque eu. Duis aute irure dolor in reprehenderit in voluptate velit esse cillum dolore eu fugiat nulla pariatur. Vestibulum erat nulla, ullamcorper nec, rutrum non, nonummy ac, erat. Aenean vel massa quis mauris vehicula lacinia. Maecenas sollicitudin. Integer vulputate sem a nibh rutrum consequat. Etiam bibendum elit eget erat. Etiam neque. Aliquam erat volutpat. Aliquam erat volutpat. +``` + +## Attachments +[sample2.pdf](sample2.pdf) + +## External Link +[Google](https://www.google.com/) + +## Protocol Citation +``` +Maria Gul, Katarina 2022. big redesign protocol Version-2. protocols.io +https://protocols.io/view/big-redesign-protocol-version-2-chddt226 +Version created by Maria Gul +``` + +## Funders Acknowledgement +``` +name \ +Grant ID +``` + +## Manuscript Citation +``` +manuscript citation test - edited +``` + +## Keywords +``` +qa, test, protocol, redesign +``` + +## License +``` +This is an open access protocol distributed under the terms of the Creative Commons Attribution License [https://creativecommons.org/licenses/by/4.0/](https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. +``` + +## Image Attribution +``` +image attribution test +``` + +## Created +``` +Oct 04, 2022 +``` + +## Last Modified +``` +Oct 04, 2022 +``` + +## Protocol Integer ID +``` +70789 +``` + +## Guidelines + +### Guidelines +``` +Lorem ipsum dolor sit amet, consectetur adipiscing elit. Integer in sapien. Praesent id justo in neque elementum ultrices. Cras elementum. +``` + +### Formula +``` +ωUp = VE / R tg(ϕ) + U sin(ϕ) +``` + +## Materials Text +``` +Lorem ipsum dolor sit amet, consectetur adipiscing elit. Integer in sapien. Praesent id justo in neque elementum ultrices. Cras elementum. Sed ac dolor sit amet purus malesuada congue. Nunc auctor. Quis autem vel eum iure reprehenderit qui in ea voluptate velit esse quam nihil molestiae consequatur, vel illum qui dolorem eum fugiat quo voluptas nulla pariatur? In convallis. Etiam quis quam. Aliquam ornare wisi eu metus. Curabitur bibendum justo non orci. Pellentesque sapien. Nullam at arcu a est sollicitudin euismod. Phasellus rhoncus. Maecenas libero. Praesent vitae arcu tempor neque lacinia pretium. Duis aute irure dolor in reprehenderit in voluptate velit esse cillum dolore eu fugiat nulla pariatur? Phasellus faucibus molestie nisl. Vivamus ac leo pretium faucibus. +``` + +- ⧫ **Vitamin K 3 P212121** + +## Safety Warnings +### Safety Warnings +``` +Lorem ipsum dolor sit amet, consectetur adipiscing elit. Integer in sapien. Praesent id justo in neque elementum ultrices. Cras elementum. Sed ac dolor sit amet purus malesuada congue. Nunc auctor. Quis autem vel eum iure reprehenderit qui in ea voluptate velit esse quam nihil molestiae consequatur, vel illum qui dolorem eum fugiat quo voluptas nulla pariatur? In convallis. Etiam quis quam. Aliquam ornare wisi eu metus. Curabitur bibendum justo non orci. Pellentesque sapien. Nullam at arcu a est sollicitudin euismod. Phasellus rhoncus. Maecenas libero. Praesent vitae arcu tempor neque lacinia pretium. Duis aute irure dolor in reprehenderit in voluptate velit esse cillum dolore eu fugiat nulla pariatur. Phasellus faucibus molestie nisl. Vivamus ac leo pretium faucibus. +``` + +## Disclaimer +``` +Lorem ipsum dolor sit amet, consectetur adipiscing elit. Integer in sapien. Praesent id justo in neque elementum ultrices. Cras elementum. Sed ac dolor sit amet purus malesuada congue. Nunc auctor. Quis autem vel eum iure reprehenderit qui in ea voluptate velit esse quam nihil molestiae consequatur, vel illum qui dolorem eum fugiat quo voluptas nulla pariatur? In convallis. +``` + +## Before Starting +### Before start +``` +Lorem ipsum dolor sit amet, consectetur adipiscing elit. Integer in sapien. Praesent id justo in neque elementum ultrices. Cras elementum. Sed ac dolor sit amet purus malesuada congue. Nunc auctor. Quis autem vel eum iure reprehenderit qui in ea voluptate velit esse quam nihil molestiae consequatur, vel illum qui dolorem eum fugiat quo voluptas nulla pariatur? In convallis. +``` + +### Section 1 +1. Lorem ipsum dolor sit amet, consectetur adipiscing elit. Integer in sapien. Praesent id justo in neque elementum ultrices. Cras elementum. Sed ac dolor sit amet purus malesuada congue. + +``` +Lorem ipsum dolor sit amet, consectetur adipiscing elit. Integer in sapien. Praesent id justo in neque elementum ultrices. Cras elementum. Sed ac dolor sit amet purus malesuada congue. Nunc auctor. Quis autem vel eum iure reprehenderit qui in ea voluptate velit esse quam nihil molestiae consequatur, vel illum qui dolorem eum fugiat quo voluptas nulla pariatur? In convallis. Etiam quis quam. Aliquam ornare wisi eu metus. +``` + +![figure](figure.jpg) + +``` +Lorem ipsum dolor sit amet, consectetur adipiscing elit. Integer in sapien. Praesent id justo in neque elementum ultrices. Cras elementum. Sed ac dolor sit amet purus malesuada congue. Quis autem vel eum iure reprehenderit qui in ea voluptate velit esse quam nihil molestiae consequatur, vel illum qui dolorem eum fugiat quo voluptas nulla pariatur. In convallis. +``` + +![figure](figure.jpg) + +- ⧫ **Vitamin K 3 P212121** \ +Nullam at arcu a est sollicitudin euismod. Phasellus rhoncus. Maecenas libero. Praesent vitae arcu tempor neque lacinia pretium. Duis aute irure dolor in reprehenderit in voluptate velit essa cillum dolore eu fugiat nulla pariatur. Phasellus faucibus molestie nisl. Vivamus ac leo pretium faucibus. + +## Section 2 +2. Lorem ipsum dolor sit amet, consectetur adipiscing elit. Integer in sapien. Praesent id justo in neque elementum ultrices. Cras elementum. Sed ac dolor sit amet purus malesuada congue. Nunc auctor. Quis autem vel eum iure reprehenderit qui in ea voluptate velit esse quam nihil molestiae consequatur, vel illum qui dolorem eum fugiat quo voluptas nulla pariatur. In convallis. + +``` +Lorem ipsum dolor sit amet, consectetur adipiscing elit. Integer in sapien. Praesent id justo in neque elementum ultrices. Cras elementum. Sed ac dolor sit amet purus malesuada congue. Nunc auctor. +``` + +- ⧫ **Riboflavin (Vitamin B2) P212121** \ +Lorem ipsum dolor sit amet, consectetur adipiscing elit. Integer in sapien. Praesent id justo in neque elementum ultrices. Cras elementum. Sed ac dolor sit amet purus malesuada congue. Nunc auctor. + +``` +Lorem ipsum dolor sit amet, consectetur adipiscing elit. Integer in sapien. Praesent id justo in neque elementum ultrices. Cras elementum. Sed ac dolor sit amet purus malesuada congue. +``` + +## Section 3 +3. Lorem ipsum dolor sit amet, consectetur adipiscing elit. Integer in sapien. Praesent id justo in neque elementum ultrices. + +- ![clock](clock.jpg) ⏰ 00:10:23. +- Nullam at arcu a est sollicitudin euismod. + +``` +Lorem ipsum dolor sit amet, consectetur adipiscing elit. Integer in sapien. Praesent id justo in neque elementum ultrices. +``` + +### Task +- ⏰ 00:00:23 +- ⧫ 125 Aliquam ornare wisi eu metus. Curabitur bibendum justo non orci. + +## Section 4 +4. Lorem ipsum dolor sit amet, consectetur adipiscing elit. Integer in sapien. Praesent id justo in neque elementum ultrices. Cras elementum. + +### Citation +``` +mar gul (2022). test citation. test citation journal. +http://dx.doi.org/10.17504/protocols.io.ewovn1n7qkqr2/v1 +``` + +⏩ +``` +Lorem ipsum dolor sit amet, consectetur adipiscing elit. Integer in sapien. Praesent id justo in neque elementum ultrices. Cras elementum. +``` + +![check](check.jpg) + +## Section 5 +5. Lorem ipsum dolor sit amet, consectetur adipiscing elit. Integer in sapien. Praesent id justo in neque elementum ultrices. + +### Required Row +``` +| **required row** | +| ---------------- | +| Test test test | +| Test test | +| Test test test | +``` + +### Unrequired Row + +``` +| **unrequired row** | +| ---------------- | +| Test test test | +| Test test | +| Test test test | +``` + +## Section 5.1 +- ⏰ 10 rpm, 30°C, 00:01:23 + +``` +Aliquam ornare wisi eu metus. Curabitur 👁️ bibendum justo non orci. Pellenst. +``` + +- ⏰ 12 rpm, 24°C, 00:14:35 shaker + +## Section 5.2 +![🔖](🔖.jpg) + +Lorem ipsum dolor sit amet, consectetur adipiscing elit. Integer in sapien. \ +Praesent id justo in neque elementum ultrices. Cras elementum. Sed ac dolor sit amet purus malesuada congue. Nunc auctor. + +- Sample: [Sample1.pdf ⧫](Sample1.pdf) + +## Section 6 + +``` +Lorem ipsum dolor sit amet, consectetur adipiscing elit. Integer in sapien. Praesent id justo in neque elementum ultrices. Cras elementum. Sed ac dolor sit amet purus malesuada congue. Nunc auctor. Quis autem vel eum iure reprehenderit qui in ea voluptate velit esse quam nihil molestiae consequatur, vel illum qui dolorem eum fugiat quo voluptas nulla pariatur. In convallis. +``` + +## Formula +``` +\[ ωUp = \frac{\ VE \ }{ \ R tg(\ \varphi \ )} + U sin(\ \varphi ) \] +``` + +## Miscellaneous + +``` +Test Test Test +Test Test 01, Test 02, Test 03 +``` + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/biochemical-analysis-of-quail-blood-s7yehpw.md b/markdown-output/biochemical-analysis-of-quail-blood-s7yehpw.md new file mode 100644 index 0000000000000000000000000000000000000000..2b1bbae60b75b98a6c859e755821252f5917a5d4 --- /dev/null +++ b/markdown-output/biochemical-analysis-of-quail-blood-s7yehpw.md @@ -0,0 +1,158 @@ +```markdown +# Goal/Experiment: +Biochemical analysis of quail blood. + +## Biochemical analysis of quail blood + +**Authors:** +Gamal Mehaisen, Ahmed O. Abass + +**Citation:** +Gamal Mehaisen, Ahmed O. Abass Biochemical analysis of quail blood. protocols.io dx.doi.org/10.17504/protocols.io.s7yehpw + +**Published:** +03 Sep 2018 + +--- + +## Protocol + +### Sample preparation: + +#### Step 1. + +1. Blood samples were collected into heparinized tubes. +2. Samples were centrifuged at 2000 xg for 10 min at 4º C. +3. The plasma was separated and stored at -20 ºC until analyzed. + +### Lipid peroxidation (Colorimetric MDA Assay Kit, ab118970, Abcam, UK): + +#### Step 2. + +1. Add 600 µL of Thiobarbituric Acid (TBA) solution to 200 µL standard and 200 µL test samples. +2. Incubate TBA-standard/TBA-sample mixture at 95 °C for 60 minutes. +3. Cool to room temperature in an ice bath for 10 minutes. +4. Pipette 200 µL from each 800 µL TBA-standard and TBA-sample reaction mixture into a 96 well microplate. +5. Measure plate immediately at OD532 nm for colorimetric assay. + +### Alanine aminotransferase (Colorimetric ALT Assay Kit, Ref-264, Spectrum Diagnostics, Egypt): + +#### Step 3. + +1. Add 0.5 mL of R1 (100 mmol Phosphate buffer, 200 mmol DL-Alanine, 6 mmol 2-Oxoglutarate, and 12 mmol Sodium Azide) to 100 µL of distilled water or test samples. +2. Mix and incubate for exactly 30 minutes at 37 °C. +3. Add 0.5 mL of R2 (2,4-dinitrophenyl hydrazine) to all tubes. +4. Mix and incubate for exactly 20 minutes at 20-25 °C. +5. Mix with 0.5 mL of sodium hydroxide (0.4 mol/L). +6. Measure absorbance of samples against reagent blank at 546 nm after 5 minutes. +7. The sensitivity of this assay is 4 U/L and the analytical range is 4-94 U/L. + +### Aspartate aminotransferase (Colorimetric AST Assay Kit, Ref-260, Spectrum Diagnostics, Egypt): + +#### Step 4. + +1. Add 0.5 mL of R1 (100 mmol Phosphate buffer, 100 mmol L-aspartate, 5 mmol 2-Oxoglutarate, 140 mmol sodium hydroxide, and 12 mmol Sodium Azide) to 100 µL of distilled water or test samples. +2. Mix and incubate for exactly 30 minutes at 37 °C. +3. Add 0.5 mL of R2 (2 mmol 2,4-dinitrophenyl-hydrazine and 8.4 % HCl) to all tubes. +4. Mix and incubate for exactly 20 minutes at 20-25 °C. +5. Mix with 0.5 mL of sodium hydroxide (0.4 mol/L). +6. Measure absorbance of samples against reagent blank at 546 nm after 5 minutes. +7. The sensitivity of this assay is 7 U/L and the analytical range is 7-89 U/L. + +### Triglycerides (GPO-PAP-enzymatic colorimetric Assay Kit, Ref-314, Spectrum Diagnostics, Egypt): + +#### Step 5. + +1. Add 1.0 mL of prepared Reagent to 10 µL of standard triglyceride (200 mg/dl) or test samples. +2. Mix and incubate for 5 minutes at 37 °C. +3. Measure absorbance of samples (Asp) and standard (Asd) against reagent blank at 546 nm within 30 minutes. +4. Triglycerides conc. (mg/dL) is calculated as (Asp/Asd) x 200. + +### Cholesterol (CHOD-PAP-enzymatic colorimetric Assay Kit, Ref-230, Spectrum Diagnostics, Egypt): + +#### Step 6. + +1. Add 1.0 mL of prepared Reagent to 10 µL of standard cholesterol (200 mg/dl) or test samples. +2. Mix and incubate for 5 minutes at 37 °C. +3. Measure absorbance of samples (Asp) and standard (Asd) against reagent blank at 546 nm within 30 minutes. +4. Cholesterol conc. (mg/dL) is calculated as (Asp/Asd) x 200. + +### Calcium (O-CPC colorimetric Assay Kit, Ref-226, Spectrum Diagnostics, Egypt): + +#### Step 7. + +1. Mix 0.5 mL of R1 (0.3 mol 2-Amino-2-methyl-1-propanol, pH 10.5) and 0.5 mL of R2 (0.16 mmol O-cresolphthalein complexone, 7 mmol 8-hydroxyquinoline). +2. Add the mixture to 10 µL of standard calcium (10 mg/dl) or to 10 µL of test samples. +3. Incubate for 5 minutes at 20-25 °C. +4. Measure absorbance of samples (Asp) and standard (Asd) against reagent blank at 578 nm. +5. Calcium conc. (mg/dL) is calculated as (Asp/Asd) x 10. + +### Phosphorus (UV colorimetric Assay Kit, Ref-294, Spectrum Diagnostics, Egypt): + +#### Step 8. + +1. Add 1.0 mL of Reagent (3.5 mmol ammonium molybdate, 750 mmol sulphuric acid, and 1% Surfactants) to 10 µL of either blank reagent (distilled water), standard reagent (5 mg/dl phosphorus) or test samples. +2. Mix and wait for 5 minutes at 37 °C. +3. Measure absorbance of samples (Asp) and standard (Asd) against reagent blank at 340 nm within 30 minutes. +4. Phosphorus conc. (mg/dL) is calculated as (Asp/Asd) x 5. + +### Total protein (Biuret colorimetric Assay Kit, Ref-310, Spectrum Diagnostics, Egypt): + +#### Step 9. + +1. Add 1.0 mL of Reagent (750 mmol sodium hydroxide, 12 mmol copper sulphate, 40.9 mmol sodium potassium tartrate, and 19.8 mmol potassium iodide) to 20 µL of either standard total protein (6 mg/dL) or test samples. +2. Mix and incubate for 10 minutes at room temperature. +3. Measure absorbance of samples (Asp) and standard (Asd) against reagent blank at 546 nm within 30 minutes. +4. Protein conc. (mg/dL) is calculated as (Asp/Asd) x 6. + +### Albumin (BCG colorimetric Assay Kit, Ref-211, Spectrum Diagnostics, Egypt): + +#### Step 10. + +1. Add 1.0 mL of Reagent (100 mmol acetate buffer, 0.27 mmol Bromocresol green, and detergent) to 10 µL of either standard albumin (4 g/dL) or test samples. +2. Mix and incubate for approximately 5 minutes at 20-25 °C. +3. Measure absorbance of samples (Asp) and standard (Asd) against reagent blank at 623 nm within 60 minutes. +4. Protein conc. (mg/dL) is calculated as (Asp/Asd) x 4. + +### Corticosterone (Chicken CORT ELISA Kit, MBS701668, MyBioSource Inc., USA): + +#### Step 11. + +1. Add 50 µL of standard and sample per well. +2. Add 50 µL Antibody to each well immediately. +3. Mix well with the pipette for 30 seconds and cover with the adhesive strip provided. +4. Incubate for 30 minutes at 25 °C. +5. Aspirate each well and wash with Wash Buffer (250µl) using a multi-channel pipette. +6. Repeat the process three times for a total of four washes. +7. After the last wash, remove any remaining Wash Buffer and blot the plate inversely against clean paper towels. +8. Add 100 µL HRP-conjugate to each well immediately and cover with the adhesive strip provided. +9. Incubate for 30 minutes at 25°C. +10. Repeat the aspiration/wash process for four times as in step 5. +11. Add 100 µL of TMB Substrate to each well. +12. Incubate for 15 minutes at 25°C, protecting from light. +13. Add 50 µL of Stop Solution to each well and gently tap the plate to ensure thorough mixing. +14. Determine the optical density of each well within 5 minutes, using a microplate reader set to 450 nm, 540 nm or 570 nm. +15. Subtract readings at 540 nm or 570 nm from the readings at 450 nm. + +### Tumor necrosis factor alpha (Chicken TNF-α ELISA Kit, MBS701522, MyBioSource Inc., USA): + +#### Step 12. + +1. Set a Blank well without any solution. +2. Add 50 µL of standard and sample per well. +3. Add 50 µL HRP-conjugate (1x) to each standard/sample wells immediately. +4. Mix well with the pipette for 60 seconds and cover with the adhesive strip provided. +5. Incubate for 40 minutes at 37 °C. +6. Aspirate each well and wash with Wash Buffer (250µl) using a multi-channel pipette. +7. Repeat the process three times for a total of four washes. +8. After the last wash, remove any remaining Wash Buffer and blot the plate inversely against clean paper towels. +9. Add 90 µL of TMB Substrate to each well. +10. Incubate for 20 minutes at 37 °C, protecting from light. +11. Add 50 µL of Stop Solution to each well and gently tap the plate to ensure thorough mixing. +12. Determine the optical density of each well within 5 minutes, using a microplate reader set to 450 nm, 540 nm or 570 nm. +13. Subtract readings at 540 nm or 570 nm from the readings at 450 nm. + +--- + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/bisulfite-pyrosequencing-protocol-for-human-sperm-n52dg8e.md b/markdown-output/bisulfite-pyrosequencing-protocol-for-human-sperm-n52dg8e.md new file mode 100644 index 0000000000000000000000000000000000000000..25ee275c2d717ea0b7f24a35e0ea59b171cec744 --- /dev/null +++ b/markdown-output/bisulfite-pyrosequencing-protocol-for-human-sperm-n52dg8e.md @@ -0,0 +1,180 @@ +```markdown +# Goal/Experiment: +The goal of this experiment is to isolate human sperm DNA, treating it with bisulfite, and performing pyrosequencing to analyze the methylation status of specific DNA regions. + +# Bisulfite Pyrosequencing Protocol for Human Sperm DNA +**Version 2** + +**Zhaoxu Lu, Mei Qiang** + +**Published: 30 Mar 2018** + +## Abstract +Citation: Zhaoxu Lu, Mei Qiang Bisulfite pyrosequencing protocol for Human sperm DNA. protocols.io dx.doi.org/10.17504/protocols.io.ns2dg8e + +## Protocol + +### Human Sperm DNA Isolation Procedure + +#### Step 1 +**Extraction buffer of sperm DNA** + +1. 21.2 ml 6mol/L guanidine thiocyanate +2. 600 µl 5mol/L NaCl +3. 1 ml 30% N-lauroylsarcosine sodium salt +4. 3 ml 1mol/l dithiothreitol (DTT) +5. 600 µl 10mg/ml proteinase K +6. 3.6 ml Doubly deionized water + +#### Step 2 +Add 150 µl of semen to a 1.5 ml micro-centrifuge tube. + +#### Step 3 +Wash with 1 ml of PBS (0.1 mol/L). + +#### Step 4 +Centrifuge at 1500 ×g for 10 min at 4°C. + +#### Step 5 +Repeat washing 2 times as described in step 3-4. + +#### Step 6 +Add 0.5 ml extraction buffer into sperm pellet. + +#### Step 7 +Place in a 65°C water bath for 12 hours. + +#### Step 8 +Cool at room temperature. + +#### Step 9 +Add 10 µl of RNase A (10 mg/ml), mix by pulse-vortexing for 15 seconds, and incubate for 10 min at room temperature. + +#### Step 10 +Briefly centrifuge the tube. + +#### Step 11 +Add 510 µl of isopropanol and centrifuge at 10000 ×g for 10 min at 4°C. + +#### Step 12 +Add 800 µl of ethanol (75%), and reverse mix for multiple times. + +#### Step 13 +Incubate for 12 hours at -20°C. + +#### Step 14 +Centrifuge at 10000 ×g for 10 min at 4°C. Then dry sample at room temperature. + +#### Step 15 +Sperm DNA is dissolved in 50 µl of Elution Buffer. + +#### Step 16 +Incubate in a 65°C water bath for 2 hours. + +### Procedure for Bisulfite Treatment + +#### Step 17 +Add 130 µl of the CT Conversion Reagent solution to 1000 ng of your DNA sample in a PCR tube. + +#### Step 18 +Place the sample tube in a thermal cycler and perform the following steps: +- 98°C for 10 minutes +- 64°C for 2.5 hours +- 4°C + +#### Step 19 +Add 600 µl of M-Binding Buffer into a Zymo-Spin IC™ Column and place the column into a provided Collection Tube. + +#### Step 20 +Load sample (from Step 2) into the Zymo-Spin IC™ Column containing the M-Binding Buffer. Close the cap and mix by inverting the column several times. + +#### Step 21 +Centrifuge at full speed (>10,000 ×g) for 30 seconds. Discard the flow-through. + +#### Step 22 +Add 100 µl of M-Wash Buffer to the column. Spin at full speed for 30 seconds. + +#### Step 23 +Add 200 µl of M-Desulphonation Buffer to the column and let stand at room temperature (20°C-30°C) for 15-20 minutes. After the incubation, spin at full speed for 30 seconds. + +#### Step 24 +Add 200 µl of M-Wash Buffer to the column. Spin at full speed for 30 seconds. Add another 200 µl of M-Wash Buffer and spin at top speed for 30 seconds. + +#### Step 25 +Add 8 µl of M-Elution Buffer directly to the column matrix. Place the column into a 1.5 ml tube. Spin briefly at full speed to elute the DNA. Add 7 µl of M-Elution Buffer and additional repeated 1-time eluting was subsequently performed. + +#### Step 26 +The DNA is ready for immediate analysis or can be stored at or below -20°C for later use. + +### PCR Amplification of Bisulfite-Treated Sperm DNAs + +#### Step 27 +All reactions are performed with provided PCR mixtures (total volume at 25 µl) provided in Table 1. Each reaction also contains 2.5 µl of CoralLoad Concentrate (10x) for checking amplicons on an agarose gel. + +| Components | Volume (µl) | Final concentration | +|------------|-------------|---------------------| +| PyroMark PCR Master Mix, 2x | 12.5 | 1x | +| CoraLoad Concentrate, 10x | 2.5 | 1x | +| Q-solution, 5x | 5 | 1x | +| Primer forward (10 uM) | 0.5 | 0.2uM | +| Primer reverse (10 uM) | 0.5 | 0.2uM | +| Template DNA | 50ng | | +| Final volume | 25 | | + +#### Step 28 +PCR and pyrosequencing primers are designed and listed in Table 2. Reverse primer is conjugated to biotin. + +| Primer | Sequence | +|--------|----------| +| DMR Forward primer | * | +| Reverse primer | * | +| Sequencing primer H19 | GTATATGGGTATTTTTTGAGGTT | +| H19 | ATAATCCGTATTCCAAAATA | +| Sequence primer | * | +| DMR-PCR product | * | +| Sequence primer | * | +| H19 | * | +| Sequencing primer H19 | * | +| H19 | * | + +#### Step 29 +The PCR conditions are as following: +- 94 °C for 15 minutes +- 45 cycles of: + - 94 °C, 30 secs + - 56 °C, 30 secs + - 72 °C, 30 secs +- Final extension step: + - 72 °C for 10 minutes + +### Pyrosequencing + +#### Step 30 +Add 40 µl of Binding Buffer, 3 µl of streptavidin-sepharose beads, and 17 µl DDW into 20 µl of PCR products. + +#### Step 31 +Seal film and shake at 1400 rpm for 10 min to room temperature. + +#### Step 32 +PCR products on streptavidin-sepharose beads are washed with ethanol (10%) for 5 seconds. + +#### Step 33 +Place sample (from Step 31) into denaturation solution for 5 seconds. + +#### Step 34 +Place sample (from Step 31) into Wash Buffer for 10 seconds for getting purified biotinylated single stranded PCR products. These single stranded PCR products are isolated using the Pyrosequencing Work Station. + +#### Step 35 +Transfer purified biotinylated single stranded PCR products into PSQ 96 Plate Low with 40 µl annealing buffer and 1.6 µl sequencing primer (10µmol/L). + +#### Step 36 +Heat PSQ 96 Plate Low at 80 °C for 2 minutes. + +#### Step 37 +Undergo pyrosequencing on a Pyromark Q96 MD pyrosequencing instrument and sequence using PyroMark Gold Q96 kit. + +#### Step 38 +The degree of methylation at each CpG site is determined using PyroMark CpG Software (Biotage AB, Uppsala, Sweden). Pyrosequencing assays are performed in duplicate in sequential runs (technical replicates), and the values show represent the mean methylation for each individual CpG site. + +**endofoutput** +``` \ No newline at end of file diff --git a/markdown-output/boston-biopharma-carestart-rapid-diagnostic-antige-bkzxkx7n.md b/markdown-output/boston-biopharma-carestart-rapid-diagnostic-antige-bkzxkx7n.md new file mode 100644 index 0000000000000000000000000000000000000000..e4d2893bb33bd3ef01dffd5f09e3de772e7a0240 --- /dev/null +++ b/markdown-output/boston-biopharma-carestart-rapid-diagnostic-antige-bkzxkx7n.md @@ -0,0 +1,155 @@ +```markdown +# Goal/Experiment: +To evaluate the efficacy and procedure of the Boston Biopharma CareStart™ COVID-19 Rapid Diagnostic Antigen Test for the detection of SARS-CoV-2. + +# Boston Biopharma CareStart™ Rapid Diagnostic Antigen Test + +**Authors:** +- tclark¹ +- Ahmad Hashem¹ +- Jun Yong Ha² +- Charlie Mize³ + +¹Boston Biopharma +²Access Bio +³Boston Biopharma + +**Date:** +- Created: Sep 07, 2020 +- Last Modified: Sep 08, 2020 + +**Keywords:** +- Covid-19 antigen test + +**License:** +This is an open access protocol distributed under the terms of the [Creative Commons Attribution License](https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. + +**DOI:** +[dx.doi.org/10.17504/protocols.io.bkzxkx7n](https://dx.doi.org/10.17504/protocols.io.bkzxkx7n) + +**Protocol Integer ID:** +- 41751 + +### Guidelines + +- For prescription and *in vitro* diagnostic use only. +- This test has not been FDA cleared or approved. +- As with all diagnostic tests, all results must be interpreted with other clinical information available to the physician. +- Immediately use after opening the test device in the pouch. +- Follow the package insert to obtain accurate results. +- Avoid excess blood or mucus on the swab specimen to prevent interference with test performance. +- Avoid touching bleeding areas of the nasopharynx when collecting specimens. +- Do not interpret test results before 10 minutes or after 15 minutes of starting the test. +- Do not use the test device package if it is damaged. +- Do not use kit contents beyond the expiration date. + +## Materials + +### CareStart™ Antigen Kit Contents + +| Contents Name | Quantity (in a kit) | Description | +|------------------------------|---------------------|-----------------------------------------------------------------------------| +| Test device | 20 each | Foil-pouched test device containing one test strip enclosed in a plastic cassette. | +| Extraction vial / cap | 20 vials and caps | The extraction vial contains 400 ml of extraction buffer solution. | +| Nasopharyngeal swab | 20 each | Swabs for nasopharyngeal specimen collection. | +| Positive control swab | 1 each | Recombinant SARS-CoV-2 nucleocapsid protein antigen dried on a foam-tipped head. | +| Negative control swab | 1 each | Blank Universal Viral Transport media (BD UVT) dried on a foam-tipped head. | +| Package insert | 1 each | Instructions for use. | +| Quick Reference Instructions (QRI) | 1 each | Quick reference instructions. | + +### Safety Warnings + +- Do not eat, drink, or smoke in areas where specimens and kit contents are handled. +- Use appropriate precautions in the collection, handling, storage, and disposal of patient samples and used kit contents. +- Dispose of used contents as biohazardous waste following federal, state, and local requirements. +- Wear nitrile or latex gloves when performing this test. +- If extraction buffer contacts the skin or eye, flush with water. +- Handle all specimens as if infectious. +- Follow established precautions against microbiological hazards. +- Sodium azide (in reagents) is harmful and may react with metals to form explosive azides. Ensure proper disposal. +- Do not interchange kit contents from different lots. +- Do not re-use any kit contents as they are for single-use only. + +## Before Starting + +- Store the test kit between 1 – 30°C. +- Do not use beyond the expiration date. +- Ensure the test device remains in the sealed pouch until use. +- Do not freeze any contents. +- Allow contents to equilibrate to room temperature (15 – 30°C) before testing. + +## Procedure + +### Temperature Equilibrium + +1. **Room Temperature** + - Allow test devices, reagents, specimens, and/or controls to equilibrate to room temperature (15 – 30°C). + +### Nasopharyngeal Swab Specimen Collection + +2. **Swab Removal** + - Remove a nasopharyngeal swab from the pouch. + +3. **Specimen Collection** + - Place the swab into one of the patient’s nostrils and advance to the posterior nasopharynx. + - Rotate the swab 3-5 times over the surface of the posterior nasopharynx. + - Remove the swab from the nostril. + +### Test Procedure + +6. **Device and Extraction Vial Preparation** + - Remove the test device and extraction vial from the pouch. + +7. **Foil Seal Removal** + - Peel off the aluminum foil seal from the extraction vial. + +8. **Insert Swab** + - Insert the swab into the extraction vial and rotate vigorously at least 5 times. + +9. **Swab Removal** + - Remove the swab while squeezing the sides of the vial to release liquid from the swab. + +10. **Close Vial** + - Close the vial with the provided cap. + +11. **Mix Sample** + - Mix the sample thoroughly by flicking the bottom of the tube. + +12. **Sample Application** + - Invert the extraction vial and hold above the sample well. + - Squeeze the vial gently to allow three drops of sample to fall into the sample well. + +13. **Reading the Result** + - Read and interpret the test results at 10 minutes (do not read after 15 minutes). + +### Interpretation of Results + +14. **Results at 10 Minutes** + - Test results should be read and interpreted no later than 15 minutes after application. + +15. **Positive Result** + - One red-colored line next to “C” and one blue-colored line next to “T” indicates a positive result. + +16. **Negative Result** + - One red-colored line only next to “C” indicates a negative result. + +17. **Invalid Result** + - If the red-colored line next to “C” is not visible, the result is invalid and the test must be re-run. + +## Limitations + +- Proper specimen collection, handling, storage, and preparation are crucial. +- The test indicates the presence of SARS-CoV-2 nucleocapsid protein from viable and non-viable virus. +- This is a qualitative test and does not provide viral concentration information. +- The test cannot rule out infections caused by other pathogens. + +### Internal Quality Control + +22. The CareStart™ COVID-19 Antigen test includes an internal procedural control that should show a red-colored line in the control region "C" to validate the test. If this line doesn't appear, the test is invalid and must be retested with a new device. + +### External Quality Control + +23. It is recommended to use external control swabs to validate each new lot shipment and user. If external control results are invalid, contact the manufacturer or distributor. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/bulk-rnaseq-delivery-c3kzykx6.md b/markdown-output/bulk-rnaseq-delivery-c3kzykx6.md new file mode 100644 index 0000000000000000000000000000000000000000..ff449f5122401b8a0c92ad9c92c4b9bd5f5847ec --- /dev/null +++ b/markdown-output/bulk-rnaseq-delivery-c3kzykx6.md @@ -0,0 +1,162 @@ +```markdown +# Goal/Experiment: +The goal of this experiment is to provide an overview of the Genomics Research Center (GRC) data delivery structure and results files for bulk RNA sequencing (RNASeq) analysis. + +# Bulk RNASeq Delivery V.4 + +**Tyler Stahl** +*Genomics Research Center* +*Version 4 - October 17, 2023* + +## Abstract +This protocol will give an overview of the GRC data delivery structure and results files. + +## Protocol Citation +Tyler Stahl 2023. Bulk RNASeq Delivery. protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vzzx9rgx1/v4 + +## License +This is an open-access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. + +## Protocol Status +Working - We use this protocol and it's working. + +## Created +October 17, 2023 + +## Analysis Overview +The RNASeq analysis follows these stages: +1. Pre-processing (quality control/filtering/trimming) +2. Alignment +3. Post-processing +4. Feature quantification +5. Differential expression analysis between sample groups using DESeq2. + +A detailed overview of the analysis can be found in the `README.txt` and `RNASeq_methods.txt` files. Below is the workflow diagram for data processing. + +![RNASeq Workflow](images/RNASeq_Workflow.png) + +## Delivery Structure +The delivery email includes three download links corresponding to: +1. Raw (fastq) files +2. Aligned (bam/bigWig) files +3. Results files + +### Download Links +- **RNA-Seq Analysis Results and QC:** [Download Link](https://grcweb.circ.rochester.edu/pickup/230821130441-10817/deliv_NHD13_GEO_results.tar.gz) (Checksum: 1e2e1da41926b5d45ef24c103c7a5f48e) +- **RNA-Seq Analysis Aligned Data:** [Download Link](https://grcweb.circ.rochester.edu/pickup/230821130431-10715/deliv_NHD13_GEO_align.tar.gz) (Checksum: 3c57a3de7218cae0ded7f842b9b2fdd7) +- **RNA-Seq Raw Data (for GEO submission):** [Download Link](https://grcweb.circ.rochester.edu/pickup/230821130423-10606/deliv_NHD13_GEO_raw.tar.gz) (Checksum: 59b130b3986ebc9174a75ef809db1ce2) + +**Note:** These URLs will expire in 10 days. + +To uncompress the delivery directory, use FREE compression software [7zip](http://www.7-zip.org). + +If you are on a PC, download compression software to unzip the folders. Macs have built-in zip software. + +### FASTQ, BAM, and Results Files +- **.fastq files:** Contain nucleotide and quality information generated from the Illumina Sequencer. +- **.bam files:** Store alignment data and mapping quality scores in a binary format. +- **Results folder:** Contains quality control information and results files from DESeq2. + +## MultiQC Report +MultiQC aggregates QC information from multiple different analysis outputs into a single interactive report. + +![MultiQC Report](images/MultiQC_Report.png) + +### General Statistics +The general statistics include: +- **Fastp:** % Duplication, GC content, % PF, % Adapter +- **Star:** % aligned, M aligned +- **Feature counts:** % assigned, M assigned +- **Salmon:** % aligned, M aligned + +Each section is detailed below: + +#### Fastp +- **Filtering statistics:** Metrics including read quality, read length, N-Content. +- **Sequencing Quality:** Phred quality scores assigned to each base. A higher Phred score means higher confidence and lower error rate. +- **N Content:** Percentage of ambiguous or unknown bases. +- **GC content:** Proportion of guanine (G) or cytosine (C) bases in the RNA sequence. + +#### STAR Alignment +- **Uniquely mapped:** Reads aligned to a single loci. +- **Mapped to multiple loci:** Reads aligned to multiple loci. +- **Mapped to too many loci:** Reads aligned to excessive locations. +- **Mapped too short:** Reads aligned to genome but fall short of the filtering metrics. +- **Unmapped: other:** Non-alignable reads. + +#### Feature Counts +- **Assigned:** Reads assigned to a genomic feature (i.e., gene). +- **Unassigned: Multi Mapping:** Reads aligning to multiple genomic features. +- **Unassigned: No Features:** Reads that could not be aligned to any defined genomic features. +- **Unassigned: Ambiguity:** Reads aligning to multiple features, categorized as `Ambiguity`. + +#### Salmon +- **Fragment length distribution:** Refers to the distribution of fragment lengths generated in the sample. + +## DESeq2 Results +Two reports in the `deSeq2` folder: +1. Star-feature (gene-level) +2. Salmon (transcript level) + +### Files in Star and Salmon Folders +- **deSeq2_counts.txt:** Raw count values. +- **deSeq2_NormCounts.txt:** Count values normalized with DESeq2's median of ratio. +- **deSeq2_rlog_NormCounts.txt:** Log of the normalized counts. + +### Comparison Files +Example file: `deSeq2_NHD13_vs_WT.txt` + +| A | B | C | D | E | F | +|--------|---------------|--------|---------|-----------|---------| +| BaseMean | log2FoldChange | stat | pvalue | padj | +| Hoxa9 | 636.168 | 2.557 | 21.331 | 5.92E-101 | 8.68E-97 | +| Pbx3 | 456.879 | 3.091 | 20.932 | 2.76E-97 | 2.03E-93 | +| Pbx1 | 401.557 | -3.27 | -16.477 | 5.38E-61 | 2.63E-57 | + +**Terms Definitions:** +- **BaseMean:** Average expression level across samples. +- **log2FoldChange:** Log2 fold change in gene's expression between conditions/groups. +- **stat:** Test statistic to assess significance of differential expression. +- **p-value:** Calculated using a negative binomial distribution. +- **padj:** Adjusted p-value for multiple testing using Benjamini and Hochberg method. + +### EnrichR Files +EnrichR is used for gene set enrichment querying four common libraries: KEGG, GO, Wiki Pathways, and ChEA. + +Note: EnrichR is not run for salmon outputs. + +Example EnrichR file: + +| Database | Term | Overlap | P-value | Adjusted-P-value | Combined Score | Genes | +|-----------|-----------------------------------|---------|---------|------------------|----------------|--------------------------------------------------| +| GO_Biological_Process_2021 | mRNA processing (GO:0006397) | 69/190 | 5.48E-22 | 2.21E-18 | 485.4247 | PUS1,PUS3,ATRX,DDX6,ZGRF1,ABCF2,ZNF148,RPL5,PUM1 | + +More info on EnrichR can be found [here](https://enrichr.maayanlab.cloud/). + +## FAQ +### What are the salmon results? +Salmon uses a different alignment algorithm, mapping reads at the transcript level rather than whole gene. + +### Why do I not see enrichR results? +There must be at least 50 differentially expressed genes for EnrichR. + +### Why is my RNASeq data showing a weak knockdown of my gene of interest despite being validated with qRT-PCR? +Discrepancies may arise due to alignment of a non-functional transcript. Viewing aligned files in a Genome Browser may help. + +### Can I remove a sample from the analysis? +It is recommended not to remove samples based on clustering alone, unless there is clear experimental reasoning. + +### Can the GRC re-analyze my RNA-Seq experiment? +Use the BulkDeSeq application in HyperGen, a custom no-code genomics analytics software. + +### What counts files do I use and where? +Use the `deSeq2_counts.txt` files within HyperGen BulkDeqSeq application. + +For Gene Set Enrichment Analysis (GSEA), use the `deSeq2_NormCounts.txt` files. Guide to get started: [GSEA Guide](https://www.protocols.io/view/gene-set-enrichment-analysis-kqdg3x67qgq25/v1) + +## Further Educational Resources +- [Genomics Biology Article](https://genomebiology.biomedcentral.com/articles/10.1186/s13059-014-0550-8) +- [DGE Workshop Lesson](https://hbctraining.github.io/DGE_workshop/lessons/04_DGE_DESeq2_analysis.html) + +**endofoutput** +``` \ No newline at end of file diff --git a/markdown-output/burden-of-rsv-in-italian-adults-protocol-for-a-sys-c5cgy2tw.md b/markdown-output/burden-of-rsv-in-italian-adults-protocol-for-a-sys-c5cgy2tw.md new file mode 100644 index 0000000000000000000000000000000000000000..04e92a6423ad43e2b2fe5d4aee8ff0c8599c8cd0 --- /dev/null +++ b/markdown-output/burden-of-rsv-in-italian-adults-protocol-for-a-sys-c5cgy2tw.md @@ -0,0 +1,77 @@ +```markdown +Goal/Experiment: +In this protocol for a systematic review, we aim to collect and analyze data on the clinical burden of RSV in Italian adults with the goal of informing and supporting National and local decision makers on the planification and implementation of future vaccination strategies. + +# Burden of RSV in Italian adults: Protocol for a Systematic Review and Meta-Analysis + +**Authors:** +Giovanna Elisa, Alexander Domnich¹, Calabrò² +¹IRCCS Ospedale Policlinico San Martino; +²Section of Hygiene, Department of Life Sciences and Public Health, Università Cattolica del Sacro Cuore, Rome, Italy + +## Abstract +Globally, respiratory syncytial virus (RSV) is a leading cause of respiratory infections and is responsible for a significant socioeconomic burden in all age groups, including adults. To date, no review systematically appraised the burden of RSV in Italian adults. Understanding country-specific burden of disease is a key driver for policy decisions on the introduction of new vaccines. Two vaccines have been recently authorized to prevent lower respiratory tract disease caused by RSV in adults. In this protocol for a systematic review, we aim to collect and analyze data on the clinical burden of RSV in Italian adults with the goal of informing and supporting National and local decision makers on the planification and implementation of future vaccination strategies. + +## Introduction +Together with seasonal influenza, respiratory syncytial virus (RSV) is a leading cause of respiratory infections and is responsible for a significant socioeconomic burden in all age groups, especially at the extremes of age. + +### Country-Specific Burden of Disease (BoD) +Recent studies have estimated that a significant number of RSV-associated hospitalizations among European adults occur annually, primarily affecting older adults aged ≥ 65 years. In the last 20 years, mortality attributable to RSV has increased among working-age and older adults. + +### Existing Research +Multiple systematic reviews have confirmed significant RSV attack rates, hospitalization, mortality, and case-fatality rates globally, although there is a substantial under-ascertainment. Available reviews have identified only up to six primary studies conducted in Italy. + +### Vaccines +Two vaccines have been recently authorized to prevent lower respiratory tract disease (LRTD) caused by RSV in adults aged ≥ 60 years. In the UK, vaccination is recommended for older adults aged ≥ 75 years. + +### Importance of Country-Specific BoD Indicators +Understanding country-specific BoD indicators is crucial for policy making and efficient implementation of new vaccines. + +## Methods +### Reporting Standards +- The PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) statement will be adopted. +- Methodological guidance from the Joanna Briggs Institute (JBI) will be consulted. + +### Eligibility Criteria +- **Types of Studies:** Observational studies (e.g., surveillance, cross-sectional, cohort, case-series) in any modality. +- **Condition of Interest:** RSV infection detected by laboratory techniques such as RT-PCR, immunofluorescence assay (IFA), cell culture, and RADT. +- **Context:** Studies conducted in Italy, in any setting and calendar period. +- **Population:** Adults defined as individuals aged ≥ 14/18 years. + +### Exclusion Criteria +- Modeling studies, pharmacoeconomic studies, studies with no original data, general population studies with no separate data for adults, and multi-country studies with no separate information for Italy. + +### Study Endpoints +- **RSV Attack Rate:** Cumulative incidence of laboratory-confirmed RSV detections. +- **RSV Prevalence:** Proportion of RSV detections to the total number of subjects tested. +- **Case Complication Rate:** Proportion of RSV-positive individuals with complications. +- **Drug Use Indicators:** Frequency of drug prescriptions among RSV-positive subjects. +- **Hospitalization Metrics:** Crude hospitalization rates, ICU admissions, in-hospital mortality, and case-fatality rates. + +### Search Strategy +The automatic search will be performed in databases such as PubMed/Medline, Biological Abstracts, and Scopus, and will follow a specific search script. A manual search and cross-referencing will also be conducted. + +### Study Selection +Records from the automatic search will be merged into a spreadsheet, and duplicates will be removed. Titles and abstracts will be screened, and full texts of potentially relevant studies will be assessed for eligibility. + +### Data Extraction and Coding +Data extracted will include: +- Full citation record +- Location and period of study +- Study design and setting +- Population and sample size +- RSV case ascertainment methods +- Endpoints of interest +- Handling of missing data + +### Critical Appraisal +The JBI checklist for prevalence/incidence studies will be used to assess the quality of included studies. Appraisals will be performed independently by two reviewers. + +### Data Synthesis +Data will be qualitatively appraised and visualized using forest plots. Quantitative synthesis will involve proportional meta-analysis using the R package "Meta" v. 6.5-0. Heterogeneity will be quantified using I² statistics. + +## Attachments +- Prot_SRMA_RSV_Ita_v131_12013.pdf + +endofoutput +``` diff --git a/markdown-output/c-sop-501-normalisation-and-pooling-of-dna-librari-c4kfyutn.md b/markdown-output/c-sop-501-normalisation-and-pooling-of-dna-librari-c4kfyutn.md new file mode 100644 index 0000000000000000000000000000000000000000..81348e78ace36fd19a351d58039bf99a571bd002 --- /dev/null +++ b/markdown-output/c-sop-501-normalisation-and-pooling-of-dna-librari-c4kfyutn.md @@ -0,0 +1,132 @@ +```markdown +# Goal/Experiment: +Normalisation and Pooling of DNA Libraries for Illumina Whole Genome Sequencing + +## C-SOP-501: Normalisation and Pooling of DNA Libraries for Illumina Whole Genome Sequencing + +### Authors +Ben, Mihir Kekre¹, Pascoe¹ +¹The Centre for Genomic Pathogen Surveillance, Oxford, United Kingdom + +--- + +### Abstract +Normalisation in next-generation sequencing (NGS) is the process of equalising the concentration of multiple DNA libraries for the purpose of multiplexing. Multiplexing helps maximize the use of expensive NGS technology, enabling parallel sequencing of hundreds to thousands of libraries on a single flowcell, thereby driving down per-sample costs. + +Uneven library concentrations from different types and qualities of samples can lead to inconsistencies in data quality. Overrepresented libraries on the flowcell waste capacity while underrepresented ones may lead to poor read depth and unreliable data. Normalisation ensures each library is equally represented and sequenced to sufficient depth. + +### Quantitation Options + +- **Quick but less accurate methods**: Spectrophotometry-based quantitation. +- **Accurate methods**: Quantitative PCR (qPCR), dependent on fragment size knowledge. + +A crucial factor influencing quantitation and normalisation accuracy is whether the method specifically counts adaptor-ligated double-stranded DNA (dsDNA) molecules. Illumina's best practice suggests using fluorometric or qPCR-based quantitation. + +--- + +### Materials + +1. Quantified and size-estimated double-stranded DNA libraries +2. Nuclease-free water or 10 mM Tris-HCl (pH 8.5) +3. Single-channel pipettes (P10, P200) with compatible tips (filter-free, sterile) +4. 96-well PCR-plate, low-profile, full skirted (ThermoFisher Scientific, Cat no. AB0800) +5. 2.0 mL microcentrifuge tubes +6. **For New England BioLabs NEBNext Library Quant Assay**: + - a. NEBNext® Library Quant Kit for Illumina (NEB, Cat no. E7630) + - b. Nuclease-free water + - c. qPCR machine + - d. Compatible qPCR plates and seals + - e. PCR strip tubes or microcentrifuge tubes + - f. Conical centrifuge tubes + +### Safety Warnings +- NEBNext Library Quant Kit safety sheets: + - [Dilution Buffer (10X)](link) + - [DNA Standard 1](link) + - [DNA Standard 2](link) + - [DNA Standard 3](link) + - [DNA Standard 4](link) + - [ROX (High)](link) + - [ROX (Low)](link) + +### Before Starting + +1. Ensure that all active workbenches are cleaned with 80% ethanol, all relevant personal protective clothing is worn, and the work area is prepared according to local GLP guidelines for molecular methods. + +2. Ensure you have the following library QC metrics using 'C-SOP-401: Quality Control (QC) of DNA Libraries for Whole Genome Sequencing': + - a. Mean total library fragment size (in bp) - output from Bioanalyzer 2100 or Tapestation 4150/4200. + - b. Mean library concentration (in nM) - output from a qPCR quantification assay. + +> **Note:** If mean library concentration is in ng/µl, convert using [this link](link). + +--- + +### Library Normalisation + +1. **Concentration normalisation**: Dilute an aliquot of the stock library in a diluent (nuclease-free water or 10 mM Tris-HCl pH 8.5). +2. For most Illumina sequencing platforms, 2–4 nM for each library is the preferred final concentration. + + **Dilution Calculation** + \[ + (C_1 \times V_1) = (C_2 \times V_2) + \] + - \(C_1 = \) concentration of stock library (nM) + - \(V_1 = \) volume of stock library to be diluted (µl) + - \(C_2 = \) desired final concentration (nM) + - \(V_2 = \) desired total volume + + Alternatively, use the [normalisation and pooling calculator](link) to compute library dilutions. + +> **Note:** Ensure pipetted volumes are not less than 4 µl (or at least 2 µl). + +**Example Dilutions** + +| Starting Concentration | Volume of Stock DNA (for 4 nM final concentration) | Volume of Diluent (for 15 µl final volume) | +|------------------------|----------------------------------------------------|-------------------------------------------| +| 15 nM | 4 µl | 11 µl | +| 20 nM | 3 µl | 12 µl | +| 50 nM* | 1.2 µl | 13.8 µl | + +*Intermediate dilution required: + +| Starting Concentration | Volume of Stock DNA (for intermediate 20 nM concentration) | Volume of Diluent (for 15 µl volume) | Volume of Intermediate 20 nM DNA (for 4 nM final concentration) | Volume of Diluent (for 15 µl final volume) | +|------------------------|-------------------------------------------------------------|-------------------------------------|-----------------------------------------------------------------|-------------------------------------------| +| 50 nM | 6 µl | 9 µl | 3 µl | 12 µl | + +3. Mix the calculated volumes of library and diluent to obtain a normalised library solution. + +--- + +### Library Pool Creation (for Multiplexed Libraries) + +1. **Volumetric pooling**: Combine equal volumes of each normalised library in a 2.0 mL microcentrifuge tube. +2. Gently pipette the contents up and down 15 times or vortex to mix thoroughly. + +> **Note:** Steps 4-8 can be combined using the [norm/pool calculator](link). + +### Library Pool Confirmatory QC + +1. Quantify the library pool using a robust qPCR assay as described in 'C-SOP-401: Quality Control (QC) of DNA Libraries for Whole Genome Sequencing' (NEBNext® Library Quant Kit for Illumina®) and compute the pool concentration in nM. + +> **Note:** +> - Ensure final pool concentration matches the expected 2-4 nM range. +> - Step 9 can be skipped under extreme budget constraints, but downstream results like clustering densities may vary. + +2. The normalised pool is ready to be denatured and sequenced. + +--- + +### Additional Information & Troubleshooting +Refer to the following resources for detailed denaturation and dilution procedures: +- [iSeq™ 100 System Guide](link) +- [MiniSeq™ System Denature and Dilute Libraries Guide](link) +- [MiSeq™ System Denature and Dilute Libraries Guide](link) +- [NextSeq™ 500/550 System Denature and Dilute Libraries Guide](link) +- [NextSeq 1000/2000 Sequencing System Guide](link) +- [HiSeq™ Systems Denature and Dilute Libraries Guide](link) + - [cBot™ System Guide](link) for HiSeq 3000/4000 +- [NovaSeq™ System Guide](link) + +This protocol is distributed under the terms of the [Creative Commons Attribution-NonCommercial-ShareAlike](link) license. +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/canary-segmentation-of-lung-adenocarcinoma-mrcc52w.md b/markdown-output/canary-segmentation-of-lung-adenocarcinoma-mrcc52w.md new file mode 100644 index 0000000000000000000000000000000000000000..d3dc8eea1b79d572d801be0faf621b05d34f4c35 --- /dev/null +++ b/markdown-output/canary-segmentation-of-lung-adenocarcinoma-mrcc52w.md @@ -0,0 +1,102 @@ +```markdown +# Goal/Experiment: +The goal of this experiment is to segment and classify lung adenocarcinomas using the Computer-Aided Nodule Assessment and Risk Yield (CANARY) system through computed tomography (CT) imaging. This involves identifying specific voxel classes that represent a spectrum of growth patterns from lepidic to invasive. + +# CANARY Segmentation of Lung Adenocarcinoma + +**Erica C. Nakajima, Ronald Karwoski, Fabien Maldonado, Srinivasan Rajagopalan, Tucker F. Johnson, Michael P. Frankland** + +## Abstract +Computer-Aided Nodule Assessment and Risk Yield (CANARY) is a novel computed tomography (CT) tool developed at Mayo Clinic (Rochester, MN) that characterizes early lung adenocarcinoma by detecting nine distinct voxel classes, representing a spectrum of lepidic to invasive growth, within an adenocarcinoma. CANARY characterization has been shown to correlate with ADC histology and patient outcomes. + +This protocol provides basic instructions for segmentation of lung adenocarcinoma on CT imaging. CANARY has been validated in lung adenocarcinomas less than 3cm in diameter. + +**Citation:** +Erica C. Nakajima, Ronald Karwoski, Fabien Maldonado, Srinivasan Rajagopalan, Tucker F. Johnson, Michael P. Frankland CANARY Segmentation of Lung Adenocarcinoma. protocols.io dx.doi.org/10.17504/protocols.io.mrcc52w +**Published:** 23 Jan 2018 + +## Protocol + +### Finding the Nodule + +#### Step 1 +Scroll up and down in axial view to identify the pulmonary nodule. Scroll through the entirety of the lungs to ensure you are not missing a nodule. (Keep the CT image in “lung view”, i.e. Do not change the image view to “soft tissue” if trying to differentiate between solid and sub-solid tissue). + +#### Step 2 +Click the axis tool (defined below) and place it over the nodule so that you can see it in all three views (axial, sagittal, and coronal). + +#### Step 3 +Click the nodule, and a box will be placed around it with the rest of the screen in red. + +#### Step 4 +Ensure the entirety of the nodule is enclosed within the box in all three views. Adjust the box dimensions by clicking and dragging the border to the desired location. + +### Establishing the Nodule Perimeter + +#### Step 5 +If the nodule abuts the pleura or mediastinal structures, exclude these tissues as much as possible from within the box. You can expand the nodule perimeter after it has been established by the software, but it can take a great deal of time to erase tissue that you do not want included in the analysis. + +#### Step 6 +The Wall function may also be used to exclude tissue from the chest wall. Draw a line along the chest wall, clicking once to create distinct points along the line. At the last point, double click to finish the line. Scroll through the CT slices that involve the lung nodule. Adjust the line by moving its distinct points to ensure that the chest wall is appropriately excluded. + +#### Step 7 +Click "Get nodule mask". + +#### Step 8 +A red ROI will appear around the perimeter of the nodule. (Pressing the letter “T” will toggle the mask on and off from the screen, but not permanently remove it.) + +#### Step 9 +Scroll through the slices and adjust the mask with the tools below. + +#### Step 10 +Use the “Nudge” function under the REFINE tab to adjust the perimeter of the nodule. +- i. Holding down the middle mouse key will enable you to change the size of the Nudge tool. +- ii. If the cursor is within the mask when you begin to nudge, it will EXPAND the mask. +- iii. If the cursor is outside of the mask when you begin to nudge, it will SHRINK the mask. +- d. The Trace function can be used to include tissue of interest into the mask. Draw a line around the tissue, connecting the start and end points of the line to the original mask. + +#### Step 11 +Once finished with all adjustments of ROI, click "Classify nodule". + +#### Step 12 +Once nodule has been classified for the first time, the "edit nodule" & "delete nodule" buttons can be used to adjust the ROI. + +### Tools + +#### Step 13 +![Tools](tools_image.png) + +#### Step 14 +Camera tool: Will take a screenshot of the CT scan and import the image to the final report generated for each nodule. + +#### Step 15 +Ruler tool: measure structures using cursor. + +#### Step 16 +Note tool: Enables you to label structures with a small yellow “sticky note” using the cursor. Right-click for options on using the note. + +#### Step 17 +Axis tool: Locks axial, sagittal, and coronal views so that you can scroll through the images in a coordinated fashion. + +### Data Extraction + +#### Step 18 +Clicking "Classify" will save the CANARY data every time in the same Excel file. + +#### Step 19 +The Excel data file can be found by opening the C drive: Temp folder: CANARY-Plus folder: “CANARY-PlusTumorStats”. + +#### Step 20 +Data from each CT case is listed in a row. + +#### Step 21 +Seed, X, Y, Z columns indicate the position of the cursor when the nodule mask was set. + +#### Step 22 +Letters V (violet), I (indigo), B (blue), G (green), Y (yellow), O (orange), R (red), C (cyan), and P (pink) represent the 9 colors shown in the “Classify nodule” analysis wheel. The numbers in these columns represent the volume (mm³) of the nodule that is assigned to each color classification. + +#### Step 23 +Ensure that the CANARY-PlusTumorStats file is closed whenever segmentation is being performed. Otherwise, new data will not import into the Excel file. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/cas9-enrichment-for-dimelo-sequencing-c6akzacw.md b/markdown-output/cas9-enrichment-for-dimelo-sequencing-c6akzacw.md new file mode 100644 index 0000000000000000000000000000000000000000..bf79822d32b0adbef9bc0d679bfd74300d7ee3ce --- /dev/null +++ b/markdown-output/cas9-enrichment-for-dimelo-sequencing-c6akzacw.md @@ -0,0 +1,163 @@ +```markdown +# Goal/Experiment: +To enrich genomic loci of interest for DiMeLo-sequencing using Cas9 technology. + +# Cas9 Enrichment for DiMeLo-sequencing + +**DOI:** [dx.doi.org/10.17504/protocols.io.5jyil8p8n7g2w/v1](https://dx.doi.org/10.17504/protocols.io.5jyil8p8n7g2w/v1) +**Authors:** Pragya Sidhwani^1^, Aaron Straight^2^ +^1^Stanford University; ^2^Stanford University, Department of Biochemistry + +**Protocol Citation:** Pragya Sidhwani, Aaron Straight 2024. Cas9 enrichment for DiMeLo-sequencing. *protocols.io* [https://dx.doi.org/10.17504/protocols.io.5jyil8p8n7g2w/v1](https://dx.doi.org/10.17504/protocols.io.5jyil8p8n7g2w/v1) + +**License:** Creative Commons Attribution License + +**Protocol status:** Working + +**Created:** December 12, 2023 + +**Last Modified:** June 19, 2024 + +**Protocol Integer ID:** 92204 + +--- + +## Abstract + +This protocol builds upon two previously published protocols: the DiMeLo-seq protocol to map protein-DNA interactions at a single molecule level using long-read sequencing and a Cas9 enrichment protocol for Nanopore sequencing to enrich for genomic loci of interest for DiMeLo-sequencing. Unique features of our protocol include: + +- Utilizing sgRNAs instead of duplexing crRNA and tracrRNA for Cas9 enrichment. +- Cas9-enrichment of DNA containing methyl-A marks. +- Multiplexing of Cas9-enriched samples. +- Potential to use >10 sgRNAs (While we have not tested this, we think our edits to step 4 enable this). + +--- + +## Introduction + +1. This protocol builds upon two previously published protocols: the DiMeLo-seq protocol to map protein-DNA interactions at a single molecule level using long-read sequencing and the Cas9 enrichment protocol for Nanopore sequencing to enrich for genomic loci of interest. Some unique features include: + - Using sgRNAs instead of duplexing crRNA and tracrRNA for Cas9 enrichment. + - Cas9-enrichment of DNA containing methyl-A marks. + - Multiplexing of Cas9-enriched samples. + - Potential to use >10 sgRNAs (While we have not tested this, we think our edits to step 4 enable this). + +2. Designing sgRNAs for Cas9 enrichment + +### 2.1 CRISPOR +We use **CRISPOR** to design sgRNAs to target the loci of interest. We have tested this protocol by cutting with 2 sgRNAs each on either end (So 4 sgRNAs total) of a 10 kb centromeric region. + +### 2.2 Synthego +Instead of duplexing a crRNA and tracrRNA, we order 1.5 nmol sgRNAs from **Synthego**, which are resuspended in 15 uL 1x TE for 100 uM. + +--- + +## Isolating pA-Hia5-treated DNA from human K562 cells + +### 3. Extract DNA that has been methylated in situ by pA-Hia5 at sites of protein-DNA interactions +**Duration:** 1h + +#### 3.1 NEB Monarch HMW DNA Extraction Kit +- Use the **NEB Monarch HMW DNA Extraction Kit**. Follow protocol for genomic DNA isolation using cell lysis buffer. Include RNase A. +- Perform lysis with 2000 rpm agitation. We have validated 2000 rpm gives N50 ~50-70 kb but if longer reads are desired, we expect 300 rpm would work. +- Apart from using a different kit, all of the steps for the long fragment DNA extraction are the same as the general protocol. To reiterate, make the following changes to the protocol: + + - **56°C incubation for lysis for 1 hour** (Not just 10 minutes). + - Agitate for 10 minutes and then keep at 56°C without agitation for 50 minutes. + +#### 3.2 Fixation +If fixation was performed, be sure to do the 56°C incubation for lysis for 1 hour (Just not 10 minutes) to reverse crosslinks. Agitate for 10 minutes and then keep at 56°C without agitation for 50 minutes. + +#### 3.3 Quantify DNA +Quantify DNA yield by **Qubit dsDNA BR Assay Kit**. We need 3-10 ug of high quality DNA in less than 24 uL of 1x TE. + +--- + +## Preparing RNPs for Cas9 enrichment + +**Duration:** 45m + +### 4. Preparing the ribonucleoprotein (RNP) complex for Cas9 enrichment + +#### 4.1 RNP Assembly +For each sgRNA, assemble the RNP complex as follows: + +- 0.4 uL IDT HiFi Cas9 (62 uM) +- 0.5 uL Synthego sgRNA (100 uM) + +Assemble each RNP for 30 minutes at room temperature, keep on ice until ready to use. + +#### 4.2 Pool and Dilute RNPs +Prior to adding to dephosphorylated DNA from step 5, pool and dilute the RNPs as follows: + +- 3.6 uL Pooled RNPs (for 4 sgRNAs) +- 5 uL 10x rCutSmart +- 41.4 uL water + +This results in 50 uL of a 100x solution enough for 4-5 samples (10-12 uL/sample). + +--- + +## Cas9-based enrichment of pA-Hia5 methylated DNA + +**Duration:** 2h 40m + +### 5. Dephosphorylate DNA isolated in step 3 + +#### 5.1 Dephosphorylation +In a PCR tube, mix the following: + +- 3 uL 10x rCutSmart buffer +- 3-10 ug of DNA +- 3 uL of NEB QuickCIP +- Water up to 27 uL + +Mix by resuspending with a cut pipet tip. + +#### 5.2 Incubation +Incubate at: + +- 37°C for 60 minutes (dephosphorylation) +- 80°C for 2 minutes (enzyme inactivation) +- Hold at 20°C + +### 6. RNP-based cleavage and A-tailing + +#### 6.1 Cleavage and A-tailing +To the dephosphorylated DNA, add the following: + +- 12 uL of pooled 100x RNP complex +- 1 uL of 10 mM dATP +- 1 uL Taq polymerase + +Mix by resuspending with a cut pipet tip. + +#### 6.2 Incubation +Incubate at: + +- 37°C for 60 minutes (Cas9 cleavage) +- 72°C for 30 minutes (monoadenylation of cut ends) +- Hold at 12°C + +### 7. Cleanup +Do a cleanup using the **Zymogen genomic DNA clean and concentrator** following the manufacturer's protocol. Elute DNA in 30 uL EB that has been pre-heated. + +--- + +## Barcoding and library prep + +**Duration:** 10m + +### 8. Barcoding +To 500 ng of each sample, add barcodes from the SQK-LSK 109 kit and 2x TA/ligase mix: + +- 38 uL DNA (500 ng) +- 4 uL barcode +- 42 uL 2x TA/ligase mix + +### 9. Cleanup and Adapter Ligation +Pool samples and do a cleanup using the Zymogen genomic DNA clean and concentrator kit into 65 uL of EB. Proceed with adaptor ligation following ONT protocol. + +--- + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/cas9-enrichment-for-nanopore-sequencing-bmi5k4g6.md b/markdown-output/cas9-enrichment-for-nanopore-sequencing-bmi5k4g6.md new file mode 100644 index 0000000000000000000000000000000000000000..8b407cb4517ad9368fc2315be9f2576cef278d77 --- /dev/null +++ b/markdown-output/cas9-enrichment-for-nanopore-sequencing-bmi5k4g6.md @@ -0,0 +1,186 @@ +```markdown +# Goal/Experiment: +This protocol is designed to help users achieve targeted enrichment for regions of interest using nanopore sequencing, providing higher coverage for myriad future analysis applications. GuideRNA components (crRNA and tracrRNA) were designed using online tools provided by Integrated DNA Technologies (IDT). + +# Cas9 Enrichment for Nanopore Sequencing V.3 + +Timothy Gilpatrick1, Isaac Lee1, James E. Graham2, Etienne Raimondeau2, Rebecca Bowen2, Andrew Heron2, Fritz J. Sedlazeck3, Winston Timp1 +1Johns Hopkins University; 2Oxford Nanopore Technologies; 3Baylor College of Medicine +Date: Sep 22, 2020 + +## Abstract +This protocol is designed to help users achieve targeted enrichment for regions of interest using nanopore sequencing, providing higher coverage for myriad future analysis applications. GuideRNA components (crRNA and tracrRNA) were designed using online tools provided by IDT. + +## External Links +- DOI: [https://doi.org/10.1038/s41587-020-0407-5](https://doi.org/10.1038/s41587-020-0407-5) + +## Protocol Citation +Timothy Gilpatrick, Isaac Lee, James E. Graham, Etienne Raimondeau, Rebecca Bowen, Andrew Heron, Fritz J. Sedlazeck, Winston Timp 2020. Cas9 Enrichment for Nanopore Sequencing. [protocols.io](https://protocols.io/view/cas9-enrichment-for-nanopore-sequencing-bmi5k4g6) + +## License +This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. + +## Created +- Created: Sep 20, 2020 +- Last Modified: Sep 22, 2020 + +## Materials + +| NAME | CATALOG # | VENDOR | +|---------------------------------------------|----------------|-------------------------| +| CutSmart Buffer - 5.0 ml | B7204S | New England Biolabs | +| Taq DNA Polymerase with Standard Taq Buffer | M0273L | New England Biolabs | +| Quick Ligation Kit - 150 rxns | M2200L | New England Biolabs | +| Quick Dephosphorylation Kit - 500 rxns | M0508L | New England Biolabs | +| TE buffer | | Thermo Fisher Scientific| +| Alt-R® S.p. HiFi Cas9 Nuclease V3 | 1081060 | IDT | +| Duplex Buffer | 11-01-03-01 | IDT | +| 1D Ligation Sequencing Kit | SQK-LSK109 | IDT | +| AmpureXP beads | A63880 | Beckman Coulter | +| dATP | D1005 | Zymo Research | + +## Before Starting +Users should design and order their own custom crRNAs. Note that there is a strong preference for reads to start on the side of the Cas9 cut containing the PAM site. The selection of guideRNAs makes a big difference in the performance of the assay, we therefore encourage using available tools to predict guideRNA on-target performance and (if applicable) off-target performance. We used the tool provided by 'Integrated DNA Technologies' to design and estimate performance of guideRNAs. + +## Procedure + +### Making Ribonucleoprotein Complex + +1. **Resuspend crRNA(s) and tracrRNA** to final concentration of 100uM in TE, pH7.5 + - (2nmol crRNA in 20uL; 5nmol tracrRNA in 50uL) + +2. **Make equimolar mix** of all crRNAs to be used by adding 0.75uL of each to new tube + +3. **Assemble guideRNA duplex:** + - 8uL Nuclease Free Water (NFW) + - 1uL 100uM tracrRNA + - 1uL crRNA mix + +4. **Heat guideRNA duplex at +95°C for 5min**, allowing to cool on bench after incubation + - (Perform steps 5 and 6 during incubation) + +5. **Dilute the 10X CutSmart buffer** 1:8 with Nuclease Free Water (NFW) to make 1.25X CutSmart Buffer + - (14uL NFW + 2uL 10X CutSmart Buffer) + +6. **Dilute the HiFi Cas9 1:5** using the 1.25X CutSmart made in step 5 + - (1uL HiFi Cas9 + 4uL 1.25X CutSmart) + - *Cas9 comes in stock of 10ug/uL or 61pM, we dilute 1:5 to prevent pipetting of very small volumes of the Cas9 enzyme in next step* + +7. **Assemble Ribonucleoprotein Complex:** + - 23uL NFW + - 2.8uL 10X CutSmart Buffer + - 3uL guideRNA duplex (from step 4) \[30pmol\] + - 1.2uL 1:5 dilution of Cas9 (from step 6) \[15pmol\] + + Mix by gentle flicking, then keep on benchtop (room temperature) for 20min. After incubation, keep the RNP on ice. Can be stored at +4°C a few days before use. + +### Dephosphorylation of Free DNA Ends +8. **Dephosphorylation Reaction:** + - 3uL 10X CutSmart Buffer + - 3ug of DNA (often in TE) + - NFW up to volume of 27uL + + Mix gently by flicking before adding enzyme: + - Add 3uL QuickCIP enzyme (from NEB Quick dephosphorylation kit) + - *Total volume = 30uL* + + Mix by gentle flicking and incubate at: + - +37°C for 10min (dephosphorylation) + - +80°C for 2min (enzyme inactivation) + - Hold at +20°C + + During incubation: + - Get out AmpureXP to allow to come to room temperature, + - Thaw 100mM ATP, vortex to mix, keep on ice after thawing. + +### Cleavage and A-tailing of DNA +9. **Make 1:10 dilution of 100mM dATP** + - (5uL 100mM dATP + 45uL NFW) + + After allowing dephosphorylated DNA (from step 8) to cool: + - Add the following components: + - 10uL Assembled RNP complex (from step 7) + - 1uL 1:10 (10mM) dATP + - 1uL Taq polymerase + - *Total volume = 42uL* + + Mix by gentle flicking and incubate at: + - +37°C for 15min (Cas9 cleavage) + - +72°C for 5min (Mono adenylation of 3' ends by Taq/dATP) + - Hold at +12°C + + During this incubation, get out the following components from SQK-LSK109 reagents to thaw: + - Ligation Buffer (LNB) (keep at room temp) + - Adaptor Mix (AMX) (keep on ice after thawing) + +### Adaptor Ligation +10. **Make “Ligation mix":** + - 20uL LNB (ligation buffer from LSK109 kit) + - 4.5uL NFW + - 10uL T4 ligase (from NEB quick ligation kit) + - 3.5uL AMX* (sequencing adaptor from LSK109 kit) + + *Note: AMX will be consumed in solution with ligase -- add right before use* + +11. **Using pipette, mix ligation mix** gently until homogenous (LNB is very viscous) + + Add 20uL of "ligation mix" to DNA*, mix by gentle flicking + *Total volume = 60uL* + + *A DNA precipitate may form at this step, adding "ligation mix" in 2-steps helps to reduce this. Formation of DNA precipitate does not appear to interfere with protocol efficiency.* + +12. Rotate ligation for 10min at Room Temperature + + During incubation, get out Long-Fragment Buffer (LFB) and Elution Buffer (EB) from LSK109 kit, keeping both at room temp (or the Short Fragment Buffer (SFB), if the target region is less than 3kb). + +13. After ligation, add equivolume (80uL) amount of TE (pH 8.0) + *New volume = 160uL* + +14. Add 0.3X Ampure (48uL if DNA is currently in 160uL) and mix by gentle flicking until homogeneous. + +15. Rotate for 5 minutes, then keep on benchtop for 5 minutes to allow Ampure beads to bind DNA. + +16. Place on magnetic tube rack, allow 2.5 minutes for beads to collect on back of tube. + +17. Remove supernatant with a pipette, taking care not to disturb the beads. + + Add 200uL of LFB to beads (or use the Short Fragment Buffer (SFB), if the target region is less than 3kb). + + Remove tube from magnetic stand and resuspend Ampure beads by gentle flicking until homogeneous. + +18. Return sample to magnetic tube rack, allowing 2.5 minutes for beads to collect. + +19. Repeat steps 17 and 18 for a second bead wash with 200uL LFB. + +20. Remove supernatant, briefly spin tube to collect any excess LFB and carefully remove remaining solution from above the beads. + +21. Add 15uL of elution buffer (EB), and keep at room temperature for 10 minutes to elute + - For longer DNA fragments, it can be helpful to increase the elution time to 30 minutes, or to elute with gentle agitation. + + During this elution, get out the following components from LSK-109 kit to thaw: + - Flush Buffer (FLB or FB), Loading Beads (LB), Sequencing Buffer (SQB) - (all at room temp) + - Sequencing Tether (SQT) - (on ice after thaw) + +### Library Prep and Sequencing +22. Return tube with sample to magnetic rack, and allow 2min for beads to collect, transfer (and keep!) ~15uL supernatant with DNA to a new tube. + +23. Perform initial priming of flow cell by loading 800uL FLB into MinION priming port. + +24. Finish library prep by adding the following components to the 15uL DNA eluate: + - 25uL SQB + - 14.5uL Loading beads (LB) + - 0.5uL SQT + - *Total volume of prepped Library: ~55uL* + + *Loading Beads are in suspension -- vortex or flick prior to use* + +25. Perform second flow cell priming with 200uL of second priming solution: (70uL SQB + 70uL NFW + 70uL FLB) + + Then immediately afterwards load sample dropwise into MinION sample loading port. + +26. Start sequencing run using MinKNOW software. + +*We commonly get low pore occupancy with 20-30 pores performing active sequencing by this method. Runs often stop producing data after 24h*. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/caspase-apoptosis-fluorometic-assay-with-tissue-ly-gyabxse.md b/markdown-output/caspase-apoptosis-fluorometic-assay-with-tissue-ly-gyabxse.md new file mode 100644 index 0000000000000000000000000000000000000000..c0a6d26632c6766d2a7046a9ec89d5157f2e112c --- /dev/null +++ b/markdown-output/caspase-apoptosis-fluorometic-assay-with-tissue-ly-gyabxse.md @@ -0,0 +1,188 @@ +```markdown +# Goal/Experiment: +The goal of this experiment is to assay the activities of various caspases (proteases) using CasPASE™ Apoptosis Fluorometric Assay, a key early indicator of apoptosis in mammalian cells. + +# CasPASE™ Apoptosis Fluorometric Assay with Tissue Lysate + +## Abstract +CasPASE™ Apoptosis Fluorometric Assay provides a simple method for assaying the activities of various caspases (proteases), a key early indicator of apoptosis in mammalian cells. + +Citation: G-Biosciences CasPASE™ Apoptosis Fluorometric Assay with Tissue Lysate. protocols.io dx.doi.org/10.17504/protocols.io.gyabxse +Published: 16 Jan 2017 + +## Guidelines + +### Introduction +CasPASE™ Apoptosis Fluorometric Assay provides a simple method for assaying the activities of various caspases (proteases) (Caspase 1-10 & 13), a key early indicator of apoptosis in mammalian cells. The assay is based on the detection of cleavage of a synthetic substrate, which has 7-amino-4-trifluoromethyl coumarin (AFC) at the C-terminal. When liberated from the peptide, AFC produces an optical change that can be detected as fluorescence at 500-550nm with the use of a fluorometer. The reaction is selectively and irreversibly inhibited by the peptide Z-VAD-FMK (fluoromethyl ketone). + +### Items Supplied + +#### Description | 50 Assay | 100 Assay +- **CasPASE™ Lysis Buffer**: 15ml | 2 x 15ml +- **2X CasPASE™ Assay Buffer**: 5ml | 5ml +- **DTT [1M, 100µl]**: 1 vial | 1 vial +- **Substrate Solution [1mM]** Y: 0.25ml | 2 x 0.25ml +- **Free Dye (AFC), [8mM]**: 0.2ml | 0.2ml +- **Inhibitor (Z-VAD-FMK) [1mM]**: 0.1ml | 2 x 0.1ml + +Y The different substrate solutions supplied with individual kits are as follows: + +#### Substrate Solutions + +| Cat. # | Description | Size | +|-------- |--------------------------------|------| +| 786-200A| CasPASE™ - 1, 4, 5 Assay | 50 Assays / 100 Assays | +| 786-200B| Ac-WEHD-AFC substrate | Assays | +| 786-201A| CasPASE™ - 2 Assay | 50 Assays / 100 Assays | +| 786-201B| Ac-VDVAD-AFC substrate | Assays | +| 786-202A| CasPASE™ - 3, 7,10 Assay | 50 Assays / 100 Assays | +| 786-202B| Ac-DEVD-AFC substrate | Assays | +| 786-203A| CasPASE™ - 6 Assay | 50 Assays / 100 Assays | +| 786-203B| Ac-VEID-AFC substrate | Assays | +| 786-204A| CasPASE™ - 8 Assay | 50 Assays / 100 Assays | +| 786-204B| Ac-LETD-AFC substrate | Assays | +| 786-205A| CasPASE™ - 9 Assay | 50 Assays / 100 Assays | +| 786-205B| Ac-LEHD-AFC substrate | Assays | +| 786-206A| CasPASE™ -13 Assay | 50 Assays / 100 Assays | +| 786-206B| Ac-LEED-AFC substrate | Assays | + +### Storage Condition +- The kit is shipped in blue ice. Upon arrival, store all the reagents at -20°C. +- When used properly, the substrates are stable for 6 months and other components for up to 1 year. + +### Additional Items Needed +- Centrifuge +- 96-well plates or Reaction Tube + +### Preparation of Kit Reagents +1. Allow the reagents to thaw into liquid form. Centrifuge the substrate, free dye, and the inhibitor vials to collect the reagent solution at the bottom of the vial. Protect from light and humidity. Allow the reagents to reach RT before opening the vial. +2. Dissolve the supplied DTT in 90µl DI water (final volume 100µl) to give 1M concentration. Store at -20°C. + +### Assay Controls +- Prepare a negative control reaction with cells not treated with the apoptosis-inducing stimulus. + +## Assay Protocol +First read the section "Preparation Before Use". The assay may be performed in a 96 well microplate or cuvette, using a fluorometer. + +- Set up the assay in duplicate and arrange the appropriate blanks and controls, such as a non-apoptotic cell lysate (negative control). A blank should be prepared to measure the substrate background and instrument drift. + +### Inhibition of Caspase Activity (Optional) +In order to establish non-specific protease activity, a control should be run with or without the caspase-specific inhibitor (Z-VAD-FMK) supplied with the kit. + +1. Reaction tubes should be prepared as described above (e.g., 50µl lysate and 50µl of 2X Assay buffer). +2. Add 1 µl of the Inhibitor (10µM final conc.), mix and incubate the reaction at 20-37°C for 30 minutes to complete the inhibition. +3. Add 5µl of 1mM conjugated substrate (50µM final concentration). +4. Mix the content of the tube and take a reading at zero time point (t = 0). +5. Incubate the assay tubes at 20-37°C. +6. Measure the reaction every 30-60 minutes until the sample measurements are complete. + +**Component | Blank | Test Sample | +--- | --- | --- | +2X Assay Buffer | 100µl | 50µl | +Test Sample/Lysate | --- | 50µl | +Inhibitor | --- | 0.5µl-1µl | + +### Fluorescent Detection of the Caspase Activity +- Read sample in a fluorometer setting at 360-390nm EXCITATION and 510-550nm EMISSION filter. +- Zero the detection scale using 1x Assay Buffer. +- Insert the most concentrated solution (4µM AFC) and adjust the setting, e.g., adjust the gain to obtain a signal near the maximum scale. If the blank signal is more than 50% of the full-scale signal, the substrate may have degraded. +- Comparison of the fluorescence from an induced/apoptotic sample with an uninduced or inhibited control allows one to determine the fold-increase in protease activity. + +### Caspase Activity Calculation +1. Generate a dye (AFC) calibration curve and determine the slope. +2. Dilute AFC solution in CasPASE™ Lysis Buffer and prepare 0, 10, 20, 40, and 80µM stock solutions. +3. Mix 190µl of CasPASE™ Lysis Buffer with each 10µl of stock solutions, as follows: + +| AFC Solution | AFC Concentration | +|---------------|--------------------| +| 10µl of 0µM AFC + 190µl of Lysis Buffer | 0.0nmole AFC (0µM) | +| 10µl of 10µM AFC + 190µl of Lysis Buffer | 0.1nmole AFC (0.5µM) | +| 10µl of 20µM AFC + 190µl of Lysis Buffer | 0.2nmole AFC (1µM) | +| 10µl of 40µM AFC + 190µl of Lysis Buffer | 0.4nmole AFC (2µM) | +| 10µl of 80µM AFC + 190µl of Lysis Buffer | 0.8nmole AFC (4µM) | + +4. Plot nmole AFC (x-axis) Vs (FU) fluorescence unit (y-axis), and determine the slope i.e., FU/nmole AFC. +5. Calculate the rate of increase in fluorescence for each sample as follows: + +\[ \Delta F / \text{minute} = \left[ \Delta F_{\text{sample}} - \Delta F_{\text{blank}} \right] / \text{minute} \] +(i.e., change in fluorescence over the length of reaction time minus the change in the fluorescence over the same length of reaction for the blank). + +6. Calculate unit of caspase activity using the following formula: Units caspase = ΔF / minute x (calibration curve slope) 𝐶 + +#### Example +Slope of calibration plot = 2500 (i.e., per nmole of AFC yields 2500 fluorescence units) +Sample: in 60 minutes fluorescence change from 0 to 800 FU, i.e., rate of fluorescence increase = 800/60. +Units Caspase in sample = (800/60) x (1/2500) = 0.00533 nmole/minute. + +### Citations +1. Lee, R.X. et al (2012) Anticancer Res. 32:3103 +2. Soong, G. et al (2012) J. Infect. Dis. 205:1571 +3. Zhang, A. et al (2006) Am. J. Physiol. Renal. Physiol. 291:F1332 + +## Before Start + +### Preparation of Kit Reagents +1. Allow the reagents to thaw into liquid form. Centrifuge the substrate, free dye, and the inhibitor vials to collect the reagent solution at the bottom of the vial. Protect from light and humidity. Allow the reagents to reach RT before opening the vial. +2. Dissolve the supplied DTT in 90µl DI water (final volume 100µl) to give 1M concentration. Store at -20°C. + +## Protocol + +### Preparation of Tissue Lysate + +#### Step 1 +- Homogenize 3-5mg tissue in 100µl Lysis Buffer + +#### Step 2 +- Centrifuge the lysate for 30 minutes at full speed in a microfuge at 4°C. + - ⏱️DURATION: 00:30:00 + +#### Step 3 +- Collect the supernatant for the assay. + +### Preparation of CasPASE™ Assay Buffer + +#### Step 4 +- Immediately before use, transfer an appropriate volume of 2X CasPASE™ Assay Buffer in a tube. + +#### Step 5 +- Add the DTT (1M) solution to the CasPASE™ Assay Buffer to achieve 5-10mM final concentration (e.g. Add 5-10µl of 1.0M DTT per 1ml of 2X CasPASE™ Assay Buffer). DO NOT ADD THE DTT TO THE STOCK SOLUTION. + +### Assay Controls + +#### Step 6 +- Prepare a negative control reaction with cells not treated with the apoptosis-inducing stimulus. + +### Assay Protocol + +#### Step 7 +- Transfer 50µl of 2X CasPASE™ Assay Buffer (containing DTT) into each tube. + - **Notes:** + - Colin Heath 13 Jan 2017: The assay may be performed in a 96-well microplate or cuvette, using a fluorometer. + - Colin Heath 13 Jan 2017: Set up the assay in duplicate and arrange the appropriate blanks and controls, such as a non-apoptotic cell lysate (negative control). A blank should be prepared to measure the substrate background and instrument drift. + +#### Step 8 +- Add 50µl of cell lysate into each tube. + - **Notes:** + - Colin Heath 13 Jan 2017: NOTE: For each assay, use lysate (50µl) obtained from at least 1x10^6 cells for fluorescence measurement. The use of fewer cells than this may reduce the observed increase in the caspase activity. + +#### Step 9 +- Add 5µl of 1mM AFC-conjugated Substrate (50µM final concentration). + +#### Step 10 +- Mix the content of the tube and take a reading at zero time point (t = 0). + +#### Step 11 +- Close the assay tubes and incubate at 20-37°C. + +#### Step 12 +- Measure the reaction every 30-60 minutes or until the measurements are significantly different from those at t=0. + +**Component | Blank | Test Sample | Negative Control** +--- | --- | --- | --- +2X Assay Buffer | 100µl | 50µl | 50µl +Test Sample/Lysate | --- | 50µl | --- +AFC-Substrate | 5µl | 5µl | 5µl +Negative Control/Lysate | --- | --- | 50µl + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/catalogaci-n-publicaciones-seriadas-biblioteca-uca-cjx3upqn.md b/markdown-output/catalogaci-n-publicaciones-seriadas-biblioteca-uca-cjx3upqn.md new file mode 100644 index 0000000000000000000000000000000000000000..45863352e6dff8066592703d6a108932e15c3524 --- /dev/null +++ b/markdown-output/catalogaci-n-publicaciones-seriadas-biblioteca-uca-cjx3upqn.md @@ -0,0 +1,363 @@ +```markdown +# Goal/Experiment: +The goal of this experiment is to provide a detailed protocol for cataloging serial publications at UCAM University Library using the Absysnet library management system. This includes the classification, registration, cataloging, and creation of a collection for serial publications. + +# Catalogación Publicaciones Seriadas Biblioteca UCAM + +## DOI +[dx.doi.org/10.17504/protocols.io.n92ldp87xl5b/v1](dx.doi.org/10.17504/protocols.io.n92ldp87xl5b/v1) + +## Author +Antonio Rex Alegría +UCAM Universidad Católica de Murcia + +## Abstract +Una Publicación Seriadas es una publicación impresa o no, editada en partes sucesivas con designaciones numéricas o cronológicas y que pretende continuarse indefinidamente. + +_La catalogación_ es el proceso de selección y descripción de los elementos que permiten identificar un documento y establecer los puntos de acceso imprescindibles para su posterior recuperación. + +## Protocol Citation +Antonio Rex Alegría 2022. Catalogación Publicaciones Seriadas Biblioteca UCAM. +*protocols.io* +[https://dx.doi.org/10.17504/protocols.io.n92ldp87xl5b/v1](https://dx.doi.org/10.17504/protocols.io.n92ldp87xl5b/v1) + +## License +This is an open access protocol distributed under the terms of the [Creative Commons Attribution License](https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. + +## Created +Dec 01, 2022 + +## Last Modified +Dec 01, 2022 + +## Protocol Integer ID +73435 + +1. La clasificación de revistas está adaptada a las titulaciones de la UCAM. + +En la Hemeroteca podemos encontrar: + +- Publicaciones Periódicas: revistas y prensa +- Publicaciones Oficiales. + +Además de esta clasificación por titulaciones hay Publicaciones Periódicas relativas a materias generales como: UCAM, Religión, Idiomas, Murcia, Catálogos y Varios. + +La solicitud de la Publicación Periódica se realiza a través del Soporte. + +La dirección de la biblioteca estudiará y en su caso aprobará las peticiones. Éstas serán aprobadas por el Servicio de Unidad de Compras quien cursaran la petición al proveedor. + +## 2. Registro + +Se registra la publicación en una hoja de Cálculo Excel (Hoja de Registro de Publicaciones Periódicas), habiendo consultado y comprobado previamente en el catálogo electrónico (OPAC) que dicha publicación no se encuentra entre los fondos de la BUCAM, y se le asigna el número de registro y la signatura correspondiente que identificará individualmente a cada una las Publicaciones Seriadas. + +### Registro de Publicaciones Seriadas en una Hoja de Cálculo de Excel. + +| | | | | +|---|---|---|---| +| **A** | **B** | **C** | **D** | +| **Fecha** | **Nº Registro** | **Título** | **Signatura** | + + +El criterio para asignar una signatura es: + +- Si la publicación seriada es una suscripción; se le asigna las tres primeras letras de la carrera a la que pertenece en mayúsculas y el número de entrada de la publicación. Ejemplo: ENF20 + +En la página Excel se rellenan los siguientes campos: + +- Fecha de entrada en la Biblioteca. +- Número de Registro. Ejemplo SE000001 +- Título. +- Signatura. +- Viva +- Muerta + +- Donación +- Intercambio +- Gratuita +- Observaciones. + +## 3. Recepción + +Se sella la publicación recibida con el sello de la Universidad y se procede a su catalogación. + +## 4. Catalogación + +La catalogación de las Publicaciones Seriadas se realiza con el Programa de Gestión Bibliotecaria “Absysnet”. + +Los pasos a seguir son los siguientes: Desde el Módulo de Catálogo, desplegar el menú principal y darle a Añadir, y seleccionamos el Tipo de Catalogación: + +Seleccionar: Publicaciones seriadas y rellenar los siguientes campos: + +### Campo 017: +Datos codificados en longitud fija: en el campo de PRIMERA FECHA, introducimos el año que aparece en el Depósito Legal. + +### Campo 022: +Número Internacional Normalizado para Publicaciones Seriadas (ISSN): En este campo introducimos el ISSN``<>``. + +### Campo 080: +Número de Clasificación Decimal Universal (CDU): Introducimos el número correspondiente de la ``CDU`` ``<>``. + +### Campo 245: +Mención de Título: En este campo incluimos el título ``<>``, subtítulo ``<>`` y las menciones de responsabilidad ``<>``. + +### Campo 260: +Publicación, Distribución, etc.: Este campo contiene información relacionada con el lugar donde se ha publicado ``<>``, la editorial ``<>``, y el año de publicación o producción de una obra ``<>``. + +### Campo 310: +Periodicidad Actual: En este campo introducimos la periodicidad de la revista ``<>``. + +### Campo 650: +Encabezamiento secundario de materia-Termino de materia. Este campo sirve para designar la temática de los documentos. Contiene un encabezamiento de materia general, se utiliza de acuerdo con la lista de encabezamientos establecida. + +### Campo 653: +Término de Indización-No controlado: Usamos este campo para indicar la carrera a la que pertenece dicho ejemplar. Ej. ``<>`` Arquitectura Técnica. + +### Campo 856: +Localización y acceso electrónicos: En este campo introducimos la localización ``<>``y acceso electrónicos ``<>``. + +### Campo 866: +Mención textual de fondos – Información general: En este campo introducimos la mención de los fondos ``<>``. + +### Ejemplo: + +``` +$aEn Hemeroteca: 1999-2002(2003)2004-07(2008)2009- +$aEn Mediateca: Acceso online (Texto completo) +$aFondo electrónico: 1996- +``` + +A continuación de forma automática nos preguntará si queremos añadir el ejemplar, rellenamos con los siguientes datos: + +- Localización: (HEM) Hemeroteca +- Tipo de ejemplar: (SE) No Prestable +- Estado: (B) Bueno +- Soporte: + - (RVE) Revista Electrónica + - (SEC) Seriada Combinada + - (SEI) Seriada Impresa + +- Procedencia: + - (COM) Compra + - (DON) Donación + - (GRA) Gratuita + - (INT) Intercambio +-Situación ejemplar: (C) Circulación +- Fecha de Registro: (Se introduce automáticamente) +- Signatura: NUT26 + +Después de añadir el ejemplar, se crea la Colección para esa Publicación Seriada. + +## 5. Creación de la Colección. + +Desde el Módulo de Series, en el apartado de Colecciones, le damos al símbolo + del menú principal para añadir una colección nueva y buscamos el título de esa revista. + +### Dar de alta una colección. + +### Datos de la colección: + +- “Código1”: Titulación a la que pertenece. +- “Fecha de Comienzo” de la colección. +- “Descripción”: Título de la revista. +- “Sucursal”: Biblioteca General. + +## 6. Datos de los ejemplares: + +- “Generar ejemplares de cada nº”: para que se generen automáticamente ejemplares por cada número que reciba. +- “Código de barras automático”: para que se asigne el código de barras automáticamente a cada uno de los ejemplares. +- “Localización”: seleccionar la sala en la que se encuentran los ejemplares. +- “Tipo de ejemplar”: seleccionar el tipo de ejemplar que le corresponde. +- “Estado”: seleccione Bueno. +- “Signatura”: escriba la signatura que corresponde a los ejemplares. +- “Procedencia”: seleccione la procedencia que corresponda a los ejemplares. +- “Situación ejemplar”: seleccione la situación del mismo. Esta puede ser C-Circulación o A-Precirculación. + Aceptamos y creamos la cronología de esa colección. + +## 7. Añadir la cronología para una colección. + +### Datos de la Cronología: + +Una Colección puede tener varias cronologías, pero sólo una de ellas estará activa. + +#### Datos de las fechas: + +- “Fecha de comienzo” +- “Fecha final”: Si la colección está cerrada. + +#### Datos de la numeración: + +- “Periodicidad”: seleccione el código de periodicidad de los números. Índices y/o suplementos de la colección. +- “Tipo de ciclo”: seleccione el tipo de ciclo del número. Pueden ser: + - Infinito + - Nº de ejemplares + - Anuales + - Meses + +- “Ciclo”: cada cuánto tiempo se produce un cambio en la numeración de la revista. Sólo lo rellenará si ha seleccionado anteriormente Nº de ejemplares o Meses en el Tipo de Ciclo. +- “Número siguiente”: a partir de qué número de la colección debe tener en cuenta la cronología que está añadiendo. +- “Modelo de texto”: texto de la numeración correspondiente a la colección que está añadiendo. +- “Texto siguiente”: posición que ocupa dentro de Texto de numeración el primer número de la colección que debe tener en cuenta la cronología que estas añadiendo. +- “Fecha siguiente”: fecha de inicio del número de la colección que debe tener en cuenta la cronología que estas añadiendo. + +## 8. Datos del volumen: + +- “Tipo de ciclo”: tipo de ciclo del número. Pueden ser: + - Nº de ejemplares + - Anuales + - Meses + - Números + +- “Ciclo”: cada cuánto tiempo se produce un cambio en la numeración del volumen. Se tendrá que rellenar en caso de haber seleccionado Nº de ejemplares o Meses en el Tipo de Ciclo. +- “Volumen siguiente”: a partir de qué volumen de la colección debe tener en cuenta la cronología que añades. +- “Modelo de visualización”: introduce la manera en que quieres que se visualice la numeración de la revista. Hay una serie de variables: + - [nn] Números + - [vn] Volumen + - [nr] Números en romanos + - [vr] Volumen en romanos + - [nt] Texto de numeración + - [m] Meses (1, 2, 3 ... 12) + - [mm] Meses (01, 02, 03 ... 12) + - [d] Días (1, 2, 3 ... 31) + - [dd] Días (01, 02, 03 ... 31) + - [aa] Año + - [aaaa] Año + +- Una vez introducida ésta información, validamos para calcular los números para esa cronología. +- Y por último aceptamos. + +## 9. Generar los números de serie. + +### Generar números de serie: + +Una vez creada la colección y añadida la cronología, tenemos que generar los números de serie para llevar un control de los mismos. + +- Primero localizamos la colección a la que queremos generar los números. +- Desde el módulo de catálogo, desplegamos el menú y seleccionamos : “Generar Números” + +1. **“Fecha de comienzo”**: se rellena automáticamente con la fecha de comienzo de la colección. Se puede cambiar la fecha por la que se quiere empezar a generar la colección. La fecha de comienzo nunca puede ser inferior a la del comienzo de la colección. + +2. **“Fecha final”**: se rellena con la fecha final de la colección, y si la colección sigue abierta se dejará en blanco. + +3. **Marcamos los números cómo “Recibido”.** + +4. **Aceptamos.** + +## 10. Recepción de ejemplares. + +El registro de los ejemplares una revista se realiza también con el Programa de Gestión Bibliotecaria “Absysnet”. + +- Desde el Módulo de Series, en Recepción de números, buscar el título de la revista a la que le vamos a añadir el ejemplar. +- Cómo ya hemos creado los números, seleccionar el número indicado y cambiar su estado a recibido, indicando la fecha prevista y la fecha de llegada. + +## 11. Catalogación de un artículo en Absysnet (Catalogación Experta) + +Desde el módulo de Recepción en Series + +Se busca el título de la Serie. + +Seleccionas el nº de la revista que se va a hacer el artículo. + +Darles a la pestaña de [Analíticas] + +Tipo de Catalogación: Catalogación MARC y aceptamos para proceder a su catalogación. + +### Cabecera: + +Hay que poner en el nivel bibliográfico: b + +### Campo 773: Asiento de la publicación principal + +Ejemplo: + +```plaintext +«t»x«LK»xEurocarne : la revista internacional del sector cárnico«LK=‘32930’.TITN.»x«n». -- 160«g».-- Octubre,2007 +``` + +(Para quesalga el enlace del artículo con esa revista, y con ese número de revista.) + +### Campo 300: Descripción física: +Poner el número de páginas del artículo. + +### Campo 245: Mención del Título: +Poner el título del artículo, y la mención de responsabilidad. + +### Campo 100: Encabezamiento Principal – Nombre de Persona: +Buscar la autoridad: + +- Si ya está, le damos a capturar. +- Si no está, hay que añadir la autoridad nueva. + +Una vez introducidos todos los datos del artículo, hay que añadir el ejemplar, con los siguientes datos: + +- Localización: (HEM) Hemeroteca +- Tipo de ejemplar: (SE) No Prestable +- Estado: (B) Bueno +- Soporte: (AIM) Artículo Impreso +- Procedencia: (COM) Compra +- Situación ejemplar: (C) Circulación +- Fecha de Registro: (Sale automática) +- Signatura: NUT26 + +El Código de Barras te lo dará automático. +Y el nº de Registro en los artículos no se pone. + +## 12. Catalogación de un artículo en Absysnet (Catalogación Asistida) + +- Desde el módulo de Recepción en Series + +- Se busca el título de la Serie. + +- Seleccionas el nº de la revista que se va a hacer el artículo. + +- Darles a la pestaña de [Analíticas] + +- Tipo de Catalogación: Catalogación Asistida. + +- Aceptar, y salen los campos a rellenar: + +### Campo 245. Mención del Título: +Añadimos el título del artículo, y sí tiene subtítulo también. + +### Campo 100. Encabezamiento Principal – Nombre de Persona. +Autor Personal: + +Pinchar en Autor Personal, para buscar el autor: + + - Si es 1 autor que ya está en la base de datos de autoridades, capturamos el autor + - Si no está, añadir la autoridad nueva para ese autor. + +### Campo 300. Descripción Física: +Ponemos las páginas del artículo. + +### Campo 773: Asiento de la publicación principal + +### Por último en la Pestaña de Encabezamiento Secundario: + +El enlace con el Documento Principal (Enlace con el título de la revista y el número del ejemplar de ese artículo.) + +Cuando hemos hecho todo esto le damos a la (v) para validar + +Una vez validado, buscamos el título del artículo en el catálogo: + +Le damos a la pestaña de modificar, y en el modo de Catalogación Experta, en la Cabecera cambiamos el nivel bibliográfico a corr. + +### Cabecera: + +- Hay que poner en el nivel bibliográfico: b +- Aceptamos y añadimos el ejemplar, con los siguientes datos: + +- Localización: (HEM) Hemeroteca +- Tipo de ejemplar: (SE) No Prestable +- Estado: (B) Bueno +- Soporte: (AIM) Artículo Impreso +- Procedencia: (COM) Compra +- Situación ejemplar: (C) Circulación +- Fecha de Registro: (Sale automática) +- Signatura: NUT26 + +### El Código de Barras te lo dará automático. + +Y el nº de Registro en los artículos no se pone. + +endofoutput +``` + diff --git a/markdown-output/cdna-synthesis-using-superscript-iii-first-strand-b64vrgw6.md b/markdown-output/cdna-synthesis-using-superscript-iii-first-strand-b64vrgw6.md new file mode 100644 index 0000000000000000000000000000000000000000..73fc59465167c1bdd45c95850a7b8273288df21f --- /dev/null +++ b/markdown-output/cdna-synthesis-using-superscript-iii-first-strand-b64vrgw6.md @@ -0,0 +1,147 @@ +```markdown +**Goal/Experiment:** + +Synthesize cDNA using SuperScript III First-Strand Synthesis System for RT-PCR to enable the detection of RNA transcripts for qPCR analysis of gene expression. + +# cDNA Synthesis Using SuperScript III First-Strand Synthesis System for RT-PCR V.3 + +**Author:** +Lynn Doran +Realizing Increased Photosynthetic Efficiency (RIPE) + +**Date:** +April 04, 2022 + +--- + +## Introduction + +The SuperScript® III First-Strand Synthesis System for RT-PCR is used to synthesize first-strand cDNA from purified total RNA. RNA targets from 100 bp to >12 kb can be detected with this system. The procedure follows the manufacturer's instructions but includes lab-specific template and reagent amounts and specifies primers and equipment for use in our lab to prepare total RNA samples for qPCR analysis of gene expression. + +**Kit:** +[SuperScript™ III First-Strand Synthesis System, ThermoFisher Catalog number: 18080051](https://www.thermofisher.com/order/catalog/product/18080051) + +**Reference:** +- Invitrogen. “SuperScript III First-Strand Synthesis System for RT-PCR.” Jan. 2013. Doc. Part No: 18080051.pps, Pub. No.: MAN001346, Rev. 3.0. Life Technologies. Lab protocol. +- Note: An inaccurate 65C 5-minute incubation between steps 12 and 13 was removed. Note about random hexamer vs oligo dT primers was included. + +**Precautions:** +- Always wear gloves and change them often to avoid RNases. +- Use RNase-free solutions. +- Use RNase-free certified, disposable plasticware and filter tips whenever possible. + +--- + +## Materials and Reagents + +- Ice +- Dry Ice +- Ice bucket +- [SuperScript™ III First-Strand Synthesis System, ThermoFisher Catalog number: 18080051](https://www.thermofisher.com/order/catalog/product/18080051) +- 0.2ml PCR tube 125 strips flat cap, USA Scientific, 1402-2500 +- Pipette, 1-10 ul, Single Channel, Variable, Eppendorf Research Plus +- Tips, pipette, 1-10 ul, TIPEDS FILTER TIP, REFILLS item #1121-2711 +- Pipette, 10-100 ul, Single Channel, Variable, Eppendorf Research Plus +- Tips, pipette, 20-200 ul, TIPEDS FILTER TIP, REFILLS item #1120-2710 +- RNase Away, spray, ThermoScientific #21-402-178 +- 70% Ethanol +- Thermal cycler, Bio-Rad T100 Thermal Cycler or equivalent +- Mini-centrifuge, Thermo Scientific 6™ Mini Centrifuge + +**Reagent Notes:** +- RNA extracted from [Qiagen RNeasy Plant Extraction Kit](https://www.qiagen.com/us/products/discovery-and-translational-research/rna/rna-preparation/rneasy-plant-mini-kit/), of equivalent quantity and quality. +- RNA used for cDNA synthesis for qPCR should be high quality. + - A260/A280 values > 1.8, ideally values approach 2.1 (note in SYBR green guide they recommend using samples with >2.0). + - No acceptable A260/A230 ratio for qPCR but higher values indicate less contamination. + - RNA integrity as indicated by Qubit RNA IQ values >5, ideally >8. + +--- + +## Procedure + +1. **Allow Reagents to Thaw and Mix:** + - Thaw completely, mix, and briefly minifuge 10 mM dNTP mix and 50 ng/ul random hexamers before use. Store on ice when not in use. + +2. **Label Tubes:** + - Label two PCR tubes per sample, RT and NRT. + +3. **Treat Glassware and Pipettes:** + - Treat with RNase away and sterilize work area with 70% ethanol before pipetting reagents. + +4. **Prepare Master Mix:** + - If performing many reactions, prepare a master mix of primer random hexamers, dNTP mix, and water. Pipette 5 µL of the prepared master mix into each sample tube, both RT and NRT. + - If only a few reactions are needed, pipette the following for 1 reaction into each tube: + +| Component | Amount for 1 rxn | Amount for 10 rxn | +|------------------------|------------------|-------------------| +| 50 ng/µl Random Hexamers | 1 µL | 10 µL | +| 10 mM dNTP Mix | 1 µL | 10 µL | +| DEPC-treated or MilliQ water | 3 µL | 30 µL | + +5. **Add RNA:** + - Add 5 µL of high-quality RNA from each sample to the respective RT and NRT tubes. + - Keep RNA samples on dry ice when not actively in use to inhibit RNases. Return samples to -80°C as soon as possible. + +6. **Incubate:** + - Incubate at 65°C for 5 minutes in the thermocycler. + +7. **Place on Ice:** + - Place on ice for a minimum of 1 minute. + +8. **Allow Reagents to Thaw and Mix:** + - Thaw completely, mix, and briefly centrifuge 10X RT buffer, 25 mM MgCl2, 0.1 M DTT, 40 U/µl RNase OUT, and 200 U/µl SuperScript III RT before use. Store on ice when not in use. + +9. **Prepare cDNA Synthesis (RT) Master Mix:** + - Prepare Master Mix adding each component in the indicated order. + +| Order | Component | Amount for 1 rxn | Amount for 10 rxn | +|-------|------------------|------------------|-------------------| +| 1 | 10X RT buffer | 2 µL | 20 µL | +| 2 | 25 mM MgCl2 | 4 µL | 40 µL | +| 3 | 0.1 M DTT | 2 µL | 20 µL | +| 4 | 40 U/µl RNase OUT| 1 µL | 10 µL | +| 5 | 200 U/µl SuperScript III RT | 1 µL | 10 µL | + +10. **Add cDNA Synthesis (RT) Master Mix:** + - Add 10 µL of the cDNA Synthesis (RT) Master Mix to each tube labeled RT. + +11. **Prepare cDNA Synthesis (NRT) Master Mix:** + - Prepare Master Mix adding each component in the indicated order. + +| Order | Component | Amount for 1 rxn | Amount for 10 rxn | +|-------|----------------------------------|-------------------|-------------------| +| 1 | 10X RT buffer | 2 µL | 20 µL | +| 2 | 25 mM MgCl2 | 4 µL | 40 µL | +| 3 | 0.1 M DTT | 2 µL | 20 µL | +| 4 | 40 U/µl RNase OUT | 1 µL | 10 µL | +| 5 | DEPC-treated or MilliQ water | 1 µL | 10 µL | + +12. **Add cDNA Synthesis (NRT) Master Mix:** + - Add 10 µL of the cDNA Synthesis (NRT) Master Mix to each tube labeled NRT. + +13. **Program Thermocycler:** + - Run all samples, both RT, and NRT: + +| Step | Time (min) | Temp (°C) | +|------|------------|-----------| +| 1 | 10:00 | 25 | +| 2 | 50:00 | 50 | +| 3 | 5:00 | 85 | +| 4 | Infinity | 4 | + +14. **Chill:** + - Chill on ice until samples are cool to the touch. + +15. **Briefly Minicentrifuge:** + - Briefly minicentrifuge all samples. + +16. **Add RNase H:** + - Add 1 µL of RNase H to each tube (both RT and NRT) and incubate in a thermocycler for 20 minutes at 37°C. + +17. **Store cDNA Samples:** + - cDNA samples can be stored at -20°C. + +--- + +**endofoutput** +``` \ No newline at end of file diff --git a/markdown-output/cecret-workflow-for-sars-cov-2-assembly-and-lineag-by72pzqe.md b/markdown-output/cecret-workflow-for-sars-cov-2-assembly-and-lineag-by72pzqe.md new file mode 100644 index 0000000000000000000000000000000000000000..c528fb097ab3bf7958e7c8dba6895699c74a008a --- /dev/null +++ b/markdown-output/cecret-workflow-for-sars-cov-2-assembly-and-lineag-by72pzqe.md @@ -0,0 +1,210 @@ +```markdown +# Goal/Experiment: +This protocol outlines the installation and execution of the Cecret workflow to assemble SARS-CoV-2 genome sequences and classify their lineage using tools such as Pangolin and Nextclade. The aim is to generate consensus sequences from Illumina reads and analyze them for lineage classification to assist in genomic surveillance and research efforts. + +# Cecret Workflow for SARS-CoV-2 Assembly and Lineage Classification V.3 + +**Erin L Young**, Technical Outreach and Assistance for States Team +*Utah Public Health Laboratory; Centers for Disease Control and Prevention* + +For technical assistance, please contact: TOAST@cdc.gov +[StaPH-B Toolkit GitHub Repository](https://github.com/StaPH-B/staphb_toolkit) + +> The opinions expressed here do not necessarily reflect the opinions of the Centers for Disease Control and Prevention or the institutions with which the authors are affiliated. This protocol content here is under development and is for informational purposes only and does not constitute legal, medical, clinical, or safety advice. Content added to protocols.io is not peer-reviewed and may not have undergone a formal approval of any kind. + +This protocol provides instructions to install and run the Cecret workflow as part of the StaPH-B Toolkit. Cecret produces SARS-CoV-2 consensus sequence assemblies from either single- or paired-end Illumina reads in fastq (or fastq.gz) format and assigns lineage classifications using Pangolin and Nextclade. This document applies to all whole-genome sequencing runs on the Illumina platform and downstream bioinformatics for public health laboratories. + +## Software Dependencies + +The Cecret and StaPH-B Toolkit require the following dependencies: +1. **Singularity** or **Docker**: Container platforms used for creating and managing containers. +2. **Python 3.6 or later**: Programming language needed to run various software tools in the workflow. +3. **Java version 8 or later**: Programming language and computing platform for running bioinformatics software. + +Ensure all dependencies are in your system's PATH environment variable. Additional instructions are provided [here](https://staph-b.github.io/staphb_toolkit/install/). + +Within certain high-performance computing environments, these software dependencies can be loaded using GNU module commands similar to: +```bash +module load Python/3.9.1 +module load java/jdk1.8.0_221 +module load nextflow/20.04.1 +module load singularity/3.5.3 +``` + +## Installing StaPH-B Toolkit + +The assembly workflow can be installed as part of the StaPH-B Toolkit using the following commands: +```bash +git clone https://github.com/StaPH-B/staphb_toolkit.git +cd staphb_toolkit/packaging/ +python3 setup.py install --user +cd ../ +export PATH=$PATH:$(pwd) +``` + +## Running Cecret Workflow + +Run the Cecret workflow to perform SARS-CoV-2 genome sequence assembly and lineage classification. The input directory for Cecret should contain a set of single or paired-end (default) fastq.gz or fastq reads from amplicon-prepared libraries. By default, Cecret is configured to use the ARTIC V3 primer set but can be customized to use other primer sets. + +In the examples below, the `--profile` argument is set to use Singularity containers, but Cecret works with Docker containers as well (`--profile docker`). + +Detailed descriptions of parameters are provided [here](https://github.com/UPHL-BioNGS/Cecret). + +### Cecret Input and Output File Paths +```plaintext +#Input Sequencing Reads File Path: +/Full_Path_to_Fastq_File_Directory/INPUT/SRR11953697 + +#Output Directory: +/Full_Path_to_Cecret_Output_Directory/SRR11953697_cecret_output +``` + +### Cecret Workflow Command +```bash +mkdir -p cachedir +SINGULARITY_CACHEDIR=/PATH/for/cache +staphb-wf cecret /Full_Path_to_Fastq_File_Directory/INPUT/SRR11953697 --output /Full_Path_to_Cecret_Output_Directory/SRR11953697_cecret_output --profile singularity +``` +*Default cache directory is `~/.singularity/cache`. Resume is available if you would like to retry an interrupted workflow.* + +### Getting/Using an Optional Configuration File +```bash +staphb-wf cecret --get_config +staphb-wf cecret /Full_Path_to_Fastq_File_Directory/INPUT/SRR11953697 --output /Full_Path_to_Cecret_Output_Directory/SRR11953697_cecret_output --profile singularity -c /Full_Path_to_Config_File_Directory/date_cecret.config +``` + +## Output Files + +Information about output files is provided below: + +### Complete Output Files Structure +```plaintext +cecret_run_results.txt # information about the sequencing run that's compatible with legacy workflows +covid_samples.csv # only if supplied initially +cecret +├── aligned +│   ├── pretrimmed.sorted.bam +│   ├── sample +│   │   └── ivar +│   │   ├── variant.png # png of variants identified via ivar +│   │   └── bcftools +│   │   └── variant.png # png of variants identified via bcftools +│   └── bedtools_multicov +│   └── sample.multicov.txt # depth per amplicon +├── consensus +│   └── sample.consensus.fa # the likely reason you are running this workflow +├── fastp +│   ├── sample_clean_PE1.fastq # clean file: only if params.cleaner=fastp +│   └── sample_clean_PE2.fastq # clean file: only if params.cleaner=fastp +├── fastqc +│   ├── sample.fastqc.html +│   └── sample.fastqc.zip +├── filter # optional: turns aligned bams into fastq files +│   ├── sample_filtered_R1.fastq +│   ├── sample_filtered_R2.fastq +│   └── sample_filtered_unpaired.fastq +├── iqtree # optional: relatedness parameter must be set to true +│   └── iqtree.treefile +├── ivar_trim +│   ├── sample.primertrim.bam # aligned reads after primer trimming. trimmer parameter must be set to 'ivar' +├── ivar_variants +│   └── sample.variants.tsv # list of variants identified via ivar and corresponding scores +├── kraken2 +│   └── sample_kraken2_report.txt # kraken2 report of the percentage of reads matching virus and human sequences +├── logs +│   ├── process_logs # for troubleshooting purposes +├── mafft # optional: relatedness parameter must be set to true +│   ├── mafft_aligned.fasta # multiple sequence alignment generated via mafft +├── nextclade +│   └── sample_nextclade_report.csv # identification of nextclade clades and variants identified +├── pangolin +│   └── lineage_report.csv # identification of pangolin lineages +├── samtools_ampliconstats +│   └── sample_ampliconstats.txt # stats for the amplicons used +├── samtools_coverage +│   ├── aligned +│   │   ├── sample.cov.hist # histogram of coverage for aligned reads +│   │   └── sample.cov.txt # tabular information of coverage for aligned reads +│   ├── trimmed +│   │   ├── sample.cov.trim.hist # histogram of coverage for aligned reads after primer trimming +│   │   └── sample.cov.trim.txt # tabular information of coverage for aligned reads after primer trimming +├── samtools_depth +│   ├── aligned +│   │   └── sample.depth.txt # read depth for each read position +│   ├── trimmed +│   │   └── sample.depth.txt # read depth for each position after trimming +├── samtools_flagstat +│   ├── aligned +│   │   └── sample.flagstat.txt # samtools stats for aligned reads +│   ├── trimmed +│   │   └── sample.flagstat.trim.txt # samtools stats for trimmed reads +├── samtools_plot_ampliconstats +│   └── sample.*.png # images corresponding to amplicon performance +├── samtools_stats +│   ├── aligned +│   │   └── sample.stats.txt # samtools stats for aligned reads +│   ├── trimmed +│   │   └── sample.stats.trim.txt # samtools stats for trimmed reads +├── seqyclean +│   ├── sample_clean_PE1.fastq # clean file: only if params.cleaner=seqyclean +│   ├── sample_clean_PE2.fastq # clean file: only if params.cleaner=seqyclean +├── snp-dists # optional: relatedness parameter must be set to true +│   └── file containing table of the number of snps that differ between any two samples +├── submission_files # optional: is only created if `covid_samples.txt` exists +│   ├── sample.genbank.fa # fasta file with formatting and header including metadata for genbank +│   ├── sample.gisaid.fa # fasta file with header for gisaid +│   ├── sample.R1.fastq.gz # renamed raw fastq.gz file +│   └── sample.R2.fastq.gz # renamed raw fastq.gz file +├── summary +│   ├── sample.summary.txt # individual results +│   └── summary.csv # tab-delimited summary of results from the workflow +└── reads + ├── sample_S1_L001_R1_001.fastq.gz # initial file + ├── sample_S1_L001_R2_001.fastq.gz # initial file +└── work # Nextflow's work directory. +└── vadr + └── vadr* +``` + +## Cecret Consensus Sequence and Summary Report Paths +```plaintext +#Consensus Sequence Path: +/Full_Path_to_Cecret_Output_Directory/SRR11953697_cecret_output/consensus + +#Summary Report Path: +/Full_Path_to_Cecret_Output_Directory/SRR11953697_cecret_output/summary +``` + +Example Summary.txt output file: + +![Summary.txt example](https://github.com/UPHL-BioNGS/Cecret) + +Additional documentation for the Cecret workflow is available [here](https://github.com/UPHL-BioNGS/Cecret). Further details about the StaPH-B Toolkit are available [here](https://staph-b.github.io/staphb_toolkit). + +Before submitting the resulting SARS-CoV-2 consensus sequence assemblies to public repositories, such as NCBI GenBank or GISAID, refer to the following documentation describing submission criteria and minimum quality control thresholds: + +- [GenBank Submission Criteria](https://www.ncbi.nlm.nih.gov/) +- [GISAID Submission Criteria](https://www.gisaid.org/) + +## Alternative Lineage Assignment + +The SARS-CoV-2 consensus sequence assembly generated by the Cecret workflow can also be uploaded to other lineage assignment software. + +### 5.1 Upload to Pangolin COVID-19 Lineage Assigner +Upload the consensus sequence for each sample to the [Pangolin COVID-19 Lineage Assigner](https://pangolin.cog-uk.io/). + +Click the 'Start analysis' button: + +![Pangolin Assigner Example](https://pangolin.cog-uk.io/) + +### 5.2 Upload to Nextclade +Upload the consensus sequence for each sample to the [Nextclade](https://clades.nextstrain.org/) clade assignment web portal. + +![Nextclade Assignment Web Portal](https://clades.nextstrain.org/) + +The Nextclade server provides clade classification as well as QC metrics and a list of amino acid substitutions. A summary output file can be downloaded with the 'Export to CSV' button. + +![Nextclade Output](https://clades.nextstrain.org/) + +`endofoutput` +``` \ No newline at end of file diff --git a/markdown-output/cell-culture-of-j774a-1-cells-cs67whhn.md b/markdown-output/cell-culture-of-j774a-1-cells-cs67whhn.md new file mode 100644 index 0000000000000000000000000000000000000000..4fc21da052d215fffbddd0a6db5e8360093f4641 --- /dev/null +++ b/markdown-output/cell-culture-of-j774a-1-cells-cs67whhn.md @@ -0,0 +1,124 @@ +```markdown +# Goal/Experiment: +Culturing J774A.1 cells to maintain a controlled environment for their growth and proliferation for subsequent experimental applications. + +# Cell Culture of J774A.1 Cells + +**Lisa Börmel¹, Sijia Liao¹, Maria Wallert¹, Stefan Lorkowski¹** +¹Institute of Nutritional Science, Friedrich Schiller University Jena + +## Abstract +The J774 tumor arose in a female BALB/c/NIH mouse with a reticulum cell sarcoma during a plasmacytoma induction program in 1968. The J774A.1 macrophages are active in antibody-dependent phagocytosis. Their growth is inhibited by dextran sulfate, p-phenylenediamine, and LPS. They synthesize large amounts of lysozyme and exhibit low cytolysis but predominantly antibody-dependent phagocytosis. Interleukin-1β is continuously synthesized by this cell line. J774A.1 cells have a doubling time of 17 hours. + +## Protocol References +1. Hirst JW, Jones GG, Cohn M. Characterization of a BALB/c Myeloma Library. Journal of Immunology 1971; 107(3):926-927. DOI: [10.4049/jimmunol.107.3.926.c](https://doi.org/10.4049/jimmunol.107.3.926.c). +2. Ralph P, Nakoinz I. Phagocytosis and cytolysis by a macrophage tumour and its cloned cell line. Nature 1975; 257(5525):393-394. DOI: [10.1038/257393a0](https://doi.org/10.1038/257393a0). +3. Ralph P, Nakoinz I. Direct toxic effects of immunopotentiators on monocytic, myelomonocytic, and histiocytic or macrophage tumor cells in culture. Cancer Research 1977; 37(2):546-550. PMID: 318922. + +## Materials + +### Equipment +- Personal protective equipment (sterile gloves, laboratory coat, safety visor etc.) +- Water bath set to 37 °C +- Microbiological safety cabinet at the appropriate containment level +- Incubator at 37 °C and 5% (v/v) CO₂ atmosphere +- Centrifuge +- Inverted microscope +- Neubauer counting chamber and cover slips or cell counter +- Freeze container (e.g., Mr. Frosty, Nalgene) +- -80 °C freezer +- Liquid nitrogen tank + +### Materials +- Cell culture flasks: 25 and 150 cm² +- Sterile serological pipette +- Sterile Pasteur pipette +- Sterile filter tips +- Sterile reaction tubes (15 and 50 ml) +- Sterile cell scraper +- Sterile cryotubes (2 ml) + +### Chemicals +- Dulbecco’s Modified Eagle’s Medium (DMEM, 4500 mg/L glucose, sterile, suitable for cell culture) pre-warmed to 37 °C +- Fetal bovine serum (Merck/Sigma Aldrich, Cat. No. S0615) +- L-Glutamine–penicillin–streptomycin solution (L-glutamine 200 mM, streptomycin 10 mg/mL, penicillin 10,000 units) +- Sterile dimethyl sulfoxide (DMSO) +- 2-Propanol + +## Procedure + +### Thaw J774A.1 Cells +1. Transfer the cryotube with the cell suspension from the liquid nitrogen tank to the cell culture on ice. + + **Note:** Wear protective goggles, gloves, and a gown when handling liquid nitrogen. + +2. Thaw the cells immediately in a 37 °C water bath until no more ice chunks are visible. + + **Note:** This procedure and the next steps must be performed quickly because the cryoprotectant (DMSO) is cytotoxic above 4 °C. + +3. Mix the thawed 1 ml cell suspension with 14 ml pre-warmed high-glucose DMEM (containing 10% v/v fetal bovine serum (FBS) and 0.1 mg/ml glutamine–penicillin–streptomycin (PSG)) in a 15 ml tube and resuspend well but gently. +4. Centrifuge the cell suspension for 10 min with 300 x g at room temperature and carefully aspirate the supernatant with a sterile Pasteur pipette. +5. Resuspend the cell pellet in 5 ml fresh DMEM (containing 10% v/v FBS and 0.1 mg/ml PSG) and transfer the cell suspension to a 25 cm² cell culture flask and incubate it in a 37 °C incubator with 5% v/v CO₂ atmosphere for growth. +6. Leave the cells in the incubator and observe cell growth every two days. At 60-80% confluency, remove supernatant, detach the adherent cells in 5 ml fresh DMEM (containing 10% v/v FBS and 0.1 mg/ml PSG) using a cell scraper. +7. Fill the cell suspension with fresh pre-warmed DMEM (containing 10% v/v FBS and 0.1 mg/ml PSG) to a total volume of 50 ml and transfer into a 150 cm² cell culture flask and incubate in a 37 °C incubator with a 5% v/v CO₂. + +### Passaging J774A.1 Cells +8. Control the cells using an inverted microscope to assess the degree of confluency and check for morphological changes or any contamination. + + **Note:** Elongated cells indicate activation, non-activated cells have a round shape. + +9. For a 150 cm² culture flask, transfer the used DMEM into a tube (50 ml) and centrifuge at 400 x g for 5 min at room temperature. + + **Note:** Perform all steps in a sterile environment to avoid contamination. + +10. Add 10 ml fresh DMEM (containing 10% v/v FBS and 0.1 mg/ml PSG) to the cell monolayer and aspirate the supernatant. + + **Note:** DMEM (containing 10% v/v FBS and 0.1 mg/ml PSG) should be pre-warmed to 37 °C. + +11. Add an additional 10 ml DMEM (containing 10% v/v FBS and 0.1 mg/ml PSG), detach cells with a cell scraper, and transfer the suspension into a new tube. +12. Label the cell culture flask with the date and the number of passages. + + **Note:** One flask can be used for 10 to 20 passages. Replace the flask if dead cells are observed. + +13. Dilute the cell suspension for passaging (at 80% confluency, choose a 1:5 dilution with fresh DMEM (containing 10% v/v FBS and 0.1 mg/ml PSG) to a total volume of 10 ml. +14. Transfer the defined amount (e.g., 2 ml at a 1:5 dilution) back into the culture flask and add 48 ml fresh DMEM (containing 10% v/v FBS and 0.1 mg/ml PSG). +15. When subcultured cells reach 60-80% confluency (normally 2-3 days), repeat cell passaging as outlined above. + +### Seeding J774A.1 Cells for Subsequent Experiments +16. Use the remaining cell suspension from the subculture for seeding cells for further experiments. +17. Use the Neubauer counting chamber (or an automated cell counter) to determine the cell number per ml. + + **Note:** 10 µl cell suspension should be pipetted under the cover slip. Usually, a 1:20 dilution of the cell suspension is needed for counting. + +18. Prepare the required volume for seeding the cell suspension with fresh DMEM containing 0.1 mg/ml PSG. + + **Calculation Example:** + + |A|B| + |-|-| + |Determined cell number in suspension|4 x 10⁶ cells/ml| + |Total volume for seeding|5 ml| + |Required cell number for seeding|3 x 10⁶ cells in 5 ml| + |Calculation:| Required cell suspension = 0.75 ml| + |Fresh high-glucose DMEM (containing 0.1 mg/ml PSG) to add| 4.25 ml| + +19. Transfer the prepared cell suspension into culture plates or cell culture flasks as required. + + **Example of Cell Density Adjustments:** + + |A|B|C|D|E|F|G| + |-|-|-|-|-|-|-| + |25 cm² flask|6-well plate|12-well plate|24-well plate|48-well plate|96-well plate| + |Cell number/flask or well|3 x 10⁶|1 x 10⁶|0.5 x 10⁶|0.25 x 10⁶|0.1 x 10⁶|0.05 x 10⁶| + |Volume/flask or well|5 ml|2 ml|1 ml|0.5 ml|0.3 ml|0.2 ml| + +20. Label the cell culture vessel with the date and passage number of the cell culture before placing it back into the 37 °C incubator with 5% v/v CO₂ atmosphere. Allow cells to settle for 24 h. + +### Freezing J774A.1 Cells +21. Prepare a freeze container (e.g., Mr. Frosty, Nalgene) with 2-propanol and cool at 4 °C. Cooling in 2-propanol ensures a slow and gentle temperature decrease (rate of -1 °C/min is optimal). +22. Prepare freezing medium: High-glucose DMEM containing 20% v/v FBS, 0.1 mg/ml PSG, and 10% v/v sterile DMSO. +23. Count the cells using a Neubauer counting chamber or cell counter, calculate cell number needed for cryotubes and freezing medium. Adjust cell concentration to 1 x 10^7 cells/ml. +24. Distribute 1 ml of the cell suspension in freezing medium into each cryotube and place in freeze container, then into a -80 °C freezer overnight. Transfer frozen cryotubes to liquid nitrogen for long-term storage next day. + +**End of output** +``` \ No newline at end of file diff --git a/markdown-output/cell-culture-of-raw264-7-cells-cqtfvwjn.md b/markdown-output/cell-culture-of-raw264-7-cells-cqtfvwjn.md new file mode 100644 index 0000000000000000000000000000000000000000..2515c65a5d7f12e8ed1ca8be40df01d70ed06c73 --- /dev/null +++ b/markdown-output/cell-culture-of-raw264-7-cells-cqtfvwjn.md @@ -0,0 +1,133 @@ +```markdown +Goal/Experiment: +The goal of this experiment is to culture the RAW264.7 cell line, which is widely used to study immune function, inflammation, and cancer biology. The protocol describes the preparation, thawing, passaging, seeding, and freezing of RAW264.7 cells. + +# Cell Culture of RAW264.7 Cells +_Sijia Liao1, Lisa Börmel1, Maria Wallert1, Stefan Lorkowski1_ + +1Institute of Nutritional Science, Friedrich Schiller University Jena + +## Abstract +RAW264.7 cells are a macrophage-like cell line derived from a male BALB/c mouse ascites tumor induced by Abelson Leukemia Virus (A-MuLV). Due to their rapid proliferation and ease of handling, the RAW264.7 cell line is widely used to study immune function, inflammation, and cancer biology. The cells have a doubling time of about 11 to 30 hours and should be cultured under specific conditions to ensure proper growth and functionality. This document details the procedures for culturing RAW264.7 cells, including growth, passaging, preparing cells for experiments, and freezing cells for long-term storage. + +## Materials + +### Equipment +- Personal protective equipment (sterile gloves, laboratory coat, safety visor etc.) +- Water bath set to 37°C +- Microbiological safety cabinet at the appropriate containment level +- Incubator at 37°C and 5% (v/v) CO₂ atmosphere +- Centrifuge +- Inverted microscope +- Neubauer counting chamber and cover slips or cell counter +- Freeze container (e.g. Mr. Frosty, Nalgene) +- -80°C freezer +- Liquid nitrogen tank + +### Materials +- Cell culture flasks: 25 and 150 cm² +- Sterile serological pipette +- Sterile Pasteur pipette +- Sterile filter tips +- Sterile reaction tubes (15 and 50 ml) +- Sterile cell scraper +- Sterile cryotubes (2 ml) + +### Chemicals +- Dulbecco's Modified Eagle's Medium (DMEM) (4500 mg/L glucose, sterile, suitable for cell culture) pre-warmed to 37°C +- Fetal bovine serum (Merck/Sigma Aldrich, Cat. No. S0615) +- L-Glutamine–penicillin–streptomycin solution (L-glutamine 200 mM, streptomycin 10 mg/mL, penicillin 10,000 units) +- Sterile dimethyl sulfoxide (DMSO) +- 2-propanol + +### Safety Warnings +**Biosafety level 2** applies to the use of the RAW264.7 cell line, as it polytropic murine leukemia virus (MLV) has been detected in the cell culture supernatant of these cells, in addition to replication-competent, ecotropic MLV. + +## Thawing RAW264.7 Cells + +1. **Transfer the cryotube** with the cell suspension from the liquid nitrogen tank to the cell culture on ice. + > **Note:** Wear protective goggles, gloves, and a gown when handling liquid nitrogen to avoid cold burns. + +2. **Thaw the cells immediately** in a 37 °C water bath until no more ice chunks are visible. + > **Note:** Perform quickly as DMSO is cytotoxic above 4°C. + +3. **Mix the thawed 1 ml cell suspension** with 14 ml pre-warmed high-glucose DMEM containing 10% (v/v) FBS and 0.1 mg/ml glutamine–penicillin–streptomycin (PSG) solution in a 15 ml tube and resuspend well but gently. + +4. **Centrifuge the cell suspension** for 10 min with 300 x g at room temperature and carefully aspirate the supernatant with a sterile pasteur pipette. + +5. **Resuspend the cell pellet** in 5 ml fresh high-glucose DMEM and transfer the cell suspension to a 25 cm² cell culture flask and place it in a 37°C incubator with 5% CO₂ atmosphere for growth. + +6. **Leave the cells in the incubator** and observe the cell growth every two days. Once the cells reach 60 to 80% confluency, remove the supernatant and detach the adherent cells in 5 ml fresh high-glucose DMEM using a cell scraper. + +7. **Fill the cell suspension with fresh pre-warmed high-glucose DMEM** to a total volume of 50 ml and transfer the whole cell suspension into a 150 cm² cell culture flask and put the flask in a 37°C incubator with a 5% CO₂ atmosphere. + +## Passaging RAW264.7 Cells + +8. **Control the cells** using an inverted microscope to assess the degree of confluency and perform morphological checks to confirm the absence of bacterial and fungal contamination. + +9. For a **150 cm² cell culture flask**, transfer the whole used DMEM in a tube (50 ml) and centrifuge at 400 x g for 5 min at room temperature. + > **Note:** Perform all steps onwards in a sterile environment to avoid contamination. + +10. **Add 10 ml fresh high-glucose DMEM** to the cell monolayer and aspirate the supernatant. + +11. **Add an additional 10 ml fresh high-glucose DMEM**, detach cells carefully with a cell scraper, and transfer the whole cell suspension into a new tube. + +12. **Label the cell culture flask** with the date and the number of passaging. + +13. **Dilute the cell suspension** (at 80% confluency) with high-glucose DMEM (1:5 dilution with a total volume of 10 ml). + +14. **Transfer the defined amount** (e.g. 2 ml at 1:5 dilution) back into the culture flask and add 28 ml fresh high-glucose DMEM and 15 ml used DMEM from subculture. + +15. When **subcultured cells reach 60 to 80% confluency**, repeat the cell passaging as outlined above. + +## Seeding RAW264.7 Cells for Subsequent Experiments + +16. Use the **remaining cell suspension** from the subculture for seeding cells for further experiments. + +17. Use the **Neubauer counting chamber** (or automated cell counter) to determine the cell number per milliliter cell suspension. + > **Note:** If using a Neubauer counting chamber, pipette 10 μl cell suspension under the cover slip and use a 1:10 to 1:50 dilution for counting the cells. + +18. **Prepare the required volume** for seeding the cell suspension with fresh high-glucose DMEM (final cell suspension should contain two-thirds fresh medium and one-third old medium). + + Example calculation: + + | A | B | + | ------------------------------ | ----------------------------- | + | Determined cell number of suspension | 5 x 10^6 cells per ml | + | Total volume for seeding | 5 ml | + | Required cell number for seeding | 2 x 10^6 cells in 5 ml | + | Calculation: | | + | Required cell suspension | 0.40 ml | + | Fresh high-glucose DMEM | 2.93 ml | + | Used culture medium to add | 1.67 ml | + +19. **Transfer the prepared cell suspension** into culture plates or cell culture flasks as required. + +| A | B | C | D | E | F | G | +|-----------|--------------------|----------------|----------------|----------------|----------------|----------------| +| 25 cm² flask | 6-well plate | 12-well plate | 24-well plate | 48-well plate | 96-well plate | +| Cell number/flask or well | 2 x 10^6 | 0.8 x 10^6 | 0.3 x 10^6 | 0.15 x 10^6 | 0.08 x 10^6 | 0.03 x 10^6 | +| Volume/flask or well | 5 ml | 2 ml | 1 ml | 0.5 ml | 0.3 ml | 0.2 ml | + +20. **Label the cell culture vessel** with the date and passage number of the cell culture. Put the seeded cells back into the 37°C incubator with a 5% CO₂ atmosphere and allow the cells to settle for 24 hours. + +## Freeze RAW264.7 Cells + +21. **Prepare freeze container**: Fill the freeze container (e.g. Mr. Frosty, Nalgene) with 2-propanol and cool it at 4°C. + +22. **Prepare freezing medium**: high-glucose DMEM containing 20% (v/v) FBS, 0.1 mg/ml PSG and 10% (v/v) sterile DMSO. + +23. **Count the cells** with a Neubauer counting chamber or cell counter, calculate the number of needed cryotubes and freezing medium (adjust cell concentration to 1x10^7 cells/ml). + +24. **Distribute the cell suspension** in freezing medium into each cryotube, place it into the freeze container, and subsequently into -80°C freezer for cooling down overnight. Next day, transfer cryotubes to liquid nitrogen for long-term storage. + +## References + +[1] Raschke WC, Baird S, Ralph P, Nakoinz I. Functional macrophage cell lines transformed by Abelson leukemia virus. Cell 1978; 15(1):261-267. DOI: [10.1016/0092-8674(78)90101-0](https://doi.org/10.1016/0092-8674(78)90101-0). PMID: 212198. + +[2] Stellungnahme der Zentralen Kommission für die Biologische Sicherheit (ZKBS) zur Neueinstufung der Zelllinie RAW 264.7 gemäß § 5 Abs. 1 GenTSV (Az. 6790-05-04-45, February 2012) + +[3] Hartley JW, Evans LH, Green KY, Nagashfar Z, Macias AR, Zerfall PM, Ward JM. Expression of infectious murine leukemia viruses by RAW264.7 cells, a potential complication for studies with a widely used mouse macrophage cell line. Retrovirology 2008; 4(5):1. DOI: [10.1186/1742-4690-5-1](https://doi.org/10.1186/1742-4690-5-1). PMID: 18177500. + +endofoutput +``` diff --git a/markdown-output/cell-free-3pga-energy-solution-8pshvne.md b/markdown-output/cell-free-3pga-energy-solution-8pshvne.md new file mode 100644 index 0000000000000000000000000000000000000000..fac7b164f1fe3af6bbd7b492425fecb156ef3659 --- /dev/null +++ b/markdown-output/cell-free-3pga-energy-solution-8pshvne.md @@ -0,0 +1,163 @@ +```markdown +# Goal/Experiment: +Energy solution for E. coli lysate based on 3PGA. Adapted from Sun 2013 and Cai 2015. Successfully implemented at the University of Edinburgh by Nadanai Laohakunakorn, LBNC-EPFL by Zoe Swank. + +# Cell-free 3PGA Energy Solution + +**Nadanai Laohakunakorn** +*University of Edinburgh* +**May 13, 2020** +**Citation:** Nadanai Laohakunakorn (05/13/2020). Cell-free 3PGA energy solution. [https://dx.doi.org/10.17504/protocols.io.8pshvne](https://dx.doi.org/10.17504/protocols.io.8pshvne) + +## Abstract +Energy solution for E. coli lysate based on 3PGA. Adapted from Sun 2013 and Cai 2015. Successfully implemented at the University of Edinburgh by Nadanai Laohakunakorn, LBNC-EPFL by Zoe Swank. + +### References +- Sun ZZ, Hayes CA, Shin J, Caschera F, Murray RM, Noireaux V (2013). Protocols for implementing an Escherichia coli based TX-TL cell-free expression system for synthetic biology. *Journal of Visualized Experiments: JoVE*. [https://doi.org/10.3791/50762](https://doi.org/10.3791/50762) +- Cai Q, Hanson JA, Steiner AR, Tran C, Masikat MR, Chen R, Zawada JF, Sato AK, Hallam TJ, Yin G (2015). A simplified and robust protocol for immunoglobulin expression in Escherichia coli cell-free protein synthesis systems. *Biotechnology Progress*. [https://doi.org/10.1002/btpr.2082](https://doi.org/10.1002/btpr.2082) + +## Materials Text +- **Amino acids** LAA21-1KT Sigma +- **Mg-glutamate** 49605-250G Sigma +- **K-glutamate** 49601-500G Sigma +- **DTT** 10708984001 Sigma +- **NTP set** R1481 ThermoFisher +- **tRNA** 10109541001 Sigma +- **CoA** C4282-10MG Sigma +- **NAD** 10127918001 Sigma +- **cAMP** A9501-1G Sigma +- **folinic acid** RHI5141-1G Sigma +- **spermidine** S9266-1G Sigma +- **PEG-8000** 89510 Sigma +- **3PGA** P8877-1G Sigma +- **HEPES** H3375-100G Sigma +- **Tris base** T1503-100G Sigma +- **KOH** +- **Mass balance** + +## Protocol + +### 1. Make Stock Solution of Amino Acids, 1000 µL at 50 mM + +#### 1.1 Weigh Each Amino Acid (Excluding Tyrosine) +Weigh each amino acid on parafilm paper, record the weight, and carefully add together in one tube. +**Alternative:** Carrying out at 10x quantities makes weighing much easier. + +| Amino acid | Weight (mg) | +|---------------|--------------| +| Alanine | 4.5 | +| Arginine | 8.7 | +| Asparagine | 6.6 | +| Aspartate | 6.7 | +| Cysteine | 6.1 | +| Glutamate | 7.3 | +| Glutamine | 7.3 | +| Glycine | 3.8 | +| Histidine | 7.8 | +| Isoleucine | 6.6 | +| Leucine | 6.6 | +| Lysine | 9.1 | +| Methionine | 7.5 | +| Phenylalanine | 8.3 | +| Proline | 5.8 | +| Serine | 5.3 | +| Threonine | 6.0 | +| Tryptophan | 10.2 | +| Valine | 5.9 | + +#### 1.2 Prepare Stock Solution in Water +Add 1000 µL of deionized water to the tube to make a 50 mM stock solution, vortex to mix, and adjust pH with KOH to ~5.2 (approximately 80 µL of 1M KOH and 920 µL dH2O required). The pH can be roughly measured by spotting 1-2 µL of the solution on appropriate pH paper. If the powder does not fully dissolve, add up to ~50 µL more of 1M KOH. pH will be ~8. Keep solution **on ice**. + +#### 1.3 Weigh Tyrosine and Prepare Stock Solution +Weigh tyrosine and add to a separate tube. + +| Amino acid | Weight (mg) | +|------------|-------------| +| Tyrosine | 9.1 | + +#### 1.4 Add KOH to Tyrosine +Add 900 µL of 1 mM KOH and dissolve as far as possible (tyrosine powder will not be entirely soluble). + +#### 1.5 Fully Dissolve Tyrosine +Add 50 µL of 15% KOH, which should fully dissolve the powder. Then add 50 µL of deionized water. Vortex well, and measure pH, which should be around ~11-12. Keep solution **on ice**. + +#### 1.6 Storage +Keep the tyrosine and the rest of the amino acids separate. If storage is required, keep at -80°C (flash-freezing with liquid nitrogen is optional). + +### 2. Make Stock Solution of Other Components + +#### 2.1 Prepare 2M Stock Solution of Tris Base +| Component | Mass to add (g) | Water to add (mL) | Final concentration | +|-----------|------------------|-------------------|---------------------| +| Tris base | 60.57 | 250 | 2 M | + +#### 2.2 Prepare 1M KOH Stock and 15% KOH Stock + +#### 2.3 Prepare Stock Solutions +Weigh out and make the following stock solutions. Four species require titration; their pH can be approximately measured by spotting 1µL on appropriate pH paper. + +| Component | Mass to add (g) | Water to add (µL) | Tris to add (µL) | Final concentration | Notes | +|-----------------------------------------|------------------|-------------------|------------------|---------------------|--------------------------------------| +| L-glutamic acid monopotassium salt (K-glutamate) | 1.219 | 1000 | — | 6 M | — | +| L-glutamic acid hemimagnesium salt (Mg-glutamate)| 0.3886 | 1000 | — | 1 M | — | +| DTT | 0.1543 | 1000 | — | 1 M | — | +| CoA | 0.0498 | 1000 | — | 65 mM | — | +| NAD | 0.1161 | to 1000 | ~90 | 175 mM | pH 7.5-8, titrate with 2M tris | +| Folinic acid | 0.0160 | 1000 | — | 33.9 mM | — | +| cAMP | 0.2139 | to 1000 | ~365 | 650 mM | pH 8, titrate with 2M tris | +| 3-PGA | 0.2604 | to 1000 | ~540 | 1.4 M | pH 7.5, titrate with 2M tris | +| HEPES | 0.4766 | to 1000 | — | 2 M | pH 8, titrate with 1M KOH (~25 µL required) | +| Spermidine | 23.55 µL (volume) | 126.45 | — | 1 M | Heat up stock solution in your hand | +| PEG-8000 | 50 | 50 | — | 50% | — | + +### 3. Preparation of Final Energy Solution (4x) +This energy solution will form 25% of the final reaction volume. + +#### 3.1 Add Components +Add the components together to produce the final energy solution, in the following order (not critical), vortexing after each addition and keeping the tube **on ice**. + +| Component | Stock (mM) | Final concentration (mM) | Volume to add (µL) | +|--------------------|------------|--------------------------|--------------------| +| HEPES | 2000 | 200 | 100 | +| Water | — | — | 114.2 | +| ATP | 100 | 6 | 60 | +| GTP | 100 | 6 | 60 | +| CTP | 100 | 3.6 | 36 | +| UTP | 100 | 3.6 | 36 | +| tRNA (in mg/mL) | 43.75 | 0.8 | 18.29 | +| CoA | 65 | 1.04 | 16 | +| NAD | 175 | 1.32 | 7.54 | +| cAMP | 650 | 3 | 4.62 | +| Folinic acid | 33.9 | 0.27 | 8.02 | +| Spermidine | 1000 | 4 | 4 | +| 3-PGA | 1400 | 120 | 85.7 | +| Amino acids | 50 | 6 | 120 | +| Tyrosine | 50 | 3 | 60 | +| PEG-8000 | 50% | 8% | 160 | +| Mg-glutamate | 1000 | 42 | 42 | +| K-glutamate | 6000 | 400 | 66.67 | +| DTT | 1000 | 1 | 1 | +| **Total** | — | — | 1000 | + +#### 3.2 Measure and Record pH +Measure and record the pH of the final solution using pH paper (should be ~8). Aliquot into storage tubes (25 µL recommended) and (optionally) flash freeze in liquid nitrogen. + +#### 3.3 Storage +Store at -80°C. + +#### 3.4 Calibration for Maximum Yield +It is possible to calibrate the energy solution for maximum yield by determining optimum concentrations for Mg-glutamate, then K-glutamate. + +- **Sun 2013 Report Confirms:** + - Final optimal concentrations of 4.5-10.5 mM Mg-glutamate, 40-160 mM K-glutamate. +- **Kwon and Jewett 2015 Report:** + - 12 mM Mg-glutamate, 130 mM K-glutamate. + +This step is not required if only functional extract is needed. + +For optimization of the energy solution, add all components apart from PEG, Mg-glutamate, and K-glutamate. This makes a solution of **731.33 µL** and can be aliquoted into 10 tubes of **73.13 µL**. + +--- + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/cellprofiler-pipeline-to-obtain-pearson-39-s-corre-cqnivvce.md b/markdown-output/cellprofiler-pipeline-to-obtain-pearson-39-s-corre-cqnivvce.md new file mode 100644 index 0000000000000000000000000000000000000000..d6b418f70060d14063021391eebab4d5c72ca4ec --- /dev/null +++ b/markdown-output/cellprofiler-pipeline-to-obtain-pearson-39-s-corre-cqnivvce.md @@ -0,0 +1,305 @@ +```markdown +# Goal/Experiment: +To quantify the intensity of endogenous TMEM55B or pRab10 in LRRK2 R1441C or VPS35 D620N MEF cells transfected with a Myc-RILPL1 plasmid, using CellProfiler to obtain Pearson's correlation coefficients. + +# CellProfiler Pipeline to Obtain Pearson's Correlation Coefficients for TMEM55B or pRab10 and RILPL1 + +**Chloe A Hecht^1,2**, **Suzanne Pfeffer^1,2** + +^1Department of Biochemistry, Stanford University School of Medicine; ^2Aligning Science Across Parkinson's Disease + +## Abstract + +We present here a CellProfiler software pipeline to quantify the intensity of endogenous TMEM55B or pRab10 in LRRK2 R1441C or VPS35 D620N MEF cells transfected with a Myc-RILPL1 plasmid detected using an anti-Myc antibody in conjunction with 4X expansion super-resolution microscopy or regular immunofluorescence microscopy. This protocol works in conjunction with Nikon Spinning disk confocal images acquired with Metamorph software (Molecular Devices, LLC) or Zeiss laser scanning confocal microscope acquired using Zen 3.4. Acquired files have a .nd or a .czi format respectively; we use the associated .TIF raw files in this pipeline. Images are maximum intensity projected from the Z-stack acquired during imaging and used for CellProfiler analysis. + +## Materials + +- Latest edition CellProfiler 4.04 (or later) +- Maximum intensity projection raw .TIF files that accompany .nd files by Metamorph +- or +- Maximum intensity projection raw .TIF files that accompany .czi files by Zen 3.4 + +## Protocol + +### 1. Overview +The method involves the following steps: +1. Import data and extract metadata from file names +2. Group individual channels from each image +3. Rescale image intensities +4. Identify primary objects as RILPL1 positive cells +5. Measure colocalization +6. Save identified masked cells +7. Export to Spreadsheet + +### 2. Load Images +Select **Images** module at the upper right and drop images as indicated. + +### 3. Metadata Extraction +In the **Metadata** tab: +- Extract metadata: Yes. + +### 3.1. Metadata Extraction Details +- **Metadata extraction method**: Extract from image file headers +- **Extract metadata from**: All images +- **Metadata data type**: Text +- Hit "Extract metadata" +- Hit "Update" to populate the metadata field + +### 4. Names and Types + +#### 4.1. For .nd files captured using Metamorph +In the **NamesAndTypes** tab: +- Assign a name to: Image matching rules +- Process as 3D? No +- Match all of the following rules: `File/Does/Contain/w1405` + - Name to assign to these images: DAPI + - Select the image type: Grayscale image + - Set intensity range from: Image metadata +- Add two more channels and repeat the above commands, naming the channels: + - `File/Does/Contain: w2488` as RILPL1 + - `File/Does/Contain: w3561` as TMEM55b +- Hit "Update" so Cellprofiler will process the commands and sort the images based on the channels. + +#### 4.2. For .czi files acquired using Zen 3.4 +In the **NamesAndTypes** tab: +- Add two more images using "add another image tab" at the bottom + +#### 4.3. Specify Image Matching Rules + +**First Image Block:** +- Assign a name to: Image matching rules +- Process as 3D? No +- Match all of the following rules +- Select the rule criteria: Metadata does HaveC matching, 0 +- Name to assign these images: TMEM55B +- Select the image type: Grayscale image +- Set intensity range from: Image metadata + +**Second Image Block:** +- Assign a name to: Image matching rules +- Process as 3D? No +- Match all of the following rules +- Select the rule criteria: Metadata does HaveC matching, 0 +- Name to assign these images: RILPL1 +- Select the image type: Grayscale image +- Set intensity range from: Image metadata + +**Third Image Block:** +- Assign a name to: Image matching rules +- Process as 3D? No +- Match all of the following rules +- Select the rule criteria: Metadata does HaveC matching, 0 +- Name to assign these images: DAPI +- Select the image type: Grayscale image +- Set intensity range from: Image metadata +- Image set matching method: Order +- Hit "update" at the bottom +- The chart should populate with values in three channels: TMEM55B, RILPL1, and DAPI. + +### 5. Define Image Groups + +In the **Groups** tab: +- **Do you want to group your images?** Yes +- **Metadata category**: treatment +- CellProfiler should sort based on the treatment as identified in the metadata file name expression tab. + +### 6. Rescaling Intensities + +Using the "+" sign at the bottom next to Adjust Modules, add **RescaleIntensity** module: +- Select the input image: RILPL1 +- Name the output image: rescale_RILPL1 +- Rescaling method: Divide each image by the same value +- Divisor value: 0.08 for R1441C MEF or 0.3 for VPS35 D620N MEF + +**Note:** +- The RILPL1 channel should be significantly oversaturated. This is so cell profiler can easily identify mycRILPL1 positive cells, and not cells without mycRILPL1 transfection. +- This can be checked by clicking "Start Test Mode" and hitting the green triangle next to the RescaleIntensity module. + +### 7. Identify Primary Objects + +Add **IdentifyPrimaryObjects** module from the "+" sign at the bottom: +- **Use advanced settings?** Yes +- Select the input image: rescale_RILPL1 +- Name the primary objects to be identified: RILPL1cell +- Diameter of objects, in pixel units: 50 - 600 for R1441C MEF or 150-700 for VPS35 D620N MEF + +**Note:** +- This will be different for each image set. +- This can be checked by clicking "Start Test Mode" and hitting the green triangle next to the IdentifyPrimaryObjects module. + +- Discard objects outside the diameter range? Yes +- Discard objects touching the border of the image? No +- **Threshold strategy**: Global +- **Threshold method**: Otsu +- Two-class or three-class thresholding? Two classes +- Threshold smoothing scale: 1.7 for R1441C MEF or 3 for VPS35 D620N MEF +- Threshold correction factor: 0.6 for R1441C MEF or 0.4 for VPS35 D620N MEF + +**Note:** +- These values will need to be optimized for each image set. +- Again, this can be checked by clicking "Start Test Mode" and hitting the green triangle next to the IdentifyPrimaryObjects module each time a parameter is changed to find the best parameters for each image set. + +- Lower and upper bounds on threshold? 0 to 1 for R1441C MEF or 0.002 to 1.0 for VPS35 D620N MEF +- Log transform before thresholding? No +- Method to distinguish clumped objects? Intensity +- Method to draw dividing lines between clumped objects? Shape +- Automatically calculate the size of the smoothing filter for declumping? Yes +- Automatically calculate the size of the smoothing filter for declumping? Yes +- Automatically calculate minimum allowed distance between local maxima? Yes +- Speed up by using lower-resolution images to find local maxima? Yes +- Display accepted local maxima? No +- Fill holes in identified objects? After both thresholding and declumping +- Handling of objects if an excessive number of objects are identified? Continue + +### 8. Measure Colocalization + +Add **MeasureColocalization** module from the "+" sign at the bottom: +- **Select images to measure**: pRab10 or TMEM55B and RILPL1 +- **Set threshold as a percentage of maximum intensity for the images**: 15 +- **Select where to measure correlation**: Within objects, RILPL1cell + +**Note:** +- This allows for colocalization to be measured only in mycRILPL1 positive cells. + +- Run all metrics? No +- Calculate correlation and slope metrics? Yes +- Select "No" for the rest of the colocalization measures + +**Note:** +- For our purposes, we were only interested in the Pearson’s Correlation coefficient. + +### 9. Save Identified Cells + +Add the **ConvertObjectstoImage** module and the **SaveImages** module from the + at the bottom: + +#### 9.1. ConvertObjectstoImage Module +- Select the input object: RILPL1cell +- Name the output image: IdentifyRILPL1 +- Select the color format: Color +- Select the colormap: Default + +#### 9.2. SaveImages Module +- Select type of image to save: Image +- Select the image to save: IdentifyRILPL1 +- Constructing file names: From image filename +- Image name for file prefix: RILPL1 +- Append a suffix to the image file name? No +- Saved file format: .tiff +- Image bit depth: 8-bit integer +- Save with lossless compression? Yes +- Output file location: Choose a folder where images should be saved +- Overwrite existing files without warning? Yes +- When to save? Every cycle +- Record the file and path information to the saved image? No +- Create subfolders in the output folder? No + +**Note:** +- These two modules allow you to save CellProfiler’s interpretations of identified cells. This is useful when checking each image to ensure a good interpretation. + +### 10. Export to Spreadsheet + +Add the **ExportToSpreadsheet** module from the + at the bottom: + +#### 10.1. ExportToSpreadsheet Module +- Select the column delimiter: Tab +- Output file location: Choose a folder where you want the images to be saved. +- Add a prefix to file names? Yes. Add the date and experiment identifier. i.e. RILPL1March_ +- Overwrite existing files without warning? Yes + +**Note:** +- This allows you to run the pipeline multiple times without having to individually ask the pipeline to rewrite a file. + +- Add image metadata columns to your object data file? Yes +- Add image file and folder names to your object data file? Yes +- Representation of Nan/Inf: NaN +- Select measurements to export? Yes + - Press the button to select measurements: Under "RILPL1cell" select: Correlation, FileName, and PathName +- Calculate the per-image mean values for object measurements? No +- Calculate the per-image median values for object measurements? No +- Calculate the per-image standard deviation values for object measurements? No +- Create GenePattern GCT file? No +- Export all measurement types? No +- Export all measurement types? Yes +- Click "Analyze Images" +- The pipeline will run and export the data to the folder previously specified + +# Expansion Microscopy TMEM55B and Myc-RILPL1 Correlation + +## 11. Overview +The method involves the following steps: +1. Import data and extract metadata from file names +2. Group individual channels from each image +3. Measure colocalization +4. Export the data + +## 12. Load Images +Select **Images** module at the upper right and drop images as indicated. + +## 13. Metadata Extraction +In the **Metadata** tab: +- Extract metadata: No. + +## 14. Names and Types + +In the **NamesAndTypes** tab: + +#### 14.1. Specify Image Matching Rules +- Assign a name to: Image matching rules +- Process as 3D? No +- Match all of the following rules: `File/Does/Contain/w1405` + - Name to assign to these images: DAPI + - Select the image type: Grayscale image + - Set intensity range from: Image metadata + +- Add two more channels and repeat the above commands, naming the channels: + - `File/Does/Contain: w2488` as RILPL1 + - `File/Does/Contain: w3561` as TMEM55b +- Hit "Update" so CellProfiler will process the commands and sort the images based on the channels. + +## 15. Define Image Groups + +In the **Groups** tab: +- **Do you want to group your images?** No + +## 16. Measure Colocalization + +Add **MeasureColocalization** module from the "+" sign at the bottom: + +#### 16.1. MeasureColocalization Module +- **Select images to measure**: TMEM55B and RILPL1 +- **Set threshold as a percentage of maximum intensity for the images**: 15 +- **Select where to measure correlation**: Across the entire image +- Run all metrics? No +- Calculate correlation and slope metrics? Yes +- Select "No" for the rest of the colocalization measures + +**Note:** +- For our purposes, we were only interested in the Pearson’s Correlation coefficient. + +## 17. Export to Spreadsheet + +Add the **ExportToSpreadsheet** module from the + at the bottom: + +#### 17.1. ExportToSpreadsheet Module +- Select the column delimiter: Tab +- Output file location: Choose a folder where you want the images to be saved. +- Add a prefix to file names? Yes. Add the date and experiment identifier. i.e. ExM_ +- Overwrite existing files without warning? Yes + +**Note:** +- This allows you to run the pipeline multiple times without having to individually ask the pipeline to rewrite a file. + +- Add image metadata columns to your object data file? Yes +- Add image file and folder names to your object data file? Yes +- Representation of Nan/Inf: NaN +- Select measurements to export? Yes + - Press the button to select measurements: Under "Image" select: Correlation, FileName, and PathName +- Calculate the per-image mean values for object measurements? No +- Calculate the per-image median values for object measurements? No +- Calculate the per-image standard deviation values for object measurements? No +- Create GenePattern GCT file? No +- Export all measurement types? No +- Export all measurement types? Yes + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/chapter-1-handling-and-housing-bmgdk3s6.md b/markdown-output/chapter-1-handling-and-housing-bmgdk3s6.md new file mode 100644 index 0000000000000000000000000000000000000000..4a0756b7650c6340124bd0a4af2d555b9c93bd78 --- /dev/null +++ b/markdown-output/chapter-1-handling-and-housing-bmgdk3s6.md @@ -0,0 +1,108 @@ +```markdown +# Goal/Experiment: +The goal of this protocol is to effectively and safely handle, transport, house, and release vultures during rehabilitation. This guide aims to ensure the welfare of the birds while minimizing stress and injury risk to both the vultures and handlers. + +# Chapter 1: Handling and Housing + +## Abstract +Rehabilitation of any injured wild animal is an honorable and laudable activity. However, in taking any animal into care, the rescuer accepts the responsibilities of ‘ownership’ for the animal, ensuring that all necessary treatment and care is provided to optimize its potential release back into the wild. It is vitally important, wherever possible, that one works closely with a local veterinary surgeon, who is prepared to aid and support when necessary (seeking additional support and advice themselves if needed). You may need to have access to appropriate veterinary services at all hours, which could mean using the services of more than one practice. It is thus worth proactively building relationships with your local veterinary practices, encouraging them to accompany you on rehabilitation courses and ensuring that all necessary resources and equipment are at hand before an incident occurs. + +## Protocol Information +**Authors:** +- Kerri Wolter + +**Published:** +- Oct 19, 2020 + +**Collections:** +- Vulture Rehabilitation Manual + +**Keywords:** +- vultures, vulture rehabilitation, vulture handling, vulture housing + +## Handling + +### Overview +For the average biologist or bird ringer, handling a vulture may be intimidating or even dangerous. Even ringers who are comfortable handling large eagles may find their hands full trying to process a vulture. There are several important differences in the best techniques used to process vultures versus other large raptors. The techniques detailed here have been refined over the past fifteen years of VulPro’s experience working with vultures in southern Africa. + +## Transportation +Transportation is potentially stressful for every bird and all care should be taken to keep the bird as calm as possible. The binding of a vulture’s feet, beaks, or wings is totally contraindicated and should never be done. If the transportation of vultures will be a common procedure, purpose-built crates are recommended. Each side has several large ventilation holes which are then covered with shade netting to reduce light and keep curious fingers out of the vulture’s reach. Each crate should be lined with a pre-cut swath of carpet. Carpets prevent the bird from sliding, are easier to clean, and are more shred-resistant than blankets. + +## Housing + +### Temporary Housing +Temporary housing should only be considered if the bird is to be housed for a few hours, up to 3 days, until the bird can be transported to a licensed vulture rehabilitation facility or more suitable housing. If the bird is to be housed longer than a few days, further arrangements must be considered. + +### Long-term Housing +Housing considerations should be made if the bird is to be housed for more than a few days. One of the most important considerations is socialization. Vultures are social creatures and they need to see, hear, and interact with other vultures of their same species. VulPro has shown that social interaction greatly increases their wellbeing and improves the chances of a positive outcome. + +#### Enclosures +Enclosures should be constructed with strong but flexible materials to prevent the birds from injuring themselves when flying around the enclosures and impacting with the sides. + +# Feeding and Maintenance + +## Enclosure Hygiene and Maintenance +- Remove left-over food twice weekly. +- Water ponds and baths must be refilled every day and cleaned every second day. + +## Feeding/Food Preparation +Vultures should be fed twice a week on whole carcasses and bone fragments. Whole carcass feeding is ideal for vultures as this provides the birds with the natural nutritional requirements as gained if living in the wild. Avoid using lead bullets which can fragment into the carcasses, contributing to lead poisoning, a significant cause of mortality in vultures. + +# Checklist: Basic Required Rehabilitation Equipment + +## Transportation +- Vulture-specific crates +- Carpets +- Disinfectant + +## Medical Equipment +- Silicone/plastic tubing around 30 cm long for oral tubing (IV fluid lines can be recycled for this purpose) +- Electrolytes (Darrow’s and Ringer’s lactate solution) +- Needles (23-21 gauge) +- Intra-venous cannulas (Jelco® 18-20 gauge) +- Syringes (60, 20, 10, 5, 2 and 1 mL, range for drugs and gavage) +- Gauze +- VetWrap +- Elastoplast tape +- Cotton wool +- Scissors +- Super glue +- F10® SC Veterinary Disinfectant spray (water-based) +- PluroGen PluroGel® +- Karbadust® or Frontline® powder +- Necrospray (or honey or other appropriate wound applications) +- Various drugs + +# Detailed Instructions for Handling and Rehabilitating Vultures + +## Catching Vultures +1. Approach the bird in such a manner as to avoid scaring it away or causing unnecessary stress. +2. Once within close reach, grab its neck from behind first, just below the jawbone. +3. Reach around the neck just below the jaw bone with pressure on the sides of the neck to avoid suffocating the bird. +4. Quickly hug the bird just above the legs, enclosing the wings in the embrace. +5. Ensure the legs stay below your arm and the wings are under control by your embrace. +6. If the bird struggles, tighten your grip using the elbows to prevent wing escape. + +## Processing Vultures +1. Always minimize the duration of vulture restraint. Prepare all necessary equipment and staff in a shaded quiet location. +2. If the bird begins to overheat, spray cool water on its neck and body and complete the process as soon as possible. +3. Complete vulture processing within 20 minutes per bird. The absolute maximum processing time should not exceed 35 minutes. +4. Avoid holding a bird in a horizontal position for more than necessary. +5. When working with a bird on a table, four people are typically required ensuring no tying, taping, or binding of the bird. + +## Releasing +1. Choose a release site and direction considering that birds prefer to take off against the wind. +2. Release from a crate at ground level, not an elevated site. +3. Lower the bird's feet to ground, allowing the bird to stand before releasing. +4. Allow the bird to walk or fly away calmly, avoiding any panic before observing for any unusual behavior that could indicate heat exhaustion or injury. + +## Figures +- **Figure 1:** A vulture crate used as a processing table. +- **Figure 2:** Proper hand placement to stabilize a vulture head. +- **Figure 3:** Proper way to hold a large vulture. +- **Figure 4:** A vulture being processed on its sternum. +- **Figure 5:** Preparing to release the vulture. +- **Figure 6:** Releasing the vulture. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/chapter-10-medications-bmjkk4kw.md b/markdown-output/chapter-10-medications-bmjkk4kw.md new file mode 100644 index 0000000000000000000000000000000000000000..2688b6d773c223e7678df1a7ff43c4cad8df6a2a --- /dev/null +++ b/markdown-output/chapter-10-medications-bmjkk4kw.md @@ -0,0 +1,120 @@ +```markdown +Goal/Experiment: +This protocol provides information about medications for vulture rehabilitation and how to calculate drug dosage. + +# Chapter 10: Medications + +**Author:** Kerri Wolter +**Collections:** Vulture Rehabilitation Manual +**Keywords:** vultures, vulture rehabilitation, vulture medication +**License:** This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. +**Created:** Sep 21, 2020 +**Last Modified:** Oct 19, 2020 +**Protocol Integer ID:** 42316 + +This protocol provides information about medications for vulture rehabilitation and how to calculate drug dosage. + +## Medications for Vulture Rehabilitation + +Below is a list of the drugs most frequently used by the authors for vulture rehabilitation. The dosages listed below are those used and recommended based on our experience. However, it is always important to consult with your veterinarian. In addition to this, always check the information insert in the drug’s package to confirm the dosage recommended by the manufacturer (as on occasions the same drug may be available at different concentrations). Medications available in your region may not be the same as in South Africa and drug trade names and concentrations will vary. **Always consult your veterinarian!** + +Some facilities deworm their vultures as a matter of routine. We recommend testing birds on admission and only deworming a vulture if it is suffering from an overwhelming burden of parasites. Wild animals typically carry a low level of parasites and build up ‘resilience’ towards them. They can cope with a low level. On occasion, if this burden is removed by deworming, animals can become immunologically ‘naïve’ and future parasite infections pose a problem. + +### Use of Antibiotics + +The decision to treat with systemic antibiotics is not to be taken lightly. Development of antibiotic-resistant bacteria is always a concern. For this reason, antibiotics should always be used judiciously. In ideal circumstances and with ideal resources, a wild vulture presenting for rehabilitation should have blood collected for a complete blood count (CBC) at minimum. See Chapter 8 for CBC reference ranges for various African vulture species. A white blood cell count greater than the upper limit of the reference range may warrant treatment with antibiotics. However, in many rehabilitation situations, performing a CBC may not be practical or possible. In these cases, consultation with a veterinarian is recommended. + +Similarly, the use of antibiotics in wound treatment should ideally be informed by the collection of a swab from the wound, which is submitted to a lab for bacterial culture and antibiotic sensitivity testing. When not practical or possible, the following should be considered in deciding whether or not systemic antibiotic treatment should be used in wound treatment: cause of the wound and age of the wound (if known), depth, surface area, and body part affected, gross contamination of the wound, and overt evidence of infection (increased warmth, redness, swelling, and/or discharge such as pus). + +With the above-mentioned considerations in mind, there are certain conditions in which antibiotic treatment is always warranted: +- Compound (open) bone fractures +- Soft tissue wounds that expose bone, tendon, and/or ligament +- Wounds that are the result of an animal bite +- Severe bumble-foot +- Severe electrical burns that require removal of necrotic tissue +- Osteomyelitis, as determined by radiography or appearance of exposed bone +- Upper respiratory infections +- Whenever more than one dose of dexamethasone is given (generally for treatment of seizures) + +### Antimicrobial Agents + +| Agent (Tradename) | Route | Dosage | Interval | Indications | +|--------------------------|-------|----------|-----------------------------|------------------------------------------------------------------------------------------------------| +| Amoxacillin/Clavulanate (Synulox®) | IM | 125 mg/kg | 1 x / day. For at least 5 days. | A good, broad-spectrum, first-line choice. Used for a wide variety of infections including open wounds. | +| Enrofloxacin (Baytril®) | IM | 15 mg/kg | 1 x / day. For at least 5 days. | A second-generation fluoroquinolone- this drug should not be reached off the shelf readily. Broad-spectrum. Useful for septicaemia, respiratory infections. Do not use in chicks. | +| Florfenicol (Nuflor®) | IM | 0.17 mg/kg | 1 x / day. Every 3 days for at least 3 treatments. | Broad-spectrum Chloramphenicol derivative. Effective against respiratory tract infections, osteomyelitis (bone infection), pododermatitis (bumblefoot) etc. | +| Ciprofloxacin (Ciprod®) | PO (oral) | 50 mg/kg | 1 x / day. In combination with Clindamycin for open bone fractures 5-14 days | Broad-spectrum. In combination with Clindamycin for open bone fractures. | +| Clindamycin | PO | 150 mg/kg | 1 x / day. 5-14 days | In combination with Ciprofloxacin for open bone fractures. | +| Tobramycin (Tobrex® eye drops) | Topical (eye drops) | 2-3 drops per eye | 1 x / day. 5-7 days | Eye ulcers and infections. | +| Ofloxacin (Exocin®) | Topical (eye drops) | 2-3 drops per eye | 3-5 x / day. For at least 5 days. | Eye infections. | + +### NSAIDs: Non-Steroidal Anti-Inflammatory Drugs + +| Agent (Tradename) | Route | Dosage | Interval | Indications | +|--------------------------|-------|----------|-----------------------------|--------------------------------------------------| +| Meloxicam (Metacam®, Mobic®) | IM | 1 mg/kg | 1 x / day. Up to 3 days. | For pain and inflammation. | + +### Analgesic Agents + +| Agent | Route | Dosage | Interval | Indications | +|--------------------------|-------|----------|-----------------------------|--------------------------------------------------| +| Butorphanol | IM | 1 mg/kg | 1 dose per day. Can repeat daily as long as required. | For pain (fractures, large wounds, electrical burns). | + +### Steroid Agents + +| Agent | Route | Dosage/Interval | Indications | +|--------------------------|-------|------------------------------------------|--------------------------------------------------| +| Dexamethasone | IM | 4mg/kg (day 1), 2mg/kg (day 2), 1mg/kg (day 3), 0.5mg/kg (days 4-5), 0.5mg/kg (day 6) | For shock/trauma and seizures. Always in combination with antimicrobial. | +| Hydrocortisone (Cortisol®) | IV, IM | Consult with your vet as this depends on the frequency and strength of seizures. | Seizures only and ONLY if the seizures are severe and only on its own without any other medication. | + +### Antiparasitic Agents + +| Agent | Route | Dosage | Interval | Indications | +|--------------------------|--------|--------|-----------------------------|--------------------------------------------------| +| Ivermectin | PO, SC | 0.2 mg/kg | Once. Can repeat in 10-15 days. | De-wormer. | +| Carbaryl (Karbadust®) | Topical | As needed | | Feather lice, mites, flat flies, and other ectoparasites. | + +### Miscellaneous Agents + +| Agent | Route | Dosage | Interval | Indications | +|--------------------------|-------|----------|-----------------------------|--------------------------------------------------| +| Ca-EDTA | IM | 35 mg/kg | 2 x / day (every 12 h) for 5 days – again do your blood test to assess. | Lead poisoning. | +| Propofol | PO | 5 mg/kg | 1 x / day up to 10-20 days. | Wing-tip oedema after electrocutions. | + +### Mineral Support Agents + +| Agent (Tradename) | Route | Dosage | Interval | Indications | +|--------------------------|-------|----------|-----------------------------|--------------------------------------------------| +| Vitamin B12 (Catosol) | IM, SC, PO | 0.5 mg/g | Once, can repeat in 7 days. | To aid with poisoning and recovery. Also post-electrocution to protect the nerves. | +| Calcium 10% solution | IM | 1 ml/kg | Daily supplement | Hypocalcaemia, lead poisoning | + +### Emergencies + +| Agent (Tradename) | Route | Dosage/Interval | Indications | +|--------------------------|-------|------------------------------------------|--------------------------------------------------| +| Atropine (Atropen®) | Half dose IV, half dose IM | 2 mg/kg + PAM at 25 mg/kg | Repeated as necessary according to clinical response, typically every 2-4 hours. | Organophosphate poisoning, Bradycardia (slow heart rate) and cardiorespiratory arrest. | + +#### Safety Warnings + +*The only vulture-safe NSAID is Meloxicam (trade names Mobic, Metacam or Petcam)! Do not use ANY other NSAID. Research has shown all other tested NSAIDs to be toxic to vultures.* + +## How to Calculate Volume of the Drug + +1. Calculate the dose required by multiplying the required dose rate (mg/kg) by the bird’s weight (kg). This gives you the dose required in mg. +2. To calculate the volume of drug required, you need to divide this value by the drug concentration (mg/ml). + +**Example:** +- Drug dosage: 20 mg/kg +- Bird weight: 10 kg +- Concentration: 50 mg/ml + +\[ 20 \times 10 = 200 \text{ mg} \] + +This is the dose of drug a 10 kg bird must receive. + +\[ 200 \div 50 = 4 \text{ ml} \] + +This is the volume of drug to be administered. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/chapter-2-stabilizing-the-bird-conducting-an-initi-bmi7k4hn.md b/markdown-output/chapter-2-stabilizing-the-bird-conducting-an-initi-bmi7k4hn.md new file mode 100644 index 0000000000000000000000000000000000000000..c3a7dcce6a93c6db3affd1e8538c54808df9b052 --- /dev/null +++ b/markdown-output/chapter-2-stabilizing-the-bird-conducting-an-initi-bmi7k4hn.md @@ -0,0 +1,181 @@ +```markdown +Goal/Experiment: +To stabilize a vulture after intake and conduct an initial clinical exam. This protocol outlines steps to stabilize a bird, assess dehydration, and administer necessary fluid therapies. + +# Chapter 2: Stabilizing the Bird, Conducting an Initial Clinical Exam + +**Author:** +Kerri Wolter + +**Date Created:** +September 20, 2020 + +**Last Modified:** +October 19, 2020 + +**Protocol ID:** +42303 + +**Collection:** +[Vulture Rehabilitation Manual](https://protocols.io/view/chapter-2-stabilizing-the-bird-conducting-an-initi-bmjl7k4hn) + +**Keywords:** +- Vultures +- Vulture rehabilitation +- Vulture stabilization +- Rehydrating a bird +- Vulture clinical exam + +**License:** +This protocol is distributed under the terms of the [Creative Commons Attribution License](https://creativecommons.org/licenses/by/4.0/). + +--- + +## Abstract + +This protocol outlines steps to stabilize a bird after intake and the conduction of an initial clinical exam. + +## Guidelines + +With experience, it is possible to begin to understand the injury and state of the bird by observing it at a distance. Pay attention to the bird’s body posture and motion: +- **Reaction speed**: How quickly do they react to you approaching? +- **Drooping head**: Is its head drooping? (A symptom of non-specific weaknesses and depression, commonly observed with poisonings) +- **Wing symmetry**: Are the wings symmetrical and held close to the body? +- **Head level**: Is it holding its head level? +- **Legs**: Is it placing its full weight on both legs or just one? +- **Distortions**: Are there obvious distortions in the limbs? +- **Blood presence**: Can you see blood? +- **Flies presence**: Are flies present? + +A bird recently undergoing a traumatic event (e.g., vehicle collision) will be in shock and in a critical condition. Initial efforts should focus on stabilization, preferably by moving to a warm, dark, and quiet location, intensive fluid therapy (preferably intravenously), and correcting hypo- or hyperthermia. Address pain relief and infection control promptly. + +## Safety Warnings + +**Medical treatment must be undertaken by a registered vet, vet nurse, or para-veterinarian. Prescribing medications, injections, and some forms of fluid administration should only be done by one of these registered individuals.** + +--- + +## Protocol Steps + +### Assessing dehydration + +1. **Observation:** + Observe the bird to assess the state of dehydration. + - **Neck Dehydration Check for Gyps Species:** + - Large patches of bare skin can indicate the level of dehydration. + - **Wrinkled skin**: indicates dehydration. + - **Smooth, plumped skin**: indicates good hydration. + + ```markdown + Healthy, hydrated skin should spring back when pinched. Tented skin indicates severe dehydration. + + Additional indicators include tacky mucous membranes, thick strands of saliva at the back of the mouth, sunken eyes, and weak pulses. + ``` + +2. **Rehydration Methods:** + - **Oral Fluids (PO fluids via tubing or gavage):** + - tubing is an invasive procedure and must be conducted carefully. + - **Subcutaneous (SC) Fluids:** + - Suitable for conscious patients. + - **Intravenous (IV) Fluids:** + - To be administered by experienced rehabilitators or vets. + +#### Oral Fluids (via tubing or gavage) + +3. **Tubing Technique:** + - Two people needed: one to hold bird upright, another to open the beak and administer fluids. + - Use caution with vulture beaks; tubing should be inserted into the crop and not the trachea. + + ```markdown + Vultures have a crop (an extendable pouch before the stomach) and can store up to 2 kg of meat. Observe for recent meal and fluid emptying into the stomach (proventriculus). + ``` + +4. One holds the bird’s head; the other administers fluids while securing head and beak. +5. Open mouth, inspect, and ensure the glottis (base of the tongue) is avoided. +6. Insert tube down the esophagus, taking care to avoid glottis. +7. Ensure the tube is down the neck; if obstructed, reassess and retry. + + ```markdown + For Gyps vultures, the tube moving down the feather-covered neck can be visually confirmed or felt. + ``` + +8. Avoid giving fluids until confirmation; Large Gyps volure or Lappet-faced vulture should receive up to 180 mL of suitable rehydration solution. + +9. Repeat tubing a few times a day if necessary. + + ```markdown + If oral therapy fails, subcutaneous or intravenous methods should be considered. + ``` + +#### Subcutaneous Fluids + +10. SC fluids provide rapid rehydration: + - Administer sterile solutions such as saline or Ringer’s lactate. + - Gyps vultures can receive up to 120 mL each time at one or more locations. + +11. Insert needle with preloaded syringe under the skin, inserted shallowly. +12. Draw back the syringe for assessment. +13. Slowly inject fluids. Multiple sites (40-70 mL each) can be administered if necessary. + +#### Intravenous Fluids + +14. **IV Fluid Therapy:** + - Suitable for critical rehydration needs and the quickest method. + - Requires vet expertise. + +15. Example: Assume 10% dehydration: + - 100 mL fluid deficit per kg body weight. +16. Replace fluid deficit in stages: + - **50% on Day 1** + - **25% on Day 2** + - **25% on Day 3** + - [...] with maintenance fluids **50 mL/kg daily**. + +Typical Cape Vulture Case: + +```markdown +10 kg bird requires 1000 mL fluid deficit (split over three days): + +| Day | Fluid | +|------------|----------------------| +| Day one | 500 mL | +| Day two | 250 mL | +| Day three | 250 mL | +| Maintenance| 500 mL daily | +Provision for fluid therapy: 100 mL at 5-minute intervals after admission. +``` +- Repeating doses with trained adherence. + +17. Administer fluids at 10 mL/kg intervals. +18. Fluid delivery with IV giving set preferred; set pace for IV drip initially every 3.5 seconds, reducing after stabilization. + +19. Ensure catheter cleanliness and secure placement. + +### Full Clinical Examination (post shock) + +19. Perform an in-depth clinical examination: + - Check dehydration (tented skin could indicate 10-15% dehydration and fluid need adjustments). + - Assess behavior and physical checks. + +20. Evaluate Physical Condition: + - **Head to Toe Examination:** + - **Beak condition**: Check for chips. + - **Eyes**: Observe for brightness and alertness. + - **Nictitating Membrane**: Stimulus reaction. + + - **Body Condition Scoring (BCS):** + - **Pectoral muscles** flanking the keel bone (breastbone), BCS 1 (thin) to 5 (obese). + +21. Examine General Health: + - **Crop**: Check food intake. + - **Wing symmetry**: Assess breaks and joint dislocations. + - **Leg functionality**: Check weight support, limping, or dislocation. + - **Open wounds**: Treat maggots. + - **Bones and tendons**: Address fractures and keep moist. + - **Parasites**: Dust with ectoparasite dust for external pests like ticks and lice. + +*endofoutput* + +``` + +This ensures all the instructions and steps in the original document are mapped clearly in markdown format, with added scientific information and careful adherence to prescribed safety protocols. \ No newline at end of file diff --git a/markdown-output/chapter-3-wound-care-bmi8k4hw.md b/markdown-output/chapter-3-wound-care-bmi8k4hw.md new file mode 100644 index 0000000000000000000000000000000000000000..68cd8a8775eadbe48f7ec2ec62c79b844bade9fc --- /dev/null +++ b/markdown-output/chapter-3-wound-care-bmi8k4hw.md @@ -0,0 +1,163 @@ +```markdown +## Goal/Experiment: +This protocol outlines the steps to properly care for wounds when rehabilitating vultures. + +# Chapter 3: Wound Care + +### Author: +Kerri Wolter +VulPro + +### Abstract: +This protocol outlines the steps to properly care for wounds when rehabilitating vultures. + +### Keywords: +vultures, vulture rehabilitation, wounds care, maggots, infection, bee stings, snake bites, bumble-foot + +### License: +This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. + +### Date Created: +Sep 20, 2020 + +### Last Modified: +Oct 19, 2020 + +### Protocol ID: +42304 + +### Collections: +Vulture Rehabilitation Manual + +--- + +## Materials: +Important items to have when treating wounds: +- F10® SC Veterinary Disinfectant spray (water based) +- Hibitane or similar (alcohol based) +- F10® Germicidal Wound Spray +- F10® Germicidal Barrier Ointment +- Necrospray (Oxytetracycline Hydrochloride) +- PluroGen PluroGel® +- Honey (preferably Manuka honey) +- VetWrap +- Granuflex® dressing or any moisture-retentive dressing +- Gauze +- Cotton wool +- Elastoplast tape or other strong fabric/athletic tape +- Super glue +- Masking tape +- Tweezers +- Sharp scissors + +### Bumble-foot Treatment: +- Toothbrush or nail brush +- DMSO (Dexamethasone ointment) +- Baytril (soak cotton wool) +- A large amount of cotton wool OR +- A spongy foam matting +- VetWrap +- Elastoplast tape +- Nuflor (inject via IM) + +--- + +## Safety Warnings: +Most medical treatment needs to be undertaken by a registered vet, vet nurse or para-veterinarian. Procedures such as prescribing medication, injections and some forms of fluid administration should only ever be done by one of the above mentioned, registered individuals. + +--- + +## Procedure: + +### Maggots: + +1. **Observation and Removal:** + - Observe all wounds for maggots and remove the maggots by physically picking them out. + +2. **Application of Honey:** + - If not all maggots can be removed by hand, liberally apply honey to the wound which will smother the maggots and draw them upward and out of the wound. + + **Note**: Ectoparasitic wound preparations, such as F10® Germicidal and Insecticidal Spray should not be used on vultures as the permethrin ingredient can be toxic to the patient. Use Necrospray and F10® Germicidal Barrier Ointment which will kill the maggots and then remove them from the wound. + +### Closed vs Open Wounds: + +3. **Distinguishing Wound Type:** + - It is important to first make the distinction between wounds that will require surgical closure (intervention) and those that will heal by second intention (the body's natural healing process). + +4. **Contaminated/Infected Wounds:** + - It is critical not to put anything inside the wound which may impede the healing process. + +5. **Antibiotics Post-Surgery:** + - Place the patient on a course of antibiotics for 48 to 72 hours, while cleaning and flushing the wound, to reduce the likelihood of post-surgery 'wound breakdown'. + +6. **Debriding the Area:** + - Debride the area meaning all the dead (necrotic) tissue should be removed. + +7. **Disinfection:** + - Next, disinfect the area with a water-based disinfectant spray such as F10® SC Veterinary Disinfectant. + +8. **Keeping the Wound Moist:** + - The wound requires a moist environment to form a 'granulation bed', a vital stage in the healing process. + +9. **Maintenance:** + - If the wound is left open, keep it clean and moist and do not allow it to dry out. + +10. **Re-bandaging:** + - Re-assess and re-bandage wounds every 2-3 days. Remove maggots if present and assess the wound daily. + +### Infection: + +11. **Signs of Infection:** + - If the wound is infected, you will see signs of inflammation. If a wound shows signs of infection, place the bird on a course of Amoxycillin-Clavulanate (Synulox) antibiotics. + +### Bee Stings/Snake and Other Animal Bites: + +12. **Stabilizing the Bird:** + - First, stabilize the bird which means it will likely need to be put on an IV drip and given antivenom. + +13. **Necrotic Skin Triage:** + - With puff adder bites and multiple bee stings, the skin will become necrotic and cannot be stitched. + +14. **Sterile Environment:** + - Keep the bird in a relatively sterile environment until the exposed wounds heal. + +15. **Carnivore Bites:** + - Carnivore bite wounds are typically highly infected. Never suture or close the wounds. Clean and administer antibiotics. + +### Bumble-foot: +Bumble-foot is the term for inflammation and/or infection of the plantar surface of the feet in vultures. It results from a pressure sore typically affecting one foot. + +16. **Prevention and Treatment:** + - The best way to treat bumble-foot is preventive. Use weight-absorbing substrates and appropriate bandaging for treatment. + +#### Step-by-step Guide to Bumble-foot Treatment: + +17. **Cleaning:** + - Clean the area vigorously with disinfectant spray and a toothbrush or nail brush. + +18. **Application:** + - Cover the surface with DMSO (Dexamethasone) ointment. + +19. **Bandaging:** + - Prepare a doughnut-shaped cotton wool bandage to reduce pressure on the bulge. + +20. **Proper Wrapping:** + - Wrap the foot with VetWrap and Elastoplast tape. + +21. **Re-assessment:** + - Replace dressing every 3 days and continue until swelling is reduced. + +22. **Topical Treatments:** + - For dry and cracked foot, apply Preparation H ointment daily for 2 weeks. + +--- + +## Figures: +![Typical bumble-foot in a bird of prey's feet](https://example.com/image1.jpg) +Figure 8: Image showing typical bumble-foot in a bird of prey's feet + +![Doughnut-shaped bandage](https://example.com/image2.jpg) +Figure 9: Doughnut-shaped bandage. Note the hole in the middle of the bandage. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/chapter-4-broken-bones-and-dislocations-bmjbk4in.md b/markdown-output/chapter-4-broken-bones-and-dislocations-bmjbk4in.md new file mode 100644 index 0000000000000000000000000000000000000000..700c898f2e7ec73e0764e26669e71b64e629859a --- /dev/null +++ b/markdown-output/chapter-4-broken-bones-and-dislocations-bmjbk4in.md @@ -0,0 +1,89 @@ +```markdown +# Goal/Experiment: +This protocol describes how to treat broken bones and dislocations in vultures. The goal is to stabilize fractures and dislocations correctly to ensure the best possible healing and rehabilitation for injured vultures. + +# Chapter 4: Broken bones and dislocations + +**Protocol Author: Kerri Wolter** + +## Abstract +This protocol describes how to treat broken bones and dislocations in vultures. + +[Download PDF Attachment](Vulture_Rehabilitation_Manual_Version_2.0.pdf) + +## Protocol Citation +Kerri Wolter 2020. Chapter 4: Broken bones and dislocations. [protocols.io](https://protocols.io/view/chapter-4-broken-bones-and-dislocations-bmjbk4in). + +## Collections +📂 **Vulture Rehabilitation Manual** + +## Keywords +vultures, vulture rehabilitation, broken bones, dislocations, wing wraps, upper wing fractures + +## License +This is an open access protocol distributed under the terms of the [Creative Commons Attribution License](https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. + +## Created +Sep 20, 2020 + +## Last Modified +Oct 19, 2020 + +## Ownership History +- **Created by:** Emily Hasser, Cornell University (Sep 20, 2020) +- **Updated by:** Kerri Wolter (Oct 01, 2020) + +## Protocol Integer ID +42307 + +## Guidelines + +### Important Considerations: +- **Compound Fractures:** If the skin is broken over any wound, the underlying tissues will likely be infected, termed a compound fracture. It is critical to keep the bone and tendon moist to prevent drying and ensure saving the limb. Immediate avian veterinary care is required. +- **Triage:** Any suspected break or dislocation must be evaluated by an avian vet urgently. This involves fluid therapy, pain relief, and antibiotics, typically along with X-ray diagnosis. +- **Stabilization:** If the bird is tripping on its wing, tape the tips of the primary feathers or strap the wing against the body carefully to avoid excess pressure and ensure proper breathing. + +### Key Points for Treatment +- All trauma cases receive fluid therapy. +- All open fractures should be treated with antibiotics (Nuflor), with assessment before administering if closed. +- Pain relief drugs like meloxicam should be given post-rehydration to avoid kidney damage in dehydrated birds. + +### Wing Wraps +1. **Choosing the Wrap:** + - Base the decision on the fracture's location. + - The wrap's function is to stabilize the joints on either side of the fracture. + - A body wrap may be necessary for proximal fractures (near the shoulder joint). + +### Upper Wing Fractures: Body Wrap +1. **Most Common Fracture Site:** + - The humerus is the most common fracture site for vultures. + - Preparation: Use VetWrap, re-rolling it before starting to ensure proper tension. + +2. **Applying the Bandage:** + - Ensure the wing is in an anatomically correct position. + - Step-by-step application: + 1. Place the wrap under the wing, below the wrist joint bend. + 2. Fold the loose end over the wing just behind the carpus. + 3. Secure this fold by wrapping over the loose end. + 4. Bring the bandage over the vulture's back towards the tail. + 5. Wrap under the opposite wing and in front of the opposite leg. + 6. Continue wrapping, ensuring the bandage does not obstruct breathing or wing movement. + 7. Secure the wrap on the back. + +3. **Securing the Bandage:** + - Ensure the bandage is not too tight and doesn't cover the vent or lie over the scapula. + - The final wrap should immobilize the injured wing securely without obstructing the crop or respiratory functions. + +### Dislocations +1. **Challenges with Dislocations:** + - Dislocations have a lower chance of recovery compared to fractures. + - Immediate action is needed. +2. **Common Sites:** + - Elbow and shoulder dislocations are frequent. + - Monitor for loss of circulation, indicated by cold distal limbs. +3. **Management:** + - Promptly present any dislocated joint to a qualified avian vet for proper treatment. + - Ensure continuous monitoring for any signs of distress or complications. + +**endofoutput** +``` \ No newline at end of file diff --git a/markdown-output/chapter-5-severe-nutritional-deficiencies-emaciati-bmjek4je.md b/markdown-output/chapter-5-severe-nutritional-deficiencies-emaciati-bmjek4je.md new file mode 100644 index 0000000000000000000000000000000000000000..867959789841d27597d3b214a8cf7d461238a26d --- /dev/null +++ b/markdown-output/chapter-5-severe-nutritional-deficiencies-emaciati-bmjek4je.md @@ -0,0 +1,95 @@ +```markdown +# Goal/Experiment: +This protocol describes the proper treatment for severe nutritional deficiencies, emaciation, and head trauma in vultures. + +## Chapter 5: Severe Nutritional Deficiencies, Emaciation, Head Trauma + +**Author**: Kerri Wolter +**Institution**: VulPro +**Date**: October 19, 2020 + +### Abstract +This protocol describes the proper treatment for severe nutritional deficiencies, emaciation, and head trauma in vultures. + +### Table of Contents + +1. [Body Condition Score](#body-condition-score) +2. [Feeding an Emaciated Bird, Probiotics and Supplements](#feeding-an-emaciated-bird-probiotics-and-supplements) +3. [Seizures](#seizures) +4. [Blindness](#blindness) + +### Guidelines +Vultures with broken wings can still walk and survive for several weeks after their injury. Many power line collision victims arrive at a rehabilitation facility emaciated and weak from spending extended periods on the ground after the collision. Emaciated, inexperienced juvenile vultures are common rehabilitation patients during the fledging season. When fledglings first leave the nest, many are not able to make it back to their colony or nest and are found grounded in areas where they are unable to fly. The risk of this occurring is greatly increased if the weather is inclement. + +### Body Condition Score + +1. **Score the body condition of all vultures:** + - Feel the amount of flesh around the keel (breastbone). + - Rate the bird using a basic scale from 1 to 5. + - Healthy birds range from 2.5 to 4. + - Any bird below 2 is considered thin. + - In severely emaciated birds, there may be little if any muscle felt around the keel, and neck vertebrae are usually prominent. + + ![Body Condition Score Diagram](assets/body_condition_score.png) + *The images above depict a cross-sectional view of the bird's keel (as though you are looking down on the bird from above). The black area represents the keel bone and the white sections are the flanking pectoral muscles.* + +### Feeding an Emaciated Bird, Probiotics and Supplements + +2. **Feeding:** + - Feed any bird presenting with a score of 2 or below daily. + - Frequently feed emaciated birds and keep them hydrated at all times. + +3. **Diet:** + - Feed emaciated birds with very small, thin pieces of muscle and fat from fresh carcasses. + - Soak these pieces in water or saline for 5 minutes prior to feeding to assist in digestion and rehydration. + +4. **Crop Caution:** + The crop is not a stomach but more akin to a shopping basket or personal pantry. There are no acids or enzymes inside the crop to stop food putrefying, so any food which does not pass on into the stomach rapidly begins to rot. + + - If the crop does not reduce in size, do not continue to feed. Ensure the meat is passing through the bird before you continue to feed. This can take up to 40 hours. + - If food is slow in passing from the crop, moisten the meat in the crop and massage it to encourage it to move on into the stomach. Do this by tubing 60 mL - 120 mL of fluids after feeding. + +5. **Probiotics:** + - Consider the administration of probiotics to any bird which is in bad condition, has had a gut infection, or been on a course of antibiotics. Administer powdered probiotics with food, or in fluid via crop tubing. A suitable dosage would be one heaped teaspoon on one or two consecutive feeds. + +6. **Supplements for Thin Birds:** + - For thin birds, especially young ones, provide bone chips, powdered calcium carbonate, or calcium and vitamin D3 liquid or powder, daily for two weeks after admission. + + *Thin individuals will also benefit from vitamin supplements for the first few days after admission. Intramuscular Catasol (multi-vitamin), Vitamin B1, and Calcium may be beneficial. It is important to use one preparation and limit it to the recommended dose rate, as overdosage can be dangerous.* + +### Seizures + +8. **Seizure Symptoms and Immediate Care:** + In vultures, seizures range from slight twitches of the head to full body convulsions. Severe, involuntary muscle contractions often result in characteristic twisting of the neck towards the back of the body. Seizures can be related to severe hypoglycemia (critically low blood glucose), toxicity (poisonings), head trauma, severe nutritional deficiencies, or general emaciation or debilitation. + + - Give emaciated, seizing birds fluids (containing 10% glucose) and force feed them, preferably with a semi-elemental critical care diet (e.g., Emeraid® carnivore care), immediately. + - If a critical care diet is not available, then a diet of minced whole carcass, with added glucose powder and vitamins is appropriate. Seizures typically stop once some nutrients and fluids have processed through their system, indicating the seizures are likely nutritional or otherwise related to toxins, head trauma, etc. + + *Unfortunately, seizures are self-perpetuating; in other words, the more seizures that occur, the higher the likelihood of another seizure occurring. If seizures are occurring frequently (every few minutes), it is important that these are controlled quickly.* + +9. **Environment and Fluid Therapy:** + - Keep any bird having seizures in a dark, calm, quiet, stable environment. + - Administer intravenous fluid therapy via a drip to all such cases. + +10. **Sedative Administration:** + - **Diazepam** (commonly known as Valium) administered at a dose of 0.5-1mg/kg by intramuscular or intravenous injection every 8-12 hours, or 2.5-5mg by mouth every 12 hours, or + - **Midazolam** administered at a dose of 0.5-1mg/kg by intramuscular or intravenous injection every 8-12 hours. + + *If sedatives are not available (these drugs are controlled veterinary medications), some cases will respond favorably to steroids (e.g., dexamethasone). Care must be taken when administering steroids to bird species due to potential immunosuppressive effects and the risk of secondary infections.* + +### Blindness + +11. **Eye Damage and Blindness Assessment:** + In view of their lifestyle (communal living, flying, and eating, and their quarry/food doesn't run away), vultures with compromised eyesight generally cope well. However, damage to the first eye often goes unnoticed and only becomes apparent when both eyes are affected. + + - Some 30% of all raptor trauma cases have suffered eye damage; in 70% of these cases, only the posterior segment is affected. + - Posterior segment damage (behind the lens where it cannot be seen without an ophthalmoscope) typically comprises hemorrhage or retinal detachment, often unappreciated without direct inspection. + + *There are many potential diagnoses for blindness, such as cataract formation, neoplasia, dry eye, uveitis, parasitic infestation, bacterial infection in the globe, penetrating lesions, glaucoma, or insect stings. In some cases, therapy can be effective.* + + - In cases of blindness not associated with trauma, the main differential cause would be lead poisoning. Generalized weakness of limbs and incoordination is typically associated with lead poisoning in birds; sometimes, acute onset blindness may be the only presenting sign. + + - If a vulture is presented with apparent blindness, submit the vulture for full ophthalmic examination by an experienced avian or ophthalmic vet. + +_endofoutput_ +``` \ No newline at end of file diff --git a/markdown-output/chapter-6-paralysis-bmjfk4jn.md b/markdown-output/chapter-6-paralysis-bmjfk4jn.md new file mode 100644 index 0000000000000000000000000000000000000000..0f01e45c2aeb7aa293d113ef0dbde2ad7bac78a4 --- /dev/null +++ b/markdown-output/chapter-6-paralysis-bmjfk4jn.md @@ -0,0 +1,84 @@ +```markdown +# Goal/Experiment: +The goal of this protocol is to assess and manage paralysis in vultures, often caused by trauma or poisoning, and to determine the appropriate course of action, including supportive care or euthanasia based on the vulture’s response to treatment. + +# Chapter 6: Paralysis + +## Author: +Kerri Wolter, VulPro + +## Date: +October 19, 2020 + +## Abstract: +Paralysis is commonly associated with traumatic injuries and poisoning (carbamate, organophosphate, and lead) cases. In cases of trauma, these typically involve birds that have collided with a power line cable, crash-landed, and impacted the ground with their chest. This tends to result in a compression fracture of the lumbar (back) vertebrae, often resulting in paralysis of the legs, while the wings remain functional. + +## Keywords: +vultures, vulture rehabilitation, paralysis, trauma, injury, poisoning + +## License: +This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. + +## Created: +September 21, 2020 + +## Last Modified: +October 19, 2020 + +## Vulture Rehabilitation Manual +[Link to Attachment](https://protocols.io/view/chapter-6-paralysis-bmjfk4jn) + +## Assessment of Paralysis Cases + +1. **Check for Pain Response:** + - Use a pair of forceps to pinch a toe or skin on the leg. + - The bird may exhibit a spinal reflex by pulling its leg up. This response only indicates nerve supply connectivity from the leg to the corresponding spinal segment and back. + +2. **Assess Deep Pain Response:** + - Look for conscious acknowledgment of the pinch, such as the bird moving its head, turning towards the stimulus, appearing alarmed, or trying to defend itself. + - These are indications that the nerve impulses have successfully traveled up the spine to the brain. + +3. **Severe Spinal Damage:** + - Lack of 'deep pain response' suggests severe spinal damage and has a grave prognosis. + - If the bird is not in too much distress, treat with fluid therapy and NSAIDs (e.g., Meloxicam) for a maximum of 48 hours. + +4. **Euthanasia:** + - If the deep pain response has not returned after 48 hours, euthanize the bird. + +5. **Initiate Supportive Care:** + - If some indication of deep pain response is observed, initiate supportive care measures. + +### Supportive Care Methods: + +#### 5.1 Physical Support: +- Sling the bird, ensuring a hole is provided for defecation to prevent soiling. + +#### 5.2 Fluid Therapy: +- Administer fluid therapy (refer to Chapter 2 for detailed procedure). + +#### 5.3 Nutritional Support: +- Provide nutritional support via force feed or tube feed at a rate of 2-3% of bodyweight per day. + +#### 5.4 Anti-inflammatory Medication: +- Administer Meloxicam following rehydration for pain relief. + +#### 5.5 Vitamin B Complex: +- Administer a Vitamin B complex to aid nerve function and healing. + +#### 5.6 Physiotherapy: +- Perform physiotherapy to prevent cramping and muscle/tendon contracture. This may include the use of a water bath. + +6. **Euthanasia Decision:** + - If there is no response or improvement within 2 weeks, consider the case hopeless and euthanize the bird. + +![Figure 10: Cape Vulture in a sling](https://your-image-url) + +End of the protocol. + +##### References: +*Original protocol citation:* +Kerri Wolter 2020, Chapter 6: Paralysis. protocols.io +[https://protocols.io/view/chapter-6-paralysis-bmjfk4jn](https://protocols.io/view/chapter-6-paralysis-bmjfk4jn) + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/chapter-7-poison-treatment-bmjhk4j6.md b/markdown-output/chapter-7-poison-treatment-bmjhk4j6.md new file mode 100644 index 0000000000000000000000000000000000000000..26d82fc30f498070efbf001ade07a1d04698f56b --- /dev/null +++ b/markdown-output/chapter-7-poison-treatment-bmjhk4j6.md @@ -0,0 +1,151 @@ +```markdown +# Goal/Experiment: +This protocol outlines the steps to take in the event of a likely poisoning incident of multiple vultures. + +# Chapter 7: Poison Treatment + +## Authors +Kerri Wolter + +## Abstract +This protocol outlines the steps to take in the event of a likely poisoning incident of multiple vultures. + +## Keywords +- Vultures +- Vulture rehabilitation +- Vulture poisoning +- Agrochemical poisoning +- Organophosphate poisoning +- Carbamate poisoning +- NSAID poisoning +- Lead poisoning + +## Guidelines + +### Agrochemical Poisoning + +#### Possible Drug Culprits: +- **DDT**: Unlikely to be a culprit as large quantities need to be ingested for lethal or sub-lethal clinical effect. + +#### Organophosphates: +- **Fenthion** +- **Methamidophos** +- **Diazinon** +- **Chlorfenvinphos** +- **Fenamiphos** +- **Cadusafos** + +#### Carbamates: +- **Aldicarb** +- **Carbofuran** (granular and liquid) +- **Methomyl** + +#### Clinical Signs: +- Incoordination, stumbling, falling over, and inability to balance (ataxia) +- High- or goose-stepping (over-exaggerated lifting of legs when walking) +- Severe seizures + +#### Organophosphate (OP) Poisoning +Specific characteristics include: + +| Chemical | Onset of Symptoms | Prognosis | Distance Birds Found from Source | +| -------- | ----------------- | --------- | -------------------------------- | +| **Fenthion** | Slow onset (~30 minutes), pupil constriction, vomiting, tremors, paralysis | Very poor | Typically within 100m | +| **Methamidophos** | Rapid onset (~5 minutes) | Very poor | Further than 50m | +| **Diazinon** | Very rapid onset (~3 to 5 minutes) | Good with no long-term effects | Within 100m | +| **Chlorfenvinphos** | Rapid onset (~5 minutes) | Poor | No long-term effects within 100m | +| **Fenamiphos** | Rapid onset | Very poor | Within 100m | +| **Cadusafos** | Slow onset (~30 minutes) | Very poor | Unlikely to move far from source | + +#### Carbamate Poisoning +Specific characteristics include: + +| Chemical | Onset of Symptoms | Prognosis | Distance Birds Found from Source | +| -------- | ----------------- | --------- | -------------------------------- | +| **Aldicarb** | Immediate onset, pupil constriction, paralysis, some vomiting, tremors, and hypothermia | Extremely poor | Within a few meters | +| **Carbofuran (granular)** | Onset 5 to 30 minutes after exposure | Less guarded for sub-lethal concentrations | At their nests with chicks dead | +| **Carbofuran (liquid)** | Immediate onset | Very poor | At the source | +| **Methomyl** | Immediate onset | Extremely poor | At the source | + +### NSAID Poisoning + +#### Possible Drug Culprits: +All NSAIDs except Meloxicam are toxic to vultures. + +Drugs Proven to be Toxic to Vultures: +- **Diclofenac Sodium** +- **Ketoprofen** +- **Phenylbutazone** +- **Flunixin** +- **Vedaprofen** +- **Carprofen** + +#### Clinical Signs: +- Dehydration (slight to severe) +- General weakness +- Drooping head +- "Zoning out" periods +- Depressed appearance +- Wings held slightly out from the body + +### Lead Poisoning +#### Background: +Lead poisoning arises from ingestion of spent ammunition and has been a longstanding issue. Incidences of lead in vulture populations vary based on region, local ecologies, etc. + +#### Levels: +- **Normal**: <10 µg/dl +- **Mild toxicity**: > 10 µg/dl +- **Consider chelation (EDTA) therapy**: > 20 µg/dl + +#### Sources of Poisoning: +- Lead bullets (common in hunting) +- Fishing tackle weights +- Residue on old agricultural gates + +#### Symptoms: +- Seizures +- Incoordination (walking, flight) +- Paralysis (severe cases) +- Emaciation and malnutrition +- Acute blindness + +#### Diagnosis: +Presence of lead particles in gastrointestinal tract on x-ray or elevated blood lead level on testing. + +## Steps in Poisoning Incident + +1. Attend the site as a "crime scene" and avoid any damage to evidence. +2. Contact local law enforcement. +3. Treat any surviving vultures, focusing on the type of poisoning: + +### Treatment of Organophosphate Poisoning + +4. **Rapid administration of antidote (critical within 24-48 hours):** +- **2 PAM (Pralidoxime Chloride)**, 50 mg/kg IV slowly over 5 to 10 min, repeated after 6 hours or as needed. +- Provide supportive care: sling, control seizures, fluid and nutritional support. + +### Treatment of Carbamate Poisoning + +5. **Antidote:** **Atropine** 2 mg/kg by IM or IV injection. If necessary, repeat treatment, increasing the dose each time until stabilized. + +### Treatment of NSAID Poisoning + +6. IV fluid therapy via drip at twice maintenance rates (100 ml/kg/day). +7. Reduce uric acid levels with **Allopurinol** 30 mg/kg twice daily by mouth until recovery. +8. Euthanize if significant clinical signs and no improvement. + +### Treatment of Lead Poisoning + +9. **Chelation (EDTA) therapy:** 35 mg/kg twice daily IM or IV for 5 days post removal of lead particles from the gastrointestinal tract. +10. If lead in gut, stabilize for 24 hours with intensive fluid and chelation therapy. +11. Remove lead under anesthesia via tilted table with body above head at 45 degrees and perform gastric lavage. +12. Do not extend chelation therapy beyond 5 days to avoid renal damage. +13. Recheck blood lead level after 7 days. If >10 µg/dl, repeat 5-day treatment. +14. Use fluid therapy (IV) to reduce kidney failure risk and treat seizure activity. + +## Conclusion + +This protocol helps in managing and treating vulture poisoning incidents, spanning agrochemical, NSAID, and lead poisoning cases. Rapid identification and adequate treatment are crucial for the survival and rehabilitation of affected vultures. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/chapter-8-managing-sick-and-injured-vulture-admiss-bmjik4ke.md b/markdown-output/chapter-8-managing-sick-and-injured-vulture-admiss-bmjik4ke.md new file mode 100644 index 0000000000000000000000000000000000000000..86311212af5a045c762aed60d0e54aca8deccd5f --- /dev/null +++ b/markdown-output/chapter-8-managing-sick-and-injured-vulture-admiss-bmjik4ke.md @@ -0,0 +1,86 @@ +```markdown +# Goal/Experiment: +To provide guidelines for managing sick and injured vulture admissions with emphasis on appropriate medical, nutritional, and biosecurity measures to optimize rehabilitation outcomes. + +--- + +# Chapter 8: Managing Sick and Injured Vulture Admissions + +## Author +- Kerri Wolter¹ +- ¹VulPro + +## Abstract +This protocol provides guidelines for managing sick and injured vulture admissions. + +## Attachments +[Vulture_Rehabilitation_Manual_Version_2.0.pdf](Vulture_Rehabilitation_Manual_Version_2.0.pdf) + +## Protocol Citation +Kerri Wolter 2020. Chapter 8: Managing sick and injured vulture admissions. [protocols.io](https://protocols.io/view/chapter-8-managing-sick-and-injured-vulture-admiss-bmjjk4ke) + +## Collections +- Vulture Rehabilitation Manual + +## Keywords +- vultures, vulture rehabilitation, injured vulture, sick vulture + +## License +This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. + +## Creation and Modification Dates +- Created: Sep 21, 2020 +- Last Modified: Oct 19, 2020 + +## Ownership History +- Sep 21, 2020 - Emily Hasser, Cornell University +- Oct 01, 2020 - Kerri Wolter + +## Introduction +It is vital to bear in mind that more birds will be saved by appropriate fluid and nutritional support than any single medical or surgical therapy. + +## Patient Monitoring and Management +Patients should at least be maintaining weight, if not gaining weight. Feeding regimes must be adjusted to account for any shortfalls. This may warrant increasing the frequency or volume of supplementary feeding. Further weight loss, despite efforts, suggests compromised health. + +### Infection Control and Biosecurity +A sick vulture can be carrying and excreting pathogens. Maintaining rigorous infection control is crucial to protect both the bird and others in the facility. + +#### Testing White Blood Cell (WBC) Counts +- A blood sample is necessary to verify infections. +- Published normal ranges for WBC counts for African vulture species: + +| Species | WBC Count Reference Interval (x10³ cells/µL) | HCT Reference Interval (%) | HCT Mean (%) | Sample Size (n) | +|---------------------------------|----------------------------------------------|----------------------------|--------------|-----------------| +| Cape vulture (Gyps coprotheres) | 3.4 - 22.9 | 37.1 - 56.9 | 45.2 | 96 | +| African White-backed vulture (Gyps africanus) | 13.45 - 19.97 | 42 - 58 | 50 | 21 | +| Ruppell's griffon vulture (Gyps rueppelli) | 6.02 - 49.87 | 31 - 53 | 43.8 | 388 | +| Griffon vulture (Gyps fulvus) | 8.0 - 29.93 | 32.5 - 50.8 | 42.0 | 41 | +| Bearded vulture (Gypaetus barbatus) | 7.06 - 15.42 | 41 - 54.2 | 47.1 | 26 | +| Cinereous vulture (Aegypius monachus) | 5.05 - 37.12 | 32 - 49 | 41.2 | 329 | +| Hooded vulture (Necrosyrtes monachus) | 5.69 - 39.29 | 27.6 - 52.5 | 40.6 | 142 | +| Lappet-faced vulture (Torgos tracheliotos) | 5.79 - 38.70 | 34 - 50 | 43.2 | 211 | + +### Record Keeping +Maintain records detailing: +- Reason for admission/cause of injury +- Origin of where the bird came from +- Clinical presentation and demeanor +- Weight and body condition on admission and at subsequent checks +- All medications administered, tests conducted, food, and fluid administered +- Response to therapy + +### Accommodation +Requirements include: +- Easy to clean and disinfect +- Quiet, dimly lit environment +- Easy access for observation (direct or by CCTV) +- Facilities for isolation + +### Handling +If a bird is in sternal recumbency, use a towel donut to prevent pressure sores. For birds with paralyzed legs, use a sling or cushion block to support the head. + +## Conclusion +Housing birds where they have access to sunshine, grass, and other vultures is beneficial once no longer in a critical condition. + +endofoutput +``` diff --git a/markdown-output/chapter-9-release-bmjjk4kn.md b/markdown-output/chapter-9-release-bmjjk4kn.md new file mode 100644 index 0000000000000000000000000000000000000000..9278f61646fc602a28f02dd58f72b2f3752b1375 --- /dev/null +++ b/markdown-output/chapter-9-release-bmjjk4kn.md @@ -0,0 +1,74 @@ +```markdown +# Goal/Experiment: +This protocol describes when, where, and how to release vultures after successful rehabilitation. + +# Chapter 9: Release + +## Abstract +This protocol describes when, where, and how to release vultures after successful rehabilitation. + +## Attachments +[Vulture_Rehabilitation_Manual_Version_2.0.pdf](Vulture_Rehabilitation_Manual_Version_2.0.pdf) + +## Protocol Citation +Kerri Wolter 2020. Chapter 9: Release. [protocols.io](https://protocols.io/view/chapter-9-release-bmjjk4kn) + +## Collections +[Vulture Rehabilitation Manual](https://protocols.io/view/vulture-rehabilitation-manual-szjdjj6) + +## Keywords +vultures, vulture rehabilitation, vulture release + +## License +This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. + +## Created +Sep 21, 2020 + +## Last Modified +Oct 19, 2020 + +## Ownership History +- Sep 21, 2020 - *Emily Hasser* - Cornell University +- Oct 01, 2020 - *Kerri Wolter* + +## Protocol Integer ID +42315 + +## Parent Protocols +Part of collection [Vulture Rehabilitation Manual](https://protocols.io) + +## Guidelines +It is vitally important that individual birds undergo an assessment prior to release. Any bird being released should be as fit as possible. Releasing a bird with an infection is a serious failure, and allowing that released bird back into a colony situation carrying contagious pathogens or infections, to potentially infect other birds in the colony, would act against the original goal of rehabilitation: to boost the overall population health and foster healthy wild individuals. + +### When is the bird ready to be released? +Only birds meeting the following list of requirements will be fit for release: +- **Symmetry of wings** when standing (both at rest and when extended). +- Birds should show **strong flight capabilities** and be able to gain height in a suitably large flight enclosure. +- **Normal body posture, coordination, reaction, and body movements** are all essential. +- The bird should be assessed in **social situations**, if possible. Normal feeding habits, including interaction with a group of other vultures at a carcass, is an important indicator of successful feeding within a colony. The age and species of vulture must be taken into account during this assessment. +- In the case of **leg injuries**, even a minor limp must be considered carefully. To be released and survive successfully, a vulture must be able to run, perch on a movable object such as the branch of a tree, and defend itself within a group situation. +- **Fully functional binocular vision** is essential to cope within a colony and interact with other vultures. Any individual with blindness in one eye, or impaired vision, should not be released. + +### Where should the bird be released? +Birds in your care have been compromised for a reason, and often this is the result of some conflict within their environment (power line collision, exposure to poison, etc.). While it may seem intuitive to release the bird at the same location it was found, this is not always in the best interests of the bird as there is a chance for re-injury. It is important to consider the release location carefully to balance finding an ecologically and socially appropriate location, while trying to avoid the initial cause of injury, all giving the individual the best possible chance of survival. + +The following must be considered for every release, on a case-by-case basis: +- **Weather** – warm days with no or little cloud cover are ideal; the conditions when thermals will be available. Rain should not be forecast for the next 24 to 48 hours, in the case that the bird does not decide to immediately leave the release site. +- **Time of day** – release birds in the late to mid-morning, if possible. Vultures are heavy-bodied, with high wing loads (body mass to wing area ratio) and will generally struggle to fly high and find thermals in the late afternoon. +- **Habitat** – ideally the habitat should be suitable for the species and within the species’ normal foraging range. For example, tree nesting-species should be released in an area with plenty of trees for perching opportunities. Colonial cliff nesting species would ideally be released near a cliff roosting or breeding location. The bird may fly away immediately, but if it does not, you need to feel comfortable with the release location if the bird decides to remain in the area for a while. +- **Human disturbance** – birds should not be released within the immediate vicinity of dense human habitation. No power lines (dangerous or safe structures) should be present for 1 km surrounding the release location. This allows the bird plenty of distance to take off and gain height without risk of collision. No dangerous power line or wind farm structures should be present in the greater ( > 1 km ) release area. If you have concerns over the safety of various power line structures, contact VulPro (see contact details in the Introduction). +- **Social considerations** – Vultures are very social creatures and choose to seek out the company of other birds. If several vultures have been rehabilitated together, it is best to release them together. It may be worth keeping a vulture which is ready for release until the other is ready, but only if the extra time held in captivity is short (several days). If one vulture will be held more than several days, it should be released as soon as possible. If possible, release the bird where other vultures will be present (i.e. a vulture restaurant, colony). At the very least, the release location should be a place where vultures are known to fly overhead on a regular basis. +- **Food availability** – Do not release a bird in a region where you suspect poison to be used. Similarly, do not release a vulture in a location where you suspect lead ammunition is used and gut piles or meat is made available for foraging birds. Well-managed vulture restaurants provide ideal, safe locations for releases. This allows the released patient time to adjust to the surroundings and feed before leaving the site, if it so chooses. + +### Steps for Releasing a Bird +1. When releasing a vulture, lower your body (see CHAPTER 1 for pictures), bringing the bird's feet to ground level, slowly allowing the bird to stand. +2. Then release your grip on its entire body and neck at the same time, stepping away to give the bird some space. Be careful never to drop or throw the bird down, nor allow the bird to fall prior to lowering it gently to ground level. +3. When releasing the bird or placing it back inside the enclosure, remain still and allow the bird to walk or fly away from you, rather than panicking it, in which case it may take off, flying into stationary objects / fences. +4. Do not force the bird to move. Allow the bird time to recover but monitor it for any unusual behavior that could be a sign of heat exhaustion or injury from handling. The bird will decide for itself what to do next; it might fly off, run or drink water. Never force the bird to move or fly after handling. Simply monitor and interfere only as a last resort if the bird appears not to be fit for release. +5. Vultures, when given the opportunity, will time their take-off to coincide with a thermal or an increase in wind strength, making the take-off easier. Birds in general prefer to take-off against the wind. Consider these factors when choosing the release site and direction. +6. Release a bird from a crate at ground level, and not from an elevated site such as the back of a vehicle. +7. Do not pull a bird out by a wing or the tail or tilt a crate to encourage a bird to exit. Give the bird time to leave the crate of its own accord. + +## endofoutput +``` \ No newline at end of file diff --git a/markdown-output/chickpea-inoculation-with-a-rabiei-for-ascochyta-b-bw9iph4e.md b/markdown-output/chickpea-inoculation-with-a-rabiei-for-ascochyta-b-bw9iph4e.md new file mode 100644 index 0000000000000000000000000000000000000000..6c997d328e73596e1fe0ecd28744dba90425870d --- /dev/null +++ b/markdown-output/chickpea-inoculation-with-a-rabiei-for-ascochyta-b-bw9iph4e.md @@ -0,0 +1,139 @@ +```markdown +# Goal/Experiment: +This experiment aims to prepare inoculation, conduct bioassay, and assess disease outcomes of *Ascochyta rabiei* isolates on a differential set of chickpea host genotypes under controlled conditions. + +# Chickpea Inoculation with *A. rabiei* for Ascochyta Blight Disease Assessment Under Controlled Conditions V.2 + +**Melody Christie1, Dr Prabhakaran Sambasivam1, Ido Bar1** + +1Griffith University + +[GRDC Ascochyta rabiei research program](#) +[Griffith University](#) + +**Melody Christie** + +## Abstract +This protocol describes the inoculation preparation, bioassay, and disease assessment of *Ascochyta rabiei* isolates on a differential set of chickpea host genotypes. + +--- + +## DOI +[https://dx.doi.org/10.17504/protocols.io.bw9iph4e](https://dx.doi.org/10.17504/protocols.io.bw9iph4e) + +## Protocol Citation +Melody Christie, Dr Prabhakaran Sambasivam, Ido Bar 2021. Chickpea Inoculation with A. rabiei for Ascochyta Blight Disease Assessment Under Controlled Conditions. *protocols.io*. [https://dx.doi.org/10.17504/protocols.io.bw9iph4e](https://dx.doi.org/10.17504/protocols.io.bw9iph4e) + +## License +This is an open access protocol distributed under the terms of the [Creative Commons Attribution License](https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. + +## Created +Aug 10, 2021 + +## Last Modified +Aug 10, 2021 + +## Protocol Integer ID +52234 + +--- + +## Materials + +### Equipment for *A. rabiei* Culture and Inoculation Work +- Burner +- Ethanol bath for utensils +- Ethanol spray +- Petri dishes for dissection +- Tape +- Forceps +- Gloves +- Kimwipes +- Lighter +- Media plates +- Paraffin film +- Permanent lab marker +- Scalpel +- Scissors +- Pipette and tips +- Tween 20 +- 10cm diameter pots +- Premium potting mix +- Trays for bioassays (to fit 10 pots) +- Plastic containers with lids to fit 10 pots +- Spray bottles (500ml) +- Haemocytometer and cover slips + +### Reagents +- *Streptomycin/Penicillin*: Antibiotics used to prevent bacterial contamination during fungal culture incubation. +- *Tween 20*: A surfactant used to enhance spore dispersion in the spray inoculum. + +--- + +## Protocol Steps + +### Inoculum Preparation (~30 minutes) + +1. **Subculturing Isolates**: Approximately 2 weeks before the planned inoculation date, subculture isolates of *A. rabiei* onto V8 media agar containing 30 mg/L of antibiotic (Streptomycin/Penicillin). Add the antibiotic after the media has been autoclaved and cooled (~45°C). Aseptically place one plug from the original culture in the middle of the plate and incubate at room temperature for 14 days. Include a positive control isolate with each batch per bioassay. + +### Sowing (~30 minutes) + +2. **Soil Preparation**: Autoclave soil (premium potting mix) before use (one day before sowing). Ensure pots have been cleaned and dried if previously used. + +3. **Sowing Seeds**: Into 10 cm diameter pots, sow five or six chickpea seeds, ensuring they are not too close to the side of the pot. Sow two pots of each chickpea genotype per isolate so that at least 10 plants can be inoculated. Sow ICC3996 and Kyabra one day earlier than the other genotypes. Fertilize with Osmocote (slow release) at the time of sowing and Aquasol once a fortnight. + +4. **Growth Conditions**: Grow plants under controlled environment conditions at ~23°C. When chickpea plants are at the 4th node stage (or as many as possible without other varieties growing too large), inoculation should occur. + +### Inoculum Preparation on Day of Inoculation (~30 minutes) + +5. **Plate Saturation**: On the day of inoculation, saturate the plates with distilled water and leave for 15 minutes. + +6. **Spore Harvesting**: Using a wire loop or glass rod, gently scrape over the surface of the plate to dislodge spores. + +7. **Spore Suspension**: Pour the solution into a 500 mL flask (filter through a piece of tissue/gauze if required). + +8. **Spore Concentration Adjustment**: Using a haemocytometer, adjust spore concentration to 1x10^5 spores/mL. In a separate container, add two to three drops of Tween 20 (0.02% v/v) per 100 mL of spore suspension. Ensure the sprayer is labeled with the isolate code. + +9. **Storage if Needed**: If the inoculum is not to be used immediately, store it in the dark in the refrigerator for up to 2 hours. + +### Inoculation (~30 minutes) + +10. **Plant Grouping**: Place pots in groups of 10 (two pots of each genotype). Separate each pot from the others for the inoculation step. Using a Preval hand-held sprayer, spray inoculum onto chickpeas until runoff (approximately 15-20 mL per plant) and return to their original position. + +11. **Incubation**: Place pots inside black plastic tubs and leave in the dark for 24 hours. + +12. **Post-Inoculation Handling**: 24 hours after inoculation, remove plants from plastic tubs and place them in a tray under grow lights. + +13. **Plant Care Post-Inoculation**: Mist plants until bead forms on leaves every three days and water twice a week (or as required). Disease assessment (phenotyping) occurs after 21 days. + +### Disease Assessment + +14. **Assessment Timing**: Disease assessments are made 21 days post-inoculation using the following scales: + +#### Disease Severity Assessment Scores 1-9 for Leaf Lesions + +| Symptoms | Score | Description | +|----------|-------|-------------| +| | 1 | No leaf symptoms. | +| ![](leaf_symptom_2.png) | 3 | Pin prick lesions (sign of potential HR defence). | +| ![](leaf_symptom_3.png) | 5 | Small, inconspicuous lesions present without pycnidia (evidence of potential initial biochemical defence/containment). | +| ![](leaf_symptom_4.png) | 7 | Individual lesions developed with darker margins and some pycnidia (evidence of initial defence breakdown). | +| ![](leaf_symptom_5.png) | 9 | Lesions with dark margin coalesced with pycnidia developed (evidence of defence breakdown as lesions are growing in size and the pathogen is able to reproduce). | + +#### Disease Severity Assessment Scores 1-9 for Stem Lesions + +| Symptoms | Score | Description | +|----------|-------|-------------| +| | 1 | No stem symptoms. | +| ![](stem_symptom_2.png) | 3 | Pin prick lesions visible on the stem (sign of potential HR defence). | +| ![](stem_symptom_3.png) | 5 | Small, inconspicuous lesions present without pycnidia (evidence of potential initial biochemical defence/containment). | +| ![](stem_symptom_4.png) | 7 | Individual lesions developed with darker margins and some pycnidia, no stem breakage visible (evidence of initial defence breakdown). | +| ![](stem_symptom_5.png) | 9 | Lesions with dark margin coalesced with pycnidia developed and stem breakage possible (evidence of defence breakdown as lesions are growing in size and the pathogen is able to reproduce). | + +## Notes +Separate notes should be taken for leaf drop and defoliation. + +--- + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/chromatin-immunoprecipitation-bjj4kkqw.md b/markdown-output/chromatin-immunoprecipitation-bjj4kkqw.md new file mode 100644 index 0000000000000000000000000000000000000000..3b55c5e5c14b9467c0c688fc6a4554b0384c2c17 --- /dev/null +++ b/markdown-output/chromatin-immunoprecipitation-bjj4kkqw.md @@ -0,0 +1,200 @@ +```markdown +# Goal/Experiment: +Chromatin Immunoprecipitation (ChIP) - The goal of this experiment is to determine the association of specific proteins with specific genomic regions in chromatin from a sample of interest. + +## Chromatin Immunoprecipitation V.1 +**Authors: Georgios I Laliotis, Philip N. Tsichlis** +**Institution: The Ohio State University** +**Date Created: August 11, 2020** +**DOI: [dx.doi.org/10.17504/protocols.io.bij4kkqw](dx.doi.org/10.17504/protocols.io.bij4kkqw)** + +## Chemicals Required + +### 1. Cytosolic Lysis Buffer (200 ml) +- 5 mM PIPES (pH 8.0) +- 85 mM KCl +- 0.5% NP40 + PI + +**Preparation:** +- PIPES 0.3 g => Adjust to pH 8.0 +- 2M KCl, 8.5 ml +- 10% NP40, 10 ml + +### 2. Nuclear Lysis Buffer (50 ml) +- 50 mM Tris (pH 8.0) +- 10 mM EDTA +- 0.5% SDS + +**Preparation:** +- 1 M Tris (pH 8.0), 2.5 ml +- 0.5 M EDTA, 1 ml +- 20% SDS, 1.25 ml + +### 3. IP Dilution Buffer (250 ml) +- 16.7 mM Tris (pH 8.0) +- 167 mM NaCl +- 1.2 mM EDTA +- 1.1% Triton X-100 +- 0.01% SDS + +**Preparation:** +- 1 M Tris (pH 8.0), 4 ml +- 0.5 M EDTA, 0.6 ml +- 5 M NaCl, 8.5 ml +- 20% Triton X-100, 12.5 ml +- 10% SDS, 250 µl + +### 4. Low Salt Wash Buffer (250 ml) +- 20 mM Tris (pH 8.0) +- 2 mM EDTA +- 150 mM NaCl +- 1% Triton X-100 +- 0.1% SDS + +**Preparation:** +- 1 M Tris (pH 8.0), 5 ml +- 0.5 M EDTA, 1 ml +- 5 M NaCl, 7.5 ml +- 20% Triton X-100, 12.5 ml +- 10% SDS, 2.5 ml + +### 5. High Salt Wash Buffer (250 ml) +- 20 mM Tris (pH 8.0) +- 2 mM EDTA +- 500 mM NaCl +- 1% Triton X-100 +- 0.1% SDS + +**Preparation:** +- 1 M Tris (pH 8.0), 5 ml +- 0.5 M EDTA, 1 ml +- 5 M NaCl, 25 ml +- 20% Triton X-100, 12.5 ml +- 10% SDS, 2.5 ml + +### 6. NS Buffer +- 50 mM Hepes +- 500 mM NaCl +- 1 mM EDTA +- 1% Triton X-100 +- 0.1% Sodium deoxycholate +- 0.1% SDS + +### 7. LiCl Wash Buffer (250 ml) +- 10 mM Tris (pH 8.0) +- 1 mM EDTA +- 250 mM LiCl +- 1% NP40 +- 1% (w/v) Sodium deoxycholate + +**Preparation:** +- 1 M Tris (pH 8.0), 5 ml +- 0.5 M EDTA, 0.5 ml +- 1 M LiCl, 72.5 ml +- 10% NP40, 50 ml +- Sodium deoxycholate, 0.25 g + +### 8. Miscellaneous +- 1.25 M Glycine, 9.38 g / 100 ml +- 1 M NaHCO3, 8.4 g / 100 ml => Aliquot (0.5 ml) and keep at -20 °C + +## Cross-linking and Lysis of the Cells +### For Cell Lines (20-50x10^6 cells) +For each antibody (Ab) to be tested, 3 plates for each cell line must be used. + +1. **Fixation:** + - Add 270 µl of 37% Formaldehyde into 10 ml culture media (P100). + - Incubate at 37 °C for 15 min. + - Add 1 ml of 1.25 M Glycine and incubate for 5 min at RT. + - Wash with cold PBS (+ PI) twice. + - Scrap cells into a new tube. + +2. **Cell Collection:** + - Centrifuge at 14000 rpm for 1 min at 4 °C to remove PBS. + - Remove supernatant. + +3. **Lysis:** + - Incubate lysis buffer at 37 °C for a few minutes to dissolve precipitates. + - Add fresh 1x protease inhibitor (10 µl from 100x stock). + - Add/Resuspend 1 ml of nuclear lysis buffer (+ PI) per 3 P100 plates => 200 µl for 1x10^6 cells. + - Manually shake the tube to break the pellet. + - Split into 2 tubes with 500 µl to help the sonication process. + +4. **Sonication:** + - Sonicate for 15 sec on/45 sec off (60 sec x 6-7 times = 6-7 min total) at 30% duty. + +5. **Centrifugation:** + - Centrifuge at 14000 rpm for 15 min at 4 °C. + - (Optional) Store supernatant at -80 °C. + +### Immunoprecipitation of Crosslinked Protein/DNA Complex +1. **Dilution:** + - Dilute 5 fold of lysate volume with IP dilution buffer in a 15 ml tube. + - Add 1x protease inhibitors in the IP dilution buffers. + - Dilute to 5 ml from initial 1 ml using 4 ml IP dilution buffers. + +2. **Pre-clearing:** + - Add 30 µl of protein G bead per tube for pre-clearing. + - Incubate for 1 hr at 4 °C with rotation. + - Centrifuge at 3000 rpm for 2 min at 4 °C. + - Transfer the supernatant into a new 2 ml tube. + +3. **Adding Antibody:** + - Add 2 µg of antibody or normal IgG per tube (for normal rabbit IgG, add 2 µl from 1 mg/mL stock). + - Incubate overnight at 4 °C with rotation. + +4. **Adding Beads:** + - Add 25 µl of protein G agarose bead per tube. + - Incubate for 2 hrs at 4 °C with rotation. + - Centrifuge for 2 min at 3000 rpm at 4 °C. + - Place beads for at least 5 min in the magnetic rack. + - Carefully remove the supernatant with WB long tips gradually. + - Add/Resuspend beads using 500 µl cold low salt wash buffer/tube, and combine tubes in one. + - Rotate for 5 min at 4 °C. + - Wash with wash buffer. + +### Wash Buffers Order: +- Cold low salt wash buffer (2x) +- Cold high salt wash buffer (2x) +- Cold LiCl wash buffer (2x) +- Cold TE buffer (3x) + +5. **Elution:** + - Elute with 200 µl elution buffer (0.1 M NaHCO3 and 1% SDS). For 1.5 ml: + - Add 1.2 ml H2O first. + - Add 150 µl 10x SDS. + - Add 150 µl 1 M NaHCO3. + - Incubate for 15 min with shaking (900 rpm) at 23 °C. + - Centrifuge at 3000 rpm for 3 min. + - Transfer supernatant to a new tube. + - Repeat steps #16-19 (Total elution vol. will be 400 µl). + +## Reverse Crosslinking and Elution of Protein/DNA Complexes +32. Add 20 µl of 5 M NaCl and 1 µl of 20 mg/ml RNase per 200 µl tube. + +**Note:** Do not forget input sample (10% lysate + 350 µl elution buffer)! + +33. Mix well manually before putting in the cycler. +34. Incubate at 65 °C in Thermo cycler for 5 hrs or overnight with 450 rpm rotation to prevent precipitation. + +35. Add 10 µl of 0.5 M EDTA, 20 µl of 1 M Tris (pH 8.0), and 2 µl of 10 mg/ml Proteinase K per tube. +36. Incubate at 45 °C for 1 hr. + +**Note:** Use QIAGEN PCR extraction kit (Or use P/C/I extraction method). + +37. Add 5 Vol. of PBI buffer (QIAGEN PCR extraction kit) and mix well—450 µl => 2.25 ml final volume. + +**Note:** Check pH => add 10 µl of 3 M sodium acetate if #23 turns orange or violet. + +38. Add sample into a column—maximum 800 µl. +39. Centrifuge at 14000 rpm for 1 min. +40. Add 750 µl of PE buffer. +41. Centrifuge at 14000 rpm for 1 min and remove the liquid from the tube. +42. Centrifuge again for 1 min at 14000 rpm. +43. Add 60 µl of EB buffer. +44. Centrifuge for 1 min at 14000 rpm. +45. Store DNA eluate at -20 °C. +46. Carry out qPCR using 2 µl elution/reaction. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/cichlid-genome-modification-malawi-cichlids-cyrqxv5w.md b/markdown-output/cichlid-genome-modification-malawi-cichlids-cyrqxv5w.md new file mode 100644 index 0000000000000000000000000000000000000000..332f8152c4b5a82c572cb4a4af9971330a66bc4d --- /dev/null +++ b/markdown-output/cichlid-genome-modification-malawi-cichlids-cyrqxv5w.md @@ -0,0 +1,203 @@ +```markdown +# Goal/Experiment: +Modification of cichlid fish via CRISPR or transgenesis + +# Cichlid Genome Modification - Malawi Cichlids +#### Forked from Cichlid Genome Modification + +Authors: Scott Juntti¹, Bethan Clark² +¹University of Maryland, ²University of Cambridge + +**Date:** +August 17, 2023 + +**Abstract:** +Here we provide a microinjection protocol for the modification of cichlid fish via CRISPR or transgenesis. This is a modified version of the protocol provided by Scott Juntti (https://www.protocols.io/view/cichlid-genome-modification-cj5wuq7e) for our Lake Malawi cichlid species for both NHEJ knock-outs and HDR knock-ins. These protocols accompany a review article on cichlid genome editing which contains more general considerations. + +**DOI:** +[dx.doi.org/10.17504/protocols.io.kqdg3xxxeq25/v1](https://dx.doi.org/10.17504/protocols.io.kqdg3xxxeq25/v1) + +**License:** +This is an open-access protocol distributed under the terms of the Creative Commons Attribution License. + +**Protocol status:** +Working - We use this protocol and confirm it's functional. + +**Created:** +August 16, 2023 + +**Last Modified:** +August 17, 2023 + +--- + +## General Considerations + +1. **Obtaining Viable Embryos** + + Obtaining viable embryos is critical for generating transgenic fish. Understanding the ovarian cycle of your cichlid species ensures timely egg collection. For example, A. burtoni has a four-week cycle, while some Malawi species have longer cycles. Maintain a cohort of females to increase the likelihood of obtaining fertilized eggs. You should be able to identify ready-to-spawn females by their physical and behavioral characteristics, such as a distended abdomen. + +## Fish Housing + +2. **Housing Setup** + + House a single male with a cohort of 7-15 females in either 80, 100, or 200-liter tanks, ensuring males and females are separated by a transparent barrier. + +## Microinjection Needle Production + +3. **Needle Preparation** + + Use a Sutter P-97 micropipette puller to make needles from borosilicate capillary tubes (GC100F-10, Harvard Apparatus). Adjust puller settings: heat 545, pull 30, velocity 50, time 200. Break the tip of the needle using a beveller to achieve an appropriate diameter (7.5-12.5 µm). + +4. **Bulk Needle Prep** + + Prepare needles in bulk but only bevel them as needed when eggs are collected. + +## Injection Station Setup + +6. **Equipment Setup** + + Use a stereomicroscope (Leica, MS5) with a 3D micromanipulator (MM33 Micromanipulator, ASI) and a Milli-Pulse Pressure Injector (MPPI-3, ASI Imaging). Set pressure to 40 psi; pulse duration to 1 ms. Confirm injection volume (1-2 nL) using phenol red solution in mineral oil. + +## Prepare Molecular Reagents + +8. **CRISPR guide RNAs synthesis** + + For synthesis of crRNA and tracrRNA plus Cas9: + [Li et al., 2023](https://liwebsite2023) + +9. **Tol2 Transgenesis Plasmids and Transposase mRNA** + + [Ma et al., 2015](https://mawebsite2015) + +## Embryo Injection Holder + +9. **Prepare Injection Holder** + + Use a 3D-printed mold for embedding embryos in 1.5% low-melting agarose. Ensure proper fit by checking for air bubbles and pre-testing molds with different teeth sizes. + +## Obtain Fertilized Embryos + +10. **Spawning** + + Remove the barrier separating males and females to initiate spawning and collect fertilized eggs. + +## Injection Mixture and Concentrations + +11. **Mixture Prep** + + - For two guides for NHEJ: + + | Reagent | Aliquot concentration (ng/μl) | Volume (μl) | Final concentration in 2.5 μl mix (ng/μl) | + |-----------|-------------------------------|-------------|-------------------------------------------| + | Cas9 | 500 | 0.5 | 100 | + | Guide 1 | 500 | 0.75 | 150 | + | Guide 2 | 500 | 0.75 | 150 | + | Texas Red | 2.5% | 0.5 | 0.5% | + + - For one guide for HDR: + + | Reagent | Aliquot concentration (ng/μl) | Volume (μl) | Final concentration in 2.5 μl mix (ng/μl) | + |-----------|-------------------------------|-------------|-------------------------------------------| + | Cas9 | 500 | 0.5 | 100 | + | Guide | 1000 | 0.75 | 300 | + | Texas Red | 2.5% | 0.5 | 0.5% | + +## Collection and Injection Process + +12. **Collect Tank Water** + + Collect water from the spawning tank but avoid Methylene Blue due to allergy concerns. + +13. **Prepare Plates** + + Fill wells with water and antibiotics (Sigma P4333) and label for tracking. + +14. **Transfer Eggs** + + Remove mold gently, and transfer embryos into holder using tank water. + +15. **Insert Eggs into Holder** + + Place embryos into individual compartments using a metal microspatula. + +## Inject and Raise Embryos + +16. **Perform Injection** + + Ensure proper pressure, focus the microscope on the chorion, and inject using a 3D micromanipulator to avoid damaging the yolk. + +17. **Assess Each Injection** + + Evaluate the injection flow, needle penetration, and embryo's response. + +18. **Transfer** + + Place embryos in individual wells post-injection, evaluating for any signs of damage. + +## Raising Embryos + +19. **Incubation** + + Initially, place embryos on an orbital shaker and change the water daily. + +20. **Track Survival** + + Regularly document embryos' survival and any emergent phenotypes. + +21. **Post-Fertilization Care** + + At 15-20 days, transfer fry to tanks for grow-out, providing food such as artemia. + +## Genotyping Injected Fish + +22. **Assess Mutations** + + Typically analyzed via Sanger sequencing of PCR from fin clip DNA at 6-12 weeks post-fertilization. + +23. **Primer Design and PCR** + + Design primers around 150 bp from the cut site. Use PCRBIO Rapid Extract Lysis Kit PB15.11, or Zymo Quick DNA/RNA Miniprep D7003. + +25. **PCR Mix** + + | Reagent | 50 μL reaction Volume | Final concentration | Notes | + |-----------------------------|-----------------------|---------------------|------------------------------------| + | 2× PCRBIO Taq Mix Red | 25 μL | 1× | see PCRBIO manual | + | Forward primer (10μM) | 2 μL | 400 nM | see optimal primer design | + | Reverse primer (10 μM) | 2 μL | 400 nM | see optimal primer design | + | Template DNA | up to 20 μL | variable | <100 ng cDNA, <500 ng genomic DNA | + | PCR grade dH₂O | up to 50 μL | final volume | - + +26. **Purify** + + Purify using QIAquick PCR Purification Kit 28104 and sequence at a facility (e.g., University of Cambridge Department of Biochemistry). + +27. **Sequence Analysis** + + Use Geneious or Synthego ICE for analysis. + +## Breeding and Analyzing Mutants + +28. **Selecting for Breeding** + + Choose animals most likely to pass mutations to the F1 generation, avoiding those with high rates of off-target effects. + +29. **Setting up Matings** + + Cross mosaic mutants or transgenes with wild-type fish to avoid off-target effects. + +30. **Collect Offspring** + + Raise offspring for PCR analysis at around 4 weeks to confirm genome editing. + +## Important Considerations + +32. **Long-term Analysis** + + Regularly cross F0 animals with wild-types to dilute off-target effects and analyze ≥2 independent mutant lines for reproducibility. + +--- + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/cloning-ecgreg-bpegreg-or-truncated-genes-into-the-ijxccpn.md b/markdown-output/cloning-ecgreg-bpegreg-or-truncated-genes-into-the-ijxccpn.md new file mode 100644 index 0000000000000000000000000000000000000000..79e9a44f7325de28f8599fa2d72c93df8e683d19 --- /dev/null +++ b/markdown-output/cloning-ecgreg-bpegreg-or-truncated-genes-into-the-ijxccpn.md @@ -0,0 +1,134 @@ +```markdown +# Goal/Experiment: +Cloning of ecGReg, bpeGReg, or truncated genes into the pTrc99A or pET-3a vectors through molecular cloning method in our laboratory. + +# Cloning ecGReg, bpeGReg, or truncated genes into the pTrc99A or pET-3a Vectors + +### Xuehua Wan, Jennifer A. Saito, Shaobin Hou, Maqsudul Alam + +> **Abstract** +> The protocol describes a molecular cloning method in our laboratory. + +## Protocol + +### DNA Amplification + +#### Step 1 + +The ecGReg, bpeGReg, or truncated genes were amplified by PCR (polymerase chain reaction). The PCR reactions were set up as below, with PfuTurbo DNA polymerase being added after a 2 minute hot start at 94 °C. This was followed by 25 or 30 cycles of 94 °C for 30 seconds, 55 °C (varies from 45 °C to 62 °C according to the Tm of primers) for 30 seconds, and 72 °C for 1-3 minutes with a final extension at 72 °C for 7 minutes. PCR products were analyzed by agarose gel electrophoresis. + +| Reagent | Volume | +| --------------------------------------- | ------- | +| DNA template | 1 µl | +| 10 × Pfu Buffer | 5 µl | +| dNTPs (10 mM) | 1 µl | +| DMSO | 2 µl | +| Primer 1 (10 pmole/µl) | 1 µl | +| Primer 2 (10 pmole/µl) | 1 µl | +| ddH2O | 38 µl | +| PfuTurbo DNA polymerase (2.5U/µl) | 1 µl | + +### PCR Purification + +#### Step 2 + +PCR products were purified using the QIAquick PCR Purification Kit (Qiagen). + +1. The 50 µl PCR product was mixed with 250 µl of Buffer PB, incubated at room temperature for two minutes, applied to a QIAquick spin column, and centrifuged for one minute at 14,000 rpm. +2. The flow-through was discarded and the bound DNA was washed twice with 750 µl Buffer PE. +3. After the flow-through was discarded, the column was centrifuged for additional one minute to remove residual ethanol. +4. The QIAquick column was placed in a clean microcentrifuge tube and air-dried for 20 minutes. +5. To elute, 30 µl of ddH2O was added to the center of the QIAquick membrane, allowed to stand for two minutes, and then centrifuged for one minute at 14,000 rpm. + +### TOPO Cloning + +#### Step 3 + +The purified blunt-end PCR product was mixed with pCR4Blunt-TOPO vector as below, and then incubated for 5-30 minutes at room temperature. + +| Reagent | Volume | +|------------------------ |------- | +| TOPO vector | 1 µl | +| ddH2O | 3 µl | +| PCR product | 1 µl | + +### Transformation of _Escherichia coli_ Competent Cells + +#### Step 4 + +1. The TOPO cloning reaction was mixed with 25 µl of TOP10 or Mach1-T1R competent cells, and then incubated on ice for 20-30 minutes. +2. The cells were heat shocked at 42 °C for 45 seconds and then placed on ice for two minutes. +3. Then, 200 µl of LB medium was added to the cells and incubated at 37 °C for one hour with shaking. +4. The cells were centrifuged at 5,000 rpm for two minutes. +5. The cell pellet was resuspended in about 50 µl of LB medium and spread on 1.2% agar plates containing the appropriate antibiotics. +6. The plates were incubated at 37 °C for 10-16 hours. + +### Plasmid Isolation + +#### Step 5 + +Plasmids were isolated using the QIAprep Spin Miniprep Kit (Qiagen). + +1. Single colonies were inoculated into 2 ml of CircleGrow medium and incubated overnight at 37 °C with shaking. +2. The cells were harvested by centrifugation and the cell pellet was resuspended with 250 µl of Buffer P1. +3. Then, 250 µl of Buffer P2 was added and the tubes were gently inverted for 4-6 times to mix. +4. After 5 minutes, 350 µl of Buffer N3 was added and mixed thoroughly by inverting the tubes. +5. The tubes were incubated at 4 °C for 10-15 minutes and then centrifuged at 14,000 rpm for 10 minutes at 4 °C to remove the precipitated cell debris, proteins, and genomic DNA. +6. The supernatant was transferred to clean tubes and centrifuged at 14,000 rpm for 15 minutes at 4 °C. +7. The supernatant was transferred to QIAprep spin columns, centrifuged at 14,000 rpm for one minute at room temperature, and the flow-through was discarded. +8. The columns were washed twice by adding 750 µl of Buffer PE and centrifuging for one minute. +9. After the second wash, the column was centrifuged for an additional one minute, transferred to a clean microcentrifuge tube, and air-dried for 45 minutes. +10. The plasmid DNA was eluted with 35 µl of Buffer EB. + +### Restriction Enzyme Digestion + +#### Step 6 + +Restriction enzyme and alkaline phosphatase were purchased from Promega. + +1. The preparative digestion reaction was set up as below and incubated in 37 °C water bath for one hour. +2. The vector was dephosphorylated by adding 1 µl of alkaline phosphatase (1U/µl) and incubating at 37 °C for an additional one hour. This was done to prevent self-ligation of partially digested vector. + +| Reagent | Volume | +|-------------------------- |------- | +| Enzyme(s) (10U/µl) each | 1 µl | +| 10X Enzyme buffer | 3 µl | +| ddH2O | 21 µl | +| DNA | 5 µl | + +### Extraction of DNA from Agarose Gels + +#### Step 7 + +DNA was purified from agarose gels using the Geneclean Spin Kit (Qbiogene). + +1. The digested DNA was run on an agarose gel and the desired bands of DNA were cut out and placed in a Geneclean Spin filter. +2. Then, 400 µl of Geneclean Spin Glassmilk was added to the filter and heated at 55 °C to melt the gel, mixing every two minutes. +3. The tube was centrifuged at 14,000 rpm for one minute and the flow-through was discarded. +4. Then 400 µl of ice-cold GeneClean Spin New Wash was added to the filter, centrifuged at 14,000 rpm for one minute, and the flow-through was discarded. +5. This washing step was repeated 3 more times. +6. The filter was centrifuged for an additional one minute, transferred to a new catch tube, and heated at 55 °C to remove residual ethanol (until Glassmilk powder became loose). +7. To elute, 15 µl of ddH2O was added to the filter, allowed to stand for 1-2 minutes, and then centrifuged for one minute. + +### Ligation to pTrc99A or pET-3a Vectors + +#### Step 8 + +The DNA purified by Geneclean was ligated overnight at 14 °C as the below. + +| Reagent | Volume | +|------------------------ |------- | +| 10X T4 DNA ligase buffer| 1 µl | +| T4 DNA ligase | 1 µl | +| DNA | 8 µl | + +### Repeat Transformation and Plasmid Isolation + +#### Step 9 + +Repeat steps 4 and 5. + +--- + +**endofoutput** +``` \ No newline at end of file diff --git a/markdown-output/cloning-shrna-oligos-into-plko-1-i8es6v.md b/markdown-output/cloning-shrna-oligos-into-plko-1-i8es6v.md new file mode 100644 index 0000000000000000000000000000000000000000..98380b6fff6bc5c115d769c9f18b327485906bc3 --- /dev/null +++ b/markdown-output/cloning-shrna-oligos-into-plko-1-i8es6v.md @@ -0,0 +1,138 @@ +```markdown +# Goal/Experiment: +Cloning shRNA Oligos into pLKO.1 + +## Abstract +This is the protocol accompanying the "pLKO.1 – TRC Cloning Vector". For information about the PLKO.1-TRC cloning vector and tips on designing shRNA oligos for pLKO.1 see Addgene's website: [http://www.addgene.org/tools/protocols/plko/](http://www.addgene.org/tools/protocols/plko/) + +Citation: Caroline LaManna Cloning shRNA Oligos into pLKO.1. protocols.io dx.doi.org/10.17504/protocols.io.cdus6v +Published: 25 Sep 2014 + +## Protocol + +### Annealing Oligos + +#### Step 1 +Resuspend oligos in ddH2O to a concentration of 20 µM. + +#### Step 2 +Add 5 µl Forward oligo. + +#### Step 3 +Add 5 µl Reverse oligo. + +#### Step 4 +Add 5 µl 10x NEB buffer 2. +- **NEBuffer 3** - 5.0 ml B7003S by **New England Biolabs**. + +#### Step 5 +Add 35 µl ddH2O. + +#### Step 6 +Incubate for 4 minutes at 95°C in a PCR machine or in a beaker of boiling water. +**Duration:** 4 minutes. + +#### Step 7 +Incubate the sample at 70°C for 10 minutes in a PCR machine. +**Duration:** 10 minutes. + +#### Step 8 +Slowly cool to room temperature over the period of several hours. +**Duration:** 3 hours. + +**Notes:** +Caroline LaManna, 21 Aug 2014 +This will take a few hours, but it is important for the cooling to occur slowly for the oligos to anneal. If using a beaker of water, remove the beaker from the flame, and allow the water to cool to room temperature. + +### Digesting pLKO.1 TRC Cloning Vector + +#### Step 9 +Mix: 6 µg pLKO.1 TRC-cloning vector (maxiprep or miniprep DNA). + +#### Step 10 +Add 5 µl 10x NEB buffer 1. +- **NEBuffer 1** - 5.0 ml B7001S by **New England Biolabs**. + +#### Step 11 +Add 1 µl AgeI. +- **AgeI** - 300 units R0552S by **New England Biolabs**. + +#### Step 12 +Bring to 50 µl with ddH2O. + +#### Step 13 +Incubate at 37°C for 2 hours. +**Duration:** 2 hours. + +#### Step 14 +Purify with Qiagen gel extraction kit, eluting in 30 µl of ddH2O. + +#### Step 15 +Digest eluate with EcoRI by mixing: 30 µl pLKO.1 TRC-cloning vector digested with AgeI. + +#### Step 16 +Add 5 µl 10x NEB buffer for EcoRI. +- **NEBuffer for EcoRI** - 10,000 units R0101S by **New England Biolabs**. + +#### Step 17 +Add 1 µl EcoRI. +- **EcoRI** - 10,000 units R0101S by **New England Biolabs**. + +#### Step 18 +Add 14 µl ddH2O. + +#### Step 19 +Incubate at 37°C for 2 hours. +**Duration:** 2 hours. + +#### Step 20 +Run digested DNA on 0.8% low melting point agarose gel until you can distinctly see 2 bands, one 7kb and one 1.9kb. + +**Notes:** +Caroline LaManna, 21 Aug 2014 +When visualizing DNA fragments to be used for ligation, use only long-wavelength UV light. Short wavelength UV light will increase the chance of damaging the DNA. + +#### Step 21 +Cut out the 7kb band and place in a sterile microcentrifuge tube. + +#### Step 22 +Purify the DNA using a Qiagen gel extraction kit. Elute in 30 µl of ddH2O. + +#### Step 23 +Measure the DNA concentration. + +### Ligating and Transforming into Bacteria + +#### Step 24 +Use your ligation method of choice. For a standard T4 ligation, mix: 2 µl annealed oligo from "Annealing Oligos" section above. + +#### Step 25 +Add 20 ng digested pLKO.1 TRC-cloning vector from the "Digesting pLKO.1 TRC Cloning Vector" section above. + +**Notes:** +Caroline LaManna, 22 Aug 2014 +If you were unable to measure the DNA concentration, use 1 µl. + +#### Step 26 +Add 2 µl 10x NEB T4 DNA ligase buffer. +- **T4 DNA Ligase Reaction Buffer** - 6.0 ml B0202S by **New England Biolabs**. + +#### Step 27 +Add 1 µl NEB T4 DNA ligase. +- **T4 DNA Ligase** - 20,000 units M0202S by **New England Biolabs**. + +#### Step 28 +Bring up to 20 µl with ddH2O. + +#### Step 29 +Incubate at 16°C for 4-20 hours. +**Duration:** 4-20 hours. + +#### Step 30 +Transform 2 µl of ligation mix into 25 µl competent DH5 alpha cells, following manufacturer’s protocol. + +#### Step 31 +Plate on LB agar plates containing 100 µg/mL ampicillin or carbenicillin (an ampicillin analog). + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/co-culture-leukemia-high-content-image-analysis-us-cxq6xmze.md b/markdown-output/co-culture-leukemia-high-content-image-analysis-us-cxq6xmze.md new file mode 100644 index 0000000000000000000000000000000000000000..3d2990194ed4eb7df55fc634d487f6e2d3a02550 --- /dev/null +++ b/markdown-output/co-culture-leukemia-high-content-image-analysis-us-cxq6xmze.md @@ -0,0 +1,82 @@ +```markdown +Goal/Experiment: +To develop a non-informatics based approach to high-content image analysis of acute leukemia cells in co-culture with mesenchymal stromal cells (MSCs) using supervised machine learning. The minimum goal is to determine absolute cell numbers for each cell class from fluorescence microscopy images of cells stained with a DNA dye. + +# Co-culture Leukemia High-Content Image Analysis using Supervised Machine Learning + +**Author:** Hayden L Bell +**Affiliations:** University of Newcastle-upon-Tyne, Dana-Farber Cancer Institute +**Published on:** July 28, 2023 +**Protocol Status:** Working +**Created:** July 26, 2023 +**DOI:** [10.17504/protocols.io.rm7vzxy52gx1/v1](https://dx.doi.org/10.17504/protocols.io.rm7vzxy52gx1/v1) + +## Disclaimer +This protocol is not intended for medical purposes. This protocol is intended for use only in a research capacity. The author/s accept no responsibility for the accuracy of data resulting from this protocol. + +## Abstract +This protocol describes an approach to high-content image analysis of acute leukemia cells in co-culture with mesenchymal stromal cells (MSCs) using supervised machine learning, leveraging open-source software applications - Cell Profiler and Ilastik. At a minimum, it aims to determine absolute cell numbers from fluorescence microscopy images of DNA-stained cells. + +## Guidelines +This example pipeline uses images from patient-derived xenograft (PDX) acute leukemia cells (AML/ALL) in co-culture with human bone marrow-derived mesenchymal stem cells (MSCs). The images are grayscale TIF images of cells assayed across a wide range of experimental conditions and live cells stained with the nucleic acid dye CyQUANT™. + +Appropriate high-content imaging systems include the Zeiss CellDiscoverer 7 and the PerkinElmer Opera. + +Quantitative data will be exported as an SQLite database. Alternatively, data can be exported as CSV file format using the ExportToSpreadsheet module. + +## Materials +- **CyQUANT™ Direct Cell Proliferation Assay, ThermoFisher Scientific, #C35011** + +## Before Start Instructions +- Two open-source applications are required: [Ilastik](https://github.com/ilastik/ilastik) and [Cell Profiler](https://github.com/CellProfiler). +- Download the project files from the Github repository available at [Image_Analysis](https://github.com/hayden-bell/Image_Analysis). Download the `BaseProject.ilp` and `BaseProject.cppipe` files before starting. +- Use high-quality fluorescence microscopy images in a lossless high-resolution file format such as TIF. + +## Protocol Steps + +### 1. Training a Supervised Machine Learning Model (Semantic Segmentation) +1. Open the Ilastik software and load the `BaseProject.ilp` project. +2. In the *Input Data* tab, load several different images (up to ~10) for training which are representative of different experimental conditions. + - For example, images from positive and negative controls whereby cell number is maximized/minimized. +3. In the *Feature Selection* tab, click **Select Features**... and ensure all 37 features are selected. +4. In the *Training* tab, ensure there are three separate Labels/classes in order as: + 1. PDX + 2. MSC + 3. Bg + 4.1 Using the Brush Cursor, manually annotate within several nuclei of each class using the respective Label class. + - Use the zoom to view the image large to ensure precision in annotation. + - Errors can be corrected using the Eraser Cursor and the image contrast can be changed using the Window Leveling tool to better visualize dimmer nuclei. + +![Example Annotation](example_annotation.png) + +5. In the *Prediction Export* panel, select **Source: Probabilities**. + 5.1 Click **Choose Export Image Settings...** and ensure the output file is in TIF format with the axis order yxc. + 5.2 Choose the Output File destination as `{dataset_dir}/probabilities/{nickname}_{result_type}.tif` +6. In the *Batch Processing* tab, click **Select Raw Data Files...** to import all of the test image data files. +7. Click **Process all files**. + +### 2. Quantifying Individual Cell Nuclei (Instance Segmentation) +8. Open the Cell Profiler software and import the `BaseProject.cppipe` pipeline (File > Import > Pipeline from File...). +9. In the *Images* module, load the probability map images generated from the Ilastik project. + - Note: Do not load the original images at this step. +10. Optional: In the *Metadata* tab, regular expressions (regex) can be used to extract meaningful data from each image filename such as plate id, well id, etc. + - By default, the pipeline will attempt to extract the well id of each image in the format A1 or A01. +11. Optional: Outlines of how well the pipeline identifies individual PDX or MSC nuclei can be visualized using the *OverlayOutlines* module. + - Select the checkpoint to enable this module and save the output by using the *SaveImages* module. +12. In the *ExportToDatabase* module, modify the Experiment name and SQLite database filename to better identify the experimental data output. + - Note: The default output location can be modified by clicking the *Output Settings* button. +13. Click **Analyze Images** to process the imported dataset and export data as SQLite database format. + +### 3. Reading the Data Output +14. Data can be read using any database software application which can open SQLite file format. + - Data can be retrieved from the `[Experiment_name]_Per_Image` data table. + + Recorded data include: + - Predicted PDX nuclei counts (Image_Count_LeukaemicNuclei) + - Predicted MSC nuclei counts (Image_Count_MSCNuclei) + - Image file name (Image_FileName_CyQ) + - Image well location (Image_Metadata_Well) + - Plus any additional data exported from separate modules or metadata extractions. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/code-barres-natif-des-amplicons-plaques-96-puits-c6b9zar6.md b/markdown-output/code-barres-natif-des-amplicons-plaques-96-puits-c6b9zar6.md new file mode 100644 index 0000000000000000000000000000000000000000..800d5769ecc82034fa00f67c748849a629d544da --- /dev/null +++ b/markdown-output/code-barres-natif-des-amplicons-plaques-96-puits-c6b9zar6.md @@ -0,0 +1,189 @@ +```markdown +# Goal/Experiment: +This experiment aims to prepare amplicons for sequencing using the Oxford Nanopore Native barcoding kit, version 14, with 96 barcodes (SQK-NBD114.96). The experiment involves using the appropriate Flow Cell (FLO-MIN114, R10.4.1) to ensure proper analysis and precise sequencing results. + +## Code-barres natif des amplicons (plaques 96 puits) + +### Authors: +- Alex Shaw1, Catherine Troman1, Joyce Akello1, Bujaki2, Javier Martin2, Nick Grassly1 + +1Imperial College London;
+2Medicine and Healthcare products Regulation Authority + +### Collaboration: +Poliovirus Sequencing Consortium + +### Abstract: +Le protocole suivant concerne la préparation des amplicons pour le séquençage à l'aide du kit de codage à barres Oxford Nanopore Native version 14 avec 96 codes à barres (SQK-NBD114.96). Lorsque vous utilisez ce kit, vous devez vous assurer d'utiliser la Flow Cell appropriée (FLO-MIN114, R10.4.1), sinon vous rencontrerez des difficultés lors du démarrage de l'analyse et les résultats de séquençage ne seront pas précis. + +Nous fournissons des estimations de ng de matériel requis pour ce protocole lors de l'amplification de la région VP1 de la poliomyélite, de la région de la capsid (PanEV) ou de l'ensemble du génome (PanPV). Si vous avez des amplicons d'une taille différente de ceux-ci, vous pouvez calculer le ng requis à l'aide d'un calculateur en ligne tel que celui-ci : Poids en quantité molaire (pour les acides nucléiques) (bioline.com). + +### Avant de commencer +Préparez environ 40 ml d'éthanol frais à 80 % avant de commencer. Cela devrait être suffisant pour l'ensemble du protocole. + +### Guidelines +Si vous avez de longs fragments, réduisez le pipetage (et le cisaillement potentiel) en feuilletant ou en inversant soigneusement les tubes ou les plaques pour mélanger les réactions et en les faisant tourner dans une centrifugeuse. + +## Materials +- Ultrapure Distilled, Nuclease Free Water +- Agencourt AmPure XP beads Catalog #A63880 +- NEBNext Quick Ligation Module - 20 rxns New England Biolabs Catalog #E6056S +- NEBNext Ultra II End Repair/dA-Tailing Module - 96 rxns New England Biolabs Catalog #E7546L +- Native barcoding kit (96) Oxford Nanopore Technologies Catalog #SQK-NBD114.96 +- NEB Blunt/TA Ligase Master Mix Catalog #M0367 +- Ultrapure BSA Ambion Catalog #AM2616 +- Nanopore Flow Cell R10.4.1 Oxford Nanopore Technologies Catalog #FLO-MIN114 + +## Preparation de l'ADN d'entree + +1. Laisser les billes AMPure se réchauffer à température ambiante, puis ajouter 0.8x le volume du mélange de réaction PCR à la réaction et pipeter doucement pour mélanger. + + - Pour une réaction PCR de 25µl, ajouter 15µl de billes AMPure XP. + + 1.1 Incuber sur un rotateur pendant 5 minutes à température ambiante. + + 1.2 Faites tourner l’échantillon et placer sur un aimant lorsqu’il soit claire et incolore. Gardez le tube sur l'aimant et pipettez le surnageant. + + 1.3 Maintenez l'aimant, lavez les billes avec 100µl d'éthanol à 80% sans perturber le granule. Retirez l'éthanol à l'aide d'une pipette et jetez. Répétez. + + 1.4 Centrifugez et replacez le tube sur l'aimant. Pipeter tout éthanol résiduel. Laissez sécher pendant 30 secondes. + + 1.5 Retirer le tube de l’étagère magnétique et remettre le granule en suspension dans 20µl d'eau sans nucléase. Incuber pendant 2 minutes à température ambiante. + + 1.6 Peler les billes sur l'aimant, puis retirer et conserver 20µl d'éluat (ou autant que possible) dans une nouvelle plaque PCR à 96 puits. + +2. Quantifier l'ADN amplifié avec un kit "Qubit Broad Range dsDNA". + + 2.1 En bref, créer un master mix de 200µl (199µl de tampon, 1µl de réactif Qubit) pour chaque échantillon + 2 standards + 10%. Pour les deux standards, ajouter 190µl du “master mix” à 10µl de standard, et pour les échantillons, ajouter 198µl du “master mix” à 2µl d’échantillon. Vortexer tous les tubes standards et échantillons et incuber à température ambiante pendant 2 minutes avant de quantifier. + + 2.2 *Important - assurez-vous de choisir le kit vaste gamme sur le Qubit* Enregistrer la concentration d'ADN pour chaque échantillon. + +3. Transférer 200 fmol d'ADN par échantillon dans une plaque à 96 puits propre et ajuster le volume à 12.5µl avec de l'eau sans nucléase. Mélangez doucement en pipettant. 200 fmol ≈ ~155ng VP1, ~584ng PanEV, ~974ng PanPV. + +4. Centrifugez brièvement dans une centrifugeuse à plaques. + +## End-prep & dA-tailing + +5. Préparez les réactifs suivants comme master mix pour le nombre d'échantillons + 10% supplémentaire : + + | A | B | + |-----------|-------------| + | Tampon de réaction Ultra II End-prep (Ultra II End-prep reaction buffer) | 1.75 | + | Mélange d'enzymes Ultra II End-prep (Ultra II End-prep enzyme mix) | 0.75 | + +6. Ajouter 2.5µl du mélange réactionnel à chaque échantillon. + +7. Mélanger par pipetage et centrifuger dans une centrifugeuse à plaques. + +8. Incuber 5 minutes à 20°C et 5 minutes à 65°C à l'aide du thermocycleur. + +## Ligation des codes-barres et mutualisation + +9. Sélectionnez un code-barres natif unique pour chaque échantillon de l'analyse et décongelez à température ambiante, puis gardez-les sur de la glace. + +10. Décongeler les billes AMPure à température ambiante. Mélanger les billes AMPure au vortex avant d'utiliser. + +11. Dans chaque puits requis d'une plaque 96 puits, ajouter : + + | A | B | + |-----------------------------------------|-------------| + | ADN préparé aux extrémités (end-prepped DNA) | 3.75 | + | Code-barres natifs | 1.25 | + | Blunt/TA Ligase Master Mix | 5 | + +12. Mélanger doucement en pipettant. Sceller la plaque et centrifuger. + +13. Incuber la plaque pendant 20 minutes à température ambiante. + +14. Ajouter 1µl d'EDTA dans chaque puits, mélanger par pipetage puis centrifuger brièvement. + +15. Regroupez tous les échantillons dans un tube de 1.5ml. + +16. Préparez les billes AMPure XP pour utilisation; remettre en suspension au vortex. + +17. Nettoyer et concentrer les échantillons regroupés en utilisant un volume de billes AMPure XP remises en suspension égale à 0.4x le volume de vos échantillons regroupés. + + 17.1 Ajouter les billes AMPure XP, pipeter pour mélanger. Incuber pendant 10 minutes à température ambiante. + + 17.2 Faites tourner l’échantillon et le granule sur une étagère magnétique lorsqu’il soit claire et incolore, puis pipettez le surnageant. + + 17.3 Gardez sur l'aimant et lavez les billes avec 700µl d'éthanol à 80%, puis retirer l'éthanol et jeter. Répétez. + + 17.4 Centrifugez et replacez le tube sur l’étagère magnétique. Pipeter tout éthanol résiduel. Laissez sécher pendant 30 secondes. + + 17.5 Remettre le granule en suspension dans 35µl d'eau sans nucléase en effleurant le tube. + + 17.6 Incuber pendant 10 minutes à 37°C, en agitant le tube toutes les 2-3 minutes pour favoriser l'élution. + + 17.7 Pelez les perles sur l’étagère magnétique jusqu'à ce qu'elles soient claires, puis conservez 35µl dans un tube propre Eppendorf DNA LoBind de 1.5ml. + +## Ligation de l'adaptateur + +18. Décongelez les réactifs comme suit: Décongelez le tampon de réaction de ligature rapide NEBNext (5X) (NEBNext Quick Ligation Reaction Buffer 5X), tampon d'élution (Elution Buffer - EB) et soit un tube de Long Fragment Buffer (LFB) pour les amplicons plus longues (<3kb) ou un tube de Short Fragment Buffer (SFB) pour maintenir toutes les différentes tailles d’amplicons à température ambiante, mélanger au vortex, centrifuger et placer sur de la glace. Faites tourner l'adaptateur natif (Native Adaptor - NA) et la ligase T4 rapide (Quick T4 Ligase), mélangez par pipetage et placez sur de la glace. + +19. Aux 30µL d'échantillon à code-barres regroupé, ajoutez les réactifs suivants : + + | A | B | + |----------|-------------------------------| + | Adaptateur natif (Native Adaptor - NA) | 5µl | + | Tampon de réaction de ligature rapide NEBNext (5X) (NEBNext Quick Ligation Reaction Buffer 5X) | 10µl | + | ADN ligase rapide T4 (NEB Quick T4 DNA Ligase) | 5µl | + +20. Mélanger en tapotant le tube et centrifuger. + +21. Incuber la réaction pendant 20 minutes à température ambiante. + +22. Remettre les billes AMPure XP en suspension au vortex, puis ajouter 20µl de billes AMPure XP à la réaction de ligature de l'adaptateur. + + 22.1 Incuber pendant 10 minutes à température ambiante sur un rotateur. + + 22.2 Faites tourner l'endroit sur une étagère magnétique, laissez les billes se sédimenter et pipetez le surnageant. + + 22.3 Laver les billes en ajoutant 125µL de LFB ou SFB, effleurer le tube pour remettre les billes en suspension, centrifuger puis remettre sur l'aimant pour granuler les billes. Pipeter le surnageant, puis répéter cette étape. + + 22.4 Faites tourner le tube et remettez-le sur l’étagère magnétique. Pipeter le surnageant résiduel. + + 22.5 Retirer le tube de l’étagère magnétique et remettre en suspension le granule dans 15µl de tampon d'élution. Effleurez doucement le tube pour remettre le culot en suspension. + + 22.6 Incuber à 37 degrés pendant 10 minutes, en effleurant doucement le tube toutes les 2-3 minutes pour favoriser l'élution de l’ADN. + + 22.7 Centrifugez puis culottez les billes sur l’étagère magnétique, et retirez et conservez 15µl d’éluat dans un tube propre de 1.5ml. Jetez les billes granulées. + +23. Quantifiez 1µl de votre bibliothèque adaptée à l'aide d'un Qubit ou tapestation. + +24. Ajoutez 20 fmol de votre bibliothèque dans un tube PCR de 0.2ml et augmentez le volume jusqu'à 12µl à l'aide du tampon d'élution. Si vous avez besoin de diluer la bibliothèque pour faciliter le pipetage, vous pouvez la diluer dans un tampon d'élution. 20 fmol = ~16ng VP1, ~59ng PanEV, ~98ng PanPV. + +## Amorcage et chargement de la Flow Cell MinION + +25. Décongelez le tampon de séquençage (Sequencing Buffer - SB), les perles de bibliothèque (Library Beads - LIB), l’attache de cellule d’écoulement (Flow Cell Tether - FCT) et un tube de flux de cellule d’écoulement (Flow Cell Flush - FCF) à température ambiante et remettez-les dans la glace une fois décongelés. + + Mélanger les tubes de tampon de séquençage (Sequencing Buffer - SB), flux de cellule d’écoulement (Flow Cell Flush - FCF) et attache de cellule d’écoulement (Flow Cell Tether - FCT) au vortex, centrifuger et remettre dans la glace. Faites tourner le LIB une fois décongelé, puis remettez-le dans la glace. + +26. Pour créer le mélange d'amorçage, ajoutez 30µL de FCT et 5µL de BSA (50mg/ml) au tube de FCF, puis mélangez par pipetage. + +27. Ouvrez le couvercle du dispositif de séquençage nanopore et faites glisser le couvercle du port d'amorçage de la Flow Cell dans le sens des aiguilles d'une montre afin que le port d'amorçage soit visible. Après avoir ouvert le couvercle d'amorçage, vérifiez s'il y a de l'air sous l'orifice. Retirez un petit volume en faisant tourner le piston de la pipette pour augmenter lentement le volume afin d'éliminer les bulles d'air (quelques µL). Vérifiez visuellement qu'il y a un tampon continu du port d'amorçage à travers le réseau de capteurs. + +28. Utilisez une pipette P1000 pour chargez 800µl du mélange d'amorçage dans la cellule à écoulement via le port d'amorçage, en évitant l'introduction de bulles d'air. Laissez une petite quantité de liquide à l'extrémité de la pointe de la pipette pour vous assurer de ne pas introduire d'air dans la cuve à circulation. Attendez 5 minutes. + +29. Mélanger le contenu du tube LIB en pipetant juste avant de l’ajouter au mélange de bibliothèque suivant dans un tube de 1.5ml: + + | A | B | + |---------------------------|-------------| + | Bibliothèque d'ADN | 12 | + | Tampon de séquençage | 37.5 | + | Billes de bibliothèque (LIB) | 25.5 | + +30. Terminez l'amorçage de la flowcell en ouvrant le couvercle du port SpotOn et en chargeant soigneusement 200µl du mélange d'amorçage dans le port d'amorçage. Comme avant, laissez une petite quantité de liquide au fond de l'embout pour éviter l'introduction de bulles d'air. + + Lors de l'ajout du mélange d'amorçage, vous pouvez voir une petite quantité de liquide monter par le port SpotOn. Si vous le faites, faites une pause et laissez le liquide refluer dans la flowcell avant de continuer à passer le mélange d'amorçage. + +31. Mélangez délicatement la librairie préparée en la pipetant. + +32. Ajouter goutte à goutte 75µL d'échantillon à la Flow Cell via le port d'échantillon SpotON. Assurez-vous que chaque goutte coule dans le port avant d'ajouter la suivante. + +33. Replacez délicatement le couvercle du port d'échantillonnage SpotON, en vous assurant que le bouchon pénètre dans le port SpotON, fermez le port d'amorçage et replacez le couvercle MinION. + +34. Ouvrez le logiciel ONT MinKNOW et suivez les étapes pour configurer et démarrer votre cycle de séquençage. Dans la section Démarrer, sélectionnez démarrer l'exécution et suivez les invites pour sélectionner le kit utilisé, définir la durée d'exécution et définir l'appel de base et le démultiplexage. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/coleta-de-dados-actigr-ficos-acttrust-bwbnpame.md b/markdown-output/coleta-de-dados-actigr-ficos-acttrust-bwbnpame.md new file mode 100644 index 0000000000000000000000000000000000000000..b7f9fe97a2dcd94afee29f1ba495958a766d364e --- /dev/null +++ b/markdown-output/coleta-de-dados-actigr-ficos-acttrust-bwbnpame.md @@ -0,0 +1,95 @@ +```markdown +# Goal/Experiment: +The goal of this experiment is to document the protocols for the collection of actigraphic data by the Interdisciplinary Sleep Research Group (GIPSO) using ActTrust devices manufactured by Condor Instruments. These protocols include pre-collection, collection, post-collection processing, and sterilization methods to ensure accurate and reliable actigraphic data collection. + +## Coleta de dados actigráficos - ActTrust V.2 + +### Author +**Daniel Vartanian** +University of Sao Paulo + +For any inquiries or suggestions, please contact us at gipso@usp.br. + +--- + +### DISCLAIMER +**FOR INFORMATIONAL PURPOSES ONLY; USE AT YOUR OWN RISK** + +The protocol content here is for informational purposes only and does not constitute legal, medical, clinical, or safety advice. The content added to [protocols.io](https://protocols.io) is not peer reviewed and may have not undergone a formal approval of any kind. Any action you take or refrain from taking using or relying upon the information presented here is strictly at your own risk. The Company, authors, or anyone else associated with [protocols.io](https://protocols.io) cannot be held responsible for the use of the information in this protocol or any affiliated services. + +--- + +### ABSTRACT +This collection brings together the protocols for the actigraphic data collection process of the Interdisciplinary Sleep Research Group (GIPSO). The process is designed for actigraphs from Condor Instruments. Some configurations may be specific to these devices. + +If you are not a member or partner of GIPSO, you may use this collection as a reference for processing your equipment. Please do not send the mentioned forms regarding this process. + +If you use GIPSO's protocols in your research, please consider citing this collection in your materials and methods section. We strive to create and maintain open access protocols for the scientific community. You can find protocol citations under metadata. + +Please, refer to the Guidelines tab to visualize a flowchart of the entire process. + +For questions or suggestions, contact us via email: gipso@usp.br. + +**DOI:** +[10.17504/protocols.io.bwbnpame](https://dx.doi.org/10.17504/protocols.io.bwbnpame) + +**Collection Citation:** +Daniel Vartanian 2021. Coleta de dados actigráficos - ActTrust. protocols.io +Version created by Daniel Vartanian + +--- + +### Keywords +Actigrafia, actimetria, sono, cronobiologia, condor instruments + +--- + +### LICENSE +This is an open access collection distributed under the terms of the [Creative Commons Attribution License](https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. + +--- + +### Created +July 04, 2021 + +### Last Modified +July 04, 2021 + +### Collection Integer ID +51278 + +--- + +## GUIDELINES + +The actigraphic data collection process of GIPSO follows the following flowchart: + +![Flowchart of the Actigraphic Data Collection Process by GIPSO](path_to_your_diagram_image.png) + +**Font:** elaborated by the author. + +_Note: The 'Volunteer Approach' step does not have a general protocol as it varies according to the study._ + +--- + +### Files + +- **Conexão do actígrafo no computador - ActTrust - V.1** + - by Daniel Vartanian, University of Sao Paulo + +- **Processamento de actígrafos pré-coleta - ActTrust - V.2** + - by Daniel Vartanian, University of Sao Paulo + +- **Processamento de actígrafos pós-coleta - ActTrust - V.2** + - by Daniel Vartanian, University of Sao Paulo + +- **Higienização/esterilização de actígrafos - ActTrust - V.1** + - by Daniel Vartanian, University of Sao Paulo + +- **Processamento de actígrafos para armazenagem - ActTrust - V.2** + - by Daniel Vartanian, University of Sao Paulo + +--- + +**endofoutput** +``` \ No newline at end of file diff --git a/markdown-output/collect-of-amoebophrya-parasite-free-living-stage-vrye57w.md b/markdown-output/collect-of-amoebophrya-parasite-free-living-stage-vrye57w.md new file mode 100644 index 0000000000000000000000000000000000000000..d9cb7f6255e90222719f396198e66f49517d4b53 --- /dev/null +++ b/markdown-output/collect-of-amoebophrya-parasite-free-living-stage-vrye57w.md @@ -0,0 +1,182 @@ +```markdown +# Goal/Experiment: +Collect Amoebophrya parasite (free-living stage) for genomic and transcriptomic analyses for better understanding of their infection cycle and impact on host organisms. + +## Collect of Amoebophrya parasite (free-living stage) for genomic and transcriptomic analyses + +### Authors +- Estelle Bigeard +- Station Biologique de Roscoff, France, CNRS + +### DOI +dx.doi.org/10.17504/protocols.io.vrye57w + +### Supporting Team +Ecology of Marine Plankton (ECOMAP) team - Roscoff + +--- + +### Abstract + +**Amoebophrya** (Syndiniales or MALVII, Alveolata) is an eukaryotic parasite which infects and kills its host. Parasitic cultures are particularly rich in bacteria that develop by degrading the organic matter released during the infection. + +![Life cycle of Amoebophrya](https://image.url.here) + +- **Uninfected**: Just the host. +- **Cytoplasm**: Initial steps of the growing endoparasitic stage inside the host. +- **Intermediate**: Feeding stage of the parasite inside the host nucleus (trophont). +- **Beehive**: Sporulating parasite. +- **Dinospores**: Free-living stage. + +### Source of Strains + +Strains originated from the Penzé estuary (North-West of France, English Channel, 48°37'N; 3°56'W). Cultures of the non-toxic dinoflagellates _Scrippsiella acuminata_ and _Heterocapsa triquetra_ were established from single cyst isolates. + +- Two reference strains (MALVII-Clade 2) were chosen: + - A25: Specialist parasite from Penzé, infecting _Scrippsiella sp._ + - A120: Generalist parasite from Penzé, infecting _Heterocapsa triquetra_ + +The strains are maintained at the Roscoff Culture Collection: + +- ID numbers: ST147=RCC1627, HT150=RCC3596, A25=RCC4383, A120=RCC4398. + +### Experimental Objectives + +- Synchronize infection of _Scrippsiella_ hosts for mass production of dinospores. +- Remove bacteria at the beginning of infection and limit their growth. +- Collect free-living dinospores without host cells or large cell debris. +- Extract genomic DNA without host contaminants for genomic analyses. +- Extract total RNA without residual DNA for transcriptomic analyses. + +### Protocol Status +- **Working** + +### Guidelines +- Protocol: EB-BM-M0-009 + +### Materials + +**Flasks:** + +| Item | Specifications | Vendor | Ref No. | +|------|----------------|--------|---------| +| Flacons T-150 CytoOne | Treated, ventilated, Green | Starlab | CC7682-4815 | +| Flacons T-225 CytoOne | Treated, ventilated | Starlab | CC7682-4822 | +| Flacons T-75 CytoOne | Non-treated, non-ventilated | Starlab | CC7672-4175 | +| Erlenmeyer | 250 and 500ml | - | - | + +**Filtration System:** + +| Item | Specifications | Vendor | Ref No. | +|------|----------------|--------|---------| +| Nalgene PSF | 250ml diameter 47mm | - | - | +| Tweezers | Millipore | - | - | +| Filters Nylon | 10μm, diameter 47mm | Millipore | NY1004700 | +| Filters PC Whatman | 10μm, diameter 47mm | VWR | 515-2089 | +| Filters PC Whatman | 5μm, diameter 47mm | VWR | 515-2087 | +| Filters PC Whatman | 3μm, diameter 47mm | VWR | 515-2086 | +| Filters PC nuclepore Whatman | 0.6μm, diameter 47mm | VWR | 515-2082 | + +**Reagents:** + +| Reagent | Concentration/Quantity | Use | Vendor | Ref No. | +|---------|------------------------|-----|--------|---------| +| PNS 100x | Penicillin 5000units, Neomycin 10mg, Streptomycin 5mg | Antibiotic | Sigma | P4083 | +| Guillard's (F/2) Marine Water Enrichment Solution | 50x | Media | Sigma | G9903 | +| Glutaraldehyde solution grade II | 25% | Fixative | Sigma | G6257-10X10ML | +| Pluronic | - | - | Sigma | P5556-100ml | +| SyBrGreen | - | DNA staining | Life Technologies | S7585 | + +### Safety Warnings +- Handle all chemicals with appropriate safety measures. +- Proper sterilization and disposal of all biohazardous wastes. + +### Procedure + +#### 1. Host and Parasite Cultures + +**Media Preparation:** +- Use F/2 medium (Guillard's Marine Water Enrichment Solution, Sigma) with filtered natural seawater. +- Add 5% (v/v) soil extract (Starr and Zeikus 1993). +- Final filtration using a 0.22 μm pore size. + +**Stock Culture Conditions:** +- Maintain at 19°C on an L:D cycle of 12:12h; 80 μEinstein m².s⁻¹. + +**_Scrippsiella acuminata_ ST 147 Host Culture:** +- Inoculate in fresh media, transferring 3/4 media and 1/4 growing exponentially. +- Transfer every Monday and Friday to maintain volume. +- Incubate at 21°C on a 12:12 L:D cycle. + +**Parasite _Amoebophrya_ Culture:** +- Inoculate by transferring 1/10 of parasite culture in host J3-J4 culture. +- Seed every Monday and Friday to increase culture volume. +- Incubate at 21°C, light cycle 12:12. + +#### 2. Sample Preparation for Cytometry +- Take 1ml from each flask at each stage. +- Fix samples with 1% glutaraldehyde/pluronic for 5 minutes. +- Freeze samples in cryotubes at -80°C. + +#### 3. Filtration and Infection Initiation (T0) + +**Microscopic Observations:** +- Cultures under epifluorescence inverted microscope (450nm blue filter). +- Monitor dense, exponentially growing host stocks and liberated, active dinospores. + +**Filtration on PC 5μm Filter:** +- Install sterile filtration funnels on vacuum flasks. +- Place a PC 5μm filter on each funnel. +- Filtrate culture by gravity. +- Pool all filtrates. + +**Infection Initiation (T0):** +- In a 600ml flask, add 300ml of host culture and 150ml filtrate. +- Incubate at 21°C with light cycle 12:12. + +#### 4. Bacteria Removal by 10μm Filtration (~J1) + +- Bacteria and dinospores are pico-sized and can be separated by 10μm filtration. +- Ensure most hosts are infected to optimize filter use. + +**Observation:** +- Ensure visible dinospores but low autofluorescence from parasites. + +**First 10μm Filtration:** +- Prepare 200ml flasks with fresh media. +- Filter culture by gravity using 10μm filters. +- Rinse and observe filters, pipette fresh media to remove host cells. + +**Second 10μm Filtration:** +- Install fresh 10μm filters. +- Realize 2nd filtration. +- Rinse and transfer newly infected host cells. + +**Add Antibiotics (PNS 100x):** +- Add 20ml PNS 100x per 400ml culture. +- Incubate at 21°C, light 12:12. + +#### 5. Parasite Culture Purification (~J4) + +- Perform 3 days after bacterial removal. + +**Observation:** +- Check low presence of host cells. + +**Filtration on 3μm Filter:** +- Install filters and filtrate by gravity. +- Each flask needs 4 filtration systems. +- Pool filtrates. + +**Genomic Approach:** +- Filtrate into 15ml Falcon tubes, flash freeze in liquid nitrogen. + +**Transcriptomic Approach:** +- Add 4ml trizol to 15ml tubes, wrap in aluminum foil. +- Filtrate with 0.6μm filters. +- Flash freeze samples within 15 minutes to avoid RNA degradation. + +--- + +`endofoutput` +``` \ No newline at end of file diff --git a/markdown-output/collection-and-processing-of-intertidal-seagrass-t-butrnwm6.md b/markdown-output/collection-and-processing-of-intertidal-seagrass-t-butrnwm6.md new file mode 100644 index 0000000000000000000000000000000000000000..5e55d47df5b66d8d07e77907d01f1f215d249337 --- /dev/null +++ b/markdown-output/collection-and-processing-of-intertidal-seagrass-t-butrnwm6.md @@ -0,0 +1,130 @@ +```markdown +# Goal/Experiment: +This protocol details the collection and processing of intertidal seagrass tissues for bacterial and fungal culturing. + +## Collection and Processing of Intertidal Seagrass Tissues for Bacterial and Fungal Culturing + +**PLOS One** + +**Cassie Ettinger** +University of California, Riverside +May 11, 2021 + +**DOI:** [10.17504/protocols.io.butnrwm6](https://dx.doi.org/10.17504/protocols.io.butnrwm6) + +**Protocol Citation:** +> Cassie Ettinger 2021. Collection and processing of intertidal seagrass tissues for bacterial and fungal culturing. *protocols.io* +> [https://dx.doi.org/10.17504/protocols.io.butnrwm6](https://dx.doi.org/10.17504/protocols.io.butnrwm6) + +### Abstract +This protocol details the collection and processing of intertidal seagrass tissues for bacterial and fungal culturing. + +## Keywords +Intertidal seagrass tissues, Bacterial culturing, Fungal culturing + +## Materials + +### Materials for Field Collections +- PVC pipe (2.375 inch diameter) +- 1L Nalgene bottles +- Coring device +- Ziploc bags + +### Materials for Sample Processing and Inoculation on Culture Media +- Flame sterilized scissors +- 1.5 mL centrifuge tubes +- 1 mL autoclaved nanopure water +- Flame sterilized tweezers +- 1 mL 0.5% NaOCl +- 95% EtOH +- 500 µL 95% ethanol +- 500 µL 70% ethanol + +### Definitions and Functions +**NaOCl (Sodium Hypochlorite):** +- A common disinfectant used for surface sterilization. + +**EtOH (Ethanol):** +- Used for sterilization purposes in various concentrations (95%, 70%). + +**Autoclaved Nanopure Water:** +- Ultrapure water sterilized under high pressure and temperature to prevent contamination. + +## Procedure + +### Field Collections (Duration: 1d 4h) +1. **Put on gloves.** + +2. **Walk to a location that is submerged (~0.5 m).** + +3. **Using a coring device, take a sample of an individual seagrass plant and its associated sediment.** + 1. Insert a clear PVC pipe with a 2.375 Inch diameter that has been modified such that one end of the pipe is cut at an angle to make insertion into the sediment easier. + 2. Once inserted, cap the PVC pipe, pull the core up, and then cap the bottom of the core. + +4. **Place core in dark box on ice until transport back to the lab.** + +5. **Replace gloves and repeat to obtain multiple cores.** + +6. **Collect nearby seawater in 1 L Nalgene bottles (or similar) as needed for media recipes.** + +7. **Using new gloves, carefully pull up additional seagrass plants as needed for media recipes and/or inoculation, place into sterile Ziploc bags, and then put on ice.** + +8. **Once back at the lab, place cores, bulk tissues, and seawater into 4 °C fridge or cold room and use as needed within 4–24 hours of collection.** + +### Sample Processing and Inoculation on Culture Media (Duration: 15m 35s) +9. **Rinse the tissue with autoclaved nanopure water to remove loosely associated sediment for ~30 seconds.** + - **Plant tissues (leaf, root, rhizome, matte).** + - Before starting, prepare plates with your favorite culture media (e.g., see Ettinger & Eisen 2020 for possible media choices previously used for seagrass-associated work). + +10. **Cut ~1 cm pieces of tissue using flame sterilized scissors.** + +11. **Place a subset of these tissue segments directly on plates using flame sterilized tweezers (1–3 segments/plate).** + +12. **Take another subset of tissue segments and place these segments into 1.5 mL centrifuge tubes with 1 mL of autoclaved nanopure water.** + +13. **Vortex the 1.5 mL centrifuge tubes for ~30 seconds.** + +14. **For a subset of segments, homogenize using a sterile pestle, while for another subset, leave the segments intact.** + +15. **Directly plate intact rinsed tissue segments on media using flame sterilized tweezers (1–3 segments/plate).** + +16. **Pipette 350 µL of wash liquid from rinsed segments or homogenized smashed tissue directly on plates.** + - As needed, you can also pipette 1:10 and 1:100 dilutions of wash liquid on plates. + +17. **If of interest, one can try to remove epiphytes from tissues through several wash steps before plating.** + - One protocol is to bleach tissues prior to plating, for example: + 1. Immerse segments for 5 minutes in 1 mL 0.5% NaOCl (~10% bleach). + 2. Then immerse in 1 mL of 95% EtOH for 1 minute. + 3. Next, immerse in 1 mL autoclaved nanopure water for 3 minutes. + 4. Finally, directly plate intact bleached tissue segments on media using flame sterilized tweezers (1–3 segments/plate). + +18. **Another, slightly longer protocol to surface clean tissues, involves:** + 1. First, immerse segments in 500 µL 95% ethanol for ~5 seconds. + 2. Then, immerse in 500 µL 0.5% NaOCl (~10% bleach) for 2 minutes. + 3. Next, immerse in 500 µL 70% ethanol for 2 minutes. + 4. Then rinse segments with autoclaved nanopure water for 1 minute. + 5. Finally, directly plate intact surface cleaned tissue segments on media using flame sterilized tweezers (1–3 segments/plate). + +### Sediment (Duration: 15m 35s) +19. **Place sediment into 1.5 mL centrifuge tubes.** + +20. **Add 1 mL of autoclaved nanopure water to tubes.** + +21. **Vortex tubes for ~30 seconds.** + +22. **Pipette 350 µL of sediment suspension directly onto plates.** + - As needed, you can also pipette 1:10 and 1:100 dilutions of sediment suspension on plates. + +### Seawater (Duration: 15m 35s) +23. **Pipette 350 µL of seawater directly onto plates.** + - As needed, you can also pipette 1:10 and 1:100 dilutions of seawater on plates. + +--- + +**License:** This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. + +**Created:** May 07, 2021 +**Last Modified:** May 11, 2021 + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/collection-of-brain-stem-for-post-mortem-diagnosis-n4sdgwe.md b/markdown-output/collection-of-brain-stem-for-post-mortem-diagnosis-n4sdgwe.md new file mode 100644 index 0000000000000000000000000000000000000000..4f5643ccc052cd88cc1ff64a7a0a4b89c17602f3 --- /dev/null +++ b/markdown-output/collection-of-brain-stem-for-post-mortem-diagnosis-n4sdgwe.md @@ -0,0 +1,181 @@ +```markdown +# Goal/Experiment: +To describe the procedure of collecting brain stem tissue from a postmortem animal for the diagnosis of lyssavirus infection by the LN34 real-time RT-PCR assay. + +# Collection of Brain Stem for Post-Mortem Diagnosis of Rabies in Animals by the LN34 Pan-Lyssavirus Real-Time RT-PCR Assay + +## Abstract +Patterns of rabies virus spread within the central nervous system suggest that a thorough examination of the brainstem is critical for rabies diagnosis. A negative finding for rabies can confidently be made only if a complete cross-section of the brainstem is examined. Incomplete or suboptimal specimens should be tested when possible. + +## Guidelines + +### Purpose +To describe the procedure of collecting brainstem tissue from a postmortem animal for the diagnosis of lyssavirus infection using the LN34 real-time RT-PCR assay. + +### Responsibility +Laboratorians will perform the procedure according to the protocol. Once sample collection is complete, the work area is disinfected, and any biohazardous waste is autoclaved or incinerated before discarding. Samples are then returned to storage. + +### Specimens +Postmortem animal brain tissue. + +## Equipment, Materials and Reagents + +1. **Necropsy Instruments** + - 1 set of instruments per sample to prevent cross-contamination. + +2. **Autoclave and/or Instrument Sterilizer** + - All instruments should be cleaned and sterilized before reuse. + +3. **Specimen Storage Containers** + - Containers must be large enough for reserved portions of brainstem. + - Glass vials and tubes are unacceptable for specimen storage due to breakage risk. + +4. **Freezer** + - Long-term storage should be at -70°C or lower. + - Frost-free freezers should not be used. + +5. **PPE (Personal Protective Equipment)** + - Heavy rubber gloves or other cut-resistant gloves. + - Laboratory gown. + - Waterproof apron. + - Surgical mask. + - Boots. + - Protective sleeves. + - Face shield. + - Disposable gloves. + +6. **Polyester Fiber-Tipped Applicator Swab** + +7. **Scalpel** + +8. **Sterile Petri Dish or Paper Towel** + +9. **Disposable Sterile Screwtop Microcentrifuge Tubes with O-ring (2 ml)** + - Add 1 ml TRIzol reagent to one tube for each sample. + - Label tubes with corresponding accessioning information. + +10. **Acceptable Surface Decontaminants** + 1. Quaternary ammonium disinfectant (1:256). + 2. 70% Isopropanol. + 3. Iodophors. + +### Definitions and Keywords + +- **BSC (Biological Safety Cabinet)**: A ventilated cabinet for safe handling of pathogens. +- **PPE (Personal Protective Equipment)**: Protective clothing and gear. +- **Room Temperature**: 20°C to 25°C (68°F to 77°F). + +### Quality Control and Corrective Action + +- Document and report any deviations or concerns to the lab supervisor. +- Monitor temperature levels on applicable equipment. +- Ensure specimens stay within acceptable temperature range. +- All samples should have unique identifiers and corresponding accession labels. + +## Protocol + +### Shipment of Samples + +#### Step 1. +- Ensure specimen transit time to the laboratory is as short as possible, preferably within 48 hours. +- Refrigeration will preserve a sample for at least 48 hours. +- Repeated freeze-thaw cycles may reduce test sensitivity and should be avoided. +- Maintain biocontainment during specimen transport. + +### Sample Sufficiency + +#### Step 2. +- Assess the condition of the sample upon arrival at the laboratory. +- A complete cross-section of the brainstem is necessary for a reliable rabies diagnosis. +- Unsatisfactory sample conditions should be reported, and tests should still be performed. + +### Sample Processing + +#### Step 3. +- Samples may be a complete carcass, an intact head, whole brain, or dissected brain tissues. +- Dissected brain tissue must include a complete cross-section of the brainstem. +- Retain the carcass for unusual samples. +- Use unique sample identifiers. + +### Sample Handling + +#### Step 4. +- Ensure proper identification of each sample. +- Handle each specimen with new disposable gloves on a clean work surface. +- Thoroughly disinfect all instruments. +- Store instruments not in use in closed storage. + +### Sample Retention + +#### Step 5. +- Retain frozen reference material in the laboratory for all test samples for 2 to 6 months. +- Maintain positive samples for controls or epidemiologic typing. +- Ensure storage containers allow complete cross-sections to be made if repeat testing is required. + +### Collection of a Cross Section of Brain Stem + +#### Step 6. +Setup: +- Thaw frozen samples just prior to testing. +- Clean and disinfect the work surface. +- Lay out a plastic-lined absorbent pad as a clean work surface. +- Store additional clean supplies outside the BSC or work area. + +#### Step 7. +- Collect brain tissue representing a full cross-section of the brainstem from both left and right sides. + 1. The brainstem is anterior to the cerebellum and continuous with the spinal cord. + 2. Tissue should include a complete cross (transverse) section of at least one area of the brainstem. + 3. For small animals, the entire brain stem may be collected. + 4. For larger animals, collection of a cross-section is suggested. + +#### Step 8. +- Collect brain impressions for DFA test if required. Remaining tissue may be used for RNA extraction with the LN34 assay. + +### Prepare Samples for RNA Extraction + +#### Step 9. +- Important: Once TRIzol reagent is added, samples cannot be used for antigen-based detection methods. Retain a portion of original tissue without TRIzol for future testing. + +#### Step 10. +- Add 200 mg of tissue (~pea size or <250 µl liquid volume) to a 2 mL screw-top microcentrifuge tube containing TRIzol reagent. Use a polyester fiber tip applicator or similar sterile object. + +#### Step 11. +- For small to medium animals, place the entire piece of brainstem (>10 mg) into the tube with 1 ml TRIzol reagent. +- Homogenize brain pieces using a bead beater, micro tissue grinder, or sterile applicator stick. + +#### Step 12. +- For larger animals, cut the cross-section of the brainstem into small pieces. +- Homogenize brain pieces and then add 200 µl homogenized tissue to 1 ml fresh TRIzol reagent. + +#### Step 13. +- Clean and disinfect the workstation, equipment, and outside of sample tubes. + +#### Step 14. +- Samples in TRIzol can be processed immediately for RNA extraction, stable at room temperature or refrigeration for several hours, and long-term storage should be at -20°C or colder. + +#### Step 15. +- Additional DFA test recommendations: [DFA Test PDF](https://www.cdc.gov/rabies/pdf/RabiesDFASPV2.pdf) +- Necropsy procedures video: available from the CDC. + +## References + +1. Manning, S.E., et al., *Human rabies prevention--United States, 2008: recommendations of the Advisory Committee on Immunization Practices*. MMWR Recomm Rep, 2008. 57 (RR-3): p. 1-28. + +2. World Health Organization, *WHO Expert Consultation on Rabies. Second report*. World Health Organ Tech Rep Ser, 2013(982): p. 1-139. + +3. World Organization for Animal Health (OIE), *Rabies (infection with rabies virus) in Manual of diagnostic tests and vaccines for terrestrial animals.* 2017. [OIE Rabies Manual PDF](http://www.oie.int/fileadmin/Home/eng/Health_standards/tahm/2.01.17_RABIES.pdf) + +## Warnings + +### Hazards and Safety Precautions +- Samples may contain infectious agents. Use, store, and dispose of them in accordance with safety regulations. +- Wear appropriate PPE. +- Follow procedures from the Biosafety in Microbiological and Biomedical Laboratories (BMBL), 5th Edition. +- Conduct all manipulations of tissues to prevent aerosolization of liquids. +- Pre-exposure rabies vaccination and regular serologic tests are required for personnel working with lyssaviruses. +- Fume hoods or biosafety hoods are recommended for odor, ectoparasites, and bone fragments. +- A class II biosafety cabinet is required if using an electric saw. +- Use and dispose of hazardous chemicals according to safety regulations and the SDS. + +`endofoutput` +``` \ No newline at end of file diff --git a/markdown-output/combined-metagenomic-metatranscriptomic-pipeline-f-d7m9k5.md b/markdown-output/combined-metagenomic-metatranscriptomic-pipeline-f-d7m9k5.md new file mode 100644 index 0000000000000000000000000000000000000000..b42e2567fd0de8215bc3e5efff6364c465eebbfd --- /dev/null +++ b/markdown-output/combined-metagenomic-metatranscriptomic-pipeline-f-d7m9k5.md @@ -0,0 +1,176 @@ +```markdown +# Goal/Experiment: +The purpose of this protocol is to set up a combined metagenomic and metatranscriptomic pipeline for analyzing host-associated microbiomes. This pipeline aims to filter DNA and RNA reads, align them to reference databases, obtain bacterial genomes, align RNA reads to these genomes, and derive pathway information to understand bacterial abundance and gene expression changes. + +# Combined Metagenomic / Metatranscriptomic Pipeline for Host-associated Microbiomes + +**Author:** Scott Daniel + +## Abstract + +### Overview: +1. **Filter DNA reads** by quality and host (e.g., for mouse gut bacteria, filter low-quality reads and reads that belong to mice). +2. **Align DNA reads** (fastq, fasta) to [Patric](https://patricbrc.org/) database (bacteria genomes) with [taxoner](https://github.com/hurwitzlab/singularity-taxoner) (a bowtie2-based Linux program) and extract bacterial genomes. +3. **Filter** out low-quality alignments to get bacterial genomes. +4. **Filter RNA reads** by quality. +5. **Align RNA reads** to bacterial genomes obtained in step 3 to get RNA counts of genes. +6. Get pathway information to get RNA counts per KEGG pathway. + +If you have treatment groups, you can see changes in bacterial abundance as well as changes in RNA counts. + +## Before Start +It is recommended to run this pipeline on university supercomputers unless you have a small set of reads and/or genomes. + +## Protocol + +### Setup + +#### Step 1. +Get sample data from GitHub (requires `git` to be installed on your system). + +```bash +git clone https://github.com/hurwitzlab/mg-sample-data.git +``` +Notes: +- Scott Daniel, 05 Sep 2017 +- This can be downloaded to your shared supercomputers (job scheduler managed) or your own laptop. The data is small enough (< 10MB) to be trivial to run on most systems. + +#### Step 2. +Edit `config.sh` with your favorite text editor to set variables (mainly directories). +```bash +vim config.sh +``` + +### QC + +#### Step 3. +Get recipe for Singularity image containing [fastqc](https://www.bioinformatics.babraham.ac.uk/projects/fastqc/) and [solexaqa](https://solexaqa.sourceforge.net/): +- This must be downloaded to a system where you have sudo privileges. + +```bash +git clone https://github.com/hurwitzlab/singularity-fastqc.git +``` + +#### Step 4. +From within `/singularity-fastqc`, run `./create_and_bootstrap.sh`. This should create a `fastqc.img`. + +```bash +cd singularity-fastqc/ && ./create_and_bootstrap.sh +``` +Safety Information: +- Ensure there are no errors (e.g., "ABORT: Aborting with RETVAL=255"). + +#### Step 5. +sftp or scp the created `fastqc.img` to your shared supercomputer directory or put it in your `/bin` directory (or another directory in your PATH). + +```bash +scp fastqc.img username@ftp.hpc.arizona.edu:/rsgrps/usergroup/username/singularity-images +``` + +#### Step 6. +Generate quality reports with fastqc (optional). + +```bash +mg-sample-data/scripts/00-fastqc-reports.sh +``` +Notes: +- Scott Daniel, 12 Sep 2017 +- This script is intended to be submitted to a PBS job scheduler but can be edited to run with other job schedulers. + +#### Step 7. +Run trim_galore (a wrapper script for cutadapt) to cut off low-quality bases and adapters (default is to cut off bases until the probability that the base call was correct is ≥99%). + +```bash +mg-sample-data/scripts/01-trim-galore.sh +``` + +#### Step 8. +Compare quality reports between steps 5 and 6. If there's not much improvement, consider re-running step 6 with different parameters. + +### Bowtie2 Prep + +#### Step 9. +Build Singularity images for this section: singularity-taxoner and singularity-tuxedo (named after the tuxedo suite of tools: bowtie2, cufflinks, etc). + +#### Step 10. +Get the GitHub repositories for building the images. + +```bash +git clone https://github.com/hurwitzlab/singularity-tuxedo.git +git clone https://github.com/hurwitzlab/singularity-taxoner.git +``` + +#### Step 11. +From within `/singularity-tuxedo`, run `./create_and_bootstrap.sh`. This should create a `bowcuff.img`. + +```bash +cd singularity-tuxedo/ && ./create_and_bootstrap.sh +``` + +From within `/singularity-taxoner`, run `./create_and_bootstrap.sh`. This should create a `taxoner.img`. + +```bash +cd singularity-taxoner/ && ./create_and_bootstrap.sh +``` +Safety Information: +- Ensure there are no errors (e.g., "ABORT: Aborting with RETVAL=255"). + +#### Step 12. +sftp or scp the created `bowcuff.img` and `taxoner.img` to your shared supercomputer directory or put them in your `/bin` directory (or another directory in your PATH). + +```bash +scp bowcuff.img taxoner.img username@ftp.hpc.arizona.edu:/rsgrps/usergroup/username/singularity-images +``` + +#### Step 13. +Run `02-taxadb-prep.pbs` to prepare the lineage file and genome fasta's for bowtie2 indexing. + +```bash +mg-sample-data/scripts/02-taxadb-prep.pbs +``` +Note: +- This script is meant to be submitted to a PBS job scheduler using the command `qsub 02-taxadb-prep.pbs` but can be edited to run with other job schedulers. + +#### Step 14. +Make Bowtie2 indices of the split multi-genome fasta files. + +```bash +mg-sample-data/scripts/03-bowtie2-build.sh +``` +Note: +- The example script will launch bowtie2 to run index building on two computers in parallel. + +### Taxoner + +#### Step 15. +Run taxoner for the DNA reads to determine species composition. + +```bash +mg-sample-data/scripts/04-taxoner.sh +``` + +### Centrifuge + +#### Step 16. +Get the Singularity container for [centrifuge](https://ccb.jhu.edu/software/centrifuge/), a classifier program for bacterial/viral species. + +```bash +git clone https://github.com/scottdaniel/singularity-centrifuge.git +``` + +Safety Information: +- If doing this step for species identification, you can optionally skip QC and definitely skip Bowtie2 Prep. + +#### Step 17. +Build the Singularity container as before. + +```bash +cd singularity-centrifuge && make img +``` + +### Warnings + +None + +## endofoutput +``` \ No newline at end of file diff --git a/markdown-output/comparative-study-in-safety-and-efficacy-of-mainte-bvq7n5zn.md b/markdown-output/comparative-study-in-safety-and-efficacy-of-mainte-bvq7n5zn.md new file mode 100644 index 0000000000000000000000000000000000000000..78c65ef7513d6b5fa8209c660a0190ae72f3e334 --- /dev/null +++ b/markdown-output/comparative-study-in-safety-and-efficacy-of-mainte-bvq7n5zn.md @@ -0,0 +1,103 @@ +```markdown +# Goal/Experiment: +Comparative study in safety and efficacy of maintenance therapies for patients with anti-neutrophil cytoplasmic antibody small vessel vasculitis: A network meta-analysis + +## Comparative study in safety and efficacy of maintenance therapies for patients with anti-neutrophil cytoplasmic antibody small vessel vasculitis: A network meta-analysis + +### Authors +- Ioannis Bellos1 +- Sophia Lionaki2 + +1National and Kapodistrian University of Athens
+2Department of Nephrology, Attikon Hospital, National and Kapodistrian University of Athens, Athens, Greece + +### DOI +[doi.org/10.17504/protocols.io.bvq7n5zn](https://doi.org/10.17504/protocols.io.bvq7n5zn) + +### Abstract +Anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis represents a potentially life-threatening disease. Induction of remission is achieved in the vast majority of patients by high-dose glucocorticoid therapy combined with cyclophosphamide or rituximab, followed by oral glucocorticoid tapering; however, relapses are common. The present network meta-analysis aims to accumulate current literature knowledge and compare the efficacy and safety of different regimens used for maintenance of remission in patients with ANCA vasculitis. + +### Keywords +- anca +- vasculitis +- maintenance +- meta-analysis + +### License +This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. + +### Creation Date +June 11, 2021 + +### Last Modified +June 11, 2021 + +### Protocol Integer ID +50687 + +## 1. Background +Anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis represents a potentially life-threatening disease characterized by vascular inflammation, endothelial, and tissue injury. + +- **ANCA (Anti-neutrophil cytoplasmic antibodies)**: Autoantibodies mainly against enzymes found in neutrophils, such as proteinase 3 (PR3) and myeloperoxidase (MPO). +- **Proteinase 3 (PR3)**: An enzyme found in neutrophils, targeted by certain ANCAs. +- **Myeloperoxidase (MPO)**: Another enzyme found in neutrophils, targeted by certain ANCAs. + +### Disease Notes +- Necrotizing vasculitis predominantly affects small vessels leading to inflammation in various tissues and organs such as lungs and glomeruli. +- High-dose glucocorticoid therapy combined with cyclophosphamide or rituximab is essential for treatment initiation and improving prognosis by limiting irreversible organ damage. + +### Relapses and Maintenance +Relapses occur frequently, with reported rates ranging from 10% to 60%. Several factors may increase relapse risk, including PR3-ANCA seropositivity and upper respiratory involvement. + +## 2. Study Design +The network meta-analysis will comply with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses Extension Statement for Reporting of Systematic Reviews Incorporating Network Meta-Analyses (PRISMA-NMA) guidelines. + +## 3. Eligibility Criteria +- **Population**: Adult patients with ANCA-associated vasculitis in complete remission including clinical phenotypes of granulomatosis with polyangiitis, microscopic polyangiitis, and renal-limited disease. +- **Exclusions**: Patients with eosinophilic granulomatosis with polyangiitis (Churg-Strauss syndrome), end-stage kidney disease, receiving renal replacement therapies. +- **Interventions**: Azathioprine, cyclophosphamide, rituximab, methotrexate, mycophenolate mofetil, leflunomide, belimumab with azathioprine. +- **Outcomes**: Relapse (any/major), relapse-free survival, overall survival, adverse effects (any serious, any serious infections, any cancer). +- **Study type**: Randomized controlled trials. +- **Exclusions**: Observational studies, review articles, in vitro studies, animal studies. + +## 4. Search Strategy +- **Databases**: Medline, Scopus, CENTRAL, Web of Science, and Clinicaltrials.gov will be systematically searched from inception. +- **Methods**: Google Scholar will be used to provide grey literature coverage. Full references will be retrieved to identify additional sources (“snowball” method). +- **Search Terms**: Combinations of MeSH terms such as "Antibodies, Antineutrophil Cytoplasmic”, and key terms like “anca”, “vasculitis”, and specific conditions like “granulomatosis with polyangiitis”. +- **Restrictions**: No date or language restrictions will be applied. + +## 5. Study Selection +The studies will be selected in three stages: +1. **Title and abstract screening** +2. **Full-text retrieval for eligibility** +3. **Final inclusion based on the exclusion criteria** + +## 6. Data Extraction +Two researchers will perform data extraction independently. Information to be extracted includes: +- Author(s) +- Publication date +- Country +- Study design +- Inclusion and exclusion criteria +- Vasculitis clinical phenotype +- MPO/PR-3 positivity +- Patients' number, sex, serum creatinine, estimated glomerular filtration rate, BVAS score, organ involvement, type of induction treatment, and necessary data for outcomes of interest. + +## 7. Quality Assessment +- **Tool**: RoB-2 tool for randomized controlled trials. +- **Domains**: Randomization, deviations from intended interventions, missing outcome data, measurement of the outcome, selection of reported results. +- **Evaluation**: Confidence In Network Meta-Analysis (CINeMA) approach for credibility, Grading of Recommendations Assessment, Development, and Evaluation (GRADE) framework for bias assessment. + +## 8. Data Analysis +- **Model**: Random-effects frequentist network meta-analytic model for pooled odds or hazard ratios. +- **Confidence Intervals**: Set at 95%. +- **Evaluation**: P-scores for ranking interventions. +- **Heterogeneity Quantification**: Inconsistency index (I2) and 95% prediction intervals (PI). + +### Evaluation of Bias +- Small-study effects by visual inspection of comparison-adjusted funnel plots. +- Transitivity assumption testing by comparing distributions of potential confounders. +- Global consistency assessed with the design-by-treatment interaction test. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/comparison-of-two-fat-emulsions-on-interleukin-1-i-bjzhkp36.md b/markdown-output/comparison-of-two-fat-emulsions-on-interleukin-1-i-bjzhkp36.md new file mode 100644 index 0000000000000000000000000000000000000000..0bb92e25660e170ae8c6a0f58c53c812b3e3b754 --- /dev/null +++ b/markdown-output/comparison-of-two-fat-emulsions-on-interleukin-1-i-bjzhkp36.md @@ -0,0 +1,93 @@ +```markdown +# Goal/Experiment: +The aim of this study is to investigate the effect of omega-3-enriched intravenous fat emulsion compared to standard MCT/LCT intravenous fat emulsion on IL-1β, IL-8 levels and fatty acid composition in infants post-gastrointestinal surgery. + +# Comparison of Two Fat Emulsions on Interleukin-1β, Interleukin-8 and Fatty Acid Composition in Infants Post Gastrointestinal Surgery: A Randomized Trial Protocol V.2 + +**Meta Herdiana Hanindita** +Doctoral Program, Faculty of Medicine, Universitas Airlangga + +**DOI:** [10.17504/protocols.io.bjzhkp36](dx.doi.org/10.17504/protocols.io.bjzhkp36) + +## Abstract +Surgical intervention in infants is associated with postoperative sepsis and severe outcomes due to an immature immune system. Gastrointestinal surgery induces excessive cytokine secretion, which may lead to increased postoperative mortality and morbidity. + +Parenteral nutrition plays a crucial role in pediatric patients undergoing gastrointestinal surgery. Intravenous lipid emulsion is an integral part of parenteral nutrition because it contains high energy density and low osmolality, becoming the main source of energy and essential fatty acids. Fatty acids are crucial for structural integrity and cell membrane fluidity, and they regulate the expression of various genes and signaling pathways during inflammation. + +The current standard type of Intravenous Fat Emulsion (IVFE) is a 50:50 mixture of medium chain triglyceride (MCT) and long chain triglyceride (LCT). This IVFE is rich in omega-6 and contains high levels of linoleic acid and alpha-linolenic acid. Omega-6 is associated with impaired cell-mediated immunity and higher risks of elevated pro-inflammatory markers. + +Some studies have indicated that omega-3 soy oil-based fat emulsion may improve patients' outcomes by modulating the inflammatory response, particularly eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), inhibiting the inflammatory pathway and generating less inflammatory eicosanoids. However, the effect of omega-3 enriched intravenous fat emulsion on IL-1β, IL-8 levels and plasma fatty acid composition in infants undergoing gastrointestinal surgery has not been comprehensively elucidated. + +### Trial Design +**Type:** Parallel randomized controlled trial +**Allocation ratio:** 1:1 +**Framework:** Superiority + +## Aim of the Study +To investigate the effect of omega-3-enriched intravenous fat emulsion compared to standard MCT/LCT intravenous fat emulsion on IL-1β, IL-8 levels and plasma fatty acid composition in infants post-gastrointestinal surgery. + +### PICO Approach: +- **P:** Patients undergoing gastrointestinal surgery +- **I:** Omega-3-enriched intravenous fat emulsion +- **C:** MCT/LCT standard intravenous fat emulsion +- **O:** + +## Outcome Measures + +| Outcome measure | Tool for measurement | Unit of measurement | +|-------------------------------|----------------------|-------------------------| +| Primary Inflammatory response IL-1 | Blood | pg/ml | +| Inflammatory response IL-8 | Blood | Pg/ml | +| Secondary Fatty Acid composition | Blood | (% of Total fatty acids)| + +### Research Question +Will the intravenous omega-3 enriched-fat emulsion make a difference in IL-1β, IL-8 and fatty acid composition compared to the standard intravenous MCT/LCT fat emulsion? + +## Protocol + +### Experimental Groups + +**A - The experimental group (ω-3-enriched intravenous fat emulsion):** +1. The parents or legal guardians of patients who fulfill the inclusion and exclusion criteria must sign informed consent after the investigator explains the study. +2. Before surgery, blood samples of patients will be drawn (3-4 cc) to examine IL-1β, IL-8 levels, and plasma fatty acid composition. +3. After surgery, patients will receive ω-3-enriched intravenous fat emulsion (SMOFlipid) for three consecutive days at 1-4 grams/kilogram/day. +4. On day three after surgery, blood samples will be drawn (3-4 cc) to examine post-treatment IL-1β, IL-8 levels, and plasma fatty acid composition. +5. All blood samples will be sent to the Prodia laboratory. + +**B - The control group (MCT/LCT standard intravenous fat emulsion):** +1. The parents or legal guardians of patients who fulfill the inclusion and exclusion criteria must sign informed consent after the investigator explains the study. +2. Before surgery, blood samples of patients will be drawn (3-4 cc) to examine IL-1β, IL-8 levels, and plasma fatty acid composition. +3. After surgery, patients will receive MCT/LCT standard intravenous fat emulsion (SMOFlipid) for three consecutive days at 1-4 grams/kilogram/day. +4. On day three after surgery, blood samples will be drawn (3-4 cc) to examine post-treatment IL-1β, IL-8 levels, and plasma fatty acid composition. +5. All blood samples will be sent to the Prodia laboratory. + +## Materials + +| Name | Catalog # | Vendor | +|-------------------|-----------|-------------| +| Interleukin-1beta | | R&D Systems | +| Interleukin-8 | | R&D Systems | + +### Materials Text + +**Primary Outcome (p): IL-1β, IL-8** +- IL-1β, IL-8 levels will be measured by Quantikine HS ELISA R&D System before surgery and three days after surgery. + +**Secondary Outcome (Fatty Acid Composition):** +- The profiling of fatty acid composition was analyzed using gas chromatography tandem mass spectrometry (GC-MS/MS) before surgery and three days after surgery. + +### Safety Warnings +- Allergy reaction: rash + +## License +This protocol is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. + +## Citation +Meta Herdiana Hanindita 2020. Comparison of Two Fat Emulsions on Interleukin-1β, Interleukin-8 and Fatty Acid Composition in Infants Post Gastrointestinal Surgery: A Randomized Trial Protocol. protocols.io [Link](https://dx.doi.org/10.17504/protocols.io.bjzhkp36) + +**Created:** Aug 20, 2020 +**Last Modified:** Aug 20, 2020 +**Protocol Integer ID:** 40713 + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/comparison-of-two-lipid-emulsions-on-interleukin-1-bknmkvc6.md b/markdown-output/comparison-of-two-lipid-emulsions-on-interleukin-1-bknmkvc6.md new file mode 100644 index 0000000000000000000000000000000000000000..1be5de709357a17bf51a5a57fcb8a039ff99fd90 --- /dev/null +++ b/markdown-output/comparison-of-two-lipid-emulsions-on-interleukin-1-bknmkvc6.md @@ -0,0 +1,102 @@ +```markdown +# Goal/Experiment: +Investigate the effect of ω-3-enriched intravenous fat emulsion compared to standard MCT/LCT intravenous fat emulsion on the levels of IL-1β, IL-8, and plasma fatty acid composition in infants who undergo gastrointestinal surgery. + +# Comparison of Two Lipid Emulsions on Interleukin-1β, Interleukin-8 and Plasma Fatty Acid Composition in Infants Post Gastrointestinal Surgery: A Randomized Trial + +**Meta Herdiana Hanindita1** + +1Universitas Airlangga + +DOI: [10.17504/protocols.io.bkmnkv6c](https://dx.doi.org/10.17504/protocols.io.bkmnkv6c) + +## Abstract +Surgical intervention in infants is associated with postoperative sepsis and severe outcomes due to an immature immune system. Gastrointestinal surgery induces excessive cytokine secretion, leading to increased postoperative mortality and morbidity. Intravenous lipid emulsion is a critical part of parenteral nutrition, with fatty acids determining structural integrity and its impact on the inflammatory response. + +The study aimed to investigate the effect of ω-3-enriched intravenous fat emulsion compared to standard intravenous fat emulsion on IL-1β, IL-8 levels, and plasma fatty acid composition in infants undergoing gastrointestinal surgery. + +## Trial Design +- **Type:** Parallel randomized controlled trial +- **Allocation ratio:** 1:1 +- **Framework:** Superiority + +## Aim of the Study +To investigate how ω-3-enriched intravenous fat emulsion impacts IL-1β, IL-8 levels, and plasma fatty acid composition compared to standard MCT/LCT intravenous fat emulsion in infants post gastrointestinal surgery. + +## Research Question +Will the intravenous omega-3 enriched-fat emulsion make a difference in IL-1β, IL-8, and fatty acid composition compared to standard intravenous MCT/LCT fat emulsion? + +## PICO Approach: +- **P (Population):** Patients underwent gastrointestinal surgery. +- **I (Intervention):** ω-3-enriched intravenous fat emulsion. +- **C (Comparison):** MCT/LCT standard intravenous fat emulsion. +- **O (Outcome):** Inflammatory response and fatty acid composition. + +## Outcomes +| Outcome | Tool for Measurement | Unit of Measurement | +|-----------------|----------------------|------------------------| +| **Primary** | | | +| Inflammatory | Blood | pg/mL | +| Response: | | | +| IL-1β, IL-8 | | | +| Fatty Acid | Blood | % Total Fatty Acid | +| Composition | | | +| **Secondary** | | | +| Laboratory | Blood | g/dL | +| parameters: | | | +| Hemoglobin | | | +| Laboratory | Blood | /microL | +| parameters: | | | +| Leukocyte | | | +| Laboratory | Blood | g/L | +| parameters: | | | +| Albumin | | | +| Laboratory | Blood | mg/L | +| parameters: | | | +| CRP | | | + +## Before Start +### Group A +#### The experimental group (ω-3-enriched intravenous fat emulsion) +1. The parents or legal guardians of patients who fulfill the inclusion and exclusion criteria will have to sign informed consent after the investigator explains the study. +2. Before surgery, blood samples of 3-4 cc will be drawn to examine IL-1β, IL-8 levels, and plasma fatty acid composition. Laboratory parameters will also be examined. +3. Post-surgery, patients will get ω-3-enriched intravenous fat emulsion (SMOFlipid) for three consecutive days in 1-4 gram/kilogram/day dosing. +4. On the third day post-surgery, blood samples will again be drawn to measure IL-1β, IL-8 levels, and plasma fatty acid composition. Laboratory parameters will be examined. +5. All blood samples will be sent to Prodia laboratory. + +### Group B +#### The control group (MCT/LCT standard intravenous fat emulsion) +1. The parents or legal guardians of patients who fulfill the inclusion and exclusion criteria will have to sign informed consent after the investigator explains the study. +2. Before surgery, blood samples of 3-4 cc will be drawn to examine IL-1β, IL-8 levels, and plasma fatty acid composition. Laboratory parameters will also be examined. +3. Post-surgery, patients will get the standard MCT/LCT intravenous fat emulsion for three consecutive days in 1-4 g/kg/day dosing. +4. On the third day post-surgery, blood samples will again be drawn to measure IL-1β, IL-8 levels, and plasma fatty acid composition. Laboratory parameters will be examined. +5. All blood samples will be sent to Prodia laboratory. + +## Materials +| Name | Catalog # | Vendor | +|-----------------|-------------|-----------------| +| Interleukin-1β | | R&D Systems | +| Interleukin-8 | | R&D Systems | + +### Materials Text +Reagents used: +- FAME Standard Mix (Supelco) +- GLC Nonadecanoic ISTD (Supelco) +- N-Hexane MS grade (Merck) +- Chloroform (Merck) +- Methanol Hyper Grade (Merck) +- Capillary Column and Helium Gas for GCMS + +## Citation +Meta Herdiana Hanindita (09/02/2020). Comparison of Two Lipid Emulsions on Interleukin-1β, Interleukin-8 and Plasma Fatty Acid Composition in Infants Post Gastrointestinal Surgery: A Randomized Trial. [protocols.io](https://dx.doi.org/10.17504/protocols.io.bkmnkv6c) + +## License +This protocol is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0). + +**Date Created:** Sep 02, 2020 +**Last Modified:** Sep 02, 2020 + +**Protocol Integer ID:** 41389 + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/complete-sample-protocol-for-pma-induced-erk-activ-gsebwbe.md b/markdown-output/complete-sample-protocol-for-pma-induced-erk-activ-gsebwbe.md new file mode 100644 index 0000000000000000000000000000000000000000..b3c6b6609a78f283f7a18a250c11f1ac7b546d47 --- /dev/null +++ b/markdown-output/complete-sample-protocol-for-pma-induced-erk-activ-gsebwbe.md @@ -0,0 +1,282 @@ +```markdown +Goal/Experiment: +The goal of this experiment is to study PMA-induced ERK activation in suspension cell lines using an In-Cell Western assay. + +# Complete Sample Protocol for PMA-Induced ERK Activation in Suspension Cell Lines + +**Developed for:** +- Aerius +- Odyssey® Classic +- Odyssey CLx +- Odyssey Sa +- Infrared Imaging Systems + +**Citation:** +LI-COR Biosciences Complete Sample Protocol for PMA-Induced ERK Activation in Suspension Cell Lines. protocols.io dx.doi.org/10.17504/protocols.io.gsebwbe + +**Published:** +26 Jun 2018 + +## Guidelines + +### Required Reagents + +#### LI-COR Reagents +- IRDye® 800CW Goat anti-Rabbit Secondary Antibody (LI-COR P/N 925-32210 or 926-32210) +- IRDye 680RD Goat anti-Mouse Secondary Antibody (LI-COR P/N 925-68070 or 926-68070) +- Odyssey® Blocking Buffer (LI-COR P/N 927-40000 or 927-50000) +- Large Western Incubation Box (LI-COR P/N 929-97301) + +#### Additional Reagents +- 1X PBS wash buffer +- Tissue culture reagents (RPMI 1640, fetal bovine serum, etc.) +- Clear or black 96-well or 384-well microplates +- Jurkat cells (ATCC®, P/N TIB-152TM) +- THP-1 monocytes (ATCC, P/N TIB-202TM) +- K-562 lymphocytes (ATCC, P/N CCL-243TM) +- Concentrated Prefer (5X) (Anatech LTD, P/N 411) +- TO-PRO®-3 (Molecular Probes, P/N T-3605) +- PMA (phorbol 12-myristate 13-acetate) (Sigma®, P/N P1585) +- DMSO (dimethyl sulfoxide) (Sigma, P/N D8418) +- ERK rabbit antibody (Santa Cruz, P/N sc-94) +- pERK mouse antibody (Cell Signaling Technology, P/N 9106) + +### Experimental Considerations + +Proper selection of microplates can significantly affect results, as each plate has its own characteristics, including well depth, plate autofluorescence, and well-to-well signal crossover. Use the following considerations for microplate selection: + +- **In-Cell Western analyses** use detection at the well surface with minimal liquid present. Avoid plates with white walls, as autofluorescence can create significant noise. +- Both flat and round-bottom plates show some plate autofluorescence, but it is relatively small compared to the actual signal. +- **Focus Offset Optimization:** If different plates are used, determine focus offset empirically by scanning a plate and comparing focus settings. + +#### Focus Offset Determination (mm) + +| Instrument | Focus Offset Determination (mm) | +|-------------------------------|---------------------------------| +| Odyssey Classic & Odyssey CLx | 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0 | +| Odyssey Sa & Aerius | 1.5, 2.0, 2.5, 3.0, 3.5, 3.95 | + +Use the same intensity settings for each scan. After reviewing the scans, use the focus offset with the highest signal-to-noise ratio for experiments. + +#### Intensity Setting Optimization + +To optimize intensity settings: + +| Instrument | Initial Intensity Setting (700/800 nm) | Intensity Settings: Weak Signal (700/800 nm) | Intensity Settings: Saturated Signal (700/800 nm) | +|-------------------|----------------------------------------|---------------------------------------------|--------------------------------------------------| +| Odyssey® Classic | 5 / 5 | 7.5 / 7.5 | 2.5 / 2.5 | +| Odyssey CLx | 5 / 5 | 7.5 / 7.5 | 2.5 / 2.5 | +| Odyssey Sa | 7 / 7 | 8 / 8 | 4 / 4 | +| Aerius | 7 / 7 | 8 / 8 | 4 / 4 | + +**Note:** Protect plates from light before imaging. Establish the specificity of your primary antibody by screening lysates. + +## Protocol + +### Cell Preparation + +**Step 1:** +Allow Jurkat (ATCC, P/N TIB-152) cell growth in a T75 flask using standard tissue culture procedures. Avoid growing cells to density greater than 2 x 10^6^ cells. + +**Step 2:** +Transfer cells in growth media to 50 mL conical tubes and centrifuge at 500 x g for 5 minutes. + +**Step 3:** +Remove media and resuspend the cell pellet in 10 mL of serum-free media (pre-warmed to 37°C). Pipet very slowly to maintain cell integrity. Transfer resuspended cells into T75 flask and place in an incubator (37 °C and 5% CO_2). + +**Step 4:** +Allow cells to settle for 30 minutes before taking a 50µL aliquot of cells for counting using a hemocytometer. + +**Step 5:** +Add the appropriate volume of serum-free media to achieve 1 x 10^6^ cells/mL (1 plate x 96 wells x 200 µL of cells/well = 20 mL/plate). + +**Step 6:** +Serum-deprive cells by placing cells suspended in serum-free media back into the incubator for an additional 3.5 hours or overnight. + +### Cell Treatment + +**Step 7:** +- Add 2 µL of DMSO to both background samples and resting cells in triplicate wells. +- Add 2 µL of 1:1 serial dilutions of PMA ranging from 0.005 to 3.2 ng/mL in triplicate wells. + +**Step 8:** +Using a multi-channel pipettor, transfer 200 µL of suspended cells (200,000 cells) per well into the wells containing 2 µL of DMSO or PMA from step 6. + +**Step 9:** +Allow incubation at 37 °C for 15 minutes. + +### Fixing/Permealbilizing Cells + +**Step 10:** +Add 50 µL of concentrated Prefer (or 25 µL of 37% formaldehyde; final concentration is 4%) into each well. + +**Step 11:** +Allow cells to fix for 20 minutes at room temperature with gentle rotation (speed 2 on The Belly Dancer®). + +**Step 12:** +Centrifuge at 1,500 RPM (332 RCF) for 10 minutes. + +**Step 13:** +Wash three times with 100 µL of 1X PBS containing 0.1% Triton® X-100 for 5 minutes each by centrifugation at 1,500 RPM (332 RCF). + +**Step 14:** +Prepare Triton Permeabilization Solution: +- 1X PBS: 495 mL +- 10% Triton X-100: 5 mL +- 1X PBS + 0.1% Triton X-100 + +**Step 15:** +Remove fixing solution. + +**Step 16:** +Using a multi-channel pipettor, add 100 µL of fresh Triton Permeabilization Solution. Add the solution by pipetting down the sides of the wells carefully to avoid detaching the cells from the well bottom. + +**Step 17:** +Centrifuge at 1,500 RPM (332 RCF) for 5 minutes. (Wash 1/3) + +**Step 18:** +Gently remove Triton Permeabilization Solution by manually pipetting. (Wash 1/3) + +**Step 19:** +Using a multi-channel pipettor, add 100 µL of fresh Triton Permeabilization Solution. (Wash 2/3) + +**Step 20:** +Centrifuge at 1,500 RPM (332 RCF) for 5 minutes. (Wash 2/3) + +**Step 21:** +Gently remove Triton Permeabilization Solution by manually pipetting. (Wash 2/3) + +**Step 22:** +Using a multi-channel pipettor, add 100 µL of fresh Triton Permeabilization Solution. (Wash 3/3) + +**Step 23:** +Centrifuge at 1,500 RPM (332 RCF) for 5 minutes. (Wash 3/3) + +### Blocking Cells + +**Step 24:** +Using a multi-channel pipettor, add 100 µL of Odyssey Blocking Buffer to each well. Add the solution by pipetting down the sides of the wells carefully to avoid detaching the cells. + +**Step 25:** +Allow blocking for 1 hour at room temperature with very gentle shaking. + +### Primary Antibodies + +**Step 26:** +Dilute antibodies in Odyssey Blocking Buffer: +- Rabbit anti-ERK antibody (1:200 dilution; Santa Cruz) +- Mouse anti-phospho-ERK antibody (1:100 dilution; Cell Signaling Technology) + +**Step 27:** +Mix the primary antibody solution well before adding to wells. + +**Step 28:** +Remove blocking buffer. + +**Step 29:** +Add 50 µL of Odyssey Blocking Buffer to the background control wells (serves as non-specific background fluorescence). + +**Step 30:** +Add 50 µL of the primary antibody solution into the rest of the wells. + +**Step 31:** +Incubate with primary antibody for 2 hours at room temperature or overnight at 4 °C with very gentle shaking. + +### Washing + +**Step 32:** +Wash plates five times with 200 µL of 1X PBS + 0.1% Tween® 20 for 5 minutes by centrifugation at 1,500 RPM (332 RCF). + +**Step 33:** +Prepare Tween® Washing Solution: +- 1X PBS: 995 mL +- 20% Tween: 5 mL + +**Step 34:** +Remove primary antibody solution. + +**Step 35:** +Using a multi-channel pipettor, add 200 µL of Tween Washing Solution. Make sure to add down the sides to avoid detaching cells. (Wash 1/5) + +**Step 36:** +Centrifuge at 1,500 RPM (332 RCF) for 5 minutes. (Wash 1/5) + +**Step 37:** +Gently remove Tween Washing Solution. (Wash 1/5) + +**Step 38:** +Using a multi-channel pipettor, add 200 µL of Tween Washing Solution. (Wash 2/5) + +**Step 39:** +Centrifuge at 1,500 RPM (332 RCF) for 5 minutes. (Wash 2/5) + +**Step 40:** +Gently remove Tween Washing Solution. (Wash 2/5) + +**Step 41:** +Using a multi-channel pipettor, add 200 µL of Tween Washing Solution. (Wash 3/5) + +**Step 42:** +Centrifuge at 1,500 RPM (332 RCF) for 5 minutes. (Wash 3/5) + +**Step 43:** +Gently remove Tween Washing Solution. (Wash 3/5) + +**Step 44:** +Using a multi-channel pipettor, add 200 µL of Tween Washing Solution. (Wash 4/5) + +**Step 45:** +Centrifuge at 1,500 RPM (332 RCF) for 5 minutes. (Wash 4/5) + +**Step 46:** +Gently remove Tween Washing Solution. (Wash 4/5) + +**Step 47:** +Using a multi-channel pipettor, add 200 µL of Tween Washing Solution. (Wash 5/5) + +**Step 48:** +Centrifuge at 1,500 RPM (332 RCF) for 5 minutes. (Wash 5/5) + +**Step 49:** +Gently remove Tween Washing Solution. (Wash 5/5) + +### Secondary Antibodies + +**Step 50:** +Dilute secondary antibody in Odyssey Blocking Buffer with 0.2% Tween 20: +- IRDye® 680RD Goat anti-Mouse (1:800 dilution) +- IRDye 800CW Goat anti-Rabbit (1:800 dilution) + +**Step 51:** +Mix the antibody solutions and add 50 µL of the secondary antibody solution to each well. Incubate for one hour with gentle shaking. + +**Step 52:** +Protect plate from light during incubation. Use a large black Western Incubation Box to protect plate from light during subsequent steps. + +**Step 53:** +Wash plates five times with 200 µL of 1X PBS + 0.1% Tween 20 at room temperature for 5 minutes by centrifugation at 1,500 RPM (332 RCF). + +### Imaging + +**Step 54:** +Remove wash solution completely from wells. For best results, scan plate immediately; plates may also be stored at 4 °C for several weeks (protected from light). + +**Step 55:** +Scan plate with detection in both 700 and 800 nm channels using an Odyssey or Aerius System. Use the following settings: + +#### Imaging Settings + +| Instrument | Resolution* | Focus Offset | Scan Quality* | Intensity Setting (700/800 nm) | Scan Time (Medium Quality) | +|--------------------|-------------|--------------|----------------|------------------------------|----------------------------| +| Odyssey Classic | 169 µm | 3.5 | medium-lowest | 5 / 5 | 7 min | +| Odyssey CLx | 169 µm | 3.5 | medium-lowest | 5 / 5 | 7 min | +| Odyssey Sa | 200 µm | 3.5 | medium-lowest | 7 / 7 | 3 min | +| Aerius | 200 µm | 3.5 | medium-lowest | 7 / 7 | 3 min | + +**Note:** Settings may require adjustment for optimal data quality. + +### Warnings +See SDS for safety and warnings. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/computational-design-of-novel-nanobodies-targeting-cy93xz8n.md b/markdown-output/computational-design-of-novel-nanobodies-targeting-cy93xz8n.md new file mode 100644 index 0000000000000000000000000000000000000000..a36880f5fe92aace8e7ced103d2c60ad448bb249 --- /dev/null +++ b/markdown-output/computational-design-of-novel-nanobodies-targeting-cy93xz8n.md @@ -0,0 +1,97 @@ +```markdown +Goal/Experiment: Computational design of novel nanobodies targeting the receptor binding domain of variants of concern of SARS-CoV-2 + +# Computational Design of Novel Nanobodies Targeting the Receptor Binding Domain of Variants of Concern of SARS-CoV-2 + +**Phoomintara Longsomboon1, Thanyada Rungrotmongkol2, Nongluk Plongthongkum1, Kitikhun Wangkanont3, Peter Wolschann4, Rungtiva P. Poo-arporn1** + +1. Biological Engineering Program, Faculty of Engineering, King Mongkut's University of Technology Thonburi, Bangkok, Thailand +2. Center of Excellence in Biocatalyst and Sustainable Biotechnology, Department of Biochemistry, Faculty of Science, and Program in Bioinformatics and Computational Biology, Graduate School, Chulalongkorn University +3. Center of Excellence for Molecular Biology and Genomics of Shrimp, Department of Biochemistry, Faculty of Science, Chulalongkorn University, Thailand +4. Institute of Theoretical Chemistry, University of Vienna, Vienna, Austria + +**DOI:** [https://dx.doi.org/10.17504/protocols.io.4r3l22q4jl1y/v1](https://dx.doi.org/10.17504/protocols.io.4r3l22q4jl1y/v1) +**Protocol Citation:** "Phoomintara Longsomboon, et al. Computational design of novel nanobodies targeting the receptor binding domain of variants of concern of SARS-CoV-2." + +**MANUSCRIPT CITATION:** +[PLoS ONE-D-23-16784]: "Computational design of novel nanobodies targeting the receptor binding domain of variants of concern of SARS-CoV-2" + +Licence: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. + +## Abstract +The COVID-19 pandemic has created an urgent need for effective therapeutic and diagnostic strategies to manage the disease caused by SARS-CoV-2. However, the emergence of numerous variants of concern (VOCs) has made it challenging to develop targeted therapies that are broadly specific in neutralizing the virus. This study aims to develop neutralizing nanobodies (Nbs) using computational techniques to effectively neutralize the receptor-binding domain (RBD) of SARS-CoV-2 VOCs. We evaluated the performance of different protein-protein docking programs and identified HDOCK as the suitable program for Nb/RBD docking with high accuracy. Using this approach, we designed 14 novel Nbs with high binding affinity to the VOC RBDs by engineering mutated amino acids that interacted with key amino acids of the RBDs. The engineered Nbs have the potential to be employed in RBD-neutralizing assays, facilitating the identification of novel treatment, prevention, and diagnostic strategies against SARS-CoV-2. + +## 1. Validation of Protein-Protein Docking Server + +1. **Prepare Protein Datasets** + - Consists of 29 nanobody (Nb) and 86 antibody (Ab) complexes with RBDs from the Protein Data Bank (PDB) (https://www.rcsb.org/) for blind docking. + +2. **Remove Heteroatoms** + - Remove heteroatoms/molecules, including metal ions, small molecules, water molecules, and His-tags, from all complexes. + +3. **Prepare Protein Chains** + - Prepare the protein chains of RBDs and ligands (Nbs or antibodies) separately using Discovery Studio software. + +4. **Remodel Missing Amino Acids** + - The missing amino acids in the protein chain are remodeled using the SWISS-MODEL expert system (https://swissmodel.expasy.org/). + +5. **Perform Blind Docking** + - Use seven protein-protein docking programs: + 1. HDOCK (http://hdock.phys.hust.edu.cn/) + 2. ATTRACT (http://www.attract.ph.tum.de/services/ATTRACT/attract.html) + 3. pyDockWEB (https://life.bsc.es/pid/pydockweb) + 4. GRAMM-X (http://vakser.compbio.ku.edu/resources/gramm/gramm/x/) + 5. PatchDock (https://bioinfo3d.cs.tau.ac.il/PatchDock/) + 6. FRODOCK (http://frodock.chaconlab.org/) + 7. ZDOCK (https://zdock.umassmed.edu/) + +6. **Calculate RMSD Values** + - The root mean square deviation (RMSD) values of the ligands (Nb or Ab) are calculated using the Discovery Studio program. + +## 2. Selection of Lead Nbs + +7. **Redocking of Nbs** + - The 29 Nbs are redocked with each targeted RBD using a blind docking method with HDOCK. + +8. **Assess Accuracy** + - Calculate the RMSD values to assess the accuracy of the docking poses of the 29 Nbs with respect to all targeted RBDs. Present the docking scores and RMSD values for each RBD in terms of the mean. + +9. **Amino Acid Sequence Analysis** + - The similarity of amino acid sequences of 29 Nbs is analyzed using the Clustal Omega server (https://www.ebi.ac.uk/Tools/msa/clustalo/). + +10. **Selection of Lead Nbs** + - The lead Nbs are selected based on the best mean docking score, lowest RMSD, and diverse amino acid sequences, which are then employed for structure-based engineering. + +## 3. Structural-based Engineering and Broad Specific Binding of Engineered Nbs + +11. **Improve Binding Affinity** + - To improve the binding affinity of Nbs to all targeted RBDs, the two lead Nbs are mutated using the site-directed mutagenesis feature on the Discovery Studio program. + +12. **Consideration of Residue Interaction** + - Nb residues that had no interaction and repulsion with RBD are considered for the mutation process. + +13. **Energy Minimization** + - The AMBER ff14SB force field is applied for structural energy minimization before the docking process of mutated Nbs. + +14. **Calculate ΔHDOCK Values** + - Calculate the ΔHDOCK value, and choose the mutated residue at a specific position that exhibited the lowest ΔHDOCK for multi-point mutation. + +15. **Broad Specific Binding** + - Investigate the broad specific binding of engineered Nbs; cross-docking between Nb and all targeted RBDs, ACE2, and other viral RBD/HA is performed using the HDOCK program. + +## 4. Physicochemical Properties Prediction of Engineered Nbs + +16. **Determine Chemical Interactions** + - The contact surface amino acids and chemical interactions are determined using the PDBsum server (http://www.ebi.ac.uk/thornton-srv/databases/pdbsum/Generate.html). + +17. **Predict Physicochemical Properties** + - The physicochemical properties are predicted using the ProtParam (ExPASy) tool (https://web.expasy.org/protparam/). + +18. **Calculate PI Value** + - PI value is calculated using the Protein-Sol web server (https://protein-sol.manchester.ac.uk/). + +19. **Calculate Total Charge** + - The total charge is calculated by PROTEIN CALCULATOR v3.4 (https://protcalc.sourceforge.net/). + +**endofoutput** +``` \ No newline at end of file diff --git a/markdown-output/conducting-dynamic-bh3-profiling-adapted-from-leta-bahmib46.md b/markdown-output/conducting-dynamic-bh3-profiling-adapted-from-leta-bahmib46.md new file mode 100644 index 0000000000000000000000000000000000000000..2f58a6ec725eae8878b92fdcfdb148f951b389ab --- /dev/null +++ b/markdown-output/conducting-dynamic-bh3-profiling-adapted-from-leta-bahmib46.md @@ -0,0 +1,166 @@ +```markdown +# Goal/Experiment: +Conducting Dynamic BH3 Profiling Adapted From Letai Lab + +## Conducting Dynamic BH3 Profiling Adapted From Letai Lab + +**Dennis Juarez1** +1University of California, Irvine + +**DISCLAIMER** + +**DISCLAIMER – FOR INFORMATIONAL PURPOSES ONLY; USE AT YOUR OWN RISK** + +Patents and intellectual property rights around BH3 profiling belong exclusively to Dana-Farber Cancer Institute. Access to these rights is possible only through an agreement with Dana-Farber Cancer Institute, and no other. Beware of commercial entities claiming access to the intellectual property rights around this technology. + +We do not claim any intellectual property rights. This protocol is our lab's specific adaptation of Dynamic BH3 profiling from the protocol provided by the Letai lab. + +The protocol content here is for informational purposes only and does not constitute legal, medical, clinical, or safety advice, or otherwise; content added to protocols.io is not peer-reviewed and may not have undergone a formal approval of any kind. Information presented in this protocol should not substitute for independent professional judgment, advice, diagnosis, or treatment. Any action you take or refrain from taking using or relying upon the information presented here is strictly at your own risk. You agree that neither the Company nor any of the authors, contributors, administrators, or anyone else associated with protocols.io, can be held responsible for your use of the information contained in or linked to this protocol or any of our Sites/Apps and Services. + +**Protocol Citation:** Dennis Juarez 2023. Conducting Dynamic BH3 Profiling Adapted From Letai Lab. protocols.io +https://dx.doi.org/10.17504/protocols.io.3byl47jaljo5/v1 + +**License:** This is an open-access protocol distributed under the terms of the [Creative Commons Attribution License](https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. + +**Protocol status:** Working. We use this protocol and it's working. + +**Created:** Dec 13, 2019 +**Last Modified:** Oct 07, 2023 +**PROTOCOL integer ID:** 30989 + +## ABSTRACT + +Dynamic BH3 Profiling is used to test cellular proximity to apoptosis after treatment by exposure of in situ mitochondria with the BH3 domain of apoptotic proteins to induce mitochondrial cytochrome C release. Cytochrome C release is determined by flow cytometric measurement of cytochrome C fluorescence in cells and corresponds with the cellular proximity to mitochondrial outer membrane permeabilization, change of which is termed "priming". + +## MATERIALS + +All material information and preparation are dutifully discussed in the Letai lab BH3 Profiling Appendix. +[bh3_profiling_guide_appendix_20170814.pdf (accessed 20220419)](bh3_profiling_guide_appendix_20170814.pdf) + +## Before Starting + +1. Allow the MEB2-P25 to equilibrate to room temperature (RT) if stored at 4 degrees. Equilibration may take 1 to 2 hours. Preferably, you can pull the bottle out the night before to allow RT equilibration. + + >**Note:** BH3 Profiling is temperature-sensitive. As temperature increases, cytochrome c release increases as well. Conduct experiments in a constant temperature room. + +2. Prepare your peptide panel by diluting your peptides in DMSO to 100x the final concentration desired at exposure. If you do not know the desired final concentration, titrate your peptides to determine a concentration that causes around 20% cytochrome c release. + + >**Note:** You can save these working dilutions in the minus 20 for at least a week. Peptides are stable and can survive a few freeze-thaws, but the chemical BH3 mimetics should be made fresh. + +### Peptide Suggested Range + +| Peptide | Suggested Range (uM) | +| ------- | -------------------- | +| BIM | 0.001-100 | +| BID | 0.01-100 | +| PUMA | 0.1-100 | +| BAD | 0.1-100 | +| NOXA | 1-100 | +| HRKy | 1-100 | +| MS1 | 0.1-10 | +| FS1 | 0.1-10 | +| ABT199 | 0.1-1 | +| WEHI-539| 0.1-1 | +| A-1331852| 0.1-1 | +| A-1155463| 0.1-1 | +| S63845 | 1-10 | + +Adapted from "A Guide to BH3 Profiling" Appendix Revision 2017-08-14 + +3. Determine the minimum concentration of digitonin to permeabilize all your cells. While the Letai BH3 Profiling Guide recommends 0.001% final concentration, this may not work for every cell due to the amount of cholesterol at the cell membrane. Test final digitonin concentrations of 0.008-0.00025%. + +### 3.1 Testing Digitonin Mediated Cell Permeabilization with Trypan Blue + + 1. Dilute the digitonin stock to 0.016% in MEB2-P25 and perform a 2-fold serial dilution five times (0.008%, 0.004%, 0.002%, 0.001%, 0.0005%) to make a panel of 6 different digitonin concentrations (at 2x final) in MEB2-P25 buffer. + + 2. Take 400,000 cells you wish to BH3 profile and resuspend in 40 uL of MEB2-P25. + + 3. Combine 5 uL of cells with 5 uL of the 6 digitonin concentrations. + + 4. Wait 5 minutes to allow permeabilization. + + 5. Add 10 uL of trypan blue, mix, and transfer to a hemocytometer. + + 6. Permeabilized cells will appear blue under the microscope. + + 7. Select the lowest concentration of digitonin that results in 100% permeabilization. + +## BH3 Peptide Exposure + +4. Count your cells and take 2,000,000 cells per treatment condition in a 1.7 mL tube use in the assay. + + >**Note:** In this protocol, we will expose 200,000 cells to 8 different peptides. The assay tolerates cell concentrations between 0.5 million per mL to 4 million per mL at the BH3 exposure stage. In this protocol, we use 2 cells million per mL. Scale up or down as necessary. + +5. Spin down the cells at 500xg for 5 minutes. + +6. During the spin, prepare your 2x peptide solutions in MEB2-P25. The peptide stocks are made at 100x and should be diluted 1:50. Prepare enough for every cell line and treatment used in the assay. + + >Minimum Total Volume= (#cell line x #treatment condition + 1)* 25 uL + + >**Note on panel:** Always include in your peptide panel DMSO only, PUMA2A, and Alamethicin to act as controls for cytochrome C. + +7. Aspirate media and resuspend with 1 mL PBS and spin down in cold centrifuge to wash the cells. + +8. If you need to stain your cells with cell surface markers, block and stain cells now then spin down before proceeding to digitonin permeabilization. + +9. During this step, dilute the digitonin to 2x of the final concentration determined in step 3.1 in MEB2-P25 buffer. + +10. Resuspend the cells in 250 uL in the diluted digitonin/MEB2-P25 solution. This is enough for ten 25uL peptide exposures, thus preparing extra. + +11. In a V-Bottom plate, place 25 uL of 2x peptide at the bottom of the well. + +12. Place 25 uL of cells on the wall closest to you. + + Gently tap after the addition to each row. + +#### 12.1 + + Mix the 25 uL of cells and 25 uL of peptide by gently tapping the sides of the plate. Don't tap too hard or you may lose sample. Proper tapping technique: keep plate on benchtop while tapping. + + Tapping will be necessary in future steps to best mix the solution. Alternatively, a reliable plate vortexer can be used to mix, however, inappropriate settings or usage may cause the solution to spill out of the well. + + >**Note:** The tapping technique is used to control the exposure of cells to peptides if using a single-channel pipet. Exact time of exposures can be kept in combination with this tapping method and a timer. Alternatively, you may add and mix the cells with a reliable multichannel pipettor. + +13. Incubate for 1 hr at room temperature. If you have highly sensitive cells, incubate for 30 minutes. Hardier cell lines may require 1.5 hr. Determine this time for your cell system. + +## Ceasing BH3 Exposure and Cytochrome C Staining + +14. During incubation, prepare Cytochrome C Stain: 1:40 human cytochrome C (Biolegend) in 10x Cytochrome C Staining buffer, you need 10uL/well, prepare extra. + +15. Fix the cells by adding 16.5uL of 8% PFA. This is a final of ~2% PFA. + + Gently tap after the addition to each row. + + >**Note:** Use the freshest available PFA. Inadequate fixation can lead to cytochrome C loss during staining. + +16. Incubate at room temp for 20 minutes. + +17. Neutralize with 16.5uL N2 buffer. + + Gently tap after the addition to each row. + +18. Wait at least 5 min. + +19. Add 10uL of 1:40 diluted Cytochrome C Staining mix to each well. + + Gently tap after the addition to each row. + +20. Seal the plate with a foil seal or clear seal if kept in the dark. + Incubate for 4 hours at room temperature (in a drawer) or overnight (16hr minimum) at 4 degrees. + +## Flow Analysis + +21. Add ~50uL PBS to increase volume to ~150uL + +22. On the cytometer, open the BH3 Profile Template and BH3 Profiling Instrument Settings. + +23. Ensure that the threshold for FSC-H is excluding junk. This assay will have notable amounts of debris. + +24. Run the DMSO control to set cytochrome c high population above the second log. + +25. Run the BIM 10uM control to check if cytochrome c low population is below the second log. + +26. Acquire 125uL. Set acquisition settings to acquire 10,000 events. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/confirmation-of-axenic-seedlings-cvmww47e.md b/markdown-output/confirmation-of-axenic-seedlings-cvmww47e.md new file mode 100644 index 0000000000000000000000000000000000000000..cf3d7913bcb0275f8a6ee7bd7a3ebccfb45a0c8d --- /dev/null +++ b/markdown-output/confirmation-of-axenic-seedlings-cvmww47e.md @@ -0,0 +1,178 @@ +```markdown +# Goal/Experiment: +To confirm the axenic (sterile) status of Sporobolus alterniflorus plant seedlings and eliminate the presence of potential contaminants in vitro by employing both culture-dependent and culture-independent methods. + +# Confirmation of Axenic Seedlings + +## Authors +- Elena L. Peredo1 +- Suzanne M Thomas1 +- Zoe Cardon1 + +1Marine Biological Laboratory + +## Abstract + +*Sporobolus alterniflorus* plants are known to be rich in endophytes. In order to ensure the success of an in vitro culturing approach, it is crucial to remove any potential contaminants. Here, we present a protocol for detecting the presence of endophytes in in vitro plants. To obtain a comprehensive assessment of the culture's axenic state, we employ both culture-dependent and culture-free approaches. + +## Materials + +### Culture-dependent Methods +- Autoclaved double distilled water. +- Micro-pestles. +- 1.7 mL Eppendorf tubes. +- Micropipettes/transfer pipettes. +- Glass spreaders. +- Petri dishes. +- Lysogeny broth (LB). +- Modified MS medium (same culture medium the plants grow in). + +### Culture-independent Methods +- PCR 0.2 mL tubes. +- Molecular grade water. +- dNTPs. +- Taq and taq buffer. +- Primers. +- Agarose. +- TAE buffer. +- SyBR green. +- Electrophoresis rig. +- Exosap-it. + +## Safety Warnings +- **Autoclave usage.** + +## Ethics Statement +- No animal or human research is involved. + +## Before Start Instructions +Please consult your institution's guidelines regarding the culturing and discarding of microorganisms isolated from natural environments. + +## Procedure + +### 1. Collection of Plant Materials to Be Tested for Presence of Bacteria and Fungi +The aim of this protocol is to test for the presence of contaminants in putative axenic *Sporobolus alterniflorus* (smooth cordgrass) seedlings. Precautions should be taken to prevent the in vitro plant material ("vitroplants") from getting exposed to secondary sources of contamination during testing, including preparing the plant materials in a tissue culture hood and using autoclaved supplies. + +#### 1.1 Prepare Plant Materials +- **Time Required:** 30 minutes +- Prepare two racks with labeled, sterile 1.7 mL Eppendorf tubes. Samples in one rack would be used for culture-dependent methods. The second rack is for culture-independent methods. +- Place in each tube the plant material to be tested. Use autoclaved scissors to cut fragments of around 2-3 cm of leaves and roots. + +### 2. Culture-dependent Methods +Different culture media are used to detect the presence of possible contaminants. + +#### 2.1 Prepare Culture Media +- **Time Required:** 2 hours + +**Lysogeny Broth Culture Medium (LB)**: +- Weigh 25g of LB broth powder and add to a 1L autoclave bottle. +- Add double distilled water for a final volume of 1L. +- Adjust pH to ~7. +- Add 10 g/L of agar. +- Autoclave for 40 minutes. +- In the hood, add ~25 mL into as many 90 mm sterile Petri dishes as needed. + +**Plant Culture Medium (MS)**: +- Add 4.41 g of powder to a 1L autoclave bottle. +- Add 30 g/L of sucrose for a final concentration of 3%. +- Add double distilled water for a final volume of 1L. +- Adjust pH to 5.8. +- Add 8 g/L of agar. +- Autoclave for 40 minutes. +- In the hood, add ~25 mL into as many 90 mm sterile Petri dishes as needed. + +**Plant Culture Medium (MS) Includes**: +- Macro- and micronutrients as described by Murashige and Skoog (1962). +- Vitamins as described by Linsmaier and Skoog (1965). +- Ferric Sodium EDTA in place of Ferrous Sulfate and Disodium EDTA. +- 1.0 mg/L Kinetin. +- 0.3 mg/L IAA. + +#### 2.2 Prepare Plant Extracts +- **Time Required:** 30 minutes +- Add 500 μL of distilled autoclaved water (or molecular-grade water) to each sterile Eppendorf tube. +- Use a sterile micro-pestle to grind the plant material to a fine paste. (Alternatively, a bead-beating approach can be used.) +- Transfer 100 μL of the supernatant to the appropriate media for culture. +- Use a glass spreader to distribute the sample on the surface of the agar. +- Incubate for 1 week using a temperature and light regimen similar to that used for growing seedlings. (We use a growth chamber temperature of 20°C, light level ~100 μE, and 16 hr: 8 hr light:dark cycles.) + +#### 2.3 Score the Presence of Bacterial or Fungal Contamination in the Culturing Plates +- **Time Required:** 1 week + +![Figure 1](images/figure1.png) +*Detection of bacterial (top, LB plates) and fungal (bottom, MS plates) contaminants in soil-grown seedlings (left and center). No contaminants were isolated from the vitroplants (right).* + +![Figure 2](images/figure2.png) +*Detection of endogenous contaminants in leaves and roots of vitroplants that still have endogenous microorganisms. On top, we present the results of inoculating media with extracts of putative axenic vitroplants. After one week, no bacterial or fungal growth was observed in the plates. At the bottom are two example plates illustrating bacterial presence detected in extracts of vitroplants that still carried endogenous contaminants. In this specific case, the assay detected bacteria both in leaves and roots.* + +### 3. Culture-independent Methods +We use a PCR-based detection method with universal primers targeting the 16S rRNA gene to detect the possible presence of endogenous bacteria. This second method complements the culture-dependent assay described above, targeting bacteria that might not be suitable to grow in the culture media defined above but that can still be present in the in vitro plants. + +In addition to the standard negative controls, we strongly encourage using positive control for amplification, including: +- Bacterial DNA (e.g. DNA from a bacterial isolate). +- Positive control for bacterial contamination of plant material (e.g. soil-grown seedlings, or plants collected in the field). + +#### 3.1 DNA Extraction +- Add the appropriate volume (μL) of DNA extraction buffer (e.g. CTAB) to each tube containing the plant material to be evaluated using the PCR-based assay. +- Use a sterile micro-pestle to grind the plant material to a fine paste. (Alternatively, a bead-beating approach can be used.) +- Follow the instructions provided by the manufacturer for your DNA extraction kit of choice (here we used [Maxwell® RSC PureFood GMO and Authentication Kit](REFERENCE LINK)). +- Determine the DNA quantity and quality using nanodrop, and/or agarose gel, and/or fluorometric quantification. +- Prepare working solutions of each DNA (~10 ng/μL). + +#### 3.2 Primers for the Amplification of the 16S rRNA Gene +We recommend using the primer set 799F-1192R targeting the 16S rRNA gene (Ma et al., 2022). This primer combination has the advantage of reducing the affinity for the 16S rRNA gene in cyanobacteria and therefore minimizes the amplification of chloroplast DNA. + +This primer combination amplifies a ~425 bp fragment of the 16S rRNA gene in bacteria. In *Sporobolus alterniflorus* it also amplifies a ~800 bp fragment, corresponding to the 16S rRNA gene in the plant mitochondrial genome. + +| Primer Name | Sequence | +|-------------|-------------------------------| +| 799F | AACMGGATTAGATACCCKG | +| 1192R | ACGTCATCCCCACCTTCC | +| 27F | AGAGTTTGATCCTGGCTCAG | +| 1492R | TACGGYTACCTTGTTACGACTT | + +#### 3.3 PCR Reaction +We selected [Platinum™ SuperFi™ DNA Polymerase (Invitrogen)](REFERENCE LINK) as the enzyme in our PCR reactions. Many other Taqs will provide successful results; however, we recommend using a high-fidelity, high-efficiency enzyme in this protocol. +| Reagent | Concentration | Volume (μL) | +|-----------|----------------|-------------| +| Buffer | 5X | 4 | +| DNA | 10 μg/μL | 2 | +| dNTPs | 2.5 mM | 0.5 | +| Primer F | 10 μM | 1 | +| Primer R | 10 μM | 1 | +| Taq | 2 U/μL | 0.2 | +| Water | | 9.5 | + +#### 3.4 PCR Conditions +- Initial denaturation at 94°C for 5 min. +- Ten cycles of a touchdown PCR. In each cycle, we decreased the annealing temperature by 0.5°C. Each cycle consisted of denaturation at 94°C for 30 sec, followed by annealing at 60°C for 30 sec, and extension at 72°C for 45 sec. +- Twenty-five cycles of denaturation at 94°C for 30 sec, followed by annealing at 55°C for 30 sec, and extension at 72°C for 45 sec. +- Final elongation at 72°C for 5 min. +- Samples were held at 4°C. + +**Abbreviated PCR Cycle Notation**: 94°C (5’) x10 [94°C(45”) 60°C** (30”) 72°C(45”)] x25 [94°C(45”) 55°C (30”) 72°C(45”)] 72°C (5’). ** Indicates that this step is a touch-down PCR. In each cycle, we decreased the annealing temperature by 0.5°C. + +#### 3.5 Agarose Detection of PCR Fragments +The expected sizes of the fragments produced in this reaction by the primer set 799F/1199R are ~400 bp (bacteria) and ~800 bp (*Sporobolus alterniflorus* mitochondria). +- Prepare a 1% agarose gel; you can use TAE 1x as the electrophoresis buffer. +- Add SyBr to the agarose for a final concentration 1x, following manufacturer instructions. +- For each PCR reaction, separate 10 μL of PCR product and mix with 2 μL of loading dye. +- Load each sample in the agarose gel. +- Use a 1 Kb ladder (or equivalent) as a size marker (follow manufacturer instructions). +- Run the gel until the bands separate. We run our gels at 100 V for 1.5-2h but these conditions will depend on the length of the gel. +- Visualize the results of the gel in a gel reader. + +![Figure 3 - Visualization of PCR fragments in an agarose gel](images/figure3.png) +*Axenicity is determined by the detection of a single 800 bp (mitochondrial) band on the gel. In this gel, we used a series of positive controls that included bacterial DNA, or non-axenic tissues including soil-grown seedlings and plants that displayed bacterial presence. We also tested for the presence of bacteria in roots and leaves of multiple vitroplants (in green).* + +#### 3.6 Sequencing +Another way of visualizing bacterial contamination in a given sample is by visual inspection of the Sanger sequencing electropherograms. +- Samples with bacterial contamination will display low-quality base-calling results up to position ~425 due to the presence of a secondary band that corresponds to the bacterial 16S rRNA gene. The rest of the electropherogram, corresponding to the sequence of the mitochondria 16S ribosomal gene, will show as high-quality base calls. +- Samples corresponding to axenic vitroplants will display sequences of >700 bp with high-quality results for all the bases, consistent with the amplification of a single target gene (the mitochondria 16S ribosomal gene). + +![Figure 4 - Sanger sequencing electropherograms](images/figure4.png) + +--- + +`endofoutput` +``` \ No newline at end of file diff --git a/markdown-output/consulta-anest-sica-para-ect-do-ics-vgje3un.md b/markdown-output/consulta-anest-sica-para-ect-do-ics-vgje3un.md new file mode 100644 index 0000000000000000000000000000000000000000..886217cb534f71f0264c5690583bc96f04e050bb --- /dev/null +++ b/markdown-output/consulta-anest-sica-para-ect-do-ics-vgje3un.md @@ -0,0 +1,248 @@ +```markdown +# Goal/Experiment: +The goal of this experiment is to provide guidelines for anesthetic consultation for Electroconvulsive Therapy (ECT) at ICS, ensuring safety and adherence to quality standards. + +# Consulta anestésica para ECT do ICS + +### Gabriel Magalhães Nunes Guimarães +Universidade de Brasília +DOI: [dx.doi.org/10.17504/protocols.io.vlge3un](https://dx.doi.org/10.17504/protocols.io.vlge3un) + +## Abstract + +Este é um guia de como deve ser a consulta anestésica para eletroconvulsoterapia. +Nossa equipe exige um padrão mínimo de qualidade destas consultas, que devem incluir informações específicas e muito relevantes para a realização de anestesias com o máximo de segurança no nosso instituto. + +Este guia se aplica para consultas por membros internos e externos à equipe de anestesiologia que atua no ICS-Brasília. Caso uma consulta por profissional externo não contenha os critérios mínimos de qualidade, não será considerada como passo realizado para a realização de ECT com nossa equipe. + +## Tags + +- eletroconvulsoterapia +- anestesia + +## External Link + +[www.ectics.com.br](http://www.ectics.com.br) + +## Protocol Status + +**Working** +We use this protocol in our group and it is working. + +## Guidelines + +Sugerimos que este protocolo seja usado apenas por médicos anestesiologistas. + +## Safety Warnings + +Este protocolo só deve ser aplicado por médicos com registro ativo no conselho regional de medicina. + +## Before Starting + +Leia ao menos uma vez todo o protocolo antes de iniciar a aplicação. + +## Steps + +### 1. Identificação + +São dados exigidos: + +- Nome completo +- Sexo biológico +- Número de identificação único (CPF, RG, etc) +- Telefone para contato +- Nome de acompanhante responsável +- Telefone de acompanhante responsável +- Data da avaliação + +### 2. Idade e dados antropométricos + +- Data de nascimento e idade +- Peso +- Altura +- IMC +- Circunferência do pescoço +- Distância tireo-mentoniana +- Distância esterno-mentoniana + +### 3. Indicações de ECT + +Listar quais das seguintes indicações de ECT se aplicam: + +- Já fez ECT antes +- Emergência psiquiátrica +- Risco de suicídio +- Catatonia +- Epilepsia +- Depressão +- Esquizofrenia +- Refratariedade ao tratamento farmacológico +- Intolerância ao tratamento farmacológico + +Informar tempo do problema (por exemplo: depressão há 5 anos, mal controlada há 2 anos). + +### 4. Antecedentes anestésicos + +- A quais procedimentos anestésicos já foi submetido, quando e quais complicações ocorreram? +- Algum problema na família com anestesia? Exemplo: hipertermia maligna. + +### 5. Doenças associadas + +Quais doenças cardiovasculares, há quanto tempo, qual o tratamento e como está o controle, com destaque para: + +1. Hipertensão arterial +2. Arritmias (exemplo fibrilação atrial, bloqueios, síndrome do QT longo) +3. Valvulopatias +4. Angina +5. Marcapasso ou CDI +6. Hipertensão pulmonar +7. Insuficiência cardíaca +8. Aneurisma de aorta +9. Aneurisma cerebral +10. Dissecção de aorta + +Quais doenças neurológicas, há quanto tempo, qual o tratamento e como está o controle, com destaque para: + +1. Alzheimer +2. Esclerose múltipla +3. Miastenia +4. Síndrome de taquicardia postural +5. Parkinson +6. Autismo +7. Demência por corpúsculos de Lewy + +Quais doenças, há quanto tempo, qual tratamento e como está o controle, nos sistemas: + +- Pulmonar (especialmente asma e DPOC) +- Renal (especialmente insuficiência renal) +- Endocrinológico (especialmente hipotireoidismo, insuficiência hepática e diabetes) +- Oftalmológico (especialmente glaucoma e colírios) +- Odontológico (especialmente lesões dentárias) +- Ortopédico (especialmente fraturas recentes ou fragilidade óssea) + +### 6. Exame físico direcionado + +Verificação de fatores de risco para ventilação difícil: + +- SAOS +- Enulência +- Bócio mergulhante +- Estenose traqueal +- Tumor que comprime traqueia +- Barba +- Fístula em via aérea + +Fatores de risco para laringoscopia ou intubação difícil: + +- Abertura de boca pequena (<3cm) +- Pescoço largo +- Distância mentoesternal <12cm +- Distância tireo-mentoniana <6cm +- Mallampati +- Extensão cervical reduzida +- Teste de mordida do lábio superior +- Acromegalia + +### 7. Risco de retardo de esvaziamento gástrico + +- Gestantes com mais de 20 semanas de gestação (5 meses); +- Diabetes; +- Doenças neurológicas; +- Opioides (tramadol, codeína, morfina, oxicodona), alguns antidepressivos (amitriptilina, nortriptilina, venlafaxina) e anticolinérgicos; +- Hérnia de hiato; +- Obesidade; +- Hipotireoidismo; +- Doença de Parkinson; +- Esclerose múltipla; +- Cirurgia bariátrica prévia; +- Acalasia ou megaesôfago chagásico; +- Tumor gástrico ou em esôfago; +- Esclerodermia; +- Refluxo na noite antes da ECT por alimentação exagerada; +- Tabagismo intenso; + +### 8. Exame cardiovascular, pulmonar, psiquiátrico e neurológico de triagem + +#### Cardiovascular + +- Ausência de sinais de alterações no exame físico cardiovascular sugestivas de doenças não diagnosticadas. +- Veias visíveis e de calibre habitual em dorso da mão e antebraço? +- Pressão arterial e frequência cardíaca. + +#### Pulmonar + +- Ausência de sinais de alterações no exame físico pulmonar sugestivas de doenças não diagnosticadas. +- Frequência respiratória, esforço respiratório, SpO2. + +#### Psiquiátrico + +- Paciente colaborativo? +- Redução significativa de cognição? +- Episódios de agressividade? + +#### Neurológico + +- Alterações em marcha? +- Alterações na deglutição? +- Rebaixamento de consciência / sedação por medicamentos? +- Problemas de memória crônicos ou agudos? + +### 9. Medicamentos + +Descrever alergias conhecidas a medicamentos e alimentos. + +- Usa ou usou IMAO? Quando interrompeu? +- Usa anticolinesterásico? Ex: Donepezila, galantemina, neostigmina, piridostigmina, edrofônio, ecotiofato. +- Usa colírio de pilocarpina? +- Submetido a quimioterapia? Qual esquema e dose? Há quanto tempo? + +Listar com detalhes medicamentos, doses e intervalos, inclusive medicamentos ocasionais (ex: rivotril em crises de ansiedade). + +### 10. Hábitos e outras informações relevantes + +- Risco de SAOS de acordo com STOP-BANG? +- Mulher em idade fértil? +- Se gestante, DUM e idade gestacional. +- Imobilidade prolongada? +- Tabagismo? Maconha? Cocaína? Etismo? Outras drogas? + +### 11. Exames complementares já disponíveis + +Listar exames e informações relevantes ou sumárias, com data dos exames. Exemplo: ECG em 12/12/12: ritmo sinusal, sem alterações. + +### 12. Necessidade de avaliações complementares + +Havendo necessidade de avaliação odontológica (ex: fraturas ou dentes em mal estado) ou oftalmológica (ex: glaucoma), dispomos de formulários de encaminhamento para estas especialidades que explicam implicações da ECT para facilitar laudo útil. + +[relatorio odonto-ECTs.docx](URL) +[parecer oftalmo-ECTs.docx](URL) + +Não havendo emergência no início da ECT (ex: não é catatonia nem há ideação suicida), solicitar: + +- Polissonografia se STOP-BANG de moderado ou alto risco; +- Cintilografia de pesquisa de esvaziamento gástrico se risco elevado de retardo de esvaziamento e não houver contraindicação (ex: gestante, que deverá realizar ecografia de antra ao critério da equipe); + +Outros pedidos de exames ou avaliações de especialistas devem ser individualizados. + +O profissional deve julgar e decidir entre uma das seguintes conclusões da consulta: + +- Benefícios de avaliações adicionais provavelmente não superam os riscos do atraso potencial no início da ECT, paciente deve iniciar a ECT quando possível; +- Benefícios de avaliações adicionais provavelmente superam os riscos do atraso potencial no início da ECT, retorno ou nova consulta deve ser agendado após avaliações; + +### 13. Processo de consentimento informado + +O processo de consentimento, quando por um avaliador externo, poderá ser feito apresentando o endereço [www.ectics.com.br](http://www.ectics.com.br) e entregando duas vias do formulário de consentimento informado para o paciente. Neste caso, o paciente deverá ler o documento em casa e levar no dia da primeira ECT para sanar todas as suas dúvidas com um profissional da equipe e poder assinar o documento. + +Quando a consulta for realizada por profissionais da equipe, todas as dúvidas sobre o termo de consentimento e explicações sobre o procedimento devem ocorrer ao final da consulta. O termo deve ser levado pelo paciente para ler novamente em casa e decidir se irá assinar, e levado ao ICS no dia da primeira ECT. + +Em casos de emergência, a assinatura do termo pode ser adiada ou o termo pode ser assinado no dia da avaliação. + +[tcle anestesia ECT ics.pdf](URL) + +### 14. Forma de preenchimento e envio da consulta + +Preferimos receber a consulta como PDF assinado (não precisa ser por e-CRM, pode ser por e-CPF). +Também aceitamos consultas em papel, neste caso, assinaturas com carimbo e CRM do médico responsável pela avaliação devem estar em todas as páginas. + +**endofoutput** +``` \ No newline at end of file diff --git a/markdown-output/criteria-to-evaluate-neurogenic-bowel-dysfunction-bidika4e.md b/markdown-output/criteria-to-evaluate-neurogenic-bowel-dysfunction-bidika4e.md new file mode 100644 index 0000000000000000000000000000000000000000..30237d4cd9f8de62addc6049a5f35b0fec1174ad --- /dev/null +++ b/markdown-output/criteria-to-evaluate-neurogenic-bowel-dysfunction-bidika4e.md @@ -0,0 +1,119 @@ +```markdown +# Goal/Experiment: +The primary goal of this research protocol is to evaluate neurogenic bowel dysfunction in children with congenital Zika syndrome (CZS). This protocol includes methods for detailed clinical anamnesis, physical examinations, and complementary examinations to confirm bowel dysfunction diagnosis, as well as management and follow-up strategies to treat bowel dysfunction in CZS children. + +# Criteria to Evaluate Neurogenic Bowel Dysfunction in Children with Congenital Zika Syndrome + +### Authors +- Valeria Azevedo De Almeida1 +- Nancy Sotero1 +- Rafael Pauletti Gonçalves1 +- Edgard Morya1 +- Lilian Lira Lisboa1 +- Lucia Maria Costa Monteiro1 +- Reginaldo Antônio de Oliveira Freitas Júnior1 + +1Alberto Santos Dumont Institute of Education and Research (ISD) + +### DOI +[dx.doi.org/10.17504/protocols.io.bidik4e](http://dx.doi.org/10.17504/protocols.io.bidik4e) + +### Protocol Citation +Valeria Azevedo De Almeida, Nancy Sotero, Rafael Pauletti Gonçalves, Edgard Morya, Lilian Lira Lisboa, Lucia Maria Costa Monteiro, Reginaldo Antônio de Oliveira Freitas Júnior 2020. CRITERIA TO EVALUATE NEUROGENIC BOWEL DYSFUNCTION IN CHILDREN WITH CONGENITAL ZIKA SYNDROME. protocols.io [dx.doi.org/10.17504/protocols.io.bidik4e](http://dx.doi.org/10.17504/protocols.io.bidik4e) + +### License +This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. + +--- + +## Abstract +Children with Congenital Zika Syndrome (CZS) present brain abnormalities compromising the integrity of their connexions with the urinary and digestive systems, making them susceptible to bowel and bladder dysfunctions. Given the lack of specifically addressed studies, this research protocol, relying on cumulative knowledge in neurogenic bowel treatment, evaluates bowel dysfunction in CZS children, admitted for follow-up at the Anita Garibaldi Center of Education and Research in Health (CEPS). + +## Guidelines + +### Inclusion Criteria +- Patients with confirmed diagnosis of CZS, according to the 2019 diagnostic criteria recommended by the Brazilian Health Ministry (BRASIL, 2019). + +### Exclusion Criteria +- Children with other malformations not related to CZS. +- Children with Genetic syndromes. +- Newborns and children younger than 1 year of age. +- Failure to sign the informed consent form. + +### Initial Assessment Steps + +1. **Detailed Clinical History (Water Intake & Food Routine)** +2. **Detailed Clinical History (Bowel Habits)** + - Frequency of bowel emptying (evacuation diary). + - Feces color and aspect using the Bristol Stool Scale. + - Use of bowel emptying maneuvers. + - Use of laxatives, suppositories, enemas. + - History of intestinal lavage. + - Issues and side effects related to constipation (hemorrhoids, anal fissures). + - Facial expression and crying during evacuation. + - Excessive retention history (less than twice per week). + - Retentive posturing. + - Retentive posturing may be attempts to withhold evacuation (e.g., standing on tiptoe). + + **2.1.** **Retentive Posturing** + - Typically involves child standing on tiptoe, holding legs and buttocks rigidly while straining. + + **2.2.** **Bristol Stool Scale** + - Visual classification measuring feces appearance and texture. Validated for Brazil (Martinez, 2012). + +3. **Physical Examination** + - General focus on the abdomen: child positioned in supine, legs extended. + - Abdominal examination checking for distension, palpable masses, especially at sigmoid colon height. + - Anus inspection. + +4. **Guidelines for Home Evolution** + - Provide evacuation diary with Bristol Stool Scale at the end of the appointment. + - Parents/guardians to fill out the diary and monitor child’s fluid intake. + +5. **Confirmation by Complementary Examinations** + - Abdominal ultrasound measuring Rectal Diameter (RD). + - RD ≥ 2.9 cm indicates bowel constipation (Joensson et al., 2008 and Burgers et al., 2013). + +### Management and Follow-Up + +#### 6.1. 1st Multiprofessional Team Consultation +- Perform 1st consultation with steps of initial assessment. +- If clinical manifestation is compatible with intestinal dysfunction, provide general guidelines for constipation. +- Begin rectal route desobstruction or pharmacological treatment as needed. + +#### 6.2. 1st Return After 8 Days +- Review of evacuation diary and ultrasound results. +- Initiate behavioral treatment. + - Instruct behavioral treatment for evacuation, home practice precautions. + +#### 6.3. Behavioral Treatment Instructions +- Instructions on water intake, bowel habits, and reeducation. + - Nutrition education led by nutritionist. + +##### 6.3.1. Guidance on Water Intake +- Reported by parents/caregivers; recommend 1 liter/day for <10kg, 1.5-2 liters/day for >10kg. +- Address consumption challenges due to CZS-related difficulties. + +##### 6.3.2. Bowel Retraining +- Guide caregivers to perform abdominal massage for 10-15 minutes using specific techniques. +- Not for patients with major bowel obstruction or recent surgery. + +#### 6.4. Physiotherapeutic Care +- Monitoring and adjusting behavioral therapy as needed, including stretching, exercise, and anorectal stimulation. + +#### 6.5. Reassessment Every 3-6 Months +- Conducted by multiprofessional team monitoring intestinal dysfunction. + +## Fluxogram +![Flowchart](https://dx.doi.org/10.17504/protocols.io.bidik4e) + +## References +- Martinez, 2012 +- Preece, 2002 +- Joensson et al., 2008 +- Burgers et al., 2013 + +--- + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/crossmatch-testing-before-blood-component-transfus-652hg8e.md b/markdown-output/crossmatch-testing-before-blood-component-transfus-652hg8e.md new file mode 100644 index 0000000000000000000000000000000000000000..c73b49267d1eba4a185d4712bc6d2f33c7e1e3b1 --- /dev/null +++ b/markdown-output/crossmatch-testing-before-blood-component-transfus-652hg8e.md @@ -0,0 +1,118 @@ +```markdown +Goal/Experiment: +The goal of this experiment is to ensure compatibility of blood before transfusion by detecting antibodies in the patient's serum/plasma that may react with donor red cells. + +# Crossmatch Testing Before Blood Component Transfusion + +**Authors:** +Grace HJ Chung¹, Mina Hur¹, Sang Gyeu Choi¹, Hyun-Kyung Lee¹, Hanah Kim¹, Hee-Won Moon¹, Yeo-Min Yun¹ + +¹Department of Laboratory Medicine, Konkuk University Medical Center and Konkuk University School of Medicine, Seoul, South Korea + +**Publication:** +This protocol accompanies the publication Chung H., Hur M., Choi S.G., Lee H., Lee S., Kim H., Moon H., Yun Y. (2019) Benefits of VISION Max automated cross-matching in comparison with manual cross-matching: A multidimensional analysis. PLoS ONE 14(12): e0226477. doi: [10.1371/journal.pone.0226477](https://doi.org/10.1371/journal.pone.0226477). + +## 1. Scope and Application +This procedure is applied for compatibility testing of all patients requiring transfusion. Routinely major crossmatch is done in which donor red cells are crossmatched with patient serum/plasma to detect incomplete antibodies in the patient serum/plasma (including indirect antiglobulin phase). Incompatible blood units should not be used for transfusion. + +## 2. Principle +Red cells possess a variety of antigens. For identifying corresponding antibodies in the patient’s sample, donor red cells are tested against the patient’s serum/plasma or serum/plasma. The reaction between a specific antigen and its specific antibody is noticed by the presence of agglutination or hemolysis. Positive reaction in any test indicates incompatibility. + +## 3. Specimen +### 3.1 Patient Preparation: None +### 3.2 Specimen +- **Tube:** Plain tube without anticoagulant +- **Type and amount:** 5 mL of venous whole blood or 2-3 mL of serum/plasma +- **Storage:** 4°C (± 2°C) +- **Processing:** Serum/plasma should be separated by g for 10 mins from whole blood, immediately. + +### 3.3 Specimen Rejection Criteria +- Patient sample older than 24 hours from specimen collection +- Patient sample without appropriate labeling (patient’s ID/name/age/gender and name of phlebotomist) +- Hemolyzed sample by visual inspection +- Not enough specimen less than 1.5 mL (as serum/plasma) + +## 4. Material and Equipment +### 4.1 Material +- **0.9% Saline:** A sterile, isotonic solution of sodium chloride in water. +- **Polyspecific Antihuman Globulin Reagent:** Contains anti-IgG and anti-C3d used for detecting antibodies bound to red cells. +- **22% Bovine Albumin:** Enhances agglutination reactions by increasing dielectric constants. +- **Anti-Human Globulin:** Reagent that detects human antibodies bound to red cells. +- **IgG Sensitized Control Cells:** Quality control cells to verify proper reactivity of antiglobulin reagents. + +### 4.2 Equipment +- Refrigerator to store samples & reagents at 4°C (± 2°C) +- Tabletop Centrifuge for spinning samples +- Serofuge: A specialized centrifuge for blood serology. +- Cell Washer: Automated for washing red cells. + +### 4.3 Glassware and Others +- Pasteur Pipettes: For transferring small volumes of liquids. +- Tubes: 12x75 mm standard lab tubes. +- Disposal Box: For biohazard disposal. +- Glass Beakers (2) +- Dropper +- Aluminum Racks: To hold serum/plasma and tubes + +## 5. Safety Warnings +General standard precautions apply. + +## 6. Procedure +### 1st Saline Phase/Room Temperature Immediate Spin +1. Saline room temperature is done to detect Major ABO incompatibility and complete (IgM) antibodies/cold antibodies like M, N, S, P, Lewis, Lutheran, etc. This crossmatching method can be done for the issuance of blood in emergencies. + - A. Take a test tube. + - B. Label with patient’s/donor ID on the tube. + - C. Prepare 2-5% red cell suspension of donor red cells. + - D. Dispense 2 drops of patient serum/plasma into the labeled tube. + - E. Add one drop of donor red cell suspension (from step C) to the tube containing patient serum/plasma. + - F. Spin immediately at 1500g for 15 seconds. + - G. Take out the tube gently. + - H. Observe for hemolysis and then for agglutination by gentle shaking the tube. + - I. Grade and record results, manually. + - J. Always continue with anti-human globulin phase, even in emergencies. But in this case, blood units can be released after this phase. + +### 2nd Albumin Phase/37°C Phase +1. Add 2 drops of bovine albumin and incubate the tube for 30-45 minutes for albumin at 37°C + - A. Take out tubes from 37°C. + - B. Spin immediately at 1500g for 15 seconds. + - C. Take out the tube gently. + - D. Observe for hemolysis and then for agglutination by gentle shaking the tube. + - E. Grade and record results, manually. + +### 3rd Antiglobulin Phase (AHG Phase) +1. Wash three times with 0.9% saline using a cell washer for 3 mins. + - A. Add 2 drops of anti-human globulin. + - B. Spin immediately at 1500g for 15 seconds. + - C. Take out the tube gently. + - D. Observe for hemolysis and then for agglutination by gentle shaking the tube. + - E. Grade and record results, manually. + +## 6. Interpretation of Results +### A. Compatible for Transfusion +- No Hemolysis / No Agglutination of red cells + +### B. Incompatible for Transfusion +- Hemolysis/Agglutination of red cells of any grade (trace, 1+,2+,3+,4+) / Mixed field. + +**Note:** +- All steps should be done immediately. +- Never use plastic tubes for crossmatch as it adsorbed IgG antibody which can lead to false-negative results. +- Shaking should be done gently. +- Hemolyzed bag should not be selected for crossmatch. +- Use clean glasswares. +- Use all reagents according to the manufacturer's advice. +- Never issue blood which is found incompatible at any phase of crossmatch. + +### Limitations +- The saline/enzyme crossmatch will not detect incomplete antibody, ensure normal donor’s red blood cell survival, and detection of antibodies connected to low-level presence of antigens (as with heterozygous expressed blood groups like Fya/Fyb). + +## 7. Documentation +1. Enter results in laboratory information system. +2. All records are initialed by the technician who performed the test and the technologist who has verified the result. + +## 8. Responsibility +1. It is the responsibility of the technician in the clinical laboratory to perform compatibility testing to demonstrate ABO compatibility and report the results. +2. If any unexpected antibody is detected, the medical officer should be informed for further investigation. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/cryostat-sectioning-of-tissues-for-3d-multimodal-m-bt8inrue.md b/markdown-output/cryostat-sectioning-of-tissues-for-3d-multimodal-m-bt8inrue.md new file mode 100644 index 0000000000000000000000000000000000000000..9d19941598144b0a45b5ea27aef3504c6376e236 --- /dev/null +++ b/markdown-output/cryostat-sectioning-of-tissues-for-3d-multimodal-m-bt8inrue.md @@ -0,0 +1,92 @@ +```markdown +# Goal/Experiment: +Protocol for sectioning flash frozen tissue that can be used for 3D IMS or MxIF experiments. + +# Cryostat Sectioning of Tissues for 3D Multimodal Molecular Imaging V.2 + +**Authors**: David Anderson, Elizabeth Neumann, Jamie Allen, Maya Brewer, Danielle Gutierrez, Jeff Spraggins +**Institution**: Vanderbilt University +**Published**: Apr 15, 2021 + +**DOI**: [https://dx.doi.org/10.17504/protocols.io.bt8inrue](https://dx.doi.org/10.17504/protocols.io.bt8inrue) + +## Abstract +### Scope +Protocol for sectioning flash frozen tissue that can be used for 3D IMS or MxIF experiments. + +### Expected Outcome +Serial sections from a tissue that IMS and MxIF can be performed on with subsequent 3D reconstruction. + +## Guidelines +1. Wear proper PPE: gloves, lab coat, and disposable sleeves. +2. Properly dispose of blades and other sharps into sharps container and tissue waste into a biohazard bag. +3. When finished, clean outer cryostat with approved germicide and inner cryostat with ethanol. + +**Note**: Since serial sections are required for multimodal analysis, it is critical that the tissues are high-quality and devoid of wrinkles, folds, or damage. + +## Materials +1. **Cryostat**: Leica CM 3050S + - **Function**: Equipment used to maintain low temperatures for sectioning tissues. +2. **Optimal Cutting Temperature Polymer (OCT)**: Fisher SH75-125D + - **Function**: Embedding medium to effectively section tissue; maintains structure at low temperatures. +3. **Cryostat Blades (disposable, high profile)**: Tissue Tek Accu-Edge, Fisher NC9527669 + - **Function**: For precise tissue sectioning. +4. **Razor Blade**: Fisher 12-640 + - **Function**: For trimming and preparing tissue orientation. +5. **Sample Holder/Disc/Chuck**: Leica 0370 08587 + - **Function**: Holds the tissue sample during sectioning. +6. **Anti-Roll Bar (Glass Insert 70mm)**: Leica 140474742497 + - **Function**: Prevents the tissue from rolling up during sectioning. +7. **Artist’s Paintbrushes** + - **Function**: Manipulate sections into position without damage. +8. **Ethyl Alcohol (200 proof)**: Pharmco-AAPR 111000200 + - **Function**: Cleaning and disinfecting instruments. +9. **Contec PREempt RTU Disinfectant Wipes**: Fisher 19 039 936 + - **Function**: For cleaning the cryostat. + +## Safety Warnings +1. Use Biosafety Level 2 precautions when using the cryostat. The inside of the cryostat is always considered infectious. +2. The blades used for cutting are extremely sharp. Use caution when inserting and removing the blade. + +## Procedure + +1. **Set the Cryostat Temperatures**: + - **Internal**: -21°C + - **Object**: -10°C to -20°C (based on tissue type) + +2. **Prepare Blade and Mount Sample**: + - Carefully remove blade from container and wipe with ethanol and a lint-free wipe. + - Insert blade and lock into place. + - Mount the sample onto the cryostat chuck using OCT in the desired orientation. Shave a flat surface onto the mounting surface using a razor blade to ensure correct orientation and good adherence. + +3. **Adjust the Stage**: + - Adjust the angle of the stage to allow for a less acute passage of the tissue section over the blade. Acute angles cause the surrounding OCT to fragment and lose structural integrity. + +4. **Trim OCT and Sectioning**: + - Trim OCT using larger section increments of 30-50 µm until the tissue becomes visible. + - Replace blades before procuring sections for imaging as trimming can cause blade dulling which diminishes section quality. + - Adjust the section thickness setting to 10µm. + +5. **Ensure Proper Sectioning**: + - Ensure that the anti-roll bar is positioned appropriately. It must not be too far towards the sample that it scrapes the tissue. + - Once sectioning becomes reproducible and consistent, obtain the number of desired sections. + +### Adding Sections to Slides + +10.1 Add subsequent serial sections to the same slide until full for 3D imaging. Take care to record any "lost" sections as they will affect subsequent data reconstruction. + +10.2 Manipulate each section into position on a target surface (slide, ITO slide) using a fine-tipped paint brush on the surrounding OCT material to prevent contamination or disruption of the tissue. + +10.3 Gently press a Teflon coated slide onto the tissues to prevent wrinkles and ensure proper mounting. + +10.4 Use heat from your gloved hand to thaw-mount sections to the target. Alternatively, use a slide warmer at 20°C to expedite drying. Once mounted, the section should be air-dried immediately before returning the sample to the cryostat or vacuum desiccator. + +**Note**: Imaging mass spectrometry analysis requires mounting onto indium tin oxide coated slides. CODEX immunofluorescence analysis requires mounting onto poly-lysine coated coverslips. + +10.5 Store tissue sections in a desiccator if undergoing immediate analysis within 24 hours or -80°C within a vacuum-sealed slide holder for long-term storage. + +## Citations +David Anderson, Elizabeth Neumann, Jamie Allen, Maya Brewer, Danielle Gutierrez, Jeff Spraggins (04/15/2021). Cryostat Sectioning of Tissues for 3D Multimodal Molecular Imaging. [https://dx.doi.org/10.17504/protocols.io.bt8inrue](https://dx.doi.org/10.17504/protocols.io.bt8inrue) + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/ctab-chloroform-isoamyl-alcohol-dna-extraction-pro-cxhexj3e.md b/markdown-output/ctab-chloroform-isoamyl-alcohol-dna-extraction-pro-cxhexj3e.md new file mode 100644 index 0000000000000000000000000000000000000000..1e2efdfad7e49c3ac9ae2aca01d65b180790bdc7 --- /dev/null +++ b/markdown-output/ctab-chloroform-isoamyl-alcohol-dna-extraction-pro-cxhexj3e.md @@ -0,0 +1,144 @@ +```markdown +# Goal/Experiment: +This experiment aims to extract high-quality DNA using the CTAB/Chloroform-Isoamyl Alcohol DNA extraction protocol. This method is commonly used for isolating DNA from various biological samples, ensuring the removal of impurities and contaminants to yield clean and stable DNA. + +## CTAB/Chloroform-Isoamyl Alcohol DNA Extraction Protocol + +**Elena L. Peredo1** +1Marine Biological Laboratory + +**ABSTRACT** +Protocol for extracting high-quality DNA. + +### DOI +[https://dx.doi.org/10.17504/protocols.io.261gednpdv47/v1](https://dx.doi.org/10.17504/protocols.io.261gednpdv47/v1) + +### Protocol Citation +> Elena L. Peredo 2023. CTAB/Chloroform-Isoamyl Alcohol DNA Extraction Protocol. **protocols.io** https://dx.doi.org/10.17504/protocols.io.261gednpdv47/v1 + +### License +This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. + +### Protocol Status +Working - We use this protocol and it's working. + +### Created +Jul 19, 2023 + +### Last Modified +Jul 20, 2023 + +### Protocol ID +85254 + +--- + +## Materials + +**Appendix, Reagents:** + +- **CTAB Buffer:** + - 100 ml of 1 M Tris, pH 8.0 + - 280 ml of 5 M NaCl + - 40 ml of 0.5 M EDTA + - 20 g of CTAB (Cetyl Trimethyl Ammonium Bromide) + - Adjust to 1 L with H2O + +- **1 M Tris, pH 8.0:** + - 121.1 g Tris + - 700 ml ddH2O + - Dissolve Tris and bring to 900 ml. + - Adjust pH to 8.0 with concentrated HCl (approx. 50 ml). + - Adjust volume to 1 L. + +- **0.5 M EDTA, pH 8.0:** + - 186.12 g EDTA + - 750 ml ddH2O + - Add about 20 g of NaOH pellets + - Slowly add more NaOH until pH is 8.0. EDTA will not dissolve until pH is near 8.0. + - Adjust volume to 1 L. + +- **5 M NaCl:** + - 292.2 g NaCl + - 700 ml ddH2O + - Dissolve and bring to 1 L. + +- **7.5 M Ammonium Acetate:** + - 144.5 g ammonium acetate + - Adjust volume to 250 ml with ddH2O + +## Procedure + +### Preparation Steps + +#### Clean the Work Area + +1. Clean working areas and surfaces using ethanol and bleach. + - *Note:* Micro pestles need pre-treatment. DNA is thermostable but might not be degraded purely by autoclaving. + +2. *Steps:* + - Treat with bleach, rinse with distilled water. + - Use high-intensity UV (crosslinker UV machine) for additional sterilization. + +#### Prepare the CTAB Buffer + +1. Prepare a fresh extraction buffer in the hood. Combine CTAB and β-mercaptoethanol (2%-4%): + +| A | CTAB | β-mercaptoethanol (2%) | +|-----|------|------------------------| +| 490 ul | 10 ul | +| 4900 ul | 100 ul | +| 9800 ul | 200 ul | + +2. CTAB Extraction Buffer Explanation: + - **β-mercaptoethanol:** Removes tannins and phenolic compounds, denatures proteins. + - More compounds like Vitamin C and PVP can be added to target specific metabolites. + +#### Washing Debris from Algal Cultures + +1. Remove cell wall debris and bacteria by washing cultures: + +*Steps:* +1. Let cells settle in an Erlenmeyer flask. +2. Slowly pour off most of the medium, retaining 5-10 ml. +3. Gently resuspend in the medium. +4. Transfer 1.5 ml of suspension to Eppendorf tubes. +5. Repeat steps for additional tubes and washing process. + +2. Spin Algae Briefly (Eppendorf tubes): + +*Steps:* +1. Spin (5000 rpm for 1 minute). +2. Remove supernatant. +3. Resuspend in 1 ml fresh medium. +4. Spin at 2500 rpm for 30 sec. +5. Remove supernatant. +6. Resuspend in fresh medium. +7. Spin last time at 5000 rpm for 1 min 30 sec. + +### Liquid Nitrogen Grinding + +1. Recover pellet and transfer. Pre-cool mortar and pestle using liquid nitrogen, proceed with grinding. + +2. Transfer to clean Eppendorf tube with 100 ul extraction buffer. + +*Note:* +- Alternative grinding: Use commercial silica sand, zirconium beads with CTAB. + +### Extraction + +1. Add 500 ul extraction buffer to 600 ul total volume. +2. Incubate samples at 55-60°C for 1 hour. +3. Add 700 ul of Chloroform:Phenol:Isoamyl alcohol, shake to emulsify. +4. Centrifuge at max speed (13-15,000 rpm) for 10 mins. +5. Pipette off aqueous phase, transfer to new labeled tube. +6. Add 4 µl RNAse A, incubate at 37°C for 30 mins. +7. Repeat steps 8-11 with centrifuge intervals. +8. Combine volumes, add cold isopropanol and freeze. +9. Centrifuge, remove supernatant, add 70% Ethanol. +10. Dry pellet, resuspend with 45 µl TE, resuspend, incubate at room temperature or 4°C overnight. + +--- + +### endofoutput +``` \ No newline at end of file diff --git a/markdown-output/culture-of-established-induced-pluripotent-stem-ce-bgbwjspe.md b/markdown-output/culture-of-established-induced-pluripotent-stem-ce-bgbwjspe.md new file mode 100644 index 0000000000000000000000000000000000000000..6e2f5c7fae66d9ef57166e3f75eec4d2d08a26ed --- /dev/null +++ b/markdown-output/culture-of-established-induced-pluripotent-stem-ce-bgbwjspe.md @@ -0,0 +1,105 @@ +```markdown +# Goal/Experiment: +To thaw, passage, and cryopreserve established feeder-free induced pluripotent stem cell (iPSC) lines using a specified protocol suitable for differentiation and CRISPR screens. + +# Culture of Established Induced Pluripotent Stem Cell Lines +### Cellular Generation and Phenotyping +**Wellcome Sanger Institute** + +*Published on: Jun 26, 2020* + +*[DOI Link](https://dx.doi.org/10.17504/protocols.io.bgbwjspe)* + +## Abstract +This protocol outlines the method for thawing, passaging, and cryopreserving established feeder-free induced pluripotent stem cell lines. The cells are maintained as aggregates and cultured on vitronectin matrix with E8 or TeSR-E8 media. They can be cultured with or without Penicillin-Streptomycin. These protocols are suitable for downstream applications including differentiation and CRISPR screens. + +## Keywords +induced pluripotent stem cells, iPSC, feeder free, feeder free iPSCs + +## License +This protocol is distributed under the terms of the [Creative Commons Attribution License](https://creativecommons.org/licenses/by/4.0/), allowing unrestricted use and reproduction, provided the original author and source are credited. + +--- + +## Guidelines +- Cells should be cultured on a vitronectin matrix using E8 or TeSR-E8 media. +- Culturing can be done with or without Penicillin-Streptomycin. +- All cell culture should be performed under sterile conditions in a biological safety cabinet. + +## Materials + +| Name | Catalog # | Vendor | +| --------------------------------------------------------------------- | ------------- | ------------------------------ | +| TeSR™-E8™ Kit for hESC/hiPSC Maintenance 1 Kit | 5990 | Stemcell Technologies | +| Dimethyl sulfoxide (DMSO) | D2650 | Sigma Aldrich | +| Penicillin Streptomycin | 15140 122 | Invitrogen - Thermo Fisher | +| Gibco™ DPBS no calcium no magnesium | 14190144 | Thermo Fisher Scientific | +| Falcon™ 15mL Conical Centrifuge Tubes | 14-959-53A | Fisher Scientific | +| Essential 8™ Medium | A1517001 | Gibco, ThermoFisher | +| Vitronectin (VTN-N) Recombinant Human Protein, Truncated | A14700 | Thermo Fisher | +| UltraPure 0.5M EDTA pH 8.0 | 15575020 | Invitrogen - Thermo Fisher | +| Falcon 50mL Conical Centrifuge Tubes | 14-432-22 | Fisher Scientific | +| Knockout serum replacement (KSR) | 10828028 | Gibco - Thermo Fisher | +| Nunc 1.8mL Cryotube External Thread Starfoot | 375418X | Scientific Laboratory Supplies Ltd | +| Corning® CoolCell® FTS30 Freezing Container for 30 x 1mL or 2mL Cryogenic Vials Green | 432008 | Corning | +| Costar® 6-well Clear TC-treated Multiple Well Plates Individually Wrapped Sterile | 3516 | Corning | +| Falcon® 6-well Clear Flat Bottom TC-treated Multiwell Cell Culture Plate with Lid Individually Wrapped Sterile | 353046 | Corning | +| Vitronectin XF™ | #07180 | Stemcell Technologies | +| Y-27632 dihydrochloride | Y0503 | Sigma Aldrich | + +--- + +## Safety Warnings +- Refer to the manufacturer's documentation and material safety data sheets (MSDS) for all products used. + +## Protocol + +### iPSC Thawing + +#### Preparation +1. Coat a 6-well plate with vitronectin and incubate as per the manufacturer's instructions. Prepare complete E8 or TeSR-E8 media as per instructions. +2. Prepare thawing media by adding rock inhibitor (Y-27632) to culture media at a final concentration of 10μM. +3. Add 8ml of thawing media to sterile 15ml falcon tube(s). + +#### Thawing +1. Partially thaw the frozen cryovial(s) of iPSC in a 37°C water bath until small ice crystals remain. +2. Add 1ml of thawing media dropwise to each cryovial. Transfer the contents to the falcon tube prepared. Tip: Keep iPSCs in small clumps. +3. Centrifuge at 120 x g, Room temperature for 3 mins. +4. Aspirate vitronectin from the labware surface and replace with 1ml of thawing media per well, ensuring surfaces do not dry out. +5. Aspirate supernatant and resuspend cells in 1ml thawing media. Plate cell suspension into wells. +6. Agitate the plate gently within a tissue culture incubator set at 37°C, 5% CO₂ for even distribution of cells. +7. Allow cells 2-24 hours to attach to the surface. Following attachment, change media to remove the rock inhibitor. Media change every 24 hours until 70-80% confluency. + +### iPSC Passaging + +#### Preparation +1. Coat 6-well plate(s) with vitronectin as per manufacturer's instructions. +2. Prepare fresh 0.5mM EDTA in DPBS (-/-) (pH 8.0) on day of use. + +#### Passaging +1. Aspirate spent medium from cells and wash with 2ml of DPBS (-/-) per well. +2. Add 1-2ml 0.5mM EDTA per well, rock dishes gently, and incubate for 4 mins at room temperature. +3. Observe under a microscope until colonies display shiny 'halos', then aspirate EDTA and add 2ml fresh culture medium. +4. Collect and dispense medium across the labware surface. Avoid creating bubbles. +5. Collect cells into a falcon tube. +6. Aspirate vitronectin from new labware surfaces and replace with 1ml pre-warmed culture medium. Plate 1ml cell suspension into each new well. +7. Agitate the plate within a tissue culture incubator set at 37°C, 5% CO₂. +8. Allow 2-24 hours for cells to attach, then media change every 24 hours until they reach 70-80% confluency. + +### iPSC Cryopreservation + +1. When colonies are compact and ~70% confluent, they are ready for freezing. Freeze one 6-well plate well into 5 cryovials. +2. Use 10% DMSO in KSR as freezing media. +3. Prepare fresh 0.5mM EDTA in DPBS (-/-) (pH 8.0) on day of use. +4. Aspirate spent medium from cells and wash with 2ml DPBS (-/-) per well. +5. Add 2ml 0.5mM EDTA per well, incubate for 4-8 mins at room temperature until colonies display 'halos'. +6. Aspirate EDTA, add 2ml culture medium, and collect cells into appropriate falcon tube. +7. Centrifuge falcon tube at 120 x g, room temperature, for 1 min. +8. Aspirate supernatant and resuspend pellet in freezing media. +9. Dispense 1ml of cell suspension into each cryovial. +10. Place vials in a CoolCell or similar container and store at -80°C for 24-48 hours before transferring to liquid nitrogen for long-term storage. + +--- + +`endofoutput` +``` \ No newline at end of file diff --git a/markdown-output/culture-of-the-severe-acute-respiratory-syndrome-c-bcduis6w.md b/markdown-output/culture-of-the-severe-acute-respiratory-syndrome-c-bcduis6w.md new file mode 100644 index 0000000000000000000000000000000000000000..3db3a8c204d91e4a929caa3d16f5fd024db58950 --- /dev/null +++ b/markdown-output/culture-of-the-severe-acute-respiratory-syndrome-c-bcduis6w.md @@ -0,0 +1,86 @@ +```markdown +# Culture of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2; f.2019-nCoV) + +## Goal/Experiment: +To culture the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of COVID-19, by inoculating a susceptible cell line with a human patient sample. + +### Authors +Alyssa Pyke, Ian M. Mackay, Frederick Moore, Andrew Van Den Hurk, Judy Northill, Mitchell Finger, Natalie Simpson, Neelima Nair, Peter Burtonclay, Peter Moore, Sarah Wheatley, Sean Moody, Sonja Hall-Mendelin, Elisabeth Gamez, Amanda De Jong, Ben Huang, Carmel Taylor, David Warrilow, Doris Genge, Glen Hewitson, Inga Sultana, Jamie McMahon, Jean Barcelon + +Public Health Virology, Forensic and Scientific Services + +Publication Date: February 12, 2020 + +DOI: [10.17504/protocols.io.bdcui56w](dx.doi.org/10.17504/protocols.io.bdcui56w) + +### Abstract +We describe a method to inoculate a susceptible cell line with a human patient sample to culture SARS-CoV-2. Clinical samples including nasopharyngeal swabs and aspirates were inoculated onto Vero C1008 (African green monkey kidney E6 cells, ATCC® CRL-1586) grown in Opti-MEM reduced serum growth medium supplemented with 3% foetal bovine serum in polystyrene, flat-sided, screw-cap 3 mL cell culture tubes. + +### Materials +- **Nunc™ Cell Culture Tubes, 3mL** + - Catalog #: 156758 + - Vendor: Thermo Fisher + +- **Opti-MEM™ I Reduced Serum Medium** + - Catalog #: 31985088 + - Vendor: Thermo Fisher + +- **Acrodisc® Syringe Filters, 0.2 µm Pore Size, 13 mm Dia** + - Catalog #: 4602 + - Vendor: Cole-Parmer + +- **Foetal Bovine Serum (FBS) Triple 0.1 µm Sterile Filtered, Australian Origin** + - Catalog #: FBS-AU-015 + - Vendor: Serana + +### Safety Warnings +- This work was conducted under PC3 laboratory conditions by experienced virologists. +- Prioritize proper PPE use and thorough training in handling biological samples and waste. + +### Preparation + +#### Specimen and Uninfected Cell Preparation +1. **Cell Culture Tubes Setup:** + - Move cell culture tubes from the 37°C incubator to a Class II Biosafety cabinet, within a PC3 laboratory environment. + - Discard growth medium from prepared Vero E6 tubes. + +2. **Sample Preparation:** + - Dilute clinical samples in Opti-MEM reduced serum growth medium without FBS, filtering through a 0.2 µm, 13 mm Acrodisc® filter. + +### Protocol + +#### Inoculation of Vero E6 Cell Monolayers +3. **Inoculation:** + - Inoculate 150-200 µL of filtered patient material onto separate confluent cell monolayers. + - Use a negative control tube with 150-200 µL of Opti-MEM alone. + + **Note:** + - Fresh specimens are best for successful viral culture. + - Absorb samples onto cells by incubating tubes for 1 hr at 37°C, then re-feed with 2 mL pre-warmed (37°C) Opti-MEM reduced serum growth medium. + - Collect a 200 µL sample at this point as a Day 0 value to test alongside other samples from Day 2 onwards. + +#### Culture Incubation and Observation +4. **Incubation:** + - Incubate cultures for 2-7 days until signs of cytopathic effect (CPE) are observed. + + **Observation:** + - Cultures inoculated with patient sample extracts with approximately 20 cycles of CPE developed signs within 3 days post-inoculation. + +![Uninfected Monolayer](https://example.com/image1.png) +_A confluent, uninfected monolayer culture of Vero E6 cells in Opti-MEM (no FBS)._ + +### Confirmation of a Successful Virus Culture +6. **Confirmation:** + - Suspect virus replication based on CPE including damage to the monolayer, cell clearing, and morphological changes. + - Confirm suspected cultures using RT-PCR assays (e.g., Corman et al., Northill et al. ORF1ab novel coronavirus reverse-transcription real-time PCR tests). + - Expect CT values of the cultured virus to be lower than those from the original sample. + + **Note:** + - For slow cultures, compare CT values of the viral extract from the first passage with RT-PCR of the original sample to differentiate between viral remnants and true replication. + +### Actions to Consider in the Absence of Obvious CPE +7. **If no CPE is observed:** + - Passage supernatants onto fresh Vero E6 monolayers up to 3 times for retesting as described in Step 6. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/culturing-chlorella-vulgaris-and-desmodesmus-quadr-bd6vi9e6.md b/markdown-output/culturing-chlorella-vulgaris-and-desmodesmus-quadr-bd6vi9e6.md new file mode 100644 index 0000000000000000000000000000000000000000..ae369e353082454caef18a63bcaced16035d28a3 --- /dev/null +++ b/markdown-output/culturing-chlorella-vulgaris-and-desmodesmus-quadr-bd6vi9e6.md @@ -0,0 +1,139 @@ +```markdown +# Goal/Experiment: +The goal of this experiment is to cultivate and maintain healthy cultures of *Chlorella Vulgaris* and *Desmodesmus Quadricauda*. This involves preparing a nutrient medium, dissociating cell clumps, counting cell density, and inoculating fresh culture medium. + +# Culturing *Chlorella Vulgaris* and *Desmodesmus Quadricauda* + +## Abstract + +This protocol describes the steps required for maintaining a healthy culture of *Chlorella Vulgaris* and *Desmodesmus Quadricauda*. It uses ½ SŠ Growth Medium (0.5 g/l). The cells are grown until saturation or when a new split is needed. At the lowest light level, the cell culture split should take place at least once every two weeks. + +External Link: [Labstep Sharelink](https://app.labstep.com/sharelink/5ac9f965-791b-4333-b17b-f57e5e1dc42d) + +## Guidelines + +Culturing the microalgae is a process of replenishing fresh growth medium and diluting the culture suspension density. The microalgae culture involves the following steps: + +- Preparing medium +- Dissociating clumped cells +- Counting the cell culture density +- Calculating the fresh medium and inoculation culture volumes +- Counting cells +- Calculating cell culture density + +## Materials + +| Name | Catalog # | Vendor | +| ------------------------------------------------------------ | --------- | ---------------------- | +| Virkon powder | 148-0201 | VWR International Ltd | +| 1/2 SŠ medium (0.5 g/l) | Various | Homemade | +| Sterile Erlenmeyer flasks with cotton wool plug and aluminium foil seal | Various | Homemade | + +## Materials Text + +- Sterile flow hood +- Watchmaker tweezers +- Serological pipettes and pipette filler + +## Safety Warnings + +- The main hazards involved in culturing cells include handling microalgae, liquid media containing a range of chemical substances, chemical bleach agents, fragile glass flasks, repetitive strain injury due to pipetting and using a tally counter for counting cells. +- Each of these hazards needs to be assessed according to local rules and regulations and appropriate measures need to be taken. +- Use personal protective equipment. Clean working areas and wipe down spills using appropriate equipment. Do not release live microalgae into the environment, use prescribed bleach or microbicides to destroy the cultures and appropriate biohazard disposal routes for contaminated consumables. + +## Before Starting + +- Ensure the ½ SŠ Growth Medium is prewarmed to room temperature. Leave it standing for at least an hour outside the fridge for it to warm up. +- Start and wipe down the laminar flow cabinet with 70% denatured ethanol solution. +- Ensure you have enough sterile culture flasks. You can use disposable flasks, like T75, or prepare glass Erlenmeyer flasks as described in [Autoclaving Erlenmeyer Flasks for Sterile Algal Cultures](#). +- Have all equipment ready for counting cells, this includes a hemocytometer and a tally counter, as described in [Counting Microalgae Culture Density](#). + +## Preparatory Steps + +1. Warm-up ½ SŠ Growth Medium (0.5 g/l) to room temperature. + - Leave it outside of a fridge for at least an hour, or leave it in a 37°C water bath for 10 minutes or so. +2. Pre-label Erlenmeyer flasks with the culture type, date, own initials, passage number, or other pieces of information as required. + - For sterilizing the Erlenmeyer culture flasks, see [Autoclaving Erlenmeyer Flasks for Sterile Microalgae Cultures](#). +3. Turn on and wipe clean the sterile hood. +4. Take out the previous culture passage from the shaker and bring it to the tissue culture room. +5. Dissociate cell aggregates on a shaker. + - Use low power setting to prevent the suspension splashing onto the cotton wool plug. + - *Desmodesmus quadricauda* cells do not require as much vortexing. Five seconds is enough. + - *Chlorella vulgaris*, when overgrown, requires much more vortexing. The more shaking the better. Long vortexing is especially important when cells are deposited along the flask sides and thus form clumps. A minute or two of shaking is not excessive! + +## Inoculating Fresh Culture Medium + +6. **Count the microalgae cell culture density** + - Handle the cultures inside a sterile hood. Pipette 10 µl of the dissociated cell culture suspension into the hemocytometer. + - For a detailed protocol see [Counting Microalgae Culture Density](#). + - Count the culture density using a hemocytometer and keep the tally. + - With *Desmodesmus quadricauda*, count each cell, not just the coenobia, i.e. one coenobium with four cells counts as four cells, one coenobium with eight cells count as eight cells. + - Calculate the cell culture density: + - *D. quadricauda* ![\rho_dq](https://latex.codecogs.com/svg.latex?%5Crho_%7Bdq%7D) \([ml^{-1}]\) + - *C. vulgaris* ![\rho_Chl](https://latex.codecogs.com/svg.latex?%5Crho_%7BChl%7D) \([ml^{-1}]\) + +7. **Calculate the dilution factors for inoculating fresh medium** + - The seed density in the freshly inoculated medium is: + - ![\rho_{seed}^{Dq} = 2\times{10^{5}} ml^{-1}](https://latex.codecogs.com/svg.latex?%5Crho_%7Bseed%7D%5E%7BDq%7D%20%3D%202%5Ctimes%7B10%5E%7B5%7D%7D%20ml%5E%7B-1%7D) for *D. quadricauda* + - ![\rho_{seed}^{Chl} = 10^6 ml^{-1}](https://latex.codecogs.com/svg.latex?%5Crho_%7Bseed%7D%5E%7BChl%7D%20%3D%2010%5E6%20ml%5E%7B-1%7D) for *C. vulgaris* + - The volume of culture \( V \) = 20 ml is the same for both species. + - The cell density of the existing culture has been calculated in the above step. + - ![\rho_{Dq}](https://latex.codecogs.com/svg.latex?%5Crho_%7BDq%7D) \([ml^{-1}]\) is the existing cell culture density for *D. quadricauda* + - ![\rho_{Chl}](https://latex.codecogs.com/svg.latex?%5Crho_%7BChl%7D) \([ml^{-1}]\) is the existing cell culture density for *C. vulgaris* + - The inoculation volume of the existing culture is calculated as follows: + - *D. quadricauda*: + \[ + V_{culture}^{Dq} = V \times \frac{\rho_{seed}^{Dq}}{\rho_{Dq}} = 20 \ ml \times \frac{2 \times 10^{5} \ ml^{- 1}}{\rho_{Dq}} + \] + - *C. vulgaris*: + \[ + V_{culture}^{Chl} = 20 \ ml \times \frac{10^6 \ ml^{- 1}}{\rho_{Chl}} + \] + - The volume of the fresh medium is then calculated by subtracting the inoculation volume (calculated above) from the total volume of the culture \( V \): + - *D. quadricauda*: + \[ + V_{medium}^{Dq} = V - V_{culture}^{Dq} = 20 \ ml - V_{culture}^{Dq} + \] + - *C. vulgaris*: + \[ + V_{medium}^{Chl} = V - V_{culture}^{Chl} = 20 \ ml - V_{culture}^{Chl} + \] + +8. **Dispense the right volumes of the fresh medium into the pre-labeled Erlenmeyer flasks**: + - *D. quadricauda*: + \[ + V_{medium}^{Dq} + \] + - *C. vulgaris*: + \[ + V_{medium}^{Chl} + \] + 8.1 Check that the fresh medium is not too cold, if it was taken out of the fridge only a short while ago. If it is cold, leave it standing for a few minutes, it will warm up quite quickly in a small volume standing on the metal surface of the laminar flow hood. + +9. **Inoculate the medium in the Erlenmeyer flasks with the existing cultures**: + - *D. quadricauda*: + \[ + V_{culture}^{Dq} + \] + - *C. vulgaris*: + \[ + V_{culture}^{Chl} + \] +10. **Place the cells back on the algal shaker**: + ![](protocol_image.png) + - Microalgae cultures on the illuminated orbital shaker. + +11. **Clean up the sterile hood and the lab space**: + - Sterilize old cultures by adding bleach or Virkon overnight. + +## References + +- [Counting Microalgae Culture Density](#) protocol +- [Autoclaving Erlenmeyer Flasks for Sterile Microalgae Culture](#) protocol +- [Preparing 1/2 SŠ Algal Inorganic Nutrient Medium](#) +- [1/2 SŠ inorganic nutrient medium](#): Recipe from the Culture Collection of Autotrophic Organisms +- [Zachelder & Šetlík, 1982](#): The original article introducing this medium + +``` + +endofoutput \ No newline at end of file diff --git a/markdown-output/culturing-i3lmns-basic-protocol-8-5u5g6y6.md b/markdown-output/culturing-i3lmns-basic-protocol-8-5u5g6y6.md new file mode 100644 index 0000000000000000000000000000000000000000..7e441d9f5ed72338b6831987e1541a8911228f94 --- /dev/null +++ b/markdown-output/culturing-i3lmns-basic-protocol-8-5u5g6y6.md @@ -0,0 +1,100 @@ +```markdown +Goal/Experiment: +To differentiate and culture induced pluripotent stem cell-derived motor neurons (i3LMNs) for extended use in research, leveraging specific media and substrate conditions to ensure optimal cell viability and differentiation. + +# iPSCs: CULTURING i3LMNs (Basic Protocol 8) + +**Authors:** Michael S. Fernandopulle, Ryan Prestil, Christopher Grunseich, Chao Wang, Li Gan, Michael E. Ward + +**Affiliations:** +1. National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland +2. Gladstone Institute of Neurological Disease, Gladstone Institutes, San Francisco, California + +**Link:** +[dx.doi.org/10.17504/protocols.io.5u5g6y6](dx.doi.org/10.17504/protocols.io.5u5g6y6) + +**Community:** +Neurodegeneration Method Development Community + +## Abstract +Induction and differentiation of i3LMNs is nearly identical to the first 3 days of differentiation for i3Neurons (see Basic Protocol 5), including identical induction medium. Following replating, differences arise including the use of Motor Neuron Culture Medium (MM) for long-term culture (Table 5), additional reagents to reduce proliferative cells if necessary, and variable options for coating polymers. + +## Materials Required +- PLO, PEI, or PDL solution (see [Table 3](#Table-3)) +- Laminin (Gibco, cat. no. 23017015) +- Freshly split or thawed 3-day differentiated i3LMNs (following completion of Basic Protocol 5) +- Motor Neuron Culture Medium (MM, see Table 5) + +### Reagents and Equipment +- **Laminin Mouse Protein, Natural** (Thermo Fisher Scientific, Catalog #: 23017015) +- **Neurobasal™ Medium** (Thermo Fisher Scientific, Catalog #: 21103049) +- **B-27 Supplement** (Gibco - Thermo Fisher, Catalog #: 17504044) +- **N-2 Supplement (100X)** (Thermo Fisher Scientific, Catalog #: 17502001) +- **MEM Non-Essential Amino Acids Solution (100X)** (Gibco, ThermoFisher, Catalog #: 11140050) +- **L-Glutamine (200 mM)** (Gibco, ThermoFisher Catalog #: 25030081) +- **CultureOne™ Supplement (100X)** (Thermo Fisher Scientific, Catalog #: A3320201) + +### Preparation +1. **Prepare in sterile biosafety cabinet; medium should then be aliquotted to add additional supplements fresh; warm to 37°C before use.** +2. **While not required, addition of BDNF and NT-3 as described for CM ([Table 4](#Table-4)) improves long-term cell health.** + +### Table 5: Motor Neuron Culture Medium (MM) +| Component | Amount per 50 ml | Final Concentration | +|-----------------------------------------|------------------|---------------------| +| Neurobasal medium | 47.5 ml | 1× | +| B27 supplement, 50x | 1 ml | 1× | +| N2 supplement, 100x | 500 µl | 1× | +| Non-essential amino acids (NEAA), 100x | 500 µl | 1× | +| L-glutamine, 100x (or Gluta-MAX) | 500 µl | 1× | +| (Optional) CultureOne supplement, 100x | 500 µl | 1× | +| Laminin (store at -80°C, stock 1 mg/ml) | 50 µl | 1µg/ml | + +## Protocol Steps + +### Coating Dishes +1. **Prepare stock solutions of PLO, PEI, or PDL (see [Table 3](#Table-3)).** +2. **Add one-half culture volume of 1× coating solution from step 1 to the tissue culture dishes to be used for plating Day 3 partially differentiated i3LMNs.** +3. **Gently tilt the plate to ensure full coverage.** +4. **Incubate dishes for at least 1 hour at Room temperature.** + - **For best results, incubate dishes overnight in a 37 °C incubator.** +5. **Aspirate coating solution.** +6. **Wash dishes with sterile water.** +7. **Repeat twice for three total washes.** + - **An additional two washes is recommended for PEI coating.** +8. **Aspirate water and let dishes dry completely in a biosafety cabinet (typically requires 30 minutes to 1 hour).** + - **To accelerate the drying process, stand the dishes on their sides and lean them against the back of the biosafety cabinet. Lids may also be left askew to allow better airflow. In particular, PEI requires complete drying to prevent toxicity.** +9. **Coated and dried dishes should be used immediately or wrapped in aluminum foil and stored at 4°C for up to 1 week.** + - **Optional: Faster neurite outgrowth and increased neuronal survival during replating may be achieved by additionally coating plates with laminin prior to plating. Dilute laminin to 15 µg/ml in IM, and add one-half culture volume of this solution to the polymer-coated, washed, and dried wells. Incubate at 37 °C for 1 hour, then plate neurons directly by adding cells in an addition one half culture volume of IM.** + +### Plating Cells +10. **If laminin coating was not performed, prepare wells of pre-coated plates with warm IM supplemented with 10 µM ROCK inhibitor, 2 µg/ml doxycycline, 1:10,000 Compound E from stock, and 1 µg/ml laminin.** + - **Additionally supplementing with 40 µM BrdU for 24 hours helps to prevent outgrowth of mitotically active cells without affecting neuronal health. Alternatively, CultureOne supplement may be added to the medium from d4 onwards.** + +11. **From frozen stock or freshly dissociated 3-day differentiated cells, resuspend (see Basic Protocol 1, thawing iPSCs) in the appropriate amount of medium. Typical cell counts and medium volumes are as follows:** + - 96-well plate (imaging): 1–5 × 10^4 cells in 100 µl medium/well. + - 8-well chamber slide (imaging): 0.3–1.5 × 10^5 cells in 250 µl medium/well. + - 6-well plate (biochemistry): 1.5–2 × 10^6 cells in 1.5 ml medium/well (supplement to 2-3 ml one day after plating). + - 10-cm dish (biochemistry): 1–1.2 × 10^7 cells in 8 ml medium (supplement to 10-12 ml one day after plating). + - 15-cm dish (biochemistry): 3–3.5 × 10^7 cells in 18 ml medium/well (supplement to 20-22 ml one day after plating). + - **Biochemistry applications typically require a high concentration of cells for a given surface area. Thus, these experiments require a greater volume of medium than would be required for an imaging experiment. However, after splitting, cells typically adhere better to a new plate with a lesser volume of medium compared to a greater volume. Thus, neurons plated on 6-well, 10-cm, or 15-cm dishes for biochemistry should be plated with 1.5 ml, 8 ml, and 18 ml of medium, respectively. These volumes should then be supplemented to the final volumes above on the day after plating.** + +### Culture Maintenance +12. **The next day (d4), aspirate medium and replace with pre-warmed MM supplemented with 1 µg/ml laminin.** + - **For full medium changes, avoid drying wells by only aspirating one dish or a few wells at a time. Initially, add the medium very slowly dropwise in the middle of the well with the plate tilted until a small pool forms in the corner, at which point add media dropwise down the well onto this pool. This helps to avoid shear forces on the edges which can cause whole wells to detach in a sheet. If BrdU was used on day 3, wash with PBS before adding medium.** + +13. **For the first 4 days (d4 to d7), check cells daily under a phase-contrast microscope, paying particular attention to cell debris and morphological changes. Medium changes should be done every 2 to 3 days by replacing one-half of the medium with fresh MM+laminin.** + - **High levels of cell debris and/or cell clumping often indicate a problem with either the dish coating or culture medium. If seen, remove half volume of culture medium and replace with full volume of medium (50 % additional medium). If additional fresh medium does not result in less debris the next day, there has likely been insufficient coating or the coating medium was expired.** + +14. **After day 7, perform half-medium changes every 4 to 7 days with complete MM+laminin for long-term maintenance.** + - **Biweekly half-medium changes can be effective in sustaining dense cultures (e.g., biochemistry applications). Weekly half-medium changes are sufficient to sustain long-term cultures at moderate densities (e.g., microscopy applications). Use the appropriate serological pipet or micropipet to slowly aspirate a measured volume from each well, and very gently replace with fresh medium. Neurons tend to dissociate from the dish easily, so any aspiration or dispensing of medium directly onto cells is not recommended. Take care to aspirate and dissociate by tilting the dish so that medium accumulates on one side. Then, aspirate/dispense with the pipet directed toward the wall of the dish (i.e., away from the cells at the bottom).** + +15. **Optional: Supplementation with astrocytes or astrocyte-conditioned medium have been shown to improve the overall health and electrophysiological activity of i3LMNs in long-term cultures, as described for i3Neurons (see Basic Protocol 6 and Support Protocol 7).** + +## Safety Warnings +Please see SDS (Safety Data Sheet) for hazards and safety warnings. + +## References +Fernandopulle, M. S., Prestil, R., Grunseich, C., Wang, C., Gan, L., & Ward, M. E. (2018). Transcript ion-factor mediated differentiation of human iPSCs into neurons. Current Protocols in Cell Biology, e51. doi:10.1002/cpcb.51 + +**endofoutput** +``` \ No newline at end of file diff --git a/markdown-output/cut-amp-run-cnx2vfqe.md b/markdown-output/cut-amp-run-cnx2vfqe.md new file mode 100644 index 0000000000000000000000000000000000000000..5d88ce9d6fcf77c83efb9079b061f08337c87950 --- /dev/null +++ b/markdown-output/cut-amp-run-cnx2vfqe.md @@ -0,0 +1,184 @@ +```markdown +# Goal/Experiment: +The goal of this experiment is to perform CUT&RUN (Cleavage Under Targets and Release Using Nuclease) to investigate protein-DNA interactions by cleaving and isolating specific segments of DNA that are bound to proteins of interest. This allows for the analysis of chromatin profiling and epigenetics. + +# CUT & RUN Protocol + +## Author: +Yunkyeong Lee +Stanford University, School of Medicine + +## Abstract +CUT & RUN protocol + +## DOI: +[dx.doi.org/10.17504/protocols.io.261ge32b7l47/v1](https://dx.doi.org/10.17504/protocols.io.261ge32b7l47/v1) + +## Protocol Citation: +Yunkyeong Lee 2023. CUT & RUN. protocols.io +[https://dx.doi.org/10.17504/protocols.io.261ge32b7l47/v1](https://dx.doi.org/10.17504/protocols.io.261ge32b7l47/v1) + +## License: +This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited + +## Protocol Status: +Working. We use this protocol and it's working. + +## Created: +Feb 06, 2023 + +## Last Modified: +Feb 06, 2023 + +## Protocol Integer ID: +76506 + +## Equipment and Reagents +### Kit Used: +EpiCypher CUTANATM ChIC/CUT&RUN kit (Kit Version 2.0, User Manual Version 2.1) - Catalog No. 14-1048, 48 ChIC/CUT&RUN samples + +### Overview of the CUTANA CUT&RUN Protocol: +1. Immobilize & permeabilize cells (or nuclei) +2. Add antibody to histone PTM or chromatin-interacting protein +3. Add & activate pAG-MNase to cleave target-DNA complex +4. Target-DNA complex diffuses out, collect supernatant +5. Extract DNA & prepare sequencing library +6. Next-generation sequencing and data analysis + +### Boxed Reagents: +**BOX 1** (at RT): +- DNA Binding Buffer (25 mL), +- DNA Wash Buffer (5 mL), +- EDTA 100 mM (500 uL), +- CaCl₂ (500 uL), +- DNA Elution Buffer (1.2 mL), +- DNA Cleanup Columns (50 Columns), +- DNA Collection Tubes (50 Tubes), +- 8-Strip Tubes (56 Tubes) + +**BOX 2** (at 4°C): +- SA Beads (40 uL) (X), +- ConA Beads (550 uL), +- Stop Buffer (1.5 mL), +- Bead Activation Buffer (12 mL), +- Pre-Wash Buffer (105 mL) + +**BOX 3** (at -20 °C): +- IgG Negative Control (10 uL), +- H3K4me3 Positive Control (10 uL), +- Spermidine (100 uL), +- pAG-MNase (48 runs), +- Spike-in DNA (100 ng), +- 5% Digitonin (100 uL) + +### Additional Reagents: +- EpiCypher 50 uL - 0.2 mL Magnetic Rack, +- Invitrogen 1.5 mL tube Magnetic Rack + +--- + +## Day 1 - Jan 14, 2023 +### 1. Buffer Preparation: Make CUT&RUN buffers fresh the day of use. + +1. **Wash Buffer Preparation:** + 1. Add 1.8 mL Pre-Wash Buffer per sample to a 50 mL conical tube labeled "Wash Buffer". + 2. Dissolve 1 protease inhibitor tablet (Roche) in 2 mL water (25X stock). Add 72 uL per sample to the Wash Buffer. Store remaining 25X stock for 12 weeks at -20°C. + 3. Dilute 1M Spermidine 1:2,000 in the Wash Buffer. Store final buffer at RT. + +2. **Cell Permeabilization Buffer Preparation:** + - Transfer 1.4 mL of Wash Buffer per sample into a new 50 mL conical tube labeled "Cell Perm Buffer". Add 5% Digitonin (1:500 dilution). + +3. **Antibody Buffer Preparation:** + - Transfer 100 uL per sample of Cell Permeabilization Buffer into a new 50 mL tube labeled "Antibody Buffer". Add 0.5 M EDTA (1:250 dilution). Store final buffer on ice. + - Store the remaining Cell Perm Buffer at 4°C overnight for Day 2 use. + +### A. Wash buffer: +- Leave at RT for use on Day 1 + - Per sample: 1.8 mL Pre-Wash Buffer, 72 uL Protease inhibitor (1X final), 0.9 uL Spermidine (0.5 mM final) + +### B. Cell Permeabilization Buffer: +- Store at 4°C overnight for use on Day 2 + - Per sample: 1.4 mL Wash Buffer, 2.8 uL Digitonin (0.01% final) + +### C. Antibody Buffer: +- Set on ice for use on Day 1 + - Per sample: 100 uL Cell Perm Buffer, 0.4 uL EDTA (2 mM final) + +### 2. Bead Activation +1. Gently resuspend the ConA Beads by pipetting. Transfer 11 uL/sample to a 1.5 mL tube for batch processing. + - 6 samples: Total 66 uL +2. Place the tube on a magnet until slurry clears and pipette to remove supernatant. +3. Add 100 uL/sample cold Bead Activation Buffer to the dried beads, and mix gently. Repeat two washes. +4. Resuspend in 11 uL/sample cold Bead Activation Buffer. +5. Aliquot 10 uL/sample of activated bead slurry into separate 8-strip tubes and keep on ice until needed. + +### 3. Cell Harvest and Binding to Beads +1. Harvest 0.5 million cells/sample in 1.5 mL tube. Centrifuge at 600 x g, 3 min at RT. Decant or pipette culture media supernatant. +2. Resuspend cells in 100 uL/sample RT Wash Buffer. Pipette to thoroughly resuspend. Centrifuge at 600 x g, 3 min at RT. Decant or pipette supernatant. Repeat wash, decant supernatant. +3. Resuspend cells in 105 uL/sample RT Wash Buffer and thoroughly pipette to mix. Aliquot 100 uL washed cells to each 8-strip tube containing 10 uL of activated beads. Vortex and/or pipette until evenly resuspended. +4. Incubate cell-bead slurry on benchtop for 10 min at RT to adsorb cells to beads. + +### 4. Antibody Binding +1. Add 50 uL cold Antibody Buffer to each sample, vortex/pipette mix. +2. Add 2 uL CUTANA H3K4 MetStat Spike-in Control dNucs to positive (H3K4me3) and negative (IgG) control antibodies. +3. Add 0.5 ug antibody (FLAG, V5, IgG, H3K4me3) to each sample and gently vortex. +4. Incubate 8-strip tubes on nutator overnight at 4°C and store Cell Permeabilization Buffer at 4°C overnight for Day 2. + +--- + +## Day 2 - Jan 15, 2023 +### 5. Antibody Binding (Continued) +1. Fill multi-channel pipette reservoir with Cell Permeabilization Buffer. +2. Place the 8-strip tubes on magnet until slurry clears. Pipette and remove supernatant. +3. Add 200 uL cold Cell Permeabilization Buffer directly to beads, pipette to remove supernatant. Repeat two washes. +4. Add 50 uL cold Cell Permeabilization Buffer to each sample, vortex and disperse clumps. + +### 6. Binding of pAG-MNase +1. Add 2.5 uL pAG-MNase (20X stock) per sample and mix gently. +2. Incubate for 10 min at RT. Place on magnet, remove supernatant. +3. Add 200 uL cold Cell Permeabilization Buffer and repeat two washes. + +### 7. Targeted Chromatin Digestion and Release +1. Add 1 uL 100 mM Calcium Chloride per sample for digestion. +2. Incubate 8-strip tubes on nutator for 2 hours at 4°C. +3. Add 33 uL Stop Buffer per sample. Mix gently and incubate with 1 uL DNase free water-diluted Spike-in DNA at 37°C for 10 min in thermocycler. +4. Place on magnet, transfer supernatant to 1.5 mL tubes and discard ConA Beads. + +### 8. DNA Purification +1. Add 420 uL DNA Binding Buffer per sample. Mix well. +2. For every sample, place a DNA Cleanup Column into a DNA Collection Tube. Load sample, centrifuge at 16,000 x g, 30 sec, RT. Discard flow-through. +3. Add 200 uL DNA Wash Buffer, centrifuge at 16,000 x g, 30 sec. Repeat wash. +4. Elute DNA with 12 uL DNA Elution Buffer. Incubate 5 min, centrifuge at 16,000 x g, 1 min, RT. +5. Quantify DNA with Qubit fluorometer. Store at -20°C. + +--- + +## Day 3 - Jan , 2023 +### 9. NGS Library Preparation +Measure DNA concentration using Nanodrop. +Blank: DNA Elution Buffer 1 uL + +--- + +## Terms and Definitions +**Pre-Wash Buffer**: A buffer to wash cells before lysis or further processing. Often includes salts and a buffer agent. + +**Protease Inhibitor**: Compounds added to samples to prevent proteases from degrading proteins. Roche is a common vendor. + +**Spermidine**: A polyamine used to stabilize DNA and RNA structures in buffers. + +**Digitonin**: A detergent that selectively permeabilizes the plasma membrane for biochemical assays. + +**EDTA**: A chelating agent that binds divalent cations like Ca²⁺ and Mg²⁺, which are essential cofactors for DNases and other nucleases. + +**ConA Beads**: Concanavalin A-coated beads used in cell sorting and protein purification. + +**pAG-MNase**: A fusion protein of Protein A and micrococcal nuclease (MNase) used in CUT&RUN for targeted DNA cleavage. + +**H3K4me3**: A specific histone modification associated with active transcription that is often used as a marker in chromatin studies. + +## Alternative Methods or Supplies +If specific reagents (e.g., ConA beads) are difficult to source, alternative magnetic beads coated with a similar lectin (e.g., WGA beads) can be used. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/cyanobacteria-growth-english-c66qzhdw.md b/markdown-output/cyanobacteria-growth-english-c66qzhdw.md new file mode 100644 index 0000000000000000000000000000000000000000..d021404ba65425430770ac4bdabb48df96ee63b6 --- /dev/null +++ b/markdown-output/cyanobacteria-growth-english-c66qzhdw.md @@ -0,0 +1,221 @@ +```markdown +# Cyanobacteria Growth (English) + +## Goal/Experiment: +The goal of this experiment is to provide a comprehensive protocol for cultivating cyanobacteria in saline aquatic environments. This protocol includes detailed information on solution compositions, fundamental calculations, and the required volumes for executing the procedure successfully. It is crucial to note that growth rates may exhibit considerable interspecific variations among different strains of cyanobacteria. + +### Authors: +- Gabriela de Assis +- Ricardo M. Borges¹ +- Ferreira¹ +- Fernanda Chagas¹ +- pauloihc¹ + +¹UFRJ, LAABio-IPPN-UFRJ + +### Date: +January 09, 2024 + +## Abstract +This document aims to present a comprehensive protocol for the cultivation of cyanobacteria in saline aquatic environments. The protocol incorporates detailed information on the composition of solutions, fundamental calculations, and required volumes for the successful execution of the entire procedure. It is pertinent to note that growth rates may exhibit considerable interspecific variations among different strains of cyanobacteria. + +## Safety Warnings +### Autoclave: +- **Personal Protection**: Always wear appropriate protective clothing, such as an apron, gloves, safety goggles, and closed shoes when operating the autoclave. +- **Work Conditions**: Ensure that the work area is well-ventilated and has easy access to emergency showers and eye wash stations. +- **Cooling**: After autoclaving, wait for the cooling cycle to complete before opening the autoclave. High pressure and temperature can cause explosions if the process is not properly completed. +- **Caution with Steam**: Be aware of the hot steam released when the autoclave is opened. Keep your face and hands away from the opening. +- **Maintenance**: Ensure that the autoclave is in good working condition and has undergone regular maintenance. + +### Chemicals: +- **Safe Handling**: Use appropriate gloves and exercise caution when handling chemicals. Avoid direct contact with skin and eyes. +- **Filtration**: When using filters, follow guidelines for the safe disposal of contaminated filters. +- **Storage**: Store all chemicals according to safety regulations and in designated chemical storage areas. + +### Cotton Plugs: +- **Careful Handling**: Handle cotton plugs carefully and ensure there are no chemical residues or contaminants on them. +- **Careful Autoclaving**: When autoclaving cotton plugs, follow autoclave instructions carefully to avoid overheating or explosions. +- **Proper Drying**: Ensure that the cotton plugs are completely dry before using them in Erlenmeyer flasks. + +### General: +- **Proper Procedures**: Strictly follow all the steps and procedures described in the protocol. Do not take shortcuts and avoid improvisation. +- **Training**: Individuals performing these procedures should be trained in laboratory safety and familiar with the associated risks. +- **Emergency Plan**: Be aware of the laboratory's emergency plan and know how to act in case of accidents. +- **Safe Disposal**: Ensure to dispose of chemical wastes and biological materials according to local regulations and laboratory guidelines. +- **Documentation**: Keep accurate records of all steps performed, including dates and relevant details. + +**Note**: Safety precautions may vary according to local regulations and the level of risk associated with the work performed in the laboratory. Therefore, it is essential to consult the specific safety guidelines of your workplace and follow best safety practices at all times. + +## Preparation Prior to Cultivation + +1. **Determine Flask Quantities**: + - **For X samples**, prepare them in quintuples (multiply by 5). + - **Prepare the External Quality Control Strain (CCRM0280)** also in quintuples. + - **Include 2 blank culture medium samples** in Erlenmeyer flasks, treated identically with medium and nutrients, but without the inoculation step. + + As an example, if comparing 3 different species, you would need a total of (3x5)+5+2=22 flasks. + +2. **Fill Out the Metadata File**: + - Sample codes + - Strain codes + - Species + - Culture media + - Start date + - Dates of nutrient addition + - Biomass collection date + - Extraction method 1 (medium polarity: Dichloromethane-Methanol) + - Extraction method 2 (high-polarity: Methanol-Water) + - Comments + + Remember to follow these steps for a complete and accurate record of the information related to the experiment. + +## Material + +3. **Required Materials**: + - 500 mL Erlenmeyer flasks + - 250 mL and 1-liter Scotch bottles + - 50 mL Falcon-type centrifuge tubes + - Sterile loops + - Reagents for preparing stock solutions (see below) + - Refrigerated Benchtop Centrifuge (Hettich Model 320R, Tuttlingen, Germany) + - SpeedVac (Christ model RVC 2-25, Osterode am Harz, Germany) at LabMeta - Chemistry Institute - UFRJ + - Lyophilizer (model L120, Liotop, Brazil) + +4. **Preparation of Stock Solutions**: + - **Stock Solution of NaNO3**: + - Add 7.5 g of NaNO3 (NEON, p# 01813) to 100 mL of distilled water + - Store the solution under refrigeration + + Use: 1 mL for each liter of medium. + + - **Stock Solution of NaH2PO4·H2O**: + - Add 0.5 g of NaH2PO4·H2O (NEON, p# 01415) to 100 mL of distilled water + - Store the solution under refrigeration + + Use: 1 mL for each liter of medium. + + - **Trace Metal Stock Solution**: + - Add the following components: + - 23 mg of ZnSO4·7H2O + - 152 mg of MnSO4·H2O + - 7.3 mg of Na2MoO4·2H2O + - 14 mg of CoSO4·7H2O + - 6.8 mg of CuCl2·2H2O + - 4.6 g of Fe(NH4)2(SO4)2·6H2O + - 4.4 g of Na2EDTA·2H2O + - Top up the volume with distilled water to 1 liter. + - Store the solution under refrigeration. + + Use: 1 mL for each liter of medium. + + - **Vitamin Stock Solution**: + - Add the following components: + - 200 mg of Thiamine + - 10 mL (0.1 g) of Biotin + - 1 mL (1 g/L) of Cyanocobalamin + - Top up the volume with distilled water to 1 liter. + - Store the solution under refrigeration. + + Use: After autoclaving the medium, add 1 mL for each liter of medium. + + - **Preparation of Nutrient Stock Solution**: + - Mix: + - 20 mL of NaNO3 Stock Solution + - 20 mL of NaH2PO4·H2O Stock Solution + - 1 mL of Vitamin Stock Solution + - 1 mL of Trace Metal Stock Solution + - Filter using a 0.22 µm filter + - Store the solution under refrigeration + +5. **Preparation of Cotton Plugs**: + - Materials needed: hydrophobic cotton, gauze, scissors, string. + - Cut a piece of gauze the size of the palm of your hand. + - Open the gauze and insert part of it into the flask. + - Place the hydrophobic cotton inside the gauze that is in the mouth of the flask. + - Continue inserting cotton until, when removing the gauze with the cotton from the flask, a "puff" sound is heard (indicating the vacuum caused by the Plug). + - Cut a piece of string. + - Tie the gauze with the string, ensuring that it is well above the cotton (without leaving space). + - Cut off the excess string and gauze. + - Check if the Plug is slightly above the mouth of the glass. + - If necessary, adjust its position. + - Autoclave: 15 minutes at 121°C. + - Dry the flask capped with the Cotton Plugs in an oven for 8-12 hours at 70°C. + + Ensure that each step is followed precisely to ensure the quality of the culture medium and subsequent procedures. + + Cotton Plugs are reused from one cultivation to the next. At the end of cultivation, it is sufficient to dry them in an oven to prevent the proliferation of fungi. + +6. **Preparation of F/2 Culture Medium**: + - For 1 liter of distilled water, add: + - 41.5 grams of marine salt for aquarium (Reef Salt) + - 1 mL of NaNO3 Stock Solution + - 1 mL of NaH2PO4·H2O Stock Solution + - 1 mL of Trace Metal Stock Solution + - Autoclave: 15 minutes at 121°C + - Wait for the medium to cool down. + - Add 0.5 mL of the Vitamin Stock Solution before use. + +## Preparation Prior to Cultivation + +11. Separate all the 500 mL Erlenmeyer flasks + - Wash them with 5% Extran. + - Rinse them thoroughly with distilled water. + + It's important to consider the number of strains and replicates to be cultivated. + +12. Prepare the Cotton Plugs to seal the cultures. + +13. Autoclave the previously washed flasks with the Cotton Plugs + - Autoclave: 15 minutes at 121°C + - Dry the flask capped with the Cotton Plugs in an oven for 8-12 hours at 70°C. + +14. Autoclave the flasks containing the F/2 culture medium + - For 22 flasks: 22 x 200 ml = 4.4 liters (rounded up to 5 liters). + - Prepare the solution in a 5-liter flask and divide into 1-liter Schott bottles. + - Autoclave: 15 minutes at 121°C. + +15. After the culture medium has cooled, add 0.5 mL of vitamin solution (previously filtered through a syringe filter: 0.22 µm) + - Note: Do not use the 0.45 µm syringe filter. + +16. Label each Erlenmeyer flask with tags containing a code (traceable in the project's sample list) and the production date. + - The production dates for each specimen, addition of nutrient solution, and biomass collections should be documented in the project's sample list. + - Ensure that each step is performed meticulously to ensure a proper start to the cultivation and reliable tracking of subsequent processes. + +## Inoculation + +17. Using sterile loops, proceed to collect biomass from a previously prepared cultivation. + - Quantity: a "tuft" approximately the size of a 50-cent coin. + - It is imperative to carry out all operations using disposable gloves and to minimize exposure to airflow. + - Properly seal the Erlenmeyer flasks with the Cotton Plugs prepared earlier. + +## Cultivation Maintenance + +18. **Nutrient Stock Solution**: + - To maintain the cyanobacteria in the logarithmic phase, it is essential to provide frequent feeding through the addition of the previously prepared Nutrient Stock Solution. + - For each cultivation, it is recommended to add 200 μL of the Nutrient Stock Solution. + +The need for nutrients in cyanobacteria can be visualized by their characteristic green color. + +## Biomass Collection and Treatment + +19. The cultivation period is set at: + - 28 days. + - With the addition of the Nutrient Stock Solution at every 7-day interval. + +20. At the end of the 28 days, from each Erlenmeyer flask, an amount corresponding visually to 80-90% of the biomass will be collected. + - We will allow each specimen to continue its growth until it becomes necessary to interrupt it. + - Initially, we can set a limit of 4 months for each specimen. + +21. Transfer the culture medium from each Erlenmeyer flask with the biomass to a properly labeled 50 mL conical centrifuge tube (Falcon type), with the label corresponding to the reference code in the sample list. + - Centrifuge: 3011 xg (4500 rpm) for 15 minutes at 4°C + - Discard the supernatant + - Continue transferring the contents of each Erlenmeyer flask, centrifuging and discarding the supernatant until complete elimination of all the aqueous content + - Record the volume of biomass obtained (photo for file) + +22. Subject each properly identified sample to a lyophilization process until dry. + - Each sample should be separately identified in clean, semi-screwed Falcon tubes (to prevent cross-contamination of the samples). + - Insert pertinent details and observations in the Metadata. + - Always store in a freezer at -20°C. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/cyanobacteria-total-lipid-extraction-ibkcakw.md b/markdown-output/cyanobacteria-total-lipid-extraction-ibkcakw.md new file mode 100644 index 0000000000000000000000000000000000000000..06c4dc56fc6a4be7bc89f1fd9a19e72829d6bf7e --- /dev/null +++ b/markdown-output/cyanobacteria-total-lipid-extraction-ibkcakw.md @@ -0,0 +1,199 @@ +```markdown +Goal/Experiment: +The experiment aims to extract total lipids from cyanobacteria for subsequent analysis, potentially by Liquid Chromatography-Mass Spectrometry (LCMS). The procedure is specified for both lab-grown cultures and liquid environmental cultures, with a separate variation for Sterivex filtered environmental samples. + +# Cyanobacteria Total Lipid Extraction + +*Dr. Steven Wilhelm, Maddie Denney* + +## Abstract +Please contact Dr. Steven Wilhelm (wilhelm@utk.edu) or Maddie Denney (mdenney5@vols.utk.edu) for additional information regarding this protocol. + +Modified from Guan, Riezman, Wenk & Riezman, 2010 + +Please note that there are two versions of this protocol. Use the one that corresponds to your sample. + +## Protocol + +### Version 1 - Lab Grown Cultures and Liquid Environmental Cultures + +#### Step 1 +Prepare lipid extraction solvent (step 2) with a 1:1 ratio of butanol and lab purified water (water-saturated butanol). + +**Reagents:** +- Butanol 71-36-3 by [Fischer Scientific](https://www.fishersci.com/) + +**Annotations:** +- This is written incorrectly. Should state: "Prepare lipid extraction solvent (step 2), 1:1 ratio butanol and lab purified water (water-saturated butanol)." + +#### Step 2 +Prepare extraction solvent with a 15:15:5:1:0.18 ratio by volume solvent composed of 95% ethanol, water, diethyl ether, pyridine, and 4.2 N ammonium hydroxide. + +**Reagents:** +- Ethanol BE-BDH1156 by [P212121](https://p212121.com/) +- Diethyl ether 60-29-7 by [Fischer Scientific](https://www.fishersci.com/) +- Pyridine 110-86-1 by [Fischer Scientific](https://www.fishersci.com/) +- Ammonium hydroxide 7664-41-7 by [Fischer Scientific](https://www.fishersci.com/) + +#### Step 3 +Grow cultures to log phase and collect 2 mL in 2 mL microfuge tube (for environmental samples, use a minimum of 500 µL). + +**Amount:** +- 2 ml + +**Notes:** +- If the sample is a liquid environmental sample, simply proceed without a growth period. + +#### Step 4 (Optional) +Remove a small aliquot (100 µL) of culture and fix with 5% by volume glutaraldehyde for later cell counts for normalizing lipid amounts. Store at 20ºC. + +**Reagents:** +- 25% Glutaraldehyde + +#### Step 5 +Pellet culture at 10,000 xg for 2 minutes. + +**Duration:** +- 2 minutes + +#### Step 6 +Remove supernatant. + +#### Step 7 +Add 1 mL extraction solvent to resuspend pellet. + +**Amount:** +- 1 ml + +#### Step 8 +Add 100 µL of 150 µm glass beads in a 1 dram vial. + +**Amount:** +- 100 µL + +**Notes:** +- Ensure proper vial dimensions. + +#### Step 9 +Vortex sample for 5 seconds. + +#### Step 10 +Incubate sample in a 60ºC water bath for 20 minutes. + +**Duration:** +- 20 minutes + +#### Step 11 +Centrifuge sample at 10,000 xg for 10 minutes in a benchtop microcentrifuge. + +**Duration:** +- 10 minutes + +#### Step 12 +Remove supernatant to a glass vial. + +**Notes:** +- All extractions for a single sample will be added to the same sample glass vial. + +#### Step 13 +Repeat steps 7-12; DO NOT add more glass beads. + +#### Step 14 +Add 150 µL water to microfuge tube. + +**Amount:** +- 150 µL + +#### Step 15 +Vigorously shake water-saturated butanol (1:1 ratio) to mix and add 300 µL to microfuge tube. + +**Amount:** +- 300 µL + +**Reagents:** +- Butanol 71-36-3 by [Fischer Scientific](https://www.fishersci.com/) + +#### Step 16 +Vortex sample for 5 seconds. + +#### Step 17 +Remove top butanol phase to sample glass vial. + +#### Step 18 +Repeat steps 15-17. + +#### Step 19 +Either immediately dry sample under N₂ for LCMS or store overnight at -20ºC or longer-term storage at -80ºC. + +### Version 2 - Sterivex Filtered Environmental Sample + +#### Step 20 +Prepare lipid extraction solvent as in steps 1-2 of version 1 above. + +#### Step 21 +Use a Sterivex cutter to open the plastic unit. Using a sterile razor blade, cut filter off the unit and cut into small pieces. Place filter pieces in a 2 mL microfuge tube. + +#### Step 22 +Add 1 mL of extraction solvent to resuspend pellet. + +**Amount:** +- 1 mL + +#### Step 23 +Add 100 µL of 150 µm glass beads. + +**Amount:** +- 100 µL + +#### Step 24 +Vortex sample very vigorously, 5 seconds -- do not want filter pieces to settle at the bottom of microfuge tube. + +#### Step 25 +Incubate sample in a 60ºC water bath for 20 minutes. + +**Duration:** +- 20 minutes + +#### Step 26 +Centrifuge sample at 10,000 xg for 10 minutes in a benchtop microcentrifuge. + +**Duration:** +- 10 minutes + +#### Step 27 +Remove supernatant to a glass vial. + +**Notes:** +- All extractions for a single sample will be added to the same glass vial. + +#### Step 28 +Repeat steps 22 and 24-27; DO NOT add more glass beads. + +#### Step 29 +Add 150 µL water to microfuge tube. + +**Amount:** +- 150 µL + +#### Step 30 +Vigorously shake water-saturated butanol to mix (1:1 ratio) and add 300 µL to microfuge tube. + +**Amount:** +- 300 µL + +#### Step 31 +Vortex sample for 5 seconds. + +#### Step 32 +Remove top butanol phase to sample glass vial. + +#### Step 33 +Repeat steps 30-32. + +#### Step 34 +Either immediately dry the sample under N₂ for LCMS or store overnight at -20ºC or longer-term storage at -80ºC. + +--- + +**endofoutput** +``` \ No newline at end of file diff --git a/markdown-output/cytof-staining-dfjb3kin.md b/markdown-output/cytof-staining-dfjb3kin.md new file mode 100644 index 0000000000000000000000000000000000000000..db1f98242776f8fef830dc24d98e57faa348af4a --- /dev/null +++ b/markdown-output/cytof-staining-dfjb3kin.md @@ -0,0 +1,126 @@ +```markdown +# Goal/Experiment: +This experiment aims to provide a comprehensive protocol for CYTOF (Cytometry by Time-Of-Flight) staining, a powerful technique for single-cell immunological assays that enables detailed and simultaneous analysis of diverse immune cell populations. + +# CYTOF Staining + +### DOI +[https://dx.doi.org/10.17504/protocols.io.14egn69nml5d/v1](https://dx.doi.org/10.17504/protocols.io.14egn69nml5d/v1) + +### Authors +- Meelad Amouzgar¹ +- Patricia Favaro¹ +- Daniel Ho¹ +- Trevor Bruce¹ +- Kausalia Vijayaragavan¹ +- Sean Bendall¹ + +¹Stanford University + +### Institution +Stanford University + +### Correspondence +Bangelo lab protocol development +Technical support email: mbosse@stanford.edu + +## Abstract +CYTOF has significantly advanced immunophenotyping, especially in exploratory settings where comprehensive characterization of immune populations is necessary but sample sizes are limited. It is a powerful tool for single-cell immunological assays, particularly for the detailed and simultaneous analysis of diverse immune cell populations. By using metal isotopes instead of fluorescent labels, mass cytometry eliminates the spectral overlap issues seen in optical flow cytometry, allowing robust analysis of around 60 individual parameters simultaneously. The technique involves increased complexity in the design, execution, and interpretation of experiments. + +## Reagents +1. Paraformaldehyde (PFA) 16% (Electron Microscopy Sciences – catalog number 15710) +2. Cisplatin Cell-IDTM Cisplatin-198Pt—100 μL (#201198 standard biotools) +3. 1x Low-Barium PBS +4. BSA +5. Sodium Azide +6. Cell Suspension Media (CSM) + +## Reagent Preparation + +### Cisplatin-198Pt Preparation +- **Storage:** 100mM stock Cisplatin-198Pt is stored at -20 °C. +- **Procedure:** + 1. Prepare 0.5μM working stock solution of Cisplatin-198Pt by carrying out two dilutions: + - Add 1μL of 100mM Cisplatin-198Pt stock + 199μL of Low-Barium PBS = 0.5mM (500μM) Cisplatin-198Pt. + - Add 1μL of 0.5mM (500μM) Cisplatin-198Pt + 999μL of Low-Barium PBS = 0.005mM (0.5μM) Cisplatin-198Pt. + 2. For final live/dead cell staining with PBMCs, use 0.025μM Cisplatin-198Pt/1 million cell suspension. + +- **Note:** Cisplatin concentration used does not reach saturation. So 0.5μM is adequate for 1-10 million cells. Cisplatin staining intensity increases with cell size (monocytes > lymphocytes, neurons > astrocytes > microglial). Cisplatin binds covalently to any proteins in dead/dying cells. + +#### Cisplatin Concentrations + +| Stock (mM) | Dilution 1 | Working Conc (μM) | Dilution 2 | Final (μM) | +|------------|------------|-------------------|------------|------------| +| 100 | 0.005 | 500 | 0.001 | 0.5 | + +### Cell Suspension Media (CSM) +- **Ingredients:** + - 1 L of 1x Low-Barium PBS + - 5g of BSA + - 200 mg of sodium azide +- **Procedure:** + 1. Mix ingredients in an appropriate container. + 2. Filter using 2x 500 mL vacuum filter unit. + +## CYTOF Staining + +### PFA Fixation +1. Add 100 μL of 16% PFA per 1 mL of media and incubate cells for 10 minutes at room temperature. +2. Wash with 4 mL of CSM 2x. +3. Pellet the cells by centrifugation at 600 x g for 5 minutes. +4. Store fixed cells in 1 mL of CSM at -80°C. + +### Barcoding Protocol +1. Transfer cells to a cluster tube (if not already). +2. Wash 1x with cold PBS, spin 5’ 600g 4°C, aspirate, vortex to re-suspend pellet. +3. Wash 1x with 1 mL cold PBS-S, spin 5’ 600g 4°C, aspirate, vortex to re-suspend pellet. +4. Cut barcode plate cover to expose only the barcode wells to be used (keep cover on until ready to pipette). +5. Using a multichannel pipette: + 1. Remove 100 μL cold PBS-S from the reservoir. + 2. Transfer the 100 μL of now PBS-S + Barcode to the corresponding sample tubes, mix up & down several times (final sample + barcode volume ~180 μL). + 3. After 15 minutes, add 700 μL CSM to quench, spin 5’ 600g 4°C, aspirate, and wash 1X with 1 mL CSM. + 4. Aspirate & re-suspend cell pellets by vortex. + 5. Pool all barcoded cells into the FACS tube for staining. + +### Surface Staining +1. 100 μL total staining, bring volume to 55 μL. +2. Add 5 μL of FC block (Human) and incubate for 10’ at RT in the shaker. +3. Make surface antibody cocktail (~40 μL total) and add to cells (100 μL total). +4. Incubate for 30’ at RT in the shaker. +5. Wash cells with 4 mL of CSM 600 x g for 5 minutes. + +### Permeabilization with Methanol +1. Add 1 mL of 4°C 100% methanol (keep on ice) (slowly). +2. Incubate for 10’ on ice. +3. Add 3 mL of CSM. +4. Pellet at 600 x g for 5 minutes. +5. Wash more 2 times. Vortex the pellet and add the panel to a final volume of 100 μL (1x test). + +### Intracellular Staining +1. Filter the intracellular antibody cocktails before adding to the cells. +2. 10,000g 1 minute prewet with CSM (if volume is not too small. 20 μL is very little). +3. Add the intracellular staining. +4. Incubate for 30’ at RT. +5. Add 3 mL of CSM. +6. Pellet at 600 x g for 5 minutes. + +### DNA Intercalator +**Iridium Intercalator: DNA1/DNA2 = Ir191/Ir192** + +**Procedure:** +1. Prepare 5x DNA intercalator solution: + - Add 1 mL of DNA intercalator solution to each tube and mix. + - Incubate either for 30 minutes at Room temperature to run at cytot the same day or overnight at 4°C. + - Store in fridge for ~a week in the intercalation solution. + +### Day of CyTOF Run +1. Wash 1x CSM and 2x Water and run on CyTOF. 600 x g/5 minutes. +2. Dilute EQ beads 1:10 in ddH2O and add 1 mL of diluted beads per 1 million cells. Usually, 1.5 mL of water/beads per tube and run 500 μL in one push at CyTOF. It is enough to visualize the titration. If you think you need more, run another push and concatenate the data afterwards. + +### Long-term Storage +1. Wash with 3 mL CMS and spin down 600 x g/5 minutes. +2. Re-suspend in CSM+10% DMSO, and put right away in -80°C. +3. You can keep it there for months if needed. Once ready, thaw and wash 1 time with CSM and 2 times with ddH2O and acquire at 600 x g/5 minutes. + +endofoutput +``` diff --git a/markdown-output/deep-brain-stimulation-dbs-implant-b9uer6te.md b/markdown-output/deep-brain-stimulation-dbs-implant-b9uer6te.md new file mode 100644 index 0000000000000000000000000000000000000000..9342e05d0d641d0804fd2a4a5a3d4f19d15d1fbf --- /dev/null +++ b/markdown-output/deep-brain-stimulation-dbs-implant-b9uer6te.md @@ -0,0 +1,102 @@ +```markdown +# Goal/Experiment: +To describe the steps for making implants used for deep brain stimulation (DBS) in rodents. + +# Deep Brain Stimulation (DBS) Implant + +**Author:** Alexandra Nelson +**Institution:** University of California San Francisco + +## Abstract + +This protocol describes the steps for making implants used for deep brain stimulation (DBS) in rodents. + +## DOI + +[dx.doi.org/10.17504/protocols.io.b9uer6te](https://dx.doi.org/10.17504/protocols.io.b9uer6te) + +## Protocol Citation + +Alexandra Nelson 2022. Deep Brain Stimulation (DBS) Implant. protocols.io +[https://dx.doi.org/10.17504/protocols.io.b9uer6te](https://dx.doi.org/10.17504/protocols.io.b9uer6te) + +## Manuscript Citation + +Jonathan S Schor, Isabelle Gonzalez Montalvo, Perry WE Spratt, Rea J Brakaj, Jasmine A Stansil, Emily L Twedell, Kevin J Bender, Alexandra B Nelson (2022) Therapeutic deep brain stimulation disrupts movement-related subthalamic nucleus activity in Parkinsonian mice eLife 11:e75253 +[https://doi.org/10.7554/eLife.75253](https://doi.org/10.7554/eLife.75253) + +## Keywords + +Mouse, Deep Brain Stimulation, Implant, Device, ASAPCRN + +## License + +This is an open access protocol distributed under the terms of the [Creative Commons Attribution License](https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. + +**Created:** May 23, 2022 +**Last Modified:** Jul 27, 2022 + +--- + +## Protocol Integer ID + +63078 + +## Twisting Wires Together + +### Materials Required +1. Gather + - Ruler + - Wire [A-M Systems #791000, .003' bare, .0055 coated, half hard, 100ft roll] (red top box, located on shelf above electronics bench) + - Small scissors + +### Procedure +2. For each electrode, take 3 wires of **13 cm**. + - **2.1** To cut wires into 13 cm, use scissors. If the tip of the wire is bent, cut it as well to ensure the wire is straight. Make many wires (~18). + - **2.2** Take a bundle of 3 and run fingers down the fiber to remove dust. + - **2.3** Line up tips on one end and hold with fingers, then fold the wires in half bringing the ends to touch and align into a tight bunch of 6 termini. + - **2.4** Pinch with a bulldog clip (green clip in Image 1) attached to a long rod (grey rod with yellow adhesive in Image 1) on the long axis and reinforce with a larger clip (black clip in Image 1). + +![Twisting wires with tetrode spinner](image1.png) + +### Continued Procedure +3. Hang on the pole upside down, putting the loop around the pole. The loop should hang 2 cm back from the end of the pole. +4. Twist counterclockwise until the wires are twisted/joined ½ cm from the top. Untwist slightly rotating clockwise, then twist again to ½ cm. +5. Use a heat gun to heat the bundled portion of the wire, slowly and smoothly working up and down for 1 min. Let cool for 30 s before removing (Heat gun 800°F, 4 fan bars). + +## Fitting into 6-pin Connector + +6. Cut the top of the loop with scissors to form 6 endings. +7. Use a razor blade to carefully scrape insulation off the wires from the end to the top of the twist. + +![Scrape here](image2.png) + +8. Separate into 2 sets of 3. +9. Check for shorts with a multimeter. + - **9.1** If there is a short, attack from both ends: + - Try trimming the bottom of the twisted wires in case the short is occurring down there. + - If the short still exists, pull apart your wires that have been stripped even further. If that fixes the problem, remove insulation down to that point again. + - Retest for shorts. + +## Fitting the Wires into the Millmax + +10. To make the millmax a "6-pin" connector, you need to cut the longer strip of female millmax: + - **10.1** Use wire cutters to cut on the 4th pin over and save those pins (in case you lose one or damage the pins when cutting). + - **10.2** Use a hemostat to remove gold pins from the millmax. You can also take a needle and push on the bottom of the millmax to push the pin up for better grip. Then pull out using a hemostat. It’s best to go vertical, or in a straight line out, minimizing side-to-side fudging. + - **10.3** Cut the skinnier portion off each gold pin using a wire cutter. Keep the thicker side for pressure fitting (see below). + +![Use for pressure fitting](image3.png) + + - **10.4** Once all 6 pins are removed, feed wires into the millmax: + - First put in one vertical side of 3, then put in the 3 on the other side. + - Then fold the wires that you threaded through all to one side forming a “pony-tail” ensuring that these wires do not touch each other or tangle during pressure fitting. + +![Pressure fit these endings into the millmax](image4.png) + + - **10.5** Trim the bundled end of wires to your desired length. Do this at an angle so that you get wires ending at varied DV levels when you insert the electrodes. + - For STN DBS we trim the insertable portion to 6-6.5 mm. + +![Final length trimming](image5.png) + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/dental-calculus-field-sampling-protocol-sabin-vers-bqecmtaw.md b/markdown-output/dental-calculus-field-sampling-protocol-sabin-vers-bqecmtaw.md new file mode 100644 index 0000000000000000000000000000000000000000..a00213775ab9bdb09ec498f2e8e1f45433450063 --- /dev/null +++ b/markdown-output/dental-calculus-field-sampling-protocol-sabin-vers-bqecmtaw.md @@ -0,0 +1,109 @@ +```markdown +## Goal/Experiment: +This protocol describes the process of sampling dental calculus from skeletal remains for biomolecular analysis. This is particularly recommended for sampling calculus from teeth attached to a jawbone, skull, or skeleton. The primary use-case is for DNA and proteomic analysis. + +# Dental Calculus Field-Sampling Protocol (Sabin version) V.2 + +### Authors +- Susanna Sabin +- James A Fellows Yates + +**Affiliation**: Max Planck Institute for the Science of Human History + +**Published**: Dec 04, 2020 + +**DOI**: [https://dx.doi.org/10.17504/protocols.io.bqecmtaw](https://dx.doi.org/10.17504/protocols.io.bqecmtaw) + +**Keywords**: +- ancient DNA +- dental calculus +- sampling +- archaeogenetics +- archaeology +- biomolecular archaeology +- skeleton +- proteomics + +### Abstract +This protocol describes how to sample dental calculus from skeletal remains for biomolecular analysis. This protocol is particularly recommended for sampling calculus from teeth attached to a jaw bone, skull, or skeleton. The primary use-case is for DNA and proteomic analysis. + +### External Links +[Warinner Group Resources](http://christinawarinner.com/resources/archaeologists/) + +#### License +This is an open access protocol distributed under the terms of the [Creative Commons Attribution License](https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. + +### Materials + +#### Essential +- Microcentrifuge tubes (1.5 or 2 ml, screw-cap or safe-lock) +- Disposable nitrile gloves (sterile, powder-free) +- Aluminum foil +- Weigh paper + +#### Reagent Sources +- Alcohol wipes or household Bleach (1:10 dilution) +- Lab-grade water (HPLC grade) +- Paper towels +- Dental scalar +- Sharpie (for Tube/Bag labeling) +- Biro Pen (for Metadata sheet writing) +- Metadata sheet (includes Sampling ID, Collection ID, Collection Storage ID, Tooth FDI Code, Surface, Pathologies, Consolidants, Other Notes) +- Camera + +#### Optional +- Copy of a picture-based short-version sampling protocol +- Sample Bags +- Weigh boats +- Personal protective gear (e.g. face mask) +- Tooth ID diagram (with FDI codes) + +### Guidelines +- Sterility: Use sterile equipment to reduce contamination. +- Gloves: Use nitrile, powder-free gloves. Avoid latex gloves as they interfere with proteomic and archaebotanical analyses. + +### Safety Warnings +- Bleach can irritate eyes and skin. Have clean water or eye wash available for emergencies. +- Dental scalars can be sharp. Handle with care. +- Calculus deposits may flick off suddenly. Protect your eyes. + +### Workstation Preparation +1. Put on two pairs of gloves (replace the outer layer after each sampling). +2. Cover the work surface with two layers of aluminum foil (replace the top layer after contact with sampling debris). +3. Sterilize all utensils using alcohol wipes or bleach solution (1:10 dilution with HPLC water). +4. Replace top layer of your gloves. + +### Sampling Preparation +5. Label the tube ready for sampling. +6. Fill in your metadata sheet with corresponding tube IDs. +7. Label all bags for placing corresponding tubes. +8. Lay out a fresh sheet of weighing paper. Create a gentle crease to ease specimen collection. +9. If possible, remove the tooth from the jaw and locate the dental calculus. + - Large calculus deposits obstruct the entire tooth. + - Photograph the tooth before and after sampling. +10. Write the sample name and/or label on paper. Place next to the weighing paper. +11. Place the tooth on weighing paper, photograph from various angles, and include a scale if possible. +12. Record the location of the calculus deposit in the metadata sheet. Note evidence of disease or consolidants if present. + +### Sampling Procedure +13. Position the specimen to scrape calculus towards the fold in weighing paper. +14. Use the dental scalar to scrape calculus downwards into the weigh paper without touching enamel. Optionally, place the weighing paper inside a weighing boat for better containment. + - Use gentle pressure initially. + - Do not use the pick of the scalar to avoid damaging enamel. +14.1. Gently tap the collected calculus into the labelled tube using the weighing paper as a guide. +14.2. If some calculus does not fall into the weighing paper, use sterile forceps to transfer it. + +### Post-Sampling Procedure +15. Securely close the tube and place it into a labelled sample bag. +16. Sterilize all used utensils with alcohol wipes or bleach. +17. Replace the top layer of your gloves. +18. Photograph the tooth again, focusing on the sampling site. +19. Record observations such as approximate calculus weight. +20. Return tooth to storage. Discard the used materials. +21. Replace gloves and repeat the process for additional samples. + +### References +- Protocol Citation: Susanna Sabin, James A Fellows Yates 2020. Dental Calculus Field-Sampling Protocol (Sabin version). protocols.io [dx.doi.org/10.17504/protocols.io.bqecmtaw](https://dx.doi.org/10.17504/protocols.io.bqecmtaw) Version created by James Fellows Yates. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/denv-2-infection-crvhv636.md b/markdown-output/denv-2-infection-crvhv636.md new file mode 100644 index 0000000000000000000000000000000000000000..533751fef67f9b932b2b1f9eb16798d40ffc2f51 --- /dev/null +++ b/markdown-output/denv-2-infection-crvhv636.md @@ -0,0 +1,180 @@ +```markdown +# Goal/Experiment: +The goal of this experiment is to describe the conditions for maintaining C6/36 HT and BHK-21 cell lines, as well as the procedure for DENV 2 infection and plaque assay. + +--- +# DENV 2 Infection + +## Authors +- Edwin Stiven Quiguanás¹ +- Delia Piedad Recalde-Reyes² + +¹Universidad del Quindío +²Corporación Universitaria Empresarial Alexander von Humboldt + +## Abstract +This protocol describes the conditions for maintaining the C6/36 HT and BHK-21 cell lines, the procedure for DENV 2 infection, and plaque assay. + +--- +## Materials + +### Equipment and Consumables +- 1.5 mL Tubes +- 2.0 mL Tubes +- 15 mL Tubes +- 50 mL Tubes +- Cryovials +- T25 and T75 Flasks +- 24-well Microplates +- Filter tips (10, 200, and 1000 µL) + +### Reagents +- **L-15 Medium**: Leibovitz's L-15 Medium with _L-glutamine_ for cell culture. +- **RPMI-1640 Medium**: RPMI medium for cell culture that contains L-glutamine and no bicarbonate. +- **Antibiotic-Antimycotic**: A solution commonly used to prevent bacterial and fungal contamination in cell cultures. +- **L-glutamine**: An essential amino acid in cell culture for protein synthesis and cell proliferation. +- **Sodium Bicarbonate**: A component to buffer the pH of the medium. +- **Fetal Bovine Serum (FBS)**: A supplement for cell culture media to provide growth factors. +- **Saline Solution**: Used for washing cells. +- **PBS 1X Buffer**: Phosphate-buffered saline solution for washing cells. + +--- +## Virus Amplification + +### Cell Line Information +Infection and amplification of DENV 2 are performed in C6/36 cells (_Aedes albopictus_ clone C6/36 CRL-1660™ ATCC) highly susceptible to Flavivirus replication. + +### Preparation of L-15 Medium +- Note: L-15 medium does not contain sodium bicarbonate. +- Each packet contains 13.7 grams for preparing 1 Liter. +- **Steps**: + 1. In a glass container, add 500 mL of sterile water and the entire content of one packet of L-15 medium. Stir using a magnetic stirrer. + 2. Once dissolved, add another 500 mL of sterile water and continue stirring. + 3. Adjust the pH to 7.6 with HCl if necessary. + 4. Sterilize the medium by filtration. + +### L-15 Medium for Initial Growth of C6/36 +Prepare 50 mL aliquots supplemented with 10% FBS. + +#### Table 1. Preparation of L-15 Medium for Initial Growth +| Reagent | Stock Concentration | Final Concentration | Volume | +|------------------|---------------------|---------------------|--------| +| L-15 | 1X | 1X | 43 mL | +| FBS | 100% | 10% | 5 mL | +| L-glutamine | 200 mM | 2 mM | 500 μL | +| Antibiotic | 100X | 1X | 500 μL | +| Trypsin Phosphate| 4X | 10% | 1.25 mL| + +--- + +### L-15 Medium for Maintaining C6/36 +Prepare 50 mL aliquots supplemented with 2% FBS. + +#### Table 2. Preparation of L-15 Medium for Maintenance +| Reagent | Stock Concentration | Final Concentration | Volume | +|------------------|---------------------|---------------------|--------| +| L-15 | 1X | 1X | 47 mL | +| FBS | 100% | 2% | 1 mL | +| L-glutamine | 200 mM | 2 mM | 500 μL | +| Antibiotic | 100X | 1X | 500 μL | +| Trypsin Phosphate| 4X | 10% | 1.25 mL| + +--- + +## Procedure for Thawing and Opening C6/36 Cell Line +1. Clean the laminar flow hood and expose all necessary materials to UV for 15-20 minutes. +2. Add 3 mL of L-15 medium supplemented with 10% FBS to a 15 mL falcon tube (refer to Table 1). +3. Retrieve the cryovial of C6/36 cells from the freezer or liquid nitrogen and thaw gently by hand. +4. Once thawed, add 1 mL of L-15 medium supplemented with 10% FBS to the cryovial, mix gently, and transfer all contents to the 15 mL tube (total volume 5 mL). +5. Centrifuge the cells at 150 g for 10 minutes at 18-20°C. +6. Discard the supernatant and resuspend the cell pellet in 5 mL of L-15 medium with 10% FBS. +7. Transfer the content to a T25 flask, label it, and incubate at 28°C without CO₂. + +--- + +## Passage of C6/36 Cells +1. Pre-warm reagents (saline solution and L-15 medium with 2% or 5% FBS) and observe cells under a microscope for morphology and confluence before passaging. +2. Remove medium from T25 flask and wash cells gently with saline solution 1-2 times. +3. Add 5 mL of L-15 medium with 2% FBS, and gently tap to detach cells from the flask without using trypsin. Transfer to a T75 flask and add 10 mL of L-15 medium with 2% FBS. Incubate at 28°C. +4. For maintenance, add fresh L-15 medium if required. + +--- + +## Cryopreservation of C6/36 Cells +1. With 90-100% confluence, prepare for freezing: + 1. Retrieve sterile cryovials and label them. + 2. Add 100 μL of DMSO (Dimethyl Sulfoxide) per cryovial. + 3. Transfer cells to a 15 mL falcon, add 5 mL of FBS and centrifuge at 800 rpm for 10 minutes. + 4. Resuspend the cell pellet in cryopreservation medium (90% FBS + 10% DMSO). + 5. Aliquot 900 μL of cells into cryovials, mix gently, and store in liquid nitrogen. + +--- + +## Infection of C6/36 Cells with DENV 2 +1. Use a T75 flask with 90-100% confluence. +2. Wash cells with saline, then add 8 mL of L-15 medium (without supplements) with 500 µL of DENV 2 (pre-thawed). +3. Incubate at 28°C for 2 hours, gently mixing every 20 minutes. +4. After incubation, add 4 mL of L-15 medium. Incubate at 28°C for 7 days. +5. Observe cells for infection and proceed with further analysis. + +--- + +## Plaque Assay of DENV 2 in BHK-21 Cells + +- **Cell Line Information**: BHK-21 cells are used with RPMI-1640 medium (L-glutamine without bicarbonate). + +### Preparation of RPMI-1640 Medium +- Note: RPMI-1640 does not contain sodium bicarbonate. +- **Steps**: + 1. Mix 500 mL of sterile water with the RPMI-1640 contents. + 2. Once dissolved, add a further 500 mL of sterile water and stir. + 3. Sterilize by filtration. + +### BHK-21 Cell Thawing and Passage +- Use same procedures as for C6/36 cells, adjusting incubation to 37°C and 5% CO₂. + +### Plaque Assay Procedure +1. Clean the laminar flow hood with 70% alcohol and expose materials to UV for 15 minutes. +2. After 24 hours of incubation, observe confluence under a microscope. +3. Discard the medium, wash wells with saline, and add 200 μL RPMI-1640 without supplements. +4. Add 200 μL of pure DENV 2 to the wells of column 1 and perform serial dilutions across the plate. +5. Incubate at 37°C, 5% CO₂ for 2 hours, occasionally mixing. +6. Post-incubation, overlay cells with preparative medium and incubate for 8 days. + +#### Table 3. Overlay Medium Preparation +| Reagent | Volume | Final Concentration | +|----------------|--------|---------------------| +| RPMI-1640 2X | 13.5 mL| 1X | +| CMC 3% | 10 mL | 1.2% | +| Antibiotic | 250 μL | 1X | +| Sodium bicarb. | 500 μL | - | +| FBS | 500 μL | 2% | + +### Post-Infection Steps +1. Perform washes to remove overlay medium. +2. Fix cells with 300 μL formaldehyde and stain with violet crystal. + +--- + +## Results and Interpretation +- **Interpretation**: + - Cell monolayer destruction due to the pure virus (column 1). + - Observations of plaque forming units (PFUs). + - Virus titer calculation in the last countable dilution. + +--- + +Protocol DOI: [10.17504/protocols.io.j8nlkwpbdl5r/v1](https://dx.doi.org/10.17504/protocols.io.j8nlkwpbdl5r/v1) + +--- + +License: [Creative Commons Attribution License](https://creativecommons.org/licenses/by/4.0/) + +--- + +Keywords: DENV, Infection, Plaque Assay + +--- + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/derisi-lab-rna-library-prep-96-well-protocol-on-ec-cpm7vk9n.md b/markdown-output/derisi-lab-rna-library-prep-96-well-protocol-on-ec-cpm7vk9n.md new file mode 100644 index 0000000000000000000000000000000000000000..a9049751f9826b335c1217e86cd747b69c6a7fea --- /dev/null +++ b/markdown-output/derisi-lab-rna-library-prep-96-well-protocol-on-ec-cpm7vk9n.md @@ -0,0 +1,348 @@ +```markdown +# Goal/Experiment: + +The goal of this experiment is to prepare high-quality sequencing libraries for metagenomic next-generation sequencing (mNGS) efficiently and cost-effectively by automating the process using the Labcyte Echo 550 acoustic liquid handler and Illumina sequencing platforms. + +# DeRisi Lab RNA Library Prep 96-well Protocol on Echo 550 + +## Authors + +- Madeline Y Mayday (Yale University) +- Miriam R Simon (UCSF School of Medicine, Department of Pediatrics) +- Lillian M Khan (UCSF School of Medicine, Department of Pediatrics) +- Eric D. Chow (UCSF School of Medicine, Department of Biochemistry & Biophysics) +- Matt S Zinter (UCSF School of Medicine, Department of Pediatrics) +- Joseph Derisi (UCSF School of Medicine, Department of Biochemistry & Biophysics) + +## Abstract + +Preparation of high-quality sequencing libraries for metagenomic next generation sequencing (mNGS) is costly and time-consuming. Reducing the overall scale and cost of mNGS library preparation is crucial. Integration of Labcyte Echo 550 for automated dispensing of reagents and utilizing sub-microliter volumes without consumable pipette tips offers significant benefits. This protocol optimizes library preparation for various sample types using ERCC spike-in RNAs, demonstrating compatibility with sequencers like Illumina iSeq and NovaSeq. + +# Guidelines + +## Pre-PCR BSL-1 Guidelines +- No amplicons allowed in this room. Thermocyclers should **NOT** be used for PCR. +- Always wear a lab coat and gloves when doing library prep or working with patient samples and isolated nucleic acid. +- Wipe down workspace with 70% ethanol and RNase-ZAP before starting work. +- Clean up lab space after work is complete. + +## Protocol Specific Guidelines +- Prepare master mix reagents in a hood of the day of use to preserve enzyme efficiency. +- Use only designated consumables and pipettes. +- Take Ampure beads out of 4°C and room temperature 30 minutes before use. + +## Equipment Guidelines + +### Vacuum Evaporators +- Varying sample dehydration times. Test minimal drying time for each, fill each 96-well PCR plate with 5 µL water and spin at 37°C-38°C until completely dry. +- Prevent excessive drying and RNA degradation by testing desired brands and using temperatures between 37°C-38°C. + +### Labcyte Echo 525 +- Ensure sufficient reagent volume for minimum and maximum working volume of source plates. +- 384 PP Plus Echo Qualified source well working volume: 20-65 µL (dead volume of ~20 µL). + +## Materials + +### Reagents +| Reagent | Vendor | Catalog Number | +|---------|--------|----------------| +| AMPure XP SPRI beads | Beckman Coulter | A63881 | +| ERCC RNA Spike-In Mix | Invitrogen - Thermo Fisher | 4456740 | +| NEBNext Ultra II RNA Library Prep Kit for Illumina | New England Biolabs | E7770S/L | +| NEBNext Adaptor for Illumina | New England Biolabs | E7337AA | +| NEB USER Enzyme | New England Biolabs | M5505S/L | + +### Plates +| Equipment | Type | Brand | SKU | Link | Specifications | +|-----------|------|-------|-----|------|----------------| +| 384-well PP 2.0 Plus Microplate Echo Qualified | Plate | Labcyte | PPL-0200 | [Link](https://www.beckman.com/supplies/echo-plates/001-14622) | Sterile | +| Echo Qualified Reservoir 2x3 Well Polypropylene Microplate | Plate | Labcyte | ER-0050 | [Link](https://www.beckman.com/supplies/echo-plates/001-11101) | RNase/DNase free | + +# Before Start Instructions + +## Prep Once: +### A. Calibrating the Vacuum Concentrator +- Test and calibrate all new plate brands by drying 5 µL water in each well to confirm plates dry within 20-30 minutes. +- Maintain temperature settings between 40-45°C (varies by model). + +### B. Calibrating Plates for Liquid Handlers +- Input and test labware definitions for all plates to confirm appropriate liquid transfer settings. + +### C. Prep ERCCs +- External RNA Control Consortium (ERCC) RNA Spike-In Mix used as control. Dilute in nuclease-free water to target stock concentration of 50 pg/µL. Store in aliquots at -80°C. + +## Prep Every Time: +### A. Prep Master Mix Calculations & Sample Sheets +- Reflect transfer volumes and destination wells by preparing a sample sheet listing the well location of each sample. + +### Prep Before PCR Amplification Step: +#### A. Barcode Plate +- Prepare a 96-well PCR plate with at least 10 µL of 5 µM unique dual indexing primers in each necessary well. Prepare before post-ligation bead clean as it is used for elution step. + +#### B. USER/PCR Plate +- Prepare a 96-well PCR plate with 10.9 µL of USER/PCR master mix in each necessary well using the Echo. Prepare before post-ligation bead clean as it is used for the final plate. + +# GeneVac + +1. Warming up GeneVac: + - Turn to "aqueous" setting, set temperature to ~40-42°C, and let run for ~30 min before use. + +2. Spin plate of sample RNA in vacuum evaporator at appropriate temperature and time settings to dry completely (37-38°C for approximately 1 hour). + +### Equipment +| Equipment | Specifications | +|-----------|----------------| +| Genevac EZ-2 | [Spec Link](https://www.spscientific.com/Products/Centrifugal_Evaporators__Sample_Concentrators/Genevac/EZ-2_Series/EZ-2_Series/) + +## Prepare Master Mix Reagents (Day 1) + +3. Prepare enough fragmentation, first strand, and second strand master mixes for each sample. + - Note: Most clinical respiratory samples do not require FastSelect depletion, verify desired sample type. + +### Equipment +| Equipment | Type | Brand | SKU | Link | Specifications | +|-----------|------|-------|-----|------|----------------| +| 384-well PP 2.0 Plus Microplate Echo Qualified | Plate | Labcyte | PPL-0200 | [Link](https://www.beckman.com/supplies/echo-plates/001-14622) | Sterile | + +### Reagents Preparation Tables + +#### Fragmentation Master Mix (1x) +| Reagent (lilac tubes) | Volume (µL per sample) | +|-----------------------|------------------------| +| ERCC (stock concentration 50 pg/µL) | 0.5 | +| First Strand Synthesis Reaction Buffer | 0.4 | +| Random Primers | 0.1 | +| **Total Reaction Volume** | **1.0** | + +#### First Strand Synthesis Master Mix (1x) +| Reagent (lilac tubes) | Volume (µL per sample) | +|-----------------------|------------------------| +| Fragmentation Reaction Volume | 1.0 | +| NEBNext First Strand Synthesis Enzyme Mix | 0.2 | +| Nuclease Free Water | 0.8 | +| **Total Reaction Volume** | **2.0** | + +#### Second Strand Synthesis Master Mix (1x) +| Reagent (orange tubes) | Volume (µL per sample) | +|------------------------|------------------------| +| First Strand Reaction Volume | 2.0 | +| Second Strand Synthesis Reaction Buffer | 0.8 | +| Second Strand Synthesis Enzyme Mix | 0.4 | +| Nuclease Free Water | 4.8 | +| **Total Reaction Volume** | **8.0** | + +### FastSelect Reagent Preparation +| Reagent | Volume (µL) | +|---------|-------------| +| RNA (USE ERCC @ 50 pg/µL) | 0.5 | +| First SS Reaction Buffer 5x (pink) | 0.4 | +| Random Primers (pink) | 0.1 | +| rRNA + Globin FastSelect (1:10) | 0.1 | +| **Total Volume** | **1.1** | + +4. Load reagents into a 384PP Plus Echo source plate by pipetting into each required well and seal with foil. Avoid bubble formation, make Echo CSV with transfer specifications. + +5. Spin Echo source plate in centrifuge for 5 minutes at 2000 rpm to remove bubbles. Reagents at room temperature before Echo transfer. + +## Dispensing Fragmentation Reagents + +6. Use Echo's "Survey" setting for 384-well source plates to determine volume in each source well ensuring volume is 20 < x < 65 µL. + +7. Use BP setting, dispense 1000 nL of fragmentation master mix (1100 nL for FastSelect) into each sample well. + - Note: Ensure no bubbles are present in the source plates. + +8. Remove sample plate and seal with foil. Gently vortex and quick spin. + +## Fragmentation Incubation (For FastSelect Only) + +9. Follow steps for fragmentation incubation with heated lid set to 105°C +| Fragmentation Incubation | Time (minutes) | +|--------------------------|----------------| +| 94°C | 1 | +| 75°C | 2 | +| 70°C | 2 | +| 65°C | 2 | +| 60°C | 2 | +| 55°C | 2 | +| 37°C | 5 | +| 25°C | 5 | + +## Dispensing First Strand Synthesis Reagents + +10. Remove foil seal, load into Echo. Dispense 1000 nL of first strand synthesis master mix into each sample well using GP setting. + +11. Remove sample plate and seal with foil. Gently vortex, then quick spin. + +### First Strand Synthesis Incubation +| Temperature (°C) | Time (minutes) | Heated Lid: 105°C | +|------------------|----------------|-------------------| +| 25 | 10 | Yes | +| 42 | 15 | Yes | +| 70 | 15 | Yes | +| 4 | Hold | No | + +## Dispensing Second Strand Synthesis Reagents + +13. Remove sample from thermocycler, remove foil, load into Echo. Dispense 6000 nL of second strand synthesis master mix into each sample well using GP setting. + +14. Remove sample plate and seal with foil. Gently vortex and quick spin. + +### Second Strand Synthesis Incubation +| Temperature (°C) | Time (minutes) | Heated Lid: Off | +|------------------|----------------|-----------------| +| 16 | 60 | Yes | +| 10 | Hold | No | + +## Nucleic Acid Purification: Hand Bead Clean + +16. Bead Clean using a 1.4x Ampure bead-to-sample ratio. Make your own SPRI beads if needed to match Ampure bead ratio. + +| Cleaning Steps | Volume, Duration | Notes | +|----------------|------------------|-------| +| 80% ethanol (fresh), beads (50 mL, RT) | 12 µL | Prepare for use | +| Add 12 µL water | appropriate vol. | Bring wells to 20 µL, enhancing elution | +| Add 28 µL beads, mix, incubate | 5 mins | Confirm mixture visually | +| Incubation on magnet | 5 mins | Transfer supernatant | +| Wash with 150 µL 80% ethanol | Pause-30s | Transfer supernatant | +| Second ethanol wash | Repeat | Remove ethanol w/p20 tip | +| Air dry beads | 10 mins | Do not overdry | +| Resuspend beads in water | 6 µL | Visually confirm mixture | +| Incubation on magnet | 3-5 mins | Transfer supernatant | + +## Prepare Master Mix Reagents (Day 2) + +17. Prepare enough end prep, adaptor ligation, USER/PCR master mixes, and adaptor dilutions. + +### Equipment +| Equipment | Type | Brand | SKU | Link | Specifications | +|-----------|------|-------|-----|------|----------------| +| 384-well PP 2.0 Plus Microplate Echo Qualified | Plate | Labcyte | PPL-0200 | [Link](https://www.beckman.com/supplies/echo-plates/001-14622) | Sterile | + +### End Prep Master Mix (1x) +| Reagent (green tubes) | Volume (µL per sample) | +|-----------------------|------------------------| +| Post-Second Strand Synthesis Bead Clean Volume | 5.0 | +| Ultra II End Prep Reaction Buffer | 0.7 | +| Ultra II End Prep Enzyme Mix | 0.3 | +| **Total Volume** | **6.0** | + +### Adaptor Ligation Master Mix (1x) +| Reagent (red tubes) | Volume (µL per sample) | +|---------------------|------------------------| +| End Prep Reaction Volume | 6.0 | +| NEBNext Ultra II Ligation Master Mix | 3.0 | +| NEBNext Ligation Enhancer | 0.1 | +| **Total Volume** | **9.1** | + +### Adaptor Master Mix (1x) +| Reagent (red tube) | Volume (µL per sample) | +|-------------------|------------------------| +| Diluted Adaptor (1:100) | 0.25 | +| **Note:** Adaptor should not cause dimers. Dilute according to input sample. + +### USER/PCR Master Mix (1x) +| Reagent (USER- white tube; Q5-blue tube) | Volume per sample (µL) | +|------------------------------------------|-------------------------| +| Adaptor Ligation Reaction Volume | 5.0 | +| Nuclease Free Water | 2.5 | +| NEB USER Enzyme | 0.9 | +| NEBNext Ultra II Q5 Master Mix | 7.5 | +| **Total Volume** | **15.9** | + +18. Load reagents into Echo source plates by pipetting into each required well and seal with foil. + - Note: End Prep and Adaptor master mixes are to be loaded into a 384 PP Plus plate. USER/PCR master mix should be loaded into a 6-Res plate. + +19. Spin Echo source plate in the centrifuge for 5 minutes at 2000 rpm to remove bubbles produced during pipetting. + +# Dispensing End Prep Reagents + +20. Use Echo’s "Survey" setting to determine volume in each source well of 384-well source plates ensuring volume is 20 < x < 65 µL. + +21. Using the BP setting, dispense 1000 nL of end prep master mix into each sample well. + - Note: Ensure no bubbles are in the source plates. + +22. Remove sample plate and seal with foil. Gently vortex, then quick spin. + +### End Prep Incubation +| Temperature (°C) | Time (minutes) | Heated Lid: >75°C | +|------------------|----------------|-------------------| +| 20 | 30 | Yes | +| 65 | 30 | Yes | +| 10 | Hold | No | + +## Dispensing Adaptor Ligation Reagents + +24. Remove sample plate from thermocycler, remove foil seal, load into Echo. Using GP setting, perform two transfers: + - **First transfer:** 3100 nL of adaptor ligation master mix. + - **Second transfer:** 250 nL of diluted adaptor to each sample well. Avoid excessive adaptor dimer. + +25. Remove sample plate and seal with foil. Gently vortex, then quick spin. + +### Adaptor Ligation Incubation +| Temperature (°C) | Time (minutes) | Heated Lid: Off | +|------------------|----------------|-----------------| +| 20 | 15 | Yes | +| 10 | Hold | No | + +## USER/PCR Setup - Reagent Plate + +27. Prepare USER/PCR master mix plate as the final destination for cDNA during post-adaptor ligation bead clean-up. + - Note: Must be done before bead clean-up. + +28. Load new sterile PCR plate into Echo destination port. Dispense 10,900 nL of USER/PCR master mix from a 6-Res plate into each sample well using GP setting. + +## Nucleic Acid Purification: Hand Bead Clean + +29. Take beads out of 4°C 30 min before bead cleaning. Clean using a 0.8x bead solution ratio. + +| Steps | Volume/Duration | Notes | +|-------|-----------------|-------| +| 80% ethanol, beads (50mL reservoir) | Prepare at RT | Fresh for use | +| Add 12.72 µL water | to increase volume at samples | ensuring enough elution | +| Add 28 µL beads, mix, incubate | 5 mins | confirm mix visually | +| Incubation on magnet | 5 mins | +| Transfer supernatant | 27.5 µL | transfer to waste | +| Ethanol wash | 2x 150 µL 80% ethanol | crucial to remove ethanol | +| Air dry beads | 10 mins | not overdrying | +| Resuspend beads | 6 µL eluent (water) | Confirm sufficient mix visually | +| Incubation on magnet | 5 mins | +| Transfer supernatant (cDNA) | to new Echo plate | transfer 31 µL | + +Note: Maintain elution volume for accurate results with Labcyte Echo. + +# Finished Libraries + +32. Finished libraries undergo quality check and equal volume pooling to determine library representation. Use this data to determine equal pooling volumes. + +### Transfer Libraries +32.1. Transfer to new 384-well Echo source plate skipping if already complete. Ensure volumes settle between 22-25 µL. + +### Pool Libraries +32.2. Pool 0.5 µL from each library into a 384-well destination plate using Echo. + - Note: Consider max working volume and avoid 12 µL per well. + +32.3. Manual combination of pool wells into a single tube. + +### Sequencing +32.4. Sequence pool using Illumina iSeq or low-throughput sequencer. + +### Representation Calculation +32.5. Calculate using equal volume pooling results to determine the library representation. + +```math +\ T = N \sum \frac{1}{x} +``` + +- *note*: Normalize based on sample read fraction percentage. + +32.6. Use normalization factor to determine even pool volume. + +### Dispense Libraries +32.7. Dispense sequenced libraries into final pool using Echo. + +32.8. Combine pool wells into one tube, mix. + +32.9. Libraries are ready for high throughput sequencer (Illumina HiSeq or NovaSeq). +``` +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/detailed-protocol-for-leptospira-isolation-from-an-ifccbiw.md b/markdown-output/detailed-protocol-for-leptospira-isolation-from-an-ifccbiw.md new file mode 100644 index 0000000000000000000000000000000000000000..735c9d0dba46e9f6d9450fe02e7d9d92f8c634fa --- /dev/null +++ b/markdown-output/detailed-protocol-for-leptospira-isolation-from-an-ifccbiw.md @@ -0,0 +1,121 @@ +```markdown +# Detailed Protocol for Leptospira Isolation from Animal Samples + +**Authors:** Gomard Yann, Lagadec Erwan, Tortosa Pablo + +**Published:** June 13, 2017 + +Goal/Experiment: +The goal of this experiment is the isolation of pathogenic Leptospira from wild animals (terrestrial small mammals, bats, and dogs). This protocol can be employed for Leptospira isolation from urine by directly proceeding to step 10. + +--- + +## Abstract +This protocol is adapted from the International Course on Laboratory methods for the Diagnosis of Leptospirosis, by Dr. R.A. Hartskeerl, Dr. H.L. Smits, Mr. H. Korver, Dr. M.G.A. Goris, and Dr. W.J. Terpstra from the Leptospirosis Reference Center in Amsterdam, the Netherlands. It is routinely used at UMR PIMIT, St Denis de La Réunion, France. + +--- + +## Guidelines +All steps should be carried out using sterile forceps. Forceps must be cleaned between samples by placing them 1 minute in a 1.0 mL sterile tube filled with ethanol 70%, and then in a 1.0 mL sterile tube containing sterile water. All microtubes, containing either kidney samples, culture media, or water/ethanol for forceps sterilization, can be placed in a single tray. + +--- + +## Before Start + +### Equipment +- An incubator at 28°C. +- A dark field microscope. +- Sterile BenchGuard. +- At least a couple of sterile forceps. +- Sterile filter tips. +- A 100 – 1000 µL micropipette. +- Sterile disposable blades. +- Sterile petri dishes. +- 500 or 1000 mL sterile units of a filtration system with a pore size of 22 µm (Stericup®, Millipore Express). +- Sterile syringe with a 0.22 µm filter. +- 1.5 – 2.0 mL sterile tubes (hereafter referred to as microtubes). +- 5.0 mL sterile culture tubes (Falcon®, Round-Bottom, polypropylene, non-pyrogenic). +- A tray for microtubes. + +### Reagents +- **Three distinct media** are used to maximize isolation culture success from various animal species: + - **(i) EMJH + AFAS:** Prepared from Leptospira Medium Base Ellinghausen-McCullough-Johnson-Harris (EMJH) powder and supplemented with Albumin Fatty Acid Supplement (AFAS). Required autoclaving and pH adjustment. + - **(ii) EMJH + AFAS + RS 1% + FCS 1%:** Same as media (i) but with added rabbit serum and fetal calf serum (both 1%). Both sera should be heat-inactivated (30 minutes at 56°C). + - **(iii) Fletcher semi-solid medium + RS 8%:** Prepared from Fletcher Medium Base powder, supplemented with heat-inactivated rabbit serum (8%). + +All media are supplemented with 5-fluorouracil (5-FU) at a concentration of 200 µg/mL. For sub-culture steps, prepare the media without 5-FU and filter using Stericup® filtration. + +- **Ethanol 70%** +- **Sterile water** (autoclaved) + +--- + +## Protocol + +### Kidney Sampling + +#### Step 1 +Following euthanasia, place one kidney in a sterile microtube containing 1.0 mL of EMJH+AFAS+5-FU. This microtube can be stored at room temperature until the end of the animal dissection. + +### Kidney Processing + +#### Step 2 +Transfer the freshly dissected kidney into a 1.5 mL tube containing 1.0 mL of ethanol 70% for 30 seconds to remove potential surface contamination. + +#### Step 3 +Transfer the kidney to a microtube containing 1.0 mL of sterile water for 30–60 seconds to remove ethanol. + +#### Step 4 +Transfer the kidney to a sterile petri dish and, using a sterile scalpel, cut a fine transversal slice (1 mm) of the kidney. + +#### Step 5 +Within this fine slice, cut a small piece containing parts of the cortex, medulla, and renal pelvis. + +#### Step 6 +Crush this small piece using a disposable scalpel for 1 to 5 minutes. Add 100 - 200 µL of EMJH+AFAS+5-FU to facilitate the slicing process. + +#### Step 7 +With a sterile 1000 µL filtered tip, suck the mixture containing finely sliced kidney tissue into a microtube containing 1.0 mL EMJH+AFAS+5-FU. Store remaining tissue in sterile microtubes at -20°C, -80°C, or in ethanol 70% for future use. + +#### Step 8 +Keep the microtube containing the sliced tissue at room temperature for 45–60 minutes (avoid direct sunlight). Mix by inversion every 15 minutes. + +#### Step 9 +Discard tips and disposable scalpel and petri dish in a proper laboratory trash container. + +### Culture + +#### Step 10 +After 45–60 minutes, mix the microtube by inversion and pipet 250 µL of the solution into three Falcon® culture tubes, each containing 2.5 mL of the three media types. For urine samples, inoculate 2–4 urine droplets into each of the three culture media. + +#### Step 11 +Incubate the tubes at 28°C. For field cultures with limited equipment, maintain tubes in a polystyrene box to limit temperature variations until reaching lab facilities. + +### Culture Checking + +#### Step 12 +After one week of incubation, place a drop of the inoculated medium onto a clean microscope slide and check for Leptospira under a dark field microscope. + +#### Step 13 +Repeat Step 12 once a week for four months. + +#### Step 14 +For positive cultures, transfer 1.0 mL of the positive culture into 3.0–5.0 mL of fresh medium without 5-FU. + +#### Step 15 +If positive cultures are contaminated, perform a sub-culture into fresh medium with 5-FU or filter through a sterile syringe with a 0.22 µm filter. + +#### Step 16 +Isolate storage: +- Freeze 1.0 mL of the culture at -80°C in a nitrogen tank. +- Inoculate a tube containing Fletcher medium + RS 8% with 100–500 µL of the culture; maintain at room temperature for six months. + +--- + +## Warnings +Users must wear personal protective equipment (PPE) necessary for handling Class II bacterial pathogens. Procedures should be carried out in a BSL2 lab, or for fieldwork, within an aseptic environment created by a Bunsen burner. Adapt PPE accordingly. + +--- + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/detection-of-arboviruses-in-salivary-glands-and-mi-cyadxsa6.md b/markdown-output/detection-of-arboviruses-in-salivary-glands-and-mi-cyadxsa6.md new file mode 100644 index 0000000000000000000000000000000000000000..2a1470d6a6fa7eee44a15a9ea2eabbb3cdc1aa01 --- /dev/null +++ b/markdown-output/detection-of-arboviruses-in-salivary-glands-and-mi-cyadxsa6.md @@ -0,0 +1,71 @@ +```markdown +# Goal/Experiment: +Detection of arboviruses in salivary glands and midgut of mosquitoes vector + +## Detection of arboviruses in salivary glands and midgut of mosquitoes vector + +**Authors:** +ISABELLA FERREIRA DA COSTA, CAROLINE PIRES SANTANA, Isabela Cinquini Junqueira, Christian Luz, Juscelino Rodrigues, Izabela Batista Melo, Suleimy Marinho Fernandes, Fabíola Fiaccadori, Valéria Christina de Rezende Feres + +**Affiliations:** +1. Laboratory of Molecular Biology and Applied Technologies for Laboratory Diagnosis, Faculty of Pharmacy, Federal University of Goiás (UFG), Goiânia, GO, Brazil +2. Laboratory of Invertebrate Pathology, Institute of Tropical Pathology and Public Health, Federal University of Goiás (UFG), Goiânia, GO, Brazil +3. Laboratory of Virology and Cell Culture, Institute of Tropical Pathology and Public Health, Federal University of Goiás (UFG), Goiânia, GO, Brazil + +**Date:** +August 7, 2023 + +**DOI:** +[10.17504/protocols.io.8epv5jxndg1b/v1](https://dx.doi.org/10.17504/protocols.io.8epv5jxndg1b/v1) + +## Abstract +Dengue, Zika, and chikungunya viruses are widely disseminated in Brazil and cause infections in humans, with similar clinical manifestations of varying intensities, with relevance in the context of public health. These diseases are mainly transmitted by mosquitoes of the genus Aedes. Monitoring the circulation of these arboviruses in vectors is crucial in epidemiological surveillance. Adult mosquitoes were dissected to extract the salivary glands and midgut, which are structures directly associated with viral replication and transmission. These structures were inoculated into cells of the C6/36 lineage of Aedes albopictus for amplification and viral isolation. + +## Safety Warnings +- Please use personal protective equipment (such as gloves, safety glasses, a lab coat, and masks) during all experiments. + +## Keywords +Arbovirus, Mosquitoes, Salivary Glands, Midgut, Cell Culture, Detection + +## Before Starting +1. Prepare pools of 2 to 10 live and engorged or non-engorged adult mosquito specimens, sorted by sex (male/female). Place the pools in 1.5 µL microtubes and store them in a freezer at -80°C. + +## Mosquitoes Dissection +1. Pinch one mosquito at a time and remove the legs. +2. Place the mosquito on a micro glass slide under the stereoscopic magnifying glass. +3. Add a drop of sterile solution of phosphate buffer saline (PBS) 1% to the micro glass slide to create a conductive medium for the separation of the desired structures. +4. With a needle, immobilize the mosquito's thorax by pinning it against the micro glass slide. +5. Using tweezers, carefully remove the entire abdomen of the mosquito, ensuring that the midgut remains attached to the immobilized thorax. +6. Use a needle to separate and section the midgut from the thorax. +7. Transfer the midgut to microtubes already prepared with 200 µL of PBS solution and store them appropriately. +8. While the mosquito is still fixed on the slide, use another needle to separate the mosquito's head from the rest of the body. This is necessary because the salivary glands, located in the anterior portion of the thorax, may partially adhere to the head. +9. Gently press the head and thorax to extract the salivary glands. +10. Using a pipette, extract the salivary glands from the medium and transfer them to the respective microtube where the intestine has already been stored. +11. Each tube will be homogenized by repeated pipetting (60 seconds). Subsequently, vortex for 60 seconds to disrupt the cells. + +## Cell Culture +1. Maintain C6/36 cells (Aedes albopictus) in culture using Leibovitz medium (L-15 Sigma-Aldrich, St. Louis, MO, USA), supplemented with 10% fetal bovine serum (FBS) (Sigma-Aldrich, St. Louis, MO, USA) and 1% Penicillin/Streptomycin (Gibco, Waltham, MA, USA) at 28°C in an incubator. +2. Monitor cell growth and wait until the cells reach approximately 80% confluence. +3. Completely discard the medium from the cell culture bottle and resuspend the cells in approximately 2 mL of L-15 medium. +4. Transfer the cells to a falcon tube (50 mL) and proceed to count them using a Neubauer chamber. +5. Prepare a solution combining the cell suspension and Trypan blue dye at a 1:4 dilution ratio. +6. Under an optical microscope, count all living cells present in the four lateral quadrants of the Neubauer chamber. +7. Calculate the average of the cells counted in the four quadrants. Then, multiply the result by the dilution factor and the Neubauer chamber factor (10^4). +8. Aliquot 500 µL of the cell suspension into each well of a 24-well cell culture plate. Incubate the cell culture plate at 28°C for 48 hours. +9. After confirming the formation of a cell monolayer, inoculate 50 µL of the samples processed as described in mosquito dissection section. Add 500 µL of L-15 medium to each well and incubate the plate for 7 days. +10. At the 7th day, freeze the plates at -80°C for at least overnight. Then, thaw the plates at room temperature to promote cell disruption and virus release. +11. Aspirate the contents of each well individually and transfer them to centrifuge tubes. +12. Centrifuge the tube at 14,000 rpm for 60 seconds at room temperature. After centrifugation, separate the supernatant and refrigerate it at -80°C for subsequent viral detection and identification through molecular biology techniques. +13. All stages of cell cultivation must be carried out in a sterile biological safety cabinet and following biosafety standards. + +## Arbovirus Molecular Identification +1. Extract the viral RNA using the silica/gel membrane column methods, utilizing a commercial kit (QIAamp Viral RNA Mini Kit, Qiagen, Hilden, Germany). +2. Perform reverse transcription and amplify the genetic material using real-time polymerase chain reaction (PCR). +3. Standardize the protocol using two methodologies for real-time RT-PCR. + + - The protocol described by Huhtamo et al. 2010 for Dengue virus (DENV) detection, which employed specific primers and a probe for the conserved 3'UTR region (Path-ID™ multiplex One-Step RT-qPCR - Applied Biosystems – Carlsbad, CA, USA). The final reaction volume was 10 µL, and the components included 2.5 µL of extracted material, 5.0 µL of Multiplex RT-PCR buffer, 1.0 µL of Multiplex Enzyme Mix, 100 nM of each primer, 200 nM of TaqMan probe, and Nuclease-Free water provided in the kit to complete the volume. + - The protocol described by Lanciotti et al. 2008 for Zika virus (ZIKV) detection using specific primers and a probe targeting the conserved region of the protein E gene (GoTaq® Probe 1-Step RT-qPCR System, Promega – USA). The final reaction volume was 10 µL, and the components included 2.5 µL of extracted material, 5.0 µL of GoTaq® Probe Master Mix with dUTP, 0.4 µL of GoScript™ RT Mix, 100 nM of each primer, 200 nM of TaqMan probe and, Nuclease-Free water provided in the kit to complete the volume. + - For the detection of nucleic acids (RNA) from ZIKV, DENV (4 serotypes), and Chikungunya virus, the commercial Kit IBMP Biomol ZDC-Zika, Dengue and Chikungunya (IBMP, Curitiba - PR - Brazil) was utilized, following the manufacturer’s instructions. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/determination-of-glomerular-filtration-rate-in-con-3fngjme.md b/markdown-output/determination-of-glomerular-filtration-rate-in-con-3fngjme.md new file mode 100644 index 0000000000000000000000000000000000000000..44bcd119173ed59097841e581612b8d890d2f3fe --- /dev/null +++ b/markdown-output/determination-of-glomerular-filtration-rate-in-con-3fngjme.md @@ -0,0 +1,93 @@ +```markdown +# Goal/Experiment: +Determination of glomerular filtration rate (GFR) in conscious mice using FITC-inulin as a tracer, to estimate kidney function by measuring inulin clearance from the bloodstream. + +# Determination of Glomerular Filtration Rate in Conscious Mice using FITC-inulin + +### Authors: +- Zhonghua Qi¹ +- Matthew D. Breyer² +¹Vanderbilt University, ²Eli Lilly Company + +**DOI:** [dx.doi.org/10.17504/protocols.io.3fngjme](dx.doi.org/10.17504/protocols.io.3fngjme) +**Consortium:** Diabetic Complications Consortium +**Tech Support:** [mcindoe@augusta.edu](mailto:mcindoe@augusta.edu) + +## Summary: +This protocol describes the procedures for estimating glomerular filtration rate in conscious mice based on Fluorescein Isothiocyanate-inulin (FITC-inulin) clearance following a single bolus intravenous injection. + +## Diabetic Complication: +- Nephropathy + +## Reference: +1. Hem, A., Smith, A.J., and Solberg, P. 1998. Saphenous vein puncture for blood sampling of the mouse, rat, hamster, gerbil, guinea pig, ferret, and mink. *Lab Anim* **32**:364-368. +2. Sturgeon, C., Sam, A.D., 2nd, and Law, W.R. 1998. Rapid determination of glomerular filtration rate by single-bolus inulin: a comparison of estimation analyses. *J Appl Physiol* **84**:2154-2162. +3. Qi, Z., Whitt, I., Mehta, A., Jin, J., Zhao, M., Harris, R.C., Fogo, A.B., and Breyer, M.D. 2003. Serial Determination of Glomerular Filtration Rate in Conscious Mice Using FITC-Inulin Clearance. *Am J Physiol Renal Physiol*. + +**External Link:** [Protocol](https://www.diacomp.org/shared/document.aspx?id=28&docType=Protocol) + +## Materials: + +| Name | Catalog # | Vendor | +|--------------------------------|-----------|---------------| +| FITC-inulin | F-3272 | Sigma Aldrich | +| HEPES | N/A | Sigma Aldrich | +| Dialysis Membrane | 132636 | Spectrum Lab | +| Closures | 132736 | Spectrum Lab | +| Syringes (1ml 5ml) | N/A | N/A | +| Needles (30 G1/2 23 G1/4) | N/A | N/A | +| Filter (0.22µm) | 8110 | Costar | +| Isoflurane drop jar | N/A | N/A | +| Isoflurane | N/A | N/A | +| Centrifuge (desktop) | N/A | N/A | + +### Reagent Preparation: +1. **5% FITC-inulin Solution**: Dissolve 100 mg of FITC-inulin in 2 ml of 0.9% NaCl by heating the solution in boiling water. +2. **500 mM HEPES Buffer**: Dissolve 59.6 g of HEPES in 500 ml of deionized water and adjust pH to 7.4 using 10N NaOH. + +## Protocol: + +### 1. Preparation of 5% FITC-inulin Solution: +1. Dissolve 100 mg of FITC-inulin in 2 ml of 0.9% NaCl by heating the solution in boiling water. +2. To remove residual FITC not bound to inulin, fill the solution into a 1000 Daltons cut-off dialysis membrane (e.g., Spectra/Pro 6, Spectrum Laboratories Inc., Rancho Dominguez, CA). +3. Put the dialysis membrane filled with FITC-inulin into 1000 ml 0.9% NaCl for 24 hours at room temperature. +4. Prior to use, sterilize this dialyzed solution by filtration through a 0.22 μm filter (e.g., Costar). + +### 2. Intravenous Injection and Blood Collection: +1. Mice are anesthetized using Isoflurane, which for approximately 20 seconds. +2. Inject 2.5% FITC-inulin (3.74 μl/g body weight) retroorbitally under anesthesia within 10 seconds. +3. After regaining consciousness, restrain the mouse inside a 50- ml centrifuge tube with large air-holes drilled in the tip. +4. The inner thigh is closely shaven and wiped with 75% ethanol to reveal the saphenous vein. Approximately 20 μl blood is collected in a heparinized capillary tube (Fisher Scientific) via venepuncture using a sterile 23 gauges syringe needle (see Hem et al.1998(1)). On average, this yields 10 μl of plasma following centrifugation (4,000 RPM, 10 min). +5. Blood is sampled via the saphenous vein at 3, 7, 10, 15, 35, 55, 75 minutes post injection of FITC-inulin. + +### 3. Determination of Fluorescence of the Sampled Plasma: +1. Since pH significantly affects FITC fluorescence value, each plasma sample is buffered to pH 7.4 by mixing 10 μl of plasma with 40 μl of 500 mM HEPES (pH 7.4). +2. Load the titrated samples onto a 96-well plate, 50 μl sample/well. Determine fluorescence using a Fluoroscan Ascent FL (Labsystems, FIN-00811 Helsinki, Finland), with 485 nm excitation, and read at 538 nm emission. + +### 4. Calculation of GFR: +A two-compartment clearance model is employed for the calculation of GFR. In the two-compartment model used, the initial, rapid decay phase represents the redistribution of the tracer from the intravascular compartment to the extracellular fluid. Systemic elimination also occurs, but the distribution process is relatively dominant during this initial phase. During the later, slower decay in the concentration of the tracer, systemic clearance of the tracer from the plasma predominates. At any given time (t), the plasma concentration of the tracer (Y) equals to: + +``` +Ae^(-αt) + Be^(-βt) + Plateau +``` +The parameters of the above equation can be calculated using a non-linear regression curve-fitting program (GraphPad Prism, GraphPad Software, Inc., San Diego, CA). GFR was calculated using the equation: + +``` +GFR = I / (A / α + B / β) +``` +where I is the amount of FITC-inulin delivered by the bolus injection; A (Span1) and B (Span2) are the y-intercept values of the two decay rates, and α and β are the decay constants for the distribution and elimination phases, respectively. + +### 5. Use of Prism: +1. Open Prism program and click "New table". +2. Input time point into "X Values" column, and the corresponding fluorescence data into "Y" column. +3. Click "Analysis" and choose "Nonlinear regression (curve fit)". +4. Click "OK", and choose "Two phase exponential decay". +5. Set "Plateau" to zero. +6. The parameters of the fluorescence decay curve are shown in a spreadsheet. SPAN1 and SPAN2 are Y intercepts, and K1 and K2 are constants of the distribution and clearance phases, respectively. + +--- + +This is an open access protocol distributed under the terms of the Creative Commons Attribution License ([https://creativecommons.org/licenses/by/4.0/](https://creativecommons.org/licenses/by/4.0/)), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/development-and-validation-of-a-multi-locus-dna-me-jwzcpf6.md b/markdown-output/development-and-validation-of-a-multi-locus-dna-me-jwzcpf6.md new file mode 100644 index 0000000000000000000000000000000000000000..8c27e995ed71d3fbb04370c3bf7d1fefb0cbc17a --- /dev/null +++ b/markdown-output/development-and-validation-of-a-multi-locus-dna-me-jwzcpf6.md @@ -0,0 +1,284 @@ +```markdown +# Goal/Experiment + +The objective of this research is to develop a multi-locus DNA metabarcoding method for forensic wildlife species identification and to evaluate the applicability and reproducibility of this approach across different laboratories. This method will assist in the identification of endangered species in complex samples such as food supplements and traditional medicines, providing detailed data on the composition of complex food products, including information on the presence of CITES-listed species. + +# Development and Validation of a Multi-Locus DNA Metabarcoding Method to Identify Endangered Species in Complex Samples + +**SOP Version 2** + +## Authors +Alfred J. Arulandhu, Martijn Staats, Rico Hagelaar, Marleen M. Voorhuijzen, Theo W. Prins, Ingrid Scholtens, Tamara Peelen, and Esther Kok + +## Abstract + +### Background +DNA metabarcoding provides great potential for species identification in complex samples. This method aids CITES (the Convention on International Trade in Endangered Species of Wild Fauna and Flora) enforcement officers by preventing the illegal trade of endangered plant and animal species. The objective of this research was to develop a multi-locus DNA metabarcoding method for forensic wildlife species identification and to evaluate the applicability and reproducibility of this approach across different laboratories. + +### Results +A DNA metabarcoding method was developed using 12 DNA barcode markers, demonstrating universal applicability across a wide range of plant and animal taxa, facilitating the identification of species in samples containing degraded DNA. This DNA metabarcoding method, based on Illumina MiSeq amplicon sequencing, has been internationally validated by 16 laboratories, proving to be highly reproducible and sensitive enough to identify species present in mixtures at 1% dry weight content. + +### Conclusion +The advanced multi-locus DNA metabarcoding method provides reliable and detailed data on the composition of complex food products, including the presence of CITES-listed species. This method improves resolution for species identification and enhances quality assurance. + +## Guidelines + +This DNA metabarcoding method combines DNA barcoding with next-generation sequencing (NGS), using universal PCR primers to mass-amplify and sequence one or more taxonomically informative DNA barcodes. The following animal DNA barcodes are used: COI-2, cytB (cytochrome b), and 16S (16S ribosomal RNA). The plant DNA barcodes used are: matK, rbcL, trnL (UAA), and ITS2. + +### Samples + +The participating laboratories are provided with ten microcentrifuge tubes containing 250 mg of powdered tissue stored in dry ice. Additionally, two tubes containing positive control DNA (Bos taurus and Lactuca sativa) are provided. All samples should be stored frozen at -20°C until processed and can be stored frozen indefinitely. + +### Required Equipment, Chemicals, Reagents, Consumables, and Materials + +#### Equipment +- 96-well thermocycler with a temperature range between 8°C and 95°C, e.g., BIO-RAD CFX PCR Thermal Cycler +- Spectrophotometer (e.g., NanoDrop) +- Thermo-Shaker for 1.5 and 2 ml tubes capable of mixing at ~1000 rpm and 65°C (mandatory) +- Centrifuge for 1.5-2 ml tubes with a capacity of ~18,000×g +- Mini-centrifuge for PCR tube strips, spinning at 10,000×g +- Vortex machine for 1.5 and 2 ml tubes +- Electrophoresis system for discriminating PCR amplification fragments between ~180 bp and ~920 bp +- Gel electrophoresis imaging UV machine +- Full set of semi-automatic pipettes +- Analytical scales (max. capacity 100 g, readability 0.001 g) +- Fridge or 4°C storage facility +- Freezer or -20°C storage facility +- 1.5 ml and PCR tube racks +- Cold block or ice + +#### Chemicals and Reagents +- Nuclease-free molecular grade water (ddH2O) +- RNase A solution (QIAGEN, Cat No./ID: 19101), 100 mg/ml (or equivalent) +- Proteinase K solution (Fermentas, Cat No./ID: E00491), 20 mg/ml (or equivalent) +- Chloroform +- Ice-cold ethanol (96%) +- Ethanol (70%) +- 100 bp DNA Ladder (e.g., Invitrogen, for gel electrophoresis) +- Agarose +- Ethidium bromide +- TBE running buffer for gel electrophoresis: + - 54 g Tris + - 27.5 g Boric acid + - 20 ml 0.5M EDTA, pH 8 +- 10X loading dye + +#### Consumables and Materials +- Centrifuge tubes (100-150 tubes 1.5 ml and 50 tubes 2 ml) +- PCR tube strips (8 tube strips, single attached domed cap, e.g., Bioplastics EU 8-tube cat no. C78201) +- Gel electrophoresis system, tray, and comb +- Micropipette filter tips (10 µl, 30 µl, 200 µl, 1000 µl) +- Small plastic spatula +- Disposable powder-free gloves +- Clean lab coat +- Aluminium foil + +### Chemicals and Reagents for CTAB DNA Isolation +- Molecular grade water (ddH2O) +- 17.5 ml CTAB extraction buffer +- RNase A solution (QIAGEN, 100 mg/ml) +- Proteinase K solution (Fermentas, 20 mg/ml) +- Chloroform +- Ice-cold ethanol (96%) +- Ethanol (70%) + +#### Primer Labelling Information +- 16S-FCOI-2-FCytB-F16S-mini-FCOI-mini-FCytB-mini-FMatk-FrbcL-FtrnL(UAA)-FITS2-FrbcL-mini-FtrnL(P6loop) +- 16S-R + +### CTAB DNA Isolation + +#### Required Reagents and Chemicals +- Molecular grade water (ddH2O) +- 17.5 ml CTAB extraction buffer +- RNase A solution (QIAGEN, 100 mg/ml) +- Proteinase K solution (Fermentas, 20 mg/ml) +- Chloroform +- Ice-cold ethanol (96%) +- Ethanol (70%) + +### Protocol for CTAB DNA Isolation + +#### Step 1 +Perform the DNA isolation for all 11 samples simultaneously, including the negative control. + +#### Step 2 +Weigh 100 +/-10 mg per sample in a 2 ml tube. + +#### Step 3 +Add 300 µl nuclease-free molecular grade water. + +#### Step 4 +Add 700 µl CTAB extraction buffer and vortex at 1000 rpm for 10 seconds. + +#### Step 5 +Add 5 µl RNase (100 mg/µl) solution and gently shake the tube. + +#### Step 6 +Incubate at 65 °C for 15 minutes to ensure efficient cell disruption in a Thermo-Shaker at 1000 rpm. + +#### Step 7 +Add 20 µl of Proteinase K solution (20 mg/ml) and shake the vial again. + +#### Step 8 +Incubate at 65 °C for 1 hour in the Thermo-Shaker. + +#### Step 9 +Centrifuge the sample at 18,000 x g for 10 minutes. + +#### Step 10 +Fill a 1.5 ml tube with 500 µl of chloroform. + +#### Step 11 +Transfer the supernatant to the tube containing chloroform and shake the vial by hand for 30 seconds. + +#### Step 12 +Centrifuge at 18,000 x g for 10 minutes. Clearly separate the two phases. + +#### Step 13 +Transfer the upper layer to a new 1.5 ml tube and add 700 µl of chloroform, then shake for 30 seconds. + +#### Step 14 +Centrifuge for 10 minutes at 18,000 x g. + +#### Step 15 +Transfer the upper layer to a new 2 ml tube. + +#### Step 16 +Add 2 volumes of CTAB precipitation buffer and mix by pipetting up and down. + +#### Step 17 +Incubate at room temperature for 1 hour. + +#### Step 18 +Centrifuge for 10 minutes at 18,000 x g and remove the supernatant. + +#### Step 19 +Add 350 µl of NaCl (1.2 M) to the precipitate DNA. + +#### Step 20 +Add 350 µl of chloroform to the tube containing DNA and NaCl, vortex for 30 seconds. + +#### Step 21 +Centrifuge for 10 minutes at 18,000 x g. + +#### Step 22 +Transfer the upper layer to a new 1.5 ml tube. + +#### Step 23 +Add 2 volumes of ice-cold ethanol (96%) and mix by gently inverting the tube several times. + +#### Step 24 +Store the samples overnight at -20 °C. + +#### Step 25 +On the next day, centrifuge for 30 minutes at 18,000 x g and remove the supernatant. + +#### Step 26 +Add 500 µl of ethanol (70%) to wash the pellet. + +#### Step 27 +Centrifuge for 5 minutes at 18,000 x g and remove the supernatant by pipetting. + +#### Step 28 +Air-dry the pellet for 1 hour. + +#### Step 29 +Add 100 µl of water and let the pellet dissolve overnight at 4 °C. + +#### Step 30 +Vortex the dissolved DNA and short-spin for 10 seconds. + +#### Step 31 +Measure the DNA concentration using a spectrophotometer (e.g., NanoDrop ND1000) according to the manufacturer's protocol. + +#### Step 32 +No DNA yield should be present in the DNA isolation negative control. + +#### Step 33 +Label (sample name, date) the 1.5 ml tubes containing the purified DNA and store at 4 °C. + +### PCR + +#### Step 34 +Prepare a PCR mixture for each DNA barcode marker primer pair in 1.5 ml tubes using the provided chemicals and volumes listed in Table 1. + +| Component | Stock Concentration | Final Concentration | Volume (µl) | Volume (µl) X 13 | +| -------------- | ------------------- | ------------------- | ----------- | ---------------- | +| HotStarTaq MM | 2X | 1X | 12.5 | 187.5 | +| Forward Primer | 10 µM | 0.2 µM | 0.5 | 7.5 | +| Reverse Primer | 10 µM | 0.2 µM | 0.5 | 7.5 | +| ddH2O | - | - | 6.5 | 97.5 | +| Total Volume | - | - | 20 | 260 | + +*Note: The mixture should be prepared 12 times, once for each DNA barcode marker.* + +#### Step 35 +Vortex the PCR mixture for 10 seconds. + +#### Step 36 +Aliquot 20 µl of PCR mixture to each well according to the scheme in Table 3. + +#### Step 37 +Aliquot 5 µl of sample DNA template (10 ng/µl) to each well. + +#### Step 38 +Prepare positive and negative control wells using provided control DNA or ddH2O. + +#### Step 39 +Place the PCR tube strips in the thermocycler block and start the PCR program with the thermal profile outlined in Table 2. + +| Thermal Cycler Steps | Temperature (°C) | Time | Cycles | +| -------------------- | ---------------- | -------- | ------ | +| Hot Start | 95 | 15 min | - | +| Denaturation | 94 | 30 sec | 5 | +| Annealing | 49.5 | 40 sec | | +| Extension | 72 | 1 min | | +| Denaturation | 94 | 30 sec | 35 | +| Annealing | 54 | 40 sec | | +| Extension | 72 | 1 min | | +| Final Extension | 72 | 10 min | - | +| Hold | 8 | Forever | - | + +#### Step 40 +Ensure the PCR run has completed successfully with no error messages. + +#### Step 41 +After the PCR run, spin down all PCR tube strips for 15 seconds using a mini-centrifuge. + +#### Step 42 +Visualize 5 µl of PCR products using 1% agarose gel electrophoresis and appropriate staining methods. Compare the samples to a 100 bp DNA Ladder. + +| Marker | Fragment Size (bp) | +| ---------- | ------------------- | +| 16S | 650 | +| COI-2 | 720 | +| CytB | 800 | +| 16S-mini | 300 | +| COI-mini | 400 | +| CytB-mini | 400 | +| matK | 920 | +| rbcL | 650 | +| trnL(UAA) | 600 | +| ITS2 | 550 | +| rbcL-mini | 250 | +| trnL(P6loop) | 180 | + +#### Step 43 +Example gel images should show no DNA bands for negative control reactions. + +#### Step 44-54 +Follow the steps for pooling, purifying, and quantifying the PCR products with detailed precautions to prevent contamination. + +### Warnings + +- Ensure enough space between tubes during DNA isolation. +- Make sure all PCR tube caps are closed when adding DNA to PCR. +- Ensure only PCR tubes corresponding to the sample is opened when pooling samples. + +## References + +Refer to the list of cited references for further reading and method verification. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/development-of-a-doubled-haploid-mapping-populatio-x3vfqn6.md b/markdown-output/development-of-a-doubled-haploid-mapping-populatio-x3vfqn6.md new file mode 100644 index 0000000000000000000000000000000000000000..12d3382088b5d7bb9401a70feacc5d0936f96273 --- /dev/null +++ b/markdown-output/development-of-a-doubled-haploid-mapping-populatio-x3vfqn6.md @@ -0,0 +1,148 @@ +```markdown +# Goal/Experiment: +Development of a doubled haploid mapping population of **Pyropia yezoensis** and measurement of the economic characters of the blade + +## Development of a doubled haploid mapping population of *Pyropia yezoensis* and measurement of the economic characters of blade + +*Version 2* + +### PLOS One + +**Linbin Huang**, **Xinghong Yan** +Shanghai Ocean University +dx.doi.org/10.17504/protocols.io.x3fvg6 + +### ABSTRACT +*Pyropia yezoensis* is one of the most valuable and widely cultivated seaweeds across the world. However, there is limited data regarding its genetic background underlying complex economic traits. The molecular genetic linkage map of *P. yezoensis* was not constructed. The most probable cause was the lack of mapping population, which was the basic of linkage analysis. In the present study, a red-type and fast-growing pigmentation mutant Py-HT and a wild-type strain Py-LS who had enough variation for traits of interest at both DNA and phenotypic level were crossed under control. The heterozygote (heterozygous conchocelis) was identified among conchocelis colonies which were developed from single zygotospore released from zygotosporangia of fertilized Py-HT blade, based on the result that 91.9% F1 gametophytic blades developed from the conchospores of heterozygous conchocelis were linearly sectored with 2-4 color sectors of parental colors. Afterwards, 57 chimeric blades with four color sectors were screened from thousands of F1 blades and 228 color sectors were separated according to the boundaries of color sectors for single culture. A single zygotospore released from zygotosporangia of one sector through selfing was selected and then developed into homozygous conchocelis, which was declared a double haploid strain. Finally, a mapping population with 148 strains obtained from 37 of the 57 four color sectored blades was developed, which was used for the construction of genetic linkage maps and analysis of quantitative trait loci of *P. yezoensis* blades. + +### EXTERNAL LINK +[https://doi.org/10.1371/journal.pone.0209128](https://doi.org/10.1371/journal.pone.0209128) + +### PROTOCOL STATUS +Working + +--- + +## Preparation of Conchocelis + +1. **Stock culture of free-living conchocelis** + - *Pyropia yezoensis* wild-type strain Py-LS and red-type pigmentation mutant Py-HT were maintained in the laboratory at 19°C under 10 μmol photons m² s⁻¹ (10:14 LD) provided by cool-white, 40-W fluorescent lamps, in a 250 ml SEBC bottle with 200 ml culture medium, which was usually renewed 50% every 10~12 months. + + - The culture medium was natural seawater collected from the East China Sea off Qushan Island enriched with MES medium (10% Volume Percent). The formula of MES medium and MP II solution is shown in Tables 1 and 2, respectively. + +Table 1. Formula of MES medium + +| Component | Quantity | +|------------------------------------------|------------| +| Sodium nitrate (NaNO₃) | 2.8 g | +| Sodium glycerophosphate | 0.4 g | +| MP II solution | 200 ml | +| Tris (hydroxymethyl) aminomethane | 4.0 g | +| Distilled water | 700 ml | + +3 M HCl was used to adjust pH to 7.6, and the final volume was 1000 ml. + +Table 2. Formula of MP II solution + +| Component | Quantity | +|-----------|----------| +| EDTA-2Na | 3.0 g | +| H₃BO₃ | 2.5 g | +| MnCl₂·4H₂O| 0.35 g | +| FeCl₃·6H₂O| 2 ml | +| ZnCl₂ | 2 ml | +| CoCl₂·6H₂O| 2 ml | +| Fe-citrate| 2 ml | +| Distilled water | 1000 ml | + +**Note:** *Pyropia yezoensis* has a biphasic life cycle that alternates between macroscopic, foliose, gametophytic blades and microscopic, sporophytic filaments (referred to as the conchocelis phase). The gametophytic blade is haploid and develops from a conchospores produced by mature conchocelis. The conchocelis, which develops from a carpospore produced by the mature gametophytic blade, is shell-boring in nature. When the conchocelis grows in either seawater or liquid culture medium, it is called free-living conchocelis. + +--- + +## Collection of Conchospores + +2. **Conchospores collection** + - A small amount of conchocelis (about 10 μg) of each strain was sampled from stock culture and incubated in a 90 mm petri dish at 23°C under 20 μmol photons m² s⁻¹ (10:14 LD) to induce the formation of conchosporangia. + + - When conchosporangia appeared, the conchocelis was transferred into a 250 ml Erlenmeyer flask containing 200 ml culture medium and cultured with aeration in an incubator at 19°C under 40 μmol photons m² s⁻¹ (10:14 LD). + + - Vinylon monofilaments (about 3 cm long) were placed in the flask for attachment of conchospores. Then, the monofilaments with attached conchospores were transferred to a new flask and cultured under the same condition to obtain conchospores gemlings (gametophytic blades). + +**General cultural conditions** +250 ml Erlenmeyer flask containing 200 ml culture medium and cultured with aeration in an incubator at 19°C under 40 μmol photons m² s⁻¹ (10:14 LD). The culture medium was renewed 50% every 5 days. + +--- + +## Cross of Blades + +3. **Crossing the blades** + - When gametophytic blades of each strain grew up to 1-2 cm long, they were detached from vinylon monofilaments and cultured individually (one blade cultured in one flask). + + - When spermatangia appeared in the blade, but before spermatia release, the upper peripheral portions of one blade of Py-HT strain and one blade of Py-LS strain were cut out and co-cultured until carposporangia appeared. + + - Individual fertilized blade was then transferred to a 500 ml flask with 450 ml culture medium and cultured until carposporangia mature. In the present study, only fertilized blade of Py-HT strain was selected for releasing carpospores. + + - The blade with mature carposporangia was dried in the shade at 19°C and immersed in a 90 mm petri dish with 50 ml culture medium for 1 hour to induce the release of carpospores. + *Note:* The blade was subcultured in the flask and used for releasing carpospores at the next several days. + + - The carpospores from Py-HT blade grew into conchocelis colonies in 90 mm petri dishes at 19°C under 20 μmol photons m² s⁻¹ (14:10 LD). When the color of conchocelis colonies became distinguishable microscopically, individual colonies with the color of wild-type were picked out by means of a finely drawn Pasteur pipette and transferred to petri dishes or test tubes for culture. + +**Note:** During the culture of carpospores and conchocelis colonies, the culture medium can only be renewed after the colonies become macroscopic. + +### Determination of Heterozygote + +4. **Determination of heterozygote** + - After approximately 3 months in culture, when wild type color conchocelis clumps grew into approx. 10 mm in diameter, they were cultured at 23°C under 30 μmol photons m² s⁻¹ (10:14 LD) for induction of conchosporangia. + +**Further steps:** + - F1 gametophytic blades developed from conchospores were detached from the monofilaments with a surgical blade. + - Color phenotypes and blade types of the F1 blades were examined microscopically. + + - More than 90% F1 blades of one conchocelis were 2-4 color-sectored blades; thus, indicating the conchocelis as heterozygote. + +**Note:** For *Pyropia yezoensis*, the blades are monoecious and could be self-fertilized, the heterozygote could only be identified through F1 blades if they are mainly color-sectored, which depended on tissue culture techniques. + +--- + +## Construction of a Doubled Haploid Population + +5. **Construction of DH population** + - Only four-color sectored mosaic blades were screened from the F1 blades. + + - Every four-color sectored blade was then cut into four color-sectors along the boundaries of adjacent color-sectors and every color-sector was cultured individually. + + - A DH strain was obtained when one of the carpospores was released from a self-fertilized color-sector and developed into a single conchocelis. + +**Further steps:** +Collection of carpospores and culture of conchocelis according to steps in **Collection of Conchospores**. + +**Note:** In *Pyropia yezoensis*, the blades are monoecious and could be self-fertilized. Therefore, the DH population can be established by self-fertilization because spermatia (male gamete) and carpogonium (female gamete) always occur diffusely on a single sector developed from one of the tetrad cells after mitosis. Through self-fertilization of a color-sector, a procedure of chromosome doubling of gamete was considered. + +--- + +## Measurement of the Economic Characters of the Blade + +6. **Measurement of economic characters** + - All the conchospores of 148 strains were collected and attached to Vinylon monofilaments. + + - Gametophytic blades of each strain developed from conchospores were cultured in a 300 ml flask with 250 ml culture medium at 19°C under 28-30 μmol photons m² s⁻¹ (10:14 LD). The medium was renewed 50% every 10 days. + + - Blades were detached from monofilaments after 30 days in culture. Then, 100 intact blades with holdfasts and tips were screened and transferred to a 600 ml flask with 500 ml culture medium (half old and half new medium) under 38-40 μmol photons m² s⁻¹. + + - After 5 days in culture, 50 intact blades (35-day old) were screened randomly, and half medium was renewed. + + - After 5 days, 30 intact blades (40-day old) were screened randomly and transferred to a 1200 ml flask with 1000 ml culture medium (half old and half new medium). The medium was renewed 50% every 5 days. + + - The length, width, and fresh weight of 40-day old blades (L40, W40, and FW40) were measured. The blade length was measured from the holdfast to the blade tip. The blade width was measured at the widest part of the blade. The fresh weight was measured after surplus surface water was sucked up with a paper towel. + + - After 10 days in culture, the length, width, and fresh weight of 50-day old blades (L50, W50, and FW50) were measured. + + - The specific growth rate of blade length, width, and fresh weight between 40th and 50th day (LGR, WGR, and FWGR) were calculated based on the formula: + + \[ LGR (\%) = \frac{\ln(L50) - \ln(L40)}{n} \times 100 \] + + Where ln was the natural logarithm, L50 and L40 were blade length at 40th and 50th day, respectively, and n was the interval of 10 days. +--- + +endofoutput +``` diff --git a/markdown-output/diaetoxil-avis-o-acheter-en-ligne-conseils-d-39-ac-cc26syhe.md b/markdown-output/diaetoxil-avis-o-acheter-en-ligne-conseils-d-39-ac-cc26syhe.md new file mode 100644 index 0000000000000000000000000000000000000000..b69ff73a2cf3ac1e3a0c68cfc6484cc291e15124 --- /dev/null +++ b/markdown-output/diaetoxil-avis-o-acheter-en-ligne-conseils-d-39-ac-cc26syhe.md @@ -0,0 +1,88 @@ +```markdown +Goal/Experiment: +To review and provide purchasing advice for Diaetoxil in 2022. + +# Diaetoxil Avis: Où acheter en ligne {Conseils d'achat 2022} + +Author: roxypins + +[DOI Reference](https://dx.doi.org/10.17504/protocols.io.36wgq7zj3vk5/v1) + +## Disclaimer + +> **Diaetoxil Avis France**: Le complément alimentaire révolutionnaire Diaetoxil Gélules, dont l'efficacité a été scientifiquement prouvée, est désormais disponible pour les personnes au régime qui souhaitent perdre du poids de la manière la plus naturelle possible tout en préservant leur santé et leur bien-être. +> +> **Diaetoxil Avis France** Contrairement à la plupart des autres régimes, le régime cétogène limite votre apport en glucides à moins de 50 g par jour. En conséquence, votre corps est poussé à brûler les graisses pour obtenir de l'énergie, ce qui accélère considérablement le processus de perte de poids. Lorsque vous suivez un régime cétogène, votre corps réagit différemment! + +![Diaetoxil 600 mg](https://healthcare24hrs.com) + +## Abstract +Diaetoxil Avis France is an innovative dietary supplement, developed to assist individuals aiming to lose weight. It is particularly beneficial for those following a ketogenic diet, which limits carbohydrate intake to under 50 grams per day. This necessitates the body to burn fat for energy, hence accelerating weight loss. + +## Key Links +- [**Visit Healthcare24hrs.com**](https://healthcare24hrs.com) +- [Diaetoxil Avis on Facebook](https://www.facebook.com/DiaetoxilAvis/) +- [Articles on IPSNews](https://ipsnews.net/business/2022/07/01/diaetoxil-avis-france-gelules-diaetoxil-erfahrungen-bezugsquellen-entgiftung-avis/) + +## Watch Our Official Video Before Buying +[Official Video](https://youtu.be/PXuIY3rz3z0) + +## What is Diaetoxil France? +Diaetoxil Avis France is a natural and effective weight loss product for various slimming situations. It does not include any nutritional component and has been introduced for safe use without adverse health impacts. + +## Benefits of Using DIAETOXIL Capsules + +1. **Reduces Bad Cholesterol (LDL)** + - Weight and obesity are linked to higher LDL cholesterol levels and lower HDL cholesterol levels. Studies show noticeable changes in cholesterol levels even with slight weight loss (5 to 10 pounds). + +2. **Delay or Reverse Type 2 Diabetes** + - Obesity increases the risk of type 2 diabetes. By reducing weight and maintaining a healthy weight, one can manage blood sugar levels. + +3. **Improves General Mood** + - Losing weight can help improve mental health, reducing despair and anxiety, connected with obesity. + +4. **Lower Blood Pressure** + - Weight loss can lower blood pressure, which is essential in preventing hypertension. + +5. **Reduce Cancer Risk** + - Obesity is linked to various cancers. Reducing weight can decrease inflammation and hormonal imbalances associated with cancer. + +## How Does Diaetoxil Work? +It is recommended to take Diaetoxil Gélules 30 minutes before the largest meal of the day to maximize effectiveness. Drinking at least 500 ml of water daily improves results. + +## Scientific Evidence +Primo Weight Reduction incorporates natural substances proven to support weight reduction and maintenance of a healthy weight. Produced in an FDA-approved facility, its formulation is rigorously tested. + +### Key Ingredients and Their Functions +- **L-Arginine**: Essential amino acid aiding in growth hormone production and vasodilation. +- **L-Carnitine**: Helps improve brain, heart, and muscle functions. +- **Gelatine**: Encasement agent derived from animals. +- **Magnesium Stearate**: Commonly used as a lubricant in capsule production. +- **Silicon Dioxide**: Prevents caking and clumping in supplements. +- **Rice Flour**: Used as a filler in some dietary supplements. + +## Usage and Dosage + +- **Consumption Method**: Take the capsule 15-30 minutes before a heavy meal with 2 glasses of water. +- **Duration**: Effects can be seen as early as three months with continuous use. For optimal results, adhere to the supplement's usage guidelines. + +## Possible Side Effects +No significant side effects have been noted. The product is safe for use as directed. + +## Conclusion +Diaetoxil Avis France offers a natural solution for weight loss without requiring a prescription. The product is suitable for anyone aiming to lose weight and improve general health while avoiding the complications associated with obesity. + +## Pricing +- One box: 49.95 euros +- Two boxes: 79.95 euros + +## Notice Before Purchase +One month's supply is recommended for beginners. Regular follow-up with a healthcare provider is advised. + +## References +- [Healthcare24hrs.com](https://healthcare24hrs.com) +- [Official Video](https://youtu.be/PXuIY3rz3z0) +- [Facebook Links](https://www.facebook.com/DiaetoxilAvis/) + +Endofoutput +``` \ No newline at end of file diff --git a/markdown-output/differentiation-of-mesenchymal-stromal-cells-to-en-ddnd25a6.md b/markdown-output/differentiation-of-mesenchymal-stromal-cells-to-en-ddnd25a6.md new file mode 100644 index 0000000000000000000000000000000000000000..11ded317df396c443fee6e6bb38ae285e2634134 --- /dev/null +++ b/markdown-output/differentiation-of-mesenchymal-stromal-cells-to-en-ddnd25a6.md @@ -0,0 +1,175 @@ +```markdown +# Goal/Experiment: +The goal of this experiment is to differentiate Mesenchymal Stromal Cells (MSCs) to Endothelial-like cells using spheroidal culture. The protocol encompasses the formation of MSC spheroids and their subsequent endothelial differentiation and analysis. + +# Differentiation of Mesenchymal Stromal Cells to Endothelial-like Cells in Spheroidal Culture V.3 + +## DOI +[dx.doi.org/10.17504/protocols.io.5jyj82m17l2w/v3](https://dx.doi.org/10.17504/protocols.io.5jyj82m17l2w/v3) + +## Authors +- Simeng Li¹ +- Isabel Arias Quiros¹ +- Guenther Eissner¹ + +¹University College Dublin + +## Abstract +In this protocol, an easy and cost-friendly method to form mesenchymal stromal cell (MSC) spheroids was specified. The MSC spheroids form in 1-3 days and are suitable for endothelial differentiation. Accutase treatment can be used to disassociate the spheroids into single cells for further analysis. Multiple growth factors were used to differentiate MSC Spheroids into endothelial-like spheroids, which express endothelial markers such as von Willebrand factors (vWF) and CD31 detected by immunofluorescence. + +## Keywords +Mesenchymal stromal cells, Spheroids culture, endothelial differentiation, Spheroids disassociation + +## Materials +| Reagent Name | Company/Brand | Catalogue number | +|------------------------------------|---------------------------|-----------------| +| 0.05% Trypsin-EDTA (1x) | Gibco | 25300-054 | +| Trypsin Neutralizing Solution | Lonza | CC-5002 | +| Recombinant human epidermal growth factor/EGF | PELOBiotech | C029-B | +| Recombinant human fibroblast growth factor (bFGF) | PELOBiotech | C046-A | +| Recombinant human vegf-a/vegf165 | PELOBiotech | C083-A | +| Propidium Iodide Solution | Sigma-Aldrich | P4864 | +| Mesencult-ACF Plus culture kit | Stemcell Technologies | 5448 | +| L-Glutamine (100x) | Gibco | 25030-024 | +| iBiD Angiogenesis Slides | ibidi | 81506 | +| 96-well, round bottom Culture plate| VWR | 392-0291 | +| Dulbecco PBS, w/o Ca++/ Mg++ | Promocell | C-40232 | +| Human serum from human male ab plasma | Sigma-Aldrich | H4522 | +| ibidi mounting medium | ibidi | 50001 | +| Trypan Blue Solution, 0.4% | Gibco | 15250061 | +| PBS, pH 7.2 | Gibco | 20012027 | +| TWEEN 20 | Sigma-Aldrich | P1379 | +| Tris Buffered Saline (TBS), 10x | Sigma-Aldrich | T5912 | +| Paraformaldehyde (PFA), 16% w/v, methanol free | Thermo Scientific Chemicals | 043368-9M | +| Bovine Serum Albumin (BSA) | Sigma-Aldrich | A3294 | +| Fetal Bovine Serum (FBS) | Sigma-Aldrich | F7524 | +| Triton X-100 | Sigma-Aldrich | T8787 | +| Olympus FV1000 confocal microscope | Olympus | / | +| Accuri C6 flow cytometer | BD Biosciences | / | + +## Protocol + +### Mesenchymal Stromal Cells (MSC) Culture + +1. **Coat Culture Flasks:** + - Coat culture flasks with Animal Component-Free Cell Attachment Substrate prior to cell seeding. + - Dilute Animal Component-Free Cell Attachment Substrate 1:15 in D-PBS (without Ca++ and Mg++) and coat the culture wares for 2 hours at room temperature (15-25°C), using a sufficient volume to cover the culture surface. + - Alternatively, coat flasks overnight at 2-8°C with flask lid sealed by parafilm. + +2. **Bring Flasks to Room Temperature:** + - Bring flasks with attachment substrate to room temperature if flasks were coated at 2-8°C. + - Remove attachment substrate completely by tilting the flask and gathering the substrate to the edge of the flask. + +3. **Wash Flasks:** + - Wash flasks once with D-PBS. The flasks are now ready to use. + +4. **Complete Medium Preparation:** + - Complete 500 mL MesenCult-ACF Plus Medium by adding 1 mL of MesenCult-ACF Plus 500X Supplement and 2 mM of L-glutamine. + +5. **Trypsinize MSCs:** + - Trypsinize MSCs with controlled treatment time. Normally 30s-1min is sufficient to detach MSC from flasks. + - Use Trypsin neutralizing solution to neutralize trypsin since there is no serum in the cell culture medium. + +6. **Count Cells and Seed MSC:** + - Count cells with Trypan Blue exclusion and seed MSC in fully-supplemented MesenCult-ACF Plus Medium. + +### Mesenchymal Stromal Cell Spheroids Formation + +7. **Resuspend MSC:** + - Split and resuspend MSC in fully-supplemented MesenCult-ACF plus medium. + +8. **Dilute Cells:** + - Count cells using Trypan blue and dilute the cells to 3x10e5/mL or 12x10e5/mL. + - Cells need to be over 95% viable to form spheroids. + +9. **Seed Cell Suspension:** + - Seed 50 µL of cell suspension to each well of a 96-well round-bottom plate. Each well will contain 15,000 cells. + +10. **Incubate MSC:** + - Keep MSC in the incubator for 1-3 days until one spheroid forms in each well. + - Depending on the size of the spheroids, they can be observed either with the naked eye or using a microscope. + - Maintain culture conditions at 37°C, 5% CO₂, and 80% humidity. + +### Endothelial Differentiation of MSC Spheroids + +11. **Endothelial Differentiation Medium Preparation:** + - Make endothelial differentiation medium by adding 10 ng/mL (final concentration) VEGF, EGF, bFGF, and 2% (v/v) human serum (HS) into fully supplemented MesenCult ACF-plus medium. + +#### MSC Spheroids Formation After 3 Days in Culture + +- Primary umbilical cord MSC and hTERT BMMMSC (an immortalized bone marrow MSC cell line) were used in the experiment. +- MSC were seeded at 60,000 or 15,000 per well. +- Visible spheroids formed in each well after 3 days of culture. However, more than one spheroid formed in some wells, especially in the wells with 60,000 MSC seeded. +- This observation suggests 15,000 cells per well is a good starting concentration to form MSC spheroids. + +12. **Remove Media:** + - Carefully remove the media from each well, avoiding the spheroid sticking at the end of the tips. + +13. **Add Differentiation Medium:** + - Add 50 µL of differentiation medium to each well. Keep some spheroids as untreated control by adding normal culture medium. + +14. **Differentiation Duration:** + - Differentiation duration can be between 5-11 days and can be determined by how advanced the differentiated cells need to be. + +### Immunofluorescence to Check Endothelial Markers of Differentiated Spheroids + +15. **Remove Medium:** + - Remove the medium in each well carefully. Before fixation and permeabilization, wash spheroids three times with PBS, 5 minutes each. + +16. **Fixation:** + - Use the same 96-well round-bottom plate for fixation. Fixation was performed with 4% PFA for 1 hour at room temperature (RT). Fixation time varies depending on the size of spheroids. + +17. **Wash with PBS:** + - Wash spheroids with 100 µL PBS per well three times, 5 minutes each. + +18. **Block Spheroids:** + - Block spheroids using blocking buffer (PBS with 3% FBS, 1% BSA, 0.5% Triton X-100, and 0.5% Tween) for 2 hours at RT on a shaker. + +19. **Primary Antibody Incubation:** + - Dilute primary antibodies 1:100–1:200 (or according to manufacturer’s instructions) in the blocking buffer. Add 50 µL of primary antibody to each well and incubate overnight at 2-8°C. Primary antibodies from different species can be used together. + +20. **Wash with PBST:** + - Remove solution from each well and wash spheroids with PBST (0.05% Tween 20 detergent in PBS) for 10 min at RT, three times. + +21. **Add Secondary Antibodies:** + - Add corresponding secondary antibodies at 1:250 in blocking buffer for 2 h at RT. + +22. **Wash with TBST:** + - Remove solution from each well and wash spheroids with TBST (0.1% Tween 20 detergent in Tris-buffered saline), 20 min each time, 3 times. + +23. **Stain with DAPI:** + - Stain spheroids with 300nM DAPI for 20 min at 37°C. Then wash the spheroids once with 50 µL PBST for 5 min. + +24. **Transfer Spheroids:** + - Use a 1 mL pipette to take the spheroid up together with the solution from each well. Transfer the spheroid to a well of ibidi μ-Slide 15 Well 3D. + +25. **Mount Spheroids:** + - Remove PBST from each well of ibidi μ-Slide, add a drop of ibidi mounting medium to mount the spheroid. Control the amount of mounting medium added, so the spheroids are located at the bottom of each well. + +26. **Imaging:** + - Image the spheroids with an Olympus FV1000 confocal microscope (Olympus Lifescience), or other suitable confocal microscope. + +### Disassociation of MSC from Spheroids for Flow Cytometry Analysis + +27. **Harvest Spheroids:** + - Harvest 10 spheroids from each condition into one 1.5 mL Eppendorf tube. Centrifuge at 300g for 4 min. Then remove and discard the supernatant. + +28. **Add Accutase:** + - Add 400 µL Accutase solution (Sigma/ Cat: 6964) to each tube and incubate the tube on a Thermomixer comfort (Eppendorf, Hamburg, Germany) at 37°C, 1400 rpm for 10 minutes. Use a 200 µL pipette to pipette up and down for ten times to further disassociate the spheroids. This is considered one disassociation cycle. + +29. **Repeat Disassociation:** + - Repeat the disassociation cycle 1-3 times more until no visible cell clumps remain in the tube. + +30. **Final Wash:** + - Wash cells once with 1 mL PBS. Centrifuge at 300g for 5 min and then cells can be resuspended in flow cytometry buffer for antibody labeling. + +## Protocol References +1. Redondo-Castro, E., et al., Generation of Human Mesenchymal Stem Cell 3D Spheroids Using Low-binding Plates. Bio Protoc, 2018. 8(16). +2. Grasser, U., et al., Dissociation of mono- and co-culture spheroids into single cells for subsequent flow cytometric analysis. Ann Anat, 2018. 216: p. 1-8. +3. Wimmer, R.A., et al., Human blood vessel organoids as a model of diabetic vasculopathy. Nature, 2019. 565(7740): p. 505-510. +4. Oswald, J., et al., Mesenchymal stem cells can be differentiated into endothelial cells in vitro. Stem Cells, 2004. 22(3):p. 377-84. +5. Gang, E.J., et al., In vitro endothelial potential of human UC blood-derived mesenchymal stem cells. Cytotherapy, 2006. 8(3): p. 215-27. +6. Konno, M., et al., Efficiently differentiating vascular endothelial cells from adipose tissue-derived mesenchymal stem cells in serum-free culture. Biochem Biophys Res Commun, 2010. 400(4): p. 461-5. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/dilution-to-extinction-experiment-protocol-cux7wxrn.md b/markdown-output/dilution-to-extinction-experiment-protocol-cux7wxrn.md new file mode 100644 index 0000000000000000000000000000000000000000..e0c2e4a412bedbfb6c43edcd64a16b94d1cc0a12 --- /dev/null +++ b/markdown-output/dilution-to-extinction-experiment-protocol-cux7wxrn.md @@ -0,0 +1,207 @@ +```markdown +# Goal/Experiment: +Dilution-to-Extinction Experiment Protocol aims to isolate single microbial cells by diluting a sample until each well in a multi-well plate statistically contains one cell. It allows for culturing and identifying clonal populations from environmental samples. + +## Dillution-to-Extinction Experiment Protocol V.2 + +**Authors:** +Michael Henson +Celeste Lanclos +Shelby J Barnes +Cameron Thrash +Jordan Coelho + +**Institution:** +University of Southern California + +**Version:** +2 - July 10, 2023 + +**DOI:** +[10.17504/protocols.io.36wgq7d2ovk5/v2](https://dx.doi.org/10.17504/protocols.io.36wgq7d2ovk5/v2) + +**License:** +Creative Commons Attribution License (CCAL) + +--- + +### Abstract +Dilution-to-Extinction Experiment Protocol + +--- + +## Materials + +### Medium Preparation + +#### Defined Media +- Acid-washed and autoclaved Pyrex screw-top bottles (Corning) + **Reference:** SJB Artificial Media Protocol DOI: [10.17504/protocols.io.rm7vzy615lx1/v1](https://dx.doi.org/10.17504/protocols.io.rm7vzy615lx1/v1) + +#### Collection and Preparation +- **10% HCl**: For acid washing collection carboys, medium preparation bottles, and incubation PTFE plates and flasks (~25 L). + - **Alternative:** 1% HCl can be used if the higher concentration is not available. +- **2.7 μm Whatman GF/D glass fiber filters (25 mm)** and housings. + - **Alternative:** Pall PES filters Supor (25mm) and housings or vacuum filtration setups. + +### Inoculum Sample Enumeration + +- **BD Accuri C6 Plus Flow Cytometer**: Flow cytometer system for counting cells (BD Biosciences) +- **SYBR Green (Life Technologies)**: DNA stain, diluted to 100x (from 10,000x stock) in TE. +- **Corning**: Non-sterile, round-bottom 96-well assay plates. + - **Alternative:** Use similar 96-well assay plates from other vendors. + +--- + +## Procedure + +### Medium Inoculation, Distribution, and Incubation + +#### Equipment Required: +- **PTFE 2.1 mL, 96-well plates**: For cultivation (Radleys, Essex). + - Acid-washed and autoclaved. +- **PTFE-coated silicon 96-well plate mats**: To prevent contamination and evaporation (Thermo Scientific). + - Acid-washed and autoclaved. +- **PIPETMAN L Multichannel P8x1200L, 100-1200 μL** (Gilson). +- **Laminar flow hood or biosafety cabinet**: For inoculation/transfer. +- **Incubator**: Capable of maintaining in-situ temperatures. + +#### Steps: +1. Using a laminar flow hood, pipette STEM medium and distribute it equally into the 96-well plates. +2. Use the PIPETMAN L multichannel pipette to add inoculum in serial dilutions. +3. Incubate the plates at room temperature (~25°C) for 2–4 weeks. + +### Large Format Enumeration +- When culturing larger volumes, transfer 200 μL aliquots using the pipette into 96-well counting plates. +- Stain samples using 2 μL SYBR Green freshly diluted in TE buffer. + +### Culture Filtering and DNA Extraction + +#### Membrane Filtration +- **Supor PES Membrane Disc Filters**: Available from multiple suppliers for filtering cultures. + +#### DNA Extraction +- **GenElute™ Bacterial Genomic DNA Kit**: For genomic DNA extraction. + +### PCR Preparation and Purification + +#### Preparation +- **Taq DNA Polymerase, recombinant** (Thermo Fisher Catalog #10342020) +- **16S rRNA Primers**: + - 27F: 5’-AGAGTTTGATCMTGGCTCAG + - 1492R: 5’-GGTTACCTTGTTACGACTT +- **MilliporeSigma**: dNTP Mix, 10mM. +- **Molecular Biology Grade Water** + +#### PCR Master Mix (Per Sample) +- Buffer: 5 μL +- Forward Primer (10 μM): 5 μL +- Reverse Primer (10 μM): 5 μL +- MgCl₂: 1.5 μL +- dNTP: 1 μL +- Taq Polymerase: 0.2 μL + +### PCR Product Purification +Following either: +- **GeneJET PCR Purification Kit** (Thermo Fisher Catalog #K0702) +- **Ampure XP beads** (Beckman Coulter Catalog #A63881) + +### Medium Preparation + +#### Defined Medium +1.1. Can be prepared in advance using the SJB Artificial Media Protocol or an alternative medium of choice. + +### Collection and Filtration + +#### Culturing Hardware Preparation +2.1. **Culturing Hardware Washing**: Use lab-grade detergent, hot/cold DI water, followed by autoclaving. +2.2. **Overnight HCl Bath** of all hardware, rinse with DI water before autoclaving. + +#### Sample Collection +3.1. Collect samples in sterile, acid-washed carboys. +3.2. Transport samples back to the lab, using appropriate measures to minimize contamination during transport. + +### Inoculum Sample Enumeration + +#### Preparation +5.1. Enumerate total cells per microliter using BD Accuri C6 Plus Flow Cytometer. +5.2. Add artificial medium controls (stained and unstained SYBR Green). +5.3. Transfer 198 μL of each sample in triplicate to 96-well plates. +5.4. Transfer 400 μL controls to 0.5 mL microcentrifuge tubes, then add 198 μL to plates. +5.5. Stain samples and controls with SYBR Green (final concentration 1x). +5.6. Incubate samples for 30 minutes in the dark. + +### Run Settings and Worklist Setup + +6.1. Use BD Accuri C6 Plus Software. +6.2. Adjust thresholds and run control samples using settings defined by the experiment. +6.3. Custom run settings tailored to applications. +6.4. Use various plot setups for evaluating counts. +6.5. SIP settings: Rinse and clean after each run. + +### Analysis and Gating + +7.1. Calculate cell concentration using plots with green fluorescence. +7.2. Apply global gate on stained artificial medium. +7.3. Customize gates for variations in signal. + +### Medium Inoculation + +8.1. Dilute cells before inoculation into deep 96-well plates. +8.2. Calculate volume and distribute evenly ensuring the cell count is optimal. + +### Inoculated Medium Distribution and Incubation + +#### Manual Distribution +9. Distribute using a P1200 multichannel pipette or automated system. +10. Manual distribution steps, including careful aliquoting, and use of silicon lids for incubation. + +#### Automated Distribution +11. Use Eppendorf epMotion 5075. +11.1. Follow manufacturer's setup instructions. + +### Large Format Enumeration + +12. Utilize bio-safe cabinet and multichannel pipettes for safe aliquoting and counting. +12.1. Use Eppendorf epMotion 5075 or similar equipment for large format processing. + +### Transferring Plate Isolates and Culture Maintenance + +13. Wells with positive cell densities (>10^4) are transferred. +14. Use appropriate volumes and media for maintaining cultures. + +### Identification through 16S rRNA PCR Amplification + +15. Use extracted genomic DNA or maintained cells. +16. Filter cultures with PES filters for DNA templates. +17. Store filters at -20°C until extraction. +18.1. Extract DNA using GenElute™ Bacterial Genomic DNA Kit. +18.2. Follow kit protocol. + +### 16S rRNA PCR Preparation and Purification + +19. Setup PCR master mix. Use 50 µL reactions. + - Master mix: 15 µL + - gDNA: 3 µL + - H₂O: 32 µL + +19.2. Thermocycler conditions: + - 105°C Lid, 50 µL sample volume. + - 94°C 3 mins, 94°C 30 secs, 50.8°C 30 secs, 72°C 2 min. + - Repeat steps for 34 cycles. Hold at 4°C. + +20. Store or purify PCR products. +21. Verify PCR product with 1.5% agarose gel. +22. Purify PCR product using either GeneJet kit or Ampure XP beads. + +### Sanger Sequencing Preparation + +23. Quantify product, set up sequencing samples as per guidelines. +23.1. Prepare forward and reverse sequences. +23.2. Follow instructions by the sequencing service. +23.3. Submit sequencing request. + +--- + +endofoutput +``` diff --git a/markdown-output/dispersed-pancreas-cw4uxgww.md b/markdown-output/dispersed-pancreas-cw4uxgww.md new file mode 100644 index 0000000000000000000000000000000000000000..fba7ee7f1cccaea4feef60b11d9ea37bd106c5e9 --- /dev/null +++ b/markdown-output/dispersed-pancreas-cw4uxgww.md @@ -0,0 +1,225 @@ +```markdown +# Goal/Experiment: +Dispersed pancreas protocols to isolate, culture, and analyze islets, ducts, and acinar cells from human pancreas samples for research purposes. + +## Dispersed Pancreas Protocols + +### Abstract +Dispersed pancreas protocols. + +### Culture Media + +#### 1. Human islets: +- **Dulbecco's Modification of Eagle's Medium (DMEM, 4.5 g/L glucose, L-glutamine, sodium pyruvate)** + - Source: Vendor-specific, e.g., Gibco +- **Fetal Bovine Serum (FBS)** at 10% + - Source: Various suppliers, heat-inactivated +- **Penicillin-Streptomycin** at 1% + - Antibiotic solution to prevent bacterial contamination +- **GlutaMAX™ supplement** at 1% + - Stable form of L-glutamine for cell culture + +#### 2. Human exocrine tissues (ducts and acinar): +- **DMEM (4.5 g/L glucose, L-glutamine, sodium pyruvate)** +- **Fetal Bovine Serum (FBS)** at 5% +- **Dexamethasone** at 1 µg/mL + - A synthetic adrenal corticosteroid +- **Soya Trypsin Inhibitor** at 0.1 mg/mL + - Inhibits trypsin and prevents autolysis of cells +- **Insulin** at 100 nM + +#### Incubation +- **37°C Cell Culture Incubator** in an atmosphere of >95% humidity + +### Dithizone (DTZ) Staining and Islet Equivalent + +#### 1. Materials and Reagents: +- **Dithizone (Sigma-Aldrich), cat # D-5130** +- **Dimethyl sulfoxide (DMSO)** +- **1mL Syringe** +- **30mm Syringe filter 0.46µm nylon membrane** +- **Microscope with a micrometer in the scope to measure the diameter of the islets** + +#### Procedure: +1. Dissolve 10 mg of DTZ in 4 mL of DMSO. +2. Add 36 mL of HBSS (or any medium without serum). +3. Aliquot the solution and keep it frozen. +4. Filter and add a few drops to the medium where islets are kept. Storage: Up to 1 week at room temperature. + +#### To Count Islet Equivalence: +1. Resuspend the islets in a known volume (25-50 mL). +2. Take three or more samples of 50 or 100 µL and put them on a dish. +3. Count all the islets and measure their diameter. +4. Convert each size range with the conversion factor to get a total number by considering the dilution factor. +5. Note that six 50 µm islets equal one 150 µm islet. + +### Disperse Islets, Ducts and Acinar Into Single Cells + +#### 1. Materials and Reagents: +- **Phosphate-Buffered Saline (PBS)** without calcium and magnesium +- **TrypLE™ Express [−] Phenol Red** +- **Water bath at 37°C** +- **Vortex** +- **Centrifuge** + +#### Procedure: +1. Collect islets, ducts, or acinar cells + medium into a 15 mL tube. Centrifuge at 1.5 rpm for 2 minutes. +2. Aspirate medium and add 10 mL of PBS. Centrifuge at 1.5 rpm for 2 minutes. +3. Repeat step 2. +4. Aspirate PBS and add TrypLE™ (e.g., 750 µL for ~1,000 islets). +5. For acinar cells: Add enough TrypLE™ to cover the tissue. +6. Water bath: 37°C for 10 minutes. Vortex tubes every 3 minutes for 10 seconds. +7. Stop TrypLE™ reaction with cold media. +8. Centrifuge at 2 rpm for 2 minutes. Resuspend cell pellet in pre-warmed medium. +9. Incubate according to protocol. + +### B-Gal Activity While Excluding For CD45/CD11B Using APC + +#### 1. Materials and Reagents: +- **Cellular Senescence Live Cell Analysis Assay Kit (Enzo) cat # ENZ-KIT 130-0010** +- **Phosphate-Buffered Saline (PBS)** without calcium and magnesium +- **Water bath at 37°C** +- **FACS Buffer: PBS with 2% FBS** +- **Normal Rat Serum** +- **Normal Goat Serum** +- **CD45** +- **CD11b** +- **Vortex** +- **Centrifuge** +- **37°C Cell Culture Incubator** +- **Flow cytometer equipped with 488 nm laser source** + +#### Procedure: +1. Collect cells + medium into a 50 mL tube. Centrifuge at 2 rpm for 2 minutes. +2. Aspirate medium and add pre-warmed medium to culture cells into a 24-well plate (1 mL per well). +3. Add 1x Cell Pre-Treatment Solution and incubate at 37°C for 2 hours. +4. Directly add SA-β-Gal Substrate to cells. Incubate at 37°C for 1 hour. +5. Collect cells into a 1.5 mL tube. Centrifuge at 2 rpm for 2 minutes. +6. Wash the stained cells two times with PBS. +7. Block for 45 minutes on ice with 100 µL per sample of FACS buffer with CD45 and CD11b. +8. Centrifuge at 2 rpm for 2 minutes and aspirate buffer. +9. Wash the stained cells two times with FACS buffer and resuspend in 400 µL of FACS buffer for flow cytometry. + +### Glucose Stimulated Insulin Secretion (GSIS) In Human Pancreatic Islets + +#### 1. Materials and Reagents: +- **Krebs Ringer Bicarbonate Hepes (KRBH)** + +##### Mixed Salts Stock Solution: +| Compound | g/L | +|-----------------|------:| +| NaCl | 35.4 | +| KCl | 1.78 | +| KH2PO4 | 0.81 | +| MgSO4.7H2O | 1.46 | +| CaCl2.2H2O | 1.85 | + +##### Sodium Bicarbonate Stock Solution: +| Compound | g/L | +|----------|------:| +| NaCl | 5.35 | +| NaHCO3 | 2.63 | + +##### Working KRBH +- **20 mL mixed salts stock solution** +- **16 mL sodium bicarbonate stock solution** +- **64 mL dH2O** +- **0.42 g NaHEPES** +- **0.1 g BSA** +- **pH 7.4** + +##### Reagents +- **45% Glucose Solution (CORNING) cat # 25-037-Cl** +- **Phosphate-Buffered Saline (PBS)** without calcium and magnesium +- **10 mL Syringe** +- **30 mm Syringe filter 0.45 µm nylon membrane** +- **37°C Cell Culture Incubator** + +#### Procedure: +1. Filter working KRBH into two 50 mL tubes (one for low and high glucose). +2. For low glucose (2.8 mmol/L): Add 56 µL of glucose solution to 50 mL KRBH; for high glucose (20.2 mmol/L): Add 404 µL of glucose solution to 50 mL KRBH. +3. Wash islets twice in low glucose KRBH; plate in 24-well at 1 mL per well and incubate for 1 hour. +4. Exchange medium; collect 800 µL for insulin; add respective glucose KRBH for 1-hour incubation at 37°C, and collect 800 µL. +5. All samples should be frozen if storing for later analysis. + +### Protocol for Human Islets + +#### Media: +- **CMRL (Invitrogen 11530-037)** +- **100X GlutaMAX (Gibco 35050-061)**: 1% +- **50X Penicillin Streptomycin Solution**: 1% +- **FBS**: 10% + +#### Day 1: +1. Combine shipped islets from transplant media into a 50 mL conical tube. +2. Centrifuge at 1000 rpm for 5 minutes. +3. Aspirate and add 20 mL of islet culture medium into a 15mm x 150mm ultra-low attachment culture dish, incubate at 37°C overnight. + +#### Day 2: +1. Transfer islet culture to a 50 mL conical tube (scrape if necessary). +2. Centrifuge at 1000 rpm for 5 minutes and wash with 5 mL PBS. +3. Centrifuge again and aspirate PBS. +4. Resume studies for GSIS or submit to dispersion. + +### GSIS of Beta-Cells: +- **Krebs-Ringer Bicarbonate HEPES** + +#### Mixed Salts Stock Solution: +| Compound | g/L | +|----------------|:------| +| NaCl | 35.4 | +| KCL | 1.78 | +| KH2PO4 | 0.81 | +| MgSO4.7H2O | 1.46 | +| CaCL2.2H2O | 1.85 | + +#### Sodium Bicarbonate Stock Solution: +| Compound | g/L | +|--------- |:------| +| NaCl | 5.35 | +| NaHCO3 | 2.63 | + +#### Working KRBH: +- **20 mL mixed salts** +- **16 mL Sodium Bicarbonate** +- **64 mL dH2O** +- **0.42g NaHEPES** +- **0.1 g BSA** +- **pH 7.4** +- Low glucose: 2.8 mmol glucose/L +- High glucose: 20.2 mmol glucose/L + +1. Wash islets in KRBH twice. +2. Incubate in 24-well plate with various glucose levels as appropriate for 1 hour. +3. Collect glucose-conditioned media for ELISA and extract DNA using 300 μL PBS. + +### FACS Sorting for Beta-Gal Positive Cells: +- Disperse cells with TrypLE at 37°C for 10 minutes. +- Use cellular senescence kit for B-Gal measurement, follow with FACS sorting for pure β-cell populations. + +### Protocol for Ki67 or HMGB1 Stain: +*Cells must be adhered to dish overnight before stain* + +#### Day 1: +1. Wash with 1x PBS-CMF. +2. Add 10% formalin for 30 minutes. +3. Wash with PBS three times. +4. Incubate in 0.3% Triton for 15 minutes. +5. Wash with 2% PBS, incubate in NDS. +6. Incubate with specific antibody dilution. +7. Store overnight at 4°C. + +#### Day 2: +1. Defrost, add secondary antibody, wash, and incubate with final stain set. +2. Mount and seal covers with DAPI mounting media. + +### Attachment and Staining Protocol for Human Pancreatic Tissue +1. Glass dishes treated with 1X PEI for 24 hours before employing. +2. Disperse cells, allow media to attach dish overnight. +3. Wash with PBS, fix with 10% formalin. +4. Incubate in 0.3% Triton for 15 minutes. +5. Wash three times with PBS, incubate with NDS serum for secondary incubations. +6. After staining incubate, finish with DAPI media. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/dissociation-of-neuronal-culture-to-single-cells-f-bh32j8qe.md b/markdown-output/dissociation-of-neuronal-culture-to-single-cells-f-bh32j8qe.md new file mode 100644 index 0000000000000000000000000000000000000000..bc9e46d47cf8a1b1724ac1c707db23657bb6f2a8 --- /dev/null +++ b/markdown-output/dissociation-of-neuronal-culture-to-single-cells-f-bh32j8qe.md @@ -0,0 +1,197 @@ +```markdown +Goal/Experiment: +Dissociation of neuronal culture to single cells for scRNA-seq (10x Genomics) + +# Dissociation of Neuronal Culture to Single Cells for scRNA-seq (10x Genomics) +**Authors**: Julie Jerber, James Haldane, Juliette Steer, Daniel Pearce, Minal Patel +**Institute**: Wellcome Sanger Institute +**DOI**: [dx.doi.org/10.17504/protocols.io.bh32j8qe](https://dx.doi.org/10.17504/protocols.io.bh32j8qe) +**Date Created**: Jun 30, 2020 +**Date Last Modified**: Jul 24, 2020 +**protocols.io Protocol ID**: 38746 + +## Abstract +This protocol outlines a method for dissociating a human pluripotent stem cell-derived neuronal culture to single cells for loading onto a Chromium 10x chip for single-cell RNA-sequencing. + +This protocol makes a distinction between early neuronal progenitors and mature neuronal cultures as additional steps and reagents are required to sufficiently dissociate the latter. These include: + +- **DNase Vial (D2)** +- **PDS Kit Papain Vial** + +**Note**: In our labs, iPS cells were undergoing a 52-day long differentiation process to dopaminergic neurons (adapted from [doi.org/10.1038/nature10648](https://doi.org/10.1038/nature10648)), and were treated under the 'mature' conditions when harvested from day 20 onward, and as 'early neuronal progenitors' on day 11. Example images of neuronal culture from our labs can be found in the Guidelines of this protocol for reference. + +This protocol assumes use of: + +- 12-well tissue culture plates (3.8 cm² surface area per well) for samples/wells being harvested for dissociation. +- A NucleoCounter®NC-200™ for purposes of cell counting. + +## Guidelines +Unless otherwise stated, all steps should be performed under sterile conditions in a CL2 biological safety cabinet. + +Refer to 10x Genomics Chromium single-cell gene expression kit guidelines for subsequent steps in loading cells. + +Below are example images showing a neuronal culture undergoing a 52-day differentiation to dopaminergic neurons. These are for reference regarding dissociation steps in this protocol that differ for early neuronal progenitors and mature neurons. Details on the protocol used can be found in the Abstract of this protocol. + +- **Day 11** of differentiation: + + ![Example of neuronal culture at Day 11 of differentiation](example_day_11.jpeg) +- **Day 30** of differentiation: + + ![Example of neuronal culture at Day 30 of differentiation](example_day_30.jpeg) +- **Day 52** of differentiation: + + ![Example of neuronal culture at Day 52 of differentiation](example_day_52.jpeg) + +## Materials +| Name | Catalog # | Vendor | +|----------------------------------------------------|------------------|------------------------------| +| Ice | | | +| DNA LoBind Tubes, 1.5 mL | 0030108051 | Eppendorf | +| DPBS | 14190 | Invitrogen - Thermo Fisher | +| Falcon™ 15mL Conical Centrifuge Tubes | 14-959-53A | Fisher Scientific | +| DMEM/F-12, GlutaMAX™ supplement | 10565018 | Thermo Fisher | +| StemPro™ Accutase™ Cell Dissociation Reagent | A1110501 | Thermo Fisher | +| Y-27632 dihydrochloride | Y0503 | Sigma – Aldrich | +| DNase Vial (D2) | LK003170 | Worthington Biochemical Corporation | +| PDS Kit Papain Vial | LK003176 | Worthington Biochemical Corporation | +| PluriStrainer Mini 40µm | 43-10040-60 | pluriSelect | +| 1.5 ml TubeOne® Microcentrifuge Tubes Natural (Sterile) | S1615-5510 | StarLab | +| Bovine Serum Albumin | A0281 | Sigma | +| Water for embryo transfer sterile filtered | W1503-500ML | Sigma Aldrich | +| 0.6ml Crystal Clear Microcentrifuge Tubes (Sterile)| E1405-0610 | StarLab | +| 12-well Falcon™ Polystyrene Microplates | 10489482 | Fisher Scientific | + +### Equipment: +- Centrifuge (for both 15mL & 1.5mL tubes) +- Pipette boy +- Sterile 5/10mL stripettes +- P1000 pipette and filter tips +- Vacuum aspirator and tips +- Microbiology Safety Cabinet (MSC) +- Light Microscope +- Scale (for making up BSA) +- Method of Cell Counting (NucleoCounter®NC-200™) +- 37°C, 5% CO2 incubator + +### Safety Warnings: +- **ROCK inhibitor (Y-27632)**: Harmful if swallowed, in contact with skin or inhaled. + +### Before Starting: +- Prepare a 10% BSA solution (100 mg/mL) by dissolving Bovine Serum Albumin powder in sterile Water for Embryo Transfer (or equivalent water for cell culture). Filter sterilize with a 0.2 µm sterilizing grade filter. + - Aliquot into sterile 0.6 mL microcentrifuge tubes. Store at -20°C. Thaw at Room temperature and resuspend prior to use. +- Pre-warm Accutase to 37°C shortly before starting (Caution: Accutase is inactivated after 45 minutes at 37°C). +- If working with mature neurons, calculate how many vials of Papain/DNase you will need given the number of wells being harvested. + +## Buffer Preparation + +### Step 1: +**Prepare Wash Buffer 1 (without DNase)** by supplementing 15mL DMEM:F12 + GlutaMAX with ROCK inhibitor (Y-27632) to a final concentration of 10 µM. + +**Note**: We make up 15mL of Wash Buffer 1 for every 6 wells of a 12-well plate to dissociate. + +For mature neurons, 1 vial DNase (D2) should be added for every 15mL of buffer prepared. However, DNase should only be added immediately prior to use of the buffer when the cells are nearing the end of their dissociation. Keep the DNase at 4°C until use. + +| Cells | DMEM/F12 + glutamax | ROCK inhibitor (Y-27632) 10mM stock | DNase (D2) | +|-----------------------|---------------------|-------------------------------------|------------| +| Neuronal progenitors (~Day 11) | 15 mL | 15 µL | Not required | +| Mature Neurons (> Day 20) | 15 mL | 15 µL | 1 vial (250 µL) | + +### Step 2: +Prepare **Wash Buffer 2** by adding 10% BSA solution (100 µg/mL) to DPBS (-/-) to a final concentration of 400 µg/mL. i.e., add 60 µL 10% BSA solution to 15 mL of DPBS (-/-). + +### Step 3: +Prepare **Dissociation Buffer for mature neurons** by combining Accutase with DPBS (-/-) at a 1:1 ratio. Add 1 vial Papain (PDS Kit) per 5mL buffer. Reconstitute the lyophilized papain with the Accutase/DPBS solution, replace the lid on the vial and invert several times ensuring all powder in the vial and on the lid is dissolved. Transfer solution back into the Dissociation Buffer tube. + +**Dissociation Buffer for neuronal progenitors** is undiluted Accutase only. + +| Cells | Accutase | DPBS (-/-) | Papain (PDS kit) | +|-----------------------|----------|------------|------------------| +| Neuronal progenitors (~Day 11) | 0.5 mL per well | Not required | Not required | +| Mature Neurons (> Day 20) | 2.5 mL | 2.5 mL | 1 vial | + +## Dissociation + +### Step 4: +Aspirate the media from the well(s) of the neuronal culture and gently add 1 mL of DPBS (-/-) without disturbing the cell layer. + +### Step 5: +Aspirate the DPBS (-/-) and add 0.5 mL of Dissociation Buffer to each well. + +### Step 6: +Transfer cells to a 37°C, 5% CO2 tissue culture incubator. Incubate for the appropriate time according to the age of the culture: + - **Early neuronal progenitors (~Day 11)**: 10-20 mins + - **Young mature neurons (~Day 30)**: 20-30 mins + - **Older mature neurons (~Day 52)**: 25-35 mins + + Check the progress of the dissociation after the indicated time. If necessary, extend the incubation period for up to 10 mins more, checking the cells every few minutes. + +### Step 7: +For dissociation of mature neurons, complete the preparation of Wash Buffer 1 within the last 5 minutes of incubation of the cells. +- Retrieve the DNase (D2) vial(s) from 4°C storage. +- Reconstitute the vial(s) by adding 250 µL Wash Buffer 1 to each vial to make a 2 mg/mL solution. Make sure to reconstitute all the powder (check the lid). Avoid vigorous mixing - do not vortex. +- Transfer the entire contents of the reconstituted vial(s) back into the Wash Buffer 1 tube. Use 250 µL (1 vial) for every 15 mL buffer. + +### Step 8: +Following incubation, inspect the cells under a microscope. + +The cells should be detaching from the plate and the cell layer should have a darkened appearance. Cells will have a rounded appearance as they dissociate and individual cells should be visible at the edges or in gaps in the cell layer. + +### Step 9: +**Optional**: Test for ease of dissociation by gently pipetting ~100 µL of the Dissociation Buffer against the cells with a P1000 pipette. If cells do not dissociate easily, extend digestion by 3 mins and repeat this step. + +### Step 10: +Add 2 mL of Wash Buffer 1 per well. + +### Step 11: +Use a P1000 pipette to repeatedly wash the buffer over the well(s) to detach the cells and dissociate them to a single-cell suspension. + +**Caution**: Sufficient dissociation is critical at this step. It is recommended to check the cell suspension under a microscope and if necessary, pipette the cell suspension up and down further until the majority is single cells. Try to avoid over-pipetting the cells as this could affect viability. + +### Step 12: +Transfer the cell suspension into a 15mL centrifuge tube capped with a 40µm cell strainer. + +### Step 13: +**Optional**: If there are residual cells in the plate, wash well(s) with 0.5 mL of Wash Buffer 1 and transfer this suspension into the tube from the previous step. + +### Step 14: +Centrifuge cells at 150 x g, Room temperature for 3 mins. + +### Step 15: +Aspirate supernatant and gently re-suspend the cell pellet in 1 mL of Wash Buffer 2 with a P1000 pipette. + +### Step 16: +Transfer the cells to a Lo-bind 1.5mL Eppendorf tube. + +**Recommendation**: We recommend preparing and thawing necessary reagents for the 10x loading procedure during these next steps if you have not already done so. + +### Step 17: +Repeat steps 14 & 15 three times in a mini centrifuge (for a total of four centrifugations). + +## Cell Counts + +**Note**: This step describes a 1 in 10 dilution for cell counting on a NucleoCounter®NC-200™. Depending on the method of cell counting and the number of cells, this dilution step may not be necessary. + +### Step 18: +Prepare a cell count sample tube for each suspension prepared by adding 450 µL of Wash Buffer 2 to a fresh Lo-bind 1.5mL Eppendorf tube. + +Take a 50 µL aliquot of the well-mixed cell suspension and add this to the 450 µL of Wash Buffer 2 in the prepared counting tube(s). + +### Step 19: +Place the cell suspension tube(s) on ice during the cell counts and following steps. + +### Step 20: +Count the cell suspension(s) on a NucleoCounter®NC-200™ and record % cell viability and viable cell concentration. Ensure less than 3% of cells are in aggregates. + +If >3% of the cells are in aggregates, triturate the cell suspension by pipetting up and down with a P1000 pipette. Carry out a second cell count on a new sample. Alternatively, dilute the sample and pass through a cell strainer, then centrifuge and resuspend in 1mL Wash Buffer 2 before repeating the cell count. + +### Step 21: +Using the cell counts, calculate the dilution of the cell suspension(s) necessary to produce the desired cell number for loading for the 10x. Dilutions should be made using Wash Buffer 2, to a final volume of 1 mL. + +### Step 22: +Keeping the cell suspensions on ice, proceed with the sample preparation and loading steps described in the 10x Genomics single-cell gene expression kit guidelines. + +### Step 23: +We recommend performing a post-10x loading cell count on the diluted cell suspension. This cell count can normally be performed without further dilution (depending on the method of cell counting). This cell count will confirm the cell number loaded for 10x and check the cell viability has not dropped significantly since the first cell count. It can also check the percentage cell aggregates has remained acceptable. + +endofoutput +``` diff --git a/markdown-output/dissolved-sulfide-concentrations-h2s-hs-s2-colorim-bd7ji9kn.md b/markdown-output/dissolved-sulfide-concentrations-h2s-hs-s2-colorim-bd7ji9kn.md new file mode 100644 index 0000000000000000000000000000000000000000..7230fa9ac506260f03306f881d09ebc9b124238c --- /dev/null +++ b/markdown-output/dissolved-sulfide-concentrations-h2s-hs-s2-colorim-bd7ji9kn.md @@ -0,0 +1,89 @@ +```markdown +Goal/Experiment: +The goal of this experiment is to measure dissolved sulfide concentrations (H2S, HS-, S2-) in water samples using a colorimetric assay with a multi-mode plate reader spectrophotometer. This method is adapted from Cline (1969) and modified for use with 96-well plates for rapid and accurate measurements. + +# Dissolved Sulfide Concentrations (H2S, HS-, S2-) Colorimetric Assay Using a Plate Reader (96-Well Plate) + +**Author**: Jian Gong +**Institution**: Massachusetts Institute of Technology +**Protocol Published Date**: Mar 25, 2020 +**Last Modified Date**: Jul 13, 2020 +**Protocol ID**: 34763 + +This protocol is published under a DOI. +[View Protocol](https://protocols.io/view/dissolved-sulfide-concentrations-h2s-hs-s2-colorim-bd7rjj9kn) + +## Abstract +This protocol describes the adaptation of a sulfide colorimetric assay for use on a multi-mode plate reader spectrophotometer (BioTek, Synergy 2), using standard 96-well plates for rapid measurements of < 400 μL of water samples. The assay measures the concentration of total sulfide species (H2S, HS-, S2-) in solution. + +Samples for this assay should be filtered (0.2 μm syringe filter) and placed in 1.5 mL Eppendorf tubes. Care should be taken to avoid oxidation of the sample by oxygen in the atmosphere by completing the first step of the assay quickly following sampling, which stabilizes the sample against further oxidation. + +## Keywords +- Total dissolved sulfide +- Colorimetric assay +- Plate reader + +## License +This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. + +## Guidelines + +### Working Principle +N,N-dimethyl-p-phenylenediamine (diamine) and ferric ion together form a stable colored complex with zinc sulfide at pH 0.35, with maximum absorbance at λ = 670 nm. This property is used to design the colorimetric assay allowing measurement of sulfide concentrations in natural waters, relatively free from the interference of salts up to 40 ‰ and sulfite up to 100 μM. Thiosulfate may inhibit the color-development of this assay. + +### Reagent Concentrations and Dilution Factors +| Sulfide Concen (μM) | Diamine Concen (g/100 mL) | Ferric Concen (g/100 mL) | Dilution Factor (mL/mL) | Path Length (cm) | +|----------------------|---------------------------|--------------------------|-------------------------|------------------| +| 1-3 | 0.1 | 0.15 | 1:1 | 10 | +| 3-40 | 0.4 | 0.6 | 1:1 | 1 | +| 40-250 | 1.6 | 2.4 | 2:25 | 1 | +| 250-1000 | 4.0 | 6.0 | 1:50 | 1 | + +### Measurement Range +Refer to Table 1 for the recommended preparation of diamine and ferric chloride reagent specific to the range of sulfide concentrations. Prepare standards and dilutions accordingly. + +Note: The spectrophotometer (BioTek Synergy 2) used in this protocol has a path length of approximately 4 mm with a 200 μL load. To ensure accurate measurement, apply a 4:10 dilution (Reagent A to sample). + +### Materials +- 1.5 mL Eppendorf® Microcentrifuge Tubes +- Corning® 96-Well EIA/RIA Assay Microplate + +### Reagents +All reagents are prepared with nanopure water and stored in 4°C in the dark. + +1. **Zinc Acetate Solution:** 0.05 M zinc acetate [Zn(CH3COO)2·2H2O, FW 219.51, 98%, Aldrich #383058] +2. **Diamine Reagent:** + - 0.4 g N,N dimethyl-p-phenylenediamine sulfate [(C6H3)2(CH3)2]2·HCl·H2SO4, FW 234.27, Aldrich #186384 + - 0.6 g Anhydrous ferric chloride (FeCl3, FW 162.20, 97%, Aldrich #157740) in 100 mL of 6 N HCl [hydrochloric acid, Aldrich #1.00318] +3. **Sulfide Standard Stock:** 0.1 M sodium sulfide nonahydrate [Na2S·9H2O, Aldrich #431648, 99.99%] + +### Standards +Prepare fresh standards by diluting stock reagents and measure their concentrations using the protocol below. + +### Safety Warnings +- Wear safety goggles and nitrile gloves. +- Dispose of waste chemicals in designated waste jars. + +## Procedure + +1. **Sample Stabilization:** + - Add 400 μL liquid sample in 1 mL of reagent A upon sampling. + - Zinc sulfide is stabilized, preventing further oxidation. + - Adjust this amount for different detection ranges if necessary. + +2. **Colored-Complex Development:** + - Transfer 900 μL of the stabilized mixture to a new tube. + - Add 15 μL reagent B. + - Mix, then react for 20 minutes in the dark. + +3. **Measurement:** + - Measure absorbance at 670 nm. + - Use BioTek Synergy 2 plate reader. + - Load quadruplêts of 200 μL mixtures on a 96-well plate. + +## References + +- Cline JD. 1969. Spectrophotometric determination of hydrogen sulfide in natural waters. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/dna-extraction-protocol-for-cryptosporidium-spps-i-r49d8z6.md b/markdown-output/dna-extraction-protocol-for-cryptosporidium-spps-i-r49d8z6.md new file mode 100644 index 0000000000000000000000000000000000000000..b2261aca324d808e290359842ca4d6215a812b70 --- /dev/null +++ b/markdown-output/dna-extraction-protocol-for-cryptosporidium-spps-i-r49d8z6.md @@ -0,0 +1,139 @@ +```markdown +# Goal/Experiment: +The goal of this experiment is to extract DNA from Cryptosporidium spp. in stool samples using an adapted protocol from the GF-1 Soil Sample DNA Extraction Kit. + +# DNA Extraction Protocol for Cryptosporidium spp. in Stool Samples (Adapted from GF-1 Soil Sample DNA Extraction Kit) + +### Authors: +Asar Khan, Sumaira Shams, Abid Ali, Saima Khan, Muhammad Iftikhar Khan + +## Abstract +The GF-1 Soil Sample DNA Extraction Kit is designed for rapid and efficient DNA purification from soil samples without the need for precipitation or organic extraction. The kit uses a high-purity, specially treated silica-based material in a column to efficiently bind DNA in the presence of high salt. The kit applies the minicolumn spin technology, using optimized buffers to ensure isolation of only DNA while removing cellular protein, humic acid, metabolites, salts, and other low molecular weight impurities during subsequent washing steps. This protocol has been used for Cryptosporidium spp. DNA extraction from stool samples with minor modifications. + +--- + +## Guidelines +- All steps should be carried out at room temperature unless stated otherwise. + +## Before Start +- Pre-set the water bath at 70°C. + +## Materials +- Centrifuges 5810 R [Eppendorf Centrifuge](https://online-shop.eppendorf.com/OC-en/Centrifugation-44559/Centrifuges-44637/5810-5810-R-PF-6322.html) +- Micropipettes tips +- GF-1 Soil Sample DNA Extraction Kit (GF-SD-025) by [Vivantis Technologies Sdn. Bhd.](https://www.vivantechnologies.com/gf-sd-025.html) +- Water bath at 70 °C +- Micropipettes (2-20 µL, 50-250 µL, 100-1000 µL) +- Eppendorf tubes (1.5 & 2.0 mL) +- Vertex mixer + +## Protocol + +### Step 1: Lysis + +1. **Lysis Buffer Preparation** + - Add 1 g of stool sample into a 2 mL microcentrifuge tube. + - Add 500 mg (0.5 g) of glass beads. + - Add 1 mL of SLX-buffer and vortex at maximum speed for 5 minutes. + - Add 100 µL of DS-buffer and vortex at maximum speed for another 3 minutes. + - Incubate at 70 °C for 10 minutes. + + **Quantities and Times**: + - 1 g stool sample + - 500 mg glass beads + - 1 mL SLX-buffer + - 100 µL DS-buffer + - 5 minutes vortex at max speed + - 3 minutes vortex at max speed + +2. **Centrifugation** + - Centrifuge at 5,000 x g for 3 minutes at room temperature. + - Move 800 µL of the supernatant to a new microcentrifuge tube. + - Add 270 µL of P2-buffer, vortex, and incubate on ice for 5 minutes. + - Centrifuge again at 14,000 x g for 3 minutes. + + **Quantities and Times**: + - 800 µL supernatant to new tube + - 270 µL P2-buffer + - 5 minutes on ice + - 3 minutes, 14,000 x g centrifugation + +### Step 2: DNA Precipitation + +1. **Isopropanol Addition** + - Add 700 µL of isopropanol and invert the tube 20-30 times. + - Centrifuge at 14,000 x g for 10 minutes. + - Discard the supernatant carefully. Invert the tube on a paper towel to drain any remaining liquid. + + **Quantities and Durations**: + - 700 µL isopropanol + - 20-30 times inversion + - 10 minute centrifugation at 14,000 x g + +### Step 3: DNA Solubilization + +1. **Elution Buffer Addition** + - Add 200 µL of Elution Buffer (EB) and mix using pulse-vortexing. + - Incubate at 70 °C for 10-20 minutes to dissolve the DNA pellet. + - Add 100 µL of HTR reagent, mix thoroughly using vortexing for 10 seconds, and incubate at room temperature for 2 minutes. + - Centrifuge at 14,000 x g for 2 minutes and transfer the supernatant to a new clean 1.5 mL microcentrifuge tube. + + **Reagents and Durations**: + - 200 µL EB + - 100 µL HTR reagent + - Incubation: 70 °C for 20 minutes, room temp for 2 minutes + - Centrifugation: 14,000 x g for 2 minutes. + +### Step 4: Protein Digestion + +1. **Proteinase-K Addition** + - Add 2 µL of Proteinase-K to each tube (1.5 mL) and vortex thoroughly. + - Incubate at 37 °C for 10 minutes. + - Add XP1-buffer (equal to the sample volume, 1.5 mL) and vortex. + + **Quantities and Conditions**: + - 2 µL Proteinase-K + - 1.5 mL XP1-buffer + - Incubation: 37 °C for 10 minutes + +### Step 5: DNA Wash and Purification + +1. **Column Purification** + - Insert columns into 2 mL collection tubes. + - Add 600 µL of sample to each column and centrifuge at 10,000 x g for 1 minute. Discard flow-through. + - Add 300 µL XP1-buffer, centrifuge at 10,000 x g for 1 minute, and discard flow-through. + - Insert columns into new 2 mL collection tubes. + - Add 700 µL SPW wash buffer, centrifuge at 10,000 x g for 1 minute, and discard flow-through. + - Repeat the SPW wash buffer step. + - Centrifuge columns at 14,000 x g for 2 minutes to remove ethanol. Place the columns into clean 2 mL Eppendorf tubes. + - Add 60 µL of Elution Buffer to the center of the column membrane and incubate at 70 °C for 5 minutes. + + **Reagents and Conditions**: + - 600 µL sample + - 300 µL XP1-buffer + - 700 µL SPW buffer (x2) + - 60 µL Elution Buffer + - Incubation: 70 °C for 5 minutes + - Centrifugation times vary by step. + +### Step 6: Elution of DNA and Storage + +1. **Final Elution** + - Centrifuge columns at 14,000 x g for 1 minute to elute DNA. + - Elute twice with 60 µL Elution Buffer. + - Store extracted DNA at -20 °C until amplification. + + **Quantities and Conditions**: + - 60 µL Elution Buffer (x2) + - Storage: -20 °C + +## Warnings +- Buffer SB contains guanidine salts, hazardous upon skin contact or ingestion. +- Always wear gloves and follow standard safety precautions. +- Do NOT disinfect guanidine or extraction waste in solutions containing bleach or acid. +- Clean items contaminated with guanidine by soaking in water and detergent before using acidic solutions or bleach. + +--- + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/dorsal-root-ganglia-stimulation-block-colorectal-a-b828ryhw.md b/markdown-output/dorsal-root-ganglia-stimulation-block-colorectal-a-b828ryhw.md new file mode 100644 index 0000000000000000000000000000000000000000..ff539309395fc1c2735ea4c87e272f14b8915efc --- /dev/null +++ b/markdown-output/dorsal-root-ganglia-stimulation-block-colorectal-a-b828ryhw.md @@ -0,0 +1,120 @@ +```markdown +# Goal/Experiment: +To test the feasibility of blocking transmission in visceral afferents by dorsal root ganglion (DRG) stimulation in an ex vivo mouse model. + +# Dorsal Root Ganglia Stimulation-Block Colorectal Afferents + +**Authors:** +Longtu Chen1, Bin Feng1 +1Department of Biomedical Engineering, University of Connecticut, Storrs, CT, United States + +**DOI:** +[dx.doi.org/10.17504/protocols.io.bp2l61rnzvqe/v1](https://dx.doi.org/10.17504/protocols.io.bp2l61rnzvqe/v1) + +To test the feasibility of transmission block in visceral afferents by DRG stimulation, we developed an ex vivo preparation in which mouse colorectum, pelvic nerve (PN), L6 DRG, and dorsal root (DR) were harvested in continuity. We conducted experiments with tissues harvested from mice receiving intracolonic treatment of 2,4,6-trinitrobenzenesulfonic acid (TNBS), a model of postinfectious Irritable bowel syndrome (IBS). We then systematically investigated the blocking effect of DRG stimulation by conducting single-fiber recordings from split L6 DRs of action potentials evoked from peripheral endings in the colorectum through colorectal distension (CRD). + +## Materials +### Animals +- **Mouse Strain:** C57BL/6 mice (C57BL/6NTac, RRID:IMSR_TAC:b6, Taconic, Germantown, NJ) + +### Reagents +- **TNBS:** Sigma-Aldrich, St. Louis, MO + - **Description:** A chemical compound used to induce inflammatory bowel disease in experimental models. +- **Dietary Gel:** NGB-1; Bio-Serv, Flemington, NJ + - **Description:** Nutrient-rich gel provided to prevent severe weight loss. +- **Isoflurane:** Hospira Inc., Lake Forest, IL + - **Description:** Used for anesthesia induction. +- **Krebs Solution:** (Custom preparation) + - **Description:** Physiological saline solution commonly used in biological research. + +### Equipment +- **22-Gauge Feeding Needle:** (#18061-22; Fine Science Tools, Foster City, CA) + - **Description:** Used for precise intracolonic TNBS administration. +- **TDT System:** Tucker-Davis Technologies (TDT), Alachua, FL + - **Description:** Used for data acquisition and electrical stimulation. +- **Custom-Built Colorectal Distension Device** + - **Description:** Utilized for applying controlled distension pressures to the colorectum. +- **Needle Electrode:** FHC, platinum-iridium + - **Description:** Used for delivering electrical stimulation. +- **Custom-Built Recording Electrodes and Perfusion Chamber** + - **Description:** Used for electrophysiological recordings and maintaining tissue viability. + +### Software +- **SigmaPlot v11.0:** Systat Software, Inc., San Jose, CA +- **MATLAB v2022:** Mathworks Inc., Natick, MA + +## Methods + +### Intracolonic TNBS Treatment +1. **Anesthesia:** + - C57BL/6 mice (8-16 weeks, 25-35 g, either sex) were anesthetized with isoflurane inhalation. + +2. **TNBS Administration:** + - Mice were transanally administered with 2,4,6-trinitrobenzene sulfonic acid (TNBS) (0.2 mL at 10 mg/mL in 50% ethanol; Sigma-Aldrich, St. Louis, MO) using a 22-gauge feeding needle (#18061-22; Fine Science Tools, Foster City, CA), and held in a head-down position (~30°) for 5 minutes to preserve TNBS in the colorectum. + +3. **Dietary Gel:** + - Provided to mice showing severe weight loss (0.5% original body weight). + +### Ex Vivo Colorectum-Pelvic Nerve-Dorsal Root Ganglia-Dorsal Root (PN-DRG-DR) Preparation +4. **Post-Treatment:** + - Mice at 7 to 14 days after TNBS treatment were used, a time span of colorectal hypersensitivity. C57BL/6 mice without TNBS treatment were used as control. + +5. **Anesthesia:** + - Mice were anesthetized by 2% isoflurane inhalation followed by intraperitoneal and intramuscular injection of a ketamine/xylazine cocktail (100/10 mg per kg weight). + +6. **Euthanasia and Perfusion:** + - Mice were then euthanized by exsanguination from the right atrium and transcardiac perfusion from the left ventricle with oxygenated (95% O2, 5% CO2) ice-cold Krebs solution. + +7. **Dorsal Pediculectomy:** + - Performed to expose the spinal cord and DRG from T12 to S1 segments. + +8. **Dissection:** + - The exsanguinated mouse carcass was placed in a dissection chamber circulated with oxygenated ice-cold Krebs solution. + +9. **Dissection and Transfer:** + - The colorectum-PN-DRG-DR were carefully dissected and transferred to a custom-built chamber consisting of a tissue chamber and an adjacent recording chamber. + +10. **Perfusion:** + - The colorectum, PN, and L6 DRG were placed in the tissue chamber perfused with oxygenated Krebs solution at 30°C, and the DR was gently pulled into the recording chamber filled with mineral oil (Fisher Scientific, East Greenwich, RI). + +11. **Fiber Splitting:** + - The L6 DR was split into fine filaments (~10 µm) for single-fiber recordings from individual afferent axons using a custom-built microwire electrode array. + +12. **Colorectal Distension (CRD):** + - The colorectum was cannulated and connected to a custom-built CRD device consisting of 4 hydrostatic columns of phosphate-buffered saline (PBS) set at 15, 30, 45, and 60 mmHg pressures, respectively. + +### Protocol for DRG Stimulation +13. **Stimulation Electrode:** + - A blunt-tipped needle electrode (FHC, platinum-iridium, tip size Φ ~25 µm) was used to deliver constant current stimulation to the caudal region of the L6 DRG. + +14. **Biphasic Stimulation:** + - Biphasic constant current stimuli (charge-balanced bipolar) generated by an IZ2H stimulator (Tucker-Davis Technologies Inc, Alachua, FL) were delivered to the L6 DRG at a frequency range from 10 to 1000 Hz. + +15. **Stimulus Pulse Width:** + - Set to either 0.1 or 0.2 ms based on the chronaxie measurement. + +16. **Stimulation Amplitude:** + - Set to be suprathreshold to evoke action potentials in single-fiber recordings. Subthreshold DRG stimulation was also tested during which no action potentials were evoked. + +17. **Stimulation Protocol:** + - Consisted of 30 pulse trains at 0.5 Hz train frequency and 0.5-second intertrain intervals (60 seconds in total). + +### Protocol for Colorectal Distension (CRD) +18. **CRD Protocol:** + - Performed before (control), immediately after, and 15 to 30 minutes after DRG stimulation (recovery). + +19. **CRD Pressure Steps:** + - Consisted of 4 ascending pressure steps of 5 and 10 seconds duration for TNBS-treated and naive mice, respectively, and 8 seconds interstep intervals (15, 30, 45, and 60 mmHg). + +### Data Processing +20. **Recorded Data Processing:** + - Recorded data were processed offline to identify individual action potentials. + +21. **Normalization:** + - The number of action potentials evoked by each CRD protocol was normalized to the number of action potentials evoked by 60 mmHg distension (=100%) in the control trial. + +**Citation:** +Longtu Chen, Bin Feng Dorsal Root Ganglia stimulation-block colorectal afferents. protocols.io [https://dx.doi.org/10.17504/protocols.io.bp2l61rnzvqe/v1](https://dx.doi.org/10.17504/protocols.io.bp2l61rnzvqe/v1) + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/dose-response-assay-for-inducible-promoters-in-syn-55wg87e.md b/markdown-output/dose-response-assay-for-inducible-promoters-in-syn-55wg87e.md new file mode 100644 index 0000000000000000000000000000000000000000..8fd8fb92e506de93ae992b34e2035fbd39ba6015 --- /dev/null +++ b/markdown-output/dose-response-assay-for-inducible-promoters-in-syn-55wg87e.md @@ -0,0 +1,98 @@ +```markdown +# Goal/Experiment: +Dose Response Assay for Inducible Promoters in *Synechocystis* sp. PCC 6803 + +## Authors: +Anna Behle1, Pia Saake1, Ilka Maria Axmann2
+1Institute for Synthetic Microbiology
2Synthetic Microbiology, CEPLAS, Heinrich Heine University Duesseldorf + +## Abstract: +Inducible promoters are an important tool for synthetic biology. They enable temporal control of gene expression, as well as controlled expression of toxic genes. This protocol describes the methodology for fluorescence-based reporter assays in the unicellular cyanobacterium *Synechocystis* sp. PCC 6803 to quantify the dose-dependent response of an inducible promoter to its inducer. + +## Guidelines: +For comparability, all cultures that are to be compared to each other should be similar in terms of optical density. To achieve this, it is important to adjust preculture optical densities until they are in the same growth phase. + +## Materials: +### Common Reagents +| Name | Catalog # | Vendor | +|----------------------------------------------|------------------|-------------------------------| +| Ethanol | - | - | +| Nuclease-free water (e.g., MilliQ or HPLC) | - | - | +| Anhydrotetracycline hydrochloride | 37919-100MG-R | Sigma Aldrich | +| L-rhamnose | W373011-100G-K | Sigma Aldrich | +| Vanillic acid | H36001-25G | Sigma Aldrich | +| 6-well plate | 92406 | Techno Plastic Products (tpp) | +| Vision Plate 96 well | 4ti-0221 | 4ti | + +### Additional Materials +- Erlenmeyer flasks, wide neck +- BG11 media +- Antibiotic stocks + - Spectinomycin 20 mg/mL + - Kanamycin 25 mg/mL + - Chloramphenicol 10 mg/mL + +## Before Starting: +Prepare all stock solutions, including one stock of the solvent the inducer is dissolved in (e.g., H2O or ethanol). + +**Important:** +If a large range of inducer concentrations is used, prepare multiple stock dilutions so that a similar amount of inducer is pipetted for each concentration. For example, if you require a final concentration of 1 µM and 1 nM, prepare 1:1000 stock solutions for both concentrations (i.e., 1 mM and 1 µM, respectively). Make sure to prepare all dilutions from the same stock solution to minimize technical errors. + +## Protocol: +### Inoculation of Preculture +1. Inoculate an appropriate amount of BG11 with your cyanobacterial strain of choice. Include appropriate controls, i.e., an empty vector control. The inoculation volume depends on the number of concentrations to be tested. For example: 6 concentrations x 3 replicates x 5 mL = 90 mL of preculture. Since the culture will be diluted ~1:3 before starting, 35 mL culture volume should be enough. Include appropriate antibiotics, especially when using plasmid-based systems, since the plasmid will be lost in the absence of selection pressure. + - Grow the culture for 3-5 days, until an OD750 of ~1 is reached. + +### Dilution of Preculture +2. Dilute precultures to an OD750 of 0.2. Include the appropriate antibiotics. This step is important for comparability. In order to compare cultures, precultures should be relatively fresh (i.e., no older than 1 week and no further than early stationary phase). Furthermore, their OD750 should be roughly the same. + - Grow preculture for 2-3 additional days, until OD750 has reached ~0.6. + +### Dilution of Main Culture +3. In a large sterile vessel, dilute the preculture to OD750 = 0.2, making sure to prepare a sufficient amount of culture. Include appropriate antibiotics. + - Aliquot this main culture into clear 6-well culture plates, with 5 mL per well and at least three biological replicates per inducer concentration. + +### Induction +4. At the end, the total volume added should be equal for each replicate. This can be achieved either by preparing one stock solution for each concentration or adding the difference in volume of the solvent used. The following three tables show the pipetting schemes for the three inducible promoters PrhaP, PL03, and PvanCC over a range of inducer concentrations. + +#### Table 1: Pipetting Scheme for Dose Response Assay with L-rhamnose as Inducer +Inducer amounts are calculated for 5 mL culture volume in a 6-well plate format. Total inducer volume per well is 100 µL. + +| L-rhamnose, final concentration (mM) | 0 | 0.01 | 0.05 | 0.1 | 0.5 | 1 | 2 | 5 | 10 | 20 | +|--------------------------------------|---|------|------|-----|-----|---|---|---|----|----| +| L-rhamnose stock solution (100 mM) | - | 10 | 10 | 10 | 100 | 100 | 100 | 1000 | 1000 | 1000 | +| µL stock solution | 0 | 5 | 25 | 50 | 25 | 50 | 100 | 25 | 50 | 100 | +| µL MilliQ water, sterile | 100 | 95 | 75 | 50 | 75 | 50 | 0 | 75 | 50 | 0 | + +#### Table 2: Pipetting Scheme for Dose Response Assay with aTc as Inducer +Inducer amounts are calculated for 5 mL culture volume in a 6-well plate format. Total inducer volume per well is 5 µL. + +| aTc, final concentration (nM) | 0 | 10 | 50 | 100 | 500 | 1000 | +|------------------------------------------|----|-----|-----|-----|-----|------| +| aTc stock solution (10 µM) | - | 10 | 50 | 100 | 500 | 1000 | +| µL stock solution | 0 | 5 | 5 | 5 | 5 | 5 | +| µL EtOH, 100 % | 5 | 0 | 0 | 0 | 0 | 0 | + +#### Table 3: Pipetting Scheme for Dose Response Assay with Vanillate as Inducer +Inducer amounts are calculated for 5 mL culture volume in a 6-well plate format. Total inducer volume per well is 5 µL. + +| Vanillate, final concentration (µM) | 0 | 100 | 200 | 500 | 1000 | 2000 | +|------------------------------------------|----|------|------|-----|------|------| +| Vanillate stock solution (100 mM) | - | 100 | 100 | 250 | 250 | 250 | +| µL stock solution | 0 | 5 | 5 | 10 | 10 | 20 | +| µL EtOH, 100 % | 40 | 35 | 30 | 30 | 20 | 0 | + +### Fluorescence Measurement +5. To monitor fluorescence development, fluorescence measurements should be performed 6h, 24h, and 48h post-induction. For each sample, pipette 150 µL into a black-walled, clear-bottom 96-well plate in technical triplicates. + +![Figure 1: Pipetting Scheme for Plate Reader Measurement](figure1.png) + +### Fluorescence Measurement Settings in the BMG Clariostar +- Top optic +- Excitation λ: 511/12 nm +- Excitation λ: 552/20 nm +- # of flashes: 20 +- To quantify cell density, measure the absorption at 750 nm. +- Shake before plate reading at 500 rpm for 30 seconds (double orbital). + +## endofoutput +``` \ No newline at end of file diff --git a/markdown-output/dota-seq-v3-1-cy45xyy6.md b/markdown-output/dota-seq-v3-1-cy45xyy6.md new file mode 100644 index 0000000000000000000000000000000000000000..555a5bcd56dfba1802014567cc3f5887b42944dd --- /dev/null +++ b/markdown-output/dota-seq-v3-1-cy45xyy6.md @@ -0,0 +1,119 @@ +```markdown +# Goal/Experiment: +Generate a single-cell sequencing library from a cell suspension using the DoTA-seq protocol. + +# DoTA-seq V3.1 +Forked from DoTA-seq V3.1 + +## Abstract +This protocol describes the process of DoTA-seq generating a single-cell sequencing library from a cell suspension. This workflow can be performed in two days, with the PCR step happening overnight. Before beginning this workflow make sure to have: +1. The necessary microfluidics devices prepared and ready to go +2. The multiplex DoTA-seq target primers validated to work together without generating large molecular weight primer dimers. + +## Guidelines +**Strongly recommend** all pre-PCR steps (setting up reagents, washing gels) to be done in a PCR Clean hood. This has two purposes: +1. Reduce PCR contamination of templates which can strongly affect single-cell PCR reactions. +2. Reduce dust contamination of reagents which can clog devices and cause failures. + +## Materials + +| Reagent | Vendor | Catalog Number | +| --- | --- | --- | +| ddPCR Supermix for probes (no dUTP) | BioRad | #1863024 | +| MetaPolyzyme | Sigma Aldrich | #MAC4L-5MG | +| Lysozyme from chicken egg white | Sigma Aldrich | #L6876 | +| HFE 7500 Perfluorinated Oil | Aldrich | #370533 | +| Perfluorooctanol | Sigma Aldrich | #370533 | +| TCEP-HCl | Gold Biotechnology | #TCEP | +| NN'-Bis(acryloyl)cystamine | Santa Cruz Biotechnology | #sc-215506 | +| Ammonium persulfate | Aldrich | #A3678 | +| TEMED (Tetramethyl-ethylenediamine) | Sigma-Aldrich | #T9281 | +| Biorad Evagreen Droplet Oil | BioRad Sciences | #1864005 | +| DNA Clean & Concentrator™-5 | Zymo Research | #D4003 | +| Axygen® 0.2 mL Maxymum Recovery® Thin Wall PCR Tubes | Corning | #PCR-02-L-C | +| NEBNext Library Quant Kit for Illumina - 100 rxns | New England Biolabs | #E7630S | +| SYBR Green | Thermo Fisher Scientific | - | +| Proteinase K solution, 20 mg/ml | Ambion | #AM2546 | +| Pre-injection buffer 10mM HEPES pH 7.5 Pluronic 0.1% | - | - | + +## Safety Information +Unpolymerized Acrylamide is toxic, handle with care and dispose of according to regulations. + +## Preparing Cells +1. **Prepare a cell suspension:** Wash twice in 1 mL of PBS 0.1% Tween20 by spinning down at 5000 x g for 1 minute. +2. **Resuspend cells:** In 100 µL PBS 0.1% Tween20. +3. **Stain cells:** Add 1 µL SYBR Green dye (10,000x) to the cells. +4. **Count cells:** Use a hemacytometer to determine the concentration using the SYBR signal. +5. **Optional:** Stain with CellBrite Fix 555 Sigma Aldrich Catalog ##30088 to get a cell membrane/wall stain. + +## Preparing Gel +1. Make Hydrogel Precursor Solution - Mix together in a tube: + - 100 µL Acrylamide Sigma Aldrich monomer in water (25 Mass / % volume) + - 15 µL NN'-Bis(acryloyl)cystamine Sigma Aldrich in Methanol (5 Mass / % volume) + - 10 µL Ammonium persulfate Sigma Aldrich (10 Mass / % volume) + - 75 µL Cell suspension diluted in PBS (total of 7e6 cells) to achieve a final concentration of 3.5e7 cells/mL +2. Vortex vigorously to mix. + +## Generate Gel Droplets +1. **Prepare and load the syringes:** Load syringes with gel sample and 600 µL Biorad Evagreen Droplet Oil Sigma Aldrich and follow the microfluidics protocol. +2. **Run pumps:** At 500 µL/hr for the gel syringe and 900 µL/hr for the oil syringe. Collect gel droplets for 20 minutes in a 1mL tube. + +## Gel Polymerization +1. **Prepare Gel Polymerization Oil:** Mix together in a tube: + - 195 µL Biorad Evagreen Droplet Oil Sigma Aldrich + - 5 µL TEMED (Tetramethyl-ethylenediamine) Sigma Aldrich +2. **Polymerize:** Add the oil to the collected droplets, invert slowly 3 times to mix, incubate at 37°C for 10 minutes. + +## Breaking out Gels from Emulsion +1. **Centrifuge and remove oil:** Pulse spin to close pack the emulsion, remove oil from the bottom. +2. **Break emulsion:** Add 200 µL Perfluorooctanol Sigma Aldrich, vortex, and wait 1 minute. Pulse spin and remove oil. + +## Lysis of Bacteria +1. **Wash gels:** Three times in 1 mL 1X PBS (No Tween) by centrifugation at 500 x g for 30 seconds. +2. **Make Enzymatic Lysis Solution:** + - 20 mg Lysozyme from chicken egg white Sigma Aldrich + - 100 µL MetaPolyzyme Sigma Aldrich (1 mg/mL) + - 900 µL 1X PBS. +3. **Incubate gels:** In lysis solution at 37°C for 2 hours. +4. **Wash gels:** Three times in 1 mL PBS 0.1% Tween20. +5. **Make SDS Lysis Solution:** + - 20 µL Proteinase K solution, 20 mg/mL + - 100 µL 10% SDS Bio-Rad Laboratories + - 880 µL TE Buffer (Tris 10mM EDTA 1mM pH 8). +6. **Incubate gels:** In SDS Lysis solution at 55°C for 1 hour, then wash three times in 1 mL TE 2% Tween-20. + +## Barcoding the Cells +1. **Wash gels:** Three times in 1 mL Pre-injection buffer 10mM HEPES pH 7.5 Pluronic 0.1%. +2. **Load gels into a syringe:** Refer to a protocol for detailed visuals or use P200 pipette for direct pipetting. +3. **Generate PCR Master Mix:** For ~10,000 cells per library: + - 25 µL ddPCR Supermix for probes (no dUTP) Bio-Rad + - 0.4 µL P7 Primer (50 µM) mixed with Barrev primer (5 µM) + - 0.4 µL P5 Primer (50 µM) with appropriate I5 index + - 0.2 µL (10 µM) DoTA-seq multiplex primer mix (10-20 nM) + - 0.5 µL 16S DoTA-seq primers (10-20 nM) + - 0.5 µL Barcode Oligo (1 pM from 500 pM stock) + - 0.25 µL DTT Merck MilliporeSigma (500 mM). + +## Final Steps +1. **Thermocycle the PCR emulsion:** + - 95°C for 5 min + - 40 cycles of: + - 95°C for 30 sec + - 72°C for 10 sec + - 60°C for 5 min + - 72°C for 30 sec + - Final incubation: + - 72°C for 10 min + - 12°C Hold + - All ramp times at 1°C per sec. +2. **Cleanup PCR reaction:** Transfer to a new tube, vortex with 20 µL TCEP-HCl Sigma Aldrich, Clean up using the DNA Clean & Concentrator™-5 kit. Elute in 50 µL Elution Buffer. +3. **Check library:** Remove primer dimers with SPRIselect reagent kit at 0.7X volume of beads, then analyze with TapeStation Agilent G2991AA. +4. **Quantify library:** Use NEBNext Library Quant Kit for Illumina. +5. **Sequence library:** Use Illumina sequencer with custom sequencing primers. + +### Notes: +- **Barcode concentrations:** May need to be measured and adjusted for accurate encapsulation. +- **Precaution:** Handle all steps under recommended safety conditions. + +endofoutput +``` diff --git a/markdown-output/double-stranded-rna-extraction-by-cellulose-ce3btgin.md b/markdown-output/double-stranded-rna-extraction-by-cellulose-ce3btgin.md new file mode 100644 index 0000000000000000000000000000000000000000..febcff03a99243fa220650041978af1ec0b1ea12 --- /dev/null +++ b/markdown-output/double-stranded-rna-extraction-by-cellulose-ce3btgin.md @@ -0,0 +1,177 @@ +```markdown +# Goal/Experiment: +The aim of this protocol is to extract viral double-stranded RNA (dsRNA) from infected grapevine plants using cellulose. + +## Double stranded RNA extraction by cellulose + +**Author**: Vahid Jalali Javaran +**Affiliation**: Département de Biologie, Centre SÈVE, Université de Sherbrooke, Sherbrooke, QC J1K 2R1, Canada +**DOI**: [dx.doi.org/10.17504/protocols.io.4r3l2odrjy1y/v1](https://dx.doi.org/10.17504/protocols.io.4r3l2odrjy1y/v1) +**License**: Creative Commons Attribution License + +### Abstract +In this protocol, the viral dsRNA extraction from infected grapevine plants is explained. + +### Keywords +dsRNA extraction, cellulose, viral dsRNAs, grapevine + +## Materials + +### Extraction Buffer +(Add ingredients in the following order and ensure each is completely dissolved before adding the next) + +- 700 ml Ultra-pure water +- 200 ml 1 M Tris buffer (pH 8.3) +- 20 ml 0.5 M EDTA solution +- 12.7 g Lithium chloride +- 15 g Lithium dodecyl sulfate +- 10 g Deoxycholic acid +- 20 g PVP 40000 (Poly(vinylpyrrolidone)) +- 10 ml Nonidet P-40 (Detergent) +- Add up to 1 liter Ultra-pure water +- Mix well + +### Potassium Acetate Buffer (5.8 M) + +- 500 ml Ultra-pure water +- 104 ml Glacial acetic acid +- 384 g Potassium acetate +- Add up to 1 liter Ultra-pure water + +### 3 M Sodium Acetate Buffer (NaOAc) (pH 5.2) + +- 60 ml Ultra-pure water +- 24.6 g Sodium acetate +- Adjust pH to 5.2 with Glacial acetic acid +- Add up to 100 ml Ultra-pure water + +### 10x STE Buffer + +- 500 ml Ultra-pure water +- 100 ml 1 M Tris (pH 8.0) +- 20 ml 0.5 M EDTA (pH 8.0) +- 58.44 g Sodium chloride +- Add up to 1 liter Ultra-pure water + +### 1x STE + +- 900 ml Ultra-pure water +- 100 ml 10x STE buffer + +### 1x STE-18 Buffer + +- 500 ml Ultra-pure water +- 100 ml 10x STE buffer +- 180 ml 100% ethanol +- Add up to 1 liter Ultra-pure water + +### Cellulose-1x STE-18 + +- 3 g Sigmacell cellulose type 101 powder (Sigma-Aldrich, S6790) +- 20 ml 1x STE-18 + +## Total Nucleic Acid Extraction + +1. **Homogenization** + - Weigh ~ 1.5 g fresh or frozen leaves. + - Place the leaves into a 50 ml capped centrifuge tube containing 8X 8-mm stainless balls (sterilized). + - Immerse the tube in liquid nitrogen for 5 minutes. + - Rapidly mount the tubes in a foam holder and move to the MiniG chamber. + - Fix the tubes correctly according to instructions. Run at 1,500 rpm for 1 minute. + +2. **Buffer Addition** + - Add 12 ml of extraction buffer and 120 μl of 2-mercaptoethanol to each sample; mix well. + - Add 8 ml of extraction buffer and 80 μl of 2-mercaptoethanol to bean tissue. + +3. **Initial Centrifugation** + - Transfer 120 μl of suspension to each sample; shake for 40 minutes at 300 rpm. + - Centrifuge at 1000 x g for 1 minute at 10°C to remove bubbles and debris. + - Decant supernatant to a new 50 ml tube. + +4. **Potassium Acetate Addition** + - Add 12 ml of 5.8 M potassium acetate; mix thoroughly. + - Centrifuge at 14,000 x g for 15 minutes at 10°C. + +5. **Filtration and Isopropanol Addition** + - Decant supernatant through 3 layers of sterilized cheesecloth (optional) into a clean 50 ml tube. + - Add 16 ml 100% isopropanol; mix well. + - Leave at -20°C for 20 minutes. + - (Safe pause point) + +6. **Pellet Formation** + - Centrifuge at 11,000 x g for 16 minutes at 4°C. + - Carefully discard supernatant. + +## dsRNA Purification by Cellulose + +7. **Initial Resuspension** + - Resuspend pellet in 20 ml STE-18; vortex. + - Centrifuge at 14,000 x g for 15 minutes at 4°C. + - Transfer to a new 50 ml tube. + +8. **Cellulose Suspension Addition** + - Add 2 ml Sigmacell cellulose suspension (0.3 g); vortex. + - Shake at 300 rpm for 15 minutes at room temperature. + - Centrifuge at 14,000 x g for 5 minutes at 20°C; discard supernatant. + +9. **Repeats** + - Resuspend pellet in 40 ml STE-18. + - Centrifuge at 14,000 x g for 5 minutes at 20°C; discard supernatant. + - Add 20 ml STE-18; suspend, then centrifuge at 14,000 x g for 5 minutes at 20°C. + - Discard the supernatant. + +10. **Ethanol Evaporation** + - Evaporate ethanol from cellulose pellet at 40°C for 15 minutes. + - Resuspend in 6 ml 1xSTE. + - Shake at 300 rpm for 15 minutes at room temperature. + - Centrifuge at 14,000 x g for 8 minutes at 20°C. + - Transfer supernatant to a new 50 ml tube (even with cellulose particles). + +## dsRNA Precipitation + +11. **Precipitation Setup** + - Add 0.6 ml 3M NaOAc and 12 ml anhydrous alcohol; mix well. + - Leave at -20°C for 20 minutes. + - (Safe pause point) + +12. **Final Centrifugation and Washing** + - Centrifuge at 11,000 x g for 15 minutes at 4°C; discard supernatant. + - Rinse pellet twice with cold 70% ethanol. + - Air dry. + - Resuspend in 300 μl TE or sterile water. + - Transfer to 0.85 μm PES filter column (Sartorius Stedim Biotech). + - Centrifuge at 13,000 x g for 30 seconds at room temperature. + - Use 100 μl TE or water to rinse; transfer the liquid to the same column. + - Centrifuge for 2 minutes. + - Total volume: 400 μl. + - Discard filter. + - Store at -80°C (safe pause point) or proceed to digestion. + + +## Reagents and Terms + +### Reagents +- **Tris Buffer (tris(hydroxymethyl)aminomethane)**: Used to maintain a stable pH during biological reactions. +- **EDTA (Ethylenediaminetetraacetic acid)**: A chelating agent that binds divalent metal ions. +- **Lithium Chloride and Lithium Dodecyl Sulfate**: Used for RNA precipitation. +- **Deoxycholic Acid**: A bile acid used as a detergent. +- **PVP (Polyvinylpyrrolidone)**: Used to prevent phenolic compounds from affecting reactions. +- **Nonidet P-40**: A non-ionic detergent used for cell lysis. +- **Potassium Acetate**: A salt used to precipitate RNA. +- **Sodium Acetate**: Used in molecular biology for precipitating nucleic acids. +- **Ethanol**: Used in the precipitation of nucleic acids. +- **Sigmacell Cellulose**: Used to bind and purify nucleic acids. + +### Terms +- **Centrifuge/Centrifugation**: The process of using centrifugal force to separate components from a liquid. +- **Resuspend**: To mix a pellet back into a liquid solution. +- **Vortex**: To mix samples quickly using a vortex mixer. + +## Alternative Methods +- If Sigmacell cellulose type 101 powder is unavailable, other forms of microcrystalline cellulose can be a substitute. +- In place of Nonidet P-40, other non-ionic detergents such as Triton X-100 can be used but validation would be needed. + +--- + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/e-coli-protein-expression-and-purification-p89drz6.md b/markdown-output/e-coli-protein-expression-and-purification-p89drz6.md new file mode 100644 index 0000000000000000000000000000000000000000..3156fa23265a71f3cde7a8c88d4461a0d997c507 --- /dev/null +++ b/markdown-output/e-coli-protein-expression-and-purification-p89drz6.md @@ -0,0 +1,164 @@ +```markdown +# Goal/Experiment: +To express and purify a recombinant protein from E. coli for enzyme assays, protein crystallography, and other applications. + +# **E. coli Protein Expression and Purification** + +**Authors:** +Diep R. Ganguly, Timothy Rhodes, Nay Chi Khin, Estee E Tee, Kai Xun Chan +The Australian National University + +**Pogson Genomics Group** +The Australian National University + +--- + +## Abstract +Protocol for recombinant protein expression in E. coli for enzyme assays, protein crystallography, etc. + +## Guidelines +This protocol will take a few days so be sure to have all buffers, cell strains, and plasmids on hand. Practice and familiarity are the best tools. Different sections do not need to be performed immediately after each other. Several stopping points allow cells to be stored at -20/-80 °C until you are ready to continue. + +Adjust volumes, taking care to use appropriate vessels to allow proper aeration (e.g., grow 800mL culture in 2L flasks). Commonly used strains include BL21 (DE3) for T7 expression (i.e., IPTG induction). + +## Materials + +| Name | Catalog # | Vendor | +|-------------------------------------------|------------------|---------------------------| +| Potassium chloride | View | P212121 | +| Petri Dish | LHPD01100 | P212121 | +| Luria-Bertani (LB) broth, makes 1L | K488 | Amresco | +| EDTA | | | +| 1.5 mL Eppendorf tubes | | | +| Electroporation System Gene Pulser XCell | | Bio-rad Laboratories | +| 37°C Incubator | | | +| DTT | D0632 | Sigma Aldrich | +| 2.5ml SDS-PAGE Sample Loading Buffer [2X] | 786-025 | G-Biosciences | +| 14ml Polystyrene Cell Culture Tubes | CT5250 | Alkali Scientific | +| 4X Bolt LDS Sample Buffer | B0007 | Invitrogen - Thermo Fisher| +| NaCl | 53014 | Sigma Aldrich | +| Heat transfer block | Z3271 | Promega | +| IPTG | IBI068.SIZE.100g | Bio Basic Inc. | +| Coomassie Blue Staining & Destaining Solution | AR0140 | Boster Bio | +| BL21(DE3) or BL21-Star(DE3) or Rosetta2(DE3) | | | +| Magnesium chloride hexahydrate | M2670 | Sigma Aldrich | +| Electroporation Cuvette 1mm | 1652089 | Bio-Rad Sciences | +| Falcon® Conical Tubes, 50 mL 500 Tubes | 38010 | Stemcell Technologies | +| Tris-HCl | AM9855 | Life Technologies | +| 28°C incubator without CO2 | | Thermo Fisher Scientific | +| Disodium phosphate | S7907 | Sigma Aldrich | +| Monopotassium phosphate | P9791 | Sigma Aldrich | +| 42°C water bath | | | +| Imidazole | I5513 | Sigma Aldrich | +| UV/Vis spectrophotometer | View | | +| GelCode™ Blue Stain Reagent | 24590 | Thermo Fisher Scientific | + +## Safety Warnings +Ensure use of appropriate aseptic technique. Use caution if using a Bunsen burner and ethanol. + +## Before Starting +Make sure you have your verified plasmid transformed into your desired E. coli strain for protein expression (e.g., BL21 Star (DE3)). Plate on selective LB media to produce positive colonies for starter cultures. Prepare all buffers described in Step 1, except make fresh IPTG stocks (ideally). + +## Prepare Buffers +1. **10X PBS:** + - Dissolve the following in 800mL distilled H₂O: + - 80 g of NaCl + - 2.0 g of KCl + - 14.4 g of Na₂HPO₄ + - 2.4 g of KH₂PO₄ + - Adjust pH to 7.4. + - Add H₂O to 1L. + - Autoclave. + - Store 10X stock at 4°C and dilute 1:10 to make 1X working stock. + +2. **Resuspension Buffer:** + - 50 mM Tris-HCl pH 8 + - 2 mM EDTA + +3. **1X Binding Buffer:** + - 50 mM NaH₂PO₄, pH 8 + - 300 mM NaCl + - 10 mM Imidazole + +4. **1X Wash Buffer:** + - 50 mM NaH₂PO₄, pH 8 + - 300 mM NaCl + - 20 mM Imidazole + +5. **1X Elute Buffer:** + - 50 mM NaH₂PO₄, pH 8 + - 300 mM NaCl + - 250 mM Imidazole + +6. **Digestion / Storage Buffer:** + - 50 mM Tris-HCl pH 8 + - 150 mM NaCl + - 20 mM KCl + +## Transformation +2. Transform desired E. coli cell strain with plasmid to be expressed using desired method (e.g., heat shock or electroporation). + +3. **For Electropotent Cells:** + - Add 0.5 - 1 μL purified plasmid to 50 μL cells (thawing on ice, 15 minutes) + - Gently flick with finger to mix + - Transfer to chilled electroporation cuvette ensuring bubbles + - Set machine to 1.8 kV, 25 μF, 200-400 Ω + - Immediately move cuvette and add 1 mL LB. Transfer to microfuge tube + - Let recover at 37°C with ~200 rpm shaking for >1 hour. + +4. **For Chemically Competent Cells:** + - Add 0.5 - 1 μL purified plasmid to 50 μL cells (Ice, 15 minutes) + - Gently flick + - Sit on ice for 30 minutes, set to 42°C heat bath (30 - 90 seconds) + - Incubate in water bath + - Return to ice (5 minutes) + - Add 1 mL LB, let recover at 37°C ~200 rpm shaking >1 hour. + +5. Plate transformed cells (~100 μL) onto selective LB media, grow O/N @ 37°C. Obtain single colonies for subsequent inoculation. + +## Protein Expression +6. Inoculate bacterial colony from selective media into LB + antibiotic. Grow O/N @ 37°C with ~200 rpm shaking. + +7. Inoculate starter culture 1:100 dilution (e.g., 0.5 mL in 50 mL LB + antibiotic). Grow O/N @ 37°C. + +8. Grow culture at 37°C and check OD₆₀₀ after 2.5 - 3 hours. + +9. When OD₆₀₀ between 0.6 - 0.8, take an aliquot as non-induced control. To remaining culture, add induction media (e.g., 100 mM IPTG). + +10. Grow for approx. 5 hours, then check OD for difference. Non-induced should be higher by at least 0.1. + +## QC Protein Induction +11. Test constructs, induction with SDS-PAGE gels for crude lysate. + +12. Spin down cells (~1 - 3 mL) at max speed 3 minutes, remove supernatant. + +13. Resuspend cells in 100 μL 1X PBS (per 1 mL culture). + +14. Calculate how much crude lysate to load onto gel. + +15. Add appropriate amount of 4X LDS, DTT, and MgCl₂. + +16. Heat sample @ 72°C for 10 minutes. + +17. Place samples on ice, spin for max speed for 15 minutes. + +18. Transfer supernatant to new tubes. + +19. Run supernatant on SDS-PAGE gel (160V, 40-45 minutes). + +20. Stain gel with Coomassie or Gel Code Blue. Alternatively, perform Western blot if antibodies available. + +## Store Cells for Purification +21. If protein induction confirmed, preserve culture for purification, spin cells at 4°C (~7000 rcf for 5 minutes). Remove supernatant. + +22. Wash cells, spin down and remove supernatant. + +23. Snap-freeze pellet in LN₂ and store at -80°C. This is a safe stopping point. + +## Protein Purification +24. Take stored cells out of -80°C and let thaw on ice. + +--- + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/ecis-data-analysis-for-stimulation-of-human-pulmon-dapt2dnn.md b/markdown-output/ecis-data-analysis-for-stimulation-of-human-pulmon-dapt2dnn.md new file mode 100644 index 0000000000000000000000000000000000000000..a732c8f10825581b658454af59cd115a11179a1f --- /dev/null +++ b/markdown-output/ecis-data-analysis-for-stimulation-of-human-pulmon-dapt2dnn.md @@ -0,0 +1,188 @@ +```markdown +# Goal/Experiment: +To analyze the data obtained from the stimulation of Human Pulmonary Microvascular Endothelial Cells (HPMECs) with human serum using ECIS (Electric Cell-Substrate Impedance Sensing). The objective is to determine the effects of these stimuli on the cells and to calculate various parameters such as Area-under-the-Curve (AUC) and differences (ΔAUC) between different serum conditions. + +# ECIS Data Analysis for Stimulation of Human Pulmonary Microvascular Endothelial Cells (HPMECs) with Human Serum V.5 + +### Michael Bokoch +*University of California, San Francisco* + +*UCSF Transplant Anesthesia Research Group (TARG)* + +--- + +### Abstract +Sera from patients in our UCSF Liver Transplant Biobank are used to stimulate human pulmonary microvascular endothelial cells (HPMECs) grown to confluency on the ECIS platform. This protocol prospectively defines the plan for data analysis from these experiments. + +### DOI +[https://dx.doi.org/10.17504/protocols.io.bp2l69erklge/v5](https://dx.doi.org/10.17504/protocols.io.bp2l69erklge/v5) + +### License +This is an open-access protocol distributed under the terms of the [Creative Commons Attribution License](https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. + +--- + +## 1. Determine Difference in Area-under-the-Curve (Normalized Resistance at 4000 Hz) +To be calculated after recording full ECIS curve after stimulation with human serum from liver transplant (LT) patients. + +**Goal:** To calculate the difference in AUC between the normalized ECIS curves generated by (a) Liver transplant serum and (b) Healthy pooled serum. + +\[ \Delta AUC = AUC_{LT \, serum} - AUC_{Healthy \, pooled \, serum} \] + +**Sign conventions:** +- Baseline resistance is normalized to 1 +- AUC of curve that drops below 1 = Negative AUC (interpreted as an increase in endothelial cell (EC) permeability) +- AUC of curve that rises above 1 = Positive AUC + +**Examples:** +- For a typical Liver serum sample that induces permeability: + - \(AUC_{LT \, serum} = \) Negative number + - \(AUC_{Healthy \, pooled \, serum} = \) Positive number + - \(\Delta AUC = \) Large negative number +- For a typical Liver serum sample that does not induce much permeability: + - \(AUC_{LT \, serum} = \) Smaller positive number + - \(AUC_{Healthy \, pooled \, serum} = \) Larger positive number + - \(\Delta AUC = \) Small negative number + +--- + +## 2. Prepare ECIS Data for Analysis + +### 2.1 Using ECIS software, normalize the resistance +- Normalization is done by dividing the resistance at each time point by the baseline resistance (plateau resistance reached just prior to stimulation with serum). This sets the baseline resistance to 1. + +### 2.2 Export "Graph Data" and import it into GraphPad Prism +- Name the data table: `raw normalized_mm-dd-yy_cell line` + +--- + +## 3. Align t=0 + +### 3.1 Duplicate X-Y tables with data from 0-6 hrs +- Name duplicates as `*_adjustedt_0-3h*` + +### 3.2 Align time to 0 +- Inspect normalized curves for a given experiment (day and cell line). +- Determine the time point (\(t_{stim}\)) where curves begin to diverge from baseline resistance = 1. +- The time point prior to that time (\(t_{stim-1}\)) will be set to 0 hrs. +- Calculate time shift in Excel: + \[ t_{adjusted} = t_{original} - t_{stim-1} \] +- Use \(t_{adjusted}\) for determination of AUC 0-3 hrs. + +--- + +## 4. Determine AUC from 0 to 3 Hours + +### 4.1 Delete data points with \(t_{adjusted} > 3\) hrs + +### 4.2 Analyze -> XY analyses -> Area under curve +- **Baseline Y = 1** +- **Minimum Peak Height =** Ignore peaks < 5% +- **Minimum Peak Width = Leave blank** +- **Peak direction = Also consider peaks that go below the baseline** + +### 4.3 Export AUC Results: Copy "Net Area" and "Std Error" to Excel +**Note:** It is not necessary to copy/paste transpose each result individually. Copy the 'Net Area' rows from the whole table and then use `=transpose()` function in Excel. + +--- + +## 5. Calculate ΔAUC, Error, and Store Data + +### 5.1 Calculate ΔAUC +\[ \Delta AUC = AUC_{LT \, serum} - AUC_{Healthy \, pooled \, serum} \] + +--- + +### 5.2 Standard Error Calculation for ΔAUC +\[ Std \, Error_{\Delta AUC} = \sqrt{(dx^2 + dy^2)} \] + +Where: +- \(dx =\) Std Error of \(AUC_{LT \, serum}\) +- \(dy =\) Std Error of \(AUC_{Healthy \, pooled \, serum}\) + +--- + +### 5.3 Upload Mean ΔAUC\(_{0 \ to \ 3}\) hr in REDCap for each patient, time point, and cell line +**REDCap variables for Cell Line \#1:** +- auc_ln1_s1 +- auc_ln1_s2 +... +- auc_ln1_s5 + +**REDCap variables for Cell Line \#2:** +- auc_ln2_s1 +- auc_ln2_s2 +... +- auc_ln2_s5 + +--- + +## 6. Predefined Comparisons for Analysis + +### 6.1 S1 vs. Healthy Serum +**Question:** Is liver serum at the start of surgery different from Healthy controls? +- Perform one sample t-test (or one sample Wilcoxon signed rank test, if the normality assumption is not met) for each ΔAUC\(_{0 \ to \ 3}\) hr time point vs. 0.0 (the value for Healthy Pooled Serum). + +### 6.2 S1 vs. S3 +**Question:** What is the effect of liver reperfusion? +- Perform paired t-test (or Wilcoxon signed rank test, if the normality assumption is not met) for each ΔAUC\(_{0 \ to \ 3}\) hr at the S1 vs. S3 time points, which were measured for each subject. + +### 6.3 Additional Pairwise Comparisons for Time Points with Missing Data (Supplementary Analysis) +**Note:** Control column should be S1, Start of Surgery for all comparisons. + +1. S1 vs. S2: Effect of surgery (dissection phase) alone with no liver reperfusion? +2. S1 vs. S4 (or S5): If there is an effect of liver reperfusion, does it get worse with more time from reperfusion? + +--- + +## 7. Supplementary Details for Each Experiment + +### 7.1 General Parameters +- **Cell line \#** +- **Passage \#** +- **Old plate (yes/no)** +- **Seeding density (cells/well) - should be 40,000** +- **Plateau capacitance at 64,000 Hz, average and st. dev. - prior to stimulation** +- **Plateau resistance at 4,000 Hz, average and st. dev. - prior to stimulation** +- **Time of stimulation - elapsed from seeding ECIS plate** + +--- + +# Equations and References: + +## Common Equations + +- **ΔAUC Calculation:** + \[ \Delta AUC = AUC_{LT \, serum} - AUC_{Healthy \, pooled \, serum} \] + +- **Standard Error for ΔAUC:** + \[ Std \, Error_{\Delta AUC} = \sqrt{(dx^2 + dy^2)} \] + +### Notes from Prism Software +- Both AUC and ΔAUC can be tabulated in a single Prism worksheet (Grouped). +- Store results in a new "Grouped" table in Prism (Mean ± SEM) named "AUC" with column labels: + - AUC, 0-3h + - ΔAUC\(_{LT - Healthy \, 0-3h}\) + +### Important Considerations +- Plot average curves with error bars from 0-6 hrs for: + - Healthy serum with positive controls (LPS, TNF, IL-1b) and negative control (Media) - one for each day of experiment. + - Healthy serum with Liver serum (S1, S2, S3, S4, S5) - one for each patient. + - Generally, there are 3 replicates per experiment, occasionally 2 replicates. +- Different cell lines, sera, and experimental days should be treated distinctly during analysis. + +--- + +**End of Document** + +--- + +**References and Further Reading** +- [Creative Commons Attribution License](https://creativecommons.org/licenses/by/4.0/) +- [GraphPad Prism Software](https://www.graphpad.com/scientific-software/prism/) +- [REDCap Software](https://projectredcap.org/) + +--- + +**endofoutput** +``` \ No newline at end of file diff --git a/markdown-output/economical-nanopore-lsk-sequencing-adapted-protoco-c6sbzean.md b/markdown-output/economical-nanopore-lsk-sequencing-adapted-protoco-c6sbzean.md new file mode 100644 index 0000000000000000000000000000000000000000..87a87631950b05ac2b4c647a0306a85cfeadacaf --- /dev/null +++ b/markdown-output/economical-nanopore-lsk-sequencing-adapted-protoco-c6sbzean.md @@ -0,0 +1,188 @@ +```markdown +Goal/Experiment: +To develop a cost-effective alternative for end repair and dA-tailing in DNA library preparation using homebrew reagents, tailored for PCR samples with an N50 of 3 kb, with a focus on optimizing reagent use and purification methods. + +# Economical Nanopore LSK Sequencing: Adapted Protocols with Homebrew Reagents V.2 + +## Authors +- Jie Hao Ou¹ +- Yin-Tse Huang² + +¹National Chung Hsing University, Taichung +²Kaohsiung Medical University + +## Abstract +This protocol introduces a cost-effective alternative for end repair and dA-tailing in DNA library preparation, particularly tailored for PCR samples with an N50 of 3 kb. By employing a homebrew end repair reagent solution, this method replaces the use of the NEBNext® Ultra™ II End Repair/dA-Tailing Module recommended by Nanopore. Optimization of reagent quantities and a bead-free purification method are combined to achieve efficient adapter ligation while minimizing costs. Notably, an extended incubation time during adapter ligation enhances efficiency. This resource provides a strategic approach for researchers aiming to customize their sequencing workflows while achieving optimal results and substantial savings. + +## Guidelines + +1. **Optimized Homebrew Approach**: This protocol features a unique homebrew end repair reagent solution as a cost-effective alternative to the Nanopore-recommended NEBNext® Ultra™ II End Repair/dA-Tailing Module. Adhere to the specified volumes meticulously to achieve successful results with your customized reagent. + +2. **Bead-Free Purification**: The protocol employs a bead-free PEG/NaCl precipitation method for efficient purification. Ensure precise centrifugation to maintain DNA pellet integrity and maximize recovery rates. + +3. **Strategic Ligation Enhancement**: An extended incubation period during adapter ligation enhances efficiency. Dedicate adequate time to this step to optimize adapter ligation results. + +## Materials + +### 1. 4X Quick Ligase Buffer + +| A | B | C | +|---|----|---------| +| 1 | H₂O | 70 ml | +| 2 | Tris-Base | 2.42 g (200 mM) | +| 3 | MgCl₂ • 6H₂O| 0.8 g (40 mM) | +| 4 | DTT | 0.6 g (40 mM) | +| 5 | PEG 6000 | 40 g (40% w/w) | +| 6 | HCl | Adjust pH to 7.6 | +| 7 | H₂O | bring the volume up to 100 ml | + +Add the materials in the following table sequentially: + +### 2. 33.5% (w/v) PEG 8000 + +### 3. NP (5M NaCl + 6.7% PEG8000) + +| A | B | C | +|---|------|---------| +| 1 | PEG 8000 | 3.35 g (6.7%) | +| 2 | NaCl | 14.61 g (5M) | + +### 4. 80% (v/v) ethanol + +### 5. 99.5% ethanol + +### 6. Elution buffer (EB, 1X TE, pH=8.0) + +### 7. LFB wash buffer + +| A | B | C | +|---|---|---------| +| 1 | 33.5% (w/v) PEG 8000 | 80 uL | +| 2 | NP | 40 uL | +| 3 | H₂O | 320 uL | + +## Safety Warnings + +### 1. Ethanol Flammability +Ethanol is highly flammable. Use caution when handling and storing ethanol solutions. Work in well-ventilated areas away from open flames, sparks, or heat sources. + +## Homebrew End Repair/dA-Tailing + +1. Add the materials in the following table sequentially. + +| Materials | Quantity | +|-------------------------|--------------------------------------| +| **DNA** | 1600 ng (for samples with N50 of 3 kb) | +| **H₂O** | Bring up to a volume of 68 uL | +| **4X Quick Ligase Buffer** | 25 uL | +| **ATP (25 mM)** | 1 uL | +| **dNTP (10 mM)** | 5 uL | +| **Taq polymerase** | 0.5 uL (2.5 U) | +| **T4 PNK** | 0.5 uL (5 U) | + +Note: Due to the high concentration of PEG in the solution, it may be slightly viscous. Gently invert several times to ensure thorough and uniform mixing. + +2. Incubate at 37°C for 30 minutes. + + Note: T4 PNK will add phosphate to the 5' ends of both strands of DNA. + +3. Incubate at 65°C for 20 minutes. + + Note: T4 PNK will be inactivated, and Taq polymerase will begin repairing DNA ends and adding A-tails. + +## Purification + +4. For the subsequent purification, commercially available spin-column or magnetic bead-based methods can be used. Here, to save costs, we will use the PEG/NaCl precipitation method. + +5. Add 15 uL of NP. + + Note: Due to the high concentration of PEG in the solution, it may be slightly viscous. Gently invert several times to ensure thorough and uniform mixing. + +6. Centrifuge at maximum speed (at least 13000 rpm) for 15 minutes. + + Note: Most of the time, the pellet is not visible to the naked eye, so pay attention to the orientation during centrifugation. + +7. Carefully remove the supernatant using a pipette. + + Note: Sometimes the pellet becomes more visible after adding ethanol. + +8. Add 200 uL of 80% ethanol. + +9. Centrifuge at maximum speed (at least 8000 rpm) for 2 minutes. + +10. Carefully remove the supernatant using a pipette. + +11. Add 200 uL of 80% ethanol. + +12. Centrifuge at maximum speed (at least 8000 rpm) for 2 minutes. + +13. Carefully remove the supernatant using a pipette. + +14. Wait for approximately 10 minutes to ensure complete ethanol evaporation. + + Note: Prevent excessive drying of the DNA pellet. Prolonged periods can result in DNA breakage. + +15. Add 25 uL of elution buffer. + + Note: Optional: Measure the DNA concentration. Normally, the DNA concentration should be >30 ng/uL. + +## Adapter Ligation + +16. **When using LNB (with ATP) provided by Nanopore** + +| Reagent | Volume | +|--------------------|-----------------------------------------| +| **DNA** | 400 ng (for samples with N50 of 3 kb) | +| **H₂O** | Bring up to a volume of 15.25 uL | +| **Ligation Buffer (LNB)** | 6.25 uL | +| **T4 Ligase** | 2.5 uL | +| **Ligation Adapter** | 1 uL | + +**When using homemade 4X Quick Ligase Buffer (without ATP)** + +| A | B | | +|---|----|---------| +| **Reagent** | **Volume** | +| **DNA** | 400 ng (for samples with N50 of 3 kb) | +| **H₂O** | Bring up to a volume of 14.25 uL | +| **4X Quick Ligase Buffer** | 6.25 uL | +| **ATP (25 mM)** | 1 uL | +| **T4 Ligase** | 2.5 uL | +| **Ligation Adapter** | 1 uL | + +Note: The reagent amounts used in this step are about 1/4 of the manufacturer's recommended volume. This means that the ligation sequencing kit, which was originally designed for 6 uses, can now be utilized for 24 uses. This adjustment is primarily due to our utilization of a bead-free purification method later on, which significantly enhances the recovery rate. + +17. Incubate at room temperature for 1 hour. + + Note: Despite the official recommendation of a 5-minute reaction, based on our own experience, a 60-minute reaction significantly improves adapter ligation efficiency. + +18. Add 3 uL of NP. + + Note: + 1. Due to the high concentration of PEG in the solution, it may be slightly viscous. Gently invert several times to ensure thorough and uniform mixing. + 2. Normally, PEG and NaCl buffer is required for DNA precipitation. However, it's evident that LNB buffer already contains a high concentration of PEG, so adding NP alone is sufficient for DNA precipitation. For more information, refer to [this protocol](https://dx.doi.org/10.17504/protocols.io.7erhjd6). + +19. Centrifuge at maximum speed (at least 13000 rpm) for at least 15 minutes. + + Note: To prevent overheating, it is recommended to use a refrigerated centrifuge. If a refrigerated centrifuge is unavailable, it is advised to perform centrifugation in two steps of 10 minutes each, with a 10-minute interval in between. + +20. Carefully remove the supernatant using a pipette. + + Note: Most of the time, the pellet is not visible to the naked eye, so pay attention to the orientation during centrifugation. + +21. Add 200 uL of LFB wash buffer. + + Note: Note that in this step, use the LFB wash buffer instead of alcohol. This is because the DNA now has motor proteins attached, and using alcohol could disrupt the motor proteins. + +22. Centrifuge at maximum speed (at least 8000 rpm) for 2 minutes. + +23. Carefully remove the supernatant using a pipette. + +24. Repeat the above washing step twice. + +25. Add 20 uL of EB buffer (provided by the Ligation Sequencing Kit). + + Note: Optional: Measure the DNA concentration. Normally, the DNA concentration should be >15 ng/uL. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/edna-fish-metabarcoding-pcr-12s-cytb-fckbiuw.md b/markdown-output/edna-fish-metabarcoding-pcr-12s-cytb-fckbiuw.md new file mode 100644 index 0000000000000000000000000000000000000000..93aad70ad402dcd369e7d24ebb4d5c38714ccd3e --- /dev/null +++ b/markdown-output/edna-fish-metabarcoding-pcr-12s-cytb-fckbiuw.md @@ -0,0 +1,114 @@ +```markdown +# Goal/Experiment: +The goal of this experiment is to perform a one-step metabarcoding PCR to amplify either mitochondrial 12S or cytochrome B regions from environmental DNA (eDNA) samples. This will help identify fish species from freshwater environments, using Illumina MiSeq for sequencing the PCR products. + +# eDNA Fish Metabarcoding PCR: 12S & cytB +**Harriet Johnson** + +## Abstract + +One-step metabarcoding PCR. Used to amplify either mitochondrial 12S or cytochrome B regions from environmental DNA sampled from freshwater to identify fish species. PCR product contains the sequencing primers ready for sequencing on the Illumina MiSeq (custom oligos required for sequencing). + +--- + +## Guidelines + +References to primer sequences and their use with this method: + +- Kocher, Thomas D., et al. "Dynamics of mitochondrial DNA evolution in animals: amplification and sequencing with conserved primers." Proceedings of the National Academy of Sciences 86.16 (1989): 6196-6200. +- Riaz, Tiayyba, et al. "ecoPrimers: inference of new DNA barcode markers from whole genome sequence analysis." Nucleic Acids Research (2011): gkr732. +- Kelly, Ryan P., et al. "Using environmental DNA to census marine fishes in a large mesocosm." PloS one 9.1 (2014): e86175. +- Hänfling, Bernd, et al. "Environmental DNA metabarcoding of lake fish communities reflects long-term data from established survey methods." Molecular ecology (2016). + +## Protocol + +### Step 1. Dual Indexed Primers: + +- **Primers Utilized:** + - 16 forward primers + - 24 reverse primers (FA, FB, RA, RB) +- **Primer Combinations:** + - Can create 384 unique combinations to identify multiplexed samples. +- **Dilution:** + - Dilute primers to 10 µM concentration with water. + +**Preparation Example:** +```plaintext + RA701 ----> RA712 + FA501 | FA508 +``` +- **Notes:** + - Multiple dilutions are recommended for creating working concentration primers to minimize the handling of stock primers and avoid contamination. + +- **Expected Amplicon Size:** + - Cytochrome B: 460 bp region (F primer: 74, R primer: 70), total: 604 bp + - 12S: 106 bp region (F primer: 67, R primer: 62), total: 235 bp + +- **Controls:** + - *Positive control:* Rhamphochromis esox (pelagic species) + - *Negative control:* Molecular grade water + +- **Summary:** + - Number of cycles reduced from 40x to 35x. + +### Step 2. PCR Reaction Setup: + +- **20 µl Reaction (Windermere Samples):** + - 10 μl Q5 2x mastermix + - 1 μl F primer + - 1 μl R primer + - 2 μl DNA + - 6 μl H2O + +- **20 µl Reaction (Scottish Loch Samples):** + - 10 μl Q5 2x mastermix + - 1 μl F primer + - 1 μl R primer + - 4 μl DNA + - 4 μl H2O + +- **35 µl Reaction (LFC Samples):** + - 17.5 μl Q5 2x mastermix + - 1.75 μl F primer + - 1.75 μl R primer + - 3.5 μl DNA + - 10.5 μl H2O + +- **Notes:** + - Use PCR strip tubes with individual lids instead of open plates. + +### Step 3. Thermal Cycling Parameters: + +- **Initial Denaturation:** + - 98°C 5 min; +- **35 Cycles:** + 1. 98°C 10 sec + 2. 58°C 15 sec + 3. 72°C 20 sec; +- **Final Extension:** + - 72°C 5 min + - Store at 10°C + +### Step 4. Visualize PCR Product via Gel Electrophoresis: + +- **Preparation:** + - 1.5g agarose + - 100 ml 0.5x TBE + - 10 μl GelRed + +- **Procedure:** + - Run at 90V for 40 mins. + - Load 2-3 μl product with 1.5 μl Easy Ladder. + +### Step 5. Triplicate Repeat: + +- **SEPA Jan/Feb 2016:** + - 20 µl reactions performed in triplicate and then pooled. + +### Warnings + +- **Custom oligos:** Required for Illumina sequencing. +- **Sequencing Library Preparation Protocol:** Refer to the Schloss library prep protocol as needed. + +**endofoutput** +``` \ No newline at end of file diff --git a/markdown-output/effect-of-supplementation-of-iron-and-enterobactin-cgevtte6.md b/markdown-output/effect-of-supplementation-of-iron-and-enterobactin-cgevtte6.md new file mode 100644 index 0000000000000000000000000000000000000000..8a3aa6a96865cdeffdb6830e056f548d157a66ca --- /dev/null +++ b/markdown-output/effect-of-supplementation-of-iron-and-enterobactin-cgevtte6.md @@ -0,0 +1,172 @@ +```markdown +# Goal/Experiment: +To screen candidate behaviour-modifying *Escherichia coli* BW25113 single-gene deletion mutants from the 'Keio Collection', to investigate their differential effects on *Caenorhabditis elegans* behaviour when supplemented with iron and enterobactin. + +# Effect of Supplementation of Iron and Enterobactin on *C. elegans* Behaviour on Keio *E. coli* Mutants (6-well plates) + +## Author +**Saul Moore** +Imperial College London, MRC London Institute of Medical Sciences (LMS) + +## DOI +[10.17504/protocols.io.dm6gpj8bpgzp/v1](https://dx.doi.org/10.17504/protocols.io.dm6gpj8bpgzp/v1) + +## Abstract +Protocol for screening candidate behavior-modifying *E. coli* BW25113 single-gene deletion mutants from the 'Keio Collection', to investigate their differential effects on *Caenorhabditis elegans* behaviour when supplemented with iron and enterobactin. + +## Materials + +### For Bacterial Culture +- 500 mL LB +- 50 mL Erlenmeyer flasks + +### For Worm Maintenance and Imaging Plates +- 1 L NGM agar (for ingredients, see protocol for making NGM agar) +- 60 mm Petri plates ('maintenance plates') +- 90 mm Petri plates ('nursery plates') +- 6-well flat bottom plates ('imaging plates') + +### Supplements +- **Iron(III) chloride (FeCl₃)**, reagent grade, 97% (Sigma-Aldrich, CAT: 157740-100G, CAS: 7705-08-0) +- **Iron(II) sulfate (Fe₂(SO₄)₃ xH₂O)**, hydrate (Sigma-Aldrich, CAT: 307718-100G, CAS: 15244-10-7) +- **Enterobactin**, from *Escherichia coli*, ≥98% (HPLC) (Sigma-Aldrich, CAT: E3910-1MG, CAS: 28384-96-5) + +### Others +- KIMTECH Science lint-free precision wipes + +### Safety Warnings +Iron and enterobactin are toxic substances, so ensure that you wear gloves and a lab coat when working with them. + +## Protocol + +### Preparing NGM Agar + Pouring Plates + +1. **Materials Needed:** + - 6-well plates ('imaging plates') + - 15 mL Falcon tubes + - 50 mL Erlenmeyer flasks + - 90 mm Petri plates ('maintenance plates') + - 150 mm Petri plates ('nursery plates') +2. **NGM Agar Preparation**: + Make 1 L normal Nematode Growth Media (NGM) agar following the protocol: + [Making normal NGM for imaging plates (Cabreiro Lab)](https://dx.doi.org/10.17504/protocols.io.6bhhaj6) + +3. **Plate Pouring**: + Pour 15 mL NGM agar into each 60 mm maintenance plate, and 35 mL NGM agar into each 90 mm nursery plate, following the protocol for Plate pouring: + [Plate pouring](https://dx.doi.org/10.17504/protocols.io.6bhhaj6). + +4. Using the Integra ViaFill, dispense 4 mL NGM agar into each well of the 6-well plates, following the protocol: + [Dispensing agar into multiwell plates](https://dx.doi.org/10.17504/protocols.io.6bhhaj6) + +5. Leave the plates on the lab bench (with lids on) until the agar has cooled and solidified (approximately 1 hour, timing depends on humidity) + +6. Measure the weight of 3 imaging plates (with lids on) and record the average plate weight on the day of pouring. + +7. Dry the imaging plates under a hood (or drying cabinet) until the plates lose between 3-5% of their original plate weight (with lids on). + +8. Store the imaging plates upside-down at 4°C until used for experiments. + +### Preparing Worms + +9. **Inoculate** 10 mL LB broth media with *E. coli* BW25113 (Keio background wild-type strain, used as negative control and for raising worms, no Kanamycin) in an Erlenmeyer flask for overnight culture following the protocol: + [Inoculating a Liquid Bacterial Culture by Priota Islam](https://dx.doi.org/10.17504/protocols.io.6bhhaj6) + +10. Place the inoculation in a shaking incubator at 37°C at 200 rpm and leave to grow overnight. + +11. Remove the BW culture from the shaking incubator and place it in a 4°C fridge until seeding. + +12. Remove the plates from storage and the BW culture from the fridge, and leave on the bench for approximately 30 minutes to acclimate to room temperature. + +13. Using aseptic technique, seed the 60 mm maintenance plates each with approximately 250 µL of BW25113 culture. + +14. Leave it under a hood until dry (with lids on, timing depends on humidity). + +15. Using a platinum pick, gently pick 30 adult N2 Bristol *C. elegans* onto each maintenance plate, and store in an incubator at 20°C. + +16. After 24 hours, remove the adult worms, leaving the eggs behind to hatch into L1 larvae. + +17. Inoculate a further 10 mL LB broth with BW25113 bacteria for overnight culture (no Kanamycin), following the protocol in step 9 and place in a shaking incubator at 37°C. + +18. After 24 hours, remove the culture from the incubator, and the 90 mm nursery plates from storage, and leave to acclimate on the bench top for 30 minutes. + +19. **Seeding Nursery Plates**: + Seed the nursery plates each with approximately 1 mL of fresh BW25113 culture. Leave under hood until dry. + +20. Wash the worms off the BW-seeded maintenance plates, into two 15 mL Falcon tubes. + +21. Perform an egg prep on worms in the Falcon tubes, following the protocol: + [Egg Prep for Bleach Synchronization (Cabreiro Lab)](https://dx.doi.org/10.17504/protocols.io.6bhhaj6) + +22. At around noon the next day, wash L1-arrested larvae off the empty plate and re-feed onto the BW-seeded nursery plates using a glass Pasteur pipette. Aim to dispense around 500 worms per plate. + +23. Incubate at 20°C for 68 hours until the worms are Day 1 adults for the experiment. + +### Preparing Bacteria + +24. Fill 2 separate Erlenmeyer flasks with 25 mL LB. Add 50 µg/mL Kanamycin to one flask, and leave the other flask without Kanamycin for the BW25113 control. + +25. Remove the required Keio frozen stock plates from -80°C containing the strains for antioxidant testing. Gently remove the aluminum film and leave it to partially thaw for a minute or so. + + > **Caution**: To avoid damaging the bacterial stocks through repeated freeze-thaw, do not let the wells completely defrost. Just enough to be able to pick up some cells with the replicator. + +26. Inoculate the Erlenmeyer flasks with the desired strains for antioxidant testing from Keio frozen stock plates, following the protocol: + [Inoculating a Liquid Bacterial Culture by Priota Islam](https://dx.doi.org/10.17504/protocols.io.6bhhaj6) + +27. Incubate the cultures overnight at 37°C in a shaking incubator at 200 rpm. + +28. Remove the overnight cultures from the incubator. Inoculate 2 more Erlenmeyer flasks for a second round of overnight cultures from the first, this time without Kanamycin (to avoid exposing the worms to the antibiotics), and incubate overnight at 37°C at 200 rpm. + +29. After 24 hours, remove the cultures from the incubator and store at 4°C until used for experiments. + +### Seeding Imaging Plates (6-well) + +30. Remove the imaging plates from 4°C storage. + +31. Ensure that imaging plates have lost approximately 3-5% of their original weight (so that they are not too wet for imaging when seeded). Place under a hood or drying cabinet until they have. + +32. Remove overnight cultures of Keio strains from 4°C storage. Using a pipette, seed 30 µL of bacterial culture into the wells of each 6-well imaging plate. + +33. Place the seeded plates under a laminar flow hood to dry for 20 minutes, then place in an incubator at 25°C (no shaking) for 7 hours 40 minutes (total lawn growth time: 8 hours). + +34. After 8 hours total growth time, remove the plates from the incubator and store at 4°C. + +### Adding Iron and Enterobactin (6-well) + +35. On the day of tracking, remove the seeded imaging plates from 4°C, and dry for 30 minutes under a laminar flow hood. + +36. **Supplement Preparation**: + Remove the iron(III) chloride, iron(III) sulfate, and enterobactin from 4°C storage. + - Prepare 100 mM and 400 mM of iron(III) chloride and iron(III) sulfate (in H₂O). + - Prepare 1 mg/mL enterobactin solution (in DMSO). + +37. Using a pipette, dispense 40 µL of iron(III) chloride and iron(III) sulfate into the desired wells (on top of the lawns) of the 6-well imaging plates (for a final concentration of 1 and 4 mM iron in 4 mL agar). + +38. Using a pipette, dispense 5 µL of enterobactin solution on top of the lawns of the desired wells in the 6-well imaging plates (for a final concentration of 1.25 µg/mL enterobactin in 4 mL agar). + +39. Leave the plates to dry in a biosafety hood for 30 minutes. + +40. Record the weight of the plates after drying (as weight at imaging). + +41. Then leave the plates on the bench (with lids on) for a further 1 hour 30 minutes (total 2 hours with the supplements added) before adding worms. + +### Picking Worms + Hydra Tracking (6-well) + +42. Prior to tracking, ensure that the imaging cave air conditioning is turned on (and there has not been a power-cut) and also empty the dehumidifier wastewater tray (see pre-imaging checklist). + +43. Remove the nursery plates of worms from the 20°C incubator. + +44. Using a platinum worm pick, carefully pick 10 Day 1 worms onto the edge of the bacterial lawns in each well of the 6-well imaging plates, then place in an incubator at 20°C until tracking (after 4 hours on food + supplements). + +45. 30 minutes prior to tracking with the Hydra rig (each run is performed every 20-30 minutes), remove 5 imaging plates at a time from the 20°C incubator and leave to acclimate in the imaging cave. + +46. Wipe the underside of the lids using KIMTECH Science lint-free precision wipes (to remove any condensation that has formed). + +47. Place the plates under the Hydra rig and record worm behavior on the bacterial food for 15 minutes (at the 4-hour timepoint, 25 fps, exposure: 25000 msec, blue-light stimulation). + +48. After tracking, discard the plates in a biological waste bin. + +49. Check tracking checklist to ensure that all videos have been saved correctly: + '/Volumes/behavgenom$/Documentation/Protocols/analysis/tracking-checklist-20210210.docx' + +endofoutput +``` diff --git a/markdown-output/effective-identification-of-dna-bound-protein-comp-bjj6kkre.md b/markdown-output/effective-identification-of-dna-bound-protein-comp-bjj6kkre.md new file mode 100644 index 0000000000000000000000000000000000000000..446453ce31a9405190cdfa38dc918ad77399fe62 --- /dev/null +++ b/markdown-output/effective-identification-of-dna-bound-protein-comp-bjj6kkre.md @@ -0,0 +1,240 @@ +```markdown +# Goal/Experiment: +Effective Identification of DNA-bound Protein Complexes using Chromatin Immunoprecipitation + +## Effective identification of DNA-bound protein complexes using Chromatin Immunoprecipitation V.2 + +**Georgios I Laliotis**1, **Philip N. Tsichlis**1 +1The Ohio State University + +**DOI**: [dx.doi.org/10.17504/protocols.io.bij6kkre](https://dx.doi.org/10.17504/protocols.io.bij6kkre) + +**Protocol Citation**: +Georgios I Laliotis, Philip N. Tsichlis 2020. Effective identification of DNA-bound protein complexes using Chromatin Immunoprecipitation. *protocols.io* [https://dx.doi.org/10.17504/protocols.io.bij6kkre](https://dx.doi.org/10.17504/protocols.io.bij6kkre) + +**License**: +This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. + +**Created**: Aug 11, 2020 +**Last Modified**: Aug 11, 2020 +**Protocol ID**: 40286 + +--- + +## Chemicals Required (1d) + +### 1. Cytosolic Lysis Buffer (200 ml) + +- **Composition:** + - 5 mM PIPES (pH 8.0) + - 85 mM KCl + - 0.5% NP40 + - PI (Protease Inhibitor) + +**Preparation:** +- 0.3 g PIPES => Adjust pH to 8.0 +- 8.5 ml 2M KCl +- 10 ml 10% NP40 + +### 2. Nuclear Lysis Buffer (50 ml) + +- **Composition:** + - 50 mM Tris (pH 8.0) + - 10 mM EDTA + - 0.5% SDS + +**Preparation:** +- 2.5 ml 1M Tris (pH 8.0) +- 1 ml 0.5M EDTA +- 1.25 ml 20% SDS + +### 3. IP Dilution Buffer (250 ml) + +- **Composition:** + - 16.7 mM Tris (pH 8.0) + - 167 mM NaCl + - 1.2 mM EDTA + - 1.1% Triton X-100 + - 0.01% SDS + +**Preparation:** +- 4 ml 1M Tris (pH 8.0) +- 0.6 ml 0.5M EDTA +- 8.5 ml 5M NaCl +- 12.5 ml 20% Triton X-100 +- 250 ul 10% SDS + +### 4. Low Salt Wash Buffer (250 ml) + +- **Composition:** + - 20 mM Tris (pH 8.0) + - 2 mM EDTA + - 150 mM NaCl + - 1% Triton X-100 + - 0.1% SDS + +**Preparation:** +- 5 ml 1M Tris (pH 8.0) +- 1 ml 0.5M EDTA +- 7.5 ml 5M NaCl +- 12.5 ml 20% Triton X-100 +- 2.5 ml 10% SDS + +### 5. High Salt Wash Buffer (250 ml) + +- **Composition:** + - 20 mM Tris (pH 8.0) + - 2 mM EDTA + - 500 mM NaCl + - 1% Triton X-100 + - 0.1% SDS + +**Preparation:** +- 5 ml 1M Tris (pH 8.0) +- 1 ml 0.5M EDTA +- 25 ml 5M NaCl +- 12.5 ml 20% Triton X-100 +- 2.5 ml 10% SDS + +### 6. NS Buffer (50 ml) + +- **Composition:** + - 50 mM Hepes + - 500 mM NaCl + - 1 mM EDTA + - 1% Triton X-100 + - 0.1% Na-deoxycholate + - 0.1% SDS + +### 7. LiCl Wash Buffer (250 ml) + +- **Composition:** + - 10 mM Tris (pH 8.0) + - 1 mM EDTA + - 250 mM LiCl + - 1% NP40 + - 1% (w/v) Deoxycholic acid + +**Preparation:** +- 5 ml 1M Tris (pH 8.0) +- 0.5 ml 0.5M EDTA +- 72.5 ml 1M LiCl +- 50 ml 10% NP40 +- 0.25 g Deoxycholic acid + +### 8. Miscellaneous + +- **1.25M Glycine:** 9.38 g / 100 ml +- **1M NaHCO3:** 8.4 g / 100 ml => Aliquot (0.5 ml) and store at -20°C + +--- + +## Cross-linking and Lysis of the Cells (6h) + +### For Cell Lines (20-50 x 10^6 cells) + +For each Ab to be tested, 3 plates for each cell line must be used. + +1. Add 270 ul of 37% Formaldehyde into 10 ml culture media (P100) +2. Incubate at 37°C for 15 min +3. Add 1 ml of 1.25 M Glycine and incubate for 5 min at RT +4. Wash with cold PBS (x2) +5. Wash with 1 ml of cold PBS (+ PI) +6. Scrap cells and collect into a new tube + - *Tip*: Scrap each plate and place it in a different tube +7. Centrifuge at 14000 rpm for 1 min at 4°C to remove PBS +8. Remove supernatant +9. Incubate lysis buffer in 37°C. For a few minutes to dissolve precipitates +10. Add fresh 1x proteinase inhibitor: 10 ul from 100x stock +11. Add/Resuspend 1 ml of nucleic lysis buffer (+ PI) per 3 P100 plates => 200 ul for 1 x 10^6 cells +12. Manually shake tube to break pellet +13. Split the 1 ml lysate into 2 tubes with 500 ul - Helps the sonication process +14. Incubate on ice for 10’ +15. Sonication: 15 sec on/45 sec off (60 sec x 6-7 times = 6-7 min on/each sample) at 30% duty +16. Centrifuge at 14000 rpm for 15 min at 4°C +17. Transfer supernatant into a new single tube + - *Can be stored at -80°C* + +--- + +## Immunoprecipitation of Crosslinked Protein/DNA Complexes (1d 6h) + +1. Dilute 5 fold of lysate volume with IP dilution buffer in a 15 ml tube +2. Add 1x protease inhibitors in the IP dilution buffers +3. Dilute to 5 ml from initial 1 ml using 4 ml IP dilution buffers +4. Split in 2x tubes for Ab and 2 for normal IgG + + **Caution**: Each tube is HALF REACTION => Final buffer concentration 0.1% SDS of nucleic lysis buffer + +5. Pre-cleaning: + - Add 30 ul of protein G bead / Ab reaction for pre-clearing per tube +6. Incubate for 1 hr at 4°C with rotation +7. Centrifuge at 3000 rpm for 2 min at 4°C +8. Transfer supernatant into a new 2 ml tube +9. Add 2 ug of antibody or normal IgG per tube (for Normal Rabbit IgG add 2 ul from 1 mg/dl. In order to make that add 8.7 ul from 11.4 mg/dl stock and 92.3 ddH2O) +10. Incubate overnight at 4°C with rotation +11. Add 25 ul of protein G agarose bead / Ab reaction per tube +12. Incubate for 2 hrs at 4°C with rotation +13. Centrifuge at 3000 rpm for 2 min at 4°C +14. Place beads at least 5 minutes in magnetic rack +15. Remove supernatant carefully with WB long tips gradually +16. Add/Resuspend beads 500 ul cold low salt wash buffer/tube and combine tubes in 1 +17. Rotate for 5’ at 4°C +18. Wash with wash buffer: + - *Order:* + - Cold low salt wash buffer (x2) + - Cold high salt wash buffer (x2) + - Cold LiCl wash buffer (x2) + - Cold TE buffer (x3) + +19. Centrifuge at 3000 rpm for 2’ +20. Place on magnetic rack for 5’ +21. Add 500 ul of buffer +22. Incubate for 5 min at 4°C with rotation +23. Centrifuge at 3000 rpm at RT (or 4°C) for 1 min +24. Place on magnetic rack for 5’ +25. Remove supernatant +26. Wash with next one etc. +27. Add 200 ul elution buffer (0.1M NaHCO3 and 1% SDS) +**Preparation for 1.5 ml:** + - 1.2 ml H2O first + - then, 150 ul 10x SDS + - finally, 150 ul 1M NaHCO3 + +28. Incubate for 15 min with shaking heating block (900 rpm) at 23°C +29. Centrifuge at 3000 rpm for 3 min +30. Put in magnetic rack for 5’ - Transfer supernatant to a new tube +31. Repeat #16-19 (Total elution vol. will be 400 ul) + +--- + +## Reverse Crosslinking and Elution of Protein/DNA Complexes (6h) + +1. Add 20 ul of 5M NaCl and 1 ul of 20 mg/ml RNase / 200 ul tube (remember the volume is 400 ul) + + **Note:** Don’t forget input sample (10% lysate + 350 ul Elution buffer)!! + +2. Mix well manually before putting into the cycler +3. Incubate at 65°C in Thermo cycler for 5 hrs or overnight with 450 rpm rotation to prevent precipitation +4. Add 10 ul of 0.5M EDTA, 20 ul of 1M Tris (pH 8.0), and 2 ul of 10 mg/ml Proteinase K per tube +5. Incubate at 45°C for 1 hr + +### QIAGEN PCR Extraction Kit (or use P/C/I extraction method) + +1. Add 5 voulmes of PBI buffer (QIAGEN PCR extraction kit) and mix well - 450 ul => 2250 ml final volume. + - **Note:** Check pH => add 10 ul 3M sodium acetate if #23 turn orange or violet color. + +2. Add sample into a column-max 800 ul +3. Centrifuge at 14000 rpm for 1 min +4. Add 750 ul of PE buffer +5. Centrifuge at 14000 rpm for 1 min and remove liquid from a tube +6. Centrifuge at 14000 rpm for 1 min again +7. Add 60 ul of EB buffer +8. Centrifuge at 14000 rpm for 1 min +9. Store DNA elution at -20°C +10. Carry out qPCR using 2 ul elution/reaction + +--- + +**endofoutput** +``` \ No newline at end of file diff --git a/markdown-output/effective-identification-of-protein-protein-intera-bqx8mxrw.md b/markdown-output/effective-identification-of-protein-protein-intera-bqx8mxrw.md new file mode 100644 index 0000000000000000000000000000000000000000..b5963be806a53fd8726b873aa623c088178349f0 --- /dev/null +++ b/markdown-output/effective-identification-of-protein-protein-intera-bqx8mxrw.md @@ -0,0 +1,105 @@ +```markdown +Goal/Experiment: Effective Identification of Protein-Protein Interaction using RIME-IP + +# Effective Identification of Protein-Protein Interaction using RIME-IP + +**Author**: George Laliotis +**Affiliation**: The Ohio State University +**Published**: Dec 21, 2020 + +**DOI**: [10.17504/protocols.io.bqx8mxrw](http://dx.doi.org/10.17504/protocols.io.bqx8mxrw) +**Protocol ID**: 45792 + +## License +This is an open access protocol distributed under the terms of the [Creative Commons Attribution License](https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. + +## Disclaimer +**DISCLAIMER – FOR INFORMATIONAL PURPOSES ONLY; USE AT YOUR OWN RISK** + +The protocol content here is for informational purposes only and does not constitute legal, medical, clinical, or safety advice, or otherwise; content added to protocols.io is not peer reviewed and may not have undergone a formal approval of any kind. Information presented in this protocol should not substitute for independent professional judgment, advice, diagnosis, or treatment. Any action you take or refrain from taking using or relying upon the information presented here is strictly at your own risk. + +## Buffer Preparation (2h) + +This is a modification of the Mohammed et al., 2016 (Nature protocols, 11(2), p.316.), that has been edited and optimized for the need of Tschilis Lab. The authors declare no conflicts of interest. + +### Reagents Preparation +- **1M Tris (pH=7.5)** + For 100mL, add 12.114g base. Adjust pH to 7.5. + +- **1M NaCl** + For 100mL, add 5.844g. + +- **50mM EDTA** + For 100mL, add 1.4612g. + +- **5mM EGTA** + For 100mL, add 0.19g. + +- **50mM DTT** + For 1.5mL, add 75µL from 1M stock. + +## Immunoprecipitation (2d) + +### Buffer Preparation +Prepare the following buffers (usually prepare 25ml of each, store at 4°C and make fresh every month) at the final concentration listed: + +#### Lysis Buffer 1 (LB1) - Will Contain Cytoplasmic Proteins +For 25mL Volume: +- 50mM Tris-HCl, pH 7.5 - add 1.25mL from 1M stock. +- 20mM NaCl - add 500µL from 1M stock. +- 1mM EDTA - add 50µL from 50mM stock. +- 0.5% NP-40 - add 125µL from stock (Sigma # 74385-1L). +- 0.25% Triton X-100 - add 62.5µL from stock. +- 10% Glycerol - add 2.5mL from stock. +- 1mM DTT - add 75µL from 50mM stock. + +#### Lysis Buffer 2 (LB2) - Wash to Remove LB1 and Cytosolic Protein +For 25mL Volume: +- 10mM Tris-HCl, pH 7.5 - add 250µL from 1M stock. +- 20mM NaCl - add 500µL from 1M stock. +- 1mM EDTA - add 500µL from 50mM stock. +- 0.5mM EGTA - add 500µL from 5mM stock. +- 1mM DTT - add 75µL from 50mM stock. + +#### Lysis Buffer 3 (LB3) +For 25mL Volume: +- 10mM Tris-HCl, pH 7.5 - add 250µL from 1M stock. +- 100mM NaCl - add 2.5mL from 1M stock. +- 1mM EDTA - add 500µL from 50mM stock. +- 0.5mM EGTA - add 500µL from 5mM stock. +- 0.1% (w/v) Sodium Deoxycholate. +- 0.5% (v/v) N-Lauroylsarcosine - add 625µL from 20% Sigma stock. +- 1x Protease Inhibitors - add 250µL from 100x stocks. + +## Day 1 + +### Antibody-Bead Conjugation Day One +1. Magnetic Beads (Pierce Cat No. # 88802), lightly vortex to resuspend. With a cut pipette tip, transfer 50-75µL (some will be lost during the washes) of beads to 1mL Eppendorf tube. Place magnetic stand and remove supernatant. +2. Add 500µL ice-cold PBS and lightly vortex to resuspend. Spin briefly with table-top microcentrifuge at 2000xg for 1’ (Tip: By working in higher speed you are destroying the magnetic beads). Place tube in magnetic stand and remove PBS wash. Repeat 2x for a total of 3 washes. +3. Add 300µL of LB3 to beads. Repeat vortex, spin and wash removal as in step 2 for a total of 3 washes. +4. Add antibody for IP (according to company specifications-5µg) to the top of the beads and incubate on ice for 10 min. Add 300µL LB3 to beads. Vortex tube lightly to resuspend beads. Rotate bead/antibody mixture at 4°C O/N. + +## Day 2 + +### Harvest Cells +1. Add media from 15cm plate at 80-90% confluent (around 2x10^7 cells) to 50mL Falcon. Wash the cells with 5mL ice-cold PBS and add to Falcon. Add 3mL ice-cold PBS and collect cells with a scraper. Add to Falcon. Rinse plate with 5-10mL ice-cold PBS and add to Falcon. +2. Pellet cells by centrifugation at 4000xg for 6 min. Aspirate media. +3. Resuspend cell pellet in 1mL of ice-cold PBS and transfer to Eppendorf tube. Rinse Falcon with 500µL of ice-cold PBS and transfer to Eppendorf. Spin in step 2. +4. Remove PBS. Wash cell pellet with 500µL PBS, spin as before and remove PBS wash. +5. Resuspend cell pellet in 750µL of LB1 with fresh cocktail inhibitor. Rotate the tube at 4°C for 10 min. Centrifuge at 14000rpm at 4°C for 6 min. Remove supernatant (cytosolic proteins) and label tube if needed for further analysis. +6. Add 500µL LB2 + inhibitors to the top of nuclei. Centrifuge at 12,000rpm at 4°C for 6 min. Remove supernatant. +7. Resuspend nuclei in 300µL LB3 + inhibitors. Sonicate cells on high 30s on/off for 5 min. Centrifuge to pellet debris max. Centrifuge to pellet debris max speed at 4°C for 10 min. Do not forget the 10% input. + +### Antibody-Beads Conjugates Washes +1. Place beads on magnetic rack and remove supernatant. You can save supernatant and run in gel along with your samples to check against fractions of beads-Ab. +2. Wash beads with 300µL of LB3, vortex lightly to resuspend, spin briefly and remove supernatant. Repeat wash 4x for a total of 5 washes. +3. Quantitate the amount of protein with Bradford prior to IP to ensure equal loading. Add the appropriate LB3 protein volume to antibody-bead conjugate. Rotate at 4°C O/N. + +## Day 3 + +### Western Blot Analysis +1. Place beads in magnetic stand and remove protein mixture. Add 500µL LB3 to beads to wash, lightly vortex, spin briefly, place in stand and remove wash. Repeat this step at least 4x for a total of 5 washes. +2. For WB analysis, add about 50µL 5x sample buffer and 2.5µL 50mM DTT. Boil in at 99°C for 10’. Place on magnetic rack and remove supernatant. This is your sample for western blot analysis. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/effective-identification-of-rna-binding-proteins-u-bjpbkmin.md b/markdown-output/effective-identification-of-rna-binding-proteins-u-bjpbkmin.md new file mode 100644 index 0000000000000000000000000000000000000000..016c3b48f6c0383fe1588278c0615b3330c9468b --- /dev/null +++ b/markdown-output/effective-identification-of-rna-binding-proteins-u-bjpbkmin.md @@ -0,0 +1,214 @@ +```markdown +# Effective Identification of RNA-binding Proteins using RNA Immunoprecipitation + +## Goal/Experiment: +The purpose of this protocol is to identify RNA-binding proteins through the use of RNA immunoprecipitation (RIP). This method involves isolating the protein-RNA complexes using specific antibodies and analyzing the bound RNA. + +## Authors: +Georgios I. Laliotis1, Philip N. Tsichlis1 +1The Ohio State University + +DOI: [dx.doi.org/10.17504/protocols.io.bjpbkmin](https://dx.doi.org/10.17504/protocols.io.bjpbkmin) + +## Protocol Citation: +Georgios I. Laliotis, Philip N. Tsichlis 2020. Effective identification of RNA-binding proteins using RNA Immunoprecipitation. protocols.io. [https://dx.doi.org/10.17504/protocols.io.bjpbkmin](https://dx.doi.org/10.17504/protocols.io.bjpbkmin). + +## License: +This is an open-access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. + +## Created: +Aug 13, 2020 + +## Last Modified: +Aug 13, 2020 + +## Protocol Integer ID: +40387 + +--- + +## Chemicals Required + +### 1. Nuclear Isolation Buffer +1.28M Sucrose, 40mM Tris-HCl (pH=7.5), 20mM MgCl₂, 4% Triton-X + +**For 100ml:** + +| Reagent | Amount | +|--------------|--------| +| 1.28M sucrose| 43.21g | +| 1M Tris-HCl 7.5 | 4ml | +| 100mM MgCl₂ | 20ml | +| 100% Triton-X| 4ml | + +### 2. R.I.P. Buffer +150mM KCl, 25mM Tris-HCl (pH=7.5), 5mM EDTA, 0.5mM DTT, 0.5% Nodidet P-40 + +**For 250ml:** + +| Reagent | Amount | +|----------------|---------| +| 150mM KCl | 2.795g | +| 1M Tris-HCl 7.5| 6.25ml | +| 50mM EDTA 8.0 | 2.5ml | +| 1M DTT | 125ul | +| 100% NP-40 | 1.25ml | +| Protease inhibitor | Add fresh every time | +| RNAase inhibitor | Add fresh every time | + +**Instructions:** +- Autoclave and keep chilled at 4°C. + +--- + +## Cross-linking and Lysis of The Cells + +### Cross-linking is suggested to make stable interactions +#### For Cell Lines +For each antibody to be tested, use 1×100mm plates. + +1. Add 270µl of 37% Formaldehyde into 10ml culture media (P100). +2. Incubate at 37°C for 15 minutes. +3. Add 1ml 1.25M Glycine and incubate 5 minutes at room temperature. +4. Wash cells with ice-cold PBS ×2. +5. Add 1ml PBS (200µl), Nuclear isolation buffer (200µl), and water (600µl). +6. Scrape cells and collect in a new tube. +7. Centrifuge at 14,000rpm for 2 minutes at 4°C. +8. Remove supernatant. +9. Add/resuspend with 1ml freshly made RIP buffer (10µl protease inhibitor + 100U/ml RNAase inhibitor). +10. Incubate on ice for 10 minutes. +11. Sonication: 15 sec on / 45 sec off (6 times for each sample). +12. Centrifuge at 14,000rpm for 15 minutes at 4°C. +13. Transfer the supernatant into a new single tube (keep 10% input and 10µl for Western blot analysis of RNA-binding protein of interest, add 10µl 2× Loading buffer + DTT, boil for 10 minutes at 99°C and perform Western Analysis). + +--- + +## Immunoprecipitation of Crosslinked Protein/RNA Complexes + +1. Preclearing: Add 30µl of protein G bead for preclearing. +2. Incubate for 1 hour at 4°C with rotation. +3. Centrifuge at 3000rpm for 2 minutes at 4°C. +4. Transfer supernatant to a new tube. +5. Add 1-5µg of antibody or IgG (negative control). +6. As positive control use anti-SNRNP70 Ab Millipore Cat. # CS203214. +7. Incubate overnight at 4°C with rotation. +8. Add 50µl Magnetic Beads and incubate with rotation for 1-4 hours at 4°C. +9. Spin beads at 3000rpm for 2 minutes at 4°C. +10. Resuspend in 500µl RIP buffer. +11. Rotate at 4°C for 5 minutes each. Repeat ×3 for a final of 4 washes. +12. Resuspend beads with 100µl RIP buffer + 1µl RNAase inhibitor (do not forget the 10% input). +13. Incubate at 70°C for 1 hour to de-crosslink samples. +14. Spin down and remove supernatant. + +--- + +## RNA Purification and Reverse Transcription + +### RNA Purification + +1. Resuspend beads in 100µl RIP buffer with 0.1% SDS and 30µg protease K. (For 1ml add 10µl from 10% SDS and 3µl from 10mg/ml protease K). +2. Incubate at a heating block at 55°C for 30 minutes with shaking. +3. Repeat step 1-2 two times. For input samples do the step once. +4. Add one volume of phenol-chloroform-isoamyl alcohol mixture and vortex to mix. Centrifuge for 1 minute to separate phases. Recover (upper) water phase (you can aspirate with no problem, 175µl). +5. Add 10µg yeast tRNA (1mg/ml), 12µl 3M sodium acetate, and 250µl ethanol to 100µl water phase and mix. Ethanol-precipitate at -80°C overnight. Note: other carriers such as linear acrylamide or glycogen (5µl from 20µg/µl stock I use Invitrogen Glycogen) can be used instead of yeast tRNA. +6. Centrifuge at 14,000rpm for 30 minutes at 4°C and discard the supernatant carefully. +7. Wash the pellet once with 80% ethanol. Centrifuge at 14,000rpm for 15 minutes at 4°C. +8. Discard the supernatant carefully and air dry the pellets. +9. Resuspend in 20µl RNAase-free water and place tubes on ice. +10. Typically, proceed to RNA quality control of the input with Nanodrop. Ideally, we expect the 260/230 ratio to be close to 2.00. + +### Reverse Transcription + +1. Label the appropriate number of PCR tubes (0.2ml) for the number of samples to be analyzed and place on ice. +2. Set the reaction below. + +**Reaction Mixture:** + +| Reagent | Volume/reaction (µl) | +|---------------------|---------------------| +| RNA | 9.0 | +| 5× RT Buffer | 4.0 | +| 20× Enzyme mix | 1.0 | +| RT Primer Mix | 1.0 | +| RNAse free-water | 5.0 | +| **Final Volume** | **20.0** | + +3. In a Thermocycler, set the following program: + +**Thermocycler Program:** + +| Step | Temperature | Time | +|--------------------|-------------|-------| +| RT Reaction | 37°C | 60min | +| Stop the Reaction | 95°C | 5min | +| Hold | 4°C | Hold | + +4. Remove the PCR Tubes. Dilute the reaction with 180µl Nuclease-free water (10× dilution). Reactions can be stored at -20°C. + +--- + +## Positive Control RT-PCR + +1. Label the appropriate number of 0.2ml PCR tubes for the number of samples to be analyzed and place on ice. + - At a minimum, there will be 4 samples to undergo PCR using the RIP Primers: cDNA from positive (anti-SNRNP70) and negative control antibody (Normal Rabbit IgG) immunoprecipitations, input, and a no template tube as a Control for DNA contamination. + - The RIP primers are specific for the human U1 snRNP gene. + **Forward Primer:** 5'-GGG AGA TAC CAT GAT CAC GAA GGT-3' + **Reverse Primer:** 5'-CCA CAA ATT ATG CAG TCG AGT TTC CC-3' + +2. Add 2µl of appropriate sample to the PCR tube and return on ice. +3. Add the following reagents: + +| Reagent | Volume/reaction (µl) | +|-------------------|----------------------| +| DNA | 2.0 | +| 10× PCR Buffer(-MgCl₂)| 10.0 | +| MgCl₂ (50mM) | 0.6 | +| 2.5mM dNTPs | 1.6 | +| RIP Primers U1 snRNA | 0.8 | +| Taq (5U/µl) | 0.4 | + +4. Set up the following PCR reaction. + +**PCR Reaction:** + +| Step | Temperature | Time | +|--------------------|-------------|---------| +| Initial Denaturation| 94°C | 3min | +| Denature | 94°C | 20sec | +| Anneal | 60°C | 30sec | +| Extension | 72°C | 30sec | +| Final Extension | 72°C | 2min | + +5. Run on 2% agarose gel. The expected size of the PCR Product is 100 base pairs. You should see bands in anti-SNRNP70 RIP and in 10% input. + +--- + +## RIP-qPCR/RIP-Seq + +1. Add 2µl of the cDNA sample to the PCR plate suitable for the real-time instrument of choice, performing in triplicates. +2. Prepare the following master mix. + +**Master Mix:** + +| Reagent | Volume/reaction (µl) | +|--------------------|----------------------| +| cDNA | 2 | +| SYBR-Green Master Mix | 12.5 | +| RT Primer Mix | 1.0 | +| RNAse free-water | 9.5 | +| **Final Volume** | **23.0** | + +3. Run on this program: + +**Program:** + +| Step | Temperature | Time | Cycles | +|--------------------|-------------|--------|--------| +| Initial Denaturation| 95°C | 10min | 1 | +| Denature | 95°C | 15sec | 40 | +| Anneal and extension| 60°C | 1min | 40 | + +--- + +endofoutput +``` diff --git a/markdown-output/effects-of-whole-body-vibrations-on-neuromuscular-beadjaa6.md b/markdown-output/effects-of-whole-body-vibrations-on-neuromuscular-beadjaa6.md new file mode 100644 index 0000000000000000000000000000000000000000..691457601c2225b6b69afb8654443de727fe6d1c --- /dev/null +++ b/markdown-output/effects-of-whole-body-vibrations-on-neuromuscular-beadjaa6.md @@ -0,0 +1,96 @@ +```markdown +# Goal/Experiment: +Investigate the origin and magnitude of neuromuscular fatigue induced by half-squat whole-body vibration. + +# Effects of Whole-Body Vibrations on Neuromuscular Fatigue + +## Abstract + +**Purpose**: The aim of the study was to investigate the origin and magnitude of neuromuscular fatigue induced by half-squat whole-body vibration. + +**Methods**: Ten young, recreationally trained adults participated in six fatiguing protocols, each consisting of several sets of 30, 60, or 180 s static squatting superimposed with vibration (WBV30, WBV60, WBV180) or without vibration (SHAM30, SHAM60, SHAM180) for a total exercise exposure of 9 minutes in each trial. Maximum voluntary contraction (MVC), level of voluntary activation (%VA), single twitch peak torque (TWPT), low- (T20) and high-frequency (T100) doublets, and low-to-high-frequency fatigue ratio (T20/100) were assessed before, immediately after, 15 and 30 minutes after each fatiguing protocol. + +## Methods + +### Materials + +| Item | Description | Vendor | Quantity | +|----------------------------------------------|----------------------------------------------------------------------------------------------|--------------------------|----------| +| DS7A HV Constant Current Stimulator | High-voltage constant current stimulator | Digitimer | 1 | +| 10 mm Ag–AgCl electrode | Cathode electrode | Controle Graphique Medical| 1 | +| ELECTRODES PERFORMANCE 50 x 100MM PIN | Electrodes | Compex | 1 | +| Isometric machine with a force transducer | Isometric dynamometer | Custom Made (MES, Maribor, Slovenia) | 1 | +| Galileo Fit | Whole body vibration platform | Novotec Medical GmbH | 1 | +| PowerLab 16/35 (PL3516) | Data acquisition hardware (DAQ) | ADInstruments | 1 | +| LabChart 7 | Data acquisition software | ADInstruments Australia | 1 | + + +### Study Design + +Each subject performed three different fatiguing exercise interventions with WBV and three exercize interventions in a SHAM condition without WBV (SHAM) to discern the effect of WBV. Each intervention consisted of a cumulative exercise period of 9 minutes divided into different sets (either 18 x 30 s or 9 x 60 s or 3 x 180 s), with 120 s rest between sets. Each intervention was randomly executed on different visits at the same daytime with at least 7 days rest in-between. + +### Equipment Calibration + +1. **Isometric machine with a force transducer**: + - Calibrated prior to each measuring session. + - Connected to PowerLab 16/35 (PL3516) DAQ running LabChart 7 (Windows XP). + - Similar calibration is used in studies by Tomazin K, et al., and García-Ramos A, et al. + - Force transducer calibrated by hanging a 20 kg weight, reading the voltage transformation to calculate the exerted torque. + +### Pre-experiment Procedures + +2. **Subject positioning**: + - Subject seated in an upright position with the trunk at 100° leaning against the backrest of the isometric dynamometer. + - Knee joint at 60° angle (0° = full extension), with a fixed shin pad above the medial malleolus. + +3. **Femoral nerve stimulation electrode placement**: + - Participants flex their hip from a seated position to palpate the iliac fossa. + - Electrode (cathode) placed into the femoral triangle with a larger self-adhesive electrode placed over the gluteal fold serving as an anode. + +4. **Femoral nerve test stimulation**: + - Electrical impulses (single, square wave, 1ms duration) elicited by a high-voltage constant current stimulator (DS7A) to trigger muscle response detected as a change in knee extensors torque. + +### Warm-up and Rest + +5. **Warm-up**: + - 6-minute warm-up routine of bench stepping (20 cm high) at a frequency of 0.5 Hz, leg exchange each minute. + +6. **Rest**: + - 2-minute rest. + +### Experiments + +7. **Stimulation Intensity Determination**: + - Stimulation intensity to elicit maximum knee extensor isometric twitch determined after warm-up. Incrementally increased stimulation intensity by 10 mA until no further torque increase observed. Supramaximal stimulus obtained by increasing the current at maximal twitch torque by a factor of 1.5. + +### PRE - Assessment (t0) + +8. **Maximum Voluntary Contraction (MVC) and Double Twitch Interpolated Techniques**: + - Subjects performed a 5 s maximal isometric voluntary knee extension. Data compared with studies by Verges S, et al., and Allen DG, et al. + + +### MVC Analysis + +8.1 **High- and Low-Frequency Doublets**: + - Torque induced by paired high- (100 Hz, 10 ms interval) and low-frequency (20 Hz, 50 ms interval) supramaximal electrical stimuli analyzed, referencing studies by Place N, et al., and Verges S, et al. + +### Interventions + +9. **Intervention Protocols**: + - Interventions on Galileo Fit and WBV platform at 26 Hz frequency in a half-squat position with 60° knee flex angle and feet 40 cm apart. + - Referenced from Ritzmann R, et al. + +### POST - Assessments + +10. **POST Assessment Intervals**: + - Post-intervention assessments performed immediately after, 15 minutes, and 30 minutes following the intervention. + +### Data Analysis + +13. **Statistical Analysis**: + - Conducted a two-way factorial ANOVA using R programming language with afex and emmeans packages for comparing the main effects (time and trial) and their interaction. + - Statistical significance set at p < 0.05 with effect sizes calculated using Cohen's d thresholds. + +--- +**endofoutput** +``` \ No newline at end of file diff --git a/markdown-output/efficient-third-generation-lentiviral-particle-pro-cqnmvvc6.md b/markdown-output/efficient-third-generation-lentiviral-particle-pro-cqnmvvc6.md new file mode 100644 index 0000000000000000000000000000000000000000..3be8fb6fa3a68552c7664488f96dcb1bbc55bc5f --- /dev/null +++ b/markdown-output/efficient-third-generation-lentiviral-particle-pro-cqnmvvc6.md @@ -0,0 +1,140 @@ +```markdown +# Goal/Experiment: +Efficient third generation lentiviral particle production. This experiment aims to optimize the production of lentiviral particles using a third-generation system that minimizes handling time, cost, and maximizes yield without compromising cell culture or viral production efficiency. + +# Efficient third generation lentiviral particle production V.4 + +## Authors: + +- Michelle Newbery¹'² +- Simon Maksour¹'³ +- Amy Hulme¹'³ +- Neville S Ng¹'² +- Mirella Dottori¹'³'⁴ +- Lezanne Ooi¹'²'⁴ + +1: Illawarra Health and Medical Research Institute, Wollongong, NSW, Australia +2: School of Chemistry and Molecular Bioscience, University of Wollongong, Wollongong, NSW, Australia +3: School of Medicine, University of Wollongong, Wollongong, NSW, Australia +4: Molecular Horizons, University of Wollongong, Wollongong, NSW, Australia + +DOI: [dx.doi.org/10.17504/protocols.io.261ge42pyv47/v4](https://dx.doi.org/10.17504/protocols.io.261ge42pyv47/v4) + +Protocol Citation: +Michelle Newbery, Simon Maksour, Amy Hulme, Neville S Ng, Mirella Dottori, Lezanne Ooi 2023. Efficient third generation lentiviral particle production. protocols.io. dx.doi.org/10.17504/protocols.io.261ge42pyv47/v4 + +[Creative Commons Attribution License](https://creativecommons.org/licenses/by/4.0/): This protocol is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. + +Protocol Status: Working - This protocol is currently in use. + +Created: Mar 07, 2023 +Last Modified: Mar 29, 2023 +PROTOCOL integer ID: 78253 + +## Keywords: +lentiviral, HEK293T, differentiation, lentivirus, overexpression, plasmid, viral particle, production + +## Abstract +The overexpression of a gene of interest by third generation lentiviral particle generation systems is a critical process in molecular biology, cell biology and gene therapy research. This protocol provides at least 6 days of consecutive viral particle collection without compromising HEK293T cell culture or viral production efficiency, and can be easily and cost-effectively reproduced in basic cell culture laboratories. + +## Materials + +### Cell Culture + +- HEK293T cell line +- FreeStyle 293 Expression Medium (Thermo Fisher Scientific #12338018) OR +- DMEM/F12 (Thermo Fisher Scientific #21331020) + GlutaMAX Supplement (Thermo Fisher Scientific #35050061) + HEPES (Thermo Fisher Scientific #15630080) with Fetal Calf Serum OR KnockOut Serum Replacement (#10828028) + +#### Optional: +- DMEM/F12 (Thermo Fisher Scientific #21331020) + GlutaMAX Supplement (Thermo Fisher Scientific #35050061) + ITS-A (Thermo Fisher Scientific #51300044) +- Protamine sulfate (Sigma-Aldrich P4505) +- DEAE-Dextran (Sigma-Aldrich #93556) +- Penicillin-Streptomycin (Thermo Fisher Scientific #15140122) + +### Transfection +- Linear 20 kDa PEI (Sigma #764965 or Polysciences #23966-1) + +### Plasticware +- Tissue culture 75 cm² or 175 cm² cell culture vessels and serological pipettes +- 40 mL high-speed ultracentrifuge tubes +- =>50 mL polypropylene containers +- 0.2 mL or 2 mL centrifuge tubes +- 0.45 µm PES pore bottle-top filter or equivalent low volume syringe and syringe filter + +All plasticware should be of tissue-culture grade sterility. + +## Safety Warnings + +All stages should be performed with appropriate safety precautions specific to local standards, which may include double gloves and disposable plastic apron PPE. Lentivirus particles can be inactivated by hypochlorite, peroxide and ethanol-based sterilisation agents, UV light, and autoclave. + +## Protocol + +### 1. Lentiviral Transfection +1. Maintain HEK293T cells in animal product-free FreeStyle 293 Expression Medium or DMEM/F12 + GlutaMAX + HEPES + 5% FBS or 5% KSR. Include 50 U/mL Penicillin and Streptomycin to reduce the risk of bacterial contamination if necessary. +2. Dissociate and subculture HEK293T at a density of >50000 cells/cm² per transfer plasmid. +3. HEK293T cultures are inherently susceptible to detachment with acidification and may require weaning at high density for several passages to be sustained throughout the 6-day generation period. Viral particle production can be performed within at least 15 passages without loss of yield. + +**Citations:** +- Ausubel LJ et al. (2012). Production of CGMP-Grade Lentiviral Vectors. BioProcess international. +- Gill KP, Denham M (2020). Optimized Transgene Delivery using Third-Generation Lentiviruses. Current protocols in molecular biology. [Link](https://doi.org/10.1002/cpmb.125) + +### 2. Plasmid Transfection +1. For each lentiviral transfer plasmid and packaging plasmid vectors, calculate reagent volumes required for 12 µg gene of interest vector, 4 µg pMDLg/pRRE (Addgene #12251), 4 µg pRSV-Rev (Addgene #12253) and 4 µg pCMV-VSV-G (Addgene #8454) and 60 µg PEI per 75 cm² of HEK293T culture. Maximal transfection efficiency by PEI complex is typically obtained with higher proportions of PEI:DNA. +2. Prepare PEI-DNA solution in 1.5 mL of DMEM/F12 and incubate for 00:05:00 at ambient temperature. + + ![Figure 1](https://dx.doi.org/10.17504/protocols.io.261ge42pyv47/v4) + **Figure 1.** 2.5-5:1 PEI:DNA provides maximal transfection efficiency. Experiment performed with a hNGN2-eGFP containing plasmid (n = 3, error presented as SEM; ** p < 0.01, *** p < 0.001, analysis by 1-way ANOVA and Holm-Sidak post-hoc multiple comparisons test). + +3. Replace HEK293T cell culture medium with at least 0.2 mL/cm² medium (e.g., 15-20 mL per 75 cm² flask), add PEI-DNA complex solution, and return to the incubator **overnight**. Note: PEI-DNA complex transfection can occur without usage of low serum transfection medium products. + +**Citation:** +- González-Domínguez I et al. (2019). Impact of physicochemical properties of DNA/PEI complexes on transient transfection of mammalian cells. New biotechnology. [Link](https://doi.org/10.1016/j.nbt.2018.09.005) + +### 3. Viral Particle Collection +1. Each day collect medium in an appropriate sized sterile container (e.g., 120 mL specimen collection tubes), and store at **4°C**. Proceed to viral particle concentration at selected endpoint. Lentiviral particle collection can be performed for at least up to 7 days. +2. We utilize viral particle concentration to avoid prolonged incubation of pluripotent or multipotent stem cells in HEK293T culture medium during viral transduction experiments. If viral particle concentration is not necessary, freeze immediately at **-80°C**. + + ![Figure 3](https://dx.doi.org/10.17504/protocols.io.261ge42pyv47/v4) + **Figure 3.** Lentivirus can be collected for at least 6 days without loss of viral titre (n = 3, error presented as SEM, analysis by 1-way ANOVA and Holm-Sidak post-hoc multiple comparisons test). + +**Citations:** +- Rahman H et al. (2013). Effects of Storage Conditions on the Morphology and Titer of Lentiviral Vectors. SFA ScholarWorks. +- Ichim CV, Wells RA (2011). Generation of high-titer viral preparations by concentration using successive rounds of ultracentrifugation. Journal of translational medicine. [Link](https://doi.org/10.1186/1479-5876-9-137) + +### 4. Lentiviral Particle Concentration +1. Sterilize ultracentrifuge tubes and appropriate sized viral particle aliquot tubes (0.2 or 1.5 mL microcentrifuge tubes). +2. Optional: Filter supernatant through a 0.45 µm pore PES bottle-top filter to remove cell debris. Alternatively, syringe filtration can be performed after lentiviral particle concentration. +3. Balance ultracentrifuge tubes within 0.1 g by weighing and adding appropriate amounts of medium. +4. Collect lentiviral particles by centrifugation of supernatant at **50000 x g**, **4°C**, **00:20:00**. +5. Carefully transfer tubes on ice, mark the position of pellet, and decant waste slowly to discard the supernatant. +6. Resuspend pellet as a 200X concentrate of total supernatant (e.g., 200 µL from 40 mL of lentiviral supernatant). +7. If cell debris was not removed by filtration prior to concentration, centrifuge lentiviral concentrate at **1500 x g**, **00:03:00**, and discard the pellet. Smaller debris can be removed with a 0.45 µm pore PES syringe filter if necessary. +8. Prepare viral particle aliquots of appropriate volumes corresponding to application and freeze at **-80°C**. Lentivirus particles are enveloped and more susceptible to degradation in comparison to unenveloped DNA viruses; however, resuspension in complete cell culture media is sufficient to prevent degradation during at least up to 4 freeze-thaw cycles. + + ![Figure 5](https://dx.doi.org/10.17504/protocols.io.261ge42pyv47/v4) + **Figure 5.** Complete medium allows lentiviral concentrate to be freeze-thawed multiple times without loss of titre in presence or absence of 10% DMSO cryoprotectant (n = 3, error presented as SEM; analysis by 1-way ANOVA and Holm-Sidak post-hoc multiple comparisons test). + +### 5. Lentiviral Titre Assay +1. Seed 10000 cells/well of intended cell type in a 96-well microplate allowing for at least 3-6 concentrations, 2-3 technical replicate wells per lentivirus and enumerating cell population, and return to incubator **overnight**. +2. Prepare a 10-fold serial dilution of lentiviruses at intended concentrations (e.g. 1:1, 1:10, 1:100, or 1:1, 1:2, 1:10, 1:20, 1:100, 1:200), add to cell culture plate (e.g. 10 µL) and return to incubator. + + ![Figure 6](https://dx.doi.org/10.17504/protocols.io.261ge42pyv47/v4) + **Figure 6.** Transduction efficiency can be enhanced in presence of low serum medium (LSM) (n = 3, error presented as SEM; *** p < 0.001, analysis by 1-way ANOVA and Holm-Sidak post-hoc multiple comparisons test). + +**Citations:** +- Balak JRA et al. (2019). Highly efficient ex vivo lentiviral transduction of primary human pancreatic exocrine cells. Scientific reports. [Link](https://doi.org/10.1038/s41598-019-51763-z) + +3. Optionally include an enhancer of transduction such as DEAE-Dextran or protamine sulfate at a concentration appropriate to cell type (e.g. 50-100 µg/mL). +4. On the same day as lentivirus addition, determine cell population in at least 2 wells. Return to incubator **overnight**. +5. The next day replace media, add transcriptional activator if applicable, and return to incubator for 2-3 days. +6. Acquire microplate fluorescent microscopy images and score fluorescent reporter or immunolabeled gene of interest. +7. Calculate functional titre based on wells with 5-40% transduction rate using the following formula: + - `TU/mL = (transduction %) x (cell population at time of transduction) / (inoculum volume in mL)` + - Note: Higher % transductions may underestimate titre due to increased rate of multiple integrations. + +### 6. Citing the Protocol +Please cite this protocol if used or adapted in any publications: +- Michelle Newbery, Simon Maksour, Amy Hulme, Neville S Ng, Mirella Dottori, Lezanne Ooi 2023. Efficient third generation lentiviral particle production. protocols.io. dx.doi.org/10.17504/protocols.io.261ge42pyv47/v4 + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/electrophysiology-from-cervical-vagus-nerve-and-gr-b9t9r6r6.md b/markdown-output/electrophysiology-from-cervical-vagus-nerve-and-gr-b9t9r6r6.md new file mode 100644 index 0000000000000000000000000000000000000000..0923407fac2ce1f1173b0daea25895b320e46cae --- /dev/null +++ b/markdown-output/electrophysiology-from-cervical-vagus-nerve-and-gr-b9t9r6r6.md @@ -0,0 +1,98 @@ +```markdown +# Goal/Experiment: +Perform electrophysiological recordings from the cervical vagus nerve and great auricular nerve in swine to characterize cuff electrodes, longitudinal intrafascicular electrodes (LIFEs), and microneurography electrodes in their ability to measure neural activity in the peripheral nervous system. + +# Electrophysiology from Cervical Vagus Nerve and Great Auricular Nerve in Swine + +**Authors:** +- Nishant Verma +- Kip Ludwig + +**Affiliation:** Department of Biomedical Engineering, University of Wisconsin - Madison + +**DOI:** [dx.doi.org/10.17504/protocols.io.j8nlkk6n5l5r/v1](https://dx.doi.org/10.17504/protocols.io.j8nlkk6n5l5r/v1) + +**Date:** June 08, 2022 + +## Introduction + +This protocol is utilized to gather data for publication purposes. The dataset is also publicly available on Pennsieve. The study characterizes various types of electrodes in their capability to measure neural activity within the peripheral nervous system. + +### Key Terms and Definitions: +- **Cuff Electrodes:** Used to wrap around nerves to record electrical activity. +- **Longitudinal Intrafascicular Electrodes (LIFEs):** Electrodes inserted into nerve fascicles to record action potentials. +- **Microneurography Electrodes:** Microelectrodes used clinically to measure muscle sympathetic nerve activity (MSNA). + +## Materials and Equipment + +### Recording Electrodes + +| Type | Description | Source | Model/Part Number | +| ------------------------------ | -------------------------------------------------------------------------------- | -------------------------------------- | -------------------------- | +| **SIM** | Electrophysiology | [Tucker-Davis Technologies](https://www.tdt.com/component/subject-interface/) | N/A | +| **Tungsten Microneurography** | 0.8-1.2 MOhm microelectrode | FHC Inc. | UNA40GCT | +| **Recording Cuff Tripole** | Platinum/silicone cuff with three cylindrical contacts | Ardiem Medical Inc. | Weber cuff 3 mm | +| **Longitudinal Intrafascicular**| Platinum iridium wire with 0.004" diameter with insulation and an exposed window ~2 mm| In-house fabricated | N/A | +| **Great Auricular Cuff** | Platinum wires (0.005" diameter) in a split silicone tube | In-house fabricated | N/A | + +### Reagents and Vendors +- **Telazol:** Anesthetic (Telazol, 6 mg/kg) +- **Xylazine:** Sedative (Xylazine, 2 mg/kg) +- **Fentanyl:** Analgesic (Fentanyl, 12-30 mcg/kg/hr) +- **Lactated Ringer’s Solution (LRS):** For intravenous fluids (Vendor: ICU medical, IM-4389) +- **Vecuronium:** Muscle paralytic (Vecuronium, 1-1.5 mg/kg/hr) + +### Institutional Animal Care and Use Committee (IACUC) Approval +1. Seek approval from your local IACUC before conducting any live animal experiments. + +## Procedure + +### Anesthesia +1. Deliver intramuscular injection of Telazol (6 mg/kg) and Xylazine (2 mg/kg) to induce sedation. +2. Ventilate the animal and maintain the surgical plane using inhaled Isoflurane (∼1-2%) and intravenous Fentanyl (12-30 mcg/kg/hr) with lactated Ringer’s solution (LRS). + + **Note:** Several hours into the experiment, if tremoring is observed, consider additional administration of Telazol (4-6 mg/kg) or intravenous Ketamine (10 mg/kg/hr). To avoid cardiac blunting effects and ensure fast adjustments, minimal isoflurane is administered. + +### Cervical Vagus Nerve Preparation + +1. Position the subject supine, use a midline approach to access the left carotid sheath. +2. Mobilize and carefully retract the carotid artery to expose the cervical vagus nerve (9-12 cm). +3. Instrument the vagus nerve with the following electrodes caudal to the superior laryngeal branching: + - Bipolar stimulation electrode + - LIFE, microneurography, and a cuff electrode with three contact recordings (spaced >4 cm from the stimulation electrode). +4. Insert a reference LIFE electrode and microneurography electrode at an equal distance from the stimulation electrode to match the recording electrodes. + +5. Record Evoked Compound Action Potentials (ECAPs) with 750 biphasic stimulation pulses at 25 Hz, 200 us pulse width with a stimulation amplitude between 0 and 10 mA. + +### Great Auricular Nerve Preparation + +1. Incise skin and subcutaneous fat from the medial posterior margin of the ramus to expose the great auricular nerve (GAN). +2. Use anatomical landmarks to identify and divide the facial nerve into its branches. +3. Motor and sensory determination of nerve branches by electrical stimulation. +4. Instrument identified sensory branches with stimulation and recording electrodes. +5. Record sensory-evoked neural activity by brushing the ear areas innervated by the GAN. + +### Electrophysiology System + +15. Use a Tucker-Davis Technologies (TDT) electrophysiology system for stimulation and recording (25 kHz data collection). +16. High impedance (microneurography) and low impedance (LIFE and cuff) recordings on separate cards with active and passive head stages. + +## Data Analysis + +### Filtering and Detection +18. Use the Python package, PyeCAP, for offline analysis. +19. Apply a high pass 1st order Butterworth filter (corner frequency: 100 Hz) and low pass Gaussian filter (corner frequency: 3 kHz). +20. Detect ECAPs by median averaging across pulses, windowed by time according to known conduction speeds. + +### Authenticity and Quantification +21. Confirm authenticity by assessing conduction speed delays in expected ranges and using Vecuronium to reduce artifacts. +22. Quantify ECAP strength by distinguishing Aβ and B-fiber ECAPs. +23. Calculate time windows for Aβ and B-fibers, and measure Root Mean Square (RMS) values over fixed-duration windows. + +### Spike Detection +24. Use the thresholding method for spike detection; calculate standard deviation (SD) and set the threshold at six times the SD plus the mean. + +--- + +`endofoutput` +``` \ No newline at end of file diff --git a/markdown-output/elispot-protocol-hv2b68e.md b/markdown-output/elispot-protocol-hv2b68e.md new file mode 100644 index 0000000000000000000000000000000000000000..986ba7728925ba5a0dbc365038e77a0cc73784d2 --- /dev/null +++ b/markdown-output/elispot-protocol-hv2b68e.md @@ -0,0 +1,196 @@ +```markdown +# Goal/Experiment: +The aim of this experiment is to perform an Enzyme-Linked ImmunoSpot (ELISPOT) assay to detect and quantify cytokine production at the single-cell level. The ELISPOT assay is highly sensitive and is widely used for immunological studies to measure the frequency of cytokine-secreting cells. + +# ELISPOT Protocol Version 2 + +*Author:* Kelsey Miller + +*Published:* 09 May 2017 + +## Guidelines + +### Solutions & Buffers + +**Note:** Do not use sodium azide in any buffers or solutions as sodium azide inactivates the horseradish-peroxidase enzyme. + +#### Phosphate Buffered Saline (PBS): +- 80.0 g NaCl +- 14.4 g Na2HPO4 +- 2.4 g KH2PO4 +- 2.0 g KCl +- Add ddH2O up to 10 L; pH to 7.2 with HCl + +#### Coating Buffer: +- Can use either: + - Sterile PBS or + - Sterile Carbonate Buffer (per ELISA protocol): + - 8.4 g Na HCO3 + - 3.56 g Na2CO3 + - Add ddH2O up to 1.0 L, pH to 9.5 + +#### PBS-Tween: +- 0.05% Tween-20 in PBS (500 µl Tween-20 in 1L PBS) + +#### Blocking Buffer (PBS-BSA): +- 1% BSA in PBS (10 g BSA-Fraction V in 1L PBS) + +#### PBS-Tween-BSA: +- 1% BSA in PBS-Tween (10 g BSA-Fraction V in 1L PBS-Tween) + +#### Tissue Culture (TC) Medium: +- As appropriate for cells being analyzed + +#### AEC Solution: +- 100 mg AEC (3-amino-9-ethyl-carbazole) in 10 ml DMF (N,N, Dimethylformamide) +- *Solution should be prepared in a glass tube in a fume hood.* + +#### AEC Buffer: +- (0.1 M Acetate): + - 148 ml 0.2 M acetic acid (11.55 ml glacial acetic acid per liter of water) + - 352 ml 0.2 M sodium acetate (27.2 g per liter of water) + - Bring up to 1L with water and adjust to pH 5.0 if required + +#### Substrate Solution: +- 800 µl AEC solution in 24 ml AEC buffer +- Filter with 0.45 µm filter and add 12 µl 30% H2O2 +- *Use immediately* + +## Protocol + +### Prepare the Plate + +#### Step 1. +Prepare the PVDF membrane 96-well ELISPOT plates (e.g., Millipore Cat. No. MAIPS-4510) by soaking them in 35% ethanol for 30 seconds. + +#### Step 2. +Wash thoroughly with PBS to remove any residual ethanol. + +**Note:** Ethanol can negatively affect cell viability and antibody binding. + +### Coat the Plate + +#### Step 3. +Dilute Low-Endotoxin/Azide-Free sterile unlabeled capture antibody (BioLegend’s LEAF™ format antibodies are specifically designed for this assay) to a final concentration of 0.5–4 µg/ml in sterile Coating Buffer and transfer 100 µl/well to a high affinity binding PVDF membrane ELISPOT plate (e.g., Millipore; Cat. No. MAIPS-4510). + +**Note:** BioLegend's LEAF™ and Ultra-LEAF™ format antibodies are specifically designed for this assay. + +#### Step 4. +Store plates overnight in humidified box at 4°C or at 37°C for ≥ 4 hours in humidified atmosphere. + +### Block the Plate + +#### Step 5. +Wash the plate 3 times with 200 µl/well of sterile PBS, gently tapping plates dry on a clean paper towel between each wash. + +**Notes** + +*Kelsey Knight 09 May 2017* + +1. Since ELISPOT plates are more delicate than ELISA plates, they should be gently tapped and washed manually. +2. Do not use an automatic plate washer, since this could compromise the integrity of the PVDF membrane. + +#### Step 6. +Add 200 µl/well of sterile Blocking Buffer. + +#### Step 7. +Seal plate and incubate at room temperature for ≥ 1 hour. + +#### Step 8. +Repeat step 5. + +### Set-Up Tissue Culture and Add Antigen or Mitogen + +#### Step 9. +Add 100 µl/well of appropriate sterile antigen or mitogen solution diluted in appropriate sterile tissue culture (TC) medium. + +#### Step 10. +Add 100 µl/well of cells diluted in sterile TC medium. Use 5 x 10^4 to 5 x 10^5 cells/well. + +**Notes** + +*Kelsey Knight 09 May 2017* + +1. The minimum number of cells should be determined in preliminary experiments. +2. When determining the optimal number of cells to use, keep in mind the expected levels of expression of the target protein. If the expression is expected to be low, use a higher number of cells. +3. If the cells can withstand the environment, use a serum-free media. Serum contains proteins that can affect results or give a high background or nonspecific signal. + +#### Step 11. +Seal plate and incubate at 37°C 5% CO2 in humidified atmosphere for the optimum stimulation period. + +**Notes** + +*Kelsey Knight 09 May 2017* + +1. BioLegend recommends a 24 hour incubation for IFNγ, IL-2, and TNFα; and a 48 hour incubation for IL-4, IL-5, and IL-10 for most activation conditions. +2. Do not shake or move the plates while the cells are culturing. This will lead to spots that are not well-defined. +3. If your cells take more than 48 hours to respond to stimulation, they can be treated with the stimulant in a separate 96-well plate prior to transferring to the ELISPOT plate. + +### Add Detection Antibody + +#### Step 12. +Wash plate 3 times with PBS, 200 µl/well. + +#### Step 13. +Wash plate 3 times with PBS-Tween, 200 µl/well. + +**Note:** Tween-20 is included in the wash buffer to aid detachment of any cells that have attached during overnight cell culture. + +#### Step 14. +Add 100 µl/well of diluted biotinylated detection antibody at 0.25-2 µg/ml in PBS-Tween-BSA. + +#### Step 15. +Seal the plate and incubate at 4°C overnight, or 2 hr at room temperature. + +### Add Avidin-Horseradish Peroxidase (Av-HRP) + +#### Step 16. +Wash plate 4 times with PBS-Tween, 200 µl/well. + +#### Step 17. +Add 100 µl per well of the Av-HRP conjugate (Cat. No. 405103) or other enzyme conjugate diluted to its pre-determined optimal concentration in PBS-Tween-BSA (usually between 1/500 – 1/2000). + +#### Step 18. +Seal the plate and incubate at room temperature for 1 – 2 hours. + +#### Step 19. +Wash plate 3 times with PBS-Tween, 200 µl/well. + +**Notes** + +*Kelsey Knight 09 May 2017* + +- Take the base off of the bottom of the plate to ensure it is thoroughly washed. This will help prevent high background, since some reagents can leak through the PVDF membrane and stick to the base or bottom of the plate. + +#### Step 20. +Wash plate 3 times with PBS, 200 µl/well. + +**Notes** + +*Kelsey Knight 09 May 2017* + +- When you are done washing, be sure to replace the base to the bottom of the plate. + +### Add Substrate + +#### Step 21. +Add 200 µl/well of fresh Substrate solution. + +#### Step 22. +Monitor spot/color development at room temperature and stop reaction by rinsing plate with tap water and vigorously flicking plate over a waste container or sink, followed by blotting on paper towels or other absorbent materials. + +#### Step 23. +Air dry plate overnight, until it is completely dry. + +**Notes** + +*Kelsey Knight 09 May 2017* + +- Spots could become sharper if the plates are stored overnight at 4°C. Wrap plates in foil prior to storing. + +#### Step 24. +Count spots manually with a dissecting microscope or using an automated image acquisition/analysis unit (plates can be analyzed for up to 3 months). + +``` + +endofoutput \ No newline at end of file diff --git a/markdown-output/enteric-neuron-activity-in-the-mouse-colon-and-res-cmjeu4je.md b/markdown-output/enteric-neuron-activity-in-the-mouse-colon-and-res-cmjeu4je.md new file mode 100644 index 0000000000000000000000000000000000000000..df91d0b933e7f054086f92580172552518962308 --- /dev/null +++ b/markdown-output/enteric-neuron-activity-in-the-mouse-colon-and-res-cmjeu4je.md @@ -0,0 +1,66 @@ +```markdown +# Goal/Experiment: +The goal of this experiment is to study calcium imaging activity in the enteric nervous system (ENS) and interstitial cells of Cajal (ICC) in isolated colons and ex vivo lumbosacral spinal cord-colon preparations from GCaMP mice. + +# Enteric Neuron Activity in the Mouse Colon and Responses to Lumbosacral Stimulation V.2 + +Kristen Smith-Edwards\ +*Mayo Clinic* + +#### Abstract +This protocol describes the steps for calcium imaging activity in the enteric nervous system (ENS) and interstitial cells of Cajal (ICC) in isolated colons and ex vivo lumbosacral spinal cord-colon preparations from GCaMP mice. + +## Isolated Colon Preparation + +1. **Euthanize Ella-GCaMP mice** + (offspring from the pairing of B6.FVB-Tg(Ella-cre)C5379Lmgd/J \[RRID:IMSR_JAX:003724; cat. 003724; Jax Labs\] and B6J.Cg-Gt(ROSA)26Sortm96(CAG-GCaMP6s)Hze/MwarJ \[RRID:IMSR_JAX:028865; cat. 028865; Jax Labs\]), by inhalation of isoflurane and thoracotomy. Remove the entire colon from mouse and place into a Sylgard-lined Petri dish (Dow Corning). Circulate oxygenated artificial cerebral spinal fluid at room-temperature (ACSF, containing in mM: 117.9 NaCl \[cat. S9888; Sigma\], 4.7 KCl \[cat. P3911; Sigma\], 25 NaHCO₃ \[catS6014; Sigma\], 1.3 NaH₂PO₄ \[cat. S8282; Sigma\], 1.2 MgSO₄•7H₂O \[cat. 230391; Sigma\], 2.5 CaCl₂ \[cat. C1016; Sigma\], 11.1 D-glucose \[cat. G5767; Sigma\], 2 sodium butyrate \[cat. B5887; Sigma\], and 20 sodium acetate \[cat. S2889; Sigma\]) with 4μM nifedipine \[cat. N7634; Sigma\] and 3μM indomethacin \[cat. I7378; Sigma\] added. Cut the colon open longitudinally and pin flat using minutien pins (Fine Science Tools; cat. 26002-20) with mucosal side facing down. A small amount of stretch should be applied when pinning the colon tissue for optimal imaging conditions. Transfer to the stage of an upright fluorescence microscope (DM6 FS, Leica) equipped with camera (Prime 95B, Photometrics, Roper Scientific) and software (Metamorph, Molecular Devices) for calcium imaging and slowly heat up circulating fluid to 35-37°C or desired temperature) using a heated water bath. + +2. **Image calcium signals** + from myenteric neurons, submucosal neurons, and interstitial cells of Cajal (ICC) in the submucosa. Set acquisition parameters to: 25ms exposure time (for 40Hz sampling rate), 800 frames, binning 2x2. Using a 20X or 40X objective lens, image spontaneous activity as well as responses to colon stimulation using a stimulus isolator (A365, World Precision Instruments) and concentric bipolar electrodes (cat. CBBJR75; FHC) placed 5 mm oral and 5 mm anal to the field of view. Using one electrode at a time, record the response to electrical stimulation by delivering 20 pulses (100μs duration) at 20Hz, set to occur 10s into the recording (CED 1401 micro4 and Spike 2 software). Then repeat using the other electrode in the same field of view, allowing 2 minutes between each stimulus. + +## Lumbosacral Colon Preparation with Spinal Cord Circuits + +3. **Euthanize mice** + by inhalation of isoflurane and thoracotomy. Perform cardiac perfusion using oxygenated ice-cold sucrose ACSF containing in mM: 234 sucrose \[cat. S0389; Sigma\], 2.5 KCL \[cat. P3911; Sigma\], 26 NaHCO₃ \[catS6014; Sigma\], 1.3 NaH₂PO₄ \[cat. S8282; Sigma\], 10 MgSO₄•7H₂O \[cat. 230391; Sigma\], 0.5 CaCl₂ \[cat. C1016; Sigma\], 11 D-glucose \[cat. G5767; Sigma\], 2 sodium butyrate \[cat. B5887; Sigma\], and 20 sodium acetate \[cat. S2889; Sigma\] and do a laminectomy to expose spinal cord. Dissect spinal cord (T12-S3), pelvic nerve and 4 cm of distal colon and place into Sylgard-lined Petri dish with circulating oxygenated ice-cold sucrose ACSF, making sure that the spinal cord and colon are still connected via the pelvic nerve. Cut the colon open longitudinally adjacent to the mesenteric border and pin flat (applying some stretch), mucosal side down, and pin spinal cord in place. Remove dura from spinal cord and isolate the dorsal and ventral roots of L6 spinal cord. Replace fluid with room-temp oxygenated ACSF with 4μM nifedipine and 3μM indomethacin added (NOT sucrose ACSF) and move preparation to the stage of an upright fluorescent microscope for calcium imaging and slowly heat up circulating fluid to 31°C (this lower temperature increases the viability of spinal cord tissue). + +4. **Place two stimulating electrodes** + on the L6 dorsal and ventral roots to stimulate either the dorsal or ventral root using the same parameters as above except 100 pulses for a total of 5 sec. To verify electrode placement, image over the L6 dorsal root ganglion to confirm activation of neurons (the majority will be activated with dorsal root stimulation, whereas approx 5-10% will be activated with ventral root stimulation). Using the same imaging parameters as above, image myenteric neuron responses to dorsal or ventral root stimulation. Ventral root stimulation should be used as a positive control to make sure the preparation is still viable and intact. To determine the role of spinal cord circuits, cut the roots away from the cord and stimulate the ends of the roots that are still connected to the nerve that innervates the colon. Stimulating ventral roots should still evoke responses, whereas dorsal root should not. + +## Lumbosacral Colon Preparation with Dorsal and Ventral Roots + +5. **Euthanize mice** + by inhalation of isoflurane and thoracotomy. Perform cardiac perfusion using oxygenated ACSF and do a laminectomy to expose spinal cord. Dissect lumbosacral spinal cord, pelvic nerve and 4 cm of distal colon and place into Sylgard-lined Petri dish with circulating oxygenated ACSF, making sure that the spinal cord and colon are still connected via the pelvic nerve. Remove dura from spinal cord and isolate the L6 spinal cord and ventral roots of spinal cord. Cut both roots at the entry point into the spinal cord and remove the spinal cord from the spinal column while leaving behind the L6 dorsal root ganglion with dorsal and ventral roots still attached. Cut the colon open longitudinally, adjacent to the mesenteric border and pin flat (applying some stretch), mucosal side down, and pin spinal column with roots in place. Move preparation to fluorescent microscope for calcium imaging and slowly heat up circulating, oxygenated ACSF (with 4μM nifedipine and 3μM indomethacin added) to 35-37°C. + +6. **Place two stimulating electrodes** + on the L6 dorsal and ventral roots to stimulate either the dorsal or ventral root using the same parameters as above except 100 pulses for a total of 5 sec. To verify electrode placement, image over the L6 dorsal root ganglion to confirm activation of neurons (the majority will be activated with dorsal root stimulation, whereas approx 5-10% will be activated with ventral root stimulation). Using the same imaging parameters as above, image myenteric neuron responses to dorsal or ventral root stimulation. Ventral root stimulation should be used as a positive control to make sure the preparation is still viable and intact. + +## Reagents and Equipment + +**Reagents:** +- **Isoflurane**: An anesthetic used to euthanize the mice. +- **ACSF (Artificial Cerebral Spinal Fluid)**: A solution that mimics the cerebrospinal fluid, used to maintain tissue viability. +- **Nifedipine**: A calcium channel blocker, used to maintain tissue viability during imaging. +- **Indomethacin**: A nonsteroidal anti-inflammatory drug, used to maintain tissue viability. + +**Equipment:** +- **Sylgard-lined Petri dish (Dow Corning)**: Used for tissue dissection and preparation. +- **Upright fluorescence microscope (DM6 FS, Leica)**: For imaging calcium signals. +- **Camera (Prime 95B, Photometrics, Roper Scientific)**: Used for capturing images during fluorescence microscopy. +- **Software (Metamorph, Molecular Devices)**: For image analysis. + +**Alternative Methods:** +- **Calcium Indicators**: Instead of GCaMP mice, synthetic calcium indicators like Fura-2 can be used if GCaMP mice are not available. +- **Brain Slice Chambers**: For maintaining viability of the spinal cord and colon, use brain slice chambers if ACSF circulation systems are not available. + +**References:** +Protocols.io: [https://dx.doi.org/10.17504/protocols.io.eowyo1e817lr2/v2](https://dx.doi.org/10.17504/protocols.io.eowyo1e817lr2/v2) + +*License*: This is an open access protocol distributed under the terms of the Creative Commons Attribution License. + +*Protocol status*: Working +*Created*: Jan 10, 2023 +*Last Modified*: Jan 11, 2023 +*Protocol integer ID*: 75078 + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/enumeration-of-bacteria-and-cyanobacteria-by-flow-j2wcqfe.md b/markdown-output/enumeration-of-bacteria-and-cyanobacteria-by-flow-j2wcqfe.md new file mode 100644 index 0000000000000000000000000000000000000000..ccf64d10bf1f2496128bce8a6c8eec8a3543bdc2 --- /dev/null +++ b/markdown-output/enumeration-of-bacteria-and-cyanobacteria-by-flow-j2wcqfe.md @@ -0,0 +1,154 @@ +```markdown +# Goal/Experiment: +The goal of this experiment is to enumerate bacteria and cyanobacteria in seawater using flow cytometry. Flow cytometry allows for precise quantification and characterization of different populations within a sample, based on their fluorescent properties and light scatter. + +# Enumeration of Bacteria and Cyanobacteria by Flow Cytometry + +## Abstract +Seawater is collected and preserved to be analyzed in the lab using an InFlux Flow Cytometer with the Spigot software package. The populations targeted for enumeration are *Prochlorococcus*, *Synechococcus*, unclassified pico-eukaryotes (cells sized around 1µm), and heterotrophic non-fluorescing bacteria. Each sample is divided in two, with one aliquot analyzed for autofluorescence and the other for heterotrophic bacteria stained with SYBR Green I. In both cases, excitation is produced using stacked 488nm and 457nm lasers. The data is analyzed using FlowJo (v10) to determine cell concentrations (cells/mL). + +## Protocol + +### Sample Collection + +#### Step 1 +Use a 15 mL polypropylene centrifuge tube to collect 10-12 mL of seawater from the Niskin bottle. + +#### Step 2 +Take a 2.0 mL subsample and store it in a 2 mL Corning cryovial (P/N 66021-974 from VWR) pre-loaded with 30 μL (15 μL per 1 mL sample) of 16% paraformaldehyde (PFA). + +- **Reagents**: Paraformaldehyde Aqueous Solution -16% [15700 by Electron Microscopy Sciences](https://www.emsdiasum.com/microscopy/products/chemicals/paraformaldehyde.aspx) + +#### Step 3 +Mix the sample well (3x inversions of the cryovial) and store it in the dark at room temperature for 15 minutes. Flash freeze in liquid nitrogen and store at -80°C. + +### Instrument Setup + +#### Step 4 +Triple rinse and fill the sheath fluid reservoir with MilliQ Water. + +#### Step 5 +Attach a Millipore Sterivex 0.22 μm In-Line Filter (P/N: SVGPL10RC) to the InFlux intake for the sheath fluid. + +#### Step 6 +Set the sheath pressure to 20.0 psi and adjust the sample pressure to yield a flow rate of approximately 30 μl/min (normally 20.1-20.2 psi). + +#### Our setup +InFlux uses a Sensirion SLI-0430 Flow Meter placed in-line between the sample line pinch valve and the nozzle assembly. The InFlux interfaces with the software Spigot (ver. 6.1.10.0) from Cytopeia. + +### Laser Alignment + +#### Step 7 +The laser sources are a 488nm 200mW laser (Coherent, 488-200 CRDH) and 457nm 300mW laser (CVI Melles Griot, 85-BLS-601), both operating at 100%. + +- Initial alignment is done using the 488 nm laser, focusing the beam first on the Red and Orange channels and then on the FSC channel to maximize precision. +- The 457 nm laser alignment is done second, and is maximized to provide the highest signal on the Red and FSC channels. + +#### Step 8 +Alignment is performed using Spherotech Ultra Rainbow Fluorescent Particles in the 1.0-1.5 μm size (P/N: URFP-10-5). Gains are adjusted to ensure that the 1 μm beads are tightly clustered near the 10^3:1 decade for FSC and Red channels, the 10^1:5 decade for the Orange channel, and the 10^1:3 decade for the Green channel. + +##### The optical pathway filters used for each channel are: +- **FSC**: 488 Blocking (For cleaning the 488 light) +- **Red**: 560LP > 610LP > 692/40BP +- **Orange**: 560LP (Rfleect) 610LP > 585/40BP +- **Green**: (Reflect) 560LP > 542/27BP + +#### Step 9 +After final alignment, let the InFlux run for 45 minutes (while back flushing) to stabilize the stream. Minor re-alignments may be necessary moving forward. + +### Sample Preparation + +#### Step 10 +Remove samples from -80°C storage after the InFlux has been fully aligned. Place samples on the bench at room temperature, in the dark, to allow them to thaw. + +- Once thawed, briefly mix the sample by vortex (less than one second), and then split into two 1 mL aliquots in 5 mL polypropylene tubes (P/N: 352063 from Corning, henceforth PP tubes). +- Store unstained samples in a 4°C refrigerator in the dark. +- **Note**: The vortex used throughout this analysis is the Vortex Genie 2 from Scientific Industries, with the vortex speed set to 7. + +### Unstained Samples + +#### Step 11 +Add 2 μL of a URFP-10-5 (1 μm beads) dilution to every unstained sample, and mix by vortex for approximately 2 seconds. + +- Prepare the bead dilution by adding 2 drops of the URFP-10-5 beads to 3 mL of MilliQ water (in one of the PP tubes), then briefly mix by vortexing. +- Store the samples in a 4°C refrigerator in the dark after the beads have been added. + +### Stained Samples + +#### Step 12 +Make a 200x working dilution of SYBR Green I. + +- Remove the 10,000x SYBR Green I stock from the -20°C freezer, and place on ice to thaw. +- Briefly (20 seconds) centrifuge to condense the SYBR Green I stock before removing the aliquot. +- Prepare the 200x working dilution by diluting 20 μL of SYBR Green I to a final volume of 1000 μL using MilliQ water filtered through a 0.1 μm Acrodisc syringe filter (VWR: P/N 28143-309). Mix briefly by vortex. +- SYBR Green I dilutions will be stable at room temperature for one day, in a 4°C refrigerator for up to five days, and in a -20°C freezer for at least a month. + +#### Step 13 +Add 5 μL of the SYBR Green I 200x working dilution to each sample aliquot for heterotroph counts and mix by vortex for approximately 2 seconds. + +#### Step 14 +Place the stained aliquots in a 4°C refrigerator for 20 minutes. Store samples at 4°C until analyzed. + +### Running Samples + +#### Step 15 +**Notes**: +- Always check the instrument to ensure collected data has low noise and few false positives in the areas of interest. +- Run unstained sample aliquots first during a normal run, as SYBR Green tends to leave a residue in the sample line. Clean out the sample line after every run. +- It may be useful to occasionally take blank samples using MilliQ water (or for stained samples, MilliQ water with 5 μL SYBR Green working dilution added) to check for noise. + +### Running Unstained Samples + +#### Step 16 +Ensure that the InFlux is triggering on FSC with a trigger level in the 10-20 range. Place the PP tube on the InFlux sampler, press Run, and equilibrate the sample for 30+ seconds until the event plots on Spigot are consistent. + +- Once the sample has equilibrated, stop the run. Optionally, cross compare the SLI-0430 Flow Meter with a mass difference method by removing the sample from the InFlux and getting the initial mass. + +#### Step 17 +Begin list file collection in Spigot, reset the Totalizer on the SLI-0430 software, and press Run on the sample. Continue collecting data until the flow meter reads 100 μL of the sample has been run. Stop the run, remove the sample, and weigh if desired. + +### Step 18 +Begin back flushing the InFlux sample line for 30+ seconds between each sample. + +### Running Stained Samples + +#### Step 19 +Change the trigger channel to Green, with a trigger level in the 40-50 range. Prepare the stained samples for the run as described in Steps 12-14, ensuring that the samples sit in the refrigerator for a minimum of 20 minutes before beginning the analysis. + +#### Step 20 +Add 5 μL of the SYBR Green I dilution to a remainder of an unstained sample and run the sample to prepare the InFlux sample line for running stained samples. + +#### Step 21 +Repeat the procedure for running unstained samples (Steps 16-17), changing only the analysis volume to 50 μL instead of 100 μL. + +### Machine Shutdown + +#### Step 22 +During any run where SYBR Green I is used, the InFlux should first be cleaned by running 5% CONTRAD 70 detergent through the sample line, followed by 70% ethanol, followed by MilliQ water until the event rate for FSC detection is less than 20. + +### Data Analysis + +#### Step 23 +Analyze the .fcs files using FlowJo (ver. 10.2). + +##### Gates are drawn for the following populations, with sequential gating indicated by the `>`: + +- **Prochlorococcus**: Red vs FSC > Orange vs FSC +- **Synechococcus**: Green vs Orange > Orange vs FSC +- **Pico-Eukaryotes**: Red vs FSC > Orange vs FSC +- **Heterotrophs** (SYBR, stained samples): Green vs FSC + +*Note: The final concentration for heterotroph counts must have the *Prochlorococcus* count subtracted from the SYBR count gated in the Green vs FSC plot, as both populations take up the DNA stain and have similar FSC and SSC signals. + +#### Step 24 +- Once raw counts have been extracted from FlowJo, determine concentrations by dividing the count by the corrected sample volume and converting to cells/mL. +- The measured volume (from the Flow Meter) must be corrected to account for dilution by PFA and beads or stain. + + | Parameter | Correction Factor | + |-------------------|------------------------------------------------| + | Unstained volumes | Divide by 1.017 (15 μL PFA + 2 μL beads) | + | Stained volumes | Divide by 1.020 (15 μL PFA + 5 μL stain) | + | Seawater volume | Use the mass difference method with a static coefficient of 0.0009718 (density of 1.026 μg/μL for seawater)| + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/enzyme-ligand-interaction-monitored-by-synchrotron-bnxfmfjn.md b/markdown-output/enzyme-ligand-interaction-monitored-by-synchrotron-bnxfmfjn.md new file mode 100644 index 0000000000000000000000000000000000000000..a5e234eff2cb138936b04ef7e9f95d7ca20286cb --- /dev/null +++ b/markdown-output/enzyme-ligand-interaction-monitored-by-synchrotron-bnxfmfjn.md @@ -0,0 +1,93 @@ +```markdown +# Goal/Experiment: +Monitor enzyme-ligand interactions using Synchrotron Radiation Circular Dichroism (SRCD) spectroscopy to understand conformational changes and ligand binding mechanisms. + +# Enzyme–Ligand Interaction Monitored by Synchrotron Radiation Circular Dichroism + +**Authors**: Ronahan Hussain, Charlotte S. Hughes, Giuliano Siligardi + +**Institute**: Diamond Light Source Ltd., Chilton, UK + +**DOI**: [dx.doi.org/10.17504/protocols.io.bnxfmfin](https://dx.doi.org/10.17504/protocols.io.bnxfmfin) + +**Abstract**: +Circular Dichroism (CD) spectroscopy is an essential tool for quickly ascertaining global conformational changes, secondary structure content, and protein folding in the far-UV region, and local tertiary structure changes in the near-UV region. This chapter provides an overview of how to perform CD experiments, including utilizing SRCD at the B23 beamline of Diamond Light Source. It covers methods, materials, and experimental designs used in ligand-binding interactions, protein stability, and UV denaturation assays. + +## Keywords +- Circular dichroism +- Ligand binding +- Titration +- Binding constant +- UV denaturation +- Protein stability +- Data processing + +## Introduction +Circular dichroism (CD) spectroscopy monitors the structural and conformational changes in proteins. Using synchrotron radiation circular dichroism (SRCD) provides several advantages by using light sources that are brighter than standard xenon lamps, enabling higher spectral resolution and sensitive measurements in both the far-UV (180–250 nm) and near-UV (250-350 nm) regions. + +### CD Terminology and Reagents: +- **Chromophores**: Specific parts of a molecule responsible for its color, absorbs light leading to electronic transitions. +- **Tryptophan, Tyrosine, Phenylalanine**: Aromatic amino acids with absorbance used in protein folding studies. +- **HEPES, MES, MOPS, PIPES**: Buffers not recommended for CD as they absorb strongly in the far-UV range. +- **SRCD**: Uses synchrotron radiation for source light which provides an intense and well-collimated beam. + +### SRCD Advantages: +1. **Higher Photon Flux**: Brighter and enables the use of smaller sample volumes. +2. **Increased Sensitivity**: Allows detection of subtle changes in protein structure. +3. **Broader Wavelength Range**: Captures data in regions traditionally inaccessible with benchtop CD instruments. + +## Materials + +### 2.1 Fused Silica Cuvettes +- **Types**: Cylindrical or rectangular cells with 0.02 cm pathlength. +- **Usage**: Allows for low-volume measurements (down to 70 µl for 1 cm pathlength). + +### 2.2 Buffer Systems Specificity +#### 2.2.1 Buffering System of Choice +- **Recommended Buffers**: + - Phosphate (≤ 25 mM) + - Tris or Tris-acetate-EDTA (TAE) (≤ 25 mM) + +- **Caution**: + - Avoid HEPES, MES, MOPS, PIPES as they interfere with far-UV measurements. + - Adjust pH using UV-transparent acids like phosphoric acid. + +#### 2.2.2 Salts +- **Chloride Ions**: Fluoride ions (F⁻) can substitute in chloride ions presence. +- **Sulfate Salts**: Preferred for their UV transparency. + +### 2.3 Protein Samples +- **Purity**: Samples should be ≥95% pure, confirmed via size exclusion chromatography (SEC). +- **Preparation**: Filtrate with 0.2 µm filter to remove particulates. + +### 2.4 CDApps Software +- **Purpose**: Data analysis for SRCD/CD spectra (.ols, .csv, .txt formats), compatible with standard software. + +## Methods +### Experiment Setup +1. **Background Measurements**: Collect and subtract to minimize noise. +2. **General Conditions**: + - **Optical Density**: Aim for 0.8. + - **Titrations**: 0.4 to 1.5 absorbance range. +3. **Buffer Optimization**: Phosphate buffer is preferred, adjust for chloride-free conditions. + +## Notes +1. **Buffer Selection**: Use low-concentration phosphate buffers, adjust pH without chloride ions. +2. **Sample Purity**: Ensure highly pure samples (>95%). +3. **Measurement Practices**: Degas buffers, use appropriate pathlengths, avoid air bubbles. +4. **Software Use**: CDApps is recommended for data processing. + +## Acknowledgement +Special thanks to Diamond Light Source for access to B23 beamline (CM12182, CM14484, CM16778, CM19680). + +## References +1. Meer, et al. (2010). Measuring circular dichroism... +2. Schwartz & Siligardi (2012). Circular dichroism beamline B23... +... +47. Johnson WC (1985). Circular dichroism and its empirical application... + +**Safety Warnings**: +For hazard information and safety warnings, please refer to the SDS (Safety Data Sheet). + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/epidural-stimulation-mapping-protocol-w6hfhb6.md b/markdown-output/epidural-stimulation-mapping-protocol-w6hfhb6.md new file mode 100644 index 0000000000000000000000000000000000000000..cbd67315ff28609d00f3fe8ab8a4bfc63ca8eb8a --- /dev/null +++ b/markdown-output/epidural-stimulation-mapping-protocol-w6hfhb6.md @@ -0,0 +1,109 @@ +```markdown +# Goal/Experiment: +To utilize epidural stimulation and measure various systems in Wistar rats after either sham transection or full T9 transection to determine optimal stimulation parameters that influence bladder activity after spinal cord injury. + +# Epidural Stimulation Mapping Protocol +**Authors:** +Charles Hubscher¹, Robert Hoey² +¹University of Louisville, ²Metrohealth Rehabilitation Institute of Ohio + +**Published:** April 22, 2021 +**DOI:** [dx.doi.org/10.17504/protocols.io.w6fhfb6](https://dx.doi.org/10.17504/protocols.io.w6fhfb6) +**PROTOCOL CITATION:** Charles Hubscher, Robert Hoey 2021. Epidural stimulation mapping protocol. protocols.io [https://dx.doi.org/10.17504/protocols.io.w6fhfb6](https://dx.doi.org/10.17504/protocols.io.w6fhfb6) + +--- + +### Keywords +- Spinal cord +- Epidural stimulation +- Bladder function + +### License +This is an open access protocol distributed under the terms of the [Creative Commons Attribution License](https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. + +### Created +January 16, 2019 + +### Last Modified +April 22, 2021 + +--- + +### Safety Warnings +- **Urethane Hazards:** Urethane (used as an anesthetic) is a known reproductive hazard. Ensure appropriate PPE (Personal Protective Equipment) is worn at all times. + +### Preparation Before Starting +Animals are implanted with the following: +- Jugular catheter +- Tracheal cannula +- Bladder catheter +- Bilateral fine wire electrodes in the External Urethral Sphincter +- Modified Medtronic S-6-5 array electrode + +--- + +### Procedure + +1. **Animal Placement:** + - Place the animal on a specially designed surgical platform with all necessary attachments. + +2. **Positioning:** + - Position the rat on its ventrum with hindlimbs taped down to prevent movement due to electrical stimulation. Secure the tail with a magnetically anchored movable arm. + +3. **Electrode Implantation:** + - Use 27g needles to implant bilateral fine wire electrodes in the external anal sphincter and bulbospongiosus muscle in males. Position EAS electrodes obliquely and bulbo electrodes transcutaneously midway between scrotum and prepuce. + +4. **Pressure Sensors:** + - Insert SPR-524 pressure sensors into the rectum (2 cm from anal verge) and the distal colon (10 cm from anal verge). Secure at base of tail using tape and connect to the data acquisition unit. + +5. **Perfusion Pump Setup:** + - Connect a perfusion pump to the catheter hub to deliver saline at 0.25 ml/min. Attach a pressure sensor syringe to detect bladder pressures, using a 60 ml syringe for saline supply. + +6. **Wiring Connections:** + - Connect all electrodes (ground wire, bilateral EUS, bilateral EAS, bilateral Bulbo) using copper duck bill clip connectors. Ensure wire is stripped and clipped to prevent signal noise. Amplify the signals using AM Systems amplifiers and send to data acquisition unit. + +7. **Weighing:** + - Place an Ohaus Scout balance under the surgical platform to collect voided material. Relay data using RS-232 connector and SPDC software to acquisition computer. + +8. **Grounding:** + - Ensure grounding of the animal, perfusion pump, and table to the electrophysiology cabinet. + +9. **Data Acquisition:** + - Use CED 1401 micro 3 system and Spike 2 version 8 software for data collection. + +10. **Config File Setup:** + - Open Spike 2 configuration file and load the setup for channels being recorded (EUS, EAS, bulbo, 2 cm probe, 10 cm probe, leaks, stim marker, keyboard input). + +11. **Stimulation Setup:** + - Connect electrical stimulation equipment to Medtronic interface. Use a grass stimulator (S88) with a current isolation unit for electrical stimulation. + +12. **Initiating Recording:** + - Start the acquisition software and turn on the perfusion pump. Monitor bladder pressure rise. + +13. **Fill-Void Cycles:** + - Allow the animal to undergo several fill-void cycles until consistent intervals between voids are achieved. + +14. **Baseline Collection:** + - Collect five baseline periods of activity with 2-minute measurements. + +15. **Stimulation Activation:** + - Activate stimulation for either 2 minutes (if fill-void cycle < 2 minutes) or until one void occurs (> 2-minute interval). + +16. **Changing Parameters:** + - Change parameters after each presentation. Test frequency (5 to 60 Hz) and intensity parameters (50 to 500 µA) in increasing fashion. Allow 2 minutes for return to baseline after each stimulation. + +17. **Data Collection:** + - Collect data on: + - Bowel function (rectal and distal colon) + - Urethral sphincter activity (EUS EMG) + - Bulbospongiosus activity (bulbo EMG) + - Anal sphincter activity (EAS EMG) + - Bladder pressure + - Urine expulsion and volume + - Electrical stimulation markers + - Additional notes via keyboard input + +--- + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/epik-cancer-mirna-panel-assay-f4wbqxe.md b/markdown-output/epik-cancer-mirna-panel-assay-f4wbqxe.md new file mode 100644 index 0000000000000000000000000000000000000000..d15562ddb61869940d6325ef8f6bfe68dcb47be8 --- /dev/null +++ b/markdown-output/epik-cancer-mirna-panel-assay-f4wbqxe.md @@ -0,0 +1,199 @@ +```markdown +# Goal/Experiment: +To perform a two-step EPIK™ Cancer miRNA Panel Assay for the detection and quantification of cancer-related microRNAs using EPIK™ miRNA specific RT-oligonucleotides and SensiSMART™ Master Mix for reverse transcription and real-time PCR amplification. + +# EPIK™ Cancer miRNA Panel Assay + +**Bioline** + +## Abstract +The EPIK™ miRNA Panel Assays protocol is a two-step process consisting of: + +1. **Reverse transcription** with miRNA-specific RT-oligonucleotides and EPIK™ cDNA synthesis kit. +2. **Real-Time PCR** using SensiSMART™ Master Mix and amplification primers. + +It is critical to follow the protocol carefully for the success of the experiment, from first-strand cDNA synthesis to real-time PCR amplification (approximately 2 hours). The procedure can be paused after first-strand cDNA synthesis, with undiluted cDNA stored at -20 °C for up to three days. + +_Citation: Bioline EPIK™ Cancer miRNA Panel Assay. protocols.io dx.doi.org/10.17504/protocols.io.f4wbqx_ +_Published: 13 Dec 2016_ + +## Guidelines + +### Kit Contents +The EPIK™ Cancer miRNA ASSAY COMPONENTS KIT includes: + +| Component | Quantity | +|-----------|----------| +| Cancer Assay Plates* | 8 x 96-well plates** | +| RT Primer Pool - lyophilized | 4 tubes (A, B, C, and D) | +| RNA Spike - lyophilized | 1 tube | +| EPIK 5x RT Buffer | 1 x 32 µL | +| EPIK RT Enzyme | 1 x 8 µL | +| 2x SensiSMART™ SYBR Master Mix | 4 x 1 mL | +| DEPC Water | 3 x 1.8 mL | + +_*For detailed definitions, see [here](www.bioline.com/mirna)._ +_**Three 96-well configurations are available depending on the real-time PCR machine used._ + +### Description + +Mature microRNAs (miRNAs) are single-stranded RNAs (~22 nucleotides long) that regulate gene expression. Detecting miRNAs is challenging due to their short lengths and sequence specificity. The EPIK™ miRNA Panel Assay offers several advantages: + +- **Highly specific**: Targets only mature miRNAs, not precursors. +- **Ultra-sensitive**: Detects miRNAs from as little as 10 pg of total RNA. +- **Wide dynamic range**: Over six logs of dynamic range. +- **Fast reaction time**: Less than 2 hours from RNA to result. + +The assay uses three specific primers and the SensiSMART™ qPCR mixes (SYBR Green), ensuring high sensitivity and specificity. + +![EPIK™ miRNA Panel Assay powered by MiRXES™ technology.](image1.png) + +### Storage + +- Store reagents at -20 °C. Avoid repeated freeze-thaw cycles. +- RNA Spike is stored at -80 °C after reconstitution. +- Equipment and reagents should not be subject to multiple freeze/thaw cycles. + +### Safety Information +Always wear suitable personal protective equipment (PPE), including a lab coat, gloves, and safety glasses. Consult the material data safety sheets (MSDSs) [here](www.bioline.com/mirna). + +## Product Specifications +- **Specificity:** Highly targeted at mature miRNAs. +- **Sensitivity:** Detects as few as 100 copies per RT reaction. +- **Reagent Compatibility:** qPCR primers lyophilized and compatible with standard real-time PCR instruments. Use white plates for better signal-to-noise ratios. + +## Figure Workflow +![Workflow for EPIK™ miRNA Cancer Assay](image2.png) + +## Equipment and Reagents to be Supplied by the User +- Nuclease-free disposable plasticware. +- Plate seals suitable for qPCR. +- Microcentrifuge for 1.5 mL tubes. +- Plate centrifuge suitable for 96-well plates. +- Cooling block or ice bucket. +- Heating block or thermocycler capable of heating at 42 °C and 70 °C. +- Vortex. + +## Real-Time PCR Machine and ROX Level +Use the correct plate type and SensiSMART™ ROX level. Different qPCR machines have different requirements. The correct use of plates and machine settings is critical for accurate results. + +## Workflow + +### Using a Single qPCR Machine +1. Perform cDNA synthesis and store all reactions at -20 °C. +2. Thaw the reactions gently before use. +3. Mix the qPCR master mix and cDNA, and add to the PCR plates. +4. Ensure all samples undergo the same number of freeze-thaw cycles. +5. Process immediately and do not freeze the prepared plates again. +6. Repeat for all 8 plates. + +### Using Multiple qPCR Machines +1. Perform all RT and qPCR reactions in parallel to ensure uniform treatment. +2. Keep track of freeze-thaw cycles for consistency. + +## Materials +- EPIK™ Cancer miRNA Hi-ROX Panel [BIO-66032 by Bioline](https://www.bioline.com). + +### Protocol + +#### First-strand cDNA synthesis +**Step 1:** +Keep components and reactions on ice during the procedure. + +**Step 2:** +Gently thaw template RNA on ice. Use up to 100 ng of total RNA per 20 µL RT reaction. Adjust concentrations using nuclease-free water. + +**Step 3:** +Reconstitute the RNA spike (synthetic miRNA control) by adding 30 µL of nuclease-free water to the tube and vortex. + +**Step 4:** +Incubate at room temperature for 5 minutes. + +**Step 5:** +Centrifuge briefly, then re-vortex. + +**Step 6:** +Reconstitute the Cancer RT Primer Pools (A, B, C, and D) by adding 20 µL of nuclease-free water. Vortex and incubate at room temperature for 5 minutes, then store at -20 °C. + +**Step 7:** +Gently thaw EPIK 5x RT Buffer and Cancer RT primer pool tubes on ice. Vortex and centrifuge briefly. + +**Step 8:** +Prepare 4 reverse transcription reactions as follows (table 1): + +| Reagent | Volume (Tube A) | Volume (Tube B) | Volume (Tube C) | Volume (Tube D) | +|---------|-----------------|-----------------|-----------------|-----------------| +| Template RNA | X µL | X µL | X µL | X µL | +| EPIK 5x RT Buffer | 4 µL | 4 µL | 4 µL | 4 µL | +| DEPC Water | 6 µL - X µL | 6 µL - X µL | 6 µL - X µL | 6 µL - X µL | +| EPIK RT Enzyme | 1 µL | 1 µL | 1 µL | 1 µL | +| RT Primer Pool | 2 µL | 2 µL | 2 µL | 2 µL | +| Total volume | 20 µL | 20 µL | 20 µL | 20 µL | + +**Step 9:** +Mix reagents by pipetting up and down, centrifuge briefly. + +**Step 10:** +Incubate reaction at 42 °C for 30 minutes. + +**Step 11:** +Heat-inactivate at 90 °C for 5 minutes. + +**Step 12:** +Store undiluted cDNA at -20 °C for up to three days if not proceeding immediately. + +#### Real-Time PCR Amplification and Detection + +**Step 13:** +Prepare qPCR reagents as follows (table 2): + +| Reagent | Volume (Plate A) | Volume (Plate B) | Volume (Plate C) | Volume (Plate D) | +|---------|-------------------|-------------------|-------------------|-------------------| +| 2x SensiSMART™ PCR Master Mix | 1000 µL | 1000 µL | 1000 µL | 1000 µL | +| DEPC water | 980 µL | 980 µL | 980 µL | 980 µL | +| cDNA reaction | 20 µL | - | - | - | +| cDNA reaction | - | 20 µL | - | - | +| cDNA reaction | - | - | 20 µL | - | +| cDNA reaction | - | - | - | 20 µL | +| Total volume | 2000 µL | 2000 µL | 2000 µL | 2000 µL | + +**Step 14:** +Remove PCR plate from foil bag and place on cold block or ice bucket. + +**Step 15:** +Peel back carrier seal, and dispense 20 µL cDNA:PCR master mix per well into the corresponding PCR plate. + +**Step 16:** +Seal plate and centrifuge briefly. + +**Step 17:** +Perform real-time PCR amplification according to the following cycling parameters (table 3): + +| Cycles | Temperature | Time | Notes | +|--------|--------------|------|-------| +| 1 | 95 °C | 10 min | Polymerase activation | +| 40 | 95 °C | 10 s | Denaturation | +| 40 | 60 °C | 30 s | Annealing/extension | + +**Step 18:** +Collect raw Ct values. Verify the control wells (E12/F12; G12/H12). Export data as an Excel file for further analysis. + +**Step 19:** +Analyze miRNA profiles for relative abundance from raw data. + +### Important Notes +- Handle RNA carefully to avoid RNase contamination. +- For best results, use purified miRNA or total RNA and not partly purified samples. +- The ISOLATE II miRNA Kits are recommended for RNA preparation. + +### Controls and Calibrators + +**RNA Spike Control:** Reconstituted RNA Spike can be used during sample isolation or reverse transcription to detect differences in RNA purification or for normalization. + +_Analysis:_ Verifying the Ct values of control wells E12/F12 and G12/H12 ensures consistency in the assay. + +#### Warnings +Follow safety guidelines and wear PPE. Detailed safety information can be found in the MSDSs available [here](www.bioline.com/mirna). + +_endofoutput_ +``` \ No newline at end of file diff --git a/markdown-output/estimating-microbial-population-data-from-optical-cgumtwu6.md b/markdown-output/estimating-microbial-population-data-from-optical-cgumtwu6.md new file mode 100644 index 0000000000000000000000000000000000000000..7bcc0eed462774dfbd1325f706ee94175cd72d9a --- /dev/null +++ b/markdown-output/estimating-microbial-population-data-from-optical-cgumtwu6.md @@ -0,0 +1,125 @@ +```markdown +# Goal/Experiment: +Estimate microbial population data from optical density (OD) measurements using a spectrophotometer. + +## Estimating Microbial Population Data from Optical Density V.2 + +**Authors**: +Pamela Yeh1, Portia M Mira1 +1Ecology and Evolutionary Biology, UCLA + +[Link to full protocol](dx.doi.org/10.17504/protocols.io.8epv5j6wj1lb/v2) + +**Abstract** +The spectrophotometer has been used for decades to measure the density of bacterial populations as the turbidity expressed as optical density – OD. However, the OD alone is an unreliable metric and is only proportionately accurate to cell titers to about an OD of 0.1. The relationship between OD and cell titer depends on the configuration of the spectrophotometer, the length of the light path through the culture, the size of the bacterial cells, and the cell culture density. We demonstrate the importance of plate reader calibration to identify the exact relationship between OD and cells/mL. We use four bacterial genera and two sizes of micro-titer plates (96-well and 384-well) to show that the cell/ml per unit OD depends heavily on the bacterial cell size and plate size. We applied our calibration curve to real growth curve data and conclude the cells/mL – rather than OD – is a metric that can be used to directly compare results across experiments, labs, instruments, and species. + +## Protocol + +### 1. Preparation +When calibrating a microtiter plate reader for microbial growth experiments, it is essential to begin with a culture in which almost all the particles (cells) can form colonies, i.e., are colony-forming units or CFU. To achieve that, a mid-log phase culture is required - one in which the culture has not begun to enter the stationary phase. + +There are two easy ways to prepare overnight cultures that are highly reproducible and contain almost exclusively viable cells. Both methods involve limiting cell growth before the stationary phase has begun. + +#### 1.1 Method 1: Using Rich Broth Media to Obtain Oxygen-Limited Cultures + - Inoculate a colony into 10 ml of your favorite broth medium in a 15 ml plug-seal centrifuge tube. + - Tighten down the plug-seal cap and allow the culture to stand overnight at the preferred growth temperature without shaking or other methods of aeration. + +#### 1.2 Using a Mineral Salts Medium +A **mineral salts medium**, such as **M9 medium**, containing a limiting concentration of a carbon source such as glucose (0.02% w/v) works well for many organisms. Inoculate a suitable volume of carbon-limited medium with a single colony and shake well overnight at the optimum growth temperature. + +- **M9 Medium**: A minimal salts medium used to grow bacteria. It typically contains salts, glucose, and a nitrogen source. (Vendor: Sigma-Aldrich) +- **Glucose**: A simple sugar used as a carbon source in culture media. (Vendor: Thermo Fisher Scientific) + +A couple of days before doing the calibration: + 1. Grow either an oxygen-limited or nutrient-limited culture. + 2. Measure the OD of that culture in 6 wells of a microtiter plate and the OD of the uninoculated medium in another 6 wells (the blanks). + 3. Subtract the mean OD of the blank wells from the mean OD of the wells containing the culture. That OD is the corrected OD of the nutrient- or oxygen-limited culture. + +#### OD Units Calculation +The OD units are OD times volume in ml. For example, a 10 ml culture with a corrected OD of 0.3 contains 3 OD units. The purpose of using OD units is to let you determine the volume of limited culture you will need for the real experiment. + +### 2. Calibration +Grow a sufficient volume of limited culture to provide at least 10 OD units. For oxygen-limited cultures, this may require several 10 ml cultures. If so, combine the cultures. Check the OD of the combined culture to see that there are about 10 OD units. + +#### 2.1 Spin Down the Culture(s) + - Pour off the supernatant and resuspend the pellet in 4.0 ml (or more) of M9 buffer with no nutrients. This volume will depend on how much is needed for the calibration. + +#### 2.2 Vortex the Resuspended Cells + - This is the zero tube which should have an expected 10 OD units. + +### 3. Serial Dilutions for OD Measurements +Prepare a series of tubes numbered 1-10, each containing 2.0 ml of M9 buffer. + +#### 3.1 Serially Transfer and Vortex + - Transfer 2.0 ml from tube zero to tube 1 and vortex to mix well. Accurate dilutions require both accurate pipetting and thorough mixing at each step. + +#### 3.2 Continue Two-Fold Serial Dilutions + - Transferring and mixing 2 ml at each step. + +#### 3.3 Fill the Wells of a 96-Well Plate and/or 384-Well Plate + - According to the plate layouts below. We use 200 µl per well for 96 well plates and 80 µl per well for 384 well plates, but you can use whatever volumes you choose. + +#### 3.4 96 Well Layout +The layouts provide 6 wells at each dilution. + +| | A | B | C | +|---|---|---|---| +| Row A | Wells 1-6 | Wells 7-12 | +| Row B | Tube 0 | Tube 1 | +| Row C | Tube 2 | Tube 3 | +| Row D | Tube 4 | Tube 5 | +| Row E | Tube 6 | Tube 7 | +| Row F | Tube 8 | Tube 9 | +| Row G | Tube 10 | Buffer Blank | +| Row H | Empty | Empty | + +#### 3.5 Read Each Plate in Your Microtiter Plate Reader + - Read each plate at 5-minute intervals for 30 minutes, shaking as usual between readings. This allows the plate reader to settle down. + +#### 3.6 Serial Dilutions and Plating for Colony Counts + - From tube zero, prepare 10^4, 10^5, 10^6, and 10^7-fold dilutions in M9 buffer so that you have at least 1.0 ml of each dilution. Again, pipette carefully and mix well at each dilution. Plate 100µl per plate onto 5 broth plates for each of the 10^4, 10^5, 10^6, and 10^7-fold dilutions. Incubate the plates at the appropriate temperature until colonies have grown. + +### 4. Analyze the Data + +#### 4.1 Colony Counts + - Count the colonies on whichever dilution has the closest to an average of about 100 colonies per plate. The viable cells per ml in the zero dilution is 10 x dilution x mean colonies per plate. + +#### 4.2 OD Readings + - Copy the 30-minute OD readings into a copy of the Calibration Calculator excel file. + +#### 4.3 96 Well Plate, Sheet 1 + - From the plate reader output file copy the 30-minute OD readings into a copy of the Calibration Calculator excel file, cells D14 through I25. Enter the dilution that gave closest to 100 colonies per plate in cell A10, and the mean number of colonies per plate for that dilution in cell B10. Cell C10 will show the cells/ml in the zero tube. Cells B14 - B24 will show the cells/ml in tubes 0 through 10. J14-J25 will show the mean ODs in tube 1-10 and the buffer blank. K14-K24 will show the mean corrected OD, i.e., mean OD - mean OD of the buffer blank. + +#### 4.4 384 Well Plate, Sheet 2 + - Similar steps as above for the 384 well plate. + +### 5. Graph Mean Corrected OD vs Cells/ml and Fit Curve +You will need a graphing program that has curve-fitting functions. + +- **DataGraph for MacOS**: [DataGraph](https://www.visualdatatools.com/DataGraph/) +- **KaleidaGraph**: [KaleidaGraph](https://www.synergy.com/) +- **GraphPad Prism**: [GraphPad Prism](https://www.graphpad.com/scientific-software/prism/) +- **MagicPlot**: [MagicPlot](https://magicplot.com/) + +Into your graphing program paste the Mean Corrected OD column down through values ≥0.005 and the corresponding Cells/ml. Plot corrected OD (X-axis) vs Cells/ml (Y-axis) and fit a polynomial of degree 4 to those points. + +### 6. Example to Illustrate Using the Calibration Calculator +- The resulting .cal file, Ecoli384.cal looks like this: + +``` +1.552e9 +-2.301e9 +1.342e9 +1.002e9 +513817 +``` + +**Note**: All information provided here is adapted for better comprehension and lab application based on the protocol by Pamela Yeh and Portia M Mira. + +--- + +**Citation**: +Pamela Yeh, Portia M Mira 2022. Estimating microbial population data from optical density. [protocols.io](https://dx.doi.org/10.17504/protocols.io.8epv5j6wj1lb/v2) + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/evaluaci-n-la-producci-n-de-tnf-alfa-por-pbmcs-est-kdkcs4w.md b/markdown-output/evaluaci-n-la-producci-n-de-tnf-alfa-por-pbmcs-est-kdkcs4w.md new file mode 100644 index 0000000000000000000000000000000000000000..0d345ab7fcf33fe7e7194e5f0c853cb6ab2cfe9e --- /dev/null +++ b/markdown-output/evaluaci-n-la-producci-n-de-tnf-alfa-por-pbmcs-est-kdkcs4w.md @@ -0,0 +1,108 @@ +```markdown +# Goal/Experiment: +Evaluación de la capacidad de neutralización del efecto de Lipopolisacárido (LPS) de inducir la producción de Factor de Necrosis Tumoral (TNFα) en Células Mononucleares de Sangre Periférica (PBMCs) tratadas con péptidos catiónicos. + +## Evaluación la producción de TNF alfa por PBMCs estimulados con LPS tratados con péptidos + +### Autores +Lily Johanna Toro, Germán Alberto Téllez Ramírez, Diana Carolina Henao, Jhon Carlos Castaño Osorio + +### Abstract +Evaluación de la capacidad de neutralización del efecto de Lipopolisacárido (LPS) de inducir la producción de Factor de Necrosis Tumoral (TNFα) en Células Mononucleares de Sangre Periférica (PBMCs) tratadas con péptidos catiónicos. + +### Publicación +Publicado en: 19 Oct 2017 + +## Guidelines +La capacidad de los péptidos para neutralizar o inhibir la producción de Lipopolisacárido (LPS) es medida a través de la capacidad de los mismos de inhibir la producción de TNFα en Células Mononucleares de Sangre Periférica (PBMCs). El cultivo de PBMCs fue realizado con 250,000 células por pozo en un plato de cultivo de 96 pozos (Cellstar, greiner bio-one, cat-N°655180), las células fueron tratadas por triplicado con 10 ng/mL de LPS (Escherichia coli, LPS 026:B6, Sigma Aldrich L2654). Se utilizan controles por triplicado con medio de cultivo y los tratamientos con diferentes concentraciones del péptido a evaluar con LPS y sin LPS. + +## Procedimiento +1. Incubar las células por 12 horas (este tiempo debe estandarizarse) a 37 °C y 5 % CO₂. +2. Colectar el sobrenadante y reemplazar con medio de cultivo celular con 44 μM de Rezazurina para evaluar la viabilidad celular. +3. Posteriormente, medir los niveles de TNFα en los sobrenadantes por ELISA (Human TNF-α ELISA MAX, BioLegend), siguiendo las instrucciones del fabricante. + +## Before start +Preparar todos los reactivos y soluciones de trabajo con un día de anticipación. + +Se recomienda realizar el ELISA de los sobrenadantes inmediatamente después de ser colectados; para esto se debe tener en cuenta que la placa debe sensibilizarse un día antes del desarrollo del ELISA. + +## Materials +- Micropipetters; Sterile tips and serological pipettes (Contributed by users) +- 96 well plates with lids (FISHER SCIENTIFIC #087722C, Contributed by users) +- 1.5 mL Eppendorf tubes (Contributed by users) +- Lipopolysaccharides from Escherichia coli O26:B6 [L2654, Sigma-Aldrich](https://www.sigmaaldrich.com/US/en/product/sial/l2654) +- RPMI 1640 Medium [11875093, Thermo Fisher Scientific](https://www.thermofisher.com/order/catalog/product/11875093) +- Antibiotic-Antimycotic (100X) [15240062, Thermo Fisher Scientific](https://www.thermofisher.com/order/catalog/product/15240062) + +### Reagents +- Human TNF-α ELISA [430204, BioLegend](https://www.biolegend.com/) +- Resazurin [189900050, Acros Organics](https://www.acros.com/) + +## Protocol + +### Cultivo de Células Mononucleares de Sangre Periférica (PBMCs) + +#### Step 1 +Cultivar en una caja de cultivo celular de 96 pozos 250,000 cel/pozo a un volumen final de 100 μL/pozo en RPMI 1640 suplementado con antibiótico antimicótico (ampicilina/estreptomicina) 1X. Dejar incubando por 12 horas, hasta que las células se establezcan. + +### Preparación de soluciones de trabajo + +#### Step 2 +Realizar diluciones de proteína, péptido o compuesto a evaluar. + +- **Solución de trabajo de LPS a 10X (100ng/mL)** + - Stock 1: preparar 1 mg de LPS en 1 mL de solución salina estéril (dividir en alícuotas de 100 μL). + - Stock 2: tomar 1 μL del stock 1 en un nuevo tubo y completar hasta 1 mL (dividir en alícuotas de 100 μL). + - Solución de trabajo 10X: tomar 1 μL del stock 2 por pozo (volumen final de 100 μL) para una concentración de 100 ng/mL. + +Mezclar las soluciones de muestra, control y LPS en tubos de 1.5 mL (10 μL LPS 10X + 10 μL tratamiento o control + 80 μL medio de cultivo). + +### Adicionar los tratamientos a los PBMCs + +#### Step 3 +1. Remover el sobrenadante lentamente de cada uno de los pozos con PBMCs. +2. Añadir 100 μL por pozo de cada uno de los tratamientos. + +Se recomienda adicionar de izquierda a derecha en el siguiente orden: +1. Medio de cultivo. +2. Muestras sin LPS. +3. Muestras con LPS. + +| | Medio de cultivo | Muestra | Muestra + LPS | LPS | +|--------|------------------|---------|---------------|-----| +| **A** | | | | | +| **B** | | | | | +| **C** | | | | | + +### Recolección de sobrenadantes + +#### Step 4 +1. Preparar tubos de 1.5 mL correspondientes a cada tratamiento. +2. Recoger los sobrenadantes por cada tubo. +3. Centrifugar a 9000g por 2 minutos, colectar los sobrenadantes y realizar el ELISA de TNFα inmediatamente. Si se van a almacenar los sobrenadantes, hacerlo a -80 °C para evitar la descongelación. + +### Verificación de viabilidad de PBMCs +Adicionar Rezazurin (indicador de metabolismo celular) a cada uno de los pozos (concentración final de 44 μM), incubar 2 horas a 37 °C y 5 % CO₂. Leer la placa por espectrofotometría a 570 y 450 nm, o por fluorescencia. + +### Análisis de los resultados + +#### Step 5 +1. Obtener dos lecturas por pozo de 570 y 450 nm. +2. Utilizar los pozos con solo células y medio de cultivo (blanco) para calcular la mediana. +3. Calcular el delta de cada uno de los pozos. +4. Graficar la curva estándar de TNFα del kit (concentraciones en eje X y delta en eje Y). +5. Aplicar la fórmula a cada tratamiento: + + \[ + Y = \frac{(A - D)}{1 + (X/C)^B} + D + \] + +Finalmente graficar Tratamientos vs Concentraciones de TNFα (pg/mL). + +## Warnings +Se debe tener mucho cuidado en el momento de hacer la extracción de PBMCs en cuanto a la manipulación de la sangre de un individuo sano (ver protocolo). + +--- + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/evaluating-intrinsic-cardiac-neural-control-of-car-bvpbn5in.md b/markdown-output/evaluating-intrinsic-cardiac-neural-control-of-car-bvpbn5in.md new file mode 100644 index 0000000000000000000000000000000000000000..88ff4ab248f5d5d93effa8ee64b967d788e83e4f --- /dev/null +++ b/markdown-output/evaluating-intrinsic-cardiac-neural-control-of-car-bvpbn5in.md @@ -0,0 +1,100 @@ +```markdown +# Goal/Experiment: +Evaluating intrinsic cardiac neural control of cardiac function using sequential ganglionated plexus ablations + +## Title: +Evaluating Intrinsic Cardiac Neural Control of Cardiac Function Using Sequential Ganglionated Plexus Ablations + +## Authors: +- Peter Hanna +- Jeffrey Ardell +- Kalyanam Shivkumar + +## Institution: +UCLA + +## DOI: +[dx.doi.org/10.17504/protocols.io.bvpbn5in](https://dx.doi.org/10.17504/protocols.io.bvpbn5in) + +## Protocol Citation: +Peter Hanna, Jeffrey Ardell, Kalyanam Shivkumar 2021. Evaluating intrinsic cardiac neural control of cardiac function using sequential ganglionated plexus ablations. + +## Keywords: +- Cardiac autonomic nervous system +- Cardiac contractility +- Cardiac electrophysiology +- Ganglionated plexus +- Intrinsic cardiac nervous system +- Ablation + +## License: +This is an open access protocol distributed under the terms of the [Creative Commons Attribution License](https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. + +## Image Attribution: +Peter Hanna, MD, PhD + +## Created: +June 09, 2021 + +## Experimental Set-up +1. **Sedate and intubate the Minipig:** + - Pre-sedate a Yucatan minipig via tiletamine-zolazepam (6 mg/Kg) intramuscular injection. + - Sedate using isoflurane (5% initiation) via nosecone mask. Intubate using a 7.0mm endotracheal tube for isoflurane (1-2% maintenance) and fentanyl (20 mcg/Kg IV). + - Monitor body temperature with either an esophageal or rectal temperature probe. + +2. **ECG Recording:** + - Record continuous 12-lead ECG data using a Prucka CardioLab. Place anterior and additional electrodes for enhanced ECG acquisition. + - Use CED system and Spike2 software for surface ECG. + +3. **Vascular Access:** + - Obtain vascular access with 7 French sheaths for arterial and 8 French sheaths for venous monitoring and administration. + +4. **Monitor Blood Gases:** + - Use an i-STAT blood analyzer every 90 minutes, adjust ventilation as needed. + +5. **Thoracotomy Procedure:** + - Perform a clamshell thoracotomy to access heart and vessels. + +6. **Anesthesia Maintenance:** + - Switch to α-chloralose with IV bolus (50mg/Kg) and continuous infusion (10 mg/Kg/h) before autonomic stimulations. + +7. **Nerve Exposure and Stimulation:** + - Dissect and free the left cervical vagus (LCV) and right cervical vagus (RCV) nerves. + - Use platinum nerve cuff electrodes connected to a Grass S88 Stimulator for nerve stimulations. + - Perform specific stimulations individually for each nerve (10 Hz, 1 ms, 0.1-15 mA). + +8. **Define Stimulus Threshold:** + - Increase vagal nerve stimulation to 3 times threshold. Understand response durations and intervals. + +### Cardiac Electrophysiologic Recording Set-up: +1. **Catheter Insertion:** + - Insert a 5 French quadripolar catheter via the right external jugular vein. Monitor electrode signals until HIS bundle capture. + - Create a pericardial cradle using suture materials. + +2. **Electrode Setup:** + - Use a custom-made 56-electrode sock on both ventricles for identifying regional activation recovery intervals (ARI). + +### Hemodynamic Recordings Set-up: +1. **Pressure Assessment:** + - Place a Millar Mikro-Tip SPR-350 pressure catheter in the left ventricle. + - Monitor and record pressures using CED software. + +## Data Collection: +1. **Baseline Measurements:** + - Record hemodynamic and electrophysiologic data after baseline setup. + - Perform vagal and sympathetic stimulations (25-30 seconds each). + +2. **System Function Evaluations:** + - Assess LV systolic and diastolic functions, and perform interval increments with quadripolar catheter. + - Perform detailed nerve dissection and stimulation sequences as described. + +### Tissue Harvest for Histological Study: +1. **Tissue Collection:** + - Obtain histologic tissues using heparin bolus, and post-study ventricular fibrillation. + - Flush heart with heparinized saline, and treat specific tissue areas. + +2. **Tissue Preparation:** + - Fix tissue in 4% PFA (overnight), wash, and store in PBS for histological studies. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/ex-vivo-generation-and-maintenance-of-human-epstei-bv5rn856.md b/markdown-output/ex-vivo-generation-and-maintenance-of-human-epstei-bv5rn856.md new file mode 100644 index 0000000000000000000000000000000000000000..8a26b68d9045eff61f9254953c7aae5eee7086a1 --- /dev/null +++ b/markdown-output/ex-vivo-generation-and-maintenance-of-human-epstei-bv5rn856.md @@ -0,0 +1,96 @@ +```markdown +# Goal/Experiment: +To generate and maintain human Epstein-Barr Virus (EBV)-specific T cells ex vivo that lack alloreactivity for potential use in vitro and in vivo studies, including applications in immunotherapy research and T cell engineering. + +# Ex vivo generation and maintenance of human Epstein-Barr Virus (EBV)-specific T cells lacking alloreactivity + +**Zaki Molvi +Memorial Sloan Kettering Cancer Center, New York, NY** + +[dx.doi.org/10.17504/protocols.io.bv5rn856](dx.doi.org/10.17504/protocols.io.bv5rn856) + +## DISCLAIMER + +_This protocol content is for informational purposes only and does not constitute legal, medical, clinical, or safety advice. The information added to protocols.io has not undergone formal approval of any kind. The protocol should not substitute for independent professional judgment, advice, diagnosis, or treatment._ + +## ABSTRACT + +T cells recognizing Epstein-Barr Virus (EBV) can be generated ex vivo from immunocompetent human subjects and maintained in vitro for extended periods. EBV T cells typically exhibit viral antigen-specificity, high cytolytic, and inflammatory capacity and, when sensitized to autologous EBV blasts, lack xeno- or allo-reactivity. EBV T cells are vital for in vitro and in vivo studies involving human T cell transduction as well as T cell-specific immunomodulatory agents such as bispecific antibodies or BiTEs. + +## DOI +[dx.doi.org/10.17504/protocols.io.bv5rn856](dx.doi.org/10.17504/protocols.io.bv5rn856) + +## PROTOCOL CITATION +``` +Zaki Molvi 2021. Ex vivo generation and maintenance of human Epstein-Barr Virus (EBV)-specific T cells lacking alloreactivity. _protocols.io_ https://dx.doi.org/10.17504/protocols.io.bv5rn856 +``` + +## LICENSE +This is an open access protocol distributed under the terms of the Creative Commons Attribution License, permitting unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. + +## CREATED +Jun 27, 2021 + +## LAST MODIFIED +Jun 27, 2021 + +## PROTOCOL INTEGER ID +51089 + +## Materials + +- **Human PBMC:** Peripheral Blood Mononuclear Cells from fresh venous blood, isolated using a density gradient medium (e.g., Ficoll-Paque). + +- **EBV B95-8 transformed B cells (BCL):** Same donor as PBMC. [Protocol Link](https://protocols.io) + +- **Human IL-2:** Interleukin-2, growth factor for T cells (Vendor: R&D Systems or similar). + +- **T cell media:** Specialized media e.g., X-Vivo 15 or -20, AIM-V, OpTimizer, supplemented with: + - 5-10% human AB serum + - 2 mM L-Glutamine + - **Basal Medium:** RPMI 1640, IMDM, supplemented with: + - 5-10% human serum + - 2 mM L-Glutamine + - Non-essential amino acids + - 2-mercaptoethanol + +- **Gamma irradiator:** For irradiating BCL. + +## Procedure + +### Day 0 - Primary Stimulation: + +1. Thaw cryopreserved or freshly isolated PBMCs from the same donor as the BCL. Determine PBMC count using viable nucleated cells with a defined border. + +2. Determine BCLs for feeder based on 4:1 ratio PBMC:BCL (Responder:Stimulator). + +3. Irradiate BCLs at 90 Gy. + +4. Adjust PBMCs to 1-2×10^6 cells/mL in T cell media. + +5. Centrifuge irradiated BCLs, resuspend in PBMC suspension. + +6. Incubate cells in appropriate-sized vessel: + - 6-12 mL in T12 flask + - 12.5-20 mL in T25 flask + - Adjust remaining for larger flasks. + +7. Incubate cells 7-10 days at 37°C. + +### Day 7-10 - Secondary Stimulation: +1. Count cells. Monitor viability. Adjust media if necessary, based on cell density. +2. Add irradiated BCL at 4:1 ratio. +3. Incubate 3 days. + +### Day 10-13 - Ongoing: +1. On day 13, add 20 U/mL IL-2. +2. Regular IL-2 addition post-stimulation. +3. Restimulate with BCL, if viability drop: increment IL-2 to stabilize. + +### Quality Control: +1. **Immunophenotype:** CD3, CD4, CD8, CD56 by flow cytometry. +2. **Cytotoxicity:** Against autologous BCLs via multiple assays including 51Cr and TUNEL. +3. **Antigen Specificity:** High specificity for antigenic peptides via assays such as ELISPOT, cytokine staining. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/examining-health-conditions-body-functions-activit-b37zqrp6.md b/markdown-output/examining-health-conditions-body-functions-activit-b37zqrp6.md new file mode 100644 index 0000000000000000000000000000000000000000..c20f01ee109ef4030545b9389caf6f2b6d1744cd --- /dev/null +++ b/markdown-output/examining-health-conditions-body-functions-activit-b37zqrp6.md @@ -0,0 +1,85 @@ +```markdown +# Examining Health Conditions, Body Functions, Activity and Participation, and Quality of Life among Adults with Learning Disabilities – Towards a Theoretical Model + +## Goal/Experiment: +The study aims at examining the characteristics of health conditions, body functions, domains of activity and participation, and Quality of Life (QoL) among adults with Learning Disabilities (LD). It further aims to understand the relationships between these factors and to suggest approaches for better identification and intervention. + +## Authors: +- Kineret Sharfi^1 +- Sara Rosenblum^2 + + **Affiliations:** + 1. Laboratory of Complex Human Activity and Participation (CHAP) Dept. of Occupational Therapy Faculty of Social Welfare & Health Sciences University of Haifa, Mount Carmel, Haifa 3498838 Israel + 2. Laboratory of Complex Human Activity and Participation (CHAP) Dept. of Occupational Therapy Faculty of Social Welfare & Health Sciences University of Haifa, Mount Carmel, Haifa 3498838 Israel + +## Introduction +Learning disabilities (LD) refer to a heterogeneous group of neurodevelopmental disorders affecting the brain's ability to process information efficiently. LD often occurs with conditions diagnosed by the medical system as Attention Deficit Hyperactivity Disorder (ADHD) and/or Developmental Co-ordination Disorder (DCD). These significantly interfere with academic or occupational performance and persist into adulthood. + +### Key Objectives: +1. **Comparison:** Compare characteristics of adults with LD to a matched control group. +2. **Examination:** Assess relationships between health conditions, body functions, activity and participation domains, and QoL in adults with LD. + +## Methodology +A comparative observational study was conducted with a matched subjects design. + +### Participants: +- **Sample:** 55 adults with LD and 55 controls matched by age, gender, education, and socio-economic status. +- **Sample Size Calculation:** Statistical power analysis indicating 0.6 effect size, 0.6 alpha, and minimum 0.6. + +#### Groups' Demographics: +| Group | Mean Age (years) | Total (Males/Females) | BMI (kg/m^2) | +|-----------|------------------|------------------------|---------------------| +| LD group | 29.58 | 55 (64% males) | 23.6 | +| Control | 31.18 | 55 (45% males) | 23.6 | + +### Data Collection Instruments: +1. **Health Conditions:** + - **WHO Health Information ICD Checklist (WHO, 2003)** +2. **Body Functions:** + - Adult ADHD Rating Scale (ASRS-V1.1) + - The Adult Developmental Co-ordination Disorders / Dyspraxia Checklist +3. **Body Functions:** + - The Sensory Profile – Adolescents/Adults version (AASP) + - The Behavioral Rating Inventory of Executive Functions – Adolescents/Adults version (BRIEF-A) + - The Mini Sleep Questionnaire (MSQ) +4. **Activity and Participation:** + - Time Organization and Participation (TOPS) + - Daily Activities Participation Scale – for Adults (DAPS-A) + - The Adults Finance Management Questionnaire (AFMQ) +5. **QoL:** + - The World Health Organization Quality of Life questionnaire (WHOQOL-BREF) + +## Results +### Health Conditions: +Adults with LD presented more frequent ADHD symptoms (45.5%). + +### Body Functions: +-LD adults: Higher sensory functions issues, executive functions, and sleep disturbances. + +### Activity and Participation: +- Organizational difficulties (20% variance in time abilities) +- Lower economic self-sufficiency + +### Quality of Life: +- Social quality (WHOQOL-BREF) and psychological (AFMQ) affected significantly. + +## Discussion +Findings indicate that combinations of body function deficiencies and mal-adaptation hinder activity and participation. A new approach is required for LD evaluation and intervention to assist adults in managing life engagements more effectively. + +## Conclusions +There is a need for a more holistic model recognizing the complex interplay of various domains affecting adults with LD. Additional research and improved intervention approaches are necessary to enhance QoL for this group. + +## Funding: +The Israeli Ministry of Science and Technology, Grant number: 3-8364 + +## Citations: +Sharfi, K., & Rosenblum, S. (2022). Examining Health Conditions, Body Functions, Activity, and Participation, and Quality of Life among Adults with Learning Disabilities – Towards a Theoretical Model. [protocols.io](https://dx.doi.org/10.17504/protocols.io.b37zprp6) + +## Related Articles: +1. Sharfi, K., Rosenblum, S. (2015). Sensory modulation and sleep quality among adults with learning disabilities. PLoS One. [https://doi.org/10.1371/journal.pone.0115518](https://doi.org/10.1371/journal.pone.0115518) +2. Sharfi, K., Rosenblum, S. (2016). Executive functions, time organization and quality of life among adults with learning disabilities. PLoS One. [https://doi.org/10.1371/journal.pone.0166939](https://doi.org/10.1371/journal.pone.0166939) + +--- + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/exposed-stimula-blood-sugar-support-reviews-scam-cd4ss8we.md b/markdown-output/exposed-stimula-blood-sugar-support-reviews-scam-cd4ss8we.md new file mode 100644 index 0000000000000000000000000000000000000000..88a42b8608d84fa067e2f8a3c7f23748670aee92 --- /dev/null +++ b/markdown-output/exposed-stimula-blood-sugar-support-reviews-scam-cd4ss8we.md @@ -0,0 +1,133 @@ +```markdown +# Goal/Experiment: +Evaluate the effectiveness, ingredients, benefits, and safety of "Stimula Blood Sugar Support", a dietary supplement aimed at managing blood sugar levels. + +## [Exposed] Stimula Blood Sugar Support Reviews - (Scam Alert) Ingredients, Side Effects. Do The Pills Work? Don't Miss This Report! + +### Abstract +If you've been feeling tired and lethargic for the past few months, yet nothing seems to help or change your symptoms, your blood sugar level may be high. + +### DOI +[dx.doi.org/10.17504/protocols.io.36wgq7w55vk5/v1](https://dx.doi.org/10.17504/protocols.io.36wgq7w55vk5/v1) + +### Document Citation +> trina solan 2022. [Exposed] Stimula Blood Sugar Support Reviews - (Scam Alert) Ingredients, Side Effects. Do The Pills Work? Don't Miss This Report!. +> **protocols.io** +> [dx.doi.org/10.17504/protocols.io.36wgq7w55vk5/v1](https://dx.doi.org/10.17504/protocols.io.36wgq7w55vk5/v1) + +### Manuscript Citation +Stimula Blood Sugar Support, an integrated and natural solution offered by i-Health Labs, is clinically proven to help lower blood sugar levels. The product also lowers bad cholesterol and, when taken regularly, strengthens heart health. + +### Keywords +- Stimula Blood Sugar Support +- Stimula Blood Sugar Support Reviews +- Stimula Blood Sugar Support Scam +- Stimula Blood Sugar Support Ingredients + +--- + +### License +This is an open access document distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. + +### Created +July 24, 2022 + +### Last Modified +July 24, 2022 + +### Document Integer ID +67442 + +--- + +## Overview + +### Stimula Blood Sugar Support +Stimula Blood Sugar Support is said to comprise heart health-boosting nutrients in precise quantities that improve insulin sensitivity and healthy blood sugar. + +### Product Information +- **Product Name:** Stimula Blood Sugar Support +- **Healthy Benefit:** Help to reduce blood sugar levels in the body +- **Ingredients:** White mulberry leaf, Bitter Melon, Berberine extract, and many more +- **Results:** 2-3 months +- **Side Effects:** No side effects reported +- **Rating:** ⭐⭐⭐⭐⭐ +- **Official Website:** [Get The Stimula Blood Sugar Support From The Official Website](https://dx.doi.org/10.17504/protocols.io.36wgq7w55vk5/v1) + +## What Is The Stimula Blood Sugar Support? +Stimula Blood Sugar Support is a dietary supplement in the form of capsules that provides precise nutritional support to control blood sugar levels naturally. It contains scientifically proven natural ingredients and is said to be sourced in high quality and purity. Additionally, it lowers bad cholesterol and, when taken regularly, bolsters heart health. This product is GMP-certified and is suitable for adults of any age. + +--- + +## Stimula Blood Sugar Support Ingredients + +### 1. White Mulberry Leaf +White mulberry leaf helps balance blood sugar and is very effective in ensuring it stays within the normal range. It also lowers bad cholesterol levels and minimizes or eliminates diabetic symptoms. + +### 2. Bitter Melon +Bitter melon is a native Asian herb that helps manage blood sugar by providing an active compound known as Charantin, which slows down carbohydrate digestion. It also lowers LDL cholesterol levels. + +### 3. Biotin and Chromium +These two nutrients are proven to help with blood sugar levels by stimulating insulin production and ensuring the cells of your body absorb glucose effectively. + +### 4. Berberine Extract +Berberine extract helps lower bad cholesterol and strengthens cardiovascular health as a potent anti-inflammatory ingredient aiding in fat metabolism. + +### 5. Cinnamon Bark Powder +Cinnamon bark powder is known for its ability to balance blood sugar levels and lower bad cholesterol. This contributes to a reduced risk of cardiovascular issues. + +### 6. Juniper Berry +Juniper berry is a natural antioxidant with anti-inflammatory properties, reducing the risk of hypertension, heart attacks, and atherosclerosis. It also helps in lowering bad cholesterol levels. + +--- + +## How Does Stimula Blood Sugar Support Work? +Stimula Blood Sugar Support works by lowering elevated blood sugar levels, facilitating energetic functioning and overall wellness. It also reduces the level of LDL cholesterol and alleviates or eliminates symptoms of diabetes. + +The formula is designed to ensure cells absorb glucose without complications. The synergistic effect of nutrients promotes a healthy heart, normal blood pressure, and good mood. Continuous daily use for 2-3 months is recommended for effective results. + +### Benefits of Stimula Blood Sugar Support +If taken correctly, the formula provides the following benefits: +- **Healthy Blood Sugar Levels:** Improves the function of insulin, promotes blood flow, and ensures free-flowing arteries. +- **Weight Loss:** Enhances metabolism, eliminates excess fat, and facilitates lean body mass. +- **Balanced Cholesterol Levels:** Bitter melon extract reduces LDL cholesterol and triglycerides. +- **Treats Insulin Resistance:** Enables cells to respond to insulin effectively, preventing glucose accumulation. + +--- + +## Side Effects of Stimula Blood Sugar Support +No known side effects are reported. The ingredients are 100% natural and high quality. It is made in the USA in a GMP-certified facility. Nevertheless, consult with a healthcare specialist to prevent any unexpected reactions, especially if you have health issues or are taking medications. Individuals under 18, pregnant, or lactating women should not use this supplement. + +--- + +## Stimula Blood Sugar Support Customer Reviews +Based on official website reviews, the supplement has helped users manage blood sugar levels and shed significant weight without adverse effects. It's credited for aiding cardiovascular and digestive function without any negative feedback or complaints. + +--- + +## Our Final Verdict on Stimula Blood Sugar Support +Stimula Blood Sugar Support helps control blood sugar levels naturally and effectively. It boasts a safety profile for managing high blood sugar and related heart health issues. With a 30-day money-back guarantee, it presents no significant risk to health and proves itself as a viable option for those seeking natural remedies. + +--- + +## Frequently Asked Questions + +### Is Stimula Blood Sugar Support safe? +Stimula Blood Sugar Support is manufactured in a GMP-certified facility with strict hygienic practices. It is free from harmful toxins or contaminants and composed of high-quality natural ingredients. + +### Are there any additional charges or subscriptions for Stimula Blood Sugar Support? +No, it is sold as a one-time payment with no additional charges. + +### What if Stimula Blood Sugar Support doesn't work for me? +It includes a 30-day money-back guarantee. If unsatisfied, you will get a full refund. + +### How to get the best results from Stimula Blood Sugar Support? +Consistent use of the supplement produces the most effective results. Consume its capsules daily for 2-3 months. + +### Where is Stimula Blood Sugar Support made? +It is manufactured in a GMP-certified facility in the United States. + +--- + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/extinction-dilution-cloning-for-isolation-of-virus-dpx5pm.md b/markdown-output/extinction-dilution-cloning-for-isolation-of-virus-dpx5pm.md new file mode 100644 index 0000000000000000000000000000000000000000..25e2217d936d49fb5ed91259c84d6d57c18471f8 --- /dev/null +++ b/markdown-output/extinction-dilution-cloning-for-isolation-of-virus-dpx5pm.md @@ -0,0 +1,100 @@ +```markdown +# Goal/Experiment: +Extinction Dilution Cloning for Isolation of Viruses Infecting Protists + +## Authors +Keizo Nagasaki and Gunnar Bratbak + +## Abstract +This is a detailed outline of the extinction dilution method. For additional information, please refer to Nagasaki, K., and G. Bratbak. 2010. Isolation of viruses infecting photosynthetic and nonphotosynthetic protists, p. 92-101. In S. W. Wilhelm, M. G. Weinbauer, and C. A. Suttle [eds.], Manual of Aquatic Viral Ecology. ASLO. Published online: 02 Dec 2015. + +## Guidelines + +### List of Materials and Reagents +- Bucket or water sampler (e.g., Van Dorn water sampler) +- Sediment sampler (e.g., Ekman bottom grab sampler) +- Centrifuge and centrifuge tubes (e.g., 15- or 50-mL Falcon tubes) +- Sterilized filter holder and membrane filters (0.2, 0.22, 0.45 or 0.8µm) +- Vacuum pump +- Sterile medium for algal culture (e.g., SWM-III, f/2) +- Sterile test tubes, pipette with tips, and vortexer (for serial dilution) +- 24-well and 96-well cell culture plates (e.g., Falcon) +- 8-channel pipette and sterile reservoir tray (for extinction dilution procedure) +- Plastic tape (for sealing culture plates to avoid drying) +- Incubator (with light and temperature control) +- Inverted microscope +- Refrigerator, freezer, deep freezer, or liquid nitrogen container + +### Scientific Terms and Reagents +- **Van Dorn Water Sampler**: A device used for collecting water samples from specific depths in a water column. +- **Ekman Bottom Grab Sampler**: A device used to collect sediment samples from the bottom of a water body. +- **Falcon Tube**: A brand of plastic conical tubes commonly used for centrifugation and storage of samples in biological research. +- **Membrane Filter (0.2, 0.22, 0.45 or 0.8µm)**: Filters used to separate particles in a sample by size, useful for sterilizing solutions or isolating microorganisms. +- **Vacuum Pump**: Equipment used to create a vacuum for filtration processes. +- **SWM-III, f/2**: Sterile media formulas used for the growth of algal cultures. + +### Cloning and Maintenance of Microalgal Viruses +1. **Detection and Recognition**: + - Look for signs of decay in the tested host algal culture (e.g., bleaching, decrease in chlorophyll a fluorescence, clearing). + - Clone the lytic factor immediately upon detection. +2. **Serial Dilution**: + - Dilute the culture lysate with sterile liquid medium. + - Perform 10-fold serial dilution steps. + - Add 100 µL aliquots to wells with exponentially growing host culture in a 96-well plate. + - Incubate under conditions optimal for host growth. + - Transfer lysates from wells showing lysis to new host cultures and repeat the procedure. + +3. **Sterilization**: + - Filter the lysate in the most diluted wells using a 0.1-µm (for RNA/DNA viruses) or 0.2-µm (for large DNA viruses) pore size filter. + - Transfer into exponentially growing host culture. + - Remove cell debris by low-speed centrifugation to use as clonal pathogen suspension. + +4. **Virus Stability**: + - Maintain viruses at 4°C in the dark to limit titer decrease. + - Consider cryopreservation methods (e.g., -196°C in 10-20% DMSO, or -70°C in 10-20% sucrose). + +### Filtration Comments +- Filtration through 0.2-0.45 µm filters and then through 0.1-0.2 µm filters is common to isolate viruses (see Table 1). +- Large viruses like Mimivirus (~750 nm) might be lost during filtration through small pore size filters. +- The extinction dilution method isolates viruses with strong lytic activity, potentially losing less dominant viruses. +- Use low virus doses to avoid unnoticed lysis and weak infections. + +### Considerations for Host Cultures +- Host cultures may vary in susceptibility based on life cycle stages and ploidy levels. +- Some cultures may show different lytic activities. +- Researchers must balance natural ecosystem virus properties and extreme host-virus systems in culture. + +Brown algal phaeoviruses reproduce only in host spores or gametes and not in vegetative cells. + +## Protocol + +### Preparation of Dilution Series +1. **Step 1**: Prepare 9 tubes (#1-#9) with 4.5 mL medium. +2. **Step 2**: Add 500 µL virus filtrate to tube #1 and vortex. +3. **Step 3**: Transfer 500 µL from tube #1 to tube #2, vortex. +4. **Step 4**: Repeat for tubes #2 to #8. + +### Preparation of Cell Culture Plates +5. **Step 5**: Pour vigorously growing algal host culture into the reservoir tray. +6. **Step 6**: Fill pipetter with 150 µL culture and add to wells in lines 1-9 of a 96-well plate. +7. **Step 7**: Empty the tray. +8. **Step 8**: Pour dilution tube #9 into the tray. +9. **Step 9**: Fill pipetter with 100 µL, add to wells in line 9. +10. **Step 10**: Empty the tray. +11. **Step 11**: Repeat steps 7-8 for tubes #8-#1. + +### Sealing and Incubation +12. **Step 12**: Seal plates with plastic tape. +13. **Step 13**: Incubate under appropriate conditions. + +### Inspection +14. **Step 14**: Use an inverted microscope to inspect plates regularly. +15. **Step 15**: Mark wells showing lysis and continue daily inspections until lysis ceases. +16. **Step 16**: Prepare a second extinction dilution with the most-diluted virus. +17. **Step 17**: Propagate virus clones from the most-diluted well in a larger volume and store appropriately. + +## Warnings +- Conduct all work in a clean bench to avoid contamination. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/extracellular-and-intracellular-ph-measurements-in-bu4knyuw.md b/markdown-output/extracellular-and-intracellular-ph-measurements-in-bu4knyuw.md new file mode 100644 index 0000000000000000000000000000000000000000..cd8cae607b6c6727020fc50c656fb36e045ccda8 --- /dev/null +++ b/markdown-output/extracellular-and-intracellular-ph-measurements-in-bu4knyuw.md @@ -0,0 +1,80 @@ +```markdown +# Goal/Experiment: +To measure extracellular and intracellular pH in the respiratory tract of mice in vivo and understand the cellular responses after Influenza A Virus (IAV) infection. + +# Extracellular and Intracellular pH Measurements in Mice Respiratory Tract in vivo V.2 + +**Authors**: Faten A Okda1, Scott Perry1, Richard Webby1, Charles Russell1 +**Affiliation**: 1. St. Jude Children’s Research Hospital, Memphis, TN. +**DOI**: [dx.doi.org/10.17504/protocols.io.bu4knyuw](https://dx.doi.org/10.17504/protocols.io.bu4knyuw) +**Created**: May 17, 2021 +**Last Modified**: May 17, 2021 + +## Abstract +This study explores the measurement of extracellular and intracellular pH in the respiratory tract of mice to better understand cellular responses after Influenza A Virus (IAV) infection. A fiber-optic pH meter is used for in vivo pH sensing. Measurements were performed before and after infection with different strains of the virus to analyze local extracellular pH and intracellular pH of immune cells. + +## Materials + +- **Microfiber optic pH meter**: Needle-type housed pH Microsensor (PreSens Precision Sensing GmbH, Regensburg, Germany) +- **Software**: PreSens Precision Sensing software (GmbH, Regensburg, Germany) +- **Buffers**: pH 4, 5, 6, 7, 8, 9 buffer solutions from isotonic solutions containing NaH₂PO₄, Na₂HPO₄, NaCl +- **Anesthetic**: Isoflurane +- **Disinfectant**: Acrylan +- **Animal Model**: Six-week-old female DBA/2J mice +- **Surgical tools**: Sterile surgical scissors and scalpel +- **Consumables**: Petri dishes, 50 mL conical tubes, 100 mm Petri dishes, 1 mL DNase solution, 40 μM strainer, cell counter, and trypan blue +- **Fluorescent dyes and antibodies**: + - pHrodo™ Red AM (Invitrogen) + - Oregon Green™ 488 + - LysoSensor Blue (Invitrogen) + - EE1 antibody (Abcam) + - RAB7 antibody (Abcam) + - DAPI +- **Software**: GraphPad Prism 9 (GraphPad, San Diego, CA) + +## Methods + +### 1. Extracellular pH Measurements in Live Mice +1. **Calibration of the pH Microsensor**: + - The fiber-optic microsensor is calibrated with pH buffer solutions from 4 to 9. +2. **Selection of Anesthetic**: + - Ten healthy mice are anesthetized with different anesthetics to determine optimal conditions for pH measurement. +3. **Insertion and Measurement**: + - The sensor is inserted into the nasal cavity, soft palate, and trachea under anesthesia. +4. **Infection and Monitoring**: + - Female DBA/2J mice infected with IAV or control solution; measurements taken at multiple time points post-infection. + +### 2. Intracellular pH Dyes Preparation and Calibration +2.1. Use pH-sensitive dyes and intracellular pH calibration buffer kits as per manufacturer’s instructions. + +### 3. Single-cell Preparations of Live, Primary Mouse Nasal, Soft Palate, and Trachea Epithelial Cells +3.1. **Tissue Preparation**: + - Mice are euthanized, tissues are dissected, processed, and placed in appropriate media. +3.2. **Processing**: + - Tissues are processed into single cells using pronase solution and centrifugation. +3.3. **Staining**: + - Cells are stained with pH-sensitive dyes and sorted using flow cytometry. + +### 4. Optimization of Intracellular pH Measurement by Flow Cytometry +4.1. Cells are stained using a combination of antibodies, pH markers, and isotype controls and incubated for 30 minutes. +4.2. Cells are washed and sorted by flow cytometry. +4.3. Live and dead cells are identified, and cells expressing specific markers are selected. + +### 5. Intracellular pH Staining and Measurements +5.1. **Cell Staining**: + - Stain 200,000–500,000 cells with pH-sensitive dyes and PowerLoad™ Concentrate. +5.2. **Cell Washing**: + - Wash cells and analyze using flow cytometry. +5.3. **Quantification**: + - Intracellular pH quantified using calibration solutions. + +### 6. Calibration and Measurement of Intracellular pH +6.1. **Using the Intracellular pH Calibration Buffer Kit**: + - Buffers of pH 4.5–7.5 used to create standard curves. +6.2. **Fluorescence Intensity Analysis**: + - Measure mean fluorescence intensity for calibration. +6.3. **Statistical Analysis**: + - Data analyzed using GraphPad Prism software for statistical significance. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/extracorporeal-membrane-oxygenation-meta-analysis-bb3tiqnn.md b/markdown-output/extracorporeal-membrane-oxygenation-meta-analysis-bb3tiqnn.md new file mode 100644 index 0000000000000000000000000000000000000000..957a434d1576cec5c500c8c132bc36d388e74539 --- /dev/null +++ b/markdown-output/extracorporeal-membrane-oxygenation-meta-analysis-bb3tiqnn.md @@ -0,0 +1,98 @@ +```markdown +## Goal/Experiment: +The goal of this study is to analyze time-to-event data of all-cause mortality on extracorporeal membrane oxygenation (ECMO) in adult patients with cardiopulmonary disease by conducting a meta-analysis. + +# Extracorporeal Membrane Oxygenation Meta-Analysis of Time-to-Event Data in Cardiopulmonary Disease in Adults + +**Authors**: Hynusuk Frank Roh, Chang-Guk Kim, Soon-Ho Chon, Jung Mogg Kim +**Date**: Feb 05, 2020 + +### ABSTRACT + +**Purpose**: +Time-to-event data of hazard ratios were used to generate a forest plot of all-cause mortality on extracorporeal membrane oxygenation (ECMO) in both overall and individual indications of cardiopulmonary disease in adults. + +**Materials and Methods**: +A systematic search was conducted in PubMed from 1975 to 2018. Among 4,121 articles, a total of 34 clinical reports comprising 20,610 patients (1,631 of whom underwent ECMO and 18,979 who did not undergo ECMO) met the inclusion criteria. + +**Results**: +The pooled hazard ratio of 1.2828 (95% confidence interval: 0.9649, 1.7054) suggests that the ECMO group was not significantly associated with a reduction in mortality compared to the no-ECMO group in overall ECMO indications. Subgroup analysis showed a significantly improved patient survival in respiratory failure with 0.6308 (0.5037, 0.7900), and a worse patient survival after lung transplantation with 2.0600 (1.6987, 2.4981), in bridging to heart transplantation with 1.4212 (1.0309, 1.9592), and after heart transplantation with 5.6365 (1.7569, 18.0827). + +**Conclusions**: +Most of the included studies are retrospective, which diminishes the significance of the findings due to a higher risk of selection bias demonstrated by the allocation of ECMO based on the patient's severity condition. + +### INTRODUCTION + +Extracorporeal membrane oxygenation (ECMO) is a method used to provide prolonged cardiac and respiratory support to persons whose heart and lungs are unable to provide an adequate amount of gas exchange or perfusion to sustain life. It is most commonly indicated in patients with cardiac or respiratory failure that is unresponsive to conventional management. + +### MATERIAL AND METHODS + +#### Identification of Studies +A systematic electronic search for articles on the US National Library of Medicine's PubMed database was conducted. Keywords included ECMO-related terms alongside mortality-related terms. + +#### Eligibility Criteria +All relevant articles were evaluated using specified inclusion and exclusion criteria, focusing on studies published in English from 1975 to 2018. Specifically, consistency with ECMO status and HR with a 95% CI or sufficient data for estimating these were required. + +#### Classification of ECMO Indications +- **Respiratory Failure**: Acute respiratory disease syndrome (ARDS) and hypercapnic respiratory failure were grouped under "respiratory failure." +- **Bridge to LTx, Intra-LTx, Post-LTx**: Classified under lung transplantation indications. +- **Bridge to HTx and VAD**: Heart transplantation and ventricular assist device groups. +- **E-CPR**: Extracorporeal cardiopulmonary resuscitation was compared with conventional cardiopulmonary resuscitation. +- **Others**: Instances where ECMO was not specified in predominant references. + +#### Data Extraction and Quality Assessment +Information such as study type, year of publication, and various metrics about ECMO and no-ECMO groups was extracted. The Newcastle-Ottawa quality assessment scale was used to assess the studies. + +#### Statistical Information +Meta-analyses were conducted using the package `meta`, examining fixed and random-effect models. Statistical significance was considered at a p-value < 0.05. + +### RESULTS + +#### Study Characteristics +34 studies qualified that included 20,610 patients in total. + +#### Literature Search +A total of 34 articles were included in the final analysis. + +#### Heterogeneities and Random-Effects Model +The heterogeneity of studies was assessed and appropriate models were selected, with a focus on explaining the interaction between ECMO and patient mortality over time. + +#### Treatment Outcomes +- **Overall Mortality**: No significant difference between ECMO and no-ECMO groups. +- **Respiratory Failure** (Subgroup): Realization of better benefits with ECMO compared to the traditional approach. +- Other specific interventions (e.g., LTx, HTx) indicated higher risks when using ECMO. + +#### Figure Legends +![](image1.png) +*Figure 1. Schematic representation of the study selection process.* + +![](image2.png) +*Figure 2. Bias assessments of RCTs. +, -, and blank space denote lower risk, high risk, and unclear risk, respectively, for the risk judgement.* + +![](image3.png) +*Figure 3A. A forest plot on the all-cause mortality of ECMO use in cardiopulmonary disease in adults. This forest plot with subgroup analyses considers an aspect of mortality for both overall and individual indications as follows: (1) Respiratory failure, (2) Bridge to LTx (lung transplantation), (3) Intra-LTx, (4) Post-LTx, (5) Cardiogenic shock.* + +![](image4.png) +*Figure 3B. Continued: (6) Bridge to HTx (heart transplantation), (7) Post-HTx, (8) Bridge to VAD (ventricular assist device), (9) E-CPR (extracorporeal cardiopulmonary resuscitation), and (10) Others.* + +![](image5.png) +*Figure 4. A funnel plot. It is used to evaluate the publication bias on the hazard ratio in relation with Figure 3.* + +### CONCLUSIONS +Based on the time-to-event data, overall, ECMO provides no advantages over alternative therapy with respect to patient mortality. The results of subgroup analyses revealed a better outcome for patients with ECMO employed for respiratory failure and a worse outcome for those where ECMO was used in post-LTx, bridge to HTx, and post-HTx settings. + +### Table + +Characteristics of studies included for systematic review and meta-analysis: + +| Study | TE | seTE | Hazard Ratio | HR | 95% CI (fixed) | 95% CI (random) | Weight (fixed) | Weight (random) | +|-----------------|-----|-------|--------------|-----|----------------|----------------|----------------|-----------------| +| Combes 2018 | ... | ... | ... | ... | ... | ... | ... | ... | +| ... | ... | ... | ... | ... | ... | ... | ... | ... | + +### Supporting Information + +- **Supplementary File 1.R**: R script for a forest plot with subgroup analyses, a funnel plot, and Pearson correlation. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/fascial-manipulation-for-musculoskeletal-disorders-cj4ruqv6.md b/markdown-output/fascial-manipulation-for-musculoskeletal-disorders-cj4ruqv6.md new file mode 100644 index 0000000000000000000000000000000000000000..b497c3c04c0822258b4f51ca03162e5fd4a951f4 --- /dev/null +++ b/markdown-output/fascial-manipulation-for-musculoskeletal-disorders-cj4ruqv6.md @@ -0,0 +1,179 @@ +```markdown +Goal/Experiment: +To synthesize all available literature on the effects/mechanisms of myofascial manipulation interventions and adverse events, to assess knowledge gaps in understanding optimal approaches, and to elucidate therapeutic mechanisms and select indications. + +# Fascial Manipulation® for Musculoskeletal Disorders: A Scoping Review + +## DOI +[dx.doi.org/10.17504/protocols.io.dm6gpjz68gzp/v1](https://dx.doi.org/10.17504/protocols.io.dm6gpjz68gzp/v1) + +**Authors:** +- Yuichi Isaji +- Daisuke Sasaki +- Yusuke Kon +- Takashi Kitagawa + +**Affiliations:** +1. Department of Rehabilitation, Anshin Clinic, Kobe, Japan. +2. Department of Rehabilitation, Iwami Medical Clinic, Masuda, Japan. +3. Department of Rehabilitation, Kugayama Hospital, Setagaya, Japan. +4. Department of Physical Therapy, School of Health Sciences, Shinshu University, Matsumoto, Japan. + +## Abstract + +**Objective:** The purpose of this scoping review is to synthesize all available literature on the effects/mechanisms of myofascial manipulation interventions and adverse events, to assess knowledge gaps in understanding optimal approaches, and to elucidate therapeutic mechanisms and select indications. + +**Introduction:** Fascial manipulation® (FM) is one of the techniques developed in recent years as a manual therapy for musculoskeletal disorders. Systematic reviews have shown the efficacy of FM for patients with musculoskeletal pain, but mechanisms and adverse events have not been thoroughly examined. + +## Inclusion / Exclusion Criteria + +**Inclusion Criteria:** +- Patients with musculoskeletal disorders with pain and dysfunction. +- Outcomes will be pain and physical function. +- Interventional trials, observational studies, or case reports without restrictions on region, race, gender, or language of the original paper. + +**Exclusion Criteria:** +- Studies involving any myofascial intervention other than FM. +- Subjects other than musculoskeletal disorders such as stroke and systematic reviews. + +## Methods + +A systematic search of PubMed, CINAHL, Web of Science, and PEDro databases using the keywords "Fascial manipulation®," "musculoskeletal disorders," and "myofascial pain" during December 2022. In the first screening step, two independent reviewers will review all titles and abstracts to exclude irrelevant articles. In the second screening step, two independent reviewers will review all full texts to exclude irrelevant articles. Outcomes will be focused on pain and physical function. + +**Search Strategy:** + +- **PubMed Search Strategy:** + ```plaintext + (((((((((((("Musculoskeletal dis*"[Title/Abstract]) OR ("Musculoskeletal dis*"[Text Word])) OR ("Musculoskeletal dis*"[MeSH Terms])) OR ("Myofascial pain"[Title/Abstract])) OR ("Myofascial pain"[Text Word])) OR ("Musculoskeletal pain"[Title/Abstract])) OR ("Musculoskeletal pain"[MeSH Terms])) OR (Pain[Title/Abstract])) OR (Pain[Text Word])) OR (Pain[MeSH Terms])) OR (Disability[Title/Abstract])) OR (Disability[Text Word])) AND (((((("Fascial manipulation®"[Title/Abstract])) OR ("Fascial manipulation®"[Text Word])) OR ("Fascial manipulation®"[MeSH Terms])) OR ("Fascial manipulation®"[Text Word])) OR ("Fascial manipulation"[Title/Abstract])) OR ("Fascial manipulation"[Text Word])) OR (FM[Title/Abstract]) + ``` + +- **Web of Science Search Strategy:** + ```plaintext + ("Musculoskeletal dis*" OR "Myofascial pain" OR "Musculoskeletal pain" OR Pain OR Disability AND ("Fascial manipulation®" OR "Fascial manipulation" OR FM)) + ``` + +- **CINAHL Search Strategy:** + ```plaintext + ((((((((((((("Musculoskeletal dis*" OR AB "Musculoskeletal dis*")) OR ("Musculoskeletal dis*")) OR ((MH "Musculoskeletal dis*")) OR ("Musculoskeletal pain" OR AB "Musculoskeletal pain")) OR ("Myofascial pain")) OR ((TI "Musculoskeletal pain")) OR ((MH "Musculoskeletal pain")) OR ((TI Pain OR AB Pain)) OR ((MH Pain+)) OR ((TI Disability OR AB Disability))) OR (Disability)) AND (((((((TI "Fascial manipulation®" OR AB "Fascial manipulation®")) OR ((TI "Fascial manipulation®")) OR (TI "Fascial manipulation") OR AB "Fascial manipulation")) OR ("Fascial manipulation"))) OR ((TI FM OR AB FM))) + ``` + +- **Physiotherapy Evidence Database (PEDro) Search Strategy:** + ```plaintext + Title and abstract: Fascial manipulation + ``` + +## Eligibility Criteria + +**Inclusion:** +- Patients with musculoskeletal disorders and pain (e.g., low back pain, neck pain, knee pain, chronic ankle instability, sacroiliac joint dysfunction, tendonitis, tendinosis, and tendinopathy). + +**Exclusion:** +- Any myofascial intervention other than FM and subjects other than musculoskeletal disorders such as stroke and systematic reviews. + +## Concept + +- **Patients:** Patients with musculoskeletal disorders with pain and dysfunction. +- **Exposure:** Fascial manipulation® [Stecco method]. +- **Outcome:** Change in pain (e.g., Visual Analog Scale, Numerical Rating Scale), disability (e.g., Knee Injury Osteoarthritis Outcome Score, Oswestry Disability Index, Harris Hip Score, Disability of The Arm, Shoulder, and Hand), quality of life (e.g., SF-36: Mos Short-Form 36-Item Health. Survey), range of motion, muscle strength. + +## Context + +There is no limitation on location, race, language, or gender. + +## Types of Sources + +- Interventional trials and observational studies (including exploratory studies). + +## Methods + +This protocol was developed based on PRISMA-P. The proposed scoping review will also be conducted in accordance with the JBI methodology for scoping reviews. + +## Study/Source of Evidence Selection + +After searching, all identified citations will be collated and uploaded to Rayyan (Qatar Computing Research Institute, Ar Rayyan, Qatar) to eliminate duplicates. In the pilot test, the title and abstract will be reviewed by two independent reviewers and evaluated against the criteria for inclusion in the review. Potentially relevant references will be retrieved in full text and details will be incorporated into Rayyan. Full text of selected citations will be evaluated in detail against the inclusion criteria by two independent reviewers. Reasons for excluding full-text evidence references that do not meet the inclusion criteria will be recorded and reported. Disagreements among reviewers at each stage of the selection process will be discussed or resolved with additional reviewers. Results of the search and study incorporation process will be reported in full and in line with PRISMA-2020 flow diagram. + +## Data Extraction + +More than two independent reviewers will use a data extraction tool they created to extract data from the publications included in the scoping review. Specific information about the participants, context, research techniques, and significant findings related to the review questions will all be included in the retrieved data. + +## Data Analysis and Presentation + +Data is presented in figures, tables, and maps in categories such as FM intervention type, study population (and appropriate sample size), intervention duration, objectives, the methodology employed, key findings (established evidence), and research gaps. + +## Acknowledgments + +None. + +## Funding + +None. + +## Conflicts of Interest + +None. + +## References + +1. Japan Fascial manipulation Association [Internet] Tokyo [cited 2022 September 6] Available from: [https://fascialmanipulation-japan.com/](https://fascialmanipulation-japan.com/) + +2. Stecco C. and Day J.A., 2010. The fascial manipulation technique and its biomechanical model: a guide to the human fascial system. International journal of therapeutic massage & bodywork, 3(1),38. + +3. Stecco, L., Basmajian, J.V. and Day, J.A., 2004. Fascial manipulation for musculoskeletal pain, 123-130. Padova: Piccin. + +4. Day, J.A., Stecco, C. and Stecco, A., 2009. Application of Fascial Manipulation® technique in chronic shoulder pain–Anatomical basis and clinical implications. Journal of bodywork and movement therapies, 13(2),128-135. + +5. Ercole, B., Antonio, S., Ann, D.J. and Stecco, C., 2010. How much time is required to modify a fascial fibrosis?. Journal of bodywork and movement therapies, 14(4),318-325. + +6. Cheatham, S.W., Lee, M., Cain, M. and Baker, R., 2016. The efficacy of instrument-assisted soft tissue mobilization: a systematic review. The Journal of the Canadian Chiropractic Association, 60(3),200. + +7. Schleip, R., Klingler, W. and Lehmann-Horn, F., 2006. Fascia is able to contract in a smooth muscle-like manner and thereby influence musculoskeletal mechanics. Journal of Biomechanics, 39(1), 488. + +8. Langevin HM. Fascia Mobility, Proprioception, and Myofascial Pain. Life (Basel). 2021 Jul 8;11(7):668. doi: 10.3390/life11070668. PMID: 34357040; PMCID: PMC8304470. + +9. Arumugam K, Harikesavan K. Effectiveness of fascial manipulation on pain and disability in musculoskeletal conditions. A systematic review. J Bodyw Mov Ther. 2021 Jan;25:230-239. doi: 10.1016/j.jbmt.2020.11.005. Epub 2020 Nov 11. PMID: 33714051. + +10. Moher D, Shamseer L, Clarke M, Ghersi D, Liberati A, Petticrew M, et al. Preferred reporting items for systematic review and meta-analysis protocols (PRISMA-P) 2015 statement. Rev Esp Nutr Humana y Diet. 2016;20(2):148–60. + +11. Ouzzani M, Hammady H, Fedorowicz Z, Elmagarmid A. Rayyan-a web and mobile app for systematic reviews. Syst Rev [Internet]. 2016;5(1):1–10. Available from: [http://dx.doi.org/10.1186/s13643-016-0384-4](http://dx.doi.org/10.1186/s13643-016-0384-4) + +12. Page MJ, McKenzie JE, Bossuyt PM, et al. Updating guidance for reporting systematic reviews: development of the PRISMA 2020 statement. J Clin Epidemiol 2021; 134: 103-12. doi:10.1016/j.jclinepi.2021.02.003 + +## Appendices + +### Appendix I: Search Strategy + +**PubMed Search Strategy:** +```plaintext +((((((((((((("Musculoskeletal dis*"[Title/Abstract]) OR ("Musculoskeletal dis*"[Text Word])) OR ("Musculoskeletal dis*"[MeSH Terms])) OR ("Myofascial pain"[Title/Abstract])) OR ("Myofascial pain"[Text Word])) OR ("Musculoskeletal pain"[Title/Abstract])) OR ("Musculoskeletal pain"[MeSH Terms])) OR (Pain[Title/Abstract])) OR (Pain[Text Word])) OR (Pain[MeSH Terms])) OR (Disability[Title/Abstract])) OR (Disability[Text Word])) AND (((((("Fascial manipulation®"[Title/Abstract])) OR ("Fascial manipulation®"[Text Word])) OR ("Fascial manipulation®"[MeSH Terms])) OR ("Fascial manipulation®"[Text Word])) OR ("Fascial manipulation"[Title/Abstract])) OR ("Fascial manipulation"[Text Word])) OR (FM[Title/Abstract]) +``` + +**Web of Science Search Strategy:** +```plaintext +("Musculoskeletal dis*" OR "Myofascial pain" OR "Musculoskeletal pain" OR Pain OR Disability AND ("Fascial manipulation®" OR "Fascial manipulation" OR FM)) +``` + +**CINAHL Search Strategy:** +```plaintext +(((("Musculoskeletal dis*" OR AB "Musculoskeletal dis*")) OR ("Musculoskeletal dis*")) OR ((MH "Musculoskeletal dis*")) OR ("Musculoskeletal pain" OR AB "Musculoskeletal pain")) OR ("Myofascial pain")) OR ((TI "Musculoskeletal pain")) OR ((MH "Musculoskeletal pain")) OR ((TI Pain OR AB Pain)) OR ((MH Pain+)) OR ((TI Disability OR AB Disability))) OR (Disability) AND ((TI "Fascial manipulation®" OR AB "Fascial manipulation®")) OR (("Fascial manipulation®"[Title/Abstract])) OR ("Fascial manipulation®"[Text Word])) OR ("Fascial manipulation"[Title/Abstract]) OR ("Fascial manipulation"[Text Word]) OR (FM[Title/Abstract]) +``` + +**Physiotherapy Evidence Database (PEDro) Search Strategy:** +```plaintext +Title and abstract: Fascial Manipulation +``` + +### Appendix II: Data Extraction Instrument + +**Data Extraction Form:** +1. Author(s) +2. Year of publication +3. Origin/Country +4. Purpose/Goals +5. Study population +6. Sample size +7. Type of intervention +8. Outcomes specifics +9. Key findings + +**endofoutput** +``` diff --git a/markdown-output/field-survey-of-the-population-dynamics-of-common-mmyc47w.md b/markdown-output/field-survey-of-the-population-dynamics-of-common-mmyc47w.md new file mode 100644 index 0000000000000000000000000000000000000000..61925266b9c08ad92f79e98b2f608cf5cae87253 --- /dev/null +++ b/markdown-output/field-survey-of-the-population-dynamics-of-common-mmyc47w.md @@ -0,0 +1,143 @@ +```markdown +# Goal/Experiment: +The aim of this experiment is to monitor the population dynamics of common ragweed (Ambrosia artemisiifolia) and to estimate all vital rates of a population, including the establishment of plants, plant survival, plant growth, reproduction, and seed survival. This survey will aid in understanding how these rates relate to plant density and individual plant size and will assist in assessing the efficacy of management interventions for this invasive species. + +## Field Survey of the Population Dynamics of Common Ragweed (Ambrosia artemisiifolia) Version 2 + +**Authors:** Suzanne T. E. Lommen, Caspar A. Hallmann, Bruno Chauvel, Melinda Leitsch-Vitalos, Gerhard Karrer, Peter Tóth, Heinz Müller-Schärer, Eelke Jongejans + +### Abstract +**Background:** Common ragweed, *Ambrosia artemisiifolia* L., is a significant invasive plant globally. Its high seed production and allergenic pollen contribute to its spread. Understanding the spatio-temporal variation in population growth of this annual species is essential for evaluating management strategies. The protocol enables detailed demographic surveys over multiple years in various habitats across a wide geographic range and time span. + +**Aim:** This field survey protocol monitors the population dynamics of common ragweed across Europe. It estimates vital rates such as plant establishment, survival, growth, reproduction, and seed survival while analyzing their relation to plant density and size. The protocol targets unmanaged field populations for baseline data but can be adapted for managed populations. + +**Methods:** +- **Plot establishment:** 0.25m² plots in common ragweed populations. +- **Monitoring:** Early growing season monitoring and seed set reassessment. Soil samples and adjacent reproductive plants are analyzed in the lab. +- **Repetition:** Same plots monitored for multiple years to estimate seed survival in the soil seed bank. +- **Replication:** Multiple years and sites; additional environmental factors measured. + +### Protocols Provided +1. List of materials +2. Site selection +3. Survey setup +4. Monitoring +5. Plant, soil, and seed sampling +6. Lab analyses (plant biomass, soil seed bank, soil texture and content, cleaning of aerial seed samples) + +### Record Forms Provided +1. Registration of site meta-data +2. Measures taken in the field and lab in formats for print and excel for data digitization + +### Optimisation +The protocol gathered data from over 50 populations in Europe (2014-2016) under the EU-funded COST Action 'SMARTER'. The current protocol is an improved version based on these field experiences. + +### Guidelines +- The study focuses on common ragweed populations established on the phenology of the species in Europe. +- Vital rates include plant establishment, survival, growth, reproduction, and seed survival. +- Analysis is based on unmanaged populations to create a baseline, with options for managed populations. +- The protocol assumes no seed movement, immigration, or emigration. +- Monitoring uses permanent 0.25 m² plots representing plant population densities. + +**Time Estimates per Year per Population:** +- Site setup and initial data collection: 1 day +- Field monitoring post-establishment: 1 day +- Seed set monitoring: 1 day +- Additional field census: 0.5-1 day +- Lab analyses: 1-2 days +- Data entry: 0.5 day + +### Before Start +- Read the detailed pdf 'Protocols...'. +- Reserve time for site selection and obtaining permission. +- Developed as a 3-year field survey under the EU COST Action 'SMARTER'. +- Joint study ended; no new data/materials are accepted currently. + +### Materials +- Basic field and lab equipment for ecological research. + +### Protocol + +#### Select a Suitable Population +**Step 1:** +- **Summary:** Ensure the population meets criteria (sufficient plant numbers and surface covered). +- **Details:** Refer to Protocol Selection & Registration Population, 'Checklist for Suitability of Site'. + +#### Registration of the Population (Record Meta-data of the Site, Only Once Per Population) +**Step 2:** +- **Summary:** Record site meta-data. +- **Details:** Refer to Protocol Selection & Registration Population, 'Registration of Populations'. +- **Record Form:** Form_Ambrosia_Population_Record (Printable version) +- **Digital Form:** DigitalForm_Ambrosia_Population_Record (Excel for digitization) + +#### Setup the Site +**Step 3:** +- **Summary:** Establish permanent plots, select soil sampling sites, and define a photo reference point. +- **Details:** Refer to Protocol Setup Site. +- **Record Forms:** Form_1_site-map, Form_2_plot-map +- **Additional Forms:** Field_sheet_1_Checklist-field-work, Field_sheet_2_Legend-to-forms + +#### Census Establishment (Census e) +**Step 4:** +- **Summary:** Monitor plots and mark individual plants for measurement. +- **Timing:** Immediately after site setup when seedlings are still small. +- **Details:** Refer to Protocol Census Establishment. +- **Record Forms:** Form_3_monitor-study-area, Form_4_monitor-plot (14+ copies required) +- **Additional Forms:** Field_sheet_1_Checklist-field-work, Field_sheet_2_Legend-to-forms, Form_2_Plot-map + +#### Optional: Extra Censi (Census x1 and x2) +**Step 5:** +- **Summary:** Additional plot monitoring at monthly intervals. +- **Details:** Refer to Protocol Census Extra. +- **Record Forms:** Form_3_monitor-study-area, Form_4_monitor-plot (14+ copies required) +- **Additional Forms:** Field_sheet_1_Checklist-field-work, Field_sheet_2_Legend-to-forms, Form_2_Plot-map + +#### Census Seed Set +**Step 6:** +- **Summary:** Reassess plots, sample soil for seed bank estimation, and sample 21 mature plants for reproduction estimates. +- **Timing:** Execute around seed set. +- **Details:** Refer to Protocol Census Seed set. +- **Record Forms:** Form_3_monitor-study-area, Form_4_monitor-plot (14+ copies required), Form_5_plants-outside-plots +- **Additional Forms:** Field_sheet_1_Checklist-field-work, Field_sheet_2_Legend-to-forms, Form_2_Plot-map + +#### Optional: Collection of Aerial Seeds +**Step 7:** +- **Summary:** Collect seeds directly from plants for future research. +- **Timing:** During Census Seed set or later. +- **Details:** Refer to Protocol Aerial Seed Collection. + +#### Optional: Extra Census (Census x4) +**Step 8:** +- **Summary:** Additional plot monitoring after the Census Seed set. +- **Details:** Refer to Protocol Census Extra. +- **Record Forms:** Form_3_monitor-study-area, Form_4_monitor-plot (14+ copies required) +- **Additional Forms:** Field_sheet_1_Checklist-field-work, Field_sheet_2_Legend-to-forms, Form_2_Plot-map + +#### Lab Analyses of Samples +**Step 9:** +- **Summary:** Process, measure, and store field samples. +- **Protocols:** + 1. Analyse plant samples - Assess biomass + 2. Analyse plot soil samples - Assess seed soil bank + 3. Analyse extra soil sample - Assess soil content and texture + 4. Process and send seed samples +- **Details:** Refer to Protocol Lab Analysis Samples. +- **Record Forms:** Form_6_soil-samples +- **Additional Forms:** Field_sheet_2_Legend-to-forms + +#### Digitize Data from Step 3-9 +**Step 10:** +- **Summary:** Digitize the collected data. +- **Details:** Data collected should be digitized in the excel file 'DigitalRecordForms...'. +- **Digital Form:** Excel file 'DigitalRecordForms...' + +#### Next Year +**Step 11:** +- Repeat steps 3-10 each year of the study. + +### Warnings +- The pollen of the study species is highly allergenic. Use personal protection during monitoring. +- The species is invasive and under quarantine in some regions. Avoid dispersing seeds post-monitoring and comply with laws for plant/seed transport. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/fin-seq-frozen-immunolabeled-nuclei-sequencing-zxbf7in.md b/markdown-output/fin-seq-frozen-immunolabeled-nuclei-sequencing-zxbf7in.md new file mode 100644 index 0000000000000000000000000000000000000000..738005a2cfd1f5d0c2fc9e5cf4c40af94e3ccea4 --- /dev/null +++ b/markdown-output/fin-seq-frozen-immunolabeled-nuclei-sequencing-zxbf7in.md @@ -0,0 +1,157 @@ +```markdown +# Goal/Experiment: +To profile specific cell types in frozen archived human central nervous system (CNS) tissue using Frozen Immunolabeled Nuclei Sequencing (FIN-Seq). + +# FIN-Seq (Frozen Immunolabeled Nuclei Sequencing) + +**Authors:** +- Ryoji Amamoto1 +- Emanuela Zuccaro2 +- Paola Arlotta3 +- Constance L. Cepko1 + +**Affiliations:** +1. Harvard Medical School +2. University of Padova +3. Harvard University + +**DOI:** [10.17504/protocols.io.zxbf7in](dx.doi.org/10.17504/protocols.io.zxbf7in) + +--- + +**Abstract:** +Thousands of frozen, archived tissues from postmortem human central nervous system (CNS) are currently available in brain banks. Single-cell and single-nucleus technologies have begun to elucidate cellular diversity in the human CNS. Single-cell and single-nucleus RNA profiling provide one method to decipher this heterogeneity. FIN-Seq is an alternative approach for profiling isolated, pre-defined cell types and can be applied to many human tissue samples. FIN-Seq uses immunohistochemical isolation of nuclei, followed by RNA sequencing. This method identifies transcripts, including low abundance ones, in specific cell types. + +**External Link:** +[BioRxiv Publication](https://www.biorxiv.org/content/10.1101/602847v1) + +**Materials Text:** + +### Buffer 1 +- 1.5M Sucrose: 2500 µL +- 1M KCl: 375 µL +- 1M MgCl2: 75 µL +- 1M Tris Buffer pH 8.0: 150 µL +- Nuclease-Free Water: 11900 µL +- **Total Volume:** 15000 µL + +**Storage:** Buffer 1 can be stored at 4°C for up to 6 months. + +### Homogenization Buffer +- Buffer 1: 968 µL +- Triton X-100 10%: 10 µL +- Protease Inhibitor (Promega G6521): 20 µL, reconstituted in DMSO +- 1mM DTT: 1 µL +- Hoechst 33342: 1 µL +- **Total Volume:** 1000 µL + +**Note:** Homogenization Buffer should be made fresh. + +### Sucrose Buffer +- 1M KCl: 2250 µL +- 1M MgCl2: 450 µL +- 1M Tris Buffer pH 8.0: 900 µL +- Nuclease-Free Water: 11400 µL +- **Total Volume:** 15000 µL + +**Storage:** Sucrose Buffer can be stored at 4°C for up to 6 months. + +### Sucrose Bed +- Sucrose Buffer: 2500 µL +- 24% Sucrose: 12500 µL +- **Total Volume:** 15000 µL + +**Storage:** Sucrose Bed can be stored at 4°C for up to 6 months. + +### Blocking Buffer +- RNase-free PBS: 10000 µL +- BSA: 50 mg + +**Note:** Blocking Buffer should be made fresh. + +--- + +**Nuclei Extraction: ~1 hour** + +1. Prepare all solutions and keep on ice with the Dounce homogenizer. +2. Fill the glass Dounce homogenizer with 1 mL of cold homogenization buffer. +3. Mince the tissue into little pieces and place in 1% PFA for 5 minutes on ice. +4. Transfer the tissue pieces into the homogenizer. With the tight pestle, homogenize with 10-15 strokes on ice. Avoid foaming. +5. Transfer the homogenate into a 5 mL polypropylene tube (Thermo Fisher 14-959-11A). +6. Carefully add 2 mL of Sucrose Bed solution to the bottom of the tube so that the homogenate is above the sucrose solution. +7. Spin at 500xg, 12 minutes, at 4°C. +8. Remove the supernatant. Add 1 mL of 4% PFA solution and resuspend pellet. Incubate for 15 minutes at 4°C with rocking. +9. Centrifuge at 2000xg for 5 minutes at 4°C. + +**Note:** Add 1 µL of RNasin Plus (Promega N2615) for every 1 mL of every solution used. Incubate for at least 10 minutes with the RNasin before use. + +--- + +**Immunolabeling: ~2 hours** + +10. Resuspend pellet with Blocking Buffer. Incubate for 15 minutes at 4°C with rocking. +11. Centrifuge at 2000xg for 5 minutes at 4°C. Remove supernatant. +12. Resuspend pellet with Primary Antibody in Blocking Buffer. (Concentration depends on primary antibody. Usually higher concentration than for immunohistochemistry is necessary.) Incubate for 30 minutes at 4°C with rocking. +13. Centrifuge at 2000xg for 5 minutes at 4°C. Remove supernatant. +14. Resuspend pellet with Blocking Buffer. Incubate for 5 minutes on ice. +15. Centrifuge at 2000xg for 5 minutes at 4°C. Remove supernatant. +16. Resuspend pellet with appropriate Secondary Antibody (1:1000) in Blocking Buffer. Incubate at 4°C for 30 minutes with rocking. +17. Resuspend pellet with Blocking Buffer. Incubate for 5 minutes on ice. +18. Centrifuge at 2000xg for 5 minutes at 4°C. Remove supernatant. +19. Resuspend pellet with Blocking Buffer. +20. Filter and proceed to FACS. + +--- + +**FACS: ~30 minutes for 200,000 nuclei** + +21. Gate based on Hoechst histogram. This step ensures that you get 2N nuclei. Make sure not to include 4N nuclei. +22. Gate using the plot with appropriate wavelengths. Often, the separation will not be as obvious as GFP or well-characterized cell surface markers. Proper controls are important. +23. FACS isolated nuclei are sorted into Blocking Buffer and kept at 4°C. + +--- + +**Decrosslinking and RNA Isolation: ~4 hours** + +25. Spin at 3000xg for 7 minutes at 4°C. +26. Remove as much supernatant as possible. +27. From the RecoverAll RNA/DNA Isolation Kit (Thermo Fisher Scientific AM1975), mix 100 µL of Digestion Buffer and 4 µL of protease for each sample. Adjust accordingly based on the volume of leftover supernatant. +28. Incubate at 50°C for 3 hours (wrap the lid with parafilm). Note that this step differs from the manufacturer's protocol. +29. The samples can be stored at -80°C indefinitely after incubation or proceed to next steps according to the kit protocol. +30. Elute in ~17 µL of UltraPure water. +31. Store RNA at -80°C. + +--- + +**cDNA Synthesis** + +32. It’s possible to run an RNA pico chip on the BioAnalyzer (Agilent), but RIN will not be accurate because rRNA is not enriched in nuclei. QuBit or BioAnalyzer can be used to estimate RNA concentration. If less than 10,000 nuclei, concentration may not be available by these methods. +33. Proceed to the SMART-Seq v4 protocol for cDNA synthesis and amplification. +34. If RNA concentration is too low for QuBit or BioAnalyzer, >16 rounds of amplification may be necessary to generate enough cDNA. HS DNA chip should be run on the BioAnalyzer after the SMART-Seq v4 protocol and after Nextera indexing to ensure proper library construction. + +--- + +**Representative FACS plots** + +![Representative FACS plots](image_link_here) + +--- + +**Professional/Scientific Terms and Reagents** + +1. **Triton X-100:** A detergent used to permeabilize cell membranes. +2. **Protease Inhibitors:** Compounds used to protect proteins from degradation. +3. **DMSO (Dimethyl Sulfoxide):** Solvent for dissolving compounds. +4. **DTT (Dithiothreitol):** Reducing agent to break disulfide bonds. +5. **Hoechst 33342:** A dye for staining DNA. +6. **FACS (Fluorescence-Activated Cell Sorting):** A specialized type of flow cytometry. +7. **PFA (Paraformaldehyde):** Fixative to preserve tissues. +8. **Blocking Buffer:** Used to prevent non-specific binding during immunolabeling. + +**Alternative Methods:** +- Substitution for some Protease Inhibitors can be sourced from alternative vendors such as Roche (cOmplete Protease Inhibitor Cocktail). +- If Hoechst 33342 dye is unavailable, DAPI can be an alternative for DNA staining. + +--- +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/fitting-pdb-files-to-saxs-data-using-foxs-web-serv-bd3di8i6.md b/markdown-output/fitting-pdb-files-to-saxs-data-using-foxs-web-serv-bd3di8i6.md new file mode 100644 index 0000000000000000000000000000000000000000..4ffbeb7a29ef66668c721a1f31b4f2b9b422e96d --- /dev/null +++ b/markdown-output/fitting-pdb-files-to-saxs-data-using-foxs-web-serv-bd3di8i6.md @@ -0,0 +1,136 @@ +```markdown +# Goal/Experiment: +Fitting PDB files to SAXS data using FOXS web server. The goal of this experiment is to predict the SAXS data for protein models in the absence of SAXS data. + +# Fitting PDB files to SAXS data using FOXS web server + +*Chris Berndsen* +James Madison University + +**Date:** February 12, 2021 +**Protocol Integer ID:** 34629 +**Created:** March 21, 2020 +**Last Modified:** February 12, 2021 + +This protocol is published without a DOI. + +## Abstract +FOXS is a server for fitting models to SAXS data. It can also predict the SAXS data for protein models in the absence of SAXS data. + +### References +1. Schneidman-Duhovny D, Hammel M, Tainer JA, and Sali A. Accurate SAXS profile computation and its assessment by contrast variation experiments. Biophysical Journal 2013, 105(4), 962-974. +2. Schneidman-Duhovny D, Hammel M, Tainer JA, and Sali A. FoXS, FoXSdock and MultiFoXS: Single-state and multi-state structural modeling of proteins and their complexes based on SAXS profiles NAR 2016. + +## License +This is an open access protocol distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. + +## Materials +- Internet access +- Known protein models +- SAXS data in the form of Intensity vs. q (optional) + +## Procedure + +### 1. Preparing .dat file + +To perform this analysis, you will need subtracted SAXS data in a .dat file that has the structure shown below: + +``` +q intensity error +0.203610E-01 0.326420E-01 0.141695E-02 +0.212060E-01 0.318680E-01 0.136604E-02 +0.217880E-01 0.320550E-01 0.134368E-02 +0.223580E-01 0.311410E-01 0.122690E-02 +0.233140E-01 0.305460E-01 0.943570E-03 +``` + +#### 1.1 Generating Subtracted SAXS Data +If you do not have subtracted SAXS data, see the protocol on SCATTER or ATSAS to generate this file. + +### 2. Loading the Data into Webserver + +#### 2.1 Navigate to the [FOXS webserver](http://modbase.compbio.ucsf.edu/foxs/) + +#### 2.2 Provide the PDB file to be fit for the Input Molecule option. +This can be provided as a PDB code, a PDB file, or a zipped file of PDB. This latter option is appropriate for fitting structures from molecular dynamics simulations. + +#### 2.3 Provide the experimental data set as a .dat file in the Experimental Profile box. + +#### 2.4 Record the input files: +- PDB file +- Experimental profile file + +#### 2.5 Advanced options can also be set. These options are useful for fitting to energy minimized structures or ones from simulations. +- **Maximal q**: If data are bad at high q, reduce this value +- **Implicit hydrogens**: Uncheck if hydrogens are in the PDB file(s) +- **Offset**: Add a constant offset when fitting. This option should be used when fitting. + +### 3. Submitting the Data + +#### 3.1 Press Submit Form and wait 1 to 30 minutes depending on the number of structures to be fitted. + +### 4. Analyzing the Fitted Profile + +#### 4.1 The results are shown in three regions: +1. Top left side shows a fit of the PDB to the data +2. Top right is visualization of the PDB +3. Bottom table showing statistics of the fit + +#### 4.2 The data table is ranked by χ^2 values which range from infinity to 1, with 1 being the target value. +Ideally, the χ^2 value would be ~1 with c1 and c2 values ~1 and no more than 4. If either of the c1 or c2 is red, this suggests some issue with the fitting. + +#### 4.3 Record the numbers of the top 5 fits. +``` ++-----+------+-----+------+-----+ +| χ^2 | c1 | c2 | Rg | file_name | ++-----+------+-----+------+-----+ +| 1 | | | | | +| 2 | | | | | +| 3 | | | | | +| 4 | | | | | +| 5 | | | | | ++-----+------+-----+------+-----+ +``` + +#### 4.4 Data Plotting +A useful visualization of the data is plotting χ^2 vs. Rg (radius of gyration). However, the table can be quite large and hard to record by hand. The code below runs in R and will "scrape" the table from the web into a .csv file. + +```r +# Packages needed to scrape +library(rvest) +library(tidyverse) + +# Assign web link to the variable "page" +page <- read_html("web link from FOXS") + +# Pull the table nodes from the page +tbl <- page %>% + html_nodes("table") %>% + # isolate the 7th table, which is the data table + .[7] %>% + # make it a table file + html_table(fill = TRUE) + +# convert the list into a data frame +tbl_df <- as.data.frame(tbl) + +# Write a csv file to the working directory. +write.csv(tbl_df, "proteinname_datasetname_FOXS.csv") +``` + +*Note:* Sometimes, the table is the 6th element in the page; if the data table does not look correct, change the .[7] to .[6]. + +#### 4.5 Record the name and location of the FOXS table as a note on this step. + +### 5. Fit File Analysis +Click on the fit file link for any structures that fit particularly well. + +#### 5.1 Record the location of the fit files as a note on this step. + +### 6. Multi-FOXS Analysis +If multiple PDB files were provided for fitting, FOXS will also run multi-FOXS, which tries combinations of PDB files to produce a better fit. This can reveal the conformational ensemble of the molecule in solution. These results can be accessed by clicking the multi-FOXS link. + +#### 6.1 Record any multi-FOXS results as a note on this step. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/fixation-and-staining-of-gemmule-hatched-ephydatia-cnzdvf26.md b/markdown-output/fixation-and-staining-of-gemmule-hatched-ephydatia-cnzdvf26.md new file mode 100644 index 0000000000000000000000000000000000000000..64f2f22fbda3a59e7fc380f851a2e0b053486b2e --- /dev/null +++ b/markdown-output/fixation-and-staining-of-gemmule-hatched-ephydatia-cnzdvf26.md @@ -0,0 +1,114 @@ +```markdown +# Goal/Experiment: +Fixation and staining of gemmule-hatched Ephydatia muelleri for fluorescence microscopy. + +## Fixation and staining of gemmule-hatched Ephydatia muelleri for fluorescence microscopy + +### Scott Nichols1 +#### 1University of Denver + +--- + +### Abstract +This protocol is intended for the preparation of gemmule-hatched freshwater sponges for imaging with an inverted scanning confocal microscope. + +### Materials +- Gemmule-hatched freshwater sponges +- 35 mm coverslip-bottom dishes with a 10 mm inner well diameter (Mattek #P35G-0-10-C). Note, you can use a different coverslip thickness, but the diameter of the inner well works with the volumes suggested in this protocol. +- Fixative [4% formaldehyde (F8775-25ML Millipore) in 95% reagent alcohol] +- PTw (1x PBS containing %0.1 Tween-20) +- Block Solution (3% Bovine Serum Albumin in PTw) +- Primary and secondary antibodies of choice (if immunostaining). We use Alexa Fluor secondary antibodies from ThermoFisher Scientific +- Stock solution of appropriate phalloidin conjugate (we use Alexa Fluor Phalloidin conjugates from ThermoFisher Scientific, with the stock solution resuspended in 1 mL methanol) +- Hoechst stock solution (10mg/mL) +- Mounting medium [either Vectashield (H-1000 Vector Laboratories) or equivalent] + +### Safety Warnings +- Work with formaldehyde in a chemical fume hood and dispose of waste appropriately. + +--- + +## Protocol + +### Plate Gemmules in Coverslip-bottom Culture Dishes + +1. **Note:** Details of cleaning and plating sponge gemmules can be found at "Growing Sponges from Gemmules". + +2. Add 3-4 mL volume of culture medium to each dish, and place 1-2 gemmules in the center of the inner well. + +3. Grow the sponges for ~1 week in the dark (this reduces the growth of Chlorella-like algal symbionts that autofluoresce, particularly in the far-red channel). + + - **Note:** Different gemmules develop at quite variable rates. If you are interested in fully developed tissues, you should wait to fix tissues until you see well-developed oscula, choanocyte chambers, and water canals. + +### Fixation and Washes + +4. Remove the culture medium from the outer well by pipetting or aspiration. Then, carefully remove the residual medium from the inner well using a p200 pipette to avoid damaging the tissue. + +5. Gently add 2 mL of fixative (4% formaldehyde in 95% alcohol) to the outer edge of the dish to avoid disrupting the sponge tissues. + + - **Safety information:** Formaldehyde should be used in a chemical fume hood to avoid breathing toxic fumes. + +6. Replace the lid to the dish and incubate at Room Temperature for 45 minutes. + +7. Remove the fixative by carefully pipetting from the outer edge of the dish only. (It is better to leave the fixative in the inner well undisturbed to avoid damaging the tissue). + +8. Add 3 mL of PTw to the outer edge of the dish, and incubate for 3 minutes at Room Temperature. Remove, and repeat. + +### Permeabilization and Blocking + +9. Add 3 mL of Block Solution to the outer edge of the dish, and incubate for 45 minutes at Room Temperature. + +### Incubation with Primary Antibodies (if immunostaining) + +10. **Note:** If you are only staining with dyes like phalloidin and DAPI/Hoechst, you can skip this step and proceed directly to the next section. + + - Dilute your primary antibody at an appropriate concentration in Block Solution. You will need 80-100 µL of diluted antibody per sample. + - **Note:** If working with a new antibody, you may consider testing a range of concentrations such as 1:50, 1:200, and 1:400 to start. + +11. Gently remove all Block Solution from the outer and inner well of your samples. It is important to remove all residual block solution so that you don't further dilute your primary antibody to an unknown extent. + +12. Add 80-100 µL of the diluted primary antibody solution to the inner well of the dish, being careful not to pipette directly onto the sponge tissue. + +13. Incubate for 1 hour (or 2h if needed) at Room Temperature. Alternatively, you can place the sample at 4°C Overnight. + +14. At the end of incubation, it is not necessary to remove the primary antibody by pipetting. Instead, simply add 3 mL of PTw to the outer edge of the dish. Incubate for 5 minutes at Room Temperature. Repeat 1x. + +### Counterstaining for DNA, F-actin + +15. Dilute Hoechst and Phalloidin stock solutions to 1:100 [final concentration], and (if antibody staining) the secondary antibody conjugate to 1:500-1:1000 [final concentration] in Block Solution. You will need to prepare at least 80-100 µL of this mixture for each sample. Protect this solution from light. + + - **Note:** Take into account the species your primary antibody was produced in. (e.g., if produced in rabbit, make sure to use a goat-anti-rabbit secondary). Also take into account the dye conjugates of the phalloidin you use, and the secondary antibody. (e.g., if using 568-phalloidin, make sure to use a 488 or 657 secondary antibody). + +16. Carefully remove the final primary antibody wash from the outer and inner wells of the dish by pipetting. + +17. Add 80-100 µL of the Hoechst/Phalloidin/secondary mixture (Staining Solution) to the inner well of the dish. + +18. Incubate in the dark, for 45 minutes at Room Temperature. + +19. It is not necessary to remove the Staining Solution from the inner well. Instead, add 3 mL of PTw to the outer well area, and incubate in the dark for 3 minutes. Repeat 1x. + +### Mounting, Storage, Imaging + +20. Remove the PTw wash from the outer and inner well area of the dish by carefully pipetting. + +21. Add 80-100 µL of mounting medium to the inner well of the dish. + + - **Note:** Mounting medium is viscous so you should cut the tip off of a 200 µL pipette for this step. + +22. Store the sample at 4°C in the dark until imaging. + + - **Note:** Sponges prepared this way are best viewed on an inverted confocal microscope with the 60-100x objectives for seeing cellular-level detail. + +--- + +## Alternative Methods +For hard-to-find or difficult supplies, consider the following alternatives: +- **Formaldehyde:** If 4% formaldehyde in 95% reagent alcohol is not available, you can prepare it by diluting concentrated formaldehyde (e.g., 37% formaldehyde solution) with the appropriate volume of alcohol. +- **PTw:** PTw can be easily prepared by adding Tween-20 to 1x PBS to a final concentration of 0.1%. +- **Blocking Solutions:** Use normal serum from the same species as the secondary antibody for blocking or other commercial blocking buffers. +- **Primary and Secondary Antibodies:** Check different suppliers like Sigma-Aldrich, Abcam, or Santa Cruz Biotechnology for alternatives. + +--- + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/flex-t-hla-class-i-elisa-protocol-mcsc2we.md b/markdown-output/flex-t-hla-class-i-elisa-protocol-mcsc2we.md new file mode 100644 index 0000000000000000000000000000000000000000..acd29fa3cc8161d48fbdf5a816606f5525eb0906 --- /dev/null +++ b/markdown-output/flex-t-hla-class-i-elisa-protocol-mcsc2we.md @@ -0,0 +1,157 @@ +```markdown +# Goal/Experiment: +Determine the detection of β2-microglobulin subunit of HLA class I complexes using Flex-T™ HLA Class I ELISA and evaluate the efficiency of peptide exchange. + +## Flex-T™ HLA Class I ELISA Protocol Version 3 + +### Abstract +The HLA class I ELISA is an enzyme immunoassay based on the detection of β2-microglobulin subunit of HLA class I complexes, after capturing the complex through the conjugated biotin. To this end, biotinylated HLA class I complex is first captured in streptavidin coated microtiter wells. Subsequently, HRP-conjugated anti-human β2-microglobulin is added to detect intact HLA class I complexes. Only intact HLA class I complexes are recognized. Peptides with high affinity binding will be clearly detected by this ELISA technique, while peptides with a moderate to low binding affinity for HLA class I provide a moderate to non-detectable signal. This protocol is designed to evaluate the efficiency of peptide exchange when using the Flex-T™ system. + +Citation: Kelsey Miller Flex-T™ HLA Class I ELISA Protocol. protocols.io +dx.doi.org/10.17504/protocols.io.mcs2we +Published: 19 Dec 2017 + +--- + +## Guidelines + +### Reagent Preparation +See Appendix 1 for recipes and recommended reagents. All reagents should be prepared or diluted immediately prior to use. Before use, bring all reagents to room temperature (18-25°C) with the exception of the HRP anti-human β2-microglobulin antibody and the ELISA positive control which have to be kept on melting ice to ensure stability. Sodium azide inactivates HRP. Do not use sodium azide-containing solutions, nor add sodium azide to the supplied materials. Centrifuge all vials before use (1 minute 3000×g at 4°C). Do not allow wells to stand uncovered or dry for extended periods between incubation steps. + +### Materials +- Coating Buffer 5X concentrate (Cat# 421701) +- Streptavidin solution at 1 mg/ml (BioLegend Cat# 280302) +- Wash Buffer 20X concentrate: PBS with 0.05% (v/v) Tween 20 (BioLegend Cat# 421601) +- Dilution Buffer 10X concentrate (1 M NaCl, 0.5M Tris, 1% BSA, 0.2% Tween 20, pH8.0. See Appendix 1) +- HRP anti-human β2-microglobulin antibody (Cat# 280303) +- Flex-T™ ELISA Positive Control (BioLegend Cat# 280301) +- Substrate Buffer, 10X concentrate (0.1 M citric acid monohydrate/tri-Sodium citrate dehydrate, pH 4.0, See Appendix 1) +- ABTS substrate solution (50X 40mM solution made from e.g. Sigma Cat#A1888, or equivalent. See Appendix 1) +- Hydrogen peroxide solution (e.g. Sigma Cat#H3410, or equivalent. See Appendix 1) +- Stop Solution (2% [w/v] oxalic acid dehydrate, e.g., Sigma Cat#247537. See Appendix 1) +- Nunc™ MaxiSorp™ ELISA Plates, Uncoated (BioLegend Cat# 423501) +- Plate sealers (BioLegend Cat# 423601) +- Deionized (DI) water + +### Equipment +- Incubator (37°C) +- ELISA plate shaker +- Wash bottle or automated microplate washer +- Microtiter plate reader for measuring absorbance at 414nm +- Adjustable pipettes to measure volumes ranging from 3µl to 1 ml +- Multichannel pipetting devices + +### Representative Data and Appendix 1 + +| | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | +|-----|-------|-------|-------|-------|-------|-------|-------|-------|-------|-------|-------|-------| +| A | H | H | Dup | Dup | P1 | Dup | P2 | Dup | P3 | Dup | P4 | Dup | +| B | M | P1 | P2 | P3 | P4 | P5 | P6 | P7 | P8 | P9 | P10 | P11 | +| C | L | P11 | P12 | P13 | P14 | P15 | P16 | P17 | P18 | P19 | P20 | Blank | +| D | Blank | P21 | P22 | P23 | P24 | P25 | | | | | | | +| E | Pos | P26 | P27 | P28 | P29 | P30 | | | | | | | +| F | Neg | P31 | P32 | P33 | P34 | P35 | | | | | | | +| G | UV | P36 | P37 | P38 | P39 | P40 | | | | | | | +| H | Blank | | | | | | | | | | | | + +- H, M, L: HLA control H, M, and L respectively +- UV: UV-illuminated conditional HLA complex in the absence of exchange peptide +- Pos: Positive exchange control peptide +- Neg: Negative exchange control peptide +- P: peptide of choice +- Blank: 1X Dilution Buffer + +--- + +## Protocol + +### Reagent Preparation + +**Step 1:** +Dilute the 5X Coating Buffer to 1X with DI water. + +**Step 2:** +Prepare Streptavidin solution to coat the plate: +- 24µl of Streptavidin solution +- 11.976 ml 1X Coating Buffer + +**Step 3:** +Calculate the amount of 1X Dilution Buffer required and prepare the solution by diluting the 10X concentrated buffer 10 times in DI water before use. The 1X Dilution Buffer can be stored for up to one week at 2-8°C. + +**Step 4:** +Prepare Wash Buffer; dilute the 20X wash buffer with DI water, at least 300ml per plate. + +**Step 5:** +Dilute concentrated HRP-conjugated antibody to 0.3µg/ml in 1X Dilution Buffer just before use. Prepare 12ml per plate. + +**Step 6:** +Prepare the substrate solution approximately 10 minutes before use: +- 10.34ml DI water +- 1.2ml of Substrate Buffer 10X concentrate +- 240µl of ABTS stock solution +- 120µl of Hydrogen peroxide solution +- Vortex to mix well +The substrate solution should be at room temperature (18-25°C) for optimal reproducible results. + +**Step 7:** +Dilute the Flex-T™ ELISA Positive Control to prepare a 2.7µM solution: +- 5µl of Flex-T™ ELISA Positive Control at 0.2mg/ml +- 2.9µl of 1X Dilution Buffer + +### Assay Procedure + +**Step 8:** +Coat the wells of the plate with Streptavidin. Add 100µl of Streptavidin solution to all wells. Seal the plate and incubate overnight (16-18 hrs) at room temperature (18-25°C). + +**Step 9:** +Discard the coating solution and wash the plate 3 times with at least 300µl Wash Buffer per well and blot residual buffer by firmly tapping plate upside down on absorbent paper. All subsequent washes should be performed similarly. + +**Step 10:** +To block non-specific binding and reduce background, add 300µl of 1X Dilution Buffer to all wells. + +**Step 11:** +Seal the plate and incubate for 30 minutes at room temperature (18-25°C). + +**Step 12:** +Prepare three dilutions of the HLA control by serial dilution in 1X Dilution Buffer. Prepare the controls fresh and keep them on melting ice until usage. + +**Step 13:** +To evaluate the outcome of UV-mediated HLA peptide exchange, dilute a small aliquot of the exchange reaction mixture 300-fold in 1X Dilution Buffer (refer to the peptide exchange step in Protocol for fluorescent Flex-T™ generation and antigen-specific CD8+ T cell staining). Mix thoroughly. + +**Step 14:** +Discard the Dilution Buffer from the plate and blot the residual buffer. + +**Step 15:** +Pipette 100µl of 1X Dilution Buffer (blank), diluted ELISA controls (H, M, L, positive, negative, and UV only), or exchange reaction mixture dilutions, into the appropriate wells (recommended in duplicate, see plate map). + +**Step 16:** +Seal the plates and incubate for 1 hour at 37°C. + +**Step 17:** +Discard the liquid from the wells and wash 3 times with 300µl of wash buffer per well. + +**Step 18:** +Add 100µl of diluted HRP-conjugated antibody. + +**Step 19:** +Seal the plates and incubate for 1 hour at 37°C. + +**Step 20:** +Discard the liquid from the wells and wash 3 times with 300µl of wash buffer per well. + +**Step 21:** +Add 100µl of substrate solution. + +**Step 22:** +Incubate for 8 minutes at room temperature (18-25°C) in the dark on a plate shaker at 400-500 rpm. + +**Step 23:** +Add 50µl of Stop Solution to all wells and read at 414nm in an ELISA reader within 30 minutes. + +**Step 24** +View BioLegend website for [Representative Data, and Appendix 1](#). + +--- + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/flex-t-tetramer-and-cell-staining-protocol-hv6b69e.md b/markdown-output/flex-t-tetramer-and-cell-staining-protocol-hv6b69e.md new file mode 100644 index 0000000000000000000000000000000000000000..d73789d9569916fccf9a3522ca05c384f1ff57da --- /dev/null +++ b/markdown-output/flex-t-tetramer-and-cell-staining-protocol-hv6b69e.md @@ -0,0 +1,117 @@ +```markdown +# Goal/Experiment: +The goal of the Flex-T™ Tetramer and Cell Staining Protocol is to generate MHC/peptide monomers using UV-induced peptide exchange. These monomers are then multimerized using streptavidin-fluorophore conjugates to create Flex-T™ reagents. These reagents can be used for staining antigen-specific T cells and for flow cytometric analysis. + +# Flex-T™ Tetramer and Cell Staining Protocol Version 2 + +**Author**: Kelsey Miller +**Published**: 09 May 2017 +**Citation**: Kelsey Miller Flex-T™ Tetramer and Cell Staining Protocol. protocols.io dx.doi.org/10.17504/protocols.io.hv6b69e + +## Abstract + +Using UV-induced peptide exchange, MHC/peptide monomers can be generated with conditional Flex-T™ monomers that harbor peptides of interest in their binding grooves. These new MHC monomers are subsequently multimerized using streptavidin-fluorophore conjugates. The resulting Flex-T™ reagents can be used for staining antigen-specific T cells and flow cytometric analysis. In humans, the MHC molecules are called HLA (Human Leukocyte Antigen). + +## Guidelines + +### Materials + +- **Phosphate buffered saline (PBS), 10X concentrate** (BioLegend Cat# 926201) +- **Conditional Flex-T™ monomers** +- **10 mM peptide solution** of choice in 100% DMSO +- **DMSO** (e.g. Sigma-Aldrich Cat#D5879) +- **50 mM D-Biotin** (e.g. Thermo Fisher, Cat#B20656) +- **10% (w/v) NaN₃** (e.g. Sigma, Cat#S2002) +- **Fluorophore-conjugated Streptavidin** (BioLegend Cat# 405203, Cat# 405207, Cat#405225 or equivalent) +- **Cell Staining Buffer** (BioLegend Cat#420201 or equivalent) +- **96-well Polystyrene Microplate, U-shape** (e.g. Falcon Cat#353077) or **5mL, 12 x 75mm tubes** (e.g. Falcon Cat#352008) +- **Plate sealers** (BioLegend Cat#423601) +- **1.5 mL tubes** (e.g. Eppendorf Cat#022364111) + +### Equipment + +- **UV lamp, long-wave UV (366 nm, 8 Watts)** (e.g. CAMAG cat# 022.9115 or Ultraviolet Crosslinker CL-1000) +- **Incubator (37°C)** +- **Centrifuge** capable of accommodating microtiter plates and tubes +- **Single and multichannel pipettes** capable of accurate delivery of variable volumes, and pipette tips + +### Precautions for Use + +- DMSO can be used to dissolve the peptides; however, do not exceed an end concentration of 10% (v/v) in the exchange reaction. +- Avoid repeated freeze-thawing. +- The Flex-T™/peptide solution needs to be kept on ice in the dark as much as possible. Do not work in front of a window. +- The use of short-wavelength (254 nm) or broad-band UV lamps is detrimental to MHC complexes. +- Centrifuge all vials before use (1 minute 3000xg at 4°C). + +## Protocol + +### Peptide Exchange + +**Step 1** +Bring all reagents to 0°C by putting them on ice. + +**Step 2** +Dilute 10 mM stock solutions of peptides of choice to 400 μM by mixing 5 μL of peptide stock solution with 120 μL PBS, and keep on ice. + +**Step 3** +Add 20 μL diluted peptide (400 μM) and 20 μL conditional Flex-T™ monomer (200 μg/mL) into a 96-well U-bottom plate. Mix by pipetting up and down. + +**Step 4** +Seal the plate; centrifuge at 3300xg for 2 minutes at 4°C to collect the liquid down. +⏲️ _Duration_: 2 minutes + +**Step 5** +Remove the seal; put the plate on ice and illuminate with UV light for 30 minutes (the distance of the UV lamp to the samples should be 2-5 cm). +⏲️ _Duration_: 30 minutes + +**Step 6** +Seal the plate; incubate for 30 minutes at 37°C in the dark. +⏲️ _Duration_: 30 minutes + +**Step 7** +To evaluate the efficiency of the peptide exchange follow the Protocol for HLA class I ELISA to evaluate peptide exchange. + +### Generation of Tetramers + +**Step 8** +Transfer 30 μL of peptide-exchanged monomer into a new plate, then add 3.3 μL of conjugated streptavidin, mix by pipetting up-and-down. Incubate on ice in the dark for 30 minutes. This is enough for about 15 tests. +⏲️ _Duration_: 30 minutes + +> **Note**: BioLegend fluorophore-conjugated streptavidin products are recommended. For 30 μL exchanged Flex-T™ monomer we suggest to use 3.3 μL of BioLegend PE-streptavidin (Cat#405203) or APC- streptavidin (Cat#405207). For BV421-streptavidin conjugate (Cat#405225) use 1.3 μL. For optimal reaction with other fluorophore-conjugated streptavidin products ensure that the monomer: streptavidin conjugate has a 6:1 molar ratio. + +**Step 9** +During the incubation, prepare blocking solution by adding 1.6 μL 50 mM D-Biotin and 6 μL 10% (w/v) NaN₃ to 192.4 μL PBS, mix by vortexing. After the incubation, add 2.4 μL of blocking solution and pipette up-and-down to stop the reaction. + +**Step 10** +Seal the plate and incubate at 2 - 8°C overnight (or on ice for 30 minutes in the dark). +⏲️ _Duration_: 30 minutes + +> **Note**: We recommend Flex-T™ to be assembled with two different streptavidin conjugates in separate reactions. This allows for two-color staining with the same tetramer allele, ensuring the highest specificity. + +### Cell Staining and Flow Cytometric Analysis + +**Step 11** +Prepare cells of interest. + +**Step 12** +Prior to performing staining, centrifuge the plate at 3300xg for 5 minutes at 4°C. Keep on ice in the dark. +⏲️ _Duration_: 5 minutes + +**Step 13** +Add 2 x 10⁶ cells to a 96-well U-bottom plate or 12 x 75 mm tubes. Adjust volume to 200 μL with Cell Staining Buffer. Add 2 μL per sample of Flex-T™ complex prepared in Steps 7 - 9, mix and incubate on ice in the dark for 30 minutes. +⏲️ _Duration_: 30 minutes + +**Step 14** +If co-staining with surface antibodies, prepare the antibody cocktail based on optimal staining concentration of each reagent. Incubate for 30 minutes on ice in the dark. +⏲️ _Duration_: 30 minutes + +**Step 15** +Wash the cells with Staining Buffer two times. Resuspend cells with Staining Buffer. + +**Step 16** +Acquire the samples with a flow cytometer and appropriate settings within 2 hours. + +> **Note**: A titration of the Flex-T™ is recommended for optimal performance. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/flow-cytometry-analysis-of-human-islet-cell-expres-kwzcxf6.md b/markdown-output/flow-cytometry-analysis-of-human-islet-cell-expres-kwzcxf6.md new file mode 100644 index 0000000000000000000000000000000000000000..7d08c0b40168e6329719add4b5a00366565eedb7 --- /dev/null +++ b/markdown-output/flow-cytometry-analysis-of-human-islet-cell-expres-kwzcxf6.md @@ -0,0 +1,142 @@ +```markdown +# Goal/Experiment: +Flow cytometry analysis of human islet cell expression of heparan sulfate (HS) and collagen type XVIII (Col18) + +--- + +## Abstract +Isolated human islets were dispersed into single cells using Accutase (Millipore), ~1500-2000 islet equivalents/ml. 20,000-65,000 islet cells were transferred to individual wells of a 96 well culture plate (CELLSTAR, Greiner Bio-one) for immediate staining for flow cytometry analysis or for culture prior to staining. For intracellular staining, isolated islet cells were fixed in 2% paraformaldehyde (Sigma-Aldrich) and permeabilized using 0.3% saponin (Sigma-Aldrich). The cells were stained with 10E4 mouse anti-human HS mAb (10E4, 1/50; US Biological/Amsbio), mouse anti-mouse Col18 mAb (1/50; Santa Cruz Biotechnol.) or the corresponding isotype control Ig (mouse IgM, or IgG2bκ; BD Biosciences) followed by goat anti-mouse Ig-R-PE (1/100; Southern Biotech). The geometric mean fluorescence ratio (GMFR) was calculated by dividing the geometric mean fluorescence intensity (GMFI) of cells stained with primary mAb by the GMFI obtained with the relevant isotype control Ig. Cells were analyzed using a BD LSRI flow cytometer and CellQuest™ Pro software (version 6.0; BD Biosciences). + +--- + +## Guidelines +10E4 anti-heparan sulfate (HS) mAb identifies highly sulfated HS localized in human beta cells but does not identify the less sulfated HS in alpha cells. + +--- + +## Before start +### Materials: +#### Prepare: +1. **2% Paraformaldehyde**: + - Add 2 g Paraformaldehyde (#P6148 Sigma) to 50 ml deionized water, a stirrer and 2 drops of 10 M NaOH. Heat at 60°C for 30 mins. Add 40 ml deionized water and 10 ml of 10x PBS and pH to 7.2. Store solution at 4°C. + +2. **PBS/3 mM EDTA**: + - 112 mg EDTA (AJAX #180) in 100 ml PBS, sterile filter using 0.2 µm disposable filter. + +3. **Beta cell culture medium**: + - RPMI 1640 (Sigma #R0883) 200 ml + - Heat-inactivated fetal calf serum (HIFCS) 20 ml + - L-Glutamine (Gibco #25030081 200 mM) 2 ml (final 2 mM) + - Penicillin G (MP Biomedicals #02194537), 0.06 mg/ml + - Streptomycin (Sigma #S9137), 0.10 mg/ml + - Neomycin (Sigma #N6386), 0.10 mg/ml + +4. **PBS/5% HIFCS**: + - 500 ml PBS + 25 ml HIFCS + +5. **PBS/5% HIFCS/0.3% saponin**: + - 100 ml PBS/5% HIFCS + 300 mg saponin (#S7900, Sigma) and sterile filter with a disposable 0.2 µm filter. + +6. **PBS/5% HIFCS/0.03% saponin**: + - 10 ml PBS/5% HIFCS/0.3% saponin + 90 ml PBS/5%HIFCS + +#### Mabs and pAbs: +1. **10E4 (anti-HS) mAb, Amsbio #370255-1** + +2. **Mouse anti-mouse collagen type XVIII (Col18A1), Santa Cruz Biotechnol #1837-46** + +3. **IgMκ , BD Biosciences #550340** + +4. **Mouse IgG2bκ , BD Biosciences #557351** + +5. **Goat anti-mouse Ig R-PE, Southern Biotech #1010-09** + +#### Other reagents/materials: +1. **Accutase**, Millipore #SCR005 + +2. **Cell culture plates**: Cellstar #650180 (Greiner Bio-one) + +3. **Cluster tubes**, Fisher Biotech #MTS-11C + +--- + +## Protocol +### Step 1 +See Guidelines, “Before starting” and “Safety Warnings”. + +### Step 2 +Centrifuge human islets at 300g for 2 min at 23°C. Pour off the supernatant. Resuspend in 25 ml PBS/3 mM EDTA. Centrifuge at 300g. + +### Step 3 +Resuspend the islets in PBS/3 mM EDTA and transfer islets to 15ml tubes, 2000 IEQ/tube. Centrifuge at 300g then carefully remove the supernatant. + +### Step 4 +Gently resuspend each pellet in 1 ml pre-thawed Accutase and place tubes in 37°C water bath for 10 mins (Note: at 4 min and 8 min, gently knock the pellet to resuspend the islets). + +### Step 5 +Dissociate the islets by pipetting up and down 10-15 times using a 1 ml single channel pipette. + +### Step 6 +Add 10 ml culture medium to each tube to terminate the Accutase reaction and centrifuge for 5 min at 300g. + +### Step 7 +Discard the supernatant, pool the cells into a single 15 ml tube and determine cell density (using hemocytometer). Adjust cell density to 100,000 - 325,000 cells/ml. + +### Step 8 +Transfer islet cells to culture plate, 20,000 - 65,000 cells in 200 µl/well. + +### Step 9 +Centrifuge cells at 300g for 3 min at 23°C. Remove supernatant by flicking. + +### Step 10 +For intracellular staining, resuspend separate wells of islet cells in 100 µl 2% Paraformaldehyde. Treat for 10 min at room temperature. Add 100 µl PBS/5% HIFCS and spin again at 300g for 3 min at 23°C. + +### Step 11 +Flick off the supernatant and wash the cells in 200 µl PBS/5% HIFCS and centrifuge at 300g for 3 min at 23°C. + +### Step 12 +Incubate cells for 30 min on ice with: +- (i) 25 µl/well of 10E4 anti-HS mAb or mouse IgM (isotype control) diluted to 20 µg/ml with PBS/5% HIFCS/0.3% saponin; +- or +- (ii) 25 µl/well of anti-Col18 mAb or mouse IgG2b (isotype control) diluted to 4 µg/ml with PBS/5% HIFCS/0.3% saponin. + +Protect from light. + +### Step 13 +Wash 2x with PBS/5% HIFCS/0.03% saponin, as for Step 9. + +### Step 14 +Incubate cells for 30 min on ice with 25 µl/well of goat anti-mouse Ig PE diluted to 5 µg/ml with PBS/5% HIFCS/0.3% saponin. Protect from light. + +### Step 15 +Wash 2x with PBS/5% FCS/0.03% saponin, as for Step 9. + +### Step 16 +Resuspend cells in 100 µl/well of PBS/5% HIFCS, transfer cells from each well to an individual cluster tube and run samples on flow cytometer. + +Excitation/emission wavelength: R-PE 565 nm/575 nm. + +### Step 17 +For cell surface staining on separate aliquots of cells, apply steps 8, 9, 12-16 (inclusive), with the exception that all washes and antibody dilutions are done in PBS/5% FCS. + +### Step 18 +Analyze cell surface or intracellular HS or Col18 staining using CellQuest™ Pro software (version 6.0; BD Biosciences). + +### Step 19 +**Note**: To determine % islet cells that are beta cells, apply steps 8 and 9. Resuspend separate well(s) of cells in 10 µM Newport Green, 100 µl/well. Incubate at 37°C for 1 hr. Add 100 µl PBS, centrifuge at 300g. Remove culture supernatant and resuspend in 100 µl PBS for flow cytometry analysis. Analyze flow cytometry data using CellQuest™ Pro software (version 6.0; BD Biosciences) to identify % of total islet cells that are Newport Green+ve beta cells. + +### Step 20 +**Warnings**: All handling of human islets is done in a Class II Biological Safety Cabinet. + +--- + +**Reference**: +Theodoraki A, Hu Y, Poopalasundaram S et al (2015) Mol Cell Endocrinol 399: 296-310. + +**Citation**: +Sarah Popp, Charmaine Simeonovic. Flow cytometry analysis of human islet cell expression of heparan sulfate (HS) and collagen type XVIII (Col18). protocols.io dx.doi.org/10.17504/protocols.io.kwzxcf6 Published: 22 Nov 2017 + +--- + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/flow-cytometry-analysis-of-mouse-islet-cell-expres-bmsyk6fw.md b/markdown-output/flow-cytometry-analysis-of-mouse-islet-cell-expres-bmsyk6fw.md new file mode 100644 index 0000000000000000000000000000000000000000..ed064d73586e410c879ff8d2b51404f4e3a9cb49 --- /dev/null +++ b/markdown-output/flow-cytometry-analysis-of-mouse-islet-cell-expres-bmsyk6fw.md @@ -0,0 +1,88 @@ +```markdown +# Goal/Experiment: +Flow cytometry analysis of mouse islet cell expression of heparan sulfate (HS), heparan sulfate proteoglycans (HSPGs), and heparanase (HPSE). + +## Flow Cytometry Analysis of Mouse Islet Cell Expression of Heparan Sulfate (HS), Heparan Sulfate Proteoglycans (HSPGs) and Heparanase (HPSE) + +- **Authors**: Sarah Popp, Sarita Dhounchak, Charmaine Simeonovic +- **Affiliation**: The Australian National University +- **Date**: September 28, 2020 +- **DOI**: [10.17504/protocols.io.bmsyk6fw](https://doi.org/10.17504/protocols.io.bmsyk6fw) +- **Protocol ID**: 42552 + +### Abstract +Isolated mouse islets were dispersed into single cells using Accutase (Millipore; 250 μl/500 islets). 40,000 islet cells were transferred to individual wells of a 96 well culture plate (CELLSTAR) for staining and flow cytometry analysis or for culture. For intracellular staining, isolated islet cells were fixed and permeabilised using BD Fix/Perm (BD Biosciences). After a blocking step, the cells were stained with primary antibodies (anti-mouse collagen type XVIII (Col18), anti-mouse CD138 (anti-syndecan-1 (SDC1)), anti-mouse CD44, anti-human HS (10E4) or HP3/17 anti-human heparanase (HPSE)) and incubated with fluorescent secondary antibodies. Events were collected using a BD LSR Fortessa flow cytometer with BD FACS DIVA software (version 8). Data was analyzed using FlowJo software (version10.0.7, TreeStar Inc.). + +### Keywords +- Mouse beta cells +- Heparan sulfate +- Collagen type XVIII +- Syndecan-1 +- CD44 +- Heparanase + +### Materials + +#### 1. Preparation +1. **BD wash buffer**: 90% (v/v) Deionized water + 10% (v/v) 10x BD stock wash solution. +2. **PBS/3 mM EDTA**: 112 mg EDTA (AJAX #180) in 100ml PBS, sterile filter using 0.2 µm disposable filter. +3. **Beta cell culture medium**: + - RPMI 1640 (Sigma R0883) 200ml + - Heat-inactivated fetal calf serum (HIFCS) 20ml + - L-Glutamine (Gibco # 25030081 200mM) 2ml (final 2mM) + - Penicillin G, MP Biomedicals #02194537, 0.06 mg/ml + - Streptomycin, Sigma #S9137, 0.10 mg/ml + - Neomycin, Sigma #N6386, 0.10 mg/ml +4. **PBS/5% HIFCS (FACs Wash buffer)**: 500ml PBS + 25ml HIFCS + +#### 2. mAbs and pAbs: + +- **Rat anti-mouse CD16/CD32 (mouse Fc block)**: BD Biosciences #553142 (0.5mg/ml) +- **10E4 (anti-HS) mAb**: Amsbio #370255-1(1mg/ml) +- **Mouse anti-mouse collagen type XVIII (Col18a1)**: Santa Cruz Biotech #1837-46 (0.2mg/ml) +- **Rat anti-mouse CD44 mAb**: BD Biosciences #553130 (1mg/ml) +- **Rat anti-mouse CD138 (SDC1) mAb**: BD Biosciences #553712 (0.5mg/ml) +- **Mouse anti-human heparanase (HPSE) mAb**: Insight Biopharmaceuticals #INS-26-1-0000-12 (150mg/ml) +- **Goat anti-mouse Ig R-PE**: Southern Biotech #1010-09 (0.5mg/ml) +- **Mouse anti-rat kappa PE**: Southern Biotech #3009-09 (0.1mg/ml) +- **Rat anti-mouse Ig FITC**: BD Bioscience #553395 (0.5mg/ml) + +#### 3. Other Reagents/Materials + +- **Accutase**: Millipore #SCR005 +- **Cell culture plates**: CELLSTAR #650180 (Greiner Bio-one) +- **Mini tubes**: Axygen/Fisher Biotech #MTS-11C +- **BD CytoFix/Cytoperm Kit**: BD Biosciences #554714 + +### Methods + +1. **Preparation Steps** + 1. Disperse isolated mouse islets into single cells using Accutase (250 µl/500 islets) at 37˚C for 10 minutes. + 2. Pipette up and down 10-15 times using a 1ml single-channel pipette. + 3. Add 10ml culture medium to terminate the reaction and centrifuge for 5 min at 249g. Discard supernatant. + 4. Resuspend in beta cell culture medium and determine cell density using a hemocytometer. + 5. Transfer islet cells to a culture plate: 4-8 x 10⁴ cells/well to a final volume of 200 µl. + +2. **Flow Cytometry Analysis** + 1. **Fixation and Permeabilisation (intracellular staining)**: + 1. Treat islet cells with 100μl BD Cytofix/Cytoperm for 10 min at room temperature. + 2. Wash with BD wash buffer and centrifuge at 249g for 3 min. + 3. Incubate with primary antibodies for 30 min on ice: + - 10E4 (20µg/ml), Col18 (4µg/ml), SDC1 (20µg/ml), CD44 (40µg/ml), HPSE (1.5µg/ml) + 4. Wash and resuspend in 100µl BD wash buffer. + 5. Run samples on a BD LSR Fortessa flow cytometer, events collected using BD FACS DIVA software. Data analyzed using FlowJo software + +3. **Surface staining** + 1. Transfer cells to wells and repeat steps 9-16, except all washes and antibody dilutions are made in FACS wash buffer. + 2. Analyse tubes using flow cytometry, identical parameters and software as before. + +### Notes +- 10E4 anti-heparan sulfate (HS) mAb identifies highly sulfated HS localised in beta cells but does not identify the less sulfated HS in alpha cells. + +### References +- Theodoraki A, Hu Y, Poopalasundaram S et al (2015) Mol Cell Endocrinol 399: 296-310. + +--- + +**endofoutput** +``` \ No newline at end of file diff --git a/markdown-output/flow-cytometry-trait-measurements-size-granularity-bfemjjc6.md b/markdown-output/flow-cytometry-trait-measurements-size-granularity-bfemjjc6.md new file mode 100644 index 0000000000000000000000000000000000000000..0f49d169c0375ce0c400124288fe50ee201e65cb --- /dev/null +++ b/markdown-output/flow-cytometry-trait-measurements-size-granularity-bfemjjc6.md @@ -0,0 +1,131 @@ +```markdown +# Goal/Experiment: +The goal of this experiment is to measure the size, granularity, and chlorophyll-a content in centric diatom cells using flow cytometry. This method employs a CytoFlex LX instrument with *Thalassiosira* spp. as the model organism. + +# Flow Cytometry Trait Measurements (Size, Granularity, and Chlorophyll-a) of Diatoms + +**Phoebe Argyle1, Jana Hinners2, Nathan Walworth3, Sinead Collins4, Naomi M. Levine3, Martina Doblin5** + +1Climate Change Cluster, University of Technology Sydney, Sydney, Australia +2Institute of Coastal Ocean Dynamics, Helmholtz-Zentrum Geesthacht, Geesthacht, Germany +3Department of Biological Sciences, University of Southern California, Los Angeles, CA, USA +4Institute of Evolutionary Biology, University of Edinburgh, Edinburgh, UK +5Climate Change Cluster, University of Technology Sydney, Sydney, Australia. Sydney Institute of Marine Science, Mosman, Sydney, Australia + +## Abstract +A method and guide for measuring size, relative granularity, and chlorophyll-a content in centric diatom cells. This method was developed on a CytoFlex LX instrument with *Thalassiosira* spp. as the model organism. + +## Publications +This protocol was used in the following publications: +1. Argyle, P. A., Walworth, N. G., Hinners, J., Collins, S., Levine, N. M., & Doblin, M. A. (2021). Multivariate trait analysis reveals diatom plasticity constrained to a reduced set of biological axes. *ISME Communications*, 1(1), 59. +2. Argyle, P. A., Hinners, J., Walworth, N. G., Collins, S., Levine, N. M., & Doblin, M. A. (2021). A high-throughput assay for quantifying phenotypic traits of microalgae. *Frontiers in microbiology*, 12, 706235. + +## Guidelines +Flow cytometry analyses are most efficient if run in the afternoon of the day of sampling. However, these samples can also be snap frozen in liquid nitrogen, stored at -80°C, and analyzed in delayed mode. + +Fluorescence of chlorophyll-a diminishes over time so ensure the time between sampling and measurement is similar for all samples being compared. + +## Materials + +### Equipment +- **CytoFLEX LX** + Type: Flow cytometer + Brand: Beckman Coulter + SKU: C40312 + Link: [Beckman Coulter](https://www.beckman.com) + Specifications: CytoFLEX LX N3-V5-B3-Y5-R3-I2 Flow Cytometer (21 Detectors, 6 Lasers) + +- **96 Well TC-Treated Microplates** + Type: Microplate + Brand: Corning® + SKU: CLS3799-1EA + Link: [Sigma-Aldrich](https://www.sigmaaldrich.com) + +- **Parafilm** + Type: Lab consumable + Brand: Parafilm M + SKU: PM996 + Link: [Amazon](https://www.amazon.com/) + Specifications: Can be purchased from many vendors + + +### Reagents +- **Paraformaldehyde 8% EM Grade aqueous solutions** + Supplier: Emgrid + Catalog: #157-8 + +- **CytoFlex Daily QC Beads** + Supplier: Beckman Coulter + Catalog: #B53230 + +- **Flow Cytometry Size Calibration Kit** + Type: Nonfluorescent microspheres + Supplier: Thermo Fisher + Catalog: #F13838 + +## Safety Warnings +- *Paraformaldehyde is toxic; ensure it is added under a fume hood or chemical safety cabinet.* + +## Sample Preparation +1. **Aliquot** + Aliquot 200 μL of live, uniform culture and transfer to a round-bottom tissue culture plate. Agitate culture gently to suspend cells to create a uniform culture. + +2. **Cell Fixation** + Fix the cells with 20 μL (10 % volume) paraformaldehyde 8% solution, resulting in a final concentration of 0.8% paraformaldehyde. Use a fume hood as this reagent is toxic. + +3. **Storage** + Secure the lid and wrap plate edges with parafilm to prevent evaporation and store at 4°C prior to analysis (analyze within 48 hours, ideally later the same day). + +## Flow Cytometry Setup +4. **Loading** + Load the plate into the flow cytometer. This protocol was developed using a CytoFLEX LX instrument. + +5. **Quality Control** + Set up the flow cytometer and run the daily Quality control (QC) protocol as per the instrument instructions. + In this case, for the Cytoflex LX, multi-spectra beads (CytoFlex Daily QC Beads Beckman Coulter Catalog #B53230) are used to ensure the correct functioning of the instrument. These beads may also be used to ensure consistency across experimental runs, as discussed in step 8. + +6. **Gain and Threshold Optimization** + Optimize gain and threshold settings for the detectors of interest in the flow cytometer. A spare aliquot of diatom culture can be used to optimize gain/threshold settings. Culture media obtained from filtering diatom culture (through a 0.22 μm filter) can also be used to identify background scatter and set thresholds. + The laser and detectors are as follows. However, this will depend on the particular instrument so exact numbers may not match if a different instrument is used. + + **Laser and Detector Settings:** + - Forward scatter area: Si-photodiode with built-in 488/8 band-pass filter (CytoFLEX Channel name: FSC-A) + - Side scatter area: 488nm (50mw), Optical Filter: 488/8 nm, (CytoFLEX Channel name: SSC-A) + - Chlorophyll a: Laser: 488nm (50mw), Optical Filter: 690/50 nm, (CytoFLEX Channel name: B690-50) + +**Thresholds:** + - FSC-A (forward scatter area): 4000 + - B690-50 (chlorophyll-a): 2000 + +## Flow Cytometry Analysis +7. **Sample Mixing and Analysis** + Samples are mixed for 6 seconds by the CytoFlex instrument before analysis. + + During development, samples were generally run at medium speed/flow rate of 30 μL per minute for 1 minute. Analysis is done on at least 200 cells, however, in most cases it would be at least 2000 cells. The abort rate (%) was <1%. In the case of a highly dense culture, a slower speed may be prudent to ensure a low abort rate and ensure the accuracy of analysis. In the case of a low-density culture, a higher flow rate or longer duration of analysis may be used to ensure an adequate number of cells are analyzed. + + Diatoms were gated visually using event density plots of chlorophyll fluorescence and forward scatter (see below). + +## Calibration Using Beads +8. **Calibration Beads** + The multi-spectra beads (CytoFlex Daily QC Beads Beckman Coulter Catalog #B53230) that are used to calibrate the instrument on startup may also be used to ensure comparability between experimental runs. + It is recommended to analyze samples from one experiment on the same day. However, if this is not possible, it is necessary to ensure that the instrument is performing similarly on the different days of analysis. + + Using the parameters and thresholds established for diatoms, the QC beads may be run as if they were a sample and gated according to their chlorophyll fluorescence, forward scatter, and side scatter. If the beads fall within the same gates on each day of analysis, it may be assumed that samples run on different days may be fairly compared. + + **Additional Beads for Linear Calibration:** + In addition to the QC beads, beads of known diameter were used to create a linear calibration of forward scatter vs. spherical size. For this protocol, beads of 2, 4, 6, 10, and 15 μm were used (Thermo Fisher Catalog #F13838). These beads can also be used to ensure the performance of the instrument is consistent across experimental runs for FSC and SSC measures. + +## Calculating Cell Size and Metrics +9. **Size Calculation** + Using the FSC measure of the size beads and the known diameters, create a linear equation to approximate the spherical size of the diatom cells. Use the median FSC value based on a minimum of 200 cells but ideally 2000 or more. + e.g., Spherical size = (FSC +275,549)/83,539 + + Spherical size may also be considered as "equivalent spherical diameter" or ESD, as the diatom cells are not actually spherical in shape. + +10. **Other Metrics** + For other metrics (granularity and chlorophyll-a), units are given as relative fluorescence units (RFU) from the flow cytometer, which can be used for comparative purposes between samples. Again, the median value is used from a minimum of 200 cells. + + It is recommended that these values be corrected against cell size (ESD), as these traits are directly correlated with cell size, so it may be useful to assess these traits independent of size. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/fluorescence-activated-nuclei-sorting-fans-on-huma-bmh2k38e.md b/markdown-output/fluorescence-activated-nuclei-sorting-fans-on-huma-bmh2k38e.md new file mode 100644 index 0000000000000000000000000000000000000000..be318ed6bcba133a4bf61fec307bfaaab1506b3f --- /dev/null +++ b/markdown-output/fluorescence-activated-nuclei-sorting-fans-on-huma-bmh2k38e.md @@ -0,0 +1,193 @@ +```markdown +# Goal/Experiment: +Fluorescence-activated nuclei sorting (FANS) on human post-mortem cortex tissue enabling the isolation of distinct neural cell populations for multiple omic profiling. + +## Fluorescence-Activated Nuclei Sorting (FANS) on Human Post-Mortem Cortex Tissue Enabling the Isolation of Distinct Neural Cell Populations for Multiple Omic Profiling + +**Authors:** Stefania S Policicchio, Jonathan P Davies, Barry Chioza, Joe Burrage, Jonathan Mill, Emma L Dempster +**Institution:** University of Exeter Medical School, Exeter, UK +**DOI:** [dx.doi.org/10.17504/protocols.io.bmh2k38e](https://dx.doi.org/10.17504/protocols.io.bmh2k38e) +**Keywords:** FANS, post-mortem brain, nuclei, flow cytometry, anti-SOX10, anti-IRF8, anti-Neun, nuclei sorting +**License:** Creative Commons Attribution License + +### Abstract +Increased understanding of the functional complexity of the genome has led to growing recognition about the role of epigenetic/transcriptional variation in the health and disease. This protocol describes a method that uses fluorescence-activated nuclei sorting (FANS) to isolate and profile nuclei from multiple different human brain cell types from frozen post-mortem tissue. This protocol can be used to robustly purify populations of neuronal (NeuN+ve), oligodendrocytes (SOX10+ve), microglia (IRF8+ve) and other glial origin nuclei (NeuN–ve/SOX10–ve/IRF8–ve). + +--- + +## Materials and Equipment +### Equipment +| Equipment | Supplier | Catalog Number | +|---------------------------------------|---------------------|----------------| +| BD FACSAria™ III Cell Sorter | BD Biosciences | 648282-23 | +| Sorvall WX 80+ Ultracentrifuge | Thermo Scientific™ | 75000080 | +| 7mL Dounce Tissue Grinder | DWK Life Sciences | 357542 | +| PA Thin-walled Ultracentrifuge Tubes | Thermo Scientific | 03699 | + +### Reagents +| Reagent Name | Supplier | Catalog Number | +|------------------------------------------|-----------------------|----------------| +| D-Sucrose (Molecular Biology) | Fisher Scientific | 10638403 | +| Calcium Chloride (CaCl2) anhydrous, granular | Sigma-Aldrich | C1016-100G | +| Magnesium Acetate (Mg(Ace)2), 1M aq. soln | Alfa Aesar | J60041 | +| UltraPure™ 0.5M EDTA, pH 8.0 | Invitrogen | 15575020 | +| UltraPure™ 1M Tris-HCl Buffer, pH 8.0 | Fisher Scientific | 15568025 | +| 1,4-Dithiothreitol (DTT) | Sigma-Aldrich | 3483-12-3 | +| Triton™ X-100 | Sigma-Aldrich | T9284 | +| UltraPure™ DNase/RNase-Free Distilled Water (ddH2O) | Fisher Scientific | 12060436 | +| Bovine Serum Albumin (BSA) | Sigma-Aldrich | A9647-500G | +| PBS Phosphate-Buffered Saline (10X) pH 7.4 | Fisher Scientific | 10722497 | +| RNasin® Plus RNase Inhibitor | Promega | PAN2615 | +| TRIzol™ LS Reagent | Invitrogen | 11538616 | +| BD FACSDiva CS&T Research Beads | BD Biosciences | 655051 | +| BD FACS™ Accudrop Beads | BD Biosciences | 345249 | +| BD FACSFlow™ Sheath Fluid 20L | BD Scientific | 342003 | +| BD FACS Clean Solution | BD Scientific | 15875858 | +| BD FACSRinse Solution | BD Scientific | 340346 | + +### Buffers and Solutions +#### Lysis Buffer (LB) +| Component | Amount | +|------------------------|---------| +| 0.32M Sucrose | 5.47 g | +| 5mM CaCl2 | 250 μL | +| 3mM Mg(Ace)2 | 150 μL | +| 0.1mM EDTA | 10 μL | +| 10mM Tris-HCl, pH 8.0 | 500 μL | +| 1mM DTT | 17 μL | +| 0.1% Triton X-100 | 50 μL | +| Adjust with ddH2O to | 50 mL | + +#### 1.8M Sucrose Solution (SS) +| Component | Amount | +|------------------------|----------| +| 1.8M Sucrose | 30.78 g | +| 3mM Mg(Ace)2 | 150 μL | +| 1mM DTT | 17 μL | +| 10mM Tris-HCl, pH 8.0 | 500 μL | +| Adjust with ddH2O to | 50 mL | + +#### 5% BSA Solution (BB) +| Component | Amount | +|------------------------|------------| +| BSA | 200 mg | +| Dissolve in 1x PBS | 4 mL | +| Optional: RNasin® Plus RNase Inhibitor | 2 μL / 1mL | + +#### Staining Buffer (SB) +| Component | Amount | +|------------------------|---------| +| 5% BSA | 400 μL | +| 10X PBS | 400 μL | +| ddH2O | 3.2 mL | + +--- + +### Antibodies Required +| Antibody | Pre-conjugated | Supplier | Catalog No | Dilution | +|-------------------------------------|----------------|---------------|------------|----------| +| Hoechst 33342 | -- | Abcam | ab228551 | 1:500 | +| Anti-SOX10 | to NL577 | R&D systems | NL2864R | 1:10 | +| Anti-NeuN | to Alexa Fluor488 | Millipore | MAB377X | 1:1000 | +| Anti-IRF8 | to APC | Invitrogen | 17-9852-82 | 1:150 | + +--- + +## Methods + +### 1. Nuclear Prep for FACS Separation (using SOX10, IRF8, NeuN, and Hoechst) +1. The protocol yields at least 1,000,000 NeuN +ve, 1,000,000 SOX10 +ve, 400,000 IRF8 +ve (when the population is present), and 200,000 triple negative (NeuN-ve/SOX10-ve/IRF8-ve) nuclei per 500 mg of frozen human post-mortem cortex tissue. + +#### 1.1 Solution and Buffer Preps +- Solutions should be kept at 4 °C or on ice. + +| Solution | Preparation Time | +|----------|-------------------| +| LB | 1 week in advance | +| SS | 1 week in advance | +| SB | Fresh each day | + +#### 1.2 Nuclei Isolation +1. Pre-cool the ultracentrifuge to 4 °C for 30 min. +2. Pre-cool all buffers and Dounce homogenisers on ice. +3. Add 1mM DTT to SS and LB. +4. Transfer 3 mL LB to homogeniser per 500 mg human brain tissue. +5. Add dissected tissue sample into homogeniser. +6. Wait 3-5 min before douncing tissue to allow it to defrost. +7. Perform homogenisation using the TIGHT pestle. +8. Transfer 8 mL SS to PA thin-walled ultracentrifuge tubes. +9. Carefully overlay with tissue homogenate (1 mL per tube). +10. Overlay with another 1 mL LB. +11. Balance opposite tubes by weight with 1x PBS. +12. Perform ultracentrifugation for 45 min. + +#### 1.3 Post-Ultracentrifugation Steps +1. Aspirate supernatant. +2. Pour off remaining supernatant without dislodging pellet. +3. Resuspend pellet in 1 mL fresh SB. +4. Let samples sit on ice for 15 min. +5. Transfer volume into 2 mL tubes. +6. Pipette up and down several times. +7. Washing step: 1.0 x g for 5 min at room temperature. +8. Discard supernatant. +9. Resuspend in fresh SB (500 μL). + +#### 1.4 Immunostaining +1. Add antibodies to 1.5 mL stained tubes: + - SOX10 (1:10 dilution) – 150 μL Ab + - NeuN Alexa488 (1:1000) – 2 μL Ab + - IRF8 APC (1:150) – 10 μL Ab +2. Incubate tubes for 1h 30min at 4 °C in the dark. +3. Washing step: 1.0 x g for 5 min at room temperature. +4. Discard supernatant. +5. Resuspend in fresh SB. + +--- + +### 2. Fluorescence-Activated Nuclei Sorting (FANS) +2.1 **General Gating Parameters** + +For each sample, load stained and unstained tubes individually for data acquisition. + +**Gating Parameters (X-axis:Y-axis)** +- FSC-A:SSC-A +- SSC-W:SSC-A +- FSC-A:DAPI-A +- DAPI-A:FITC-A +- DAPI-A:PE-A +- DAPI-A:APC-A +- FITC-A:PE-A + +--- + +### 2.2 Data Recording Settings +In line with the experiment design, FSC, SSC, DAPI, FITC, APC and PE are the parameters for which voltage values may need to be adjusted. + +**Settings:** +- **Events to Record:** 3000 +- **Event to Display:** 1000 +- **Flow Rate:** 1.0 + +--- + +### 2.3 Sample Collection +1. Use LoBind Tubes for collection. +2. Store at -80 °C or use collected samples directly for downstream applications. + +**Notes:** +- 1 µg of genomic DNA is expected from 500 mg tissue. +- IRF8+ve population may be detectable in 5-10% of the sample. + +--- + +### 3. General Recommendations for Users +- Perform new experiment steps including setting the initial parameters and adjusting for optimal recognition of desired nuclei. Consistency is key across experiments. +- Efficiency and abort rates should adhere to given parameters. + +--- + +### References +1. BD FACSAria III User's Guide + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/fluorospot-assay-bpspmndn.md b/markdown-output/fluorospot-assay-bpspmndn.md new file mode 100644 index 0000000000000000000000000000000000000000..c36c89dbaa9da073b0e0b82a25d892087370f26d --- /dev/null +++ b/markdown-output/fluorospot-assay-bpspmndn.md @@ -0,0 +1,166 @@ +```markdown +# Goal/Experiment: +The goal of this experiment is to collect and assay mouse anti-human cells using a Fluorospot assay. + +# Fluorospot Assay + +### Abstract +This protocol details the steps required to collect and assay mouse anti-human cells using a fluorospot assay. + +### DOI +[dx.doi.org/10.17504/protocols.io.bspsmndn](https://dx.doi.org/10.17504/protocols.io.bspsmndn) + +### Protocol Citation +cecilia, Alessandro Sette 2021. Fluorospot Assay. **protocols.io** [https://dx.doi.org/10.17504/protocols.io.bspsmndn](https://dx.doi.org/10.17504/protocols.io.bspsmndn) + +### Keywords +- Antibodies +- Fluorospot + +### License +This is an open access protocol distributed under the terms of the [Creative Commons Attribution License](https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. + +### Created +- Nov 17, 2020 + +### Last Modified +- Jul 19, 2021 + +### Ownership History +- Nov 17, 2020 Megan Freund +- Dec 01, 2020 Yaqian Xu + +### Protocol Integer ID +- 44591 + +# Materials Text + +### Reagents +- IPFL for Fluorospot (Mabtech) +- Methanol +- Sterile water +- PBS (GIBCO BRL 10010-023) +- Culture media +- Peptide pools (1 and 2) +- PHA +- Stimuli +- BSA +- Fluorescence enhancer-II +- Paper towels + +### Antibodies +- Mouse anti-human IFN-γ antibody Clone 1-D1K (Mabtech) +- Mouse anti-human IL-5 antibody TRFK5 (Mabtech) +- Mouse anti-human IL-10 antibody + +### Detection Antibodies +- IFN-γ: 7-B6-1-FS-BAM +- IL-5: 5A10-WASP +- IL-10: 12G8 + +### Fluorophores +- IFN-γ: anti-BAM-490 +- IL-5: anti-WASP-640 +- IL-10: SA-550 1:200 + +### Equipment/Consumables +- Pipettes +- Pipette tips +- Centrifuge +- Incubator +- 96-well plates +- Fluorospot reader + +### Safety Warnings +See the Safety Data Sheet (SDS) for all safety hazards and warnings. + +### Procedure + +#### Day 1 + +1. **Coat plates with antibodies**. + 1. Perform all work in the laminar flow hood. + 2. Use IPFL for Fluorospot (Mabtech). + 3. Prewet plates with 50 μl 70% MeOH per well. + 4. Discard MeOH and wash the plate 3 times with 100 μl sterile water. + 5. Coat plates with 50 μl antibody diluted in PBS per well (according to table below): + +| Antibody | Starting Conc. | Final Conc. | Amount | Dilutions | +|----------|----------------|-------------|--------|-----------| +| Mouse anti-human IFN-γ antibody Clone 1-D1K Mabtech | 1 mg/ml | 5 µg/ml | 25 µl | 1:200 | +| Mouse anti-human IL-5 antibody TRFK5 Mabtech | 1 mg/ml | 5 µg/ml | 25 µl | 1:200 | +| Mouse anti-human IL-10 antibody | 1 mg/ml | 10 µg/ml | 50 µl | 1:100 | +| PBS: GIBCO BRL 10010-023 | - | - | 5 ml | - | + +2. Leave plates **overnight** at 4°C. + +#### Day 2 + +3. **Block plates** + 1. Discard coating antibody and tap on paper. + 2. Wash with 100 μl PBS per well 3x. + 3. Add 100 μl culture media. + 4. Incubate at 37°C for 1 hour. + +4. Harvest cells from culture wells (14-day expansion) with pipette (pipetting up and down), centrifuge, and count. + +5. **Prepare stimuli dilutions**: + +| Stimuli | Stock Conc. | Working Conc. (2X final conc.) | V stimuli (µL) | V media (µL) | +|---------|-------------|--------------------------------|-----------------|--------------| +| Neg | 100% | - | - | - | +| PHA | 1 mg/ml | 20 µg/ml | - | - | +| Peptide pool | 10 µg/ml | - | - | - | + +6. Plate 50 μl per well each of stimuli and PBMCs. +7. Incubate plates for 24 hours at 37°C, 5% CO₂. + +8. **Plate layout**: + +| | 1-3 | 4-6 | 7-9 | 10-12 | +|-------|------------------|------------------|----------------------|-------------------------| +| **A** | Peptide pool 1 | Peptide pool 2 | - | - | +| **B** | PHA | PHA | - | - | +| **C** | Neg for peptide pool 1 | Neg for peptide pool 2 | - | - | + +#### Day 3 + +9. Remove cells by emptying the plate and wash 5 times with 200 μl PBS per well (plate washer). +10. Dilute the detection antibodies, per full plate: + +| Component | Starting Conc. | Final Conc. | Amount | Dilutions | +|-----------|-----------------|-------------|--------|-----------| +| IFN-γ: 7-B6-1-FS-BAM | - | - | 50 μl | 1:200 | +| IL-5: 5A10-WASP | - | - | 50 μl | 1:200 | +| IL-10: 12G8 | 1 mg/ml | 2 µg/ml | 20 μl | 1:500 | +| PBS-0.1% BSA | - | - | 10 ml | - | + +11. Add 100 μl per well and incubate for 2 hours at room temperature in the dark. + +12. Wash 5 times with 200 μl PBS per well. + +13. Dilute the fluorophores, per full plate: + +| Component | Starting Conc. | Final Conc. | Amount | Dilutions | +|-----------|-----------------|-------------|--------|-----------| +| IFN-γ: anti-BAM-490 | - | - | 50 μl | 1:200 | +| IL-5: anti-WASP-640 | - | - | 50 μl | 1:200 | +| IL-10: SA-550 1:200 | - | - | 50 μl | 1:200 | +| PBS-0.1% BSA | - | - | 10 ml | - | + +14. Incubate at room temperature in the dark for 1 hour. + +15. Wash 5 times with 200 μl PBS per well. + +16. Empty the plate and add 50 μl Fluorescence enhancer-II per well and leave the plate for 15 minutes at room temperature. + +17. Empty the plate and remove residual fluorescence enhancer by firmly tapping the plate against clean paper towels. Do not wash in the sink. Remove the underdrain (the soft plastic under the plate). + +18. Leave the plate in the dark to dry; the plate should be completely dry before analysis. + +19. Inspect and count spots in a Fluorospot reader. + +20. Store plate in the dark at room temperature. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/fpcount-protocol-full-protocol-bztsp6ne.md b/markdown-output/fpcount-protocol-full-protocol-bztsp6ne.md new file mode 100644 index 0000000000000000000000000000000000000000..39a79c51cf31c89be70daf038c2eb0c453b73890 --- /dev/null +++ b/markdown-output/fpcount-protocol-full-protocol-bztsp6ne.md @@ -0,0 +1,162 @@ +```markdown +# Goal/Experiment: +The goal of this experiment is to provide a complete protocol for fluorescent protein calibration. This includes expression, purification, quantification, and calibration of fluorescent proteins using Thermo's HisPur Cobalt Resin and corresponding microplate reader analysis. The resultant data is analyzed using the R package FPCountR. + +# FPCount protocol - Full protocol + +**Authors:** +Eszter Csibra1, Guy-Bart Stan1
+1Imperial College London
+December 05, 2021 + +DOI: [10.17504/protocols.io.bztsp6ne](https://dx.doi.org/10.17504/protocols.io.bztsp6ne) + +FPCount is a complete protocol for fluorescent protein calibration, consisting of: + +1. **FP expression/purification using Thermo's HisPur Cobalt Resin.** +2. **FP concentration determination in a microplate reader.** +3. **FP fluorescence quantification in a microplate reader.** + +Results can be analysed with the corresponding R package, FPCountR. + +--- + +## Summary + +1. Expression +2. Harvesting/Washing +3. Lysis +4. Fractionation +5. Gel1: Verification of Expression/Fractions +6. Purification +7. Gel2: Verification of Purification +8. Protein concentration and buffer exchange +9. Quantification of FP concentration (part1) +10. Quantification of FP fluorescence +11. Quantification of FP concentration (part2) +12. Protein Storage +13. Calibration of Plate Reader + +## External Manuals, Protocols and Guides: + +- [HisPur_Cobalt_Resin, 10mg/ml capacity](https://www.thermofisher.com) +- [Batch and Spin Cup Methods](https://www.thermofisher.com) +- [HisPur™ Cobalt Resin](https://www.thermofisher.com) +- [Pierce™ Spin Columns - Snap Cap](https://www.thermofisher.com) +- [Amicon columns](https://www.merckmillipore.com) +- [ThermoFisher Protein assay compatibility table](https://www.thermofisher.com) +- [microBCA assay / Protein by BCA in plates](https://www.thermofisher.com) + +## Troubleshooting + +- **Protein expression yields low:** Lower temperature, increase incubation time, increase/decrease inducer. Try different strain. +- **Protein largely in insoluble fraction:** Lower temperature, decrease inducer. Consider adding a solubilizing fusion tag. +- **Protein purification yields low:** Extend elution fractions, use larger culture fraction for purification, increase binding/washing time. +- **Protein in elutions not pure:** Increase washes and/or add 1M NaCl to washes. + +## Reagents and Supplies + +- **Expression:** + - Luria Broth (LB), antibiotics, inducer +- **Cell lysis:** + - 1M Tris pH 7.5, filter-sterilised + - 2M NaCl, filter-sterilised + - ThermoFisher Pierce Protease Inhibitor Tablets (EDTA-free) + - Lysozyme (e.g., ThermoFisher 89833) + - DNase I (MP Biomedicals 219006210) + - Sonicator (e.g., Q125 QSonica) +- **Purification:** + - 1M imidazole, filter-sterilised + - HisPur Cobalt resin + - Spin columns + - Amicon spin-columns for buffer exchange (10K or appropriate cutoff) +- **Verification:** + - SDS-PAGE reagents or pre-made gels (e.g., Protean PreCast Gels) + - Coomassie blue stain (e.g., InstantBlue, Sigma) +- **Quantifications:** + - Microplates: 96-well, UV-transparent for A280 or clear for fluorescence + - BSA standards (ThermoFisher 23209, MicroBCA kit) + +--- + +## Protocol + +### 1. Expression +**Day 1: Overnight culture setup:** +- 50ml LB +- 50ul Cam +- 50ul arabinose 0.02% +- Glycerol stock scrape of transformant +- 30°C, 250rpm, overnight expression + +### 2. Harvesting and Washing +**Day 2:** +Buffers: +- **Wash buffer = T50N150**: 50 mM Tris-Cl pH 7.5, 150 mM NaCl +- **Resuspension buffer = T50N300+pi**: 50 mM Tris-Cl pH 7.5, 300 mM NaCl, protease inhibitors +- **Lysis Buffer = T50N300+pi**: lysozyme 1X (100ug/ml), DNase I (1000U/ml), MgCl2 stock, 2X Laemmli’s buffer + +Procedure: +- Prechill falcon tubes, prechill cells after incubation +- Resuspend cultures in wash buffer, centrifuge, suspend pellets in lysis buffer +- Sonicate: 50% amplitude, 10s on/off, 2 min + +### 3. Lysis +**Day 2: Pre-chill the microfuge to 4°C.** + +- **Lysis by Sonication:** + - Sonicate: 50% amplitude, 10s on/off, 2min. + - DNase I treatment (if using A280 assay later): DNase I = 31 kDa sensitive to vortexing + - DNase I stock: 1000 U/ml in ddH2O, prepare working solution for final 50U/ml + +### 4. Fractionation +**Day 2**: Spin out the insoluble fraction. +- Divide lysate into prechilled eppies, spin at 16K x g, 30min, 4°C +- Transfer soluble fractions to new tubes + +### 5. Gel1: Verification of Expression/Fractions +**Day 2**: +- Gel loading: Colour prestained protein markers, soluble fractions, and insoluble fractions. +- Run gel: 10mA per gel till dye front reaches bottom. +- Stain overnight, rinse, and image. + +### 6. Affinity Purification +Nickel and Cobalt resins: NiNTA and HisPur +- HisPur Cobalt resin: better specificity than NiNTA, 10mg/ml capacity. +- Use Pierce Spin Columns: max volume 300ul = 3mg protein max + +**Day 2 contd: Resin Handling:** +- Prepare HisCobalt resin +- Equilibrate resin using BB solution (Binding Buffer), repeat 3x with 15’ RT incubation. + +### 7. Gel2: Verification of Purification +**Day 3**: +- Check purification using lysate vs. flowthrough vs elution. +- Load gel: follow Gel1 steps for loading, running, staining, and imaging. + +### 8. Protein concentration and buffer exchange +**Day 2 contd**: +- Concentrate elution fractions using Amicon Ultra 10k columns, recover, resuspend to required concentration. + +### 9. Quantification of FP concentration (part1) +**Day 2 contd**: +- Set up protein quantitation assays: microBCA, A280, and ECmax. +- Prepare BSA standards for parallel treatment. + +### 10. Quantification of FP fluorescence +**Day 3 contd**: +- Measure fluorescence using clear plates, set up fluorescence channels accordingly. + +### 11. Quantification of FP concentration (part2) +**Day 3 contd**: +- Finish with microBCA assay, employ colorimetric reaction and measure at 562nm. + +### 12. Protein Storage +**Day 4**: +- Store proteins in the fridge, protected from light, stable for up to 4 weeks. + +### 13. Calibration of Plate Reader +- Use FPCountR package for analyzing the data, converting fluorescence units to FP molecules. + +`endofoutput` +``` \ No newline at end of file diff --git a/markdown-output/fpcount-protocol-short-protocol-bzt6p6re.md b/markdown-output/fpcount-protocol-short-protocol-bzt6p6re.md new file mode 100644 index 0000000000000000000000000000000000000000..5fecdf4260b215d07d9fe5cf812fbeeaa04d2b47 --- /dev/null +++ b/markdown-output/fpcount-protocol-short-protocol-bzt6p6re.md @@ -0,0 +1,215 @@ +```markdown +# Goal/Experiment: + +This experiment involves the complete protocol for fluorescent protein calibration. The protocol encompasses expression and purification of fluorescent proteins (FPs) using HisPur Cobalt Resin, their concentration determination and fluorescence quantification using a microplate reader. The results can be analyzed using the corresponding R package, FPCountR. + +## FPCount Protocol - Short Protocol + +**Authors:** +- Eszter Csibra +- Guy-Bart Stan + +Imperial College London + +DOI: [10.17504/protocols.io.bzt6p6re](https://dx.doi.org/10.17504/protocols.io.bzt6p6re) + +--- + +## Summary + +1. Expression +2. Harvesting/Washing +3. Lysis +4. Fractionation +5. Purification +6. Protein concentration and buffer exchange +7. Quantification of FP concentration (part1) +8. Quantification of FP fluorescence +9. Protein storage +10. Calibration of Plate Reader + +--- + +## External Manuals, Protocols, and Guides + +- [HisPur Cobalt Resin (10mg/ml capacity)](https://www.thermofisher.com/order/catalog/product/89965) +- [Batch and Spin Cup Methods](https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0015641_HisPur_Cobalt_Resin_10026212_PIs.pdf) +- [Pierce Spin Columns - Snap Cap](https://www.thermofisher.com/order/catalog/product/69705) +- [Amicon columns](https://www.millipore.com) + +--- + +## Troubleshooting + +- **Protein expression yields low:** + - Lower temperature, increase incubation time, increase inducer/decrease inducer. Try different strain. +- **Protein mostly in insoluble fraction:** + - Lower temperature, decrease inducer. Try different strain. + - Refolding proteins from insoluble fraction is possible but not recommended. + - Adding a solubilizing fusion tag (e.g., maltose-binding protein). +- **Protein purification yields low:** + - Concentrate existing elution fractions. + - Increase binding time or number of elutions/elution time. + - Adjust imidazole concentration in binding/elution buffer. + - Increase washes/add 1M NaCl. + - Adjust His tag position or linker length. + +--- + +## Reagents and Equipment + +1. **Expression:** + - Luria Broth (LB), antibiotics, inducer +2. **Cell lysis:** + - 1M Tris pH 7.5, filter-sterilized (f/s) + - 2M NaCl (f/s) + - Thermo Fisher Pierce Protease Inhibitor Tablets (EDTA-free) + - Lysozyme (store as powder or 500X stock) - e.g., ThermoFisher 89833 + - Sonicator (e.g., Q125 QSonica) + - DNase I (1,000 U/ml) - e.g., MP Biomedicals 219006210 +3. **Purification:** + - 1M imidazole (f/s) + - HisPur Cobalt Resin + - Spin columns - e.g., Amicon spin columns for buffer exchange (10K or appropriate cutoff) +4. **Quantifications:** + - 96-well plate (e.g., Corning 3370 or Greiner 655090) + - Plate reader + - Multipipette (optional); P200 multichannel + +f/s = filter-sterilized + +--- + +## Step-by-Step Protocol + +### 1. Expression (Day 1) + +**Overnight culture set-up:** +- 50 ml LB +- 50 ul cam +- 50 ul arabinose 0.02% +- Glycerol stock scraping of BL21/pS381_ara_His-FP transformant (or equivalent) +- 30°C, 250 rpm, spread overnight expression... + +### 2. Harvesting and Washing (Day 2) + +**Buffers:** +- **Wash buffer = T50N150** + - 50 mM Tris-Cl pH 7.5, 150 mM NaCl + - Doesn't need protease inhibitors +- **Resuspension buffer = T50N300+pi** + - 50 mM Tris-Cl pH 7.5, 300 mM NaCl, protease inhibitors + +### 3. Lysis (Day 2) + +**Prep for next stage:** +- Pre-chill the microfuge to 4°C + +**Lysis by Sonication:** +- Stand falcon in ice +- Sonicate: 50% amplitude, 10s on/off, 2 min +- Check the solution goes from turbid to clear. + +**DNase I Treatment (optional):** +- Prepare DNase I stock: 1,000 U/ml DNase I in ddH2O +- Add to lysates in T50N300 + - 50 U/ml DNase I final + - 5 mM final CaCl2 + - 50 mM final MgCl2 +- Mix thoroughly +- Incubate for 30 min at 4°C + +### 4. Fractionation (Day 2) + +- Spin out soluble fraction +- Split 2 ml lysates into pre-chilled eppies +- Spin for 30 min at 16 Kg, 4°C + +**Result:** +- Transfer SOLUBLE fractions to new tubes + +### 5. Affinity Purification (Day 2 contd) + +- Take HisCobalt resin out, shake gently +- Add 600 ul 50% resin to spin column +- Spin 1', 1,000 g to remove storage buffer +- Add binding buffer (BB) T50N300+pi, 10 mM imidazole +- Cap, mix, uncap, discard flowthrough, repeat 3X + +### Binding (Day 2 contd) + +- Add 10mM imidazole to samples before adding to column +- Add 600ul FP lysate to column +- Bind at RT for 15-90 min, flicking to mix every 5-10 min +- Spin 1', 1,000 g, keep flowthrough +- Repeat binding 1X-7X + +**Washing:** +- Add 300-600 ul BB, mix, uncap, spin, discard flowthrough +- Repeat 5X + +**Elution:** +- Add elution buffer (EB) T50N300, pi, 150 mM imidazole +- Mix, incubate 5', spin, transfer elutions to fresh eppies +- Repeat 3X + +--- + +### 6. Protein Concentration (Day 3) + +- Do a buffer exchange into T5N15+pi before next steps +- Elute and concentrate + +--- + +### 7. Plate Reader Calibration for FP (Day 3) + +- Allow using FPCountR package +- Perform 1 fluorescence assay - ECmax assay +- Need 100ul elution of FP to be calibrated. + +### 8. Quantification of FP Concentration (Day 3 contd) + +- Prepare FP dilutions (200 ul) +- 200 ul clear - ECmax assay/fluorescence assay + +**Make Protein Dilutions in Eppies:** + +| dilution | FP (ul) | Buffer (ul) | Vol left (ul) | +|----------|---------|-------------|---------------| +| 1 | 100 elution | 900 | 500 | +| 2 | 500 prev dilution | 500 | " | +| 3-11 | " | " | " | + +**Fill standard clear 96-well plates:** + +| A | B | C | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | +|---|---|---|---|---|---|---|---|---|---|---|---|---|----|----| +| FP1 | row1 | A | dilution1 (neat) | dilution2 (1:2) | d3 | d4 | d5 | d6 | d7 | d8 | d9 | d10 | d11 | buffer | + +### 9. Measure Absorbance - ECmax assay + +- Prewarm plate reader to growth temperature. +- Scan absorbance A200-1000. + +### 10. Quantification of FP Fluorescence (Day 3 contd) + +**Measure fluorescence in clear plate:** + +| A | instrument | plate | lid | scan | time taken | +|---|------------|-------|-----|------|------------| +| 1 | instrument1 | clear | seal | FP-dependent channel1 | 10 min | +| 2 | instrument1 | clear | seal | FP-dependent channel2 | 10 min | + +### 11. Protein Storage + +- Store proteins in the fridge, protected from light for up to 4 weeks. + +### 12. Analysis - Calibration of Plate Reader + +- Aim: Relate FP molecules to fluorescence units. +- Use FPCountR package for data analysis. + +## endofoutput + +``` \ No newline at end of file diff --git a/markdown-output/fresh-frozen-tissue-staining-with-codex-tagged-ant-2qpgdvn.md b/markdown-output/fresh-frozen-tissue-staining-with-codex-tagged-ant-2qpgdvn.md new file mode 100644 index 0000000000000000000000000000000000000000..7e65a321c5bbd6291e9b91a9118b80c757bf56f2 --- /dev/null +++ b/markdown-output/fresh-frozen-tissue-staining-with-codex-tagged-ant-2qpgdvn.md @@ -0,0 +1,162 @@ +```markdown +# Fresh Frozen Tissue Staining with CODEX Tagged Antibody Panel + +## Goal/Experiment: +The goal of this experiment is to perform fresh frozen tissue staining using the CODEX technology, which utilizes oligo-labeled antibodies, specialized fluorescent probes, and a companion instrument to achieve single-cell resolution fluorescence data across multiple spatial parameters within a single tissue section. + +## Authors +Yury Goltsev, Nikolay Samusik, Julia Kennedy-Darling, Salil Bhate, Matthew Hale, Gustavo Vazquez, Sarah Black, Garry Nolan + +## Abstract +CODEX is a technology that uses oligo-labeled antibodies, specialized fluorescent probes, and a companion instrument alongside a standard fluorescence microscope to create single-cell resolution fluorescence data across a multitude of parameters within spatial context in a single tissue. CODEX was developed by Yury Goltsev and Nikolay Samusik in the laboratory of Garry Nolan at Stanford. The technology is being commercialized by Akoya Biosciences. + +## External Link +A more detailed version of this protocol is available here: [Detailed Protocol](https://www.cell.com/cell/pdf/S0092-8674(18)30904-8.pdf) + +## Guidelines +- Ensure that tissues never dry out after the rehydration step. +- Never pipet directly on top of tissue. Dispense liquids on the corner of the coverslip. +- Coverslips are best handled with the bent-tip tweezers found in the materials section. + +## Materials +| PRODUCT | PROVIDER | CATALOG NUMBER | +|--------------------------|------------------------|-----------------| +| Coverslip Staining Jar | Ted Pella | 21040 | +| Bent tip forceps | Fine Science Tools | 11251-33 | +| 6-well TC plates | VWR | 10861-554 | +| 16% PFA | EMS | 15710 | +| 1X DPBS | Thermofisher | 14190144 | +| Drierite | Fisher Scientific | 23-116582 | +| Acetone | Sigma-Aldrich | 650501 | +| Methanol | Sigma-Aldrich | 34860 | +| Staining Buffer 1 | Akoya | RGT2001EA | +| Staining Buffer 2 | Akoya | RGT2001EA | +| Staining Buffer 3 | Akoya | RGT2001EA | +| Blocking Component 1 | Akoya | RGT2001EA | +| Blocking Component 2 | Akoya | RGT2001EA | +| Blocking Component 3 | Akoya | RGT2001EA | +| Blocking Component 4 | Akoya | RGT2001EA | +| Fixative (F) aliquots | Akoya | RGT2001EA | +| CODEX tagged antibodies | Akoya | RGT2000EA | + +## Before Starting +- Antibody staining volumes should be determined by prior titration experiments. A good start is 1μl per 200μl cocktail. +- Create a humidity chamber by re-purposing a pipet tip box. Remove the tray with holes that holds the tips, place a wet paper towel in the bottom of the box, replace the tray, and cover with the lid. + +## Tissue Preparation +1. **Preparation:** + - Begin with fresh frozen tissues sectioned onto 22mm² poly-lysine coated coverslips and stored at -80°C. + - Add a 5mm layer of drierite granules on the bottom of a pipet tip box. + - Place a piece of kimwipe on top of the drierite. + - Retrieve the coverslips from the freezer using bent-tip tweezers and place them tissue-side-up on the kimwipe. + - Scratch with the tip of the tweezers on the OCT layer to identify which side of the coverslip has the tissue. + - Close the box and let sit at room temperature (RT) for 2 minutes. + +2. **Acetone Incubation:** + - Transfer coverslips to a coverslip staining jar containing acetone. + - Incubate at RT for 10 minutes. + +3. **Air-Drying:** + - Arrange coverslips on a paper towel, tissue side up, and allow to air-dry for 2 minutes. + +4. **First Rehydration:** + - Fill the wells of a 6-well TC plate with ~5ml Staining Buffer 1 and transfer the coverslips into the wells. + - Incubate for 2 minutes. + +5. **Second Rehydration:** + - Fill the wells of another 6-well TC plate with ~5ml Staining Buffer 1 and transfer the coverslips into the wells. + - Incubate for 2 minutes. + +6. **Fixation Preparation:** + - Prepare fixing solution in a conical tube by making a 10-fold dilution of 16% PFA in Staining Buffer 1. + - Each coverslip will require 2ml of fixing solution. + +7. **Fixation Incubation:** + - Add 2ml fixing solution to new wells and transfer the coverslips into the fixing solution. + - Incubate for 10 minutes. + +8. **Post-Fixation Rehydration:** + - After fixation, transfer the coverslips to wells containing ~5ml Staining Buffer 1. + +9. **Equilibration:** + - Transfer the coverslips to new wells containing ~5ml Staining Buffer 2. + - Allow to equilibrate for at least 2 minutes. + +## Antibody Cocktail Preparation +10. The antibody cocktail is a combination of all the antibodies in the panel plus CODEX Blocking Buffer. +11. Prepare 200μl CODEX Blocking Buffer for each coverslip to be stained: + - 180μl Staining Buffer 2 + - 5μl Blocking Component 1 + - 5μl Blocking Component 2 + - 5μl Blocking Component 3 + - 5μl Blocking Component 4 + +12. The total volume per coverslip is 200μl. +13. Combine antibodies and CODEX Blocking Buffer and gently mix with pipet. + +## Staining +14. **Application:** + - Remove coverslips from Staining Buffer 2, dab the corner on a kimwipe to remove most of the liquid, place tissue side up on top of the humidity chamber, and pipet 190μl antibody cocktail on top. + - Incubate at RT for 3 hours. + +## Post-Processing of Stained Tissue +15. **Initial Stain Fixation:** + - Take coverslips, dab a corner on a kimwipe to remove most of the liquid, and place into wells containing ~5ml Staining Solution 2. + - Incubate for 2 minutes. + +16. **Washing:** + - Transfer coverslips to new wells with Staining Solution 2 for a total of 2 washes. + - Incubate for 2 minutes. + +17. **Final Fixing Solution Preparation:** + - Prepare fixing solution in a conical tube by making a 10-fold dilution of 16% PFA in Staining Buffer 3. + - Each coverslip will require 2ml of fixing solution. + +18. **Final Fixation:** + - Add 2ml fixing solution to new wells and transfer the coverslips into the fixing solution. + - Incubate for 10 minutes. + +19. **Slushy Ice Bath:** + - Place a 6-well TC plate on a bed of slushy ice. + - Allow the plate to chill for 2 minutes, then add ~5ml ice cold (4°C) methanol to one well per coverslip. + +20. **Transport to PBS:** + - After the 10-minute fixation, transfer the coverslips to wells containing ~5ml PBS. + - Transfer the coverslips to new wells containing PBS, for a total of 3 washes. + +21. **Ice Cold Methanol Incubation:** + - Transfer the coverslips to the ice cold methanol. + - Incubate for 5 minutes. + +22. **Final Fixative Aliquots:** + - After the 5-minute incubation, transfer the coverslips to wells containing ~5ml PBS. + - Transfer the coverslips to new wells containing PBS, for a total of 3 washes. + - Prepare 200μl Final Fixative Solution for each coverslip: + - One aliquot of CODEX Final Fixative (F) yields 1ml Final Fixative Solution for 5 coverslips. + - Cut aliquots from the Final Fixative (F) strip, allow to thaw, and quickly spin down. + - Pipet 20μl Final Fixative (F) into 1ml PBS. Gently mix with pipet. + +23. **Final Stain Fixing:** + - Rinse and dry humidity chamber tray to remove any residual antibodies from the staining. + - Take the coverslips, dab the corners on a kimwipe, and place them on the humidity chamber. + - Add 190μl Final Fixative Solution on top of each coverslip, taking care to not pipet directly on the tissue. + - Ensure that the entire tissue is covered in solution. + - Incubate for 20 minutes. + +24. **Post-Final Fixation:** + - After the 20-minute fixation, transfer the coverslips to wells containing ~5ml PBS. + - Transfer the coverslips to new wells containing PBS. + - Transfer the coverslips to new wells containing PBS, for a total of 3 washes. + +25. **Storage:** + - Store the stained and fixed samples in a 6-well TC plate in ~5ml Staining Buffer 3 at 4°C for up to 2 weeks. + +| Final Fixative Solution | 1-5 coverslips | 6-10 coverslips | +|-------------------------|----------------|------------------| +| PBS | 1000μl | 2000μl | +| (F) Aliquots | 20μl | 40μl | + +--- + +**endofoutput** +``` \ No newline at end of file diff --git a/markdown-output/frozen-tissue-nuclei-extraction-for-10xv3-snseq-bi62khge.md b/markdown-output/frozen-tissue-nuclei-extraction-for-10xv3-snseq-bi62khge.md new file mode 100644 index 0000000000000000000000000000000000000000..f335e7331e162d0828f9940ae3314c073a1586ce --- /dev/null +++ b/markdown-output/frozen-tissue-nuclei-extraction-for-10xv3-snseq-bi62khge.md @@ -0,0 +1,152 @@ +```markdown +# Goal/Experiment: +Extraction of nuclei from frozen tissue in preparation for single-nuclei sequencing (droplet-based/10X). + +# Frozen Tissue Nuclei Extraction (for 10xV3 snSEQ) V.2 + +## Abstract +Protocol for extraction of nuclei from frozen tissue in preparation for single-nuclei sequencing (droplet-based/10X). + +**This protocol is based strongly on a similar extraction protocol from the [McCarroll lab](https://mccarrolllab.org/).** + +## Protocol Citation +Carly Martin, Abdul Abdul, Charles Vanderburg, Naeem Nadaf, Ashley Feirrera, Evan Macosko 2020. Frozen Tissue Nuclei Extraction (for 10xV3 snSEQ). *protocols.io*. dx.doi.org/10.17504/protocols.io.bi62khge + +## Keywords +nuclei extraction, isolation, frozen, single-nuclei sequencing + +## Nuclei Extraction Protocol + +### 1. Nuclei Extraction Protocol, Optimized for Small Tissue Pieces +- **Macosko Lab, Stanley Center, Broad Institute** + +### 2. Dissociation Buffer: DB +Prepare a stock of 500ml of Dissociation Buffer (DB) using ultrapure nuclease-free water and these reagents: +- Na₂SO₄: 5.83 g +- K₂SO₄: 2.615 g +- Glucose: 0.905 g +- HEPES: 1.2 g +- MgCl₂: 2.5 ml + +### 3. Extraction Buffer: ExB +- DB: 15 ml +- Kollidon VA64: 150 mg +- TX-100: 150 µl (final concentration 1%) +- 10% BSA: 15 µl (final concentration 0.01%) +- RNase inhibitor (Lucigen): 1 tube (10,000 units) + +### 4. Wash Buffer: WB (30 ml per sample) +- DB: 30 µl +- 10% BSA: 30 µl +- RNase inhibitor (Lucigen): 200 µl (10,000 units) + +### 5. FACS Capture Buffer (20 µl per sample) CDB +- DB: 20 ml +- 10% BSA: 20 µl +- RNase inhibitor (Lucigen): 1 tube (10,000 units) + +### 6. 5% BSA–DB for FACS (200 µl per sample) + +### 7. Procedure +- Chill all buffers to 4°C before use. +- Pre-cool centrifuge to 4°C. +- Chill all tubes and well plates on ice for 20 minutes before use. +- Perform all steps on ice. + +### 8. Preparation Steps +- Chill a 26-gauge needle, 40µm cell strainer, and syringe at 4°C for at least 20 minutes. + +### 9. Coating and Washing +- Coat a 12 well culture plate with 1% BSA, wash with DB, and fill wells with 1ml ExB buffer. + +### 10. Tissue Sample Wash +- Wash frozen tissue sample out of the PCR tube with 150µl ExB buffer and deposit into well of the well plate. + +### 11. Trituration +- Pipette 1ml volume up and down with a 1ml Rainin tip (#30389212) without creating froth/bubbles 20x, wait 2 minutes, and repeat 4-5 times (check dissociation at 5th pass). + +### 12. Needle Passing +- Pass entire volume twice through a 26-gauge needle into the well. + +### 13. Transfer Extracted Sample +- Transfer extracted sample (~1ml) into a 50ml falcon tube, pre-coated with 1% BSA. Add 30ml of wash buffer to dissociate, then split the volume between two pre-coated falcon tubes (15+ ml each). + +### 14. Centrifugation +- Spin down at 600xg for 10 minutes at 4°C. + +### 15. Supernatant Aspiration +- Aspirate supernatant until 500µl remains in each tube. Pool these two half samples to 1ml. + +### 16. Filtration +- Pass through a pre-cooled 40µm cell strainer (gravity only) into a new, clean, pre-cooled falcon tube. + +### 17. Transfer Filtrate +- Transfer (and measure volume of) filtrate to a 1.5ml Eppendorf tube (pre-cooled). + +### 18. Staining +- Stain the filtered nuclei by adding DAPI (Thermo, #62248) at 1:1000 dilution. + +### 19. Preparing Collection PCR Tubes +- Coat a 0.2ml PCR tube with 5% BSA. Use a chilled 96 well FACS plate (Sony M800 FACSorter) to capture nuclei. Pre-fill the PCR tube with 20 µl of CDB to act as a cushion. + +### 20. FACS Enrichment for Singlets +- Use FACS with 6 forward scatter gain of 1% on DAPI gate in “purity” mode. Do not spin down after FACS unless gentle spin protocol is required. + +### 21. FACS Enrichment Settings +- Use a chilled (-20°C) 96-well cold block for FACS collection. Use a Sony SH800 sorter with a 70µm chip, with these settings: + +| Parameter | Setting | +| ----------------- | ---------------- | +| Laser (405nm) | On | +| Laser (488nm) | On | +| Laser (561nm) | On | +| Laser (638nm) | On | +| FSC | 4 | +| BSC | 27.0% | +| FL1 | 40.0% | +| FL2 | 40.0% | +| FL3 | 40.0% | +| FL4 | 40.0% | +| FL5 | 40.0% | +| FL6 | 40.0% | +| Sample Pressure | 6 | +| Threshold Value | 1.00% | +| Forward Window Ext| 50 | +| Back Window Ext | 50 | + +### 22. Calculate Nuclei Concentration +- Use a pipette to measure the sample volume in each tube post FACS. +- Prepare a 1:10 dilution of 18µl chilled DB with 2µl of nuclei PCR tube. +- Count nuclei with a Fuchs–Rosenthal Hemocytometer (16 chambers): + - Visualize, count all 16 large squares, calculate the average. + - Multiply by 10 (accounting for dilution), then by 5 (FC hemocytometer factor). + - This gives your final concentration in nuclei/µL. + +### 23. Proceed to 10X Protocol +- For 10X v3, the input nuclei volume is 46.6µL, and the maximum concentration you can input is 364 nuclei/µL. +- Input 17K nuclei into 46.6µL mix for maximum data output. +- If fewer nuclei are collected than required, follow gentle spin protocol to increase nuclei concentration. + +### 24. Gentle Spinning Protocol + +#### 25. Preparation +- Use a 0.5ml Axygen tube in a centrifuge (Labnet PrismR), coat with BSA, drain and cool on ice. + +#### 26. Adding Nuclei +- Add your fixed nuclei to the cooled tubes to a 200 µL max volume. + +#### 27. Centrifugation +- Spin at 4°C in a mini-fuge: + - 1 minute @ 200 xg, + - 1 minute rest, + - 1 minute @ 200 xg, + - 1 minute rest. + +#### 28. Aspirate Top +- Carefully aspirate top to desired concentration without losing nuclei. + +#### 29. Gently Resuspend +- Resuspend nuclei before adding to 10X solution by gentle up and down mix (2-3 times). + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/frozen-tissue-nuclei-extraction-v2-7xchpiw.md b/markdown-output/frozen-tissue-nuclei-extraction-v2-7xchpiw.md new file mode 100644 index 0000000000000000000000000000000000000000..035eb10fcdeb3a1fe9511093204b7683abe15635 --- /dev/null +++ b/markdown-output/frozen-tissue-nuclei-extraction-v2-7xchpiw.md @@ -0,0 +1,109 @@ +```markdown +# Goal/Experiment: +Extraction of nuclei from frozen tissue for single-nuclei sequencing, employing droplet-based/10X technology. + +# Frozen Tissue Nuclei Extraction (v2) + +**Authors**: Carly Martin¹, Abdul Abdul¹, Charles Vanderburg¹, Naeem Nadaf¹, Ashley Feirrera¹, Evan Macosko¹ +**Affiliation**: ¹Broad Institute +**Date**: Oct 08, 2019 +**Link**: [dx.doi.org/10.17504/protocols.io.7xchpiw](https://dx.doi.org/10.17504/protocols.io.7xchpiw) +**Reviewer**: Velina Kozareva + +**Abstract** +Protocol for extraction of nuclei from frozen tissue in preparation for single-nuclei sequencing (droplet-based/10X). + +This protocol is based strongly on a similar extraction protocol from the [McCarroll lab](https://mccarrolllab.org/). + +### Materials and Reagents + +**Disassociation Buffer** +Below are the components and preparation details: + +| Reagent | MW | Concentration (mM) | For 1L (g or mL) | For 2L (g or mL) | +|------------------------|---------|--------------------|------------------|------------------| +| Na₂SO₄ (Sodium Sulfate) | 142.04 | 82 | 11.65 | 23.3 | +| K₂SO₄ (Potassium Sulfate)| 174.26 | 30 | 5.23 | 10.46 | +| Glucose | 180.2 | 10 | 1.81 | 3.62 | +| HEPES | 238.3 | 10 | 2.39 | 4.78 | +| MgCl₂·6H₂O | 203.31 | 5 | 5 mL (1M stock) | 10 mL (1M stock) | + +**Disassociation Buffer + Extraction Additives** +- Dissociation Buffer +- 1% Kollidon VA64 +- 1% Triton X100 +- 1:40 RNase-inhibitor + +### Protocol Steps + +1. **Make Disassociation Buffer** + Prepare 50 mLs per sample: + Combine the reagents as specified in the table above. + +2. **Prepare Extraction Buffer** + Prepare 3 mLs per sample: + Combine the disassociation buffer with 1% Kollidon VA64, 1% Triton X100, and 1:40 RNase-inhibitor. + +3. **Chill Buffers** + - Chill all buffers to 4°C and maintain on ice when in use. + +4. **Prepare Equipment** + - Cold block dissecting tray, maintained at -20°C. + - Chilled clean razor blades and glass slides. + - 3 mL syringe with a 26 1/2 gauge needle, chilled to 4°C. + - 12-well tissue-culture plate with well-bottoms colored with a marker, placed on ice. + - Place 2 mL of chilled extraction buffer in the first well. + - Falcon tube with ~50 mL chilled DB (Dissociation Buffer). + - DAPI (1:1000) (diluted from [ThermoFisher D1306](https://www.thermofisher.com/order/catalog/product/D1306)). + - 50 µm Eppendorf tube filter, chilled to 4°C (available [here](https://us.sysmex-flowcytometry.com/consumables/celltrics-filters/1445/sterile-single-pack-celltrics-filters-50)). + - Two 50 mL tubes placed on ice. + - c-Chip FR hemocytometer. + +5. **Collect Supplies for FACS** + - 96-well cold block, chilled to -20°C ([Eppendorf](https://www.daigger.com/eppendorf-pcr-coolers-14616-group)). + - 200 µL 5% BSA-DB. + - 20 µL collection buffer: DB with 1:40 RNase-inhibitor (prepare immediately before FACS session). + +6. **Begin Extraction (All Steps with Cold Buffers on Ice)** + - Chop the tissue on ice, place it immediately into the extraction buffer. + - Shave the frozen sample on the glass slide, removing excess frost/damaged tissue. Prepare 2 mL cold extraction buffer by pipetting up and down 20 times. + +7. **Detergent Incubation and Trituration** + - Perform mechanical trituration by pipetting up and down 20 times with a 1000 µL pipette approximately every 2.5 minutes over a 10-minute incubation period. + +8. **Mechanical Dissociation** + - Use the chilled needle/syringe to express the sample for final mechanical dissociation. Repeat if necessary. + +9. **Large Volume Wash** + - Extract nuclei into an empty 30 mL tube, add chilled DB to ~30 mL total volume. + +10. **Split Sample** + - Divide the nuclei into two 50 mL conical tubes (~15 mL each). + +11. **Centrifuge** + - Spin at 600 rcf in a swinging bucket centrifuge for 10 minutes at 4°C. + +12. **Remove Supernatant** + - Carefully aspirate ~13 mL wash buffer with a serological pipette, avoid disturbing the pellet. Leave 500-300 µL in the bottom. Resuspend the nuclei carefully. + +13. **Filter Nuclei Clumps** + - Use a 40 µm eppendorf tube filter to filter the nuclei sample into an epi tube. + +14. **Stain DNA** + - Add 1 µL DAPI to 1 mL nuclei sample, incubate and place the nuclei into a FR hemocytometer to check concentration and quality. + +15. **FACS Enrichment for Single Nuclei** + - Use a 70 µm chip on a Sony SH800 sorter with specific settings. + +16. **Prepare Collection PCR Tubes** + - Coat PCR tube walls with 5% BSA-DB, wash with 200 µL DB, and add 20 µL collection buffer. + - FACS into the tube using a 1% FSC threshold on the DAPI peak. + +17. **Calculate Nuclei Concentration** + - Create a 1:10 dilution and count nuclei using a hemocytometer. Multiply average by 10 (dilution factor) and then by 5 (hemocytometer factor) to get the concentration in nuclei/µL. + +18. **Preparation for 10X Sequencing** + - Maximum 17000 nuclei, with post-loss input of 10000. Max conc. for 10X v3: 364 n/µL; input vol.: 46.6 µL. + +### endofoutput +``` \ No newline at end of file diff --git a/markdown-output/full-contact-microbiology-a-k-a-diatom-transformat-fzrbp56.md b/markdown-output/full-contact-microbiology-a-k-a-diatom-transformat-fzrbp56.md new file mode 100644 index 0000000000000000000000000000000000000000..cc590320685d5f65d83487ebf98ebc7318d47efa --- /dev/null +++ b/markdown-output/full-contact-microbiology-a-k-a-diatom-transformat-fzrbp56.md @@ -0,0 +1,167 @@ +```markdown +# Goal/Experiment: +The aim of this experiment is to perform diatom transformation via bacterial conjugation, specifically transforming Phaeodactylum tricornutum using E. coli as the conjugating agent. + +# Full Contact Microbiology (a.k.a Diatom Transformation via Bacterial Conjugation) Version 2 + +Jeric Harper + +## Abstract +Citation: Jeric Harper Full Contact Microbiology (a.k.a Diatom Transformation via Bacterial Conjugation). protocols.io https://www.protocols.io/view/full-contact-microbiology-a-k-a-diatom-transforma-fzrbp56 Published: 18 Oct 2016 + +## Before start + +- Make sure you are using fresh _E. coli_ cells streaked for isolation on LB + antibiotics no more than 1 week from -80°C cryostock. + +## Protocol + +### Diatom cultivation (liquid) +**Step 1.** + +Grow the diatom culture to mid-log phase (approximately 8.0E^6 cells/ml for _Phaeodactylum tricornutum_ grown on F/2 media or 5.0 E^7 cells/ml when grown on BG-11). We have investigated transformation efficiency throughout the growth curve and found this to be the sweet spot. + +**Cultivation Conditions:** +- Cultivation in 0.2 um-filtered L1 or BG-11 media prepared using 32ppt seawater collected from the Gulf of Mexico. +- 80 - 100 µmol photons m^-2 s^-1 provided by Philips Daylight Delux 40 watt T12 fluorescent tubes. +- Semi-continuous operation in 1L bubble column bioreactors (500 - 800 ml working volume). +- 0.2 µm filtered air containing 1% CO2 provides aeration/agitation at 0.2 vvm. + +> **Note:** The original protocol plated the culture (250uL of 1.0E8 cells/ml) on 1/2 strength L1, 1% agar plates for 4 days prior to transformation. +> We have been investigating liquid cultivation because many diatom species do not survive on agar. + +### Prepare _E. coli_ starter culture +**Step 2.** + +Grow 1 mL of _E. coli_ culture containing both the mobility plasmid (Pta-MOB) and carrier plasmid, overnight (16-20 hrs) in LB+antibiotics, for each planned transformation. (We grow them at 37°C at 270 rpm in a shaking incubator.) + +**Duration:** 16:00:00 + +> **Annotation:** In my experience it has proven beneficial to perform conjugations with multiple donors carrying THE SAME construct. 2/3 worked beautifully, one failed completely. +> _- Jernej Turnsek, 19 Oct 2016_ + +### Outgrow _E. coli_ +**Step 3.** + +On the day of transformation, use the overnight culture to inoculate 50 mL of fresh LB+antibiotic, 1:50 dilution, for each planned transformation. +- Grow to an OD600 of 0.8 - 1.0 (37°C with 270 rpm shaking). +- This takes about 3-4 hours. + +**Duration:** 03:00:00 + +> **Annotation:** The authors of The original protocol reported that the OD600 range is flexible. Transformation success has been seen within OD600 range of 0.4 to 1.2. +> _- Jeric Harper, 06 Oct 2016_ + +### _P. tricornutum_ cell concentration +**Step 4.** + +During the 3-4 hours the _E. coli_ culture is growing, measure the _Phaeodactylum tricornutum_ cell concentration with a FlowCam or haemocytometer to calculate the required volume needed to collect 2.5E^8 cells for each transformation. + +**Duration:** 00:05:00 + +### Concentrate the diatom and _E. coli_ cultures +**Step 5.** + +For each transformation, centrifuge 50 mL of _E. coli_ culture and the required _Phaeodactylum tricornutum_ volume at 4000 x g for 10 minutes at 4°C. +- Resuspend _E. coli_ pellet in 500 µL of SOC medium. +- Resuspend _P. tricornutum_ pellet in 500 µL of L1 medium. + +> **Note:** The diatom and _E. coli_ cultures should be centrifuged at around the same time to minimize the amount of time they spend concentrated. + +**Duration:** 00:10:00 + +> **Annotation:** The original protocol scraped the agar plates the culture was initiated on using 500uL F/2, then adjusted the volume to attain 5.0E^8 cells/ml. We have found no difference so far in transformation efficiency between liquid- and plate-initiated cultures. +> _- Jeric Harper, 04 Oct 2016_ + +### Conjugation +**Step 6.** + +In a 1.5 mL tube mix 200 µl of _E. coli_ cells with 200 µl of _Phaeodactylum tricornutum_ cells. + +Negative control: In a 1.5 mL tube mix 200 µl of SOC medium with 200 µl of _Phaeodactylum tricornutum_ cells. + +**Note:** Incubate and treat the negative control plates identically to conjugation plates. + +> **Annotation:** The authors of The original protocol suggested spreading the mixture near, but not touching, the edges of the plate, as this mixture will be scraped and replated in 2 days. N concentration seems to be important. We have found greater transformation success in using 0.5x BG-11 medium made with seawater instead of 0.5x L1 medium. +> _- Jeric Harper, 04 Oct 2016_ + +### Conjugation +**Step 7.** + +Spread the mixture (400 µL) on [Conjugation Plates](https://www.protocols.io/view/full-contact-microbiology-a-k-a-diatom-transforma-fzrbp56#flename-conjugation-plates). (0.5x BG-11 with 5% LB and 1% agar). + +> **Annotation:** The authors of The original protocol suggested spreading the mixture near, but not touching, the edges of the plate, as this mixture will be scraped and replated in 2 days. +> +> N concentration seems to be important. We have found greater transformation success in using 0.5x BG-11 medium made with seawater instead of 0.5x L1 medium. +> +> _- Jeric Harper, 06 Oct 2016_ + +### Conjugation +**Step 8.** + +Incubate plates for 90 minutes at 30°C in the dark. + +**Duration:** 01:30:00 + +> **Annotation:** _P. tricornutum_ can survive transformation temperatures up to 32°C. At 34°C and above survivorship is severely hampered. +> _- Jeric Harper, 06 Oct 2016_ + +### Conjugation +**Step 9.** + +Move plates to light incubator (18°C and 100 µmol photons m^-2 s^-1) for 2 days. + +**Duration:** 48:00:00 + +> **Annotation:** According to [Diner et. al](https://www.protocols.io/view/full-contact-microbiology-a-k-a-diatom-transforma-fzrbp56#flename-Diner-et-al), the conjugation occurs during the 2-day incubation. Transformation efficiency increased as incubation time increased. +> _- Jeric Harper, 06 Oct 2016_ + +### Selection +**Step 10.** + +Collect cells by adding 1 mL of L1 medium. Use a cell scraper to concentrate cells and medium to one side of the plate. Transfer resuspended cells to a 1.5 ml microcentrifuge tube with a P1000 pipette and filter tips. + +![Conjugation](image02.png) + +> **Annotation:** It is important to use filter tips as small pieces of agar that are accidentally scraped up can cause the pipette to cavitate resulting in contamination. We will often first add 500uL to collect the majority of the cells from the plate into the 1.5mL tube, then repeat with an additional 500ul to collect the remainder. +> _- Jeric Harper, 04 Oct 2016_ + +### Selection +**Step 11.** + +Spread 200 µl of the cell suspension on a [Selection Plate](https://www.protocols.io/view/full-contact-microbiology-a-k-a-diatom-transforma-fzrbp56#flename-selection-plate). + +> **Annotation:** Dilution of the selection volume (using L1 as makeup volume) can help facilitate colony enumeration if 200µl results in too many colonies. To maximize the number of colonies, the entire volume of resuspended cells can be plated to multiple plates. +> Collect 1 µL of the cell suspension and dilute 1:1000 for FlowCam cell count. +> _- Jeric Harper, 06 Oct 2016_ + +### Selection +**Step 12.** + +Incubate at 18°C and 100 µmol photons m^-2 s^-1 until colonies appear. + +### Colony identification +**Step 13.** + +After a minimum of 8-12 days, untransformed _Phaeodactylum tricornutum_ cells die off, and colonies of transformed cells begin to appear – in some cases, this can take 3-4 weeks. + +![Colony Identification](image04.png) + +Alternatively, selection can be done in liquid BG-11 [Selection media](https://www.protocols.io/view/full-contact-microbiology-a-k-a-diatom-transforma-fzrbp56#flename-selection-media) using eGFP as a reporter and sorted using FACS. + +![eGFP expression](image05.png) + +> **Annotation:** For plate selection, use [ImageJ protocol](https://imagej.nih.gov/ij/) for colony enumeration. +> +> Calculate transformation efficiency using: +> +> Efficiency = (number of colonies on plate) ÷ (selection volume cell density (cell/mL) x volume (mL) put on selection plate) +> +> Note: Be sure to include a dilution factor in the calculation if it was used in plating or counting! +> _- Jeric Harper, 06 Oct 2016_ + +**Step 14.** + +This protocol was modified from [the original procedure](https://www.protocols.io/view/full-contact-microbiology-a-k-a-diatom-transforma-fzrbp56#flename-original-procedure) and correspondence with the authors. + +## endofoutput +``` diff --git a/markdown-output/fun-drops-cbd-gummies-stay-active-with-fun-drops-c-b8werxbe.md b/markdown-output/fun-drops-cbd-gummies-stay-active-with-fun-drops-c-b8werxbe.md new file mode 100644 index 0000000000000000000000000000000000000000..d27199f3b5475e6e53bded8c6e02672c0fbcced5 --- /dev/null +++ b/markdown-output/fun-drops-cbd-gummies-stay-active-with-fun-drops-c-b8werxbe.md @@ -0,0 +1,131 @@ +```markdown +# Goal/Experiment: +To review the efficacy and benefits of Fun Drops CBD Gummies for male enhancement and ED (erectile dysfunction) based on the 2022 reviews. + +# Fun Drops CBD Gummies - Stay active With Fun Drops CBD Gummies Male Enhancement - 2022 Reviews, Is It Really Work? + +## Fun Drops CBD Gummies + +DOI: [dx.doi.org/10.17504/protocols.io.36wgq7m83vk5/v1](https://dx.doi.org/10.17504/protocols.io.36wgq7m83vk5) + +Fun Drops CBD Gummies 2022. 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Strict quality control is necessary when using enzymes and other molecular biology reagents. + +Quality control for functionality involves incubating the enzymes or PCR master mix in PCR reaction mixtures and testing their ability to amplify a DNA template region. Results are recorded based on the presence or absence of amplification by bands on an electrophoresis agarose gel. + +### Keywords +- Quality control tests for locally manufactured enzymes +- Quality control tests for molecular biology reagents +- Functionality of polymerase enzyme +- Functionality of DNA loading dye + +### License +This is an open access protocol distributed under the terms of the Creative Commons Attribution License [Creative Commons Attribution License](https://creativecommons.org/licenses/by/4.0/). + +### Guidelines +Before carrying out quality control on products, research should be carried out on the possible assays that apply for that particular product and availability of resources. + +## Materials and Equipment +### Reagents +- DNA polymerase (either cellular reagent or purified enzyme or PCR master mixes) +- Control DNA polymerase (known to work) +- Forward Primer (20 pMol) +- Reverse Primer (20 pMol) +- PCR grade water +- Enzyme reaction buffer +- dNTP Mix (if necessary) +- Agarose (electrophoresis grade) +- TBE running buffer + +### Materials/Equipment +- Ice +- PCR machine (mini PCR-blueGel) +- Pipettes (P-10, P-20, and P-200) +- Sterile Pipette tips (10 µl and 20 µl) +- Bowl +- Sterile 0.2 ml PCR tubes +- Electrophoretic gel tank and components (BlueGel) +- Waste container + +### Safety Warnings +- Wear protective clothing and all recommended laboratory PPE. +- PPE protects from accidental spills or splashes that may be dangerous to the eye or skin. + +### Before Starting +Clean and disinfect all work surfaces with a 1:10 dilution of bleach followed by 70% alcohol. + +## Functionality Test Procedure + +### 1. DNA Polymerase Enzyme and PCR Master Mix +After protein expression confirmation through SDS PAGE, perform the functionality test: + +#### Pipetting: +1. Thaw all reagents on ice in a bowl. +2. Label reaction tubes (PCR tubes) based on the number of samples and controls (negative and positive controls). + +#### Polymerase Enzyme Type: +1. **Cellular Reagent Enzyme:** Rehydrate with 30 µl sterile PCR grade water, flick tube, and keep on ice. +2. **Pre-purified Polymerase/Master Mix:** Remove from freezer and keep on ice. + +In each PCR tube, pipette and combine the following components: + +| | A | B | C | D | E | +| --- | --- | --- | --- | --- | --- | +| PCR Component | Test Sample - 1x PCR Master Mix | Test Sample - DNA Polymerase Enzyme | Negative Control (for OpenVent Enzyme) | Positive Control (for OpenVent Enzyme) | +| PCR water | Variable to 20 µl | Variable to 20 µl | Variable to 20 µl | Variable to 20 µl | +| dNTP (10 mM) | / | 0.4 µl | 0.4 µl | 0.4 µl | +| PCR buffer (10x) | / | 2 µl | 2 µl | 2 µl | +| Forward primer | 1 µl | 1 µl | 1 µl | 1 µl | +| Reverse primer | 1 µl | 1 µl | 1 µl | 1 µl | +| DNA template | 1 µl | 0.5-1 µl | / | 0.5-1 µl | +| Test enzyme | 17 µl | 1 µl | 1 µl | / | +| Control enzyme | / | / | / | 1 µl | + +1. **Negative Control:** All PCR components, no DNA template. +2. **Positive Control:** Include commercial DNA polymerase enzyme. + +#### Thermocycling: +1. Connect a thermocycler and set the protocol as follows: + +| | Temperature (°C) | Duration | +| --- | --- | --- | +| Initial Denaturation | 95 | 120 sec | +| Denature | 95 | 30 sec | +| Anneal | 50-65 | 20 sec | +| Extend | 72 | Variable | +| Final Extension | 72 | 120 sec | + +2. Cycle for 35-40 cycles (0.5 kb Lambda-50 ng/µl). + +### 2. Preparing Agarose Gel for Electrophoresis +Follow this [protocol](#) to prepare and cast the gel. + +### 3. Loading the Gel Wells +1. Pipette 3-5 µl DNA ladder into the first well. +2. Pipette negative control to avoid bubbles, then: +3. Mix amplicons with DNA loading dye at 1:5 ratio and load into subsequent wells. +4. Run gel for 15-30 minutes at 48 V in TBE buffer. + +### 4. Visualization and Interpretation of Gel Results +1. Visualize the gel using a UV transilluminator or blue light transilluminator. +2. With the DNA ladder, evaluate if the amplification product is as expected. Sharp, distinct bands indicate the test reagent is functioning properly. + - If no bands are seen, the reagent is non-functional. + - Record results for further actions. + +`endofoutput` +``` \ No newline at end of file diff --git a/markdown-output/fundis-ont-v14-nanopore-adapter-ligation-for-funga-db4w2qxe.md b/markdown-output/fundis-ont-v14-nanopore-adapter-ligation-for-funga-db4w2qxe.md new file mode 100644 index 0000000000000000000000000000000000000000..47803ce842e6bd2919c9af1b218dc27818961907 --- /dev/null +++ b/markdown-output/fundis-ont-v14-nanopore-adapter-ligation-for-funga-db4w2qxe.md @@ -0,0 +1,108 @@ +```markdown +# Goal/Experiment: +Ligate nanopore adapters to a DNA library prepared from fungi, using the Oxford Nanopore Flongle and associated reagents. + +# FUNDIS ONT V14 Nanopore Adapter Ligation for Fungal DNA Barcoding Flongle 10.4.1 + +## Authors: +- Stephen Douglas Russell¹ +- Harte Singer² + +¹The Hoosier Mushroom Society +²FUNDIS + +## Abstract +This protocol describes the process of adding nanopore adapters to an A-tailed DNA library. The reaction involves combining specific reagents followed by bead cleanup. + +### Tested with: +- Flowcells: Flongle 10.4.1 +- Ligation Kit: V14-LSK114 + +**Time required**: ~45 minutes +**Adapted from**: [dx.doi.org/10.17504/protocols.io.dm6gpb5zdlzp/v5](https://dx.doi.org/10.17504/protocols.io.dm6gpb5zdlzp/v5) + +## Materials + +### Reagents +| Reagent | Vendor | Catalog Number | Price per reaction | +| -------- | ------ | -------------- | ------------------ | +| Ligation Sequencing Kit V14 | Oxford Nanopore Technologies | SQK-LSK114 | $115.74 per MinION run; $57.87 per Flongle run | +| NEBNext Quick Ligation Module | New England Biolabs | E6056S | $18.05 per MinION run; $9.03 per Flongle run | +| HighPrep™ PCR Clean-up System | MagBio Genomics Inc. | AC-60005 | $0.047 per reaction | + +### Consumables +- Eppendorf DNA LoBind 1.5mL tubes +- 10μL pipette tips +- 100-200μL pipette tips +- Magnetic beads from NEBNext kit should be used + +### Equipment +- PCR tube rack +- Vortex mixer +- Mini centrifuge +- PCR cleanup magnet +- 10μL pipette +- 100μL pipette +- Hula mixer (optional) +- Quantus or Qubit Fluorometer (optional) + +## Protocol + +### Step 1: Reagent Preparation +Ensure that all reagents from the dA tailing step are on ice. + +### Step 2: Mixing Reaction Components +In a 1.5 mL DNA LoBind tube, mix the following: +1. 30 μL dA Tailed DNA +2. 12.5 μL Ligation Buffer (LNB) +3. 5 μL NEBNext Quick T4 DNA Ligase +4. 2.5 μL Ligation Adapter (LA) + +Pipette mix 10-20 times between each addition. + +### Step 3: Spin Down +Spin down with a mini centrifuge for 2 seconds. + +### Step 4: Incubate +Incubate the reaction for 10 minutes at room temperature. + +### Step 5: Add Magnetic Beads +Vortex to resuspend AMPure XP (AXP) magnetic bead stock. Add 20 μL of resuspended beads to the reaction. + +### Step 6: Incubate Bead Reaction +Incubate for 5 minutes at room temperature. + +### Step 7: Spin Down and Pellet +Spin down for 2 seconds with a mini centrifuge and pellet on a magnet for 2 minutes. Pipette off the supernatant. + +### Step 8: Wash Beads +Add 125 μL Short Fragment Buffer (SFB). Resuspend and spin down for 2 seconds. Pellet for 2 minutes. Remove and discard supernatant. + +### Step 9: Repeat Step 8 +Repeat the wash step with SFB. + +### Step 10: Pellet Drying +Spin down for 2 seconds and place back on the magnet. Allow to dry for ~30 seconds (do not over-dry). + +### Step 11: Elution +Remove from magnet and resuspend in 7 μL Elution Buffer (EB). Incubate for 10 minutes. + +### Step 12: Final Elution Cleanup +Pellet on a magnet until the eluate is clear and colorless for at least 1 minute. Remove and retain 7 μL eluate containing the DNA library. + +### Step 13: Quantification +Quantify 2 μL of your ligated library using the Qubit fluorometer. Adjust the concentration accordingly to achieve desired fmol/μL for sequencing. + +Recommended loading is between 5 fmol - 10 fmol of the final prepared library. + +For example, for 22ng/μL: +- 22ng/μL * 5μL = 110ng DNA +- 110ng/10ng (17 fmol DNA) = 55 μL total volume +- Initial volume is 5 μL, add 50 μL molecular water + +## Alternatives +- **NEBNext Quick Ligation Module**: If unavailable, alternative T4 DNA ligase kits with comparable performance can be considered. +- **Hula mixer**: If unavailable, mixing can be done manually with similar efficiency. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/gdna-extraction-from-sterivex-filters-ijccciw.md b/markdown-output/gdna-extraction-from-sterivex-filters-ijccciw.md new file mode 100644 index 0000000000000000000000000000000000000000..9e3f62835f73b79698809c205e767c5e76e7d345 --- /dev/null +++ b/markdown-output/gdna-extraction-from-sterivex-filters-ijccciw.md @@ -0,0 +1,156 @@ +```markdown +# Goal/Experiment: +The goal of this experiment is to extract genomic DNA (gDNA) from Sterivex filters. + +# gDNA extraction from Sterivex filters + +## Authors +Sophie Jurgensen, Alison Buchan, Gary LeCleir + +## Abstract +For inquiries, contact Dr. Alison Buchan (abuchan@utk.edu). + +## Citation +Sophie Jurgensen, Alison Buchan, Gary LeCleir gDNA extraction from Sterivex filters. protocols.io dx.doi.org/10.17504/protocols.io.jjcciw +Published: 06 Aug 2017 + +## Protocol + +### Preparation + +#### Step 1. +1. Plug the outflow port of the Sterivex cartridge using cha-seal clay. +2. Ensure that the incubator is set to 65°C. +3. Place the rotator in the incubator. + +**Reagents and Equipment:** + +- **Cha-seal tube sealing compound**: [CSX43510 by Medline](http://www.medline.com) +- **Sterivex™ filter unit without filling bell**: [SVGP01050 by Emd Millipore](http://www.emdmillipore.com) +- **Labquake™ Rotisserie Hybridization Rotator**: [M906150Q by Thermo Fisher Scientific](http://www.thermofisher.com) + +### Lysis + +#### Step 2. +1. Add 1.7 mL of CTAB extraction solution to each cartridge using a needle and syringe. Dispense the liquid slowly and avoid air bubbles. + +**Reagents and Equipment:** + +- **CTAB extraction solution**: [C2190 by Teknova](http://www.teknova.com) +- **3ml syringe**: [BD 309586 by BD Biosciences](http://www.bdbiosciences.com) +- **22 gauge needle**: [Z192473 by Sigma Aldrich](http://www.sigmaaldrich.com) + +**Notes:** +- Dispense liquid slowly to avoid air bubbles that will clog the port. +- **Date**: Sophie Jurgensen 20 Jun 2017 + +#### Step 3. +1. Add 65 µL of Proteinase K (10mg/mL) and 65 µL of Lysozyme (10mg/mL) to each cartridge using a needle and syringe. These may be combined and added to each tube in a single aliquot. + +**Reagents and Equipment:** + +- **Proteinase K**: [EO0491 by Thermo Fisher Scientific](http://www.thermofisher.com) +- **Lysozyme**: [12671-19-1 by Sigma Aldrich](http://www.sigmaaldrich.com) + +**Notes:** +- **Date**: Sophie Jurgensen 20 Jun 2017 + +#### Step 4. +1. Add 162 µL of filter-sterilized SDS (10%) to each tube. Invert to mix. + +**Reagents and Equipment:** + +- **SDS, 10% Solution**: [AM9822 by Life Technologies](http://www.lifetechnologies.com) + +#### Step 5. +1. Cap the open end of each cartridge with a luer lock cap and incubate in a rotary agitator at 65°C for 2 hours. + +### Lysis + +#### Step 6. +1. Label 2 sets of 2 mL centrifuge tubes with the sample name. One set will be kept and frozen (-20°C) while the other set will be used for further extraction. + +#### Step 7. +1. Using a 3 mL luer-lock syringe, attach to Sterivex cartridge and draw out fluid. +2. Depress 2 mL of sample material into the first storage 2 mL centrifuge tube, then pipette out 800 µL of this solution into another labeled 2 mL tube for further extraction. +3. Place the first 2 mL tube into the freezer. +4. Start the cool-down sequence in the microcentrifuge (set to 4°C). + +### Precipitation + +#### Step 8. +1. Add 800 µL of phenol:chloroform:isoamyl alcohol (PCI, 25:24:1, pH 8.0) to each tube and vortex to mix. Do this step in a fume hood. + +**Reagents and Equipment:** + +- **UltraPure™ Phenol:Chloroform:Isoamyl Alcohol (25:24:1, v/v)**: [15593031 by Thermo Fisher Scientific](http://www.thermofisher.com) + +**Notes:** +- **Date**: Sophie Jurgensen 20 Jun 2017 + +#### Step 9. +1. Centrifuge at 4°C, 10,000 rpm for 5 minutes. +2. Transfer the upper aqueous phase to a new 2 mL centrifuge tube. + +**Notes:** +- Be careful to not aspirate the interface between the aqueous phases. Do not attempt to remove all of the upper aqueous phase. +- **Date**: Sophie Jurgensen 20 Jun 2017 + +#### Step 10. +1. Add 800 µL of chloroform:isoamyl alcohol (CI, 24:1) to each tube. Invert and vortex to mix thoroughly, then centrifuge at 15,000 rpm for 5 minutes. Transfer the upper aqueous phase to a new 2 mL centrifuge tube. + +**Reagents and Equipment:** + +- **Chloroform:isoamyl alcohol 24:1**: [C0549 by Sigma Aldrich](http://www.sigmaaldrich.com) + +**Notes:** +- Repeat this step. The second time, transfer the upper aqueous phase to a 1.5 mL centrifuge tube. +- **Date**: Sophie Jurgensen 20 Jun 2017 + +#### Step 11. +1. Add 450 µL of room temperature isopropanol (100%) to each tube, inverting gently to mix. +2. Incubate at room temperature for 2 hours to overnight. + +**Reagents and Equipment:** + +- **Isopropanol** + +**Notes:** +- **Date**: Sophie Jurgensen 20 Jun 2017 + +#### Step 12. +1. Centrifuge the tubes at 10,000 rpm for 20 minutes. +2. Carefully decant by pouring liquid out of the tube and into a small clean petri dish, then blot tubes dry using a paper towel on the benchtop. You can also aspirate the liquid using a pipette. The petri dish serves to catch the DNA pellet if it is accidentally poured out during this step. + +### Purification + +#### Step 13. +1. Add 1.4 mL of 70% ethanol to each tube and gently invert several times to mix. +2. Centrifuge at 10,000 rpm for 5 minutes. +3. Pour off ethanol. + +#### Step 14. +1. Dry tubes in laminar hood for 10-15 minutes or until completely dry. Note that pellets may become dislodged from sides of the tube, so take care not to invert tubes. + +#### Step 15. +1. Add 50 µL of sterile Nuclease-free ultra-pure water to each tube and gently pipet to dissolve DNA. It may be helpful to either pre-heat the water to 50°C or to incubate the tubes at 37°C for 1-2 hrs to facilitate dissolution. + +**Reagents and Equipment:** + +- **Ultrapure Distilled, Nuclease Free Water** + +**Notes:** +- **Date**: Sophie Jurgensen 20 Jun 2017 + +#### Step 16. +1. Measure the DNA concentration using NanoDrop and freeze samples at -80°C. Record 260/280 ratio as well as DNA concentration. A 260/280 ratio of 1.8–2 is considered "pure" DNA. + +**Reagents and Equipment:** + +- **NanoDrop spectrophotometer**: [ND-1000 by Thermo Fisher Scientific](http://www.thermofisher.com) + +**Notes:** +- **Date**: Sophie Jurgensen 25 Jul 2017 + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/gel-free-mirna-illumina-library-preparation-protoc-e52bg8e.md b/markdown-output/gel-free-mirna-illumina-library-preparation-protoc-e52bg8e.md new file mode 100644 index 0000000000000000000000000000000000000000..36aa658832d40c159c078bf400d056bf39dd1f80 --- /dev/null +++ b/markdown-output/gel-free-mirna-illumina-library-preparation-protoc-e52bg8e.md @@ -0,0 +1,234 @@ +```markdown +# Goal/Experiment: +The goal of this experiment is to prepare high-quality miRNA libraries using the Illumina platform without the need for PAGE-gel size selection by following the Gel-Free miRNA Illumina Library Preparation Protocol using the TailorMix Gel-Free miRNA Sample Preparation Kit. + +# Gel-Free miRNA Illumina Library Preparation Protocol by TailorMix Gel-Free miRNA Sample Preparation Kit + +Authors: Karen Yip, Kelvin Chan, Danny Lee + +## Abstract +This is a magnetic bead-based protocol enabling the generation of high-quality Illumina miRNA libraries without the need for PAGE-gel size selection. + +Our protocol enables the discovery and profiling of miRNAs from various organisms and tissues via the Illumina sequencing platform. The unique TailorMix reagents and workflow have been developed for simplicity and reproducibility without sacrificing quality or yield. + +## Features +- **No need for PAGE-gel size selection** + - Final miRNA libraries are cleaned up with magnetic bead-based protocol. + +- **Low input requirement** + - Compatible with as little as 150ng total RNA input. + +- **User-friendly workflow** + - Libraries can be prepared in a single day with less than one hour of hands-on time. + +- **Comprehensive sample prep kit** + - Most components are supplied as ready-to-use mixtures which improves consistency and reproducibility. + +## Guidelines + +![TailorMix miRNA Sample Preparation Overview](image.png) + +### Best Practices +- Always wear gloves and use sterile techniques. +- Set up reactions using sterile non-stick nuclease-free tubes. +- Place samples and reagents on ice or on a chilled cooler block at all times and avoid extended pauses. +- Reagents should be prepared using RNase-free components. +- Prepare an extra 10% mixture when running multiple samples. +- Avoid repeated freeze-thaw cycles. + +## RNA Input +The TailorMix Gel-Free miRNA Sample Preparation protocol has been optimized using 900 ng of purified high-quality human kidney total RNA as input and confirmed with as low as 150ng RNA input. Because miRNA populations vary among different tissue types and species, the use of total RNA from other tissue or species may require optimization. Low yield libraries may require gel-based size selection for optimal results (See the Appendix, Table 1 for suggestions on Library Size Selection Methods). + +## Sample Pooling Guidelines +The TailorMix Gel-Free miRNA Sample Preparation kit is capable of multiplexing up to 96 samples into a single lane of an Illumina flow cell. While processing multiple samples in parallel, use a unique index primer for each sample at the PCR step. Samples can be pooled before or after the library purification step. Contact SeqMatic for more information about the 96-reaction sample preparation kit. + +## Protocol + +### Step 1. 3' Adapter Ligation + +1. Thaw Mix C400 from -20°C storage. Allow it to equilibrate to room temperature for a minimum of 30 minutes before use. +2. Pre-heat the thermal cycler to 70°C and pre-heat another thermal cycler to 25°C if available. +3. Denature the RNA Sample by assembling the following components in a sterile 200 μL PCR tube on ice: + + | Reagent | Volume (µL) | + |------------|-------------| + | RNA Sample | 6 | + | Mix A400 | 2 | + | **Total** | **8** | + +4. Vortex mix thoroughly and incubate at 70°C for 1 minute and then place the tube on ice. +5. Set up the following 3’ Adapter Ligation reaction on ice: + + | Reagent | Volume (µL) | + |------------------------------|-------------| + | Denatured RNA mix from Step 4| 8 | + | Mix B400 | 2 | + | Mix C400 | 6.5 | + | **Total** | **16.5** | + + > **Note:** Mix C400 is a highly viscous reagent. Handle with care and pipette slowly to ensure the correct amount of Mix C400 is dispensed for each reaction. + +6. Vortex mix thoroughly and pulse spin. Incubate at 25°C for 1 hour. + +### Step 2. Ligation Product Clean Up + +1. Vortex the TailorMag Purification Beads (TPB) until they are evenly suspended. +2. Prepare 80% ethanol for rinse step. +3. Add 30 μL of TPB with each 3’-adapter ligated sample from Step 6. Vortex mix thoroughly and incubate at room temperature for 15 minutes. + + | Reagent | Volume (µL) | + |-------------------------------------------|-------------| + | 3’-adapter ligated sample from Step 6 | 16.5 | + | TailorMag Purification Beads (TPB) | 30 | + | **Total** | **46.5** | + + > **Note:** Do NOT perform strong centrifugation because it will separate TPB from the sample. + +4. Place the sample tube on the magnetic stand at room temperature for 5 minutes, or until the solution clears up. +5. Carefully remove and discard 40 µL of the supernatant. + +> **Note:** Sample recovery may be affected if the TPB pellet is disrupted. + +6. Keep sample tube on the magnetic stand. Gently rinse the TPB pellet with 150 μL of 80% ethanol without disrupting the TPB pellet. Discard the rinse solution. + + > **Tip:** Point pipette tip towards opposite direction as the TPB pellet. Gently pipette the 80% ethanol up and down once, then discard the rinse solution. + +7. Air dry sample tube at room temperature. + +> **Note:** TailorMag Purification Beads are dried within 5 to 15 minutes at room temperature. Proceed to Step 14 when the appearance of the TPB pellet turns form glossy/shiny (wet) to matte (dry). Sample recovery may be affected if beads are over-dried and appear powdery. + +8. Remove sample tube from the magnetic stand. Add 7 μL of nuclease-free water to the dried TPB pellet. Vortex to resuspend and pulse spin. Incubate sample resuspension at room temperature for 2 minutes. + +### Step 3. 5' Adapter Ligation + +1. Set up the following 5' Adapter Ligation reaction on ice: + + | Reagent | Volume (µL) | + |------------------------------------|-------------| + | 3’ Adapter Ligated RNA from Step 14| 7 | + | Mix D400 | 3 | + | Mix E400 | 2 | + | **Total** | **12** | + + > **Note:** Presence of TPB does not interfere with the enzymatic reaction. + + > **Note:** To minimize the presence of artifact products, add Mix D400 and Mix E400 to the sample in consecutive steps. + +2. Vortex mix thoroughly and pulse spin. Incubate at 25°C for 1 hour and then place the tube on ice. + +### Step 4. cDNA Synthesis +1. Pre-heat the thermal cycler to 50°C. +2. Set up the following cDNA Synthesis reaction on ice: + + | Reagent | Volume (µL) | + |----------------------------------------------|-------------| + | 3’ and 5’ Adapter Ligated RNA from Step 16 | 12 | + | Mix F400 | 2 | + | Mix G400 | 1 | + | **Total** | **15** | + + > **Note:** Presence of TPB does not interfere with the enzymatic reaction. + +3. Vortex mix thoroughly and pulse spin. Incubate at 50°C for 1 hour and then place the tube on ice. + + > **Safe Stopping Point:** First strand cDNA could be stored at -20°C for up to seven days. + +### Step 5. PCR Amplification +> **Note:** This protocol has been optimized using 900 ng of purified high-quality human kidney total RNA as input. Because miRNA populations vary among different tissue types and species, the use of total RNA from other tissues or species may require additional optimization. + +1. Set up the following PCR reaction in a fresh sterile 200 µl PCR tube on ice: + + | Reagent | Volume (µL) | + |-----------------------------------|-------------| + | First strand cDNA from Step 19 | 15 | + | Mix H400 | 13 | + | PCR Primer | 1 | + | Index Primer* | 1 | + | **Total** | **30** | + + > **Note:** Presence of TPB does not interfere with the enzymatic reaction. + +2. Vortex mix thoroughly and pulse spin. Amplify the samples in the thermal cycler using the following PCR cycling conditions: + + - 95°C for 10 minutes + - 15 cycles of: + - 95°C for 5 seconds + - 60°C for 15 seconds + - 72°C for 1 minute + - 72°C for 5 minutes + - Hold at 4°C + + > **Safe Stopping Point:** PCR products could be stored at -20°C for up to seven days. + +3. PCR yield can be monitored by running an Agilent BioAnalyzer High Sensitivity DNA assay using a dilution of 1 µL of PCR product and 9 µL of nuclease-free water. A typical result shows a distinct peak at approximately 140bp (Figure 2). + + > **Note:** See Appendix A for more detailed description of BioAnalyzer High Sensitivity DNA assay profile of the PCR products. + + > **Note:** The BioAnalyzer High Sensitivity DNA assay has a 10% deviation on sizing accuracy. + +![BioAnalyzer High Sensitivity DNA assay of PCR Product (10x diluted) from Human Kidney Tissue Total RNA](figure2.png) + +### Step 6. Library Size Selection Methods +The TailorMix Gel-Free miRNA Sample Preparation protocol enables the generation of micro RNA libraries from as low as 150ng Human kidney total RNA (Figure 3 and Figure 4). However, miRNA populations vary among different samples, and the use of total RNA from other tissue or species may cause variations in PCR profiles. + +Gel-free size selection is suitable for libraries with a strong 140bp library product peak compared to the 120bp artifact product peak. It is recommended to use the PAGE-size selection approach for low-yield libraries (weak 140bp library product) and libraries that have a strong 120bp artifact product peak. See Table 2 in the Appendix for examples. + +![TailorMix Gel-Free miRNA libraries from Human Kidney Tissue Total RNA, BioAnalyzer High Sensitivity DNA assay profiles](figure3.png) + +### Step 7. Gel-Free Library Purification +> **Note:** Sample volume may change after PCR. To ensure purification efficiency, bring sample volume back to 30 μL before starting Gel-Free Purification steps if necessary. + +1. Vortex the TailorMag Purification Beads (TPB) until they are evenly re-suspended. +2. Prepare 80% ethanol for rinse step. +3. Add TPB to each sample in the PCR tube according to the following table. Vortex mix thoroughly and pulse spin. Incubate at room temperature for 5 minutes. + + | Reagent | Volume (µL) | + |-----------------------------------|-------------| + | PCR from Step 21 | 30 | + | TailorMag Purification Beads (TPB)| 30 | + | **Total** | **60** | + + > **Note:** Do NOT perform strong centrifugation because it will separate TPB from the sample. + +4. Place the sample tube on the TailorMag PCR-tube magnetic stand at room temperature for 5 minutes, or until the solution clears up. DO NOT DISCARD SUPERNATANT. +5. Keep sample tube on the magnetic stand. Transfer 55 μL of the clear supernatant to fresh sample tubes. + + > **Note:** Do not disrupt the TPB pellet. Contamination of TPB pellet to the next step may affect final library quality. + +6. Add TPB to the clear supernatant from Step 27. Vortex mix thoroughly and pulse spin. Incubate at room temperature for 5 minutes. + + | Reagent | Volume (µL) | + |-----------------------------------|-------------| + | Clear Supernatant from Step 27 | 55 | + | TailorMag Purification Beads (TPB)| 11 | + | **Total** | **66** | + + > **Note:** Do NOT perform strong centrifugation because it will separate TPB from the sample. + +7. Place the sample tube on the magnetic stand at room temperature for 5 minutes, or until the solution clears up. +8. Remove and discard 60 µL of the supernatant. + + > **Note:** Sample recovery may be affected if the TPB pellet is disrupted. + +9. Keep sample tube on the magnetic stand. Gently rinse the TPB pellet with 150 µL of 80% ethanol without disrupting the TPB pellet. Discard the rinse solution. + + > **Tip:** Point pipette tip towards opposite direction as the TPB pellet. Gently pipette the 80% ethanol up and down once, then discard the rinse solution. + +10. Air dry sample tube at room temperature. + + > **Note:** TailorMag Purification Beads are dried within 5 to 15 minutes at room temperature. Proceed to Step 33 when the appearance of the TPB pellet turns from glossy/shiny (wet) to matte (dry). Sample recovery may be affected if beads are over-dried and appear powdery. + +11. Remove sample tube from the magnetic stand. Add 27 μL of TE buffer to the dried TPB pellet. Vortex to resuspend and pulse spin. Incubate resuspension at room temperature for 2 minutes. + +### Step 8. Library Validation + +1. Use of an Agilent Technologies 2100 Bioanalyzer is recommended as a quality control analysis of your sample library. Use 1 µL of resuspended library from Step 33 on a High Sensitivity DNA chip to check the size, purity, and concentration of the sample. + + > **Note:** The BioAnalyzer High Sensitivity DNA assay has a 10% deviation on sizing accuracy. + + > **Note:** If high percentage of 120bp peak remains, use PAGE size selection gel to extract the 140bp micro RNA library. See Table 2 in the Appendix for references. + +![TailorMix Gel-Free miRNA libraries from Human Kidney Tissue Total RNA, BioAnalyzer High Sensitivity DNA assay profiles](figure3.png) + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/gene-regulatory-network-bm6rk9d6.md b/markdown-output/gene-regulatory-network-bm6rk9d6.md new file mode 100644 index 0000000000000000000000000000000000000000..7292bf2ff4da4883e02f1ad02687e9a36742193a --- /dev/null +++ b/markdown-output/gene-regulatory-network-bm6rk9d6.md @@ -0,0 +1,81 @@ +```markdown +# Goal/Experiment: +To identify key regulatory factors and differentially connected genes (DCGs) involved in the control mechanisms underpinning complex traits during the biological transition of Atlantic salmon maturation using a gene regulatory network (GRN) approach. + +# Gene Regulatory Network + +**Amin R Mohamed1, Antonio Reverter1, James Kijas1** + +1. **CSIRO** +2. **Citation:** [Amin R Mohamed, Antonio Reverter, James Kijas (10/10/2020). Gene Regulatory Network. protocols.io](https://dx.doi.org/10.17504/protocols.io.bm6rk9d6) +3. **DOI:** [https://dx.doi.org/10.17504/protocols.io.bm6rk9d6](https://dx.doi.org/10.17504/protocols.io.bm6rk9d6) + +## Abstract +Gene regulatory networks (GRNs) provide a platform for integrating multiomic data and can be used to characterize the dynamics of perturbations during biological transitions such as puberty and other complex traits. We used a multiomics approach, which has the power to identify the control mechanisms underpinning complex traits. We also utilized a Systems Biology approach to co-analyze genes with evidence of differential behavior using seven categories that included expression (DEGs), changed methylation at gene bodies (DMGs) or promoters (DMPs) and differential chromatin accessibility (DACs) into gene network. To focus the analysis towards investigation of key regulators, we also performed regulatory impact factor (RIF) analysis. This used co-expression correlation between TFs and their target differentially expressed genes to identify master regulator TFs. + +### References +- Argelaguet, R. et al. (2019). Multi-omics profiling of mouse gastrulation at single-cell resolution. [Nature](https://doi.org/10.1038/s41586-019-1825-8), 566, 655-662. +- Lloyd-Price, J. et al. (2019). Multi-omics of the gut microbial ecosystem in inflammatory bowel diseases. [Nature](https://doi.org/10.1038/s41586-019-1825-8), 569, 655-662. +- Reverter, A. & Chan, E.K.F. (2008). Combining partial correlation and an information theory approach to the reversed engineering of gene co-expression networks. Bioinformatics, 24, 2491-2497. +- Reverter, A., Hudson, N. J., Nagaraj, S. H., Perez-Enciso, M. & Dalrymple, B. P. (2010). Regulatory impact factors: unraveling the transcriptional regulation of complex traits from expression data. Bioinformatics, 26, 896-904. +- Shannon, P. et al. (2003). Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Res, 13, 2498–2504. + +## Protocol Information +**Keywords:** systems biology, multiomics, regulatory, networks + +**License:** Creative Commons Attribution License + +**Created:** Oct 08, 2020 + +**Last Modified:** Oct 10, 2020 + +**Protocol Integer ID:** 42929 + +## Guidelines +1. This analysis requires obtaining results from multiomic experiments. +2. Genes need to be filtered to have 2000 genes for network construction using PCIT. +3. Attribute table is required to assign genes to the different analysis. + +## Before Starting +Prepare gene expression matrix with all samples and normalized (log2FPKM) expression values for all genes from different analyses. + +## Protocol + +### Step 1: Master Regulator Analysis +Master regulator analysis was performed using regulatory impact factor (RIF) metrics to identify key regulators contributing to differential expression during tissue comparison at T4 vs T1 in Atlantic salmon. + +**References:** +- Reverter et al., 2010 +- Mohamed et al., 2018 + +**Procedure:** +1. Perform co-expression concepts where regulators were contrasted against unique lists of genes that were differentially expressed. +2. Apply a threshold of ±2.57 standard deviation from the mean to determine significance. +3. Identify significant regulators (n=305) across various tissues: pituitary, ovary, and liver. + +### Step 2: Gene Network Analysis +Selected genes from different omics analyses were used as nodes with significant connections (edges) identified via the Partial Correlation and Information Theory (PCIT) algorithm. + +**References:** +- Reverter and Chan, 2008 + +**Procedure:** +1. Include DEGs, DMGs, DMPs, DACs, alongside key transcription factors identified by RIF. +2. Use normalized expression data (at least 0.2 FPKM). +3. Utilize **UpSetR** R package for analysis. [UpSetR Vignette](https://cran.r-project.org/web/packages/UpSetR/vignettes/basic.usage.html). + +### Step 3: Differential Connectivity Analysis +Two networks were constructed to explore connectivity during maturation onset: +1. T1 (pre-maturation): 12 samples. +2. T2, T3, T4 (post-maturation): 36 samples. + +**Procedure:** +1. Calculate number of connections for each gene in both networks. +2. Compare networks to identify differentially connected genes (DCGs). + +**Final Analysis:** +- Subnetworks based on top trio genes and regulators (TFs). +- Differential connectivity analysis for control vs post-maturation samples. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/general-genotyping-of-myzus-persicae-using-pcr-wit-c4tnywme.md b/markdown-output/general-genotyping-of-myzus-persicae-using-pcr-wit-c4tnywme.md new file mode 100644 index 0000000000000000000000000000000000000000..0e8b5fdd24cb26330865d2a64c63ce51a9a9e4c3 --- /dev/null +++ b/markdown-output/general-genotyping-of-myzus-persicae-using-pcr-wit-c4tnywme.md @@ -0,0 +1,131 @@ +```markdown +# Goal/Experiment: +General Genotyping of Myzus persicae using PCR with Microsatellite Markers and Gel Electrophoresis + +## General Genotyping of Myzus persicae Using PCR with Microsatellite Markers and Gel Electrophoresis V.2 + +**Authors:** +- Mariska M. Beekman1,2 +- Bas J. Zwaan1 +- Marcel Dicke2 +- Eveline Verhulst2 +- Bart Pannebakker2 + +1 Laboratory of Genetics, Wageningen University and Research, Wageningen, The Netherlands +2 Laboratory of Entomology, Wageningen University and Research, Wageningen, The Netherlands + +### Disclaimer +There was a mistake in the previous version. Primers should be used in a concentration of 0.2uM instead of 0.2mM as stated in the old version. + +## Abstract +This protocol can be used to 'roughly' genotype green peach aphids, _Myzus persicae_ (Sulzer), based on microsatellites using PCR and gel electrophoresis with high percentage agarose gels. Please note this protocol does not result in exact allele information as it uses gel-electrophoresis for allele identification and comparisons instead of capillary-electrophoresis. Therefore, allele sizes are judged by eye and thus results are prone to human error as differentiating between identical and different-length alleles might be a challenge. + +Microsatellites are regions in the genome with repeating DNA sequences. This protocol targets microsatellites with di-repeats, meaning they contain repeats of two different bases, for example GAGAGAGAGA. Due to slippage during DNA replications, the number of repeats in a microsatellite region can change over time at a rate higher than that of alternative regions in the genome. The number of repeats an organism has for a certain microsatellite region represents a specific allele. The allele sizes of multiple microsatellite regions can be determined to distinguish an organism's multi-locus genotype (MLG). Because _M. persicae_ reproduces by parthenogenesis, you can use microsatellites to determine which aphids are clone-mates (aphids coming from the same parthenogenetic ancestor), as these will have the same MLG. + +### Important Note: +This protocol can be used to check if various samples of _M. persicae_ have similar or distinct MLGs. This protocol on its own can not be used to assign a specific genotype to a sample. + +## Materials + +### 1. Chelex and Proteinase K Based DNA Extraction +- Pipettes and pipette tips +- Eppendorf tubes +- Centrifuge +- Heat block or water bath +- Ultrapure water +- Chelex 100 resin (Bio-Rad, Hercules, CA, USA) +- Proteinase K (20 mg/mL; Promega, Southampton, UK) + +### PCR +- Pipettes and pipette tips +- PCR machine +- PCR tubes +- Eppendorf tubes +- Ultrapure water +- Primers (see table below) +- QIAGEN Multiplex PCR Master Mix (206143; Qiagen, Venlo, the Netherlands) OR a different DNA polymerase + dNTPs + potentially additional MgCl2 +- DNA of the aphids to be tested +- DNA of the aphid genotype of interest (reference/positive control) + +## Primers + +| A | B | C | D | +| ------- | ------------------------ | --------- | -------------------- | +| Primer | Sequence | Multiplex | Reference | +| M37_F | GTGTGAGTAAGTCGTATTG | 1 | Sloane et al., 2001 | +| M37_R | TTTGATTATGTACCTGTGC | 1 | " | +| M40_F | ACACGCATACAAGAATAGGG | 2 | " | +| M40_R | AGAGGAGGCAAGGTGAAAC | 2 | " | +| M86_F | TCCACTAGACCTCAACAC | 2 | " | +| M86_R | ATTTATTATGTCGTTCCGCC | 2 | " | +| myz2_F | TGGCGAGAGAGAAGACCTCG | 1 | Wilson et al., 2004 | +| myz2_R | TCGGAAGAGACAGACATCGAGA | 1 | " | +| myz9_F | AACCTCACCCTCGTGAGTTCG | 2 | " | +| myz9_R | CTTGGATGTGTTGGGGGTGC | 2 | " | + +**Table 1:** Primers to target different microsatellite loci, M37/M40/M86/myz2/myz9, in the _Myzus persicae_. Markers with the most information (most alleles and the largest size differences between alleles) are M86, myz2 and myz9. The least information comes from marker M37. Primers in the same multiplex can be combined in a single PCR reaction. + +## Methods + +### 1.1 DNA Extractions (1 h 12 m) +You can use any preferred protocol to extract DNA from the aphids. You don’t need a lot of high quality DNA. Extract DNA from single aphids (preferably adult, non-damaged, non-parasitized). + +#### Chelex and Proteinase K Based DNA Extraction +1. Make a **5% (v/v) Chelex 100 resin solution** in ultrapure water. +2. For each aphid sample, add **100 µL** of the 5% Chelex solution in an Eppendorf tube and add **2.5 µL** Proteinase K. +3. When extracting DNA from aphids stored in ethanol, ensure the ethanol evaporates completely before adding the aphid to the Eppendorf tube. +4. Grind up the aphid in the Chelex and Proteinase K solution. +5. Vortex and spin down the tube. +6. Incubate the tubes for at least **1 hour** at **56 °C** (can also be done overnight) and subsequently heat to **98 °C for 8 minutes** to inactivate the proteinase K. +7. Cool down on ice. +8. Centrifuge the tubes at high speed for **4 minutes**. The DNA will be in the supernatant. Be careful not to take any Chelex beads while pipetting off the supernatant, as Chelex resin will inhibit downstream PCR reactions. +9. Dilute DNA at **33% (v/v)** in ultrapure water and use immediately, or store either short-term at **4 °C** or long-term at **-20 °C** until further use. + +### 1.2 PCR Reactions +PCR reactions are carried out in **5 µL** volumes containing: +- 1x QIAGEN Multiplex PCR Master Mix (containing dNTPs, HotStartTaq DNA polymerase and 3 millimolar (mM) MgCl2 as final concentration) +- 0.2 micromolar (µM) of each primer +- 1 µL Chelex extracted DNA (which was diluted at 1/3) + +It is possible to use different DNA polymerases, but the final product might be more smeared and less clear. When using a different DNA polymerase it might also be necessary to increase the [Mg²⁺] to ensure all primers anneal during PCR. + +#### Master Mix +1. Prepare a master mix for the PCR reactions containing all ingredients except for the DNA. +2. Include a negative control in which no DNA is added. +3. Include at least one positive control for the _M. persicae_ genotype of interest. +4. If the positive control is not included or fails, it will not be possible to confirm if the collected aphids have the genotype of interest. Including a DNA sample of a _M. persicae_ genotype that is different from the genotype of interest would be better. + +### 1.3 Thermocycling Protocol PCR Machine + +| A | B | C | +| ------- | -------- | ------ | +| Temperature | Duration | Cycles | +| 95 °C | 15 min | | +| 94 °C | 30 s | 30x | +| 57 °C | 60 s | | +| 72 °C | 45 s | | +| 72 °C | 5 min | | +| 12 °C | ∞ | | + +**Table 2:** Thermocycling conditions for the PCR when using the QIAGEN Multiplex PCR Master Mix + +### 1.4 Gel Electrophoresis +The PCR products will be visualized on a **3.5-4% agarose gel**. + +1. Make a **3.5-4% agarose gel**. Stir to dissolve the agarose, boiling may help. Stain the gel and let it set properly. +2. Add loading dye to the PCR products and load them onto the gel. Start with about **1.5 µL**. +3. The gel should run for 1.5-2 hours at 90 volts. Adjust time and voltage as necessary for clear separation. +4. Each row of the gel should contain a sample of the aphid genotype of interest. + +## Examples of Results + +Below is an example of results obtained using primers M86 and myz9 in multiplex. The first and last columns are a 100bp step ladder. All other columns are single aphid samples. + +**Careful:** Ensure the 100, 200, 300 fragments of the ladder are sufficiently separated. + +**Figure 1:** A 3.5% agarose gel showing different _Myzus persicae_ genotypes, visualized by amplifying microsatellite markers M86 and myz9 in multiplex. + +**Figure 2:** Same as for figure 1. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/generating-ct-cut-off-values-using-gblocks-gene-fr-c8pyzvpw.md b/markdown-output/generating-ct-cut-off-values-using-gblocks-gene-fr-c8pyzvpw.md new file mode 100644 index 0000000000000000000000000000000000000000..4bf17745af71a554657085659a2b57fad30868b8 --- /dev/null +++ b/markdown-output/generating-ct-cut-off-values-using-gblocks-gene-fr-c8pyzvpw.md @@ -0,0 +1,128 @@ +```markdown +# Goal/Experiment: +Generating Ct cut-off values using gBlocks gene fragments. This experiment aims to resuspend, dilute, and perform qPCR on gBlocks gene fragments to generate standard curves, which will be used to determine Ct cut-off values for the S. Typhi gene targets (ttr, tviB, staG) and the HF183 bacteroides rRNA gene. + +# Generating Ct cut-off values using gBlocks gene fragments V.2 + +**Authors**: Dilip Abraham¹, Nick Grassly², Catherine Troman³, Jonathan Rigby³ +¹CMC Vellore, India; ²Imperial College London; ³Imperial College London, UK +**Project**: Typhoid Environmental Surveillance +**Date**: Feb 05, 2024 + +### Abstract +The following protocol describes the resuspension, dilution, and qPCR of gBlocks gene fragments. gBlocks gene fragments are synthesized double-stranded DNA oligos, which can be used for standardization. The standard curves generated by the gBlocks in a triplex qPCR are used to determine a Ct cut-off value for the *S. Typhi* gene targets (*ttr, tviB, staG*) and the HF183 bacteroides rRNA gene. + +### Materials + +| Item | Vendor | Catalog Number | +|--------------------------------------------------------------|------------------------------------|---------------------------------------| +| TE Buffer | Contributed by users | | +| gBlocks gene fragments | See protocol | | +| Takyon Low ROX Probe 2x MasterMix dTTP | Eurogentec Catalog | #UF-LPMT-B0701 | +| Nuclease free water | Contributed by users | | +| qPCR DNA Extraction and Inhibition Control CY5-QXL670 | Eurogentec Catalog | #RT-SPCC-Q02 | + +### gBlocks Details + +gBlocks gene fragments are synthesized double-stranded DNA fragments containing the sequence for the amplicon of interest, in this case for *ttr, staG, tviB* in *S. Typhi*, and *HF183* bacteroides. + +| Gene target | Size (bp) | gBlock sequence (5' - 3') | +|-------------|-----------|-------------------------------------------------------------------------------------------------------------------------| +| ttr | 125 | GAAACGCGTAACGGA... | +| staG | 138 | CGGGCGAAGTAGCAG... | +| tviB | 125 | CTTGATTTCGACTTCC... | +| HF183 | 132 | GGGACATTGAGTTACC... | + +*Table1: Sequences for gBlocks gene fragments for S. Typhi gene targets and HF183.* + +### Resuspending and diluting the gBlocks + +1. gBlocks are supplied as a lyophilized pellet. Resuspend in TE Buffer to achieve a stock of 10ng/μL. Information on the ng provided, OD260, and molecular weight are given on the spec sheet provided with the gBlocks. + +**Tools for Calculations:** +- [Integrated DNA Technologies - Resuspension Calculator](https://www.idtdna.com) +- [Oligo Analyzer](https://www.idtdna.com) + +2. Once resuspended, check the concentration via Qubit or Nanodrop. + + 2.1 If the concentration is not 10ng/μL, carry out your first dilution in step 4 to make it 1ng/μL. + +3. Create serial dilutions of your stock solution by adding 2μL of stock into 18μL of nuclease-free water. Create 12 dilutions to form a series of 12 concentrations. Perform at least 10 replicates split over at least two days. + + 4.1 For example, performing four replicates of 12 dilutions three times on three separate days. This would be two plates each day to include all targets. + +### qPCR and generating a standard curve + +5. Prepare the triplex qPCR mastermix described below (or singleplex for HF183): + +| Reagent | Volume per reaction (μL) | +|------------------------|--------------------------| +| ttr_F (20uM) | 0.25 | +| ttr_R (20uM) | 0.25 | +| ttr_P (5uM) | 0.5 | +| tviB_F (20uM) | 0.5 | +| tviB_R (20uM) | 0.5 | +| tviB_P (5uM) | 1 | +| staG_F (20uM) | 0.5 | +| staG_R (20uM) | 0.5 | +| staG_P (5uM) | 1 | +| 2x Mastermix with ROX | 12.5 | +| Nuclease free water | 2.5 | + +*Table2: Mastermix composition for triplex S.Typhi qPCR. Primer and probe sequences are provided in the qPCR protocol in the TyphoidES workspace.* + +| Reagent | Volume per reaction (μL) | +|-----------------------------|--------------------------| +| HF183_F | 0.5 | +| HF183_R | 0.5 | +| HF183_P | 1 | +| 10x Control Mix (Eurogentec)| 2.5 | +| 2x Mastermix with ROX | 12.5 | +| Nuclease free water | 3 | + +*Table3: Mastermix composition for the singleplex HF183 reaction. Primer and probe sequences are provided in the qPCR protocol in the TyphoidES workspace.* + +6. Aliquot 20μL of master mix for each reaction in a 96-well plate. Add 5μL of gBlock dilution to each reaction. Ensure that although the reaction is designed as a triplex, you only put one target gBlock in each reaction. + +7. Seal the plate carefully, then spin down briefly to gather all reagents at the bottom of the wells and remove bubbles. + +8. Load the plate into the real-time PCR machine after setting it up appropriately and carry out cycling using the following conditions: + +| Cycle | Temperature (°C) | Duration | +|-------|-------------------|-----------| +| 1 | 50 | 2 minutes | +| 1 | 95 | 2 minutes | +| 40 | 95 | 15 seconds| +| 40 | 60 | 30 seconds| +| 40 | 72 | 30 seconds| + +*Table4: Cycling conditions for all qPCR reactions.* + +### Analysis - determining Ct cut-off + +9. The limit of detection (LOD_95) is the genome copy number/μL and associated Ct value at which a qPCR amplification would be observed 95% of the time. This can be calculated from the results of the dilution series using PROBIT analysis. + + 9.1 We have provided an Excel file to calculate the LOD_95 for you from your data [resource](https://protocols.io/private/hybridfirecalc/). Make sure you allow macros to be run. You will also need to enable the Microsoft Solver add-in. Instructions for doing so are [here](https://support.microsoft.com/en-us/office/load-the-solver-add-in-in-excel-612926fc-d53b-46b4-872c-e24772f078ca). + + Alternatively, you can use the statistical programming language R to fit the PROBIT curve. Example code: + + ```r + #fit the profit curve + mod=glm(Ct_bin ~ log_conc, data=subset(gblocks, target=="ttr"), family=binomial(link="probit")) + summary(mod) + + #calculate the LOD_95 in log concentration (GC/uL) + LOD_est_ttr=(qnorm(0.95)-mod$coefficients[1])/mod$coefficients[2] + + #predict the LOD_95 Ct and range + LOD_Ct_ttr=predict(lm(Ct ~ log_conc, data=subset(gblocks, target=="ttr")), newdata=list(log_conc=LOD_est_ttr), interval="prediction") + ``` + + 9.2 To calculate the GC/μL from the qPCR Ct values of actual samples, you can use the equation: + + log GC/μL = (Ct - intercept) / slope + + where slope and intercept are from the linear regression of the Ct value on log GC/μL generated from the standard curves (e.g., as given in the Excel spreadsheet or from the linear model `lm` fit in R). + +endofoutput +``` diff --git a/markdown-output/generation-of-glioblastoma-spheroid-model-using-an-chv9t696.md b/markdown-output/generation-of-glioblastoma-spheroid-model-using-an-chv9t696.md new file mode 100644 index 0000000000000000000000000000000000000000..3db29181dfa5ce21cc5c144855287352a2fdeb66 --- /dev/null +++ b/markdown-output/generation-of-glioblastoma-spheroid-model-using-an-chv9t696.md @@ -0,0 +1,123 @@ +```markdown +# Goal/Experiment: +To generate glioblastoma spheroid model using an orbital shaker, which can mimic in vivo glioblastoma behavior and facilitate drug testing and therapeutic development. + +## Generation of Glioblastoma Spheroid Model using an Orbital Shaker + +DOI: [dx.doi.org/10.17504/protocols.io.8epv5jz861lb/v1](dx.doi.org/10.17504/protocols.io.8epv5jz861lb/v1) + +### Authors +- Wannawat Khotchawan¹ +- Chanchao Lorthongpanich² +- Pakpoom Kheolamai³ +- Sith Sathornsumetee⁴ +- Surapol Issaragrisil⁵ + +¹ Department of Immunology, Faculty of Medicine Siriraj Hospital, Mahidol University, Thailand +² Siriraj Center of Excellence for Stem Cell Research, Faculty of Medicine Siriraj Hospital, Mahidol University, Thailand +³ Division of Cell Biology, Faculty of Medicine, Thammasat University, Thailand +⁴ Siriraj Center of Research Excellence Management, Faculty of Medicine Siriraj Hospital, Mahidol University, Thailand +⁵ Division of Hematology, Department of Medicine, Faculty of Medicine Siriraj Hospital, Mahidol University, Thailand + +### Abstract +Glioblastoma multiforme (GBM) is a highly aggressive brain tumor with a high fatality rate. The need for new treatments is urgent. This protocol outlines a method to generate GBM spheroids from the U251 GBM cell line using an orbital shaker. These 3D models better represent in vivo tumor behaviors and drug responses, providing a useful platform for therapeutic studies. + +### Keywords +- Glioblastoma +- Orbital shaker +- Spheroid +- U251-MG + +### License +This protocol is distributed under the terms of the Creative Commons Attribution License. + +### Materials +#### Generation of Three-Dimensional (3D) Glioblastoma Spheroids +1. **U251 MG cell line human** (Sigma Aldrich) +2. **DMEM High Glucose**: Dulbecco's Modified Eagle Medium (Thermo Fisher Scientific) +3. **Fetal Bovine Serum (FBS)** +4. **75-cm2 cell culture flask** +5. **Sterile serological pipettes and pipette tips** (1000 µl) +6. **Transfer pipettes** +7. **Trypan blue** +8. **Hemocytometer** +9. **Non-treated flat-bottom 6-well plate** (Sigma Aldrich) +10. **Orbital shaker** +11. **Tissue culture hood** +12. **Humidified incubator** (37 °C, 5% CO₂) + +#### Detection of Apoptosis in Glioblastoma Spheroid Model +1. **Protein lysis buffer** +2. **12% Bis-Tris polyacrylamide gels** +3. **Electrophoresis machine** +4. **Trans-Blot® SD Semi-Dry Cell** (Bio-rad) +5. **Rabbit Anti-Caspase-3 antibody** (Cell Signaling) +6. **Goat anti-rabbit antibody-HRP** (Merck) +7. **Anti-β-actin-HRP** (Cell Signaling) +8. **Immobilon Western chemiluminescent HRP substrate** +9. **Biomolecular imager** + +#### Assessment of Drug Response in Glioblastoma Spheroid Model +1. **DMEM High Glucose**: Dulbecco's Modified Eagle Medium (Thermo Fisher Scientific) +2. **Fetal Bovine Serum (FBS)** +3. **Temozolomide** (Sigma Aldrich) +4. **Orbital shaker** +5. **Tissue culture hood** +6. **Humidified incubator** (37 °C, 5% CO₂) +7. **Confocal microscope** + +### Methods +#### Generation of Three-Dimensional (3D) Glioblastoma Spheroids +1. **Cell Culture**: + - Culture U251-MG cells in DMEM-high glucose medium with 10% fetal bovine serum in a 75-cm2 flask. + - Allow cells to grow for 3-4 days until they reach 70–80% confluence. + +2. **Cell Counting**: + - Perform cell counting using a trypan blue exclusion assay to determine cell number and viability. + - Adjust the concentration to 1×10⁶ cells/ml. + +3. **Spheroid Formation**: + - Add 2 ml of culture medium (2×10⁶ viable cells) into one well of a non-coated flat-bottom 6-well plate. + +4. **Incubation**: + - Set the orbital shaker in a CO₂ incubator at 37 °C and 5% CO₂ atmosphere. + - Place the cell culture plate on the orbital shaker, set the speed to 120 rpm, and culture overnight. + +5. **Collection**: + - The resulting GBM spheroids will be ready for the experiment the following morning. + +#### Detection of Apoptosis in Glioblastoma Spheroid Model +7. **Protein Extraction**: + - Collect spheroids for protein extraction as per manufacturer's instructions. + +8. **Lysis**: + - Lyse spheres using cell lysis buffer. + +9. **Electrophoresis**: + - Separate extracted proteins using electrophoresis on 12% Bis-Tris polyacrylamide gels and transfer them onto PVDF membranes. + +10. **Immunoblotting**: + - Perform immunoblotting with primary antibodies against Caspase-3, Cleaved-caspase-3, and horseradish peroxidase-conjugated secondary antibodies. + - Image the blot using a fluorescence scanner. + +#### Assessment of Drug Response in Glioblastoma Spheroid Model +11. **Drug Treatment**: + - Culture GBM spheroids in U251-MG medium containing 0, 25, 50, and 100 µM of Temozolomide, a standard GBM drug. + +12. **Incubation**: + - Place the cell culture plate onto the orbital shaker, adjust the speed to 120 rpm, and culture overnight in an incubator at 37 °C and 5% CO₂. + +13. **Observation**: + - Observe spheroid morphological changes and dead cells after treatment with Temozolomide for 24 hours. + +### Notes +- If the procedure and technique are correctly followed, a sphere should form after placing the plate overnight on an orbital shaker. + +### Acknowledgements +- Wannawat Khotchawan (Grant ID: Faculty of Medicine Siriraj Hospital, Mahidol University) +- Chanchao Lorthongpanich (Grant ID: National Research Council of Thailand N41A640154) + +--- + +**endofoutput** +``` \ No newline at end of file diff --git a/markdown-output/generic-protocol-for-environmental-health-systemat-biktkcwn.md b/markdown-output/generic-protocol-for-environmental-health-systemat-biktkcwn.md new file mode 100644 index 0000000000000000000000000000000000000000..470dc0cafe785bfc2e4db4e744a0b4c84fc1bdc0 --- /dev/null +++ b/markdown-output/generic-protocol-for-environmental-health-systemat-biktkcwn.md @@ -0,0 +1,172 @@ +```markdown +# Goal/Experiment: +The goal of this experiment is to follow the COSTER (Conduct of Systematic Reviews in Toxicology and Environmental Health Research) recommendations for conducting systematic reviews in environmental health. This protocol template assists researchers with planning and executing systematic reviews to convert COSTER from a checklist into an actionable sequence for research teams. + +# Generic Protocol for Environmental Health Systematic Reviews Based on COSTER Recommendations + +**Paul Whaley** +*Lancaster University* +*In Development* +[dx.doi.org/10.17504/protocols.io.biktkcwn](https://dx.doi.org/10.17504/protocols.io.biktkcwn) + +## Abstract +A protocol template to help researchers follow the [COSTER recommendations](https://doi.org/10.1016/j.envint.2020.105926) for the conduct of systematic reviews. This instance covers the planning steps of a systematic review and will assist with writing up the systematic review protocol. + +- Intent: Convert COSTER from a checklist into a sequence of actions for a research team. +- Completion: Cite the parent manuscript and this protocol when registering or submitting to a journal. + +**External Link:** +[Environment International Article](https://www.sciencedirect.com/science/article/pii/S016041202031881X) + +### Protocol Citation +Whaley, Paul. "Generic Protocol for Environmental Health Systematic Reviews Based on COSTER Recommendations." *protocols.io*. +[dx.doi.org/10.17504/protocols.io.biktkcwn](https://dx.doi.org/10.17504/protocols.io.biktkcwn) + +### Manuscript Citation +Whaley, Paul, Elisa Aiassa, Claire Beausoleil, Anna Beronius, et al. "Recommendations for the Conduct of Systematic Reviews in Toxicology and Environmental Health Research (COSTER)." *Environment International* 143: 105926. +DOI: 10.1016/j.envint.2020.105926 + +--- + +## Keywords +- Systematic review +- Environmental health +- Toxicology +- Protocol + +## License +This protocol is distributed under the terms of the [Creative Commons Attribution License](https://creativecommons.org/licenses/by/4.0/), permitting unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. + +--- + +## Securing Capacity, Competencies, and Tools + +### 1. Assess Team's Competence + +| Competency | Team Member(s) (initials) | +|------------|---------------------------| +| Information science (e.g., search strategies) | | +| Evidence appraisal methods (e.g., bias assessment) | | +| Statistical methods | | +| Domain/subject expertise | | +| Systematic review methods | | + +### 2. Identify Information Management Tools + +| Information Management Component | Tools or Packages | +|----------------------------------|-------------------| +| Reference manager | | +| Knowledge management tool | | +| Systematic review software | | +| Statistics software and packages | | +| AI support tools (e.g., for screening) | | + +### 3. List Potential Conflicts of Interest + +- Potential conflicts (both financial and non-financial) must be disclosed to understand motivations. +- Use ICMJE Conflict of Interest Disclosure Forms. + +| Author | ICMJE COI Summary | +|--------|-------------------| + +--- + +## Setting the Research Question ("Problem Formulation") + +### 4. Demonstrate Need for a New Review + +#### 4.1 Describe the scientific value of the question(s). +#### 4.2 Describe the importance to stakeholders. +#### 4.3 Summarize relevant primary research and evidence syntheses. + +### 5. Articulate Scientific Rationale for Each Question + +- Develop a theoretical framework describing the biological plausibility of the relationship being investigated. + +### 6. Define Research Objective Using PECO Elements + +- PECO: Population, Exposure or Intervention, Comparator, Outcome + +--- + +## Defining Eligibility Criteria and Screening Evidence + +### 7. Define Eligibility Criteria for Each Component + +| PECO Element | Description of Eligibility Criteria | +|--------------|-------------------------------------| +| Eligible populations | | +| Eligible exposures | | +| Eligible comparators | | +| Eligible primary outcomes | | +| Eligible secondary outcomes | | +| Eligible study designs | | + +### 8. Define Screening Points + +- Determine stages for screening: title and abstract, full text, or both. + +### 9. Include All Relevant, Publicly-Available Evidence + +- COSTER recommends including grey literature, except when methodological information for internal validity appraisal is insufficient. + +### 10. Include Relevant Evidence Irrespective of Language + +| Languages to be Included in Systematic Review | | +|-----------------------------------------|---| + +### 11. Do Not Exclude Multiple Reports of the Same Research + +--- + +## Methods for Synthesizing and Evaluating Evidence + +### 12. Design "Characteristics of Included Studies" Table + +### 13. Design and Pilot Data Extraction Forms + +### 14. Define Risk of Bias Assessment Methods + +- Use the FEAT mnemonic (Focus, Extent, Application, Transparency). + +| Tool Selected | Studies Applied | Modifications Made | Validation Method | +|---------------|-----------------|--------------------|-------------------| + +--- + +## Methods for Synthesizing Included Studies + +### 15. Define Methods for Synthesizing Included Studies + +| Synthesis Component | Planned Methods | +|---------------------|-----------------| +| Qualitative/narrative methods | | +| Quantitative methods | | +| Conditions for combining studies in analyses | | +| Choice of effect measure | | +| Assessment of heterogeneity | | +| Effect modifiers for subgroup analysis | | +| Transformation of scales into common measures | | +| Assessment of publication bias | | +| Impact of risk bias on synthesis | | + +--- + +## Methods for Integrating Evidence + +### 16. Piloting and Assessing Confidence in Synthesis + +### 17. Plan for Multiple Streams of Evidence + +--- + +## Registering and Publishing the Protocol + +### 18. Create a Permanent Public Record of Review Intent +### 19. Secure Peer-Review and Feedback on Draft Protocol +### 20. Publish Final Protocol Version in a Public Archive + +--- + +`endofoutput` +``` \ No newline at end of file diff --git a/markdown-output/genetic-diversity-and-population-structure-of-dome-umkeu4w.md b/markdown-output/genetic-diversity-and-population-structure-of-dome-umkeu4w.md new file mode 100644 index 0000000000000000000000000000000000000000..7783bd0d72bb8b6fa1dfeaf4dffc085d856c0467 --- /dev/null +++ b/markdown-output/genetic-diversity-and-population-structure-of-dome-umkeu4w.md @@ -0,0 +1,74 @@ +```markdown +# Goal/Experiment: +To characterize the genetic structure of domestic and wild reindeer populations using a genome-wide bovine genotyping array (BovineHD BeadChip). + +# Genetic Diversity and Population Structure of Domestic and Wild Reindeer (Rangifer tarandus L. 1758): A Novel Approach Using BovineHD BeadChip + +**Authors**: Veronika Ruslanovna Kharzinova, Arsen Vladimirovich Dotsev, Tatiana Evgenievna Deniskova, Anastasiya Dmitrievna Solovieva, Valeriy Ivanovich Fedorov, Kasim Anverovich Layshev, Tatiana Mikhailovna Romanenko, Innokentiy Mikhailovich Okhlopkov, Klaus Wimmers, Henry Reyer, Gottfried Brem, Natalia Anatolevna Zinovieva. + +## Abstract +Reindeer (*Rangifer tarandus* L. 1758) are crucial for the Russian Far North, offering nutrition for 18 ethnicities. This study uses a genome-wide bovine genotyping array (BovineHD BeadChip) to characterize the genetic structure of domestic and wild reindeer populations. Samples were taken from the Taymyr Peninsula and the taiga/tundra regions of the Sakha Republic (Yakutia). Results demonstrated strong genetic differentiation between domestic and wild reindeer populations, aiding genetic improvement strategies and conservation programs. + +**External Links**: +- [PLOS One Publication](https://doi.org/10.1371/journal.pone.0207944) + +## Materials and Methods + +**Samples Collected**: 135 reindeer (61 wild, 74 domestic) + +### Collection Details +- **Wild populations**: Taymyr Peninsula (27 individuals) and taiga/tundra regions of Yakutia (34 individuals). +- **Domestic populations**: Sakha Republic (Yakutia), Murmansk region, and Nenets Autonomous District. + +**Sampling Period**: 2014–2017 under Federal Scientific Programs. + +### Safety Warnings and Ethical Considerations +This study complies with ethical guidelines ensuring no harm to endangered species. Approvals were obtained from relevant authorities under the L.K. Ernst Federal Science Center for Animal Husbandry protocol (2018/1). + +### Sample Collection and Preparation of Genomic DNA + +1. **Genomic DNA Extraction**: + - **Reagent**: Nexttec columns (Nexttec Biotechnology GmbH, Germany) + - **Quality Checks**: + - **Electrophoresis**: 1% agarose gels + - **Quantification**: Qubit 3.0 fluorometer (Thermo Fisher Scientific, Wilmington, DE, USA) + - **Purity**: NanoDrop-2000 (Thermo Fisher Scientific, Wilmington, DE, USA) + +### SNP Genotyping and Quality Control + +2. **Genotyping**: + - **Platform**: Illumina BovineHD Genotyping BeadChip (Illumina, Inc. San Diego, USA) + - **Quality Control**: + - Samples with call rates below 90% were excluded. + - Additional filters for SNPs: + - >10% missing genotypes + - Minor allele frequency <5% + - Non-autosomal mapping + - Deviation from Hardy-Weinberg equilibrium (p≤1×10-6) + - **Software**: PLINK v1.07 + +### Genetic Diversity and Differentiation Analysis + +3. **Analysis Metrics**: + - **Diversity**: + - Observed Heterozygosity (Ho) and unbiased expected Heterozygosity (He) + - Inbreeding Coefficient (FIS) + - Rarefied Allelic Richness (Ar) + - **Tools**: R packages “diveRsity” and “STAMPP”, PLINK v1.07, and R package “ggplot2”. + +### Genetic Structure Analysis + +4. **Software and Tools**: + - **STRUCTURE v2.3.4**: + - For population structure analysis + +5. **TreeMix Analysis**: + - **Software**: TreeMix v1.13 + - **Analysis**: + - Jackknife blocks of 10 SNPs for calculating standard errors and p-values of population splits and gene flow. + - Visualization using R. + +--- + +**Endofoutput** +``` \ No newline at end of file diff --git a/markdown-output/genometrakr-wgs-protocol-collection-and-workflow-f-ckfkutkw.md b/markdown-output/genometrakr-wgs-protocol-collection-and-workflow-f-ckfkutkw.md new file mode 100644 index 0000000000000000000000000000000000000000..d8379fa5eec851bc55a49a99a1960500034de632 --- /dev/null +++ b/markdown-output/genometrakr-wgs-protocol-collection-and-workflow-f-ckfkutkw.md @@ -0,0 +1,165 @@ +```markdown +# Goal/Experiment: +The goal of this experiment is to provide a comprehensive workflow for Whole Genome Sequencing (WGS) using the Illumina MiSeq platform. This includes protocols for both wet lab and dry lab processes, from sample extraction to submission to the National Center for Biotechnology Information (NCBI). + +# GenomeTrakr WGS Protocol Collection and Workflow for MiSeq V.2 + +## DOI +[dx.doi.org/10.17504/protocols.io.3byl4bwyjvo5/v2](https://dx.doi.org/10.17504/protocols.io.3byl4bwyjvo5/v2) + +## Authors +- Tina Pfefer¹ +- Julie Haendiges¹ +- Maria Balkey¹ +- Ruth Timme¹ + +¹US Food and Drug Administration + +## Technical Support +Email: [genomeTrakr@fda.hhs.gov](mailto:genomeTrakr@fda.hhs.gov) + +--- + +## Abstract +Here we have created a collection of all the protocols used for WGS using the MiSeq, in order, from sample extraction to NCBI submission. + +## This collection has three sections: +1. **WGS Wet lab workflow for Illumina MiSeq** +2. **Dry lab workflow for sequence QC and NCBI submission - Direct Submission** +3. **Dry lab workflow for sequence QC and NCBI submission - PulseNet labs** + +### Associated Protocols: +- [Querying the NCBI database for GenomeTrakr data](https://www.protocols.io/view/querying-the-ncbi-database-for-genometrakr-data-pwdm656e5zdw/) +- [NCBI data curation protocol - SOP for editing GenomeTrakr submissions](https://www.protocols.io/view/ncbi-data-curation-protocol-sop-for-editing-genom-pzod68e8o14r/) + +### Protocol Citation +Tina Pfefer, Julie Haendiges, Maria Balkey, Ruth Timme 2022. GenomeTrakr WGS Protocol Collection and Workflow for MiSeq. _protocols.io_ [dx.doi.org/10.17504/protocols.io.3byl4bwyjvo5/v2](https://dx.doi.org/10.17504/protocols.io.3byl4bwyjvo5/v2) + +### Keywords +GenomeTrakr, whole genome sequencing, enteric pathogens, surveillance, MiSeq + +### License +This is an open access protocol distributed under the terms of the [Creative Commons Attribution License](https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. + +--- + +## Wet Lab + +### 1. WGS Wet Lab Workflow for Illumina MiSeq + +#### 2. DNA Extraction +- **Protocol:** Manual DNA Extraction using Qiagen DNeasy Blood and Tissue Kit + - **Created by:** Julie Haendiges + - **Details:** [Preview Protocol](https://www.protocols.io/view/manual-dna-extraction-using-qiagen-dneasy-blood-an-xyz) + +#### 3. DNA Quantification +- **Protocol:** DNA Quantification using the Qubit Fluorometer + - **Created by:** Julie Haendiges + - **Details:** [Preview Protocol](https://www.protocols.io/view/dna-quantification-using-the-qubit-fluorometer-xyz) + +#### 4. Library Preparation +- **Protocol:** Illumina DNA Prep (M) Tagmentation Library Preparation for use on an Illumina MiSeq Sequencer + - **Created by:** Julie Haendiges + - **Details:** [Preview Protocol](https://www.protocols.io/view/illumina-dna-prep-m-tagmentation-library-preparati-xyz) + +#### 5. Sequencing +- **Protocol:** Procedure for Operation and Maintenance of the Illumina MiSeq for Whole Genome Sequencing + - **Created by:** Julie Haendiges + - **Details:** [Preview Protocol](https://www.protocols.io/view/procedure-for-operation-and-maintenance-of-the-illu-xyz) + +--- + +## Dry Lab - Direct Submission + +### 6. Dry Lab Workflow for Sequence QC and NCBI Submission - Direct Submission +The following protocols are also included in a Springer Methods book chapter collection: [Utilizing the Public GenomeTrakr Database for Foodborne Pathogen Traceback](https://www.protocols.io/view/using-public-genometrakr-database-for-foodborne-pat-abcd) + +#### 7. Check Sequence Quality +- **Protocol:** Quality control assessment for microbial genomes: GalaxyTrakr MicroRunQC workflow + - **Created by:** Ruth Timme + - **Details:** [Preview Protocol](https://www.protocols.io/view/qc-assessment-microbial-genomes-xyz) + +#### 8. Populate BioSample AND SRA Metadata Templates +- **Protocol:** Guidance for populating GenomeTrakr metadata templates (BioSample and SRA) + - **Created by:** Ruth Timme + - **Details:** [Preview Protocol](https://www.protocols.io/view/fill-genometrakr-metadata-templates-xyz) + +#### 9. Submit Sequence and Metadata to NCBI +- **Protocol:** NCBI submission protocol for microbial pathogen surveillance + - **Created by:** Ruth Timme + - **Details:** [Preview Protocol](https://www.protocols.io/view/ncbi-submission-for-microbial-pathogen-surveillance-xyz) + +#### 10. Update, Retract, or Replace These Records in NCBI Databases, if Necessary +- **Protocol:** NCBI data curation protocol - SOP for editing GenomeTrakr submissions + - **Created by:** Ruth Timme + - **Details:** [Preview Protocol](https://www.protocols.io/view/ncbi-data-curation-sopr-xyz) + +--- + +## Dry Lab - PulseNet Submission + +### 11. Dry Lab Workflow for Sequence QC and NCBI Submission - PulseNet Labs + +#### 12. Check Sequence Quality +- **Note:** Follow the CDC’s guidance for assessing QC in BioNumerics + +#### 13. Populate NCBI Template +- **Note:** For NCBI submission for fields not included in the BioNumerics Template, please remember to include the name of the laboratory sequencing the isolates and the surveillance effort name in the `sequenced_by` and `project_name` fields as described in Step 5 of this protocol. +- **Protocol:** Populating NCBI template for submissions using BioNumerics v7.6 + - **Created by:** Maria Balkey + - **Details:** [Preview Protocol](https://www.protocols.io/view/populating-ncbi-template-for-submissions-using-biono-xyz) + +#### 14. Submit Sequence and Metadata to NCBI +- **Note:** Follow the CDC’s guidance for NCBI submissions through BioNumerics. + +#### 15. Update, Retract, or Replace These Records in NCBI Databases, if Necessary +- **Protocol:** NCBI data curation protocol - SOP for editing GenomeTrakr submissions + - **Created by:** Ruth Timme + - **Details:** [Preview Protocol](https://www.protocols.io/view/ncbi-data-curation-sopr-xyz) + +--- + +## End of Document +``` + +--- + +# Professional/Scientific Terms and Reagents Explained + +### Reagents +1. **Qiagen DNeasy Blood and Tissue Kit:** + - **Function:** This kit is used for the extraction of high-quality DNA from blood, tissues, cells, and other samples. + - **Vendor:** Qiagen + +2. **Qubit Fluorometer:** + - **Function:** This instrument is used for the quantification of DNA, RNA, and protein. + - **Vendor:** Thermo Fisher Scientific + +3. **Illumina DNA Prep (M) Tagmentation:** + - **Function:** Tagmentation is a process used in next-generation sequencing library preparation that fragments and tags DNA with sequencing adapters. + - **Vendor:** Illumina + +### Alternative Methods for Hard to Find Supplies +1. **Qiagen DNeasy Blood and Tissue Kit:** + - **Alternative:** Zymo Quick-DNA Miniprep Plus Kit + +2. **Qubit Fluorometer:** + - **Alternative:** NanoDrop Spectrophotometer + +3. **Illumina DNA Prep (M) Tagmentation:** + - **Alternative:** Nextera DNA Flex Library Prep kit + +### Equipment +1. **Illumina MiSeq Sequencer:** + - **Function:** A next-generation sequencing platform used for whole genome and targeted sequencing. + - **Vendor:** Illumina + +### Annotations +1. **NCBI (National Center for Biotechnology Information):** + - A division of the National Institutes of Health that houses a series of databases relevant to biotechnology and biomedicine. + +2. **BioSample and SRA (Sequence Read Archive) Metadata Templates:** + - Templates provided by NCBI for organizing and submitting metadata associated with sequence data to NCBI databases. + + +endofoutput \ No newline at end of file diff --git a/markdown-output/gibson-assembly-master-mix-assembly-e2611-imsupm.md b/markdown-output/gibson-assembly-master-mix-assembly-e2611-imsupm.md new file mode 100644 index 0000000000000000000000000000000000000000..83cc22ef3a7069055189d8e047b6b00bac370b40 --- /dev/null +++ b/markdown-output/gibson-assembly-master-mix-assembly-e2611-imsupm.md @@ -0,0 +1,109 @@ +``` +Goal/Experiment: +The goal of the experiment is to perform Gibson Assembly using the Gibson Assembly® Master Mix (E2611) to successfully assemble multiple DNA fragments into a vector. + +# Gibson Assembly® Master Mix – Assembly (E2611) +## New England Biolabs + +**Abstract** +This is the protocol for the Gibson Assembly using the Gibson Assembly® Master Mix (E2611). + +**Citation**: New England Biolabs Gibson Assembly® Master Mix – Assembly (E2611). protocols.io dx.doi.org/10.17504/protocols.io.cjxupnm + +**Published**: 26 Jan 2015 + +## Guidelines + +### Optimal Quantities + +NEB recommends a total of 0.02–0.5 pmols of DNA fragments when 1 or 2 fragments are being assembled into a vector and 0.2–1.0 pmoles of DNA fragments when 4–6 fragments are being assembled. Efficiency of assembly decreases as the number or length of fragments increases. + +**Equation to Calculate pmols:** +``` +pmols = (weight in ng) x 1,000 / (base pairs x 650 daltons) +``` + +**Examples:** +- 50 ng of 5000 bp dsDNA is about 0.015 pmols. +- 50 ng of 500 bp dsDNA is about 0.15 pmols. + +The mass of each fragment can be measured using the NanoDrop instrument, absorbance at 260 nm, or estimated from agarose gel electrophoresis followed by ethidium bromide staining. + +Optimized cloning efficiency is 50–100 ng of vectors with 2–3 fold of excess inserts. Use 5 times more of inserts if size is less than 200 bps. Total volume of unpurified PCR fragments in Gibson Assembly reaction should not exceed 20%. + +--- + +## Overview: + +Gibson Assembly was developed by Dr. Daniel Gibson and his colleagues at the J. Craig Venter Institute and licensed to NEB by Synthetic Genomics, Inc. It allows for successful assembly of multiple DNA fragments, regardless of fragment length or end compatibility. It has been rapidly adopted by the synthetic biology community due to its ease-of-use, flexibility, and suitability for large DNA constructs. + +Gibson Assembly efficiently joins multiple overlapping DNA fragments in a single-tube isothermal reaction. The Gibson Assembly Master Mix includes three different enzymatic activities that perform in a single buffer: +- **Exonuclease** creates single-stranded 3' overhangs that facilitate the annealing of fragments that share complementarity at one end (overlap region). +- **Proprietary DNA polymerase** fills in gaps within each annealed fragment. +- **DNA ligase** seals nicks in the assembled DNA. + +The end result is a double-stranded fully sealed DNA molecule that can serve as a template for PCR, RCA, or other molecular biology applications, including direct transformation. The method has been successfully used for Gibson’s group and others to assemble oligonucleotides, DNA with varied overlaps (15–80 bp), and fragments hundreds of kilobases long. + +--- + +## Overview of the Gibson Assembly Cloning Method + +1. **Design primers to amplify fragments (and/or vector) with appropriate overlaps.** +2. **PCR amplify fragments using a high-fidelity DNA polymerase.** +3. **Prepare linearized vector by PCR amplification using a high-fidelity DNA polymerase or by restriction digestion.** +4. **Confirm and determine concentration of fragments and linearized vector using agarose gel electrophoresis, NanoDrop™ instrument, or other methods.** +5. **Add fragments and linearized vector to Gibson Assembly Master Mix and incubate at 50°C for 15 minutes to 1 hour, depending on the number of fragments being assembled.** +6. **Transform into E. coli or use directly in other applications.** + +--- + +# Notes: + +### 1. General Notes: +- NEBuilder® to design PCR primers with overlapping sequences between the adjacent DNA fragments for their assembly into a cloning vector. + +### 2. Usage Notes: +To ensure the successful assembly and subsequent transformation of assembled DNAs, NEB recommends the following: + +- **Cells**: Transformation efficiency of competent cells can vary, impacting perceived assembly efficiency. Electroporation can increase transformation efficiency significantly. +- **DNA**: PCR product purification is not necessary if the total volume of PCR products is 20% or less of the Gibson Assembly reaction volume. Higher volumes may reduce efficiency due to PCR buffer carryover. +- **Insert**: When assembling fragments into a cloning vector, concentration of assembly fragments should be 2–3 times higher than the vector. For assemblies of 3 or more fragments, use equimolar ratio of fragments. +- **Biology**: Certain DNA structures may be selected against by E. coli, resulting in poor transformation or small colonies. + +--- + +## Materials + +- **Gibson Assembly Master Mix - 10 rxns [E2611S by New England Biolabs](https://www.neb.com/products/e2611) + +--- + +## Protocol + +### Step 1. +Set up the following reaction on ice: + +| **Component** | **2-3 Fragment Assembly** | **4-6 Fragment Assembly** | **Positive Control** | +|-----------------------------------|---------------------------|----------------------------|-----------------------| +| Total Amount of Fragments | 0.02-0.5 pmol | 0.2-1 pmol | 10 µl | +| Gibson Assembly Master Mix (2X) | 10 µl | 10 µl | 10 µl | +| Deionized H2O | 10-X µl | 10-X µl | 0 | +| **Total Volume** | 20 µl | 20 µl | 20 µl | + +### Step 2. +Incubate samples in a thermocycler at 50°C for 15 minutes when 2 or 3 fragments are being assembled or 60 minutes when 4-6 fragments are being assembled. + +### Step 3. +Store samples on ice or at -20°C for subsequent transformation. + +--- + +## References: +1. Gibson, D.G. et al. (2009). Nature Methods. 343-345. +2. Gibson, D.G. et al. (2010). Nature Methods. 901-903. +3. Barnes, W.M. (1994). Proc. Natl. Acad. Sci.. 91, 2216-220. + +--- + +**Endofoutput** +``` \ No newline at end of file diff --git a/markdown-output/gibson-assembly-protocol-e5510-imss45.md b/markdown-output/gibson-assembly-protocol-e5510-imss45.md new file mode 100644 index 0000000000000000000000000000000000000000..bb734057d0fd5a1f603acbea7908a905b345e22d --- /dev/null +++ b/markdown-output/gibson-assembly-protocol-e5510-imss45.md @@ -0,0 +1,121 @@ +```markdown +# Goal/Experiment: +The goal of this experiment is to perform Gibson Assembly using the Gibson Assembly® Cloning Kit (E5510) from New England Biolabs. This protocol is designed to efficiently join multiple overlapping DNA fragments in a single-tube isothermal reaction. + +# Gibson Assembly® Protocol (E5510) + +## Abstract + +This is the protocol for the Gibson Assembly using the Gibson Assembly® Cloning Kit (E5510). + +Citation: New England Biolabs Gibson Assembly® Protocol (E5510). [protocols.io](dx.doi.org/10.17504/protocols.io.cdms45) +Published: 27 Jan 2015 + +## Guidelines + +### Optimal Quantities + +NEB recommends using a total of 0.02–0.5 pmols of DNA fragments when 1 or 2 fragments are being assembled into a vector and 0.2–1.0 pmols of DNA fragments when 4–6 fragments are being assembled. Efficiency of assembly decreases as the number or length of fragments increases. + +To calculate the number of pmols of each fragment for optimal assembly, you can use NEB's online tool, [NEBioCalculator](https://nebiocalculator.neb.com), or the following formula: + +```plaintext +pmols = (weight in ng) x 1,000 / (base pairs x 650 daltons) +``` + +- 50 ng of 5000 bp dsDNA is about 0.015 pmols. +- 50 ng of 500 bp dsDNA is about 0.15 pmols. + +The mass of each fragment can be measured using the NanoDrop instrument (absorbance at 260 nm) or estimated from agarose gel electrophoresis followed by ethidium bromide staining. + +Optimized cloning efficiency is 50–100 ng of vectors with 2–3 fold of excess inserts. Use 5 times more inserts if size is less than 200 bps. Total volume of unpurified PCR fragments in Gibson Assembly reaction should not exceed 20%. + +## Overview + +### Introduction + +Gibson Assembly was developed by Dr. Daniel Gibson et al. from the J. Craig Venter Institute and licensed to NEB by Synthetic Genomics, Inc. It allows for successful assembly of multiple DNA fragments regardless of fragment length or end compatibility. It has been rapidly adopted due to its ease-of-use, flexibility, and suitability for large DNA constructs. + +Gibson Assembly efficiently joins multiple overlapping DNA fragments in a single-tube isothermal reaction. The Gibson Assembly Master Mix includes three different enzymatic activities that function in a single buffer: + +- **Exonuclease:** Creates single-stranded 3' overhangs for annealing of complementary DNA ends. +- **DNA Polymerase:** Fills in gaps within the annealed fragments. +- **DNA Ligase:** Seals nicks in the assembled DNA. + +The end result is a double-stranded DNA molecule suitable for various molecular biology applications, including direct transformation. + +### Method + +#### Overview of the Gibson Assembly Cloning Method + +1. Design primers to amplify fragments (and/or the vector) with appropriate overlaps. +2. PCR amplify fragments using a high-fidelity DNA polymerase. +3. Prepare the linearized vector by PCR amplification using a high-fidelity DNA polymerase or restriction digestion. +4. Confirm and determine the concentration of fragments and linearized vector using agarose gel electrophoresis, a NanoDrop™ instrument, or another method. +5. Add fragments and linearized vector to Gibson Assembly Master Mix and incubate at 50°C for 15 to 60 minutes depending on the number of fragments being assembled. +6. Transform the reaction into NEB 5-alpha Competent E. coli (provided) or use directly in other applications. + +### Notes: + +1. Use the [NEBuilder® tool](https://nebuilder.neb.com) to design PCR primers with overlapping sequences between adjacent DNA fragments for their assembly into a cloning vector. +2. The kit is shipped on dry ice. Upon arrival, store it at -80°C. After first use, store the kit components at indicated temperatures. +3. To ensure successful assembly and subsequent transformation of assembled DNAs: + - **DNA:** PCR product purification is not necessary if total PCR products volume in the assembly reaction is 20% or less of the total volume. Higher volumes may reduce efficiency. Column purification can enhance both Gibson Assembly and transformation efficiency. Use ddH2O, TE, or other dilution buffers for dissolving DNA. + - **Insert:** When assembling fragments into a cloning vector, concentration should be 2–3 times higher than the vector. + - **Transformation:** NEB 5-alpha Competent E. coli is recommended for assemblies under 20 kb. For other competent cells, use the suggested protocol notes. + - **Electroporation:** To increase transformation efficiency with electrocompetent cells using Gibson Assembly Master Mix, dilute the reaction 3-fold and use 1 µl for transformation. + +## References + +1. Gibson, D.G. et. al. (2009). Nature Methods. 343-345. +2. Gibson, D.G. et. al. (2010). Nature Methods. 901-903. +3. Barnes, W.M. (1994). Proc. Natl. Acad. Sci.. 91, 2216-220. + +## Materials + +- **Gibson Assembly Cloning Kit:** 10 reactions (E5510S) by [New England Biolabs](https://www.neb.com) + +## Protocol + +### Step 1 + +Set up the following reaction on ice: + +| | 2-3 Fragment Assembly | 4-6 Fragment Assembly | Positive Control | +|-----------------------|-----------------------|-----------------------|------------------| +| Total Amount of Fragments | 0.02-0.5 pmol X µl | 0.2-1.0 pmol X µl | 10 µl | +| Gibson Assembly Master Mix (2X) | 10 µl | 10 µl | 10 µl | +| Deionized H2O | 10-X µl | 10-X µl | 0 µl | +| **Total Volume** | 20 µl | 20 µl | 20 µl | + +### Step 1.1 + +**DNA Fragments** +Insert fragments as necessary to meet desired assembly requirements. + +### Step 1.2 + +**Gibson Assembly Master Mix (2X)** + +### Step 1.3 + +**Deionized H2O** + +### Step 2 + +Incubate samples in a thermocycler at 50°C for 15 minutes when 2 or 3 fragments are being assembled or 60 minutes when 4-6 fragments are being assembled. + +### Step 3 + +Store samples on ice or at -20°C for subsequent transformation. + +### Step 4 + +Transform NEB 5-alpha Competent E. coli cells (provided with the kit) with 2 µl of the assembly reaction, following the transformation protocol. + +## Additional Info + +Control reagents are provided for 5 experiments. Greater numbers of fragments may require additional Gibson Assembly Master Mix. Optimized cloning efficiency uses 50-100 ng of vectors with 2-3 fold of excess inserts. Unpurified PCR fragments should total no more than 20% of the reaction. + +**endofoutput** +``` \ No newline at end of file diff --git a/markdown-output/global-standards-for-biosimilars-rv3d68n.md b/markdown-output/global-standards-for-biosimilars-rv3d68n.md new file mode 100644 index 0000000000000000000000000000000000000000..bf35c7121da5521fdfd3645e07ffe68cc166189f --- /dev/null +++ b/markdown-output/global-standards-for-biosimilars-rv3d68n.md @@ -0,0 +1,47 @@ +```markdown +# Goal/Experiment: +The goal of this document is to understand the global standards for biosimilars, focusing on their development, regulation, and market access in different regions, with specific emphasis on Europe and America. + +# Global Standards for Biosimilars +**Author:** Bella Smith +**Published:** 23 Jul 2018 + +## Abstract + +### Introduction +As biopharmaceutical technology becomes more widely used in the treatment of diseases such as rheumatology, cancer, and other chronic diseases, professionals in the pharmaceutical industry predict that half of the newly approved drugs will soon come from biomedicine. This has also boosted the pharmaceutical industry's interest in developing biosimilars. The opportunity to sell biopharmaceuticals that have passed the patent protection period is becoming more attractive, especially since many biopharmaceuticals will lose patent protection in the coming years. + +Biogenerics are new copies of original biological products approved by law. Unlike common generic drugs defined by US Food and Drug Administration (FDA), which are small-molecule products, biosimilars are macromolecules made from biological organisms. Biosimilars require clinical trials due to their sensitivity to production processes and the potential health risks associated with impurities or differences in molecular makeup. + +Patients and doctors may be hesitant to adopt biosimilars due to concerns about efficacy and safety. Regulations are essential to reduce these concerns and ensure confidence in biosimilars. + +## Current Regulations in Europe and America + +### Europe +The European Medicines Agency (EMEA) has distinguished between chemical generics and biosimilars due to the complexity of biological and biotech derivatives. In 2006, the European Medicines Agency introduced regulations for biosimilars, providing a 10-year data protection period for generics and biologics for biosimilars. These regulations require extensive testing before approval. + +The EU's guidelines acknowledge differences between biosimilars and existing biologics in raw materials and manufacturing processes. Subtle differences may affect the safety and effectiveness of biosimilars. Therefore, the European Medicines Agency requires pre-clinical comparison tests between generic and original drugs. + +### America +In March 2010, the Affordable Health Care for America Act provided a means of approval for biosimilar drugs. According to the Act, biosimilars are products highly similar to referenced patented drugs with no meaningful clinical differences. The Act mandates consistent clinical trial results for both biosimilars and their reference drugs. + +New regulations give original pharmaceutical companies 12 years of market access protection and establish a framework for biosimilar approvals. However, there is debate surrounding the 12-year protection period, with some arguing it hinders biosimilar development. + +## Global Regulatory Outlook +Pharmaceutical companies globally are advancing in the field of biosimilars. India and some other countries have approved biosimilars based on existing regulatory requirements, while regions like Europe, Canada, USA, Japan, South Korea, and China have established special approval regulations. + +A few years after Sandoz, a Novartis subsidiary, entered the biosimilars market, many biosimilars have been approved in Europe. Indian companies like Ranbaxy, Dr. Reddy’s Laboratories and Biocon aim to enter the Western biosimilar market despite the regulatory and market access barriers. + +Biosimilars, including monoclonal antibodies, are a growing field. Decision Resources found that biological products made up a significant portion of important drugs in 2007. The future of biosimilars depends on the development of biopharmaceuticals and regulatory approvals in major markets. + +### Key Definitions and Terms +- **Biosimilars:** Biogeneric drugs evaluated mainly on analytical studies and clinical data to verify they have no meaningful differences from the original biologics in terms of safety, purity, and potency. +- **Biopharmaceuticals:** Therapeutic products containing biological substances derived from living organisms. +- **European Medicines Agency (EMEA):** An agency responsible for the scientific evaluation of medicines developed by pharmaceutical companies for use in the EU. +- **Affordable Health Care for America Act:** Legislation signed into US law in 2010 aiming, among other things, to provide a process for the approval of biosimilar drugs. + +## Conclusion +The development and regulation of biosimilars involve ensuring their safety and efficacy are comparable to original biologics. Understanding global regulatory outlooks and market dynamics is crucial for the advancement of biosimilars and biopharmaceuticals. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/glucofreeze-symptoms-of-high-blood-sugar-level-b9qvr5w6.md b/markdown-output/glucofreeze-symptoms-of-high-blood-sugar-level-b9qvr5w6.md new file mode 100644 index 0000000000000000000000000000000000000000..77bdf103a3e3ab4df10bd34a052cfd4bbc340e2d --- /dev/null +++ b/markdown-output/glucofreeze-symptoms-of-high-blood-sugar-level-b9qvr5w6.md @@ -0,0 +1,109 @@ +```markdown +Goal/Experiment: +The goal of the experiment is to evaluate the effectiveness of GlucoFreeze, a dietary supplement, in managing high blood sugar levels. + +# GlucoFreeze: Symptoms Of High Blood Sugar Level! + +## Product Name: GlucoFreeze + +- **Category:** Blood Sugar Support +- **Healthy Benefits:** Helps to maintain a healthy blood sugar level +- **Ingredients:** Vitamin C, Vitamin E, Juniper Berries +- **Item Form:** Capsules +- **Net Quantity:** 60 Capsules +- **Age Range:** Adults +- **Dosage:** 2 Capsules per day +- **Results:** 2-3 months +- **Side Effects:** No major side effects reported + +Official website - [Click Here to Order Glucofort](#) + +This GlucoFreeze review will provide detailed information about how effective this supplement is in balancing blood sugar levels. GlucoFreeze is a trusted nutritional enhancement supplement that can help maintain healthy blood sugar levels. It also improves the user's overall health. Diabetes can lead to other serious diseases such as heart disease, stroke, and kidney disease. Diabetes can be managed by more than just diet changes or the use of pharmaceutical drugs. + +### Disclaimer +FOR INFORMATIONAL PURPOSES ONLY; USE AT YOUR OWN RISK + +The protocol content here is for informational purposes only and does not constitute legal, medical, clinical, or safety advice. It is not peer-reviewed and may not have undergone formal approval of any kind. The information presented in this protocol should not substitute for independent professional judgment, advice, diagnosis, or treatment. + +## What is GlucoFreeze? +GlucoFreeze is a dietary supplement made with natural ingredients that help control the user's blood sugar levels by increasing healthy blood sugar levels. All ingredients in the supplement are scientifically evaluated and proven safe. This dietary supplement can increase overall health and reduce the risk of developing type 2 diabetes by improving metabolic activity and reducing symptoms associated with diabetes. + +### How does GlucoFreeze work? +GlucoFreeze works by supporting healthy blood sugar levels without causing diabetes. Scientific studies show that blood sugar imbalances stem from insulin levels in the pancreas and liver fat accumulation. High blood pressure and kidney disease can also be caused by liver fat accumulation in excess. + +GlucoFreeze helps reduce fat by increasing metabolism, which increases stamina, energy, and endurance. This supplement also helps to melt small amounts of fat, controlling blood sugar and reducing diabetes risk. + +## Benefits of GlucoFreeze +The **GlucoFreeze supplement** has numerous benefits, including: + +- Balancing blood sugar levels +- Assisting in the recovery of metabolic processes +- Reducing weight gain and promoting a healthy lifestyle +- Eliminating harmful fats and improving blood circulation +- Improving cardiovascular health, lowering risks of diabetes and stroke +- Increasing insulin response +- Promoting healthy glucose metabolism and immune system enhancement +- Reducing stress and increasing energy + +## GlucoFreeze Ingredients + +### Vitamin C +Vitamin C, included in GlucoFreeze, is known to reduce blood sugar levels in type 2 diabetes. It also lowers the risk of developing other conditions in diabetics by improving general health. + +### Vitamin E +Vitamin E has antioxidant and anti-inflammatory properties, protecting healthy cells from damage, enhancing insulin sensitivity, and improving immune response. It also fights malignant development and infections. + +### Chromium +Chromium regulates glucose levels, supported by numerous studies. It boosts glucose regulation in clinical trials and enhances energy levels. + +### Burseraceae Plant +This plant has been medicinally used for over 1000 years in Indian tribes. It helps prevent non-alcoholic fatty liver disease by reducing fat deposits. + +### Juniper Berries +Juniper Berries are antioxidants and anti-inflammatory agents that protect cells and lower blood cholesterol and sugar levels. They are anti-diabetic. + +### Licorice root Extract +Licorice root controls blood sugar levels with anti-diabetic and anti-inflammatory properties. It is beneficial for metabolic disorders. + +## Side Effects of GlucoFreeze +GlucoFreeze is safe with no harmful additives or artificial ingredients. It is thoroughly tested for safety, but consulting a doctor is advisable for pregnant or nursing women, those with medical conditions, or individuals under 18 years. + +## GlucoFreeze Dosage + +- **Dosage:** 2 tablets daily + +This dosage gradually lowers blood sugar levels to normal with regular use, supports weight loss by melting fat, and provides energy for an active lifestyle. + +## GlucoFreeze Results and Longevity +GlucoFreeze can be used to reduce blood sugar without medication, showing results within 2-3 months. The supplement’s effects can last up to a year with a healthy diet and regular exercise. + +## Is GlucoFreeze Legit or Not? +GlucoFreeze reviews confirm it as effective in boosting healthy blood sugar. It is FDA-approved, safe, and effective. Positive reviews support its benefits. + +### Pros: +- Identifies abnormal blood sugar levels +- Reduces body weight +- Promotes insulin creation +- Reduces harmful fat +- Boosts metabolism and overall feeling +- Lowers heart disease risk +- Controls blood sugar levels +- Prevents type 2 diabetes and cardiovascular disease +- Increases energy levels +- Lowers cholesterol +- Enhances liver function +- Increases blood flow and reduces fatigue +- Improves immune system + +### Cons: +- Only available through official website to avoid scams + +## Where Can I Buy GlucoFreeze? +The only purchase source is the company's website. It’s safe, effective, and improves cognitive abilities without exposing users to scams. Each capsule contains 30 days’ worth of nutritional content. + +## GlucoFreeze Review - Conclusion +GlucoFreeze is effective for type 2 diabetics, safe, and made of all-natural ingredients. It helps focus, increases cognitive abilities, and improves management and analytical skills. The supplement contains no harmful ingredients and has known side effects. It’s a safe and easy solution for lowering blood sugar. + +[DOI](http://dx.doi.org/https://dx.doi.org/10.17504/protocols.io.3byl4br9rvo5/v1) +``` +endofoutput \ No newline at end of file diff --git a/markdown-output/glycemic-effect-of-post-meal-walking-compared-to-o-72chqaw.md b/markdown-output/glycemic-effect-of-post-meal-walking-compared-to-o-72chqaw.md new file mode 100644 index 0000000000000000000000000000000000000000..fed6803f890b7cadd4996cfdbf2594dc92f26883 --- /dev/null +++ b/markdown-output/glycemic-effect-of-post-meal-walking-compared-to-o-72chqaw.md @@ -0,0 +1,48 @@ +```markdown +# Goal/Experiment: +The goal of this study was to determine the effect of post-meal walking on glycemic control compared with one prandial insulin injection in type 2 diabetic patients who failed basal insulin treatment. + +# Glycemic Effect of Post-Meal Walking Compared to One Prandial Insulin Injection in Type 2 Diabetic Patients Treated with Basal Insulin: A Randomized Controlled Cross-Over Study + +## Authors +- Onnicha Suntronlohanakul +- Chatchalit Rattarasarn +- Atiporn Ingsaithit +- Chatvara Areevut +- Sunee Saetung + +## Abstract +Studies demonstrate that post-meal walking decreases postprandial hyperglycemia in type 2 diabetic patients, but it has never been tested with the active treatment comparator. The objective of this study was to determine the effect of post-meal walking on glycemic control compared with one prandial insulin in type 2 diabetic patients who failed basal insulin. A randomized controlled cross-over study of post-meal walking or one prandial insulin was done in type 2 diabetic patients who were being treated with basal insulin between May 2017 and March 2018. In post-meal walking group, patients walked after meal for 15-20 minutes one meal a day every day for 6 weeks. In prandial insulin (basal plus) group, one prandial insulin was injected before breakfast or main meal with rapid-acting insulin. The primary outcome was a difference in HbA1c reduction in post-meal walking compared with basal plus groups. Fourteen patients completed the study. By intention-to-treat analysis, HbA1c was reduced by -0.05 (range: 1.08 to 0.74) and -0.19 (range: 0.8 to 0.56) % in post-meal walking and basal plus groups respectively. By per-protocol analysis, post-meal walking and basal plus groups decreased HbA1c by 0.13 (range: 0.74 to 1.08) and 0.2 (range: 0.56 to 0.8) %, respectively. There was no significant differences in HbA1c reduction from baseline in each group and between groups in both intention-to-treat and per-protocol analysis. Fructosamine levels were decreased by 17.5(-5:59 to 43) and 10(15:10 to 40) μmol/L, respectively at 3 and 6 weeks in post-meal walking group whereas the respective changes in basal plus group were 12.5(-17 to 64) and 17.5(-28 to 38) μmol/L and there was no significant difference between groups. In conclusion, although post-meal walking might be as effective as one prandial insulin to improve glycemic control in type 2 diabetic patients who failed basal insulin but the magnitude of reduction was small. A longer-term study with a larger sample size or with a different walking protocol is required. + +## External Link +[https://doi.org/10.1371/journal.pone.0230554](https://doi.org/10.1371/journal.pone.0230554) + +## References +1. American Diabetes Association. Pharmacologic approaches to glycemic treatment: Standards of medical care in diabetes. Diabetes Care 2018; 41(Suppl 1): S73-S85. +2. Davies MJ, D'Alessio DA, Fradkin J, Kernan WN, Mathieu C, Mingrone G, et al. Management of hyperglycemia in type 2 diabetes. A consensus report by the American Diabetes Association (ADA) and the European Association for the Study of Diabetes (EASD). Diabetologia 2018; 61: 2461-2498. +3. American Diabetes Association. Lifestyle management: Standards of medical care in diabetes—2018. Diabetes Care 2018;41(Suppl 1): S38-S50. +4. Colberg SR, Sigal RJ, Yardley JE, Riddell MC, Dunstan DW, Dempsey PC, et al. Physical activity/exercise and diabetes: a position statement of the American Diabetes Association. Diabetes Care 2016; 39: 2065-2079. +5. DiPietro L, Gribok A, Stevens MS, Hamm LF, Rumpler W. Three 15-min bouts of moderate postmeal walking significantly improves 24-h glycemic control in older people at risk for impaired glucose tolerance. Diabetes Care. 2013; 36: 3262-3268. +6. Erickson ML, Little JP, Gay JL, McCully KK, Jenkins NT. Postmeal exercise blunts postprandial glucose excursions in people on metformin monotherapy. Journal of App Physiol, 2017; 123: 444-450. +7. Erickson ML, Little JP, Gay JL, McCully KK, Jenkins NT. Effects of postmeal exercise on postprandial glucose excursions in people with type 2 diabetes treated with add-on hyperglycemic agents. Diabetes Res Clin Pract 2017; 126: 240-247. +8. Pahra D, Sharma N, Ghai S, Hajela A, Bhansali S, Bhansali A. Impact of post-meal and one-time daily exercise in patient with type 2 diabetes mellitus: a randomized crossover study. Diabetol Metab Syn 2017; 9: 64. +9. Reynolds AN, Mann JI, Williams S, Venn BJ. Advice to walk after meals is more effective for lowering postprandial glycaemia in type 2 diabetes mellitus than advice that does not specify timing: a randomised crossover study. Diabetologia 2016; 59: 2572-2578. +10. Richter EA, Ploug T, Galbo H. Increased muscle glucose uptake after exercise: no need for insulin during exercise. Diabetes 1985; 34: 1041-1048. +11. van Dijk J-W, Venema M, van Mechelen W, Stehouwer CD, Hartgens F, van Loon LJ. Effect of moderate-intensity exercise versus activities of daily living on 24-hour blood glucose homeostasis in male patient with type 2 diabetes. Diabetes Care 2013; 36: 3448-3453. +12. Colberg SR, Zarrabi B, Bennington L, Navae A, Somma CT, Swain DP, et al. Postprandial walking is better for lowering the glycemic effect of dinner than pre-dinner exercise in type 2 diabetic individuals. Journal of the American Medical Directors Association. 2009; 10: 394-397. +13. An H-S, Jones GC, Kang S-K, Welk GJ, Lee J-M. How valid are wearable physical activity trackers for measuring steps? European journal of sport science. 2017; 17: 360-368. +14. Case MA, Burwick HA, Volpp KG, Patel MS. Accuracy of smartphone applications and wearable devices for tracking physical activity data. J Am Med Assoc 2015; 313: 625-626. +15. Ferguson T, Rowlands AV, Olds T, Maher C. The validity of consumer-level activity monitors in healthy adults worn in free-living conditions: a cross-sectional study. Int J Behav Nutr Phys Act 2015; 12: 42. +16. Kooiman TJ, Dontje ML, Sprenger SR, Krijnen WP, van der Schans CP, de Groot M. Reliability and validity of ten consumer activity trackers. BMC Sports Sci Med Rehabil 2015; 7: 24. +17. Tully MA, McBride C, Heron L, Hunter RF. The validation of Fitbit Zip™ physical activity monitor as a measure of free-living physical activity. BMC Res Notes. 2014; 7: 952. +18. Lankisch M, Ferlinz K, Leahy J, Scherbaum W. Introducing a simplified approach to insulin therapy in type 2 diabetes: a comparison of two single-dose regimens of insulin glulisine plus insulin glargine and oral antidiabetic drugs. Diabetes Obes Metab 2008; 10: 1178-1185. +19. Lunde MS, Hjellset VT, Hostmark AT. Slow post meal walking reduces the blood glucose response: an exploratory study in female Pakistani immigrants. J Immigr Minor Health. 2012; 14: 816-822. +20. Monnier L, Colette C, Rabasa-Lhoret R, et al. Morning hyperglycemic excursions: a constant failure in the metabolic control of non–insulin-using patients with type 2 diabetes. Diabetes Care 2002; 25: 737-741. +21. Owens DR, Luzio SD, Sert-Langeron C, Riddle M. Effects of initiation and titration of a single pre-prandial dose of insulin glulisine with continuing titrated insulin glargine in type 2 diabetes: a 6-month ‘proof-of-concept’ study. Diabetes Obes Metab 2011; 13: 1020-1027. + +## Attachments +- [Thai_protocol.doc](link-to-document) +- [Eng_protocol.docx](link-to-document) + +``` +endofoutput \ No newline at end of file diff --git a/markdown-output/gradient-pcr-with-dmso-by6kpzcw.md b/markdown-output/gradient-pcr-with-dmso-by6kpzcw.md new file mode 100644 index 0000000000000000000000000000000000000000..9e6f0f354be186ee2e407b87317631a95506a407 --- /dev/null +++ b/markdown-output/gradient-pcr-with-dmso-by6kpzcw.md @@ -0,0 +1,113 @@ +```markdown +# Goal/Experiment: + +This experiment aims to utilize Gradient PCR with Dimethyl Sulfoxide (DMSO) for confirming genes or DNA of interest from a template and to amplify the required amount of genes to larger amounts. The gradient is used to find the best annealing temperature, and DMSO is required for the Pfu polymerase to work properly. + +## Gradient PCR with DMSO + +### Ashwinuday +* Indian Institute of Science Education and Research, Pune + +###### October 18, 2021 + +[dx.doi.org/10.17504/protocols.io.by6kpzcw](https://dx.doi.org/10.17504/protocols.io.by6kpzcw) + +#### iGEM IISER Pune India 2021 + +### Abstract: + +This protocol is designed for confirming genes or DNA of interest from a template using PCR and to amplify the required amount of genes. The gradient method is employed to find the best annealing temperature, and DMSO is used to ensure efficient functioning of the Pfu polymerase. + +## Materials and Reagents: + +- **Milli-Q ultrapure water** +- **Pfu Buffer (10X)**: A high-fidelity DNA polymerase buffer. +- **Forward Primer (20µM)** +- **Reverse Primer (20µM)** +- **dNTPs (2.5mM)**: Deoxynucleotide triphosphates required for DNA synthesis. +- **Pfu Polymerase** +- **Template DNA (100ng/µL)** +- **DMSO (Dimethyl Sulfoxide)**: Helps in reducing DNA secondary structures. +- **TAE Buffer (1X)**: Tris-acetate-EDTA, used for maintaining a stable pH during electrophoresis. +- **Agarose** +- **EtBr (Ethidium Bromide)**: An intercalating agent used for staining nucleic acids. +- **Thermal Cycler** +- **Agarose gel electrophoresis setup** + +### Safety Note: + +**EtBr and related consumables should be handled with gloves. Items not classified for the gel should not be touched with gloved hands.** + +## Procedure: + +1. **Prepare Reagents:** + + Prepare the working stock solution of all reagents. If stored, take them out from the refrigerator and thaw on ice. + +2. **Mix Preparation:** + + Prepare the following mixes in total 50 µL. Prepare 5 test samples for each of the following percentages of DMSO (0, 5, 10) with varying annealing temperatures: + + | Item | -ve control (0% DMSO) | Test (0% DMSO) (*5) | -ve control (5% DMSO) | Test (5% DMSO) (*5) | -ve control (10% DMSO) | Test (10% DMSO) (*5) | + |-------------------------------|-----------------------|---------------------|-----------------------|---------------------|-----------------------|----------------------| + | Ultrapure water (from Milli-Q)| 40 µL | 39 µL | 37.5 µL | 36.5 µL | 35 µL | 34 µL | + | Pfu Buffer (10X) | 5 µL (1X) | 5 µL (1X) | 5 µL (1X) | 5 µL (1X) | 5 µL (1X) | 5 µL (1X) | + | Forward Primer (20 µM) | 1 µL (0.4 µM) | 1 µL (0.4 µM) | 1 µL (0.4 µM) | 1 µL (0.4 µM) | 1 µL (0.4 µM) | 1 µL (0.4 µM) | + | Reverse Primer (20 µM) | 1 µL (0.4 µM) | 1 µL (0.4 µM) | 1 µL (0.4 µM) | 1 µL (0.4 µM) | 1 µL (0.4 µM) | 1 µL (0.4 µM) | + | dNTPs (2.5 mM) | 2 µL (0.1 mM) | 2 µL (0.1 mM) | 2 µL (0.1 mM) | 2 µL (0.1 mM) | 2 µL (0.1 mM) | 2 µL (0.1 mM) | + | Pfu Polymerase | 1 µL | 1 µL | 1 µL | 1 µL | 1 µL | 1 µL | + | Template (100 ng/µL) | - | 1 µL (100 ng) | - | 1 µL (100 ng) | - | 1 µL (100 ng) | + | DMSO | - | - | 2.5 µL | 2.5 µL | 5 µL | 5 µL | + +3. **PCR Execution:** + + Perform PCR using a Thermo cycler with the following temperature settings (all temperatures in degrees Celsius): + + | Temperature (°C) | Time | + |------------------|-------------| + | 95 | 5 min | + | 95 | 30 secs | + | 51, 53, 55, 57, 59| 40 secs | *(Gradient)* + | 72 | 2 min | + | 72 | 5 min | + + - Set steps 2, 3, and 4 to repeat 30 times (or up to 35 times). + + - The PCR product can be run using gel electrophoresis or stored at -20°C for later analysis. + +4. **Gel Electrophoresis Preparation:** + + **Safety Note:** Handle all items that interact with the gel and EtBr with gloves. Ensure gloved hands are not used to touch anything not designated for gel use. + +5. **Agarose Gel:** + + To prepare the 1% agarose gel, mix 0.5 g of agarose in 50 mL of 1X TAE buffer and heat well to form a clear solution. Cool the mixture until it is bearable to touch and then carefully add 2 µL of EtBr and mix well by swirling. + +6. **Gel Casting:** + + Pour the gel mix into a gel cast with the chosen comb (based on well number and size) without creating bubbles. If bubbles form, remove them with a tip immediately after pouring. + +7. **Loading Samples:** + + After the gel solidifies, place it in the buffer tank with enough buffer to cover the gel and carefully remove the comb. Load approximately 10 µL of each sample with 1X loading dye into the wells of the gel. Load a 1 kb ladder in the first lane. Run the gel at 100V. + +8. **Stopping the Gel:** + + Stop the current when the dye front crosses 75% of the gel length. + +9. **Imaging:** + + Take the gel out (with gloved hands), remove all buffer, and image the gel under UV light. Compare sample bands with the ladder to estimate the bands of interest. + +10. **Analysis:** + + Check bands corresponding to samples from the temperature showing good intensity and correct size for choosing the annealing temperature for further PCRs. + +#### References: + +Ashwinuday 2021. Gradient PCR with DMSO. protocols.io. https://dx.doi.org/10.17504/protocols.io.by6kpzcw + +*This protocol is distributed under the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.* + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/granulate-formulation-protocol-bkrpkv5n.md b/markdown-output/granulate-formulation-protocol-bkrpkv5n.md new file mode 100644 index 0000000000000000000000000000000000000000..11287d2fb806657f6173e1968a22c4277891315a --- /dev/null +++ b/markdown-output/granulate-formulation-protocol-bkrpkv5n.md @@ -0,0 +1,112 @@ +```markdown +Goal/Experiment: Develop a granulate formulation of bacterial inoculants for agricultural use, specifically to enhance soil fertility, plant growth, and suppression of phytopathogens. + +# Granulate Formulation Protocol + +_Author: Andreea S_ +_University of Groningen_ + +**DOI:** [dx.doi.org/10.17504/protocols.io.bkrpkv5n](https://dx.doi.org/10.17504/protocols.io.bkrpkv5n) + +**iGEM Groningen 2020** + +## Abstract + +**Bioformulation** is used in agriculture for various reasons, including soil fertility, plant growth promotion, and the suppression of phytopathogens. The bacterial inoculants are applied as a formulated product like powder, spray, or pellet. This is a green strategy that is being developed as a less harmful method to protect crops other than pesticides. In the paper published by Alvarez et al, 2016, they developed a talc-based powder formulation based on _Bacillus B25_ spores and evaluated some of its characteristics, like shelf life and efficacy against the pathogenic fungus _Fusarium verticillioides_. + +> **Reference:** +> Martínez-Álvarez, J. C., Castro-Martínez, C., Sánchez-Peña, P., Gutiérrez-Dorado, R., & Maldonado-Mendoza, I. E. (2016). Development of a powder formulation based on _Bacillus cereus sensu lato_ strain B25 spores for biological control of _Fusarium verticillioides_ in maize plants. *World Journal of Microbiology and Biotechnology, 32*(5), 75. [https://doi.org/10.1007/s11274-015-2000-5](https://doi.org/10.1007/s11274-015-2000-5) + +## Key Terms and Definitions + +- **Colony Forming Units (CFU):** A unit used in microbiology to estimate the number of viable bacteria or fungal cells in a sample. It depends on their ability to multiply under controlled conditions. + - **Reference:** + El-Hassan and Gowen, 2006. [https://doi.org/10.1111/j.1439-0434.20](https://doi.org/10.1111/j.1439-0434.20) + +- **Fatty Acid Analysis:** A characterization process for fats and oils to determine the total fat content and identify strains like _Bacillus mycoides_ in soil and their survival with the granulate formulation. + - **Reference:** + Friedrich von Wintzingerode et al. (1997). *FEMS Microbiology Ecology, 24*(3). [https://doi.org/10.1111/j.1574-6941.1997.tb00437.x](https://doi.org/10.1111/j.1574-6941.1997.tb00437.x) + +## Bacterium Inoculum + +1. **Grow a single colony of bacteria:** + - In an assay tube with **5 mL** of Luria Broth (LB) medium. + +2. **Incubate:** + - Orbital shaker at 200 rev.min⁻¹ at **30 °C** for **18:00:00** hours. + +3. **After bacterial growth:** + - Take a **500 mL** Erlenmeyer flask and add **100 mL** of LB medium. + +4. **Add bacterial culture:** + - **1 mL** of the culture ( **1% (v/v)** ) in the flask and incubate at **30 °C** and 200 rev.min⁻¹ for **24:00:00** hours, until an optical density of close to 1 is obtained. + +## Spore Production + +5. **Add to a 500 mL Erlenmeyer flask:** + - **100 mL** of Difco Sporulation Medium (DSM). + +6. **Sterilize DSM medium:** + - Autoclave at **121 °C** and 1.5 psi for **00:15:00** hours. + +7. **Add to sterilized DSM medium:** + - **1 mL** each: + - 1 M **Ca(NO₃)₂** + - 10 mM **MnCl₂.4H₂O** + - 1 mM **FeSO₄** + +8. **Inoculate:** + - **1x10⁶** c.f.u. ml⁻¹ of the bacterial strain. + - Keep at **30 °C** and 200 rev.min⁻¹ for **72:00:00** hours. + +## Powder Formulation + +9. **Mix:** + - Talc (primary carrier) with carboxymethyl-cellulose (CMC; 1% w/w), CaCO₃ (15% w/w), and glucose (0.25% w/w) in powder form. + +10. **Autoclave mixture:** + - At **121 °C** and 15 psi for **00:15:00** hours. + +11. **Combine with spore suspension:** + - Dry mixture at **55 °C** for **36:00:00** hours. + +12. **Pulverize:** + - Using sterile porcelain mortar and pestle. + +13. **Package:** + - In plastic bags and store at room temperature. + +## CFU Determination + +14. **Estimate CFUs:** + - Optical Density (OD) of spore suspension using a tube-reading spectrophotometer (OD adjusted to 1.978, corresponding to 8.5 - 10¹⁰ CFU/mL at 600nm absorbance). + +15. **Air-dry formulation:** + - Sterile aluminum foil in pans for **24:00:00** hours with occasional stirring in laminar airflow cabinet. + +16. **Sieve:** + - Dried formulations (35% moisture content of **B. mycoides**) passed through a 250μm mesh to attain desired particle size. + +17. **Packaging:** + - Sterilized polypropylene bags, seal, and store at room temperature. + +18. **Count CFUs:** + - Estimate the number of viable propagules using standard dilution plating method. + +## STD Dilution Method + +19. **Preparation:** + - Take **1 g** aliquots of dried powder and place in **99 mL** sterile PBST solution (PBS + 0.05% (v/v) Tween 20). + - Stir magnetically at high speed for **00:15:00** hours. + - Dilute suspension and take **0.2 mL** for plating on Nutrient Agar (NA) media. + +## Fatty Acid Analysis + +20. **Procedure:** + - Perform saponification, methylation, and extraction to obtain fatty acid methyl esters (FAME) from wet biomass. + +21. **Separate FAME:** + - Using microbial identification system, with automatic integration and calculation of fatty acid names and percentages by Microbial ID. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/growing-drosophila-gut-bacteria-hheb33e.md b/markdown-output/growing-drosophila-gut-bacteria-hheb33e.md new file mode 100644 index 0000000000000000000000000000000000000000..92bbe0641063fc19ea254b05272393be5b41962e --- /dev/null +++ b/markdown-output/growing-drosophila-gut-bacteria-hheb33e.md @@ -0,0 +1,89 @@ +```markdown +# Goal/Experiment: +The goal of this experiment is to grow and culture Drosophila gut bacteria. This involves the preparation of bacterial media, as well as the establishment of solid and liquid bacterial cultures. + +# Growing Drosophila Gut Bacteria +**Authors:** Zita Santos, Patrícia Francisco, Ricardo Leitão-Gonçalves, Margarida Anjos, Célia Baltazar, Ana Paula Elias, Gabriela Tondolo Fioreze, Pavel M. Itskov, Matthew D. W. Piper, Carlos Ribeiro + +## Abstract +This protocol is part of the manuscript: Gonçalves et al. Commensal bacteria and essential amino acids control food choice behavior and reproduction. PLoS Biology. 2017 Apr 18. + +We acknowledge the help of the Won-Jae Lee laboratory and the Leulier laboratory for assistance in developing these protocols. + +*Citation:* Zita Santos, Patrícia Francisco, Ricardo Leitão-Gonçalves, Margarida Anjos, Célia Baltazar, Ana Paula Elias, Gabriela Tondolo Fioreze, Pavel M. Itskov, Matthew D. W. Piper, Carlos Ribeiro. Growing Drosophila gut bacteria. protocols.io. dx.doi.org/10.17504/protocols.io.hbeb33e +*Published:* 25 Apr 2017 + +## Guidelines +The following bacterial species and strains (kindly provided by François Leulier, IGFL, France and Won-Jae Lee, SNU, South Korea) were used in this study: +- Lactobacillus plantarum^WJL^ +- Lactobacillus brevis^EW^ [1] +- Acetobacter pomorum [1] +- Commensalibacter intestini^A911T^ [1] +- Enterococcus faecalis [2] + +All bacterial media and bacterial manipulations are performed in a laminar flow hood. + +## Media Preparation +### Solid Media +- **MRS**: Mix 51 g/l of MRS (Sigma-Aldrich, #69966), 4 ml/l of Tween 20 (Sigma-Aldrich, #P9416) and 15 g/l agar (Difco, #214530) with milliQ filtered water. Autoclave the medium for 15 minutes at 121°C and pour into Petri dishes. +- **Mannitol medium**: Mix 3 g/l Bacto peptone (Difco, #0118-17), 5 g/l yeast extract (Difco, #212750), 25 g/l D-Mannitol (Sigma-Aldrich, #M1902) and 15 g/l agar (Difco, #214530) with milliQ filtered water. Autoclave the medium for 15 minutes at 121°C and pour into Petri dishes. +- **LB**: Mix 35 g/l of LB Broth with Agar (Lennox) (Sigma Aldrich #L2897) with milliQ filtered water. Autoclave the medium for 15 minutes at 121°C and pour into Petri dishes. + +### Liquid Media +- **MRS**: Mix 51 g/l of MRS (Sigma-Aldrich, #69966) and 4 ml/l of Tween 20 (Sigma-Aldrich, #P9416) with milliQ filtered water. Autoclave the medium for 15 minutes at 121°C. +- **Mannitol medium**: Mix 3 g/l Bacto peptone (Difco, #0118-17), 5 g/l yeast extract (Difco, #212750), and 25 g/l D-Mannitol (Sigma-Aldrich, #M1902) with milliQ filtered water. Autoclave the medium for 15 minutes at 121°C. +- **LB**: Mix 20 g/l of LB Broth (Sigma-Aldrich #L3022) with milliQ filtered water. Autoclave the medium for 15 minutes at 121°C. + +## References +1. Ryu J-H, Kim S-H, Lee H-Y, Bai JY, Nam Y-D, Bae J-W, et al. Innate Immune Homeostasis by the Homeobox Gene Caudal and Commensal-Gut Mutualism in Drosophila. Science. 2008;319: 777-782. doi:10.1126/science.1149357 +2. Cox CR, Gilmore MS. Native Microbial Colonization of Drosophila melanogaster and Its Use as a Model of Enterococcus faecalis Pathogenesis. Infect Immun. 2007;75: 1565-1576. doi:10.1128/IAI.01496-06 + +## Before Start +All bacterial media and bacterial manipulations are performed in a laminar flow hood. + +## Protocol +### Start Bacterial Cultures on Plates +#### Step 1. +Prepare MRS, Mannitol, and LB plates according to the [Guidelines](#guidelines). + +*Note:* All steps should be performed in a laminar flow hood and all reagents and material should be sterile. + +#### Step 2. +Start the bacterial cultures by streaking out: +- *Lactobacilli* in MRS agar plates +- *A. pomorum* and *C. intestini* in Mannitol agar plates +- *E. faecalis* in LB agar plates + +Using a 1 µl sterile loop, streak from bacterial stocks that have been kept at -80°C in 50% glycerol. + +#### Step 3. +Incubate: +- MRS plates (*Lactobacilli*) at 37°C, for 48 h +- Mannitol plates (*C. intestini^A9117^* and *A. pomorum*) at 30°C, for 24-48 h +- LB plates (*E. faecalis*) at 37°C for 24 h + +*Note:* These plates can be kept at 4°C if not used immediately. + +### Liquid Bacterial Cultures +#### Step 4. +Prepare liquid MRS, Mannitol and LB media according to the [Guidelines](#guidelines). + +#### Step 5. +Pick a single colony to start liquid culture using a 1 µl sterile loop. Wash the loop with the bacteria in the different liquid media as follows: +- Culture *Lactobacilli* in 10 ml of liquid MRS medium using 14 ml culture tubes (Thermo Scientific,#150268), at 37°C, for 24 h without agitation. +- For *C. intestini*, use 20 ml of liquid mannitol medium in 50 ml tubes (Falcon). For *A. pomorum*, use 200 ml of medium in 500 ml flasks (Sigma Aldrich #CLS4985500). Culture *C. intestini* and *A. pomorum* at 30°C, for 48h using 170 rpm agitation. +- Culture *E. faecalis* in 200 ml of liquid LB medium in 500 ml flasks (Sigma Aldrich #CLS4985500), at 37°C for 24 h using 220 rpm agitation. + +#### Step 6. +Inspect the morphology of the colonies on the plates to ensure the purity and health of the bacterial culture. + +If you are doing consecutive experiments, start a new solid culture when preparing the liquid cultures. Pick a colony from the cultures on the plates and proceed as described in Step 2. + +*Note:* Remember to include all the corresponding media without any bacterial inoculation as controls for possible media contaminations. + +We found these bacteria to have very reproducible growth rates, having ODs close to the references that we describe in calculating the internal bacterial load of flies protocol. If these ODs are much higher than the reference values, it likely means that a contamination occurred. If they are much lower, it is likely to mean that the bacteria are no longer healthy. In these cases, we advise that you start a new solid culture from the frozen bacterial stock. + +--- + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/growing-freshwater-sponges-from-gemmules-in-the-la-cnj2vcqe.md b/markdown-output/growing-freshwater-sponges-from-gemmules-in-the-la-cnj2vcqe.md new file mode 100644 index 0000000000000000000000000000000000000000..435119ce04c2564c73e38795bd2deec68cdf64da --- /dev/null +++ b/markdown-output/growing-freshwater-sponges-from-gemmules-in-the-la-cnj2vcqe.md @@ -0,0 +1,90 @@ +```markdown +# Goal/Experiment: +Growing freshwater sponges from gemmules in the laboratory + +## Growing freshwater sponges from gemmules in the laboratory +**Scott Nichols1** +1University of Denver + +**Abstract** +This is a basic protocol for growing freshwater sponges from gemmules in the laboratory. We specifically developed this protocol for working with *Ephydatia muelleri*, but have used it for other species as well. This protocol is good for cleaning gemmules, and removing contaminating protists, fungi, and bacteria. + +### Guidelines + +#### Guidelines + +Gemmules are temperature sensitive. Keep gemmule stocks at 4°C to maintain their longevity. There are published reports of defined media for growing sponges, but we typically use store-bought spring water, or sterile-filtered lakewater; both work well. + +#### A note about gemmule storage: + +Field-collected gemmules should be stored at 4°C in sterile-filtered or autoclaved lake water. Keep gemmules collected from different adults in separate containers to ensure that you don't accidentally mix different species, and to ensure that gemmules used in an experiment have the same genetic background. If stored in a deli-style fridge, place gemmules in an opaque container to limit the growth of algae. + +Anecdotally, we have observed that gemmules remain viable longer if stored in a minimal volume of water, just covering their surface. + +### Materials + +**Materials** + +- Freshwater sponge gemmules +- Autoclaved lake water or spring water +- 10 cm petri dish +- 40 or 70 µm cell strainer +- 50 mL conical tubes +- Any format cell culture plates/dishes (6-well, 12-well, 24-well) +- Hydrogen peroxide +- 100x Antibiotic-Antimycotic (Sigma-Aldrich A5955-100ML) +- p1000 pipette and tips + +**Optional** + +- Incubator set at 25-28°C +- Stereomicroscope +- 50 mg/mL Kanamycin or 100 mg/mL Ampicillin + +### Before Start Instructions + +**Before Start** + +Gemmules can be collected seasonally throughout the world. There are published records of freshwater sponge distributions, but it is also possible to use citizen science apps such as iNaturalist to identify locations where they have been reported. A map of freshwater sponge reports in the United States can be found at the following link: [iNaturalist - Freshwater sponges of the United States](https://www.inaturalist.org/projects/freshwater-sponges-of-the-united-states). + +## Gemmule sterilization + +1. Place a 40-70 µm cell strainer into a clean 10 cm Petri dish filled with cold, lake/spring water. +2. Cut off the end of a p1000 pipette tip with scissors to increase the size of the opening. Using the trimmed pipette tip, transfer the isolated gemmules into the cell strainer. + +### Gemmule sterilization: sterilize with hydrogen peroxide, then rinse + +3. Prepare a 60 mL solution of 1% hydrogen peroxide in lake water and place in a 50 mL conical tube (fill to the very top of the tube). +4. Transfer the cell strainer containing gemmules into the hydrogen peroxide solution and incubate for 5 minutes. + - **Note**: Lift the cell strainer up and then submerge it again a few times to agitate the gemmules periodically through incubation. +5. Remove the cell strainer from the hydrogen peroxide solution and rinse very thoroughly by placing under a flowing tap of RO water for at least 1 minute. + - **Note**: This step is essential to remove all traces of hydrogen peroxide and bubbles attached to the surface of gemmules that will cause them to float. + +### Gemmule sterilization: select gemmules for use + +6. Place the cell strainer into a clean 10 cm Petri dish containing lake/spring water. +7. Using a new p1000 tip, transfer the gemmules from the cell strainer to the surrounding dish. + - **Note**: This is an opportunity to spread the gemmules out and separate them from remaining debris leftover from the parent tissue. Also, at this point you can discard floating gemmules; even if they are viable they will not attach when plated. + +## Gemmule plating: sterilize in Anti-Anti for 24-48 hours + +8. You can plate gemmules in essentially any dish. The limiting factor is the water volume; a single gemmule grows best in at least 500 µL of water. If you need to grow gemmules in a smaller format, such as in a 96-well culture dish, it is possible but you have to perform frequent water changes (maybe even twice daily). + - **Note**: Even though you have sterilized the gemmules with hydrogen peroxide, it is still possible for cultures to contain contaminating fungi. To prevent fungal growth, dilute the antibiotic-antimycotic (Anti-Anti) to 1x final concentration in lake/spring water. +9. Place the desired amount of spring/lake water containing Anti-Anti in the culture dish where you wish to grow the sponges, and then add the number of gemmules you wish to grow, and place at room temperature. + - **Note**: We have successfully grown *Ephydatia* at temperatures ranging from 15-30°C. +10. Incubate in Anti-Anti for 24 - 48 hours, then replace with pure lake/spring water, or with lake/spring water containing another antibiotic such as 50 µg/mL kanamycin or 100 µg/mL ampicillin (depending upon your experimental goals). + - **Note**: Anti-Anti seems to slow gemmule hatching and may affect tissue development. + +## Gemmule plating: position gemmules in the center of the culture well + +11. Sponges typically start to hatch between 72 and 96 hours after plating, at which point they attach to the culture well/dish and cannot be moved. +12. It is important to make sure the gemmules are positioned according to your experimental needs (usually towards the center of the well to enable imaging). If placed near the edge of the well/dish, the sponges may grow vertically and be difficult/impossible to image. A trick for moving gemmules to the center of the well is to swirl the dish for several seconds to create a vortex within the well. + +### Gemmule plating: Mature sponges are best used between days 7-10 + +13. If the sponges are grown in at least 500 µL of lake/spring water per gemmule it is usually not critical to change the solution over the course of a 7-10 day experiment. +14. If you wish to limit autofluorescence for subsequent imaging studies, it is essential to grow the sponges in a dark incubator or drawer to limit the growth of intracellular *Chlorella*-like algae. +15. Without feeding, sponges cannot be maintained long beyond 10 days usually. As they age, their tissues often retract and/or the entire sponge will migrate away from the gemmule capsule, leaving behind sponging fibers and spicules. We avoid working with sponges at this stage. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/growth-of-pseudomonas-fluorescence-sbw25-in-48-wel-cgy9txz6.md b/markdown-output/growth-of-pseudomonas-fluorescence-sbw25-in-48-wel-cgy9txz6.md new file mode 100644 index 0000000000000000000000000000000000000000..1e3a9eb73ac79d0d52f912ed77f48a5be0e278ed --- /dev/null +++ b/markdown-output/growth-of-pseudomonas-fluorescence-sbw25-in-48-wel-cgy9txz6.md @@ -0,0 +1,101 @@ +```markdown +# Goal/Experiment: +To culture and measure the growth of *Pseudomonas fluorescens* SBW25 in 48-well microplates using the BioTek Epoch 2 plate reader. + +# Growth of *Pseudomonas fluorescens* SBW25 in 48-Well Plate Format + +**Rosemarie Wilton** +*Argonne National Laboratory* + +[DOI: dx.doi.org/10.17504/protocols.io.j8nlkwewdl5r/v1](dx.doi.org/10.17504/protocols.io.j8nlkwewdl5r/v1) + +## Abstract + +This protocol provides a method for culturing *Pseudomonas fluorescens* SBW25 in 48-well microplates. The protocol is specific for use with a BioTek Epoch 2 plate reader, but other plate readers can be used with appropriate modifications. One advantage of the Epoch 2 instrument is the ability to run with the plate lid at a higher temperature than the well contents. This prevents condensation on the lid and resulting noise in the OD readings. + +This protocol is suitable for many *Pseudomonas* strains. + +## Materials + +- **48-well cell culture microplates** (Greiner Bio-One 677180) + - Note: If using an Epoch2 microplate reader, the physical maximum plate height is 23.5 mm. The Greiner plates (with lids) have a maximum height of 22 mm. +- **50 mL Bio-Reaction tubes** (CELLTREAT 229475) +- **LB medium**: + - 10 g Tryptone + - 5 g Yeast Extract + - 10 g NaCl + - Add 1 L deionized water + - Sterilize by autoclaving at 15 psi, 121-124°C for 20 minutes +- **M9 Minimal Medium (1L)**: + 1. To 800 mL H₂O, add the following in the order below, mixing in between: + - 0.1 mL 1M CaCl₂ + - 0.18 mL 100 mM FeSO₄ * (optional, reduces fluorescent siderophore secretion in *Pseudomonas* strains. A 100 mM stock of FeSO₄ should be aliquoted quickly and frozen to prevent oxidation.) + - 2.0 mL 1M MgSO₄ + - 100 mL 10X M9 minimal salts + 2. Add carbon source, and add H₂O to bring the final volume up to 1 L. + 3. Filter Sterilize. + +## 10X M9 Minimal Salts (1L) + +| Ingredient | Amount (g/L) | +|------------------------------------|--------------| +| Disodium phosphate (anhydrous) | 67.8 | +| Monopotassium phosphate | 30 | +| Sodium chloride | 5 | +| Ammonium chloride | 10 | + +* Note: A 5X stock could be prepared instead. + 1. Dissolve the above (or 112.8 g M9 Minimal Salts, 5X, Sigma M6030) in 1L of distilled water. + 2. Autoclave in liquid cycle or filter sterilize. + +## Protocol + +### Culturing *Pseudomonas fluorescens* SBW25 + +#### DAY 1: Overnight Culture +1. Inoculate 5 mL LB in a vented or loosely capped culture tube (e.g., CELLTREAT 50 mL Bio-Reaction tube), from a glycerol stock or with a single colony from a plate. +2. Grow overnight at 30°C with shaking at 250 rpm. + +#### DAY 2: Four Hour Culture +- **Note:** *Pseudomonas* cells typically have a long lag time if transferred from a stationary-phase overnight culture directly to minimal medium. The additional four-hour culture period in fresh LB allows the cells to return to the exponential growth phase, and minimizes the subsequent lag upon transfer to minimal medium. + +1. Dispense 10 mL LB into vented or loosely capped 50 mL culture tube. +2. Inoculate with 250 μL overnight culture. +3. Incubate for four hours at 30°C with shaking at 250 rpm. +4. Pellet cells by centrifugation, 4000 rpm for 5 minutes. +5. Pour off supernatant and resuspend in 5 mL M9 salts. +6. Collect cells by centrifugation, and repeat M9 salts wash step. +7. Resuspend pellet in 5 mL sterile M9 salts. +8. Remove a small aliquot to measure OD600 on a spectrophotometer. + +### Plate Setup and Data Collection + +1. Prepare M9 minimal medium containing desired carbon source (M9-C) and filter sterilize. Dilute washed cells to a final OD600 of 0.05 to 0.1 OD with M9-C. +2. Dispense 600 uL/well to 48-well plate and cover with the supplied lid. Each unique well condition or sample type should be performed in triplicate. + +- **Note:** If long incubation periods are anticipated (>24 hours), fill edge wells with M9-C and only use the 24 inner wells for culturing. This will minimize evaporation during long culture times. + +3. Start up the Gen5 software on the Epoch2 system and place the plate in the drawer. Enter the protocol shown below; run times and sampling intervals can be adjusted as needed. + +#### 48-Well Plate Notes: +Notice that we have left the “Use Lid” box unchecked (even though we use the lid). This is because the Default 48 well plate plus lid is higher than the instrument max of 23.5 mm. To prevent error message, leave lid box unchecked or generate a custom plate description for the Greiner plate (System>Plate Types>View/Modify). + +### Adjusting the Well and Lid Temperature: +The Epoch 2 allows the lid to be run at a higher temperature to avoid condensation. This is set in the Gradient box below. This should be set to 1 or 2°C. + +### Adjusting the Shaking Mode and Speed: +![Shake Setup](images-shake-setup.png) + +### Adjusting the Read Step: +Edit the Read Speed to bring up the second window. Increase the delay time to 250 msec to allow wells to stabilize before reading. + +![Read Setup](images-read-setup.png) + +### Measurement Options +- Delay after plate movement: 250 msec +- Measurements per data point: 8 + +Once the protocol has been set up, start the run and monitor data collection over time. The run can be stopped at earlier time points without losing the data. + +`endofoutput` +``` \ No newline at end of file diff --git a/markdown-output/gynecomastia-surgery-b9hgr33w.md b/markdown-output/gynecomastia-surgery-b9hgr33w.md new file mode 100644 index 0000000000000000000000000000000000000000..d604cf5281655bd0e4fee9b15e9c4d5170da077f --- /dev/null +++ b/markdown-output/gynecomastia-surgery-b9hgr33w.md @@ -0,0 +1,60 @@ +```markdown +Goal/Experiment: +This document provides a comprehensive review and analysis of current surgical practices and techniques for the treatment of gynecomastia, a condition characterized by the development of excess breast tissue in males. The purpose is to evaluate and summarize the various surgical interventions, their outcomes, and associated complications, based on a detailed audit of existing literature. + +# Gynecomastia Surgery + +**Citation**: +habedih 2022. gynecomastia surgery. [protocols.io](https://dx.doi.org/10.17504/protocols.io.5jyl896y8v2w/v1). + +## Abstract +Gynecomastia is a condition characterized by the development of male breast tissue, affecting a significant portion of the male population. This comprehensive audit aims to dissect current surgical practices and patterns related to gynecomastia grade and severity. The review encompasses 17 investigations using PubMed and MEDLINE data sets, emphasizing key metrics like gynecomastia grade, surgical intervention, complication rates (hematoma, seroma, infection), and surgical channel use. + +## Key Findings +- **Patient Sample**: 1112 patients treated for gynecomastia. +- **Techniques**: Skin-saving mastectomy, often combined with liposuction. +- **Complications**: Hematoma (5.8%), Seroma (2.4%); higher rates in channel usage cases. +- **Significance**: Grade III gynecomastia showed higher error rates in studies. + +## Introduction +Gynecomastia, an enlargement of the male breast due to ductal, stromal, or fatty tissue proliferation, affects 32%-65% of men, with 84.5%-100% of patients reporting satisfactory correction. The condition is prevalent across various age groups, notably during neonatal, adolescent, and older adult stages. + +## Aetiology +Common causes include: +- **Idiopathic**: Unknown origin. +- **Medication**: Spironolactone, ketoconazole, calcium channel blockers. +- **Pathological**: Liver diseases, testicular/adrenal tumors, hypogonadism. + +## Initial Assessment +Involves: +- **History**: Symptoms, family medical history, presence of areola issues, lateral assessment. +- **Physical Examination**: Testicular examination, hepatomegaly assessment. +- **Imaging**: Ultrasound, ultrasound-guided biopsy, two-sided mammography. + +## Classification +- **Simon Classification (1973)**: Evaluates gynecomastia based on breast volume. +- **Rohrich Classification (2003)**: Focuses on tissue extraction needs. + +## Methodology +An extensive review of articles from 2000 to 2020 examined 219 articles, narrowing down to 17 relevant studies. Factors reviewed included: +- Patient gynecomastia grade. +- Type of surgical intervention. +- Complication rates such as hematoma and seroma. +- Areola complex issues. + +### Table 1: Simon Classification Grading + +| Grade | Description | +|-------|-----------------------------------| +| I | Minor breast enlargement, no excess skin | +| IIa | Moderate breast enlargement, no excess skin | +| IIb | Moderate breast enlargement, excess skin | +| III | Significant breast enlargement, excess skin | + +## Conclusion +An individualized approach to gynecomastia surgery, considering patient grade and preferences, can yield optimal results. The favored technique varies based on the severity and specific characteristics of the gynecomastia grade, with a consistent need for careful evaluation and customized treatment strategies. + +**DOI**: [dx.doi.org/10.17504/protocols.io.5jyl896y8v2w/v1](https://dx.doi.org/10.17504/protocols.io.5jyl896y8v2w/v1). + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/hep-tile-hbv-whole-genome-sequencing-nanopore-prot-dgj83urw.md b/markdown-output/hep-tile-hbv-whole-genome-sequencing-nanopore-prot-dgj83urw.md new file mode 100644 index 0000000000000000000000000000000000000000..adb3f6616a9a82cd5ba6e2552af67fc76ef796c4 --- /dev/null +++ b/markdown-output/hep-tile-hbv-whole-genome-sequencing-nanopore-prot-dgj83urw.md @@ -0,0 +1,248 @@ +```markdown +# Goal/Experiment: +To perform whole genome sequencing of Hepatitis B virus (HBV) using nanopore technology by following the HEP-TILE tiled amplicon protocol. + +# HEP-TILE: HBV Whole Genome Sequencing (Nanopore Protocol) +*Forked from [ARTIC SARS-CoV-2 sequencing protocol v4 (LSK114)](https://doi.org/10.17504/protocols.io.baktidi6)* +## DOI +[dx.doi.org/10.17504/protocols.io.5jy18zbedl2w/v1](https://dx.doi.org/10.17504/protocols.io.5jy18zbedl2w/v1) + +**Sheila Lumley1, Chris Kent2, Josh Quick2, Philippa Matthews3** + +1. University of Oxford +2. University of Birmingham +3. The Francis Crick Institute + +[ORCID iD: Sheila Lumley](https://orcid.org/) + +- License: [Creative Commons Attribution License](https://creativecommons.org/licenses/by/4.0/) +- Protocol status: Working - This protocol works and has been validated +- Created: April 09, 2024 +- Last Modified: July 25, 2024 +- Protocol Integer ID: 102752 +- Keywords: Hepatitis B virus, Whole genome sequencing, HBV, Tiled amplicon, Nanopore sequencing + +--- + +## Abstract +This protocol describes the HEP-TILE tiled amplicon protocol for whole genome sequencing of Hepatitis B virus (HBV) on the nanopore MinION. + +We developed a pan-genotypic (genotypes A-J) HBV scheme using an early version of PrimalScheme3, a web-based primer design tool for developing multiplex primer schemes. PrimalScheme3 is a successor to PrimalScheme, with a number of changes made to enable us to generate an overlapping (tiled) amplicon scheme which covered the circular HBV genome, utilizing a number of discrete primers at each position to handle intraspecies diversity. + +[Primer sequences are available here.](https://github.com/quick-lab/primerschemes/blob/main/primerschemes/hbv/600/v2.1.0/primer.bed) + +The amplicons can also be fragmented and sequenced on Illumina platforms. + +--- + +## Materials + +| Component | Supplier | Part Number | +|----------------------------------------------------|----------------|--------------------| +| HEP-TILE primers hbv/600/v2.1.0 | IDT | See links below | +| Q5 Hot Start High-Fidelity 2X Master Mix | NEB | M0494 | +| Nuclease-free water (100 mL) | NEB | B1500 | +| SPRI-select beads | Beckman | B23318 | +| Ethanol | | | +| NEBNext Ultra II End Repair/dA-tailing module | NEB | E7546 | +| Blunt/TA Ligase Master Mix | NEB | M0367 | +| NEBNext Quick Ligation Module | NEB | E6056S | +| Native Barcoding Kit 24 V14 or | ONT | SQK-NBD114.24 | +| Native Barcoding Kit 96 V14 | ONT | SQK-NBD114.96 | +| Native Barcoding Auxiliary Kit V14 (optional) | ONT | EXP-NBA114 | +| Short Fragment Buffer Expansion Kit (optional) | ONT | EXP-SFB001 | +| Flow Cell Priming Kit (optional) | ONT | EXP-FLP004 | +| Flow Cell Wash Kit (optional) | ONT | EXP-WSH004 | +| R10.4.1 flow cells | ONT | FLO-MIN114 | +| Bovine serum albumin (50mg/ml) | Invitrogen | | +| AMPure XP beads | Beckman | A63881 | +| Qubit dsDNA HS Assay Kit | Thermo | Q32854 | + +Oligos should be ordered individually from [this repository](https://github.com/quick-lab/primerschemes/tree/main/primerschemes/hbv/600/v2.1.0). + +--- + +## Protocol Steps + +### Sample Preparation +1. Prepare between 11 and 95 DNA samples plus 1 negative control of nuclease-free water per library. If previously frozen, mix by briefly vortexing and pulse spin to collect liquid. Keep samples on ice at all times. + +> **Note:** +> A positive control can also be included which may be the NIBSC HBV control or a high-titre clinical sample which can be diluted. This can help monitor run performance. + +### Primer Pool Preparation +2. If making up primer pools from individual oligos, fully resuspend lyophilized oligos in 1xTE to a concentration of 100 µM, vortex thoroughly and spin down. +3. Sort all odd regions primers into one or more tube racks. Add 5 µL of each odd region primer to a 1.5 mL Eppendorf tube labeled "Pool 1 (100 µM)". Repeat the process for all even region primers for Pool 2. These are your 100 µM stocks of each primer pool. +> **Note:** +> Primers should be diluted and pooled in the **mastermix** cabinet which should be cleaned with decontamination wipes and UV-sterilized before and after use. + +4. Dilute 100 µM pools 1:10 in molecular grade water to generate 10 µM primer stocks. +> **Note:** +> Primers are used at a final concentration of 15 nM per primer. In this case hbv/600/v2.1.0 pools have 69 primers in pool 1 and 63 primers in pool 2. The requirement is ~2.5 µL primer pool (10 µM) per 25 µL reaction. + +> **Note:** +> Make up multiple 100 µL aliquots of 10 µM primer dilutions and freeze them in case of degradation or contamination. + +### Multiplex PCR +5. Set up PCR reactions in strip-tubes or plates as follows. Gently mix by pipetting and pulse spin the tube to collect liquid at the bottom. + - **Reaction 1:** + - Q5 Hot Start High-Fidelity 2X Master Mix: 12.5 µL + - V3 Pool 1 (10 µM): 2.5 µL + - Nuclease-free water: 7.5 µL + - **Total:** 22.5 µL + - **Reaction 2:** + - Q5 Hot Start High-Fidelity 2X Master Mix: 12.5 µL + - V3 Pool 2 (10 µM): 2.5 µL + - Nuclease-free water: 7.5 µL + - **Total:** 22.5 µL + +6. Add 2.5 µL cDNA to each reaction, gently mix by pipetting and pulse spin the tube to collect liquid at the bottom. +> **Note:** +> Up to 10 µL DNA can be added to each PCR reaction (in place of water) to improve amplification of low titre samples. + +7. Set up the following program on the thermal cycler: + - **Heat Activation:** 98°C for 00:00:30 (1 cycle) + - **Denaturation:** 98°C for 00:00:15 (35 cycles) + - **Annealing:** 65°C for 00:05:00 (35 cycles) + - **Hold:** 4°C indefinitely (1 cycle) + +### Bead Clean Up +8. Label strip-tubes/plate and combine the following volumes of each PCR reaction per sample: 25 µL. + + | Component | Volume | + |-----------------------|---------| + | Pool 1 PCR reaction | 25 µL | + | Pool 2 PCR reaction | 25 µL | + | **Total:** | 50 µL | + +9. Add 40 µL SPRI beads to 50 µL pooled PCR product for an 0.8x clean up. Gently mix and incubate for 00:05:00 at room temperature. + - Place on a magnetic rack and incubate for 00:02:00 or until the beads have completely pelleted and the supernatant is clear. Remove and discard the supernatant. + - Perform an ethanol wash by adding 100 µL room temperature 70% ethanol to bathe the pellet, remove and discard ethanol without touching the bead pellet. Repeat the ethanol wash (step 9.2). Pulse centrifuge to remove residual ethanol. + - Allow to air dry for 1-2 minutes until the pellet loses its shine. Do not over-dry. + - Re-suspend pellet in 20 µL H2O, mix gently and incubate for 2 minutes at room temperature. + - Place on a magnetic rack, transfer the supernatant to clean labeled tubes. + - Quantify using a Qubit or Quantus. + +### End Preparation +10. In a new PCR strip-tube/plate set up the following reaction for each sample, normalizing to 80 ng amplicon DNA input per sample: + + | Component | Volume | + |-----------------------------------------|---------| + | Clean PCR product from previous step | Up to 8.3 µL | + | Ultra II end prep reaction buffer | 1.2 µL | + | Ultra II end prep enzyme mix | 0.5 µL | + | Nuclease-free water | Total 10 µL | + + - Incubate at room temperature for 00:15:00. + - Incubate at 65°C for 00:15:00. + - Incubate on ice for 00:01:00. + +### Native Barcoding +11. In a new PCR strip-tube/plate set up the following reaction for each sample: + + | Component | Volume | + |-------------------------------------|---------| + | End-preparation reaction mixture | 1 µL | + | NBXX barcode | 1.25 µL | + | Blunt/TA Ligase master mix | 5 µL | + | Nuclease-free water | 2.75 µL | + | **Total:** | 10 µL | + + - Incubate at room temperature for 00:20:00. + - Add EDTA and mix thoroughly: + + | EDTA Cap Color | Volume per Well | + |-----------------|-----------------| + | Clear Cap EDTA | 1 µL | + | Blue Cap EDTA | 2 µL | + +> **Note:** +> EDTA concentration varies by cap color supplied by ONT. + +11.4. Pool all one-pot barcoding reactions together in a new 1.5mL Eppendorf tube. +> **Note:** +> If processing: +> - 12-24 samples, pool 10 µL from each native barcoding reaction. +> - 48 samples, pool 5 µL from each native barcoding reaction. +> - 96 samples, pool 2.5 µL from each native barcoding reaction to avoid exceeding 240 µL. + +11.5. Add an equal volume of water to the pooled barcoded samples. +11.6. Add AMPure beads (supplied with ONT kit) for a 1.5x clean up. + + | Component | Volume | + |------------------------|----------| + | Pooled barcoded samples| 240 µL | + | Water | 240 µL | + | Beads | 720 µL | + + - Mix by vortexing and pulse centrifuge to collect liquid at the bottom of the tube. Incubate for 00:05:00 at room temperature. + +### Adaptor Ligation +12. Set up the following Native Adapter (NA) ligation and clean-up with SFB. +13. In a new 1.5mL eppendorf or PCR tube set up the following adapter ligation reaction: + + | Component | Volume | + |------------------------------------|---------| + | Barcoded amplicon pool | 30 µL | + | NEBNext quick ligation reaction buffer (5X) | 10 µL | + | Adaptor mix (NA) | 5 µL | + | Quick T4 DNA ligase | 5 µL | + | **Total:** | 50 µL | + + - Incubate at room temperature for 00:20:00. + - Perform a bead clean up. + +15. Add 50 µL AMPure beads (supplied with ONT kit) to the sample tube. Mix by vortexing and pulse centrifuge to collect all liquid at the bottom. Incubate for 00:05:00 at room temperature. + - Place on a magnetic rack and incubate for 00:02:00 or until the beads have pelleted and the supernatant is clear. Remove and discard the supernatant, being careful not to touch the bead pellet. + - Add 250 µL SFB and resuspend beads by pipette mixing. Pulse centrifuge to collect all liquid and place on the magnet. Remove supernatant and discard. Repeat to perform a second SFB wash. + - Add 200 µL room-temperature 70% ethanol to bathe the pellet. Remove and discard ethanol without touching the bead pellet. Pulse centrifuge to remove residual ethanol using a P10 pipette. + - Incubate for 00:01:00 or until the pellet loses its shine. Be careful not to over-dry. + - Resuspend pellet in 15 µL EB (ONT), mix gently and incubate for 00:02:00. + - Place on a magnet and transfer sample to a clean 1.5mL Eppendorf tube ensuring no beads are transferred. + +16. Quantify the final library using a fluorometer such as a Qubit or Quantus. +> **Note:** +> Final library can be now stored at 4°C for up to a week if needed; otherwise proceed directly to MinION sequencing. + +--- + +## Flow Cell Check and Priming +17. Refer to ONT documentation for images explaining how to check, prime, and load the flow cell. +18. Complete a flow cell check using MinKNOW user interface and MinION. +19. Prepare the flow cell priming mix: + + | Component | Volume | + |-----------------------------------|----------| + | Flow cell flush (FCF) | 1170 µL | + | Bovine serum albumin (BSA) 50mg/mL| 5 µL | + | Flow cell tether (FCT) | 30 µL | + | **Total:** | 1205 µL | + + - Slide the flow cell priming port cover clockwise to open, and set pipette to 200 µL. Insert tip into priming port, turn the wheel showing 220-230 µL to draw back 20-30 µL of buffer. Visually check for continuous flow across sensor array. + - Load 800 µL of the priming mix into the flow cell via the priming port, avoiding air bubbles. Wait for five minutes. + +## MinION Sequencing +20. Thoroughly mix the library beads by pipetting immediately before use. +21. In a new 1.5mL Eppendorf DNA LoBind tube, prepare the library for loading. + + | Reagent | Volume | + |-------------------------|--------| + | Sequencing buffer (SB) | 37.5 µL| + | Library beads (LIB) mixed just before use | 25.5 µL| + | DNA library + H2O | 12 µL | + | **Total:** | 75 µL | + +22. Complete flow cell priming and load the library (approx. 37.5 µL) into the SpotON sample port. +23. Mix the prepared library gently by pipetting just prior to loading. +24. Add half of the library to the flow cell via the SpotON sample port in a dropwise manner. +25. Gently replace the SpotON sample port cover. +26. Place the light shield over the flow cell and close the device lid. +27. Start the sequencing run using MinKNOW. + +## Protocol References +Pre-print coming soon: "Whole genome sequencing of hepatitis B virus (HBV) using tiled amplicon (HEP-TILE) and probe-based enrichment on Illumina and Nanopore platforms." + +--- + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/herp-haploid-engineering-and-replacement-protocol-drq55v.md b/markdown-output/herp-haploid-engineering-and-replacement-protocol-drq55v.md new file mode 100644 index 0000000000000000000000000000000000000000..ccea24b6940cad704ceb369cbcbdb8984c436263 --- /dev/null +++ b/markdown-output/herp-haploid-engineering-and-replacement-protocol-drq55v.md @@ -0,0 +1,162 @@ +```markdown +# Goal/Experiment: +The goal of this experiment is to provide a guide to using the Haploid Engineering and Replacement Protocol (HERP) cassettes for genome editing in Saccharomyces species. This includes preparation of selection and counterselection media, culturing and transformation for HERP cassette insertion and subsequent replacement. + +# HERP: Haploid Engineering and Replacement Protocol for Saccharomyces + +*Authored by: William G. Alexander, Drew T. Doering, and Chris Todd Hittinger* + +**Abstract:** +This protocol is derived from: Alexander WG, Doering DT, and Hittinger CT (2014) "*High-Efficiency Genome Editing and Allele Replacement in Prototrophic and Wild Strains of Saccharomyces.*" *Genetics* 198:859-866; doi:10.1534/genetics.114.170118 + +Please refer to the [full manuscript](https://www.genetics.org/content/198/3/859) for additional details. + +The purpose of this document is to provide an easy-to-follow guide for using HERP cassettes. This involves preparing selection and counterselection media, culturing, transformation for HERP cassette insertion, and counterselective replacement. + +Published: 06 Oct 2015 + +--- + +## Guidelines + +### C. Inserting the HERP Cassettes + +#### 1) Design Primers with Overhangs that Target the Cassette to Your Desired Locus + +* The 5' overhangs dictate where the cassette will be integrated, and the length needed depends on the species manipulating (e.g., 40 bp for *S. cerevisiae*, *S. paradoxus*, *S. uvarum*, & *S. eubayanus*, 50 bp for *S. mikatae*, and 70 bp for *S. kudriavzevii*). +* The 3' ends amplify the cassette from either the primer or yeast genomic DNA. The cassette construction with adjacent I-SceI recognition site provides advantages over plasmid constructs. In yeast, SCE1 is actively repressed while growing on glucose, preventing leaky nuclease expression. + +## Protocol + +### Preparing Media: Yeast Extract-Peptone-Glycerol + Antifolates (YPGly+AF) + +#### Step 1. +Add the following components to a 2-L Erlenmeyer flask: + +* 10 g yeast extract +* 20 g peptone +* 5 g sulfanilamide +* 50 mg hypoxanthine +* 18 g agar +* 900 mL ddH2O + +#### Step 2. +Mix to dissolve as much as possible. (Agar and sulfanilamide won't dissolve until heated). + +#### Step 3. +Autoclave for no more than 20 minutes on a liquid cycle. + +#### Step 4. +Once autoclaved, cool to 50°C in a water bath, then add the following and mix: +* 5 g thymidine +* 200 mg methotrexate +* 100 mL 50% (v/v) glycerol, sterilized + +*Nota Bene:* The standard practice for adding compounds after autoclaving is to dissolve them as a solvent, filter, then add to the media, or directly add the solids to the cooled media due to their quantity. + +#### Step 5. +Pour 20 mL into plastic petri dishes and allow them to set. You have now made YPGly+AF media. + +### Preparing Media: Synthetic Complete + 5-fluorodeoxyuridine (SC+FUdR) + +#### Step 6. +In a 2-L Erlenmeyer flask, make 1 L of Synthetic Complete agar using your preferred formulation. + +#### Step 7. +Autoclave, then cool to 50°C in a water bath. + +#### Step 8. +Dissolve 55 mg of FUdR into 1.1 mL of ddH2O and filter sterilize. + +#### Step 9. +Add 1 mL of FUdR solution to cooled SC agar and mix. + +#### Step 10. +Pour 20 mL into plastic petri dishes and allow them to set. You have now made SC+FUdR agar. + +*Nota Bene:* An alternate method to make SC+FUdR plates is to make a 1000x stock solution of 50 mg/mL FUdR in water, filter, then spread enough concentrate onto the surface of a premade SC plate to bring the final concentration to 50 µg/mL. + +### Inserting the HERP Cassettes + +#### Step 11. +Design primers with overhangs that target the cassette to your desired locus. (Refer to guidelines for details.) + +#### Step 12. +Amplify the HERP cassette of choice using your targeting primers and a high-fidelity polymerase such as New England Biolabs' Phusion system. + +#### Step 13. +Culture your strain by inoculating 50 mL of YPD media with enough overnight culture to bring the OD600 to 0.2-0.25. Shake at the optimal temperature for your strain or species. + +#### Step 14. +Shake at the optimal temperature until the culture's OD600 reaches 0.85-1.0. + +#### Step 15. +Harvest the cells by centrifugation in a 50-mL conical vial at 3000 RPM for 5 minutes. + +#### Step 16. +Remove supernatant, wash with 25 mL water, spin at 3000 RPM for 5 minutes. + +#### Step 17. +Remove supernatant and suspend cells in 1 mL of water. + +#### Step 18. +Aliquot 100 µL cell suspension to microcentrifuge tubes, spin for 30 seconds at max speed, remove supernatant. + +#### Step 19. +Add the following reagents to each cell pellet IN ORDER: + +* 240 µL 50% polyethylene glycol, average MW 4000, filter sterilized +* 36 µL 1 M lithium acetate, filter sterilized +* 5 µL 20 mg/mL boiled sonicated salmon sperm DNA +* 79 µL HERP cassette PCR product or control (water) + +#### Step 20. +Suspend cell pellet in transformation mixture and heat shock. + +*Nota Bene:* For optimal transformation efficiency, determine the most transformants for your species or strain through empirical testing. + +#### Step 21. +After heat-shocking, spin reactions for 30 seconds at max speed, remove supernatant, suspend cells in 600 µL of YPD. + +#### Step 22. +Transfer to glass culture tubes and spin for 3 hours in a culture wheel at the optimal temperature. + +#### Step 23. +Spread 200 µL of recovered cells on each of three YPGly+AF plates. Only one 200 µL volume of control reaction needs to be plated. Incubate at optimal temperature. Colonies will appear in 3-10 days. + +#### Step 24. +Streak colonies out to fresh YPGly+AF plates. Analyze by amplifying the target locus via PCR and/or sequencing across the insertion junction. + +### Counterselective Replacement of the HERP Cassette + +#### Step 25. +Confirm HERP cassette insertion by phenotypically confirming sensitivity to FUdR. Spot 1,000 cells onto SC+FUdR plates multiple times. + +#### Step 26. +Once confirmed, inoculate the strain in 50 mL of 2X YPA100 +4% galactose to reach OD600 of 0.2-0.25. + +#### Step 27. +Once OD600 of 0.85-1.0 is reached, repeat steps C4 to C6, except replace the HERP cassette PCR product with desired replacement PCR product. + +#### Step 28. +Once heat shock is completed, remove the supernatant and suspend in 600 µL water. + +#### Step 29. +Spread 200 µL onto each of three SC plates. + +#### Step 30. +Incubate at optimal temperature for 24 hours. + +#### Step 31. +After 24 hours, incubate plates at 4°C for one hour. + +#### Step 32. +Lightly replicate plates to SC+FUdR plates. + +#### Step 33. +Re-replicate to fresh FUdR plates no more than once a day to reduce background. Colonies will appear in 2-5 days. + +--- + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/heterologous-protein-expression-in-e-coli-bdjti4nn.md b/markdown-output/heterologous-protein-expression-in-e-coli-bdjti4nn.md new file mode 100644 index 0000000000000000000000000000000000000000..57e90db0a1adc39f394a74a72b6698392cfe588f --- /dev/null +++ b/markdown-output/heterologous-protein-expression-in-e-coli-bdjti4nn.md @@ -0,0 +1,232 @@ +```markdown +# Goal/Experiment: +The goal of this experiment is to express heterologous (recombinant) proteins in *Escherichia coli* (E. coli), enabling the subsequent purification of these proteins for use in enzyme assays, crystallography, and other applications. + +# Heterologous Protein Expression in *E. coli* V.5 + +**Authors**: Diep Ganguly, Timothy Rhodes, Nay Chi Khin, Estee E Tee, Kai Xun Chan +**Affiliation**: The Australian National University + +**DOI**: [dx.doi.org/10.17504/protocols.io.bdtj4nn](dx.doi.org/10.17504/protocols.io.bdtj4nn) + +**Protocol Citation**: +Ganguly, D., Rhodes, T., Khin, N. C., Tee, E. E., & Chan, K. X. (2020). Heterologous protein expression in *E. coli*. *protocols.io*. + +## Abstract +Protocol for recombinant protein expression in *E. coli* for protein purification, enzyme assays, protein crystallography, etc. + +**Keywords**: heterologous protein expression, E. coli + +**License**: +This work is distributed under the Creative Commons Attribution License, which allows unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. + +**Created**: March 12, 2020 + +**Last Modified**: June 19, 2020 + +**Protocol ID**: 34131 + +## Guidelines +This protocol will take a few days; ensure all buffers, cell strains, and plasmids are ready. Steps may be paused by storing cells at -20/-80 °C. +Adjust volumes to ensure appropriate aeration during culture growth. Typically, BL21 (DE3) strains for T7 expression are used. + +## Materials + +| Name | Catalog # | Vendor | +| --------------------------------------- | --------------------------- | ---------------------------- | +| Potassium chloride | P212121 | P212121 | +| Petri Dish | LHPD01100 | P212121 | +| Lysozyme from chicken egg white | L6876 | Sigma Aldrich | +| Luria-Bertani (LB) broth, makes 1L | K488 | Amresco | +| EDTA | | Sigma Aldrich | +| cOmplete™, EDTA-free Protease Inhibitor | 05056489001 | Sigma Aldrich | +| 1.5 mL Eppendorf tubes | | | +| Electroporation System Gene Pulser XCell | | Bio-rad Laboratories | +| 37°C Incubator | | | +| DTT | D0632 | Sigma Aldrich | +| 14mL Polystyrene Cell Culture Tubes | CT5250 | Alkali Scientific | +| 4X Bolt LDS Sample Buffer | B0007 | Invitrogen - Thermo Fisher | +| NaCl | 53014 | Sigma Aldrich | +| IPTG | IB0168.SIZE.100g | Bio Basic Inc. | +| BL21(DE3) or BL21-Star(DE3) or Rosetta2(DE3) | | | +| Magnesium chloride hexahydrate | M2670 | Sigma Aldrich | +| Electroporation Cuvette 1mm | 1652089 | BioRad Sciences | +| Falcon® Conical Tubes, 50 mL 500 Tubes | 38010 | Stemcell Technologies | +| Tris-HCl | AM9855 | Life Technologies | +| 28°C incubator without CO2 | S7907 | Thermo Fisher Scientific | +| Disodium phosphate | S7912 | Sigma Aldrich | +| Monopotassium phosphate | P9791 | Sigma Aldrich | +| 42°C water bath | | | +| Imidazole | I5513 | Sigma Aldrich | +| UV/Vis spectrophotometer | | | +| GelCode™ Blue Stain Reagent | 24590 | Thermo Fisher Scientific | +| Q125 Sonicator | Part #Q125 | Qsonica | + +## Safety Warnings +Ensure use of appropriate aseptic technique. Be cautious if using a bunsen burner and ethanol. + +## Before Starting +Verify your plasmid is transformed into the desired *E. coli* strain, e.g., BL21 Star (DE3). Plate these strains on selective LB media to generate starter cultures. Prepare all described buffers except fresh IPTG stocks. Add DTT fresh to buffers before use. + +## Procedure + +### 1. Buffer recipes + +- **Auto-induction TB medium (alternatively, liquid LB media for IPTG induction)**: + - 5 g/L yeast extract + - 20 g/L tryptone + - 85.5 mM NaCl (5 g/L) + - 22 mM KH₂PO₄ (2.99 g/L) + - 42 mM Na₂HPO₄ (5.96 g/L) + - Supplement fresh: 0.6% glycerol, 0.05% glucose, 0.2% lactose + +- **10X PBS**: + - Dissolve the following in 800 mL H₂O: + - 80 g of NaCl (1.37 M) + - 2.0 g of KCl (27 mM) + - 14.4 g of Na₂HPO₄ (100 mM) + - 2.4 g of KH₂PO₄ (18 mM) + - Adjust pH to 7.4 + - Add H₂O to 1L + - Autoclave + + Store 10X stock at 4°C; dilute 1:10 to make 1X working stock. Supplement with 0.05 - 0.1% Tween20 if necessary. + Alternatively, use TBS-T from the [Cold Spring Harbor Protocols](http://cshprotocols.cshlp.org/content/2015/3/pdb.rec085670.full?text_only=true). + +- **Re-suspension buffer**: + - 50 mM Tris-HCl pH 8 + - 2 mM EDTA + +- **Cell lysis buffer**: + - 50 mM NaH₂PO₄ pH 8 (or ~1-2 pH units away from the isoelectric point (pI) of expressed protein) + - 500 mM sodium chloride + - 10 mM imidazole + - 0.5% Triton X-100 + - 10% glycerol + - 2 mM DTT (add fresh before use) + - 1X Protease-inhibitor cocktail (add fresh before use) + +- **Denaturing buffer**: + - 8 M Urea + - 4% CHAPS + - 35 mM Tris-HCl pH 8 + - Adjust pH to 7.5 if necessary (warm to 40°C for dissolving urea) + +### 2. Transformation (approximately 12h + 1h) +- Transform *E. coli* with plasmid using chosen method (heat shock or electroporation). + + **For electropotent cells**: + - Add 0.5 - 1 µL purified plasmid to 50 µL cells (thaw on ice, 15 minutes) + - Gently flick to mix + - Transfer to chilled 1mm electroporation cuvette without bubbles + - Set machine to 1.8 kV, 25 µF, 200-400 Ω + - Add 1 mL LB to cuvette, transfer contents to microfuge tube + - Recover at 37°C with ~200 rpm shaking for >1 hr + + **For chemically competent cells**: + - Add 0.5 - 1 µL purified plasmid to 50 µL cells (thaw on ice, 15 minutes) + - Gently flick to mix + - Sit on ice for 30 minutes, then heat shock + - Incubate at 42°C water bath for 30 - 90 seconds + - Return to ice for 5 minutes + - Add 1 mL LB, recover at 37°C with ~200 rpm shaking for >1 hr + +### 3. Plate recovered cells +- Plate 100 µL of transformed cells on selective LB media +- Grow overnight (O/N) at 37°C (approx. 12h) +- Adjust volume to obtain single colonies + +### 4. Pick single colony for inoculation +- Inoculate for 12h O/N at 37°C in 3-5 mL LB/TB medium with antibiotic, shaking at 200-250 rpm + +### 5. Protein Expression Setup +- Inoculate larger culture from starter culture at 1:50 dilution in auto-induction TB medium or LB medium with lactose +- Ensure culture aeration + +### 6. Induce protein expression (approx. 12h) +- **Using LB with IPTG**: + - Grow at 37°C until OD₆₀₀ = 0.8, add 0.2 - 0.4 mM IPTG + - Grow cultures overnight at desired temperature +- **Using auto-induction medium**: + - Grow overnight or until OD₆₀₀ = 2 + +### 7. Pellet Cells +- Centrifuge at 4°C, 7,000 rcf for 10 minutes +- Store an aliquot at -20°C + +### 8. Remove supernatant and resuspend in buffer +- Pellet cells in re-suspension buffer, discard supernatant, snap-freeze in liquid nitrogen (LN₂) and store at -80°C + +## Quality Control of Protein Expression + +### 9. Test for expression +- Test an aliquot of induced culture for expression levels + +### 10. Pellet culture +- Centrifuge at max speed for 3 minutes + +### 11. Resuspend cells +- Resuspend in 100 µL 1X PBS (per 1 mL culture) +- Store lysate at -20°C + +### 12. Add loading agents +- **Appropriate amounts** per sample: + - 4X LDS sample buffer + - DTT to final concentration: 50 mM + - MgCl₂ to final concentration: 100 mM + +### 13. Calculate lysate volume +- Based on OD₆₀₀ and concentration factor (CF) + +\[ +\text{CF} = \frac{\text{volume of culture}}{\text{volume of resuspension}} +\] +- To load: + +\[ +\mu\text{L to load} = \frac{\text{180}}{\text{CF}/\text{OD}} +\] + +### 14. Heat sample +- 72°C for 10 minutes in water bath + +### 15. Spin samples +- On ice for 5 minutes, spin for 15 minutes at max speed + +### 16. Transfer supernatant +- Avoid "sticky" DNA coating the tube + +### 17. Run on SDS-PAGE +- Perform Coomassie staining or Western blot + +## Solubilisation + +### 18. Isolate protein +- Re-suspend cells in lysis buffer +- Ensure lysis buffer has >1 pH units away from pI + +### 19. Transfer to tube +- Transfer to microfuge tube, retain on ice at -20°C + +### 20. Sonicate samples +- Q125 ultrasonicator (125w, 20 kHz, 60% amp, 3x30s pulses) +- Keep on ice during between rounds + +### 21. Spin at high speed +- 16,000g for 30 minutes at 4°C + +### 22. Recover supernatant +- Retain the pellet at -20°C for insoluble fraction + +### 23. Process insoluble fraction +- Re-suspend pellet in denaturing buffer +- Sonicate and centrifuge as per soluble fraction + +### 24. Perform Bradford Assay +- Determine protein concentration for soluble and insoluble fraction + +### 25. Run SDS-PAGE +- Stain gel with Coomassie or Western blot + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/high-density-cultivation-of-synechocystis-sp-pcc-6-9cgh2tw.md b/markdown-output/high-density-cultivation-of-synechocystis-sp-pcc-6-9cgh2tw.md new file mode 100644 index 0000000000000000000000000000000000000000..d4d01556710d8db254f05a59bcd08941dac81e26 --- /dev/null +++ b/markdown-output/high-density-cultivation-of-synechocystis-sp-pcc-6-9cgh2tw.md @@ -0,0 +1,115 @@ +```markdown +# Goal/Experiment: +The objective of this experiment is the high-density cultivation of Synechocystis sp. PCC 6803 using the HDC 6.10B system (CellDeg) developed for photoautotrophic microorganisms. + +# High Density Cultivation of Synechocystis sp. PCC 6803 using the HDC 6.10B system (CellDeg) V.2 + +## Authors: +- Oliver Mantovani¹ +- Dennis Dienst² +- Pia Lindberg² + +¹ Universität Rostock +² Department of Chemistry - Microbial Chemistry, Ångström Laboratory, Uppsala, Sweden + +## Abstract +The CellDeg® high-density cultivation system is a revolutionary culturing system for photoautotrophic microorganisms. The culturing vessels come with a semi-permeable membrane on the bottom that allows the diffusion of CO₂, while a membrane in the lid (on top of the vessel) facilitates the passage of O₂. By placing the vessels on top of a highly concentrated carbonate buffer reservoir with high CO₂ partial pressure in combination with constant agitation, nutrient-rich media, and high light intensities, the system allows the obtainment of precedingly unparalleled cell densities. + +This protocol has been established in the Lindberg lab at Ångström laboratory (Uppsala University) for highly efficient sesquiterpenoid production with Synechocystis sp. PCC 6803 using a dodecane overlay as in situ extractant. The protocol has been proven successful for small-scale screening procedures over time periods of up to one week. + +For more information: [Link](http://celldeg.com) + +## Materials + +| Name | Catalog # | Vendor | +| -------------------- | --------- | ------------------- | +| Potassium carbonate | - | P212121 | +| Potassium bicarbonate| 237205 | Sigma – Aldrich | +| HDC 6.10B Starter Kit| - | CellDeg, CD media | + +## Preparation of Precultures +### 1. Example: 6 Well Plate Precultures +- **Materials:** + - Standard polystyrene 6 well plates. + - 3 mL standard BG11 medium ([Protocol Link](dx.doi.org/10.17504/protocols.io.wj5fqc6)) with strains of *Synechocystis sp. PCC 6803*. + - Appropriate antibiotics. + +- **Procedure:** + 1. Prepare standard polystyrene 6 well plates. + 2. Inoculate 3 mL BG11 medium with strains of *Synechocystis sp. PCC 6803*. + 3. If metal induction (e.g., Cu²⁺ and/or Co²⁺) is required, use BG11 with modified trace metal composition. + 4. Add appropriate antibiotics. + 5. Place the 6 well plates on a standard orbital shaker (e.g., 'Standard analog shaker, Model 5000 (VWR, orbit: 25 mm, frequency: 120 rpm)). + 6. Incubate at 30 °C under constant light intensities of 50-100 µmol photons · m⁻² · s⁻¹. + 7. After ~4 days, proceed to Step 2. + +## Inoculation of Celldeg Cultures +### 2. Prepare Experimental Cultures from Precultures + +- **Procedure:** + 1. Measure OD₇₅₀ in a spectrophotometer (e.g., plate reader). + 2. Calculate the volume needed for the inoculation of 8 mL Celldeg culture. + + **Example:** + - Measured OD₇₅₀ of preculture = 1.2. + - Desired OD₇₅₀ in Celldeg vessel = 0.3. + - Desired volume in Celldeg vessel = 8.0 mL. + - Calculation: + \[ + V(\text{preculture}) = \frac{0.3 * 8.0\, \text{mL}}{1.2} = 2.0\, \text{mL} + \] + + 3. Centrifuge preculture (e.g., 2.0 mL) for 5 min at 2500 g at room temperature. + 4. Resuspend pellets in 8 mL CD medium ([Media Link](dx.doi.org/10.17504/protocols.io.2bxgapn)) including appropriate antibiotics. + 5. If desired, supply cultures with inducer molecule (higher inducer concentrations might be required under HD conditions). + + **Example:** + - For Cu²⁺-mediated induction of the *P\_petE* promoter: add 4 µM CuSO₄ every second day. + - For Co²⁺-mediated induction of the *P\_coaT* promoter: add 30 µM CoCl₂ every day. + + 6. Transfer cell culture to CellDeg vessel. + 7. Add 2 mL of dodecane to the cultures (only if in situ extraction is desired). + + **Note:** The CellDeg vials, since CO₂ is provided from the bottom, do not need a large headspace for gas exchange, and can be filled generously (the ~25 mL vials can easily accommodate 10 mL cultures). If no dodecane overlay is added, 10 mL culture volume should be used to quantitatively minimize evaporation effects. + +## Celldeg System Setup +### 3. Bicarbonate-carbonate Buffer (Reservoir Preparation) + +- **Table: Ingredients of CellDeg carbonate buffer reservoir for a CO₂ partial pressure of 90 mbar at 20°C** + + | Ingredient | Concentration (M) | Concentration (g/L) | + | ---------- | ----------------- | ------------------- | + | KHCO₃ | 3 | 270.31 | + | K₂CO₃ | 3 | 41.41 | + +- **Procedure:** + 1. Dissolve KHCO₃ 900 mL H₂0. + 2. Dissolve K₂CO₃ 100 mL H₂0. + 3. Due to the high final concentrations, gentle heating (≤ 40 °C) of the solutions can accelerate the complete dissolution of the salts (in particular KHCO₃). + 4. You can easily up- or downscale the buffer amounts to a mixing ratio of 3M KHCO₃: 3M K₂CO₃ is 9:1. + 5. Ensure the vessel is tightly closed before you stir and heat. + 6. As the solution doesn’t get in contact with the cultures, it doesn’t need to be sterilized. + +## Vessel Kit Assembly +### 4. Assembly Procedure +1. Depending on the size of the CellDeg system, the reservoir container has to be filled by 20% of the total volume by the concentrated carbonate solution. Fill a standard reservoir with 200 mL bicarbonate-carbonate buffer from step 3. +2. Attach the filled vessels from Step 2 to the tray on top of the reservoir. + +## Celldeg Cultivation +### 5. Cyanobacteria Culturing + +- **Procedure:** + 1. Place the assembled CellDeg system on an orbital shaker, e.g., IKA KS 130 basic orbital shaker (orbit ø = 4 mm) and shake at 320 rpm + 2. Incubation Chamber: Versatile Environmental Test Chamber (Sanyo) w/o humidifier. + 3. Set the temperature to 30 °C. + 4. Sequence of increasing light intensities: + - 250 µmol photons * m⁻² * s⁻¹ (0h-24h), + - 490 µmol photons * m⁻² * s⁻¹ (24h-48h), + - 750 µmol photons * m⁻² * s⁻¹ (tp 48h-xh) + - **Note:** Light intensities were measured with a Licor LI-185B quantum meter and values are sums of multi-directional measurements. + 5. The bicarbonate-carbonate buffer in the reservoir should be replaced after 4 days of culturing. + + **Note:** The incubation chamber used here is not optimized for this cultivation type and has an upper limit in light intensities. Following the manufacturer’s recommendations - particularly regarding the quality of the light source - should distinctly improve the yields. + +_endofoutput_ +``` \ No newline at end of file diff --git a/markdown-output/high-quality-dna-extraction-protocol-from-recalcit-i8jchun.md b/markdown-output/high-quality-dna-extraction-protocol-from-recalcit-i8jchun.md new file mode 100644 index 0000000000000000000000000000000000000000..9bf7249e81ff1954460a0f80fa914fc89a850bd4 --- /dev/null +++ b/markdown-output/high-quality-dna-extraction-protocol-from-recalcit-i8jchun.md @@ -0,0 +1,154 @@ +```markdown +# High quality DNA extraction protocol from recalcitrant plant tissues + +## Goal/Experiment: +This protocol aims to remove secondary compounds inhibiting restriction enzyme digestion of genomic DNA from recalcitrant plant tissues such as Xanthorrhoea, Eucalyptus, Eremophila, Spyridium, and various Malvaceae, Rutaceae, Orchidaceae, and Fabaceae species. + +## Abstract +This protocol was developed to remove secondary compounds that inhibit restriction enzyme digestion of Xanthorrhoea genomic DNA. It successfully obtains high-quality, high-weight DNA from other recalcitrant plant tissues, including Eucalyptus, Eremophila, Spyridium, and various Malvaceae, Rutaceae, Orchidaceae, and Fabaceae species. + +Based on a modified CTAB protocol from Doyle and Doyle (1990), with modifications from Shepherd and McLay (2011) and Tibbits et al. (2006). + +## Guidelines +This protocol requires standard molecular laboratory equipment and chemicals. Three critical solution additives for removing inhibiting secondary compounds from Xanthorrhoea tissue include: +- STE +- NaCl:BSA (Sodium Chloride:Bovine Serum Albumin) +- NaAC (Sodium Acetate) + +These additives may vary for different plant tissues, but all are used in this lab for obtaining clean DNA from a wide variety of families. + +It is possible to include the NaAC wash step with the isopropanol precipitation, but performing it at the end seems to produce better quality DNA. + +## Before Starting + +### Reagents +1. Tris-HCl pH 8 (1M) +2. EDTA (0.5M) +3. NaCl (5M) +4. STE +5. CTAB (Cetyltrimethylammonium bromide) +6. NaCl:BSA 5:1 +7. Sodium acetate (2.4 M) +8. Tris-HCl pH 8 (10 mM) +9. 70% ethanol + +## Protocol + +### Prepare STE (fresh is best, keep for 2 weeks maximum) + +#### Step 1: +| Volume for 50 samples | Volume for 25 samples | +|---------------------------|-------------------------| +| 4g sucrose | 2g sucrose | +| 1.5 mL Tris-HCl pH 8 (1M) | 0.75 mL Tris-HCl pH 8 (1M) | +| 5 mL EDTA (0.5M) | 2.5 mL EDTA (0.5M) | +| H2O to 50 mL | H2O to 25 mL | + +### Prepare CTAB (fresh is better) + +#### Step 2: +| | Start Conc M. | Final conc M. | for 50 mL solution | +|-------------------------|------------------|-----------------|-----------------------| +| Tris-HCl pH 8 | 1 M | 100 mM | 5 mL | +| NaCl | 5 M | 1.4 M | 14 mL | +| EDTA | 0.5 M | 50 mM | 5 mL | +| CTAB | 2% | | 1g | +| PVP (Polyvinylpyrrolidone) | 2% | | 1g | +| H2O | | | To 50 mL | + +Mix on a stir plate (unheated) until the solution is clear. + +### Prepare NaCl:BSA Additive + +#### Step 3: +Make 100x (≈4%) BSA from powder in H2O. Combine 5M NaCl with 100x BSA in a 5:1 ratio. +This can be frozen for long-term storage. + +### Preheat CTAB + +#### Step 4: +Add 600 μL of CTAB per sample to a falcon tube. In fumehood, add 2 μL of BME (2-Mercaptoethanol) per mL of CTAB, and 4 μL of Proteinase K per reaction. Warm at 65°C. + +### Grind tissue + +#### Step 5: +Grind 20-50 mg of plant tissue using your preferred grinding method (e.g., liquid nitrogen and grinding sand for sclerophyllous tissue). Place into labeled 1.7 mL eppendorf tube. + +### STE Step + +#### Step 6: +Add 1 mL of STE to tissue. Vortex thoroughly. Spin at 5000 rpm for 10 minutes. Discard supernatant. + +### Addition of CTAB + +#### Step 7: +In a fumehood, add 500 uL of pre-warmed CTAB solution to each tube. Add 100 uL of NaCl:BSA solution. Vortex and incubate at 65°C for at least an hour (up to overnight). + +### First Chloroform Step + +#### Step 8: +Add 450 μL of chloroform to each tube. Shake to mix and release built-up gas using a kimwipe. Spin at maximum speed for 5 minutes. + +### Removing Supernatant + +#### Step 9: +Using a P200 set to 190 μL, remove the top aqueous phase (usually between 300-400 μL) into a new eppendorf tube. Avoid the solid layer. + +### Second Chloroform Step + +#### Step 10: +Add 450 μL to the removed aqueous phase solution. Shake to mix and release built-up gas using a kimwipe. Spin at maximum speed for 5 minutes. + +#### Step 11: +Using a P200 set to 190 μL, remove the top aqueous phase into a new eppendorf tube. + +### Isopropanol DNA Precipitation + +#### Step 12: +Add 500 μL of room temperature isopropanol. Mix by gently inverting five times. If desired, add 110 μL sodium acetate (3M, pH 5.2), mix and incubate at room temperature for 20 minutes. Follow Steps 13-16 to avoid precipitation. + +### Isopropanol Centrifugation + +#### Step 13: +Spin for 10 minutes at maximum speed with eppendorf tubes facing inwards. + +### Ethanol Wash and Centrifugation + +#### Step 14: +Add 500 μL of 70% ethanol. Try to dislodge the pellet from the tube bottom through pipetting or flicking. Centrifuge for 5 minutes at maximum speed. Carefully pour off the ethanol. + +### Drying Pellet + +#### Step 15: +Dry pellets for at least two hours to overnight by leaving tubes open on a bench/in fumehood, with kimwipes securely placed over them. Alternatively, wick away remaining ethanol using kimwipes. + +### Resuspension in Tris-HCl 10 mM + +#### Step 16: +Add 50 μL of 10 mM Tris-HCl pH 8 and allow DNA to resuspend for at least an hour before use. + +### Sodium Acetate Wash + +#### Step 17: +Add 200 μL of 100% ethanol and 36.25 μL of NaAC (2.4 M) to each tube. Mix by inverting. Centrifuge for 10 minutes at maximum speed. Carefully pour off supernatant. + +### Ethanol Wash + +#### Step 18: +Carefully pour off supernatant. Add 1000 μL of 70% ethanol. Try to dislodge the pellet through pipetting or flicking the tube. Centrifuge for 3 minutes at maximum speed. Carefully pour off the ethanol. + +### Drying Pellet + +#### Step 19: +Dry pellets for at least two hours to overnight by leaving tubes open on the bench/in fumehood with kimwipes securely placed over them. + +### Resuspension of Pellet + +#### Step 20: +Add 50-100 μL of 10 mM Tris-HCl pH 8 and allow DNA to resuspend for at least an hour before use. Check quality and quantity using spectrophotometry. + +## Warnings +General chloroform extraction safety applies. The inclusion of BME (2-Mercaptoethanol) requires the use of a fumehood. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/high-sensitivity-low-input-mirna-illumina-library-e55bg86.md b/markdown-output/high-sensitivity-low-input-mirna-illumina-library-e55bg86.md new file mode 100644 index 0000000000000000000000000000000000000000..b5abbc6af76f1420b8cb82e6af482ff0dd407db0 --- /dev/null +++ b/markdown-output/high-sensitivity-low-input-mirna-illumina-library-e55bg86.md @@ -0,0 +1,253 @@ +```markdown +# Goal/Experiment: +The goal of this experiment is to generate high sensitivity miRNA libraries for Illumina sequencing using the TailorMix miRNA Sample Preparation Kit (Version 2). + +# High Sensitivity (Low Input) miRNA Illumina Library Preparation Protocol by TailorMix miRNA Sample Preparation Kit (Version 2) Version 6 + +Authors: Karen Yip, Kelvin Chan, Danny Lee + +## Abstract + +This protocol is designed for high sensitivity miRNA library generation for the Illumina sequencing platform. Our protocol and the enhanced reagent kit enable the discovery and profiling of small RNAs from a variety of sources including FFPE, exosome, serum, and whole blood. The TailorMix workflow is designed for ease of use, enabling library preparation in a single day. + +## Features + +- **User Friendly Workflow**: Libraries can be prepared in a single day. +- **No Additional Reagents Necessary**: All reaction enzymes and buffers are provided. +- **Stress Free Gel Purification**: [TailorCut Gel Extraction Tool Set](https://example.com) is included for easy gel excision and purification. +- **Easy to Use**: Reagents are supplied as ready-to-use mixtures which improves consistency and reproducibility. +- **Ultra High Sensitivity**: Prepare miRNA libraries from any source with as little as 10 ng of total RNA input. + +**Citation**: Karen Yip, Kelvin Chan, Danny Lee High sensitivity (low input) miRNA Illumina library preparation protocol by TailorMix miRNA Sample Preparation Kit (Version 2). [protocols.io](https://dx.doi.org/10.17504/protocols.io.e55bg86). +**Published**: 16 Jun 2016 + +## Guidelines + +### Figure 1 + +TailorMix miRNA Sample Preparation V2 Overview + +![Figure 1](path/to/figure1.png) +*Figure 1: Overview of the steps involved in the TailorMix miRNA Sample Preparation.* + +## Best Practices + +- Always wear gloves and use sterile technique. +- Set up reactions using sterile non-stick nuclease-free tubes. +- Place samples and reagents on ice or on chilled cooler block at all times and avoid extended pauses. +- Reagents should be prepared using RNase-free components. +- Prepare an extra 10% mixture when running multiple samples. +- Avoid repeated freeze/thaw cycles. + +## RNA Input + +This protocol has been optimized using 10 - 100 ng of purified high-quality human kidney total RNA as input. Because miRNA populations vary among different tissue types and species, the use of total RNA from other tissue or species may require optimization. You may also use isolated miRNA as the starting material. + +## Sample Pooling Guidelines + +The TailorMix miRNA Sample Preparation kit is capable of multiplexing up to 96 samples into a single lane of an Illumina flow cell. While processing multiple samples in parallel, use a unique index primer for each sample at the PCR step. Samples can be pooled before or after the library purification step. + +### Citations & References + +Niu, Jinzhi, et al. "In vivo study of Dicer-2-mediated immune response of the small interfering RNA pathway upon systemic infections of virulent and avirulent viruses in Bombus terrestris." *Insect biochemistry and molecular biology* 70 (2016): 127-137. + +## Materials + +- [TailorMix miRNA Sample Preparation Kit V2 TM302](https://example.com) by Contributed by users + +## Protocol + +### Step 1: 3’ Adapter Ligation + +1. Thaw Mix C300 from -20°C storage. Allow it to equilibrate to room temperature for a minimum of 30 minutes before use. +2. Pre-heat the thermal cycler to 70°C and pre-heat another thermal cycler to 25°C if available. +3. Denature the RNA Sample by assembling the following components in a sterile 200 µL PCR tube on ice: + +| Reagent | Volume (µL) | +|------------|-------------| +| RNA Sample | 6 | +| Mix A300 | 2 | +| **Total** | **8** | + +4. Vortex mix thoroughly and incubate at 70°C for 1 minute and then place the tube on ice. +5. Set up the following 3’ Adapter Ligation reaction on ice: + +| Reagent | Volume (µL) | +|---------------------------|---------------| +| Denatured RNA mix from step 4 | 8 | +| Mix B300 | 2 | +| Mix C300 | 6.5 | +| **Total** | **16.5** | + +*Note*: Mix C300 is a highly viscous reagent. Handle with care and pipette slowly to ensure the correct amount of Mix C300 is dispensed for each reaction. + +6. Vortex mix thoroughly and pulse spin. Incubate at 25°C for 1 hour. + +### Step 2: Ligation Product Clean Up + +1. Vortex the TailorMag Purification Beads (TPB) until they are evenly suspended. +2. Prepare 80% ethanol for the rinse step. +3. Add 30 μL of TPB with each 3’-adapter ligated sample from Step 6. Vortex mix thoroughly and pulse-spin. Incubate at room temperature for 15 minutes. + +| Reagent | Volume (µL) | +|--------------------|---------------| +| 3’-adapter ligated sample from Step 6 | 16.5 | +| TailorMag Purification Beads (TPB) | 30 | +| **Total** | **46.5** | + +*Note*: Do NOT perform strong centrifugation because it will separate TPB from the sample. + +4. Place the sample tube on the magnetic stand at room temperature for 5 minutes. +5. Carefully remove and discard 40 µL of the supernatant. + +*Note*: Sample recovery may be affected if the TPB pellet is disrupted. + +6. Keep sample tube on the magnetic stand. Gently rinse the TPB pellet with 150 µL of 80% ethanol without disrupting the TPB pellet. Discard the rinse solution. + +*Tip*: Point pipette tip towards the opposite direction as the TPB pellet. Gently pipette the 80% ethanol up and down once, then discard the rinse solution. + +7. Air dry sample tube at room temperature. + +*Note*: TailorMag Purification Beads are dried within 5 to 15 minutes at room temperature. Proceed to Step 14 when the appearance of the TPB pellet turns from glossy/shiny (wet) to matte (dry). Sample recovery may be affected if beads are over-dried and appear powdery. + +8. Remove sample tube from the magnetic stand. Add 7 µL of nuclease-free water to the dried TPB pellet. Vortex to resuspend and pulse spin. Incubate sample resuspension at room temperature for 2 minutes. + +### Step 3: 5’ Adapter Ligation + +1. Set up the following 5’ Adapter Ligation reaction on ice: + +| Reagent | Volume (µL) | +|------------------------------------|---------------| +| 3’ Adapter Ligated RNA from step 18| 7 | +| Mix D300 | 3 | +| Mix E300 | 2 | +| **Total** | **12** | + +2. Gently pipette mix thoroughly and incubate at 25°C for 1 hour and then place the tube on ice. + +### Step 4: cDNA Synthesis + +1. Pre-heat the thermal cycler to 50°C. +2. Set up the following cDNA Synthesis reaction on ice: + +| Reagent | Volume (µL) | +|------------------------------------|----------------| +| 3’ and 5’ Adapter Ligated RNA from Step 16 (contains TPB) | 12 | +| Mix F300 | 2 | +| Mix G300 | 1 | +| **Total** | **15** | + +3. Vortex mix thoroughly and pulse spin. Incubate at 50°C for 1 hour and then place the tube on ice. + +*Safe Stopping Point*: First strand cDNA could be stored at -20°C for up to seven days. + +### Step 5: PCR Amplification + +*Note*: This protocol has been optimized using 10 - 100 ng of purified high-quality human kidney total RNA as input. Because miRNA populations vary among different tissue types and species, the use of total RNA from other tissue or species may require optimization. + +An alternative PCR protocol is available for increasing libraries from low yield samples. See the Appendix A for details. + +1. Set up the following PCR reaction in a fresh sterile 200 μL PCR tube on ice: + +| Reagent | Volume (µL) | +|-------------------------------|---------------| +| First-strand cDNA from Step 19 (contains TPB) | 5 | +| Mix H300 | 18 | +| PCR Primer | 1 | +| Index Primer* | 1 | +| **Total** | **25** | + +2. Vortex mix thoroughly and pulse spin. Amplify the samples in the thermal cycler using the following PCR cycling conditions: + + - 98°C for 30 seconds + - 15 cycles of: + 1. 98°C for 5 seconds + 2. 60°C for 15 seconds + - 72°C for 1 minute + - 72°C for 5 minutes + - Hold at 4°C + +*Safe Stopping Point*: PCR products could be stored at -20°C for up to seven days. + +3. PCR yield can be monitored by running an Agilent BioAnalyzer High Sensitivity DNA assay using a dilution of 1 μL of PCR product and 9 μL of nuclease-free water. A typical result shows a distinct peak at approximately 140bp (Figure 2). + +*Note*: See Appendix B for a more detailed description of BioAnalyzer High Sensitivity DNA assay profile of the PCR products. + +**Figure 2**: BioAnalyzer High Sensitivity DNA assay of PCR Product (10x diluted) from Human Kidney Tissue Total RNA + +![Figure 2](path/to/figure2.png) + +### Step 6: Library Purification + +1. Determine the volume of TBE buffer needed and dilute 5X TBE Buffer to 1X for use in gel electrophoresis. +2. Assemble the gel electrophoresis apparatus. +3. Mix 2 μL of Custom Ladder with 2 µL of Hi-Density TBE Sample Buffer. +4. (Optional) Mix 1 µL of 100bp DNA ladder with 1 µL of Hi-Density TBE Sample Buffer. +5. Add 2.5 µL of Hi-Density TBE Sample Buffer to 25 µL of PCR product and pipet mix thoroughly. +6. Load 25 µL of the PCR product-Sample Buffer mix into one well in the middle of the 8% PAGE gel. Refer to Figure 3 for an example. +7. Load 2 µL of the custom ladder and dye mix into the neighboring wells of the PCR products. + +*Note*: Always bracketing each PCR product lane with two custom ladder lanes to ensure precise excision of the miRNA band. + +8. (Optional) Load 2 µL of the 100bp DNA ladder and dye mix into a separate well. +9. Run the gel for 75 minutes at 145V and immediately remove the gel from the apparatus. + +*Note*: Performance of electrophoresis apparatus varies. Optimization of the setting may be needed for sufficient band separation. + +### Step 7: Recover Purified Library + +1. Prepare TE buffer with 0.1% Tween-20: + +| Reagent | Volume (µL) | +|---------------|---------------| +| TE buffer | 9,990 | +| Tween-20 | 10 | +| **Total** | **10,000** | + +2. Open the gel cassette and stain with 1 µg/mL ethidium bromide solution according to the manufacturer’s instructions. +3. Place the gel on a UV Transilluminator and observe the banding pattern (Figure 3). + + **(Alternative):** Stain gel with Sybr Gold according to the manufacturer’s instructions and observe the banding pattern on a Dark Reader Transilluminator. + +4. Place the gel breaker tube into a sterile 1.5 mL microcentrifuge tube. +5. The 140bp band represents the highest concentration of micro RNA library. To excise the 140bp band, align the center of the gel cutter tool with the 140bp band of the custom ladder (Figure 4). Press down firmly into the gel and excise the gel fragment. + + *Note:* The 150bp band represents a combination of micro RNA and other small RNA species (see Appendix B, Q10 if a strong 150bp band is observed). To include the 150bp band in the extraction, align the bottom of the gel cutter tool with the 140bp band of the custom ladder (Figure 5). Press down firmly into the gel and excise the gel fragment in between the two custom ladder markers. + + See Appendix B for a more detailed description of the gel bands. + + **Figure 3**: Micro RNA Library PCR products on 8% TBE gel. + + ![Figure 3](path/to/figure3.png) + + **Figure 4**: Close up of gel cutting position for miRNA libraries. + + ![Figure 4](path/to/figure4.png) + +6. Insert the gel cutter tool containing the gel slice into the gel breaker tube. +7. Pulse-spin the gel cutter and gel breaker assembly in a minifuge. Make sure the gel slice is collected in the gel breaker tube. Remove gel cutter from the assembly and discard. +8. Add 30 μL of TE buffer with 0.1% Tween-20 to the gel breaker tube containing the gel slice. +9. Centrifuge the gel breaker assembly in a bench top centrifuge at maximum speed (approximately 13,000x G) for two minutes at room temperature. Ensure that all of the gel has moved through the holes into the collection tube. +10. Elute the micro RNA library by shaking the tube at 600 rpm at room temperature overnight. + + *Note:* Do NOT heat up gel buffer mix. + +11. To collect the micro RNA library, spin the gel mix at maximum speed (approximately 13,000x G) for 2 minutes. +12. With a P10 pipette, gently transfer 20-25 µL of eluate from gel mix to a fresh 1.5ml tube. + +### Step 8: Library Validation + +1. Use of an Agilent Technologies 2100 Bioanalyzer is recommended as a quality control analysis of your sample library. Use 1 μL of resuspended construct from step 47 on a High Sensitivity DNA chip to check the size, purity and concentration of the sample. + +*Note*: Remnant of gel loading dye may appear as a high molecular weight peak on the Bioanalyzer profile. Presence of the dye would not affect performance of the micro RNA library during the sequencing run. + +*Note*: The BioAnalyzer High Sensitivity DNA assay has a 10% deviation on sizing accuracy. + +**Figure 6**: BioAnalyzer High Sensitivity DNA assay of Gel Purified Library from Human Kidney Tissue Total RNA Sample. + +![Figure 6](path/to/figure6.png) + +--- + +**endofoutput** +``` diff --git a/markdown-output/high-throughput-assay-for-screening-fungal-isolate-cjaeuibe.md b/markdown-output/high-throughput-assay-for-screening-fungal-isolate-cjaeuibe.md new file mode 100644 index 0000000000000000000000000000000000000000..2ac3353f0fd7906fc6c3523dbf7753788d6eeaa0 --- /dev/null +++ b/markdown-output/high-throughput-assay-for-screening-fungal-isolate-cjaeuibe.md @@ -0,0 +1,172 @@ +```markdown +# Goal/Experiment: +This protocol was developed for the screening of fungal isolates against polyphenolic compounds to test their capacity for detoxification. + +# High-throughput Assay for Screening Fungal Isolates against Polyphenolic Compounds + +**Authors**: +Jana M U'Ren1, Megan Nickerson1 +1University of Arizona + +**Abstract**: +This protocol was developed for the screening of fungal isolates against polyphenolic compounds to test their capacity for detoxification. + +**Protocol Citation**: +Jana M U'Ren, Megan Nickerson 2022. High-throughput Assay for Screening Fungal Isolates against Polyphenolic Compounds. protocols.io +[https://protocols.io/view/high-throughput-assay-for-screening-fungal-isolate-cjaeuibe](https://protocols.io/view/high-throughput-assay-for-screening-fungal-isolate-cjaeuibe) + +## Media and Solution Preparation- Aspergillus nidulans Defined Media + +1. **Combine**: + - 900mL nanopure water + - 50mL 20X Sodium nitrate salts + - 1mL Trace elements +2. **Adjust pH**: + - Bring the pH to 6.5. +3. **Add Carageenan**: + - Add 2 grams of Carageenan Type II (0.2% final concentration). +4. **Autoclaving**: + - Autoclave for 30 minutes on the liquid cycle and allow to cool to room temperature. Store at 4°C. + +## Media and Solution Preparation- Glucose Solution + +5. **Prepare Glucose Solution**: + - In a 15 mL conical tube, add 3.2g of glucose. +6. **Dissolution**: + - Add enough nanopure water to bring the volume to 10 mL and vortex to mix until completely dissolved. +7. **Sterilization**: + - In a biosafety cabinet, filter-sterilize and make 1 mL aliquots. + +## Media and Solution Preparation- Polyphenol Solutions (excluding Vanillin) + +8. **Prepare Polyphenol Stock**: + - Prepare 3 5mL tubes with 3 concentrations of each polyphenol dissolved in autoclaved nanopure water. + - **Note**: Concentrations (when diluted in wells): 2000µg/mL, 1000µg/mL, and 500µg/mL. + **Actual concentrations of stock**: 40,000 µg/mL, 20,000 µg/mL, and 10,000 µg/mL. +9. **Inoculation Preparation**: + - For inoculating two plates, dissolve 0.08g, 0.04g, and 0.02g of each polyphenol in 2mL of autoclaved nanopure water. Vortex thoroughly to mix until completely dissolved. +10. **Sterilization**: + - In a biosafety cabinet, using the 0.2µm disc filters, filter sterilize 1mL of each solution into a sterile 1.5mL tube. + +## Media Solution Preparation- Vanillin + +11. **Vanillin Preparation**: + - Due to solubility issues, the amount of vanillin used has to be scaled down. For inoculating two plates, dissolve 0.02g, 0.01g, and 0.005g of vanillin in 2mL of autoclaved nanopure water. Vortex thoroughly to mix until completely dissolved. +12. **Sterilization**: + - In a biosafety cabinet, using the 0.2µm disc filters, filter sterilize 1mL of each solution into a sterile 1.5mL tube. + +## Media and Solution Preparation- Lignin + +13. **Lignin Preparation**: + - Due to solubility issues, the amount of lignin used has to be scaled down. For inoculating two plates, dissolve 0.02g, 0.01g, and 0.005g in 20mL nanopure water in glass jars. +14. **Autoclaving**: + - Autoclave on Liquid30 cycle. +15. **Aliquot**: + - In a biosafety cabinet, aliquot into 2mL tubes. + +## Inoculate Fungal Cultures on Cellophane (to be done ahead of time) + +16. **Inoculation Procedure**: + - Inoculate ___ 60mm MEA plates + cellophane with ___ isolates and incubate at room temperature until growth is sufficient to proceed. List isolates in your notebook. + - **Example**: 24 plates with 4 isolates (6 plates per isolate). + - Allow to grow until sufficient mycelium for inoculation (e.g., 1 week). + +## Prepare Liquid Fungal Inoculum for Spectrophotometer (all steps done in biosafety cabinet) + +17. **Medium Preparation**: + - Using a serological pipette, add 20mL of *A. nidulans* defined carrageenan media to blender cup (20 mL is the minimum amount needed for blender cup to work). + +18. **Harvesting Mycelium**: + - Cut mycelium closely around the original inoculum to remove the agar square from the center of the cellophane. Carefully scrape the mycelium from the cellophane, without cutting into the cellophane, and gather into a ball using the broad scalpel. Pick up the mycelium ball using the fine forceps and place in blender cup. + +19. **Blending**: + - Blend four times at one-second pulses. Check fragment sizes to ensure sufficient blending. + +20. **Alternative Harvesting Method**: + - OR, once isolate has colonized entire plate, pipette 1-5mL of sterile water onto plate. Use sterilized rubber policeman to gently scrape the hyphae off the surface of the media. Using a wide-bore pipette tip, collect the liquid and place a sterile blender cup. + +21. **Final Volume Adjustment**: + - Using serological pipette, add 0.2% carrageenan type II media until final volume is 20mL in blender cup (e.g., 20mL final volume - ___mL collected from plate = ___mL 0.2% carrageenan media to add). + +22. **Blending**: + - Blend four times at one-second pulses. + +23. **Transfer to Cuvettes**: + - Transfer 1000µL of blended culture (while pipette mixing) with a 5mL pipette to three spectrophotometer cuvette replicates. + +24. **Blank Sample**: + - Transfer 1000µL of *A. nidulans* defined carrageenan media from 5mL tube into cuvette (this will be the blank cuvette). + +## Read Samples in Spectrophotometer (at 600nm) + +25. **Blanking the Spectrophotometer**: + - Place blank cuvette with just 1000µL of *A. nidulans* defined carrageenan media into reading position (with the cuvette side with the arrow closest to the screen) and press the 0A/100%T button to blank the sample. + +26. **Reading the Sample**: + - Place sample in reading position and press the "arrow passing through the cuvette" button. Record absorbance. + +27. **Interpretation of Results**: + - A final reading of >0.44 is needed to reach the appropriate hyphal fragment density. If the absorbance of your sample is lower than this, harvest more hyphae from plates and repeat blending procedure in a new blender cup. + +## Dilute Blended Culture + +28. **Dilution Calculation**: + - When a reading is >0.44 you need to dilute the blended inoculum with *A. nidulans* defined carrageenan media to yield 4 mL of culture inoculum with an absorbance of 0.22. Use a 5 mL conical tube for the dilution. + - **Example**: If a sample has an absorbance of 0.50, add 1.32mL of blended culture from blender cup to 1.68mL of 0.2% carrageenan type II media where 0.5(X)= 0.22(3mL) so X=1.32 ml + +## Inoculate 96-well Plates (All steps to be performed in biosafety cabinet) + +29. **Addition of Phenol Solution**: + - Once dissolved, add 5µL of sterilized phenol solution to the respective wells. + +30. **Control Wells**: + - Add 5µL of autoclaved nanopure water to controls (colored purple; media without phenols but with fungi). + +31. **Inoculation Suspension Preparation**: + - Combine 1.8mL of blended culture at absorbance 0.44 and 12.5mL of *A. nidulans* defined carrageenan media. Mix well by inversion to evenly distribute the hyphal fragments in the media. + +32. **Addition of Glucose Solution**: + - Add 0.89 mL volume glucose solution to a final concentration of 2% glucose. Immediately proceed to next step so that hyphae don’t settle in the media. + +33. **Inoculate Wells with Glucose**: + - Inoculate 95µL of GLUCOSE CONTAINING solution for wells corresponding to the proper isolate +glucose. Also inoculate the corresponding positive control wells. (*do not inoculate wells for the media only controls in Row A*). + - **Note**: Do NOT push pipette plunger past the last stop because this will create bubbles, which will cause readings to be inaccurate. If bubbles form try to pop with a sterile pipette tip. + +34. **Plate Preparation**: + - Place lid on the plate and parafilm for transport. Immediately measure the plate at 595, 660, and 750nm (cell density only) on plate reader. + +35. **Post-reading Storage**: + - After reading, re-parafilm plate and place in plastic bag with wet paper towels to keep plates from drying out. Be careful to keep the plate upright at all times. + +36. **Light Exclusion for Storage**: + - Store plates in an aluminum foil lined container to keep out the light. + +37. **Periodic Readings**: + - Read plates every 3 days for a total of 24 days (tentative, depending on isolate growth). + +38. **Final Observations**: + - After 24 days, record visual observations and take pictures of plates. + +## Inoculate 96-well Plates WITHOUT Glucose (All steps to be performed in biosafety cabinet) + +39. **Addition of Phenol Solution**: + - Once dissolved, add 5µL of sterilized phenol solution to the respective wells. + +40. **Control Wells**: + - Add 5µL of autoclaved nanopure water to controls (colored purple; media without phenols but with fungi). + +41. **Inoculation Suspension Preparation**: + - Combine 2.0mL of blended culture at absorbance 0.44 and 14.0mL of *A. nidulans* defined carrageenan media. Mix well by inversion to evenly distribute the hyphal fragments in the media. + +42. **Immediate Proceeding**: + - Immediately proceed to next step so that hyphae don’t settle in the media. + +43. **Inoculate Wells without Glucose**: + - Inoculate 95µL solution per well corresponding to the proper isolate + no glucose. Also inoculate the corresponding positive control wells (*do not inoculate wells for the media only controls in Row A*). + - **Note**: Do NOT push pipette plunger past the last stop because this will create bubbles, which will cause readings to be inaccurate. If bubbles form, try to pop with a sterile pipette tip. + +44. **Plate Preparation**: + - Place lid on the plate and parafilm for transport. Immediately measure the plate at 595, 660, and 750nm (cell density only) on plate reader. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/hmw-gdna-purification-and-ont-ultra-long-read-data-b55tq86n.md b/markdown-output/hmw-gdna-purification-and-ont-ultra-long-read-data-b55tq86n.md new file mode 100644 index 0000000000000000000000000000000000000000..a78ccc28f62cac37fe166e56a3860f945c88a0fa --- /dev/null +++ b/markdown-output/hmw-gdna-purification-and-ont-ultra-long-read-data-b55tq86n.md @@ -0,0 +1,152 @@ +``` +# Goal/Experiment: +The aim of this experiment is to purify high-molecular-weight genomic DNA (HMW gDNA) from mammalian cells and generate ultra-long reads (N50 >100 kbp) using Oxford Nanopore technology on the PromethION platform. This protocol enhances yields and read lengths from previous methods, providing about 30-50 Gb of data with an N50 ~150 kbp per PromethION flow cell. + +# HMW gDNA Purification and ONT Ultra-Long-Read Data Generation v.3 + +*Glennis Logsdon1* +University of Washington + +Mar 09, 2022 + +DOI: [dx.doi.org/10.17504/protocols.io.b5stq86n](https://dx.doi.org/10.17504/protocols.io.b5stq86n) + +## Reagents +- **Tris-EDTA, pH 8.0** (Ambion Catalog #AM9849) +- **RNase A** (Qiagen Catalog #19101) +- **Proteinase K** (Qiagen Catalog #19131) +- **UltraPure Phenol:Chloroform:Isoamyl Alcohol (25:24:1, v/v)** (Thermo Fisher Scientific Catalog #15593031) +- **Ammonium Acetate (5 M), RNase-free** (Invitrogen Catalog #AM9070G) +- **200 Proof Ethanol, pure** (Sigma Aldrich Catalog #E7023) +- **Hexamminecobalt(III) Chloride** (Alfa Aesar Catalog #A15470) + +### Kits +- **Ultra-Long DNA Sequencing Kit (SQK-ULK001)** (Oxford Nanopore Technologies Catalog #SQK-ULK001) + +### Disposables +- **DNA LoBind Tubes, 1.5 mL** (Eppendorf Catalog #0030108051) +- **DNA LoBind 2.0 mL PCR Clean Eppendorf Tubes** (Eppendorf Catalog #0030108078) +- **Maxtract high-density tubes (25 x 50 mL)** (Qiagen Catalog #129073) +- **Disposable Inoculating Loops and Needles, Flexible Loop, Volume: 10 µL, Yellow** (Thermo Fisher Catalog #22363600) +- **Glass Beads, 3 mm** (Scientific Laboratory Supplies Ltd Catalog #D0668501) +- **Monarch Bead Retainers** (New England Biolabs Catalog #T3004L) + *Alternative:* Cut an Eppendorf 1.5 mL tube 2-3 mm from the bottom to make a bead retainer. + +### Made-up Buffers +Note: All buffers should be filter-sterilized with a 0.22 µm filter prior to use + +#### Lysis Buffer +- 10 mM Tris-Cl (pH 8.0) +- 0.1 M EDTA (pH 8.0) +- 0.5% w/v SDS + +#### PEGW Buffer +- 10% PEG-8000 +- 0.5 M NaCl + +## Procedure + +### 1. Cell Collection and Lysis +1. Freeze down 2-7 x 10⁷ cells as a cell pellet, and store at -80°C. +2. When ready to purify the DNA, thaw the cell pellet on ice (usually takes ~30 mins). +3. While cells are thawing, add RNase A to the lysis buffer at a final concentration of 20 µg/mL. This must be done fresh each time. Keep the lysis buffer + RNase A solution at room temperature until ready to use. +4. Resuspend thawed cells in ice-cold TE (pH 8.0) at a concentration of 5 x 10⁷ cells/mL on ice. +5. Transfer the cell suspension to a 125-mL glass Erlenmeyer flask. + - Ensure cells are well-dispersed on the inner surface of the flask to minimize clump formation. +6. Quickly add 10 mL of lysis buffer + RNase A for each mL of cell suspension, drop-wise in a circular motion, and swirl to mix. +7. Incubate the cell suspension for 1 hr at 37°C. + +### 2. Proteinase K Treatment +8. Add proteinase K to a final concentration of 200 µg/mL, in a drop-wise manner. + - For 10 mL of cell suspension, add 100 µL Proteinase K. +9. Swirl the flask to mix the enzyme gently into the viscous cell lysate. +10. Incubate the lysate in a water bath for 2 hrs at 50°C, swirling twice per hour. +11. Cool the solution to room temperature (RT). + +### 3. Phenol-Chloroform Extraction +12. Add the viscous lysate to a 50-mL conical tube. + - Use a 10-mL serological pipette at low speed for ease. +13. Add an equal volume of ultra-pure phenol:chloroform:isoamyl alcohol (~10-11 mL) to the tube containing lysate. +14. Gently mix the two phases by slowly turning the tube end-over-end for 10 mins on a tube mixer. + *Alternative:* Use a roller apparatus for 1 hr if phases have not emulsified. +15. Pour the lysate into a Maxtract tube containing a high-density gel. +16. Spin in a centrifuge at 4000 rpm for 10 mins. +17. Pour the aqueous phase into a new 50-mL conical tube. +18. Repeat steps 13-17. + +### 4. Ethanol Precipitation +23. Add 0.4 volume of 5M ammonium acetate to the purified DNA, gently swirling for ~20 mins. + - Mix gently as longer mixing preserves DNA integrity. +24. Add 2 volumes of ethanol at RT and gently swirl to mix (~1 hr). + - Mix gently to get DNA into the solution with salt and ethanol. +25. Store the precipitating DNA solution overnight at 4°C. + - Overnight storage yields purer DNA for sequencing. +26. Remove the precipitate in one piece from the ethanol using a disposable inoculating needle shaped into a U and place into a 2-mL DNA LoBind tube. +27. Wash the DNA precipitate 2x with 1 mL 70% ethanol, centrifuging at max speed (~15,000 rpm) for 15 secs. +28. Remove ethanol traces; air dry briefly without over-drying. + - Desiccated DNA is hard to dissolve. +29. Add 750-1000 µL of EB + 0.02% Triton-X100 to rehydrate DNA, incubating at 4°C for 2 days untouched. + +### 5. ONT Library Preparation with SQK-ULK001 Kit (1 hr 15 min) +Note: Following the SQK-ULK001 protocol published by Oxford Nanopore Technologies. +30. Pre-warm heat block to 75°C. +31. Thaw Fragmentation Mix (FRA), FRA Dilution Buffer (FDB), and Rapid Adapter F (RAP F). Spin briefly and keep on ice. +32. Add 244 µL HMW gDNA and 6 µL FRA Buffer to a 1.5 mL Eppendorf DNA LoBind tube on ice. +33. Mix diluted FRA by vortexing. +34. Add 250 µL of diluted FRA to 750 µL of extracted DNA. Stir gently while adding to ensure even distribution. +35. Immediately mix reaction by pipetting 10x with a wide-bore tip. +36. Incubate as follows: + 1. RT for 5 mins + 2. 75°C for 5 mins + 3. RT for >10 mins +37. Add 5 µL of RAP F with a regular pipette tip. Use a P1000 tip by mixing. Inversion can be used. +38. Incubate for 30 mins at RT. + +### 6. Library Clean-Up with Glass Beads (1 hr) +Note: Following Matt Loose’s NEMO bead clean-up protocol. +40. Add 3 glass beads to 2-mL Eppendorf DNA LoBind tube. + - Sterilize by autoclaving/storing in 70% ethanol. +41. Add an equal volume of 10 mM CoHex to the DNA solution (1000 µL). +42. Rotate tube with vertical rotator at 9 rpm for 5-10 mins. + - If rotator not available, manually invert the tube slowly. +43. Invert the tube by hand 3x to ensure DNA binding. +44. Discard supernatant without disturbing DNA bound on beads. +45. Wash glass beads with 1 mL of PEGW buffer and invert 2-3x slowly. Incubate 3 mins at RT. +46. Discard most supernatant. +47. Wash glass beads with 500 µL of PEGW buffer, inverting 2-3x. Incubate 3 mins at RT. +48. Discard supernatant. +49. Pulse-spin tube and remove buffer traces under beads with pipette. +50. Quickly pour beads to a new 2-mL LoBind tube and add 225 µL EB Buffer. + - Do not let DNA dry. Pour buffer into a separate tube pre-stored. +51. Incubate library 30 min at 37°C. + - Leave overnight at RT. +52. Insert bead retainer into a 1.5 mL tube; pour beads into it, centrifuge at 12,000 x g for 1 min. +53. Incubate library at least 30 min at RT. +54. Store library at 4°C or proceed to load onto a flow cell. + +### 7. Flow Cell Priming and Loading +56. Let PromethION flow cell equilibrate to RT for ~10 mins. +57. Place the flow cell on PromethION, run platform QC. +58. Thaw SQB, PT, and FB at RT, mixing by vortexing and spin down. +59. Prepare library mix: + - 75 µL SQB + - 75 µL DNA library + +Total = 150 µL +60. Wait 30 mins and mix by wide-bore tip. +61. Prepare flow cell priming mix (1170 µL FB + 30 µL PT). +Total = 1200 µL +62. Load 500 µL of priming mix via flow cell port. Wait 5 mins. +63. Load 150 µL DNA library with P1000, followed by 500 µL priming mix via inlet port. +64. Immediately load remaining 150 µL. +65. Ensure no air bubbles or gaps between loads. +66. Let sit 10 mins, start sequencing. +67. Wash flow cell & reload library every 24 hrs for up to 3 loads per flow cell. + +## Citation +Glennis Logsdon, HMW gDNA Purification and ONT Ultra-Long-Read Data Generation. [DOI](https://dx.doi.org/10.17504/protocols.io.b5stq86n) + +``` + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/how-to-culture-human-bronchial-epithelial-cells-as-4u5gwy6.md b/markdown-output/how-to-culture-human-bronchial-epithelial-cells-as-4u5gwy6.md new file mode 100644 index 0000000000000000000000000000000000000000..c1c8b140cf11a1966c5708d319edcb5440b95cc3 --- /dev/null +++ b/markdown-output/how-to-culture-human-bronchial-epithelial-cells-as-4u5gwy6.md @@ -0,0 +1,88 @@ +```markdown +# Goal/Experiment: +To culture and differentiate Human Bronchial Epithelial Cells (HBECs) as spherical structures known as bronchospheres or airway organoids. + +## How to Culture Human Bronchial Epithelial Cells as Airway Organoids + +### Abstract +Human bronchial epithelial cells (HBECs) are typically cultured at the air-liquid interface (ALI) to functionally recapitulate the human airway. ALI cultures are formed by plating primary HBECs onto porous cell culture inserts and allowing the cells to achieve confluence prior to removing the culture medium from the apical surface of the cells. However, the requirement of porous culture inserts can limit the application of ALI culture to smaller scale experiments, thus largely precluding high-throughput drug screening of differentiated epithelial cultures. Alternatively, HBECs may be cultured and differentiated as spherical aggregates, providing a means for high-throughput study of the differentiated human airway. This protocol describes a method for the culture of differentiated HBECs as spherical structures known as bronchospheres or airway organoids. + +### External Link +[https://www.stemcell.com/sphere-culture-method-mucociliary-differentiation-primary-human-bronchial-epithelial-cells-lp.html?utm_source=protocolsio&utm_medium=referral](https://www.stemcell.com/sphere-culture-method-mucociliary-differentiation-primary-human-bronchial-epithelial-cells-lp.html?utm_source=protocolsio&utm_medium=referral) + +### Materials + +| Name | Catalog # | Vendor | +|-----------------------------|-----------|-----------------------| +| Trypan Blue | 07050 | Stemcell Technologies | +| PneumaCult™-ALI Medium | 05001 | Stemcell Technologies | +| Hydrocortisone Stock Solution | 07925 | Stemcell Technologies | +| Heparin Solution | 07980 | Stemcell Technologies | +| Trypsin-EDTA (0.05%) | 07910 | Stemcell Technologies | +| Corning® Matrigel® | 354277 | Corning | +| Soybean Trypsin Inhibitor | T6522 | Millipore Sigma | + +### Before Starting +The instructions below describe an optimized procedure for use with 24-well tissue culture plates. If using alternative cultureware, adjust volumes accordingly. Human bronchial epithelial cells (HBECs) can be cultured in a serum-free and BPE-free expansion medium (e.g., PneumaCult™-Ex) in T-25 flasks according to the instructions on the Product Information Sheet (PIS). The following procedure should be initiated with HBECs (P1-P4) that are approximately 70-90% confluent in PneumaCult™-Ex. + +### Preparation of Reagents and Materials +1. Prepare PneumaCult™-ALI Complete Base Medium by adding 50 mL PneumaCult™-ALI 10X Supplement to 450 mL PneumaCult™-ALI Basal Medium. This Complete Base Medium can be aliquoted and stored at -20°C for up to 6 months. Avoid additional freeze/thaw cycles. + - **Note**: The PneumaCult™-ALI Medium kit contains: + - PneumaCult™-ALI Basal Medium + - PneumaCult™-ALI 10X Supplement + - PneumaCult™-ALI Maintenance Supplement + +2. Prepare PneumaCult™-ALI Maintenance Medium by adding the following components per 1 mL PneumaCult™-ALI Complete Base Medium: + - 10 μL PneumaCult™-ALI Maintenance Supplement + - 2 μL Heparin Solution + - 5 μL Hydrocortisone Stock Solution + - **Note**: Prepare only enough PneumaCult™-ALI Maintenance Medium needed for immediate use. + +3. Prepare a sufficient volume of a 40% Matrigel® solution by promptly mixing the following components (will require 500 μL/ well of a 24-well plate): + - 300 μL cold PneumaCult™-ALI Maintenance Medium + - 200 μL cold Matrigel® + +4. Aliquot 500 μL 40% Matrigel® solution per well of a 24-well plate. + +5. Incubate the plate at 37°C in a humidified incubator at 5% CO₂ for 30 minutes to allow for the Matrigel® layer to solidify. Steps 6 through 12 can be completed during this 30 minute incubation. + +### Preparation of a Single Cell Suspension +6. Prepare a sufficient volume of a 5% Matrigel® solution by mixing the following components (require 500 μL/well of a 24-well plate): + - 475 μL cold PneumaCult™-ALI Maintenance Medium + - 25 μL cold Matrigel® + +7. Warm 5% Matrigel® solution, Ca²⁺/Mg²⁺-free PBS, 0.025% Trypsin-EDTA (1/2 dilution of 0.05% Trypsin-EDTA) and 1 mg/mL Soybean Trypsin Inhibitor to 37°C. + +8. Wash each T-25 flask of HBECs twice with 2 mL warm Ca²⁺/Mg²⁺-free PBS. Aspirate the Ca²⁺/Mg²⁺-free PBS and add 2 mL warm 0.025% Trypsin-EDTA to each flask. + +9. Incubate flask at 37°C for 3 - 5 minutes, until the cells can be dislodged with gentle tapping of the flask. Neutralize the Trypsin-EDTA by adding an equivalent volume of 1 mg/mL warm Soybean Trypsin Inhibitor to each T-25 flask. + +10. Collect the cell suspension into a 15 mL conical tube and centrifuge at 350 x g for 5 minutes. + +11. Remove the supernatant and resuspend the cell pellet in 1 - 2 mL warm 5% Matrigel® solution. + +12. Perform a viable cell count using Trypan Blue and a hemocytometer. + +### Airway Organoid Culture +13. Prepare cells at a concentration of 180,000 cells/mL in 5% Matrigel® solution. Plate 500 μL cell solution per well of a 24-well plate (90,000 cells/well or approximately 50,000 cells/cm²). + - **Note**: This seeding density has been optimized for use with P4 cells. If using lower passage number cells, the optimal seeding density may be lower. + +14. Incubate at 37°C, 5% CO₂ in a humidified incubator. Medium changes should be performed three times per week (e.g., Monday/Wednesday/Friday) by gently aspirating the medium above the semi-solid Matrigel® layer and replacing with 500 μL fresh warm 5% Matrigel® solution. + +### Figures +#### Figure 1 +![Figure 1](https://path/to/figure1/image) +Morphology of bronchospheres generated in PneumaCult™-ALI Medium. + +#### Figure 2 +![Figure 2](https://path/to/figure2/image) +Histological staining of bronchospheres reveal the presence of goblet cells and ciliated cells. +- **Top Panels:** H&E staining of representative bronchospheres. Black arrows indicate beating cilia pointing towards the lumen. +- **Bottom Panels:** PAS staining of representative bronchospheres. Red arrows indicate goblet cells. + +### Video +[Visualization of Bronchospheres](https://path/to/video) +Light microscope visualization showing a bronchosphere cultured in PneumaCult™-ALI. This video shows mucus in the lumen being swirled around by the beating of synchronized cilia lining the lumen of the bronchosphere. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/human-brain-vascular-pericytes-hbvp-culture-and-pl-brxjm7kn.md b/markdown-output/human-brain-vascular-pericytes-hbvp-culture-and-pl-brxjm7kn.md new file mode 100644 index 0000000000000000000000000000000000000000..f4d7a4c8c34644bc0e3759cf2637644bd517fc58 --- /dev/null +++ b/markdown-output/human-brain-vascular-pericytes-hbvp-culture-and-pl-brxjm7kn.md @@ -0,0 +1,78 @@ +```markdown +Goal/Experiment: +To culture Human Brain Vascular Pericytes (HBVP) suitable for several research techniques including immunohistochemistry, flow cytometry, and protein or RNA analysis. + +# Human Brain Vascular Pericytes (HBVP) Culture and Plating V.3 + +## Abstract +This protocol implements the culture of Human Brain Vascular Pericytes (HBVP) suitable for several research techniques including immunohistochemistry, flow cytometry, and protein or RNA analysis. + +## Keywords +Pericytes, Human Brain Pericytes, Cell Culture, Neuroscience + +## Guidelines +Please read the whole protocol before starting the procedure. + +## Materials Text + +### Reagents +- **Human Brain Vascular Pericytes (HBVP)**: ScienCell (Cat#1200) +- **Cell Culture Flask**: Corning® 75cm² U-Shaped Canted Neck Cell Culture Flask with Vent Cap (Cat# 430641U) +- **Pericyte Complete Medium (PCM)** for 50 mL: + - **Dulbecco's Modified Eagle Medium (DMEM)**: Wisent + - **Fetal Bovine Serum (FBS)**: Wisent + - **Penicillin-Streptomycin (10000 U/mL)**: Gibco - Thermo + - **Pericyte Growth Supplement**: ScienCell (Cat#1252) +- **Trypsin-EDTA Solution**: Sigma (Cat#T4049) +- **Dulbecco's Phosphate-Buffered Saline (DPBS)**: Gibco - Thermo, Fischer (Cat#14190144) +- **Matrigel**: Corning (Cat#356231) +- **Syringe Filter**: Filterpou S, PES, pore size: 0.2 μm, for sterile filtration (Sarstedt, Cat# 83.1826.001) +- **Pre-treated German Glass Coverslips**: Emsdiasum (Cat#72291-03) + +### Safety Warnings +- All reagents and instruments employed in this protocol must be sterile. +- Cell manipulation must be performed in a sterile/decontaminated cell culture hood. + +## Protocol + +### Before Starting +- Ensure all reagents are pre-warmed to 37°C before contact with cells. +- Exposure of cultured pericytes to cold reagents results in cell stress and death. + +### Cell Culture (30 minutes) +1. Prepare and warm 50 mL of Pericyte Complete Medium (PCM) at 37°C for 30 minutes. +2. Retrieve HBVP from -130°C and place them at 37°C for 2 minutes. Minimize exposure time to 37°C to just liquefy aliquots. +3. Place 1 mL of HBVP (~500,000 cells) in a 75cm² Cell Culture Flask filled with 9 mL of PCM. +4. Perform gently crosswise movements to spread the cells over the container’s surface. +5. Incubate the cells at 37°C until reaching a confluence of 70-80%. + +### Glass Coverslip Preparation +6. If immunohistochemistry is required: + - Coat Glass Coverslips with 100 μL or 200 μL of Poly-D-Lysine solution (1 mg/mL) and incubate at 37°C for 1 hour. + - Wash with sterile DPBS then coverslips are ready for cell plating. + +7. Slowly thaw aliquoted Matrigel at -20°C. +8. Dilute 1:30 the Matrigel in ice-cooled DPBS and filter the solution using a sterile syringe and 0.2 μm filter. +9. Place cold Glass Coverslips (stored at -20°C) in each well and add 1 mL 70% ethanol for 5 minutes. +10. Add 200 μL (12 wells plate) or 400 μL (6 wells plate) of filtered Matrigel, incubating at 37°C for 30 minutes. +11. Wash wheels three times with PCM and subsequently aspirate the media. + +### Cell Detachment (35 minutes) +12. Warm up PCM, DPBS, and Trypsin-EDTA solution at 37°C for 30 minutes. +13. When cells have reached 70-80% confluence, wash 1 time with DPBS. +14. Add 3 mL of Trypsin-EDTA solution and incubate at 37°C for 5 minutes for detachment. +15. Add 9 mL of PCM to stop the reaction and pipette to ensure complete cell detachment. +16. Suction the whole culture medium and place into a 50 mL sterile Falcon tube. +17. Centrifuge at 1000 rpm, for 5 minutes at room temperature. +18. Carefully remove the supernatant and resuspend the cell pellet in PCM. +19. Estimated cells at this confluence in a 75cm² flask is ≈ 6-7 million cells. + +### Cell Plating +20. Plate or freeze cells: + - To freeze, resuspend cells in PCM without FBS, add 20% v/v Dimethyl Sulfoxide (DMSO), and freeze at -130°C. +21. Add 750 mL of PCM to each well containing the Matrigel Coated Glass Coverslips. +22. Add 250 mL of PCM-suspended pericytes. +23. Incubate at 37°C until the desired confluence is achieved. Confluence in 6 or 12 wheels plates can be achieved in 1-3 days. + +## Endofoutput +``` \ No newline at end of file diff --git a/markdown-output/human-ovarian-tissue-procurement-and-processing-fo-c4uwywxe.md b/markdown-output/human-ovarian-tissue-procurement-and-processing-fo-c4uwywxe.md new file mode 100644 index 0000000000000000000000000000000000000000..ac6c259d33546ddf487e36a612ec8a41263d37ae --- /dev/null +++ b/markdown-output/human-ovarian-tissue-procurement-and-processing-fo-c4uwywxe.md @@ -0,0 +1,126 @@ +```markdown +# Goal/Experiment: +The goal of this experiment is to profile senescent cells in human ovarian tissue in vivo and ex vivo using the LATTICE microphysiological platform. This includes generating a molecular signature of Doxorubicin-induced cellular senescence in both static and fluidic culture conditions, which will be mapped to human ovaries across an aging series. + +# Human Ovarian Tissue Procurement and Processing for Ovarian Explant Cultures (Static and Fluidic Conditions) + +**Authors:** +- hannah.anvari¹ +- pooja.devrukhkar¹ +- hannes.campo¹ +- francesca.e.duncan duncan¹ + +¹Northwestern Medicine OBGYN, Northwestern University + +--- + +## Abstract +The Cellular Senescence Network (SenNet) was established to map senescent cells in the human body. Our goal is to profile senescent cells in the human ovary in vivo and ex vivo using a microphysiological platform called LATTICE. Human ovarian tissue collected from women undergoing bilateral salpingectomy or hysterectomies without ovarian neoplasia is cultured in Doxorubicin (0.1 µg/ml) for 24 hours and up to 11 days in either static or fluidic culture conditions. + +--- + +## Guidelines + +### Guidelines for Researchers Working with Human Specimens +Researchers must adhere to all Northwestern Medicine/Northwestern University safety and training protocols including but not limited to: + +1. Biosafety Certification +2. Bloodborne Pathogens Certification +3. Working with Formaldehyde Certification +4. Collaborative Institutional Training Initiative (CITI program) certification + +--- + +## Materials + +1. ORIGIO Handling IVF Medium (ORIGIO, #83100060) +2. Disposable Scalpel, Sterile, No. 10 and No. 22 (Fisher Scientific, 12-460-456) +3. 15 mL conical tubes (Fisher Scientific, 2610L35) +4. Gibco™ DPBS, calcium, magnesium (Fisher Scientific, 14-190-250) +5. 60 mm Dish, Non-Treated, Corning™ Falcon™ Bacteriological Petri Dishes with Lid (Fisher Scientific 08-772-12) +6. Stadie-Riggs tissue slicer (discontinued) +7. Stadie-Riggs tissue slicer/microtome blades (Thomas Scientific 6727C18) +8. Stadie-Riggs tissue slicer blade handle (Thomas Scientific 6727C25) +9. Plastic Discs (Fisher Scientific, 1018001) +10. MEM Alpha (1X) + GlutaMAX (Thermo, 32561037) +11. Human serum albumin (Cooper Surgical Inc, ART-3003) +12. Fetuin (Sigma, F3385-25G) +13. Insulin-Transferrin-Sodium Selenite Supplement (Sigma-Aldrich, I1884-1VL) +14. Doxorubicin (Fisher Scientific, 22-521-0) +15. Millicell Cell Culture Insert, 12 mm, hydrophilic PTFE, 0.4 µm (Millipore Sigma, PICM01250) +16. Falcon® 24-Well Flat-Bottom Plate, Tissue Culture-Treated (Fisher Scientific, 353047) +17. Invitrogen™ RNase-free Microfuge Tubes (ThermoFisher Scientific, AM12400) + +--- + +## Safety Warnings +Researchers will wear personal protective equipment (PPE) including gloves, mask, eye protection, and lab coat when working with human specimens. + +--- + +## Ethics Statement +Human ovarian tissue procurement and processing for ovarian explant cultures adhere to the approved IRB protocol NU12G09 for the collection of human ovarian tissue through Northwestern Medicine. + +--- + +## Processing Human Ovarian Tissue for Ovarian Explant Cultures + +1. **Transportation and Preparation** + - Ovarian tissue collected through the IRB-approved Reproductive Tissue Library at Northwestern Medicine. + - Cross-sections 3-5 mm in depth are processed within 24 hours of surgery. + - Transport in biohazard labeled cooler bag (A) and on ice (2-4°C) (B). + - Transfer tissue to ORIGIO Handling medium (C). + +2. **Measurement and Cutting** + - Measure cross-sections for length, width, and thickness (D). + - Mark approximate anatomy of the cortex and medulla (E). + - Cut along the cortex-medulla border, leaving 2-3 mm between the edge and cut surface (F). + - Slice using Stadie-Riggs tissue slicer, yielding 0.5 mm thick slices (G, H). + +3. **Manual Sectioning** + - Manually section slices into 1 mm × 1 mm squares (I) in ORIGIO Handling Medium (J). + - Submerge in ORIGIO Handling medium to prevent drying before loading into cell culture inserts. + +--- + +## Ovarian Explant Cultures + +### Media Preparation +1. Equilibrate growth media for 4-6 hours before receiving tissue (MEM Alpha, GlutaMAX, recombinant FSH, fetuin, Insulin-Transferrin-Sodium Selenite supplement, human serum albumin). +2. Load tissue into a 24-well plate (400 µL growth media per well, fill remaining wells with PBS). + +### LATTICE Plate Setup +1. Load donor wells with 1.5 mL media, tissue wells with 400 µL media, acceptor wells empty. +2. Allow media exchange of ~40 µL every hour between wells. + +3. **Tissue Placement** + - Transfer 1 mm × 1 mm pieces to new 60 mm dish with ORIGIO Handling medium. + - Section tip of 1000 µL pipette for placing tissue into transwells. + - Load tissues into culture transwells using pipette technique (A). + +4. **Culture Conditions** + - Culture at 37°C with 5% CO2 for 1-11 days. + +5. **Senescence Induction** + - Add Doxorubicin to the media at 0.01 µg/ml concentration for the first 24 hours. + +6. **Post-Exposure Procedures** + - Wash tissues twice with standard growth media. + - Perform media changes for static and LATTICE plates: + - Static: Half-volume changes every other day (200 µL). + - LATTICE: Daily media changes and flow rate checks (1.5 mL) + +7. **End-of-Culture Handling** + - Remove tissues from inserts, carefully peel away mesh. + - Fix tissues or prepare for freezing. + +--- + +Fig. 1: Ovarian tissue preparation stages. +Fig. 2: LATTICE plate media flow setup. +Fig. 3: Ovarian explant culture setup. + +--- + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/human-ra-gastruloid-induction-from-pluripotent-ste-dfta3nie.md b/markdown-output/human-ra-gastruloid-induction-from-pluripotent-ste-dfta3nie.md new file mode 100644 index 0000000000000000000000000000000000000000..36c857c26117a24802f0818dcb8a494fc2a6ae79 --- /dev/null +++ b/markdown-output/human-ra-gastruloid-induction-from-pluripotent-ste-dfta3nie.md @@ -0,0 +1,111 @@ +```markdown +Goal/Experiment: +To induce the formation of human RA-gastruloids from pluripotent stem cells using a combination of retinoic acid and other reagents. Gastruloids serve as a powerful in vitro model for studying early human development. + +# Human RA-gastruloid Induction from Pluripotent Stem Cells + +**DOI**: [dx.doi.org/10.17504/protocols.io.261ge5epog47/v1](https://dx.doi.org/10.17504/protocols.io.261ge5epog47/v1) + +## Author +**Nobuhiko Hamazaki** +University of Washington + +## Protocol Citation +Nobuhiko Hamazaki 2024. Human RA-gastruloid induction from pluripotent stem cells. protocols.io [https://dx.doi.org/10.17504/protocols.io.261ge5epog47/v1](https://dx.doi.org/10.17504/protocols.io.261ge5epog47/v1) + +## License +This is an open-access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. + +## Protocol Status +Working + +## Keywords +Embryo model, Stem cell, Gastruloid + +## Disclaimer +**DISCLAIMER – FOR INFORMATIONAL PURPOSES ONLY; USE AT YOUR OWN RISK** + +The protocol content here is for informational purposes only and does not constitute legal, medical, clinical, or safety advice, or otherwise; content added to protocols.io is not peer reviewed and may not have undergone a formal approval of any kind. Information presented in this protocol should not substitute for independent professional judgment, advice, diagnosis, or treatment. Any action you take or refrain from taking using or relying upon the information presented here is strictly at your own risk. + +## Abstract +Gastruloids are a powerful in vitro model of early human development. They are composed of all three germ layers but do not morphologically resemble post-implantation human embryos. This protocol utilizes an early pulse of retinoic acid (RA) along with Matrigel to induce human gastruloids. This induction process robustly forms human gastruloids with posterior embryo-like morphological structures. Silico staging using single-cell RNA-seq reveals human RA-gastruloids are more advanced than other models, comparable to E9.5 mouse and CS11 cynomolgus monkey embryos. This protocol leverages chemical and genetic perturbations to affirm WNT and BMP signaling in somite formation and neural tube length regulation, making RA-gastruloids a scalable model for early human embryogenesis studies. + +## Materials + +- **StemPro Accutase** (Thermo, A1110501) +- **Wash Media** + - DMEM/F-12 (Thermo, 11320033) + 0.1% BSA (Thermo, 15260037) +- **Reagents** + - 10 μM Y-27632 (Selleck, S1049) + - Nutristem hPSC XF medium (Biological Industries, 05-100-1A) + - 30mM CHIR99021 (CHIR, Millipore, SML1046) + - 100mM Retinoic Acid (RA) stock (Millipore Sigma, R2625) + - Essential 6 medium (Thermo, A1516401) + - Matrigel (Corning, 354230) + - Non-adhesive 96-well plate (e.g., Nunclon™ Sphera™ 96-Well, Nunclon Sphera-Treated, U-Shaped-Bottom Microplate) + - Vitronectin (Gibco, A14700) + +## Notes +- Store aliquots in -20°C and test before use. +- For Matrigel: Make 10 μL aliquots and store them in -20°C +- For RA stock: Make 200 μL aliquots, store them in -20°C, and dilute x100 in PBS before use. + +## Day 0: Passage Human PSCs Onto Vitronectin-Coated NutriStem + +1. Coat wells with Vitronectin and incubate them at 37°C for at least 15 minutes. +2. Dissociate pre-treated ESCs. +3. Aspirate media from wells, wash wells with PBS(-), and aspirate PBS (-). +4. Add 500 μL Accutase to each well and incubate them at 37°C for 4 minutes. +5. Quench the reaction by adding 2 mL of wash media containing 10 μM Y-27632 and pipette up and down ~10 times to dissociate cells. +6. Transfer to a new 15 mL tube and centrifuge for 3 minutes at 300 g. +7. Suspend in NutriStem containing 10 μM Y-27632 and spread 2 x 10⁴ cells per 1/12 well onto each vitronectin-coated well. + +## Day 1-3: Pre-treatment of ESCs (CHIR Stimulation) + +**Day 1** + +8. Change the medium to fresh NutriStem containing 5 μM Y-27632. + +**Day 2** + +9. Change the medium to fresh NutriStem containing 4 μM CHIR. + +**Day 3** + +10. Change the medium to fresh NutriStem containing 4 μM CHIR + 500 nM RA. + +## Day 4-9: Human RA-gastruloid Induction + +**Day 4** + +11. Prepare the following reagents: + - Accutase: 500 μL for 1/12 well. + - Wash media + 10 μM Y-27632: 2 mL for 1/12 well. + - Essential 6 medium + 1 μM CHIR + 5 μM Y-27632: 50 μL per well + 10%. + +12. Aspirate media from wells and wash with PBS(-). Aspirate PBS (-). +13. Add 500 μL Accutase to wells and incubate at 37°C for 4 minutes. +14. Quench the reaction by adding 2 mL Wash+Y media. Pipette up and down ~10 times to dissociate cells. +15. Transfer to a new 15 mL tube and centrifuge for 3 minutes at 300 g. +16. Wash again with 2 mL Essential6 + 1 μM CHIR + Y and centrifuge again. +17. Resuspend cells in 1-2 mL Essential6 + Y medium and count cells with Countess. +18. Transfer the necessary number of cells (4000 cells/well) into a reservoir containing a medium (50 μL/well). +19. Spread cells with a multi-channel pipette. +20. Incubate them in a 37°C incubator. + +**Day 5 (+24h from RA-gastruloid induction)** + +21. Add 150 μL Essential 6 medium to each well. + +**Day 6 (+48h from RA-gastruloid induction)** + +22. Carefully remove 150 μL medium and add 150 μL of fresh Essential 6, containing 100 nM RA and 5% Matrigel. + +**Day 9 (+120h from RA-gastruloid induction)** + +23. Fully elongated RA-gastruloids with neural tube and segmented somites should be observed. + +--- + +**endofoutput** +``` diff --git a/markdown-output/human-thymus-single-cell-dissociation-protocol-tei-bx8sprwe.md b/markdown-output/human-thymus-single-cell-dissociation-protocol-tei-bx8sprwe.md new file mode 100644 index 0000000000000000000000000000000000000000..c1e12774d72a6159d426963b70f4666ec7e0cc24 --- /dev/null +++ b/markdown-output/human-thymus-single-cell-dissociation-protocol-tei-bx8sprwe.md @@ -0,0 +1,227 @@ +```markdown + +## Goal/Experiment: + +The goal of this experiment is to dissociate human thymus tissue into single cells for subsequent analyses, such as flow cytometry or single-cell RNA sequencing. The procedure includes tissue preservation, enzymatic dissociation, and cell sorting techniques like FACS and MACS for enrichment. + +# Human Thymus Single Cell Dissociation Protocol - Teichmann Lab + +**Authors**: Jonggeun Park, Veronika Kedlian, Chenqu Suo, Liam Bolt, Alexander Steemers, Nadav Yayon, Sarah Teichmann + +**Affiliations**: +1. Wellcome Sanger Institute, Wellcome Genome Campus, Hinxton, Cambridge, UK +2. European Molecular Biology Laboratory, European Bioinformatics Institute, EMBL-EBI, Wellcome Trust Genome Campus, Hinxton, UK +3. Cavendish Laboratory, University of Cambridge, Cambridge, UK + +**Contributions**: Jonggeun Park, Veronika Kedlian, and Chenqu Suo contributed equally. + +**DOI**: [dx.doi.org/10.17504/protocols.io.bx8sprwe](https://dx.doi.org/10.17504/protocols.io.bx8sprwe) + +**Note**: This protocol is shared as open-science with no guarantee it will work, although we strongly believe it will. + +**Funding**: Chan Zuckerberg Initiative, Grant ID: CZIF2019-002445 + +--- + +## Materials and Reagents + +### Tick List Before Starting +1. RPMI +2. PBS +3. EDTA +4. DMSO +5. FBS +6. RNAlater +7. Cryovials +8. Petri dishes +9. Scalpels and handles +10. Sharp forceps +11. Cell strainer (70µm) +12. RBC lysis buffer, eBioscience™ (00-4333-57) + +### Enzymes +1. **Liberase TH stock (2.5 mg/mL)** - Roche, 05401135001 + - *Function*: Breaks down extracellular matrix for cell dissociation. +2. **Trypsin – EDTA (0.25%)** - Gibco, 25200056 + - *Function*: Digesting proteins binding cells together. +3. **DNaseI (10000X)** - Roche, 4716728001 + - *Function*: Degrades DNA released from damaged cells to reduce viscosity. +4. **Collagenase type IV (stock 50mg/ml current stock might be different)** - Gibco, 17104019 + - *Function*: Breaks down collagen in tissues. + +### Other Components +1. Cell counting: C-Chips and Trypan Blue +2. 5’v2 10x GEM Kit (+Beads) +3. Sony Sorting Chip - (130 µm nozzle size) + +### For Magnetic Cell Sorting +1. **MidiMACS Separator** (Miltenyi Biotec) +2. **LS columns [MACS]** (Miltenyi Biotec) [# 130-042-401](https://www.miltenyibiotec.com/GB-en/products/macs-cell-separation/columns/ls-columns.html) +3. **CD45 MicroBeads** (human 130-045-801) 2mL total (10 µL per 10⁷ cells) +4. **CD326 (EpCAM) MicroBeads** (human 130-061-101) + +### Same Day Buffers - Make Fresh + +#### DM1 - Digestion Media I +- **9.35 mL RMPI** +- **640 µL Type IV collagenase stock** (stock 50mg/ml from freezer -20ºC, final conc. 3.2mg/ml) +- **10 µL DNase I stock aliquot** (use at 1:1000 dilution from -20ºC) +- Prewarm in water bath + +#### DM2 - Digestion Media II +- **9.79 mL RMPI** +- **200 µL Liberase TH** (2.5mg/ml stock, 50X, final 50 µg/ml) +- **10 µL DNase I stock aliquot** +- Prewarm in water bath + +#### Stop Solution +- **2% FBS in RPMI** (40 mL RPMI + 800 µL FBS), Keep on ice + +#### FACS Buffer +- **49.55 mL PBS** +- **250 µL 100% FBS** +- **200 µL 0.5 M EDTA** (final 0.5% FBS + 2mM EDTA), Keep on ice + +--- + +## Working Protocol + +1. **Sample Arrivals**: + - Measure weight and size. + - Take note of time of arrival, patient number, and storage type. + +2. **Histology (Optional)**: + - Divide sample two ways: ½ for histology, ½ for dissociation. + - 1/3 for snap freezing + - 1/3 for OCT mounting + - 1/3 for FFPE + +3. **Tissue Dissociation**: + - Divide sample 4 ways: + - **1/4** for 10% DMSO 90% FBS freezing (small chunks of 3-4 mm). + - Keep small part for RNAlater – place 1 mL of RNAlater in cryovial, add sample, top up with more RNAlater, store in -80ºC freezer. + - **1/4** for collagenase type IV digestion (DM1). + - **2/4** for liberase TH digestion (DM2). + +4. **Preparation and Processing**: + 1. Prepare hood (wipe with 70% EtOH); bring sterile petri dishes and scalpels and digestion solution. + 2. Place tissue in petri dish (in hood), remove any attached fat. + 3. Mince the tissue and put it into two 50 mL Falcon tubes filled with DM1 or DM2 (based on tissue size 2-3 cm²). + - Add the appropriate volume of digestion media (if tissue is big, add 40mL). + - Do not use pipet tips (sticky tissue), use a scalpel. + - Wash scalpel with digestion solution to move remaining tissue. + 4. Incubate at 37°C on the rotator (middle speed). Check tissue every 10 minutes. + - Do not exceed 1 hour. + - If under-digested, top up with double concentration of liberase & DNaseI. + 5. Filter through strainer, settling undigested tissue at the bottom. Add 10mL stop solution. + - Label the cells "with DM1" or "with DM2". + - Cells digested with DM1 will be banked (in 90% FBS/10% DMSO) **AFTER SORTING**. + - Cells digested with DM2 will be FACS sorted for EpCAM+ cells (i.e., thymic epithelial cells). + +5. **Processing Digest**: + - Combine undigested tissue from DM1 and DM2. + - Add 15 mL Trypsin – EDTA (0.25%) pre-warmed at 37ºC for further digestion (limit to 15 min). + - Add DNase I (1000X concentration). + +6. **Incubation**: + - Incubate at 37ºC on the rotator (middle speed), checking every 10 minutes. + +7. **Centrifugation**: + - Spin down tubes from digestion I and II at 500g for 5 min at 4ºC to pellet cells. + +8. **RBC Lysis Buffer**: + - Leave at room temperature. + +9. **Resuspension**: + - If cells appear red, resuspend pellet in 10mL 1x RBC lysis buffer. Incubate for 3-5min at RT. + - Top up with PBS to 40 mL. Strain through strainer. + - Centrifuge at 500g for 5 min. + +10. **Final Resuspension**: + - Resuspend in 5 mL stop solution. Count cells. + - Cells digested with trypsin will be FACS sorted for CD45- (stromal cells). + +--- + +## MACS Enrichment + +### Prepare Solutions & Equipment: +1. **MACS Buffer**: PBS + 0.5% BSA + 2 mM EDTA, keep cold and degas. +2. **LS MACS columns and separators** +3. **CD45 MicroBeads**, human (130-045-801) 2mL total (10 µL per 10⁷ cells) +4. **CD326 (EpCAM) MicroBeads**, human (130-061-101) + +1. **Cell Number Determination** + - Use cells up to 10⁷ with 20 µL of MicroBeads. + - Use cells up to 10⁸ with 500 µL of buffer. + +2. **Separation and Centrifugation** + - Resuspend pellet in 80 µL buffer per 10⁷ cells. + - Add 20 µL of CD45/EPCAM MicroBeads per 10⁷ cells. Mix well and incubate for 15 mins (CD45) and 30 mins (EPCAM) at 4ºC in the refrigerator. + - Wash cells by adding 1-2 mL buffer per 10⁷ cells and centrifuge at 300g for 10 mins. + - Discard supernatant. + - Resuspend up to 10⁸ cells in 500 µL buffer. + +3. **Enrichment and Depletion** + - Prepare two LS columns. + - Rinse with 3 mL degassed buffer, discard effluent. + - Apply CD45 and EPCAM stained cell suspensions onto respective columns. + - **CD45-** fraction: Collect cells that pass through. + - Wash columns with 3 mL buffer three times (CD45) or four times (EPCAM). + - Collect washing buffer (**CD45-** fraction). + +4. **Elution** + - Place column on collection tube and add 5 mL MACS buffer. + - Immediately flush out fraction with magnetically labelled cells. + - **EPCAM+** fraction. + +5. **Spin Down** + - Resuspend in small volume and count. + +--- + +## FACS Staining and Sorting + +Spin down cells from digestion II and trypsin, resuspend in FACS buffer: +- **EpCAM+ need 50M (500µL)** +- **CD45- take all cells from trypsin digest** + +### FACS Staining +1. Vortex bottle of Ultra Comp Beads beforehand. +2. Add FACS buffer and cells. +3. Add 5 µL TruStain. Mix and incubate at 4ºC for 10 mins. +4. Add mix of antibodies (2 µL each), incubate 30 mins. + - Dilute DAPI stock solution 1:100, add 2 µL DAPI per 100 µL sample. + - Incubate for 15 mins. +5. Add 1 mL FACS buffer, centrifuge at 300g for 3 mins. +6. Resuspend in 1 mL FACS buffer. +7. Use 15 mL tubes with 1 mL 2% FBS in PBS buffer for collection. + +### FACS Buffer Table + +| FACS buffer | CD45 BV785 | EPCAM PE | Cells | TruStain | DAPI | +|-------------|------------|----------|-------|----------|------| +| 39 µL | 2 µL | 2 µL | 50 µL | 5 µL | 2 µL | + +### Aim +- **40,000 EpCAM+ cells** +- **40,000 CD45- cells** + +--- + +## Appendix + +### Reconstitution for Liberase TH +Reconstitute the lyophilized enzyme with injection-quality sterile water or tissue dissociation buffer. Avoid serum and inhibitors. Place on ice and agitate to dissolve. + +- **2 mL (5 mg with 2.5 mg/mL)** +- **10 mL (50 mg with 5 x 400 µL aliquots)** + +| Weight | Stock concentration | Vol. water | Aliquots | +|--------|---------------------|------------|------------| +| 5mg | 2.5mg/ml | 2ml | 5 x 400µl | + +--- + +endofoutput +``` diff --git a/markdown-output/human-tissue-nuclei-isolation-protocol-2021-10-18-bzmgp43w.md b/markdown-output/human-tissue-nuclei-isolation-protocol-2021-10-18-bzmgp43w.md new file mode 100644 index 0000000000000000000000000000000000000000..7f24234e42e0cdaaab075effc8b7ddf6490340fe --- /dev/null +++ b/markdown-output/human-tissue-nuclei-isolation-protocol-2021-10-18-bzmgp43w.md @@ -0,0 +1,132 @@ +```markdown +# Goal/Experiment: +Homebrew protocol to isolate nuclei from human frozen brain tissue. + +# Human Tissue Nuclei Isolation Protocol + +## Authors +Jose.Bras¹, lee.marshall¹, Kimberly Paquette¹ +¹Van Andel Institute, Grand Rapids, MI, USA + +DOI: [dx.doi.org/10.17504/protocols.io.bzmgp43w](https://dx.doi.org/10.17504/protocols.io.bzmgp43w) +**Citation**: Jose.Bras, lee.marshall, Kimberly Paquette. 2021. Human_Tissue_Nuclei_Isolation_Protocol_2021_10_18. protocols.io. [https://dx.doi.org/10.17504/protocols.io.bzmgp43w](https://dx.doi.org/10.17504/protocols.io.bzmgp43w) + +## Date +Oct 29, 2021 + +## Stock Solutions + +### 10% Triton X-100 (100mL) +| Solution | Final Conc | Volume | Notes | +|---------------|------------|--------|------------------| +| Triton X-100 | 10% | 10mL | | +| Ultrapure Water | Fill to: | 100mL | | + +### 1M MgCl₂ (100mL) +| Solution | Final Conc | Volume | Notes | +|----------------------|------------|---------|--------------------------------------------------| +| MgCl₂ (FW 203.31) | 1M | 20.331g | 203.31g/1000mL = 1M \| 5.08275g/100mL = 1M | +| Ultrapure Water | Fill to: | 100mL | | + +## Working Solutions +*Use PBS without Calcium Chloride and Magnesium Chloride* + +### PBSTA (3mL) without Triton X-100 – store at 4ºC for up to 1 month +| Solution | Final Conc | 1 RXN | 5.2 RXN | Notes | +|---------------------|------------|--------|---------|----------------------------------------------------| +| 10x PBS | 1x | 300uL | 1560uL | | +| 1M MgCl₂ | 3mM | 9uL | 46.8uL | | +| D-Sucrose (342.29) | 0.3M | 0.3081g| 1.602g | | +| ** RNasein (add on day) | 0.4 U/uL | 30uL | 156uL | | +| Ultrapure Water | Fill to: | 3mL | 15.6mL | Add most of the Ultrapure water, then PBS, | + then MgCl₂, then Sucrose. Vortex to dissolve | + add RNasein before using | + +### 1.4M PBS Cushion (8mL) – make fresh weekly +| Solution | Final Conc | 1 RXN | 5.2 RXN | Notes | +|---------------------|------------|--------|---------|----------------------------------------------------| +| 10x PBS | 1x | 0.8mL | 4.16mL | | +| 1M MgCl₂ | 3mM | 24uL | 124.8uL | | +| D-Sucrose | 1.4M | 3.8336g| 19.935g | | +| 10% Triton X-100 | 0.1% | 80uL | 416uL | | +| Ultrapure Water | Fill to: | 8mL | 41.6mL | Add half of the Ultrapure water, then PBS, | + then MgCl₂, then Sucrose. Vortex to dissolve, | + then add 10% Triton, then add Ultrapure water | + +### Nuclei Wash and Resuspension Buffer – make fresh daily +| Solution | Final Conc | 1 RXN | 5.2 RXN | Notes | +|----------------------------|------------|-------|---------|------------------------------------------------| +| 10x PBS | 1x | 400uL | 2080uL | | +| 50mg/uL Ultrapure BSA | 1% | 800uL | 4160uL | (40mg) | +| ** RNasein (add on day) | 0.2 U/uL | 20uL | 104uL | | +| Ultrapure Water | Fill to: | 4mL | 20.8mL | Add most of the Ultrapure water, then PBS, | + then BSA. Tilt to dissolve, do not vortex | + +## Protocol + +1. **Homogenize** + - Transfer tissue to FACS tube. + - Add 2mL of PBSTA to the FACS tube containing the tissue. + - Homogenize on ice with hand-held mixer at lowest setting, 5 sec on, 5 sec off. Repeat for a total of 4 times (do not create bubbles, check no debris after homogenizing) store on ice. + - Add 40uL of 10% TX100 (final conc 0.2%), pipette mix 10 times with p1000. Incubate for 5min on ice. For each sample leave 2min space between. + - Repeat for each sample a-c for each sample. + +2. **Extract nuclei and filter onto gradient** + - Transfer the 2mL of PBSTA/tissue to a Dounce on ice. + - Dounce 10 times gently to release the nuclei, do a half-turn at the top and bottom movement, do not create bubbles by never lifting the Dounce out of the volume at all. + - Filter the nuclei through a Miracloth (Cakbiochem, #475855) onto the 8mL of 1.4M PBS Cushion. + - Add 1mL of PBSTA to the Dounce to resuspend any remaining nuclei from the sides of the Dounce, transfer these through the same Miracloth onto the 1.4M PBS Cushion. + - Repeat for each sample a-e for each sample. + +3. **Sucrose Gradient Centrifuge** + - Do not disturb the gradient. + - Centrifuge nuclei through the gradient @3000 x g for 30 min at 4ºC. + - Take out of centrifuge immediately when finished, should see a condensed white interface. + +4. **Resuspend Nuclei** + - On ice, remove the top layer first using a p1000. + - Then remove the interface (cell debris) using a p1000. + - Once the cell debris is removed, continue to use a p1000 to remove all supernatant, switch to a p200 tip to remove the final 200uL supernatant. Make sure to remove all the supernatant without touching the pellet. + - Resuspend the pellet in 210uL of Nuclei Wash and Resuspension Buffer, pipette 10 times using a p200, then transfer to LoBind tube on ice. + - Further resuspend the nuclei into singlets by pipette 5 times using a p200 against the wall of the epi. + - Repeat for each sample a-e for each sample. + +5. **Count Nuclei** + - Transfer 10uL of nuclei into a fresh epi on ice. + - Add 10uL of Trypan Blue into the 1:2 nuclei dilution, resuspend 10 times and count nuclei numbers using a Countess. + +6. **Wash Nuclei 1** + - After incubation, add 900uL of Nuclei Wash Buffer to the 200uL nuclei, pipette 5 times with p1000. + - Centrifuge nuclei @500 x g for 5min at 4ºC. + - Discard most of the supernatant using a p200 tip, leaving 20uL of supernatant and pellet. Carefully not to disturb the pellet. + - Pipette 10 times using a p200 to resuspend the pellet. + +7. **Wash Nuclei 2** + - Add 1000uL of Nuclei Wash Buffer to the 100uL nuclei, pipette 5 times with p1000. + - Centrifuge nuclei @500 x g for 5min at 4ºC. + - Discard most of the supernatant using a p200 tip, leaving 20uL of supernatant and pellet. Carefully not to disturb the pellet. + - Pipette 10 times using a p200 to resuspend the pellet. + +8. **Wash Nuclei 3** + - Add 1000uL of Nuclei Wash Buffer to the 100uL nuclei, pipette 5 times with p1000. + - Centrifuge nuclei @500 x g for 5min at 4ºC. + - Discard most of the supernatant using a p200 tip, leaving 20uL of supernatant and pellet. Carefully not to disturb the pellet. + - Pipette 10 times using a p200 to resuspend the pellet. + - Add 100uL of Nuclei Wash Buffer, pipette 10 times using a p200 to resuspend the pellet. + +9. **Count Nuclei** + - Add 5uL of nuclei to 5uL of Nuclei Wash Buffer in a fresh epi on ice. + - Add 10uL of Trypan Blue resuspend 10 times and count nuclei numbers using a Countess and take photos on the EVOS Level 3. + +10. **Flow Cytometry** + - Add 20uL of nuclei to 180uL of Nuclei Wash Buffer in a fresh epi on ice. + - Flow QC and Count Nuclei Dilution Protocol. + +11. **Dilute Nuclei to 1000 nuclei/uL** + - Add 100,000 nuclei into 100uL of Nuclei Wash Buffer. Snap Freeze leftover Nuclei Protocol. + +12. **Snap Freeze leftover nuclei** + - Use liquid nitrogen to snap freeze leftover nuclei and store in -80ºC freezer. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/hydrop-atac-v1-0-b4xvqxn6.md b/markdown-output/hydrop-atac-v1-0-b4xvqxn6.md new file mode 100644 index 0000000000000000000000000000000000000000..6156e76023f10dade68502495995a12a4b36918e --- /dev/null +++ b/markdown-output/hydrop-atac-v1-0-b4xvqxn6.md @@ -0,0 +1,223 @@ +```markdown +# Goal/Experiment: +To perform and document the protocol for HyDrop-ATAC, a method that combines microfluidics and hydrogel beads to conduct assay for transposase-accessible chromatin using sequencing (ATAC-seq) on single cells. This involves preparing nuclei, microfluidic setup, tagmentation, bead preparation, PCR amplification, and sequencing prep. + +# HyDrop-ATAC v1.0 V.3 + +**Authors**: +- Florian De Rop¹,² +- Suresh Poovathingal¹,² +- Stein Aerts¹,² + +¹VIB-KU Leuven Center for Brain & Disease Research;
+²Laboratory of Computational Biology, Department of Human Genetics, KU Leuven + +## Citation +Florian De Rop, Suresh Poovathingal, Stein Aerts 2022. HyDrop-ATAC v1.0.
+DOI: [10.17504/protocols.io.b4vxqxn6](https://dx.doi.org/10.17504/protocols.io.b4vxqxn6) + +**Keywords**: atac-seq, ATAC, TN5, hydrogel, hydrop, hydrop-atac, droplet microfluidics, microfluidic, transposase, single-cell + +## Preparation Stage (20m) + +### 1. User Manual Sheet +The Adaptive Table to streamline execution, adjusting buffer and mix concentrations based on input parameters—including cell counts and emulsion volume. + +- [Adaptive table for HyDrop-ATAC: 20210825p_USER_HyDrop_ATAC_v1_7_1.xlsx](#) +- [Price calculation for HyDrop-ATAC: 20210827_supp_methods_table_hydrop_pricecalc.xlsx](#) +- [Reagent list: reagents.xlsx](#) +- [Oligonucleotides: 20210712_supp_methods_table_hydrop_oligonucleotide_list.xlsx](#) + +For further questions: hydrop.aertslab@gmail.com + +## Setting up the Microfluidic Framework (20m) + +### 2.1 Boot up Droplet Genomics on the Onyx System (5m) +- Ensure correct syringe diameters on Onyx program. +- Connect laptop/tablet to Onyx. + +### 2.2 Fill Syringes and Connect Tubing (5m) +- Fill two 3 mL syringes with ~1 mL fresh mineral oil using a 1 mL pipette. +- Connect a ~15 cm piece of 1 mm inner diameter tubing to each syringe. + +### 2.3 Reagent RAN Biotech Emulsion (3m) +- Fill a 3 mL syringe with ~1.5 mL RAN Biotech emulsion oil using a pipette tip. + +### 2.4 Inspect and Setup the HyDrop Microfluidic Chip (2m) +- Inspect chip under a microscope. + +### 2.5 Connect Tubing and Prepare Collection Tubes (5m) +- Connect a piece of 1 mm tubing (~7 cm) to the outlet. +- Prepare 250 uL PCR tubes. + +## Preparing Tagmented Nuclei (20m) + +### 3.1 Thaw Reagents +- BSA 10% +- Digitonin 5% +- Phusion HF Buffer +- dNTPs 25m +- DTT 1M +- Pitstop 50mM (3 µL) +- Pitstop 1mM (3 µL) + +### 3.2 Pellet Cells and Prepare ALB & ANWB (25m) + +#### Table: ATAC Lysis Buffer (ALB) +| Reagent | Vol (uL) | Stock | Final | +| ------------- | -------- | ----- | --------- | +| dH2O | 388.5 | | | +| PBS-BSA | 50 | 10% | 1% | +| Pitstop | 35 | 1000 | 70 uM | +| Tris-HCl | 5 | 1000 | 10 mM | +| NaCl | 5 | 1000 | 10 mM | +| Tween 20 | 5 | 10% | 0.10% | +| NP-40 | 5 | 10% | 0.10% | +| MgCl2 | 1.5 | 1000 | 3 mM | +| Digitonin | 5 | 1% | 0.01% | +| **Total** | 500 | | | + +#### Table: ATAC Nuclei Wash Buffer (ANWB) +| Reagent | Vol (uL) | Stock | Final | +| ------------- | -------- | ----- | --------- | +| dH2O | 2167.5 | | | +| PBS-BSA | 250 | 10% | 1% | +| Tris-HCl pH 7.4 | 25 | 1000 | 10 mM | +| Tween 20 | 25 | 10% | 0.10% | +| NaCl | 25 | 1000 | 10 mM | +| MgCl2 | 7.5 | 1000 | 3 mM | +| **Total** | 2500 | | | + +### 3.3 Isolate and Prepare Nuclei +- Perform single-cell suspension, wash twice in PBS. +- Pellet (300 x g, 5m, 4°C), resuspend in ALB. + +### 3.4 Perform Tagmentation (1h) +Prepare: +- 4X ATAC TD + +#### Table: ATAC Reaction Mix +| Reagent | Vol (uL) | Stock | Final | +| ------------- | -------- | ----- | --------- | +| 4X ATAC TD | 12.5 | 400% | 100% | +| PBS | 26-x | | | +| Nuclei | x | | | +| Tn5 | 4 | 62.5 | 5 ng/uL | +| Pitstop | 3.5 | 1000 | 70 uM | +| Tween 20 | 2 | 2.50% | 0.10% | +| Digitonin | 2 | 0.25% | 0.01% | +| **Total** | 50 | | | + +## Reagent Preparation (20m) + +### 4.1 Prepare Bead Lysis Buffer (BLB) +| Reagent | Vol (uL) | Stock | Final in mix | Final in Droplet | +| ------------- | -------- | ----- | ------------ | ---------------- | +| dH2O | 3493.205128 | | | 50 mM | +| KCl | 467.9487179 | 3000 | 280.7692308% | 50 mM | +| Tris-HCl (pH 7.4) | 701.9230769 | 1000 | 140.3846154% | 25 mM | +| Tween 20 | 280.7692308 | 10% | 0.005615384615% | 0.10% | +| MgCl2 | 56.15384615 | 1000 | 11.23076923% | 2 mM | +| **Total** | 5000 | | | | + +### 4.2 Load and Prepare Beads +- Thaw frozen HyDrop-ATAC beads, rinse in BLB, repeat wash steps. +- Load beads into mineral-oil pipette tip for microfluidic processing. + +### 5. Nuclei/PCR Mix Preparation +Prepare mix without enzymes first to ensure volume: +- Centrifuge (300 xg, 5 min, 4°C), resuspend in 200 µL 1% BSA, repeat. +- Resuspend in 20 µL, add Nuclei mix. + +#### Table: Nuclei/PCR Mix +| Reagent | Vol (uL) | Stock | In PCR mix | In droplet | +| -------------- | -------- | ----- | ---------- | ------------ | +| Phusion HF | 48.6666667 | 500% | 122% | 100% | +| dH2O | 86.05-x | | | | +| Optiprep | 30 | 100% | 15% | | +| dNTPs | 9.73333333 | 25 | 1.21666667 | 1 mM | +| Nuclei | x | | 121.666667 | 100 c/uL | +| DTT | 7.3 | 1000 | 36.5 | 30 mM | +| Phusion HF | 6.08333333 | 2 | 0.06083333 | 0.05 U/uL | +| Deep Vent Poly | 6.08333333 | 2 | 0.06083333 | 0.05 U/uL | +| ET SSB | 6.08333333 | 0.5 | 0.01216667 | 0.01 ug/uL | +| **Total** | 200 | | | | + +### 5.1 Load Nuclei/PCR Mix +Identical to bead loading process. + +## HyDrop Run (20m) + +### 6. Priming HyDrop Run (10m) +Priming flows: +- Cells: 200 µL/h +- Beads: 200 µL/h +- Oil: 100 µL/h + +Flow gets adjusted later to: +- Cells: 600 µL/h +- Beads: 150 µL/h +- Oil: 950 µL/h + +### 6.2 Run and Collect Samples +- Replace waste tube with collection tubes. + +## Library Prep Stage (1h 45m) + +### 7. Linear PCR and Clean-up (PCR 1) +- Pre-amplify libraries using the following program: + +| Temp | Time | +| ---- | --------- | +| 72°C | 5 mins | +| 98°C | 3 mins | +| 98°C | 10 s (x15)| +| 63°C | 30 s | +| 72°C | 1 min | +| 4°C | Hold | + +### 7.2 Add GITC Buffer and Droplet Breaking (10m) +| Reagent | Stock | Vol (uL) | Final | +| ----------- | ----- | -------- | ------ | +| GITC | 6 | 834 | 5 M | +| Tris HCl - 7.4 | 1 | 50 | 50 mM | +| EDTA | 0.5 | 50 | 25 mM | +| dH2O | - | 66 | | + +### 7.3 Produce EB-DTT-Tween and EB-DTT +| Reagent | Stock | Vol (uL) | Final | +| ----------- | ----- | -------- | ------ | +| DTT | 1000 | 2 | 10 mM | +| Tweem | 10 | 2 | 0.1% | +| EB | - | 196 | | + +### 7.4 PCR2 Mix Preparation +| Reagent | Vol (uL) | Stock | Final | +| ----------- | -------- | ----- | ------ | +| KAPA | 50 | 200% | 100% | +| Library | 30 | | | +| Hyi7 | 10 | 10 | 1 uM | +| Hyi5 | 10 | 10 | 1 uM | +| **Total** | 100 | | | + +PCR program: +| Temp | Time | +| ---- | ---------- | +| 95°C | 3 mins | +| 98°C | 10 s (x12) | +| 72°C | 30 s | +| 72°C | 1 min | +| 4°C | Hold | + +### 7.5 Double-sided Ampure Purification +- 0.4 volumes Ampure beads (40 µL) to PCR mix. +- Transfer supernatant to new tube, add 0.8 volumes (80 µL) Ampure beads. +- Wash and air-dry. + +### 7.6 Quantification and Sequencing +- Perform Qbit and Bioanalyzer. + +![Bioanalyzer Result](#) + +## endofoutput +``` \ No newline at end of file diff --git a/markdown-output/hydrop-rna-v1-0-c53gy8jw.md b/markdown-output/hydrop-rna-v1-0-c53gy8jw.md new file mode 100644 index 0000000000000000000000000000000000000000..d6c7baddae58d027deb171b697773b6960824850 --- /dev/null +++ b/markdown-output/hydrop-rna-v1-0-c53gy8jw.md @@ -0,0 +1,267 @@ +```markdown +# Goal/Experiment: +Perform HyDrop-RNA for single-cell RNA sequencing using a step-by-step protocol to ensure accurate and efficient encapsulation and analysis of cells. + +## HyDrop-RNA v1.0 V.5 + +**Florian De Rop1,2, Suresh Poovathingal1,2** +**Stein Aerts1,2** + +**1** VIB-KU Leuven Center for Brain & Disease Research +**2** Laboratory of Computational Biology, Department of Human Genetics, KU Leuven + +### Abstract +Step-by-step protocol for performing HyDrop-RNA. The duration of each step assumes an experienced protocol user. For a first-time user, we recommend doubling the expected time for each step. + +### Keywords +scRNA-seq, hydrop, hydrogel, rna-seq, single-cell, single cell, droplet microfluidics, HyDrop, HyDrop-RNA, ASAPCRN + +## List of Reagents and Oligonucleotides + +- **Reagents XLSX:** [Download](reagents.xlsx) +- **Oligonucleotides XLSX:** [Download](20210712_supp_methods_table_hydrop_oligonucleotide_list.xlsx) + +For any questions or remarks, please contact us at hydrop.aertslab@gmail.com. + +## Microfluidics Preparation + +### 1. Setting up the Microfluidic Framework + +**Note:** These steps should happen in advance before the run can be started, as time between preparation of the cell resuspension in RT mix and actual encapsulation needs to be minimized to preserve cell viability. + +#### 1.1. Boot up Droplet Genomics onyx. (5m) +- Check syringe diameters in the Onyx program. +- Connect the laptop or tablet to Onyx. + +#### 1.2. Prepare mineral oil syringes. (10m) +- Fill two 3 mL syringes with ~1 mL fresh mineral oil each using a 1 mL pipette. +- Connect ~15 cm piece of 1 mm inner diameter tubing to each syringe, connect to the microfluidic chip. +- Using a 6 mm and 1 mm biopsy needle, prepare PDMS plug and connect syringe tubing through the PDMS to the pipette tip. +- Fix syringes in DG Onyx pump. + +#### 1.3. Fill a syringe with RAN Biotech emulsion oil. (5m) +- Fill a 3 mL syringe with ~1.5 mL of RAN Biotech emulsion oil. +- Connect to tubing and insert into DG Onyx pump. + +#### 1.4. Prepare microfluidic chip. (5m) +- Take a HyDrop High Aspect 65/80 microfluidic chip and inspect channels under a microscope. +- Fix the chip to the moving platform. + +#### 1.5. Connect outlet tubing. (5m) +- Connect a short piece of tubing to the outlet. +- Position DNA lo-bind 1.5 mL eppendorf for waste collection. + +#### 1.6. Final setup check. +- Verify fire risk prevention seating for samples during longer runs. + +## Reagent Preparation + +### 2. Preparing the Reagents (1h 30m) + +#### 2.1. Bead Lysis Buffer (BLB) preparation: +- **Ingredients and concentrations:** + +| A | B | C | D | E | F | +|-----------|---------|-------|------|----------|------| +| BLB | Stock | Volume (uL) | Final | Final in drop | Unit | +| Tris-HCl (pH 7) | 1000 | 625 | 125 | 25 | mM | +| NaCl | 5000 | 150 | 150 | 30 | mM | +| MgCl2 | 1000 | 62.5 | 12.5 | 2.5 | mM | +| Tween-20 | 100 | 200 | 4 | 0.8 | % | +| TX-100 | 10 | 375 | 0.75 | 0.15 | % | +| Glycerol | 100 | 1250 | 25 | 5.00 | % | +| BSA | 10 | 500 | 1 | 0.2 | % | +| dH2O | - | 1837.5| - | - | - | + +- Wash beads with BLB. + +#### 2.2. Load beads into pipette (15m) +- Prepare mineral-oil filled pipette. Ensure no air bubbles. +- Aspirate beads using flow rate 5000-10000 uL/hour. + +#### 2.3. Prepare single-cell suspension (30m) +- Resuspend cells in 0.04% to 1% BSA in PBS. +- Aim for a concentration of at least 1M/mL. + +#### 2.4. RT Mix preparation (30m) + +| A | B | C | D | E | F | +|---------------|------|-------|-----|-----|-----| +| RT Mix | Stock| Volume (uL) | Final | Final in droplet | Unit | +| Maxima RT buffer | 5 | 44.2 | 1.3 | 1 | X | +| dNTP | 10 | 15.3 | 0.9 | 0.75| mM | +| DTT | 1000| 4.3 | 25 | 20 | mM | +| GTP | 100 | 2.2 | 1.3 | 1 | mM | +| Optiprep | 100 | 25.5 | 15 | 15 | % | +| RNase Inhibitor | 40 | 5.5 | 1.3 | 1 | U/ul| +| Max H- RT | 200 | 12.8 | 15 | 12 | U/ul| +| TSO-Dropseq | 250 | 8.5 | 12.5| 10 | uM | +| PEG-8K | 40 | 18.7 | 4.4 | 3.5| % | +| Cells in 1% BSA-PBS | - | 21 | - | - | - | +| dH2O | - | 10 | - | - | - | + +Proceed to HyDrop run immediately with cells on ice. + +## HyDrop Run Stage + +### 3. HyDrop Run (2h 30m) + +#### 3.1. Load cells (30m) +- Follow the same loading steps as beads but for RT mix. +- Adjust flow rates. + +#### 3.2. Priming and running the microfluidic system. +- Flow rates: + +| Component | Flow rate (uL/h) | +|-----------|------------------| +| Cells | 600 | +| Beads | 150 | +| Oil | 950 | + +#### 3.3. Collect emulsions (stabilize). (15m) +- Use 200 uL PCR tubes to collect samples. + +#### 3.4. Ending the run. +- Emulsion collection calculations and tube switching. + +## Reverse Transcription + +### 4. Reverse Transcription (2h) +1. Transfer emulsions to PCR block following this program: + +| Step | Temperature | Time | +|-------------|-------------|-------| +| Cycle 1 | 42 C | 90 min| +| Cycle 2 | 50 C | 2 min (x11)| +| Cycle 3 | 42 C | 2 min | +| Cycle 4 | 85 C | 5 min | +| Hold | 4 C | inf | + +While RT is on, prepare GITC buffer and other components for later steps. + +## cDNA Clean-up Stage + +### 5. Droplet Breaking and Purification (3h) + +#### 5.1. GITC Buffer preparation: + +| A | B | C | D | E | +|-------------|-------|------|------|-----| +| GITC Buffer | Stock | Vol (uL) | Final| Unit | +| GITC | 6 | 834 | 5 | M | +| Tris HCL 7.4| 1 | 50 | 50 | mM | +| EDTA | 0.5 | 50 | 25 | mM | +| dH2O | - | 66 | - | - | + +- Add 125 uL of droplet breaking solution to each sample. +- Incubate and remove the oil phase. + +#### 5.2. EB-DTT Tween and EB-DTT preparation (20m) + +| A | B | C | D | E | +|-----------|---------|---------|---------|-----| +| EB-DTT-Tw | Stock | Volume (uL) | Final | Unit| +| DTT | 1000 | 2 | 10 | mM | +| Tween 20 | 10 | 2 | 0.1 | % | +| EB | - | 196 | - | - | + +| A | B | C | D | E | +|-----------|---------|---------|---------|-----| +| EB-DTT | Stock | Volume (uL) | Final | Unit| +| DTT | 1000 | 2 | 10 | mM | +| EB | - | 198 | - | - | + +- Proceed with 0.9X Ampure XP purification as per instructions (15m). + +#### 5.3. Exo1 Clean-up (15m) +- Prepare Exo1 mix per sample: + +| A | B | C | D | +|--------------------|-----------|-------|------| +| Library | Stock | V (uL)| Final| +| NEBuffer 3.1 | 10X | 4 | 1X | +| Thermolabile Exo-I | - | 4 | - | +| Library | - | 30 | - | +| dH2O | - | 2 | - | + +- Incubate at 37 C, denature. + +### 6. IS-PCR (1h 15m) +- Reaction mix: + +| A | B | C | +|---------------------|--------|--------| +| Stock | V (uL) | Final | +| 2X KAPA HiFi PCR mix| 50 | - | +| Library | 40 | - | +| SMART PCR primer (10 uM) | 10 | - | + +- Amplification program. +- Purification with SPRI beads. + +### 7. NEB Ultra Library Preparation (3h 45m) + +#### 7.1. Protocol preparation (45m) +- Thaw the Ultra II FS Reaction Buffer. +- Prepare fragmentation mix: + +| A | B | Volume (uL)| +|-----------------------|-----------|-------------| +| (yellow) NEBNext Ultra II FS Reaction Buffer| - | 7 | +| (yellow) NEBNext Ultra II FS Enzyme Mix | - | 2 | +| cDNA sample | - | x | +| EB buffer | - | 26-x | +| Total | - | 35 | + +- Incubate and proceed with ligation. + +### 7.2. Fragmentation and purification (30m) +- Mix and Incubate. + +### 7.3. Ligation preparation and incubation (45m) +- Ligation mix as follows: + +| A | B | Volume (uL)| +|------------------------------------|-----------|-------------| +| Fragmented DNA | - | 35 | +| (red) NEBNext Ultra II Ligation Master Mix | - | 30 | +| (red) NEBNext Ligation Enhancer | - | 1 | +| (red) NEBNext Adaptor for Illumina | - | 2.5 | +| Total | - | 68.5 | + +Centrifuge and perform time-controlled incubations. + +### 7.4. SPRI purification post-ligation (15m) +- Follow standard purification steps after ligation (28.5 uL EB buffer). + +### 7.5. Final PCR preparation (45m) + +| A | B | C | D | E | +|------------------|--------|-------|------|-------| +| Stock | Volume | Final | Unit | | +| KAPA HiFi | 50 | 100 | % |- | +| Hyj7-X | 10 | 1 | uM |- | +| Hyj5-TS-X | 10 | 1 | uM |- | +| Library | 30 | - | | +| Total | 100 | - | - |- | + +- PCR program, SPRI purification. + +### 7.6. Final Quality Control (30m) +- Qubit and BioAnalyzer for quality assessment. + +## End of Protocol +The library is now ready for sequencing. + +*** + +For detailed information on reagents and equipment vendors, ensure access to the reagent and oligonucleotide lists. + +## Protocols References +- Protocol Citation: Florian De Rop, Suresh Poovathingal, Stein Aerts 2023. HyDrop-RNA v1.0. protocols.io https://dx.doi.org/10.17504/protocols.io.dm6gpwqjljzp/v5 + +__License:__ This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/identifying-an-active-promoter-in-b-saltans-using-shzeb76.md b/markdown-output/identifying-an-active-promoter-in-b-saltans-using-shzeb76.md new file mode 100644 index 0000000000000000000000000000000000000000..b76bc646d654f0a1b15beb6b7a2a001b074c6334 --- /dev/null +++ b/markdown-output/identifying-an-active-promoter-in-b-saltans-using-shzeb76.md @@ -0,0 +1,152 @@ +```markdown +# Identifying an Active Promoter in B. saltans Using Luciferase Assay + +## Goal/Experiment: +The goal of this experiment is to identify an active promoter in B. saltans using the luciferase assay by performing PCR amplification, gel electrophoresis, restriction enzyme digest, ligation, bacterial transformation, and screening. + +--- + +### Abstract +**Before starting, prepare the following:** +- Measure the Luciferase plasmid concentration using NanoDrop and Qubit. +- Design the PCR primers for the gene of interest with the RE (restriction enzyme) sequence at the 5’. +- Add 3 to 4 nucleotides at the end of the RE sequence. These bps will be removed during fragment digestion. +- Order TOPO 10 chemically competent coli cells and other kits for digestion, ligation, and gel purification. + +--- + +### Protocol: + +#### Day 1 + +**1. PCR Amplification:** + +*Materials:* +- DNA of B. saltans (10-100 ng/µl) +- Forward and reverse primers with RE sequence +- Master Mix (2x NEB Taq 2x) +- Thermocycler + +*Procedure:* +- Prepare DNA samples and primers. +- PCR Profile: + 1. 95°C for 30 sec + 2. 95°C for 30 sec + 3. 61°C for 30 sec + 4. 68°C for 80 sec + 5. Repeat steps 2-4 for 20 cycles + 6. 68°C for 5 min + 7. Hold at 10°C + +*Primers:* +- Forward: 8683-8713 + ``` + CTAGAAGCTTGAAAGAAGCTGAAGCCGCTGTGTGTG + ``` +- Forward: 6315-6332 + ``` + CTAGAAGCTTGGCATTGGTGAAGGAAGGAGCAGAT + ``` +- Reverse: 9603-9634 + ``` + TACAGTGTTACTTCTGCGATTCGAGTTGAGTTGGAGG + ``` + +**2. Gel Electrophoresis:** +- Check the PCR product using gel electrophoresis. + +**3. PCR Purification:** +- Purify the PCR products using column purification or beads. Elute in ~15 µl volume for a total of 75 µl. + +**4. Restriction Enzyme Digest:** +- Digestion for both plasmid and PCR product: + - 750 ng luciferase plasmid. + - X µl vector or insert. + - 1.7 µl 10X Reaction Buffer. + - 1.5 µl restriction enzyme Hind III (10 U/µl). + - Y µl water to bring volume to ~17 µl. + - Digest for at least 3-4 hours. +- Treat with Cap Alkaline Phosphatase (CAP): + - 1/10 volume of 10X CAP Buffer, 1 µl enzyme. + - Incubate at 37°C for 1 hour. + - Heat inactivate at 65°C for 10 minutes. +- Prepare mixture with gel loading buffer (bromophenol blue, glycerol). Run on 1% agarose gel. +- Place digested fragments in 1.5 ml centrifuge tube, freeze at -20°C for at least 10 minutes or longer. + +**5. Cleanup Gel Fragment Using Sigma GenElute Kit:** +- Melt tubes with fragments (56°C heat block for 5-10 minutes). +- Breakup gel with small pipette tip or spatula. +- Add 100 µl TE Buffer, centrifuge at max speed for 10 sec. +- Transfer column to new tube, centrifuge at max speed for 10 min. +- Measure recovered gel volume, add 1/10 volume of sterile 3M Sodium Acetate, then 2 volumes of 100% ethanol, 1 µl 20 mg/ml glycogen. +- Freeze overnight at -20°C. + +--- + +#### Day 2 + +**1. Ethanol Precipitation:** +- Centrifuge ethanol precipitation 10-20 minutes at max speed, pour ethanol without losing DNA pellet. +- Add 200 µl of 70% ethanol, centrifuge 3 minutes, remove ethanol without losing pellet. +- Air dry for 10-15 minutes. +- Suspend DNA pellet in 15 µl TE Buffer, let sit at room temperature for 30 minutes. +- Pipette up and down to ensure it's suspended. + +**2. Ligation Reaction:** +*Materials:* +- Restriction digest CAP treated vector and insert. +- 5X T4 Ligase Buffer. +- T4 HC Ligase enzyme (Invitrogen). + +*Procedure:* +- To a tube (total volume ~10 µl): + - X µl vector + - Y µl insert + - 2 µl 5X Ligase Buffer + - Z µl water + - 0.4 µl T4 ligase enzyme +- Incubate at room temperature for 1-3 hours. +- PCR of the ligation mix (optional) to check vector and insert integration. + +**3. Bacterial Transformation:** +*Materials:* +- TOPO-10 cells (Chemically Competent E. coli) +- SOC media +- LB + Ampicillin plates + +*Procedure:* +- Mix 1 µl ligation, dilute to 5 µl, transform cells. +- Alternately, use 1 µl concentrated ligation. +- Cold-shocked cells on ice. +- Heat shock 35 seconds at 42°C water bath. +- Incubate with 210 µl SOC media for 1 hour at 37°C. +- Spread on LB + Ampicillin plates. +- Grow overnight (not more than 16 hours). +- Save bacterial glycerol stock. + +--- + +#### Day 3 + +**1. Screening:** +- Prepare LB broth with ampicillin in equal number of tubes. +- Pick and transfer white colonies using a sterile toothpick to PCR Master Mix for amplification. Repeat for all white colonies. +- PCR with plasmid forward primer and gene reverse primer. +- Gel electrophoresis to confirm inserts. + +--- + +#### Day 4 + +**1. Plasmid Quantification:** +- Prepare overnight culture in LB broth. +- Use Zymo Midi Prep Kit for plasmid extraction. +- Measure concentration using Qubit (200-300 ng/µl usually). +- PCR confirmation with the plasmid forward/insert reverse primer. + +--- + +**Citation:** +Fatma Gomaa, ZhuHong Li, Roberto Docampo, Peter Girguis, Virginia Edgcomb. Identifying an active promoter in B. saltans using Luciferase assay. protocols.io. dx.doi.org/10.17504/protocols.io.she2b76. Published: 09 Aug 2018. +``` +endofoutput \ No newline at end of file diff --git a/markdown-output/illumina-amplicon-sequencing-using-two-step-pcr-cn4vvgw6.md b/markdown-output/illumina-amplicon-sequencing-using-two-step-pcr-cn4vvgw6.md new file mode 100644 index 0000000000000000000000000000000000000000..306ddd882cd16f83db51be11e61b5bfc29643ac7 --- /dev/null +++ b/markdown-output/illumina-amplicon-sequencing-using-two-step-pcr-cn4vvgw6.md @@ -0,0 +1,184 @@ +```markdown +# Goal/Experiment: +The aim is to provide a framework for undertaking two-step amplicon sequencing using the Illumina MiSeq platform at UKCEH-Wallingford, allowing researchers to add their own modifications to suit novel applications. + +# Illumina Amplicon Sequencing using Two-Step PCR + +**Authors:** +Lindsay Kate Newbold, Joetay, Jonathan Warren, Daniel S. Read, Kerry Walsh, Amy Thorpe +**Affiliation:** +UK Centre for Ecology & Hydrology + +## Abstract +This document provides the information needed for performing two-step amplicon sequencing utilizing the Illumina MiSeq platform. This protocol sets a foundation that researchers can adapt to fit specific needs. + +## Nextera Index Plates - Prepared prior to Step 1 PCR + +### 1. Order Indexing Primers +- **Primers:** Illumina Nextera adapter i5 (forward primer), i7 (reverse primer) + - i5 Example Sequence: `AATGATACGGCGACCACCGAGATCTACAC` + - i7 Example Sequence: `CAAGCAGAAGACGGCATACGAGAT` + - Vendor examples: IDT, Sigma Genosys, or MWG +- **Concentration:** 0.5 µM, desalt-purified, 10 µM +- **Index Barcodes:** Example sequences for unique 8bp barcodes, allocation given, creating 384 unique barcode combinations. + +### 2. Prepare Index Plate Masters +- **Volume:** 250 µL of each diluted primer stock (10 µM) per well, transferred to Deep-Well microtiter plates. +- **Method:** Manual or via liquid handling robot. + +### 3. Make PCR Plate Clones +- **Storage:** Prepare clones from the master plates, dispense 5 µL mixed primer per well, label, seal, and store at -20°C. + +## Step I: Amplicon PCR with Modified Primers + +### 4. Standardized Dilution Plate of Template DNA +- **Goal:** Dilute template DNA from extraction protocol to standard concentration using Nanodrop or Qubit BR assay. +- **Concentration Equation:** + - \( \text{Desired Concentration (ng/µL)} \times \text{Final Volume} = \text{Amount of DNA to Add} \) + - \( \text{Final Volume} - \text{Amount of DNA to Add} = \text{Amount of Water to Add} \) + +### 5. Target Primer Design + +| Source Reference | Universal Primer Name | Target Group | Pre-Adapter | Sequencing Primer Sequence | Specific Locus Primer | Combined Sequence | +|------------------|-----------------------|--------------|-------------|----------------------------|-----------------------|-------------------| +| Walters et al. (2015) | 515F | Bacteria | TCGTCGGCAGC GTC | AGATGTGTATAAGAGACAG | GTGYCAGCMGCCGCGGTA A | TCGTCGGCAGCGTCGAGTGTGTATAAGAGACAGGTGYCAGCMGCCGCGGTAA | +| Walters et al. (2015) | 806R | Bacteria | GTCTCGTGGGCTCGG | AGATGTGTATAAGAGACAG | GGACTACHVGGGTWTCTAAT | GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGGACTACHVGGGTWTCTAAT | +| Ihrmark et al. (2012) | ITS7F | Fungi | TCGTCGAGCTC | AGATGTGTATAAGAGACAG | GTGARTCATCGAATCTTG G | TCGTCGAGCTCGAGATGTGTATAAGAGACAGGTGARTCATCGAATCTTTGG | +| Ihrmark et al. (2012) | ITS4R | Fungi | GTCTCGGGCGCTCGG | AGATGTGTATAAGAGACAG | TCCTCCGCTTATTGATATGC | GTCTCGGGCGCTCGGATGTGTATAAGAGACAGTCCTCCGCTTATTGATATGC | +| Mangot et al. (2012) | NSF563 | Eukaryotes | TCGTCGGCAGCTC | AGATGTGTATAAGACAG | CCGGGTATTCCCAGTCCC A | TCGTCGGCAGCTCAGATGTGTATAAGAGACACCCCGATCCGTCCCACCACTC | +| Mangot et al. (2012) | NSR951 | Eukaryotes | GTCTCGTGGGCGG | AGATGTGTATAAGAGACAG | TTGGYRRGACATCCGTTCCG C | GTCTCGTGGGCGGAGATGTGTATAAGAGACAGTTGGYRRGACATCCGTTCCGC | +| Kelley et al. (2018) | rbcL-646F | Diatoms | TCGTCGAGGCTC | AGATGTGTATAAGAGACAG | ATGGCTGGAACAAGCTTC | TCGTCGAGGCTCGAGATGTGTATAAGAGACAGATGGCTGGAACAAGCTTC | +| Kelley et al. (2018) | rbcL-998R | Diatoms | GTCTCGGGCCTCGG | AGATGTGTATAAGAGACAG | GATCACCATTTAACWCAACTG | GTCTCGGGCCTCGGAGATGTGTATAAGAGACAGGATCACCTTCCACCACTG | + +### 6. Order Primers for Combined Sequence +- **Concentration:** 0.5 µM, desalt-purified, 100 µM. + +### Table 2: Step I PCR Reagents + +| Reagent | Per Sample | Per Plate | +|---------------------|------------|-------------| +| Molecular Grade Water | 40.5 µL | 4050 µL | +| 5X Buffer, High GC Buffer, 10 mM dNTPs | 1 µL each | 100 µL each | +| Q5 Taq Polymerase | 0.5 µL | 50 µL | +| Primer F, Primer R | 0.5 µL each | 50 µL each | +| DNA Template (10 ng/mL) | 2 µL | Added separately | + +### 8. Prepare PCR Master Mix +- Mix reagents on ice in sterile conditions. +- Add 48 µL of Mastermix to each well of a 96-well PCR plate. + +### 9. Seal Plates +- Use PCR film, then centrifuge. + +### 10. Initiate PCR Reaction +- **Machine Recommended:** Bio-Rad C1000 Touch Thermal Cycler. +- **PCR Program for 16S, 18S, ITS:** + - Denature: 95°C, 2 min + - Denature: 95°C, 15s + - Anneal: 55°C (16S), 52°C (ITS), 30s + - Extend: 72°C, 30s + - Repeat 30 cycles + - Final Extension: 72°C, 10 min +- **PCR Program for rbcL:** + - Repeat above steps but 35 cycles, Final Extension for 5 min. + +## Gel Electrophoresis + +### 11. Verify PCR Products + +#### 11.1 Prepare Electrophoresis Tank and Casting Tray + +| Setup | Tank Type | Capacity | +|---------|-----------------|-------------------------------| +| Mini GT | 28 samples + ladder | 50 ml agarose mix | +| Midi GT | 58 samples + ladder | 100 ml agarose mix | +| Sub-Cell GT | 100 samples + ladder | 250 ml agarose mix | + +#### 11.2 Prepare 1% Agarose Gel +- **Solution:** 1g agarose powder in 100 ml 1X TBE buffer. +- **Dissolve:** Microwave for 2 min, cool to room temperature. + +#### 11.3 Add Ethidium Bromide or Gel Red +- Mix and pour into casting tray. + +#### 11.4 Place Combs and Allow to Set +- Set for 45 min. + +#### 11.5 Prepare Samples +- **Loading Buffer:** 5 µL of PCR product with 1 µL Bromophenol Blue. +- **Hyper Ladder:** Add 5 µL Hyper Ladder 1KB. + +#### 11.6 Load Samples and Run Electrophoresis +- Set to run for 45 min at 90V. + +#### 11.7 Imaging +- Use Bio-Rad Gel Doc System. + +## PCR Cleanup + +### 12. Cleanup Amplicon PCR Products +- **Kit Recommended:** ZymoZR-96 Kit. +- **Alternative:** MultiScreen-PCR96 Filter Plate. + +## Step II: Nextera Indexing PCR + +### 13. Use Cleaned PCR Product +- **Volume:** 1-10 µL, combined with reagents to 25 µL total volume. + +### Table 3: Step II PCR Reagents + +| Reagent | Per Sample | Per Plate | +|---------------------|------------|------------| +| Molecular Grade Water | 7.25 µL | 725 µL | +| 5X Buffer, High GC Buffer, 10 mM dNTPs | 5 µL each | 500 µL each | +| Q5 Taq Polymerase | 0.25 µL | 25 µL | +| Primer Array Mix | 5 µL | Added separately | +| PCR Template | 2 µL | Added separately | + +### 14. Index PCR Program +- **Machine Recommended:** Bio-Rad C1000 Touch Thermal Cycler. +- **Program Steps:** + - Denature: 95°C, 2 min + - Denature: 95°C, 15s + - Anneal: 55°C, 30s + - Extension: 72°C, 30s + - Repeat for 8 cycles. + +## Normalization + +### 15. Normalize PCR Products +- **Kit Recommended:** Norgen NGS Normalization 96-Well Kit. + +### 16. Use Normalized Product +- **Volume:** 5 µL from each sample to form pooled library. + +### Vacuum Concentrate +- **Volume Reduction:** 30 µL pooled library. + +### Gel Electrophoresis Validation +- **Agarose Gel Preparation:** 50 ml 1% agarose with wide comb. +- **Run Conditions:** 45 min, 90V. + +### DNA Extraction +- **Kit Recommended:** QIAquick Gel Extraction Kit by QIAGEN. + +## Quantification + +### 20. Quantify dsDNA Library +- **Kit Recommended:** Invitrogen Qubit 1X dsDNA HS Quantification Kit. + +### 21. Calculate Molar Concentration +- **Tools Recommended:** Bioline Calculator for Amplicon Sizes. + +## Sequencing on V2 or V3 MiSeq +- **Cartridge Preparation:** V3 600 cycle, thaw at room temperature. +- **NaOH Preparation:** 0.1 Molarity (fresh). +- **Library Dilution:** Detailed steps provided. +- **Load Reagent Cartridge:** 600 µL of final library solution in well 17. +- **Flow Cell Preparation:** Clean thoroughly, load into the MiSeq. +- **Run Parameters:** Ideal metrics provided for a 90% run. + +**Note:** Perform post-run wash as per manufacturer's recommendations. + +**endofoutput** +``` \ No newline at end of file diff --git a/markdown-output/image-based-3d-cell-culture-cytotoxicity-assay-72shqee.md b/markdown-output/image-based-3d-cell-culture-cytotoxicity-assay-72shqee.md new file mode 100644 index 0000000000000000000000000000000000000000..d816fee4a2d6e04ebea1cdb263dcce673df3fd96 --- /dev/null +++ b/markdown-output/image-based-3d-cell-culture-cytotoxicity-assay-72shqee.md @@ -0,0 +1,186 @@ +```markdown +# Goal/Experiment: +To explore an in vitro 3D cell culture system to study T cell-mediated cytotoxicity by using multicellular tumor spheroids and assess the effect of T cell manipulation on T cell cytotoxicity with potential to apply findings to a mouse model. + +# Image-based 3D Cell Culture Cytotoxicity Assay + +**Authors:** +Elinor Gottschalk, Bulent Arman Aksoy, Pinar Aksoy, Eric Czech, Jeff Hammerbacher +**Institution:** +Medical University of South Carolina + +*Hammer Lab* +**Tech Support:** +Phone: +1 843 792 4527 +Email: arman@hammerlab.org + +*Date:* October 14, 2019 + +**DOI:** [10.17504/protocols.io.72shqee](https://dx.doi.org/10.17504/protocols.io.72shqee) + +## Abstract +This protocol describes an image-based 3-dimensional (3D) cell culture cytotoxicity assay using multicellular tumor spheroids sensitized with hgp100 peptide and killing them with pmel-1 T cells or the apoptosis-inducing antibiotic, Staurosporine. Our goal is to explore an in vitro 3D cell culture system to study T cell-mediated cytotoxicity as a potential way to more rapidly and relevantly test the effect of T cell manipulation on T cell cytotoxicity before moving to the mouse model. + +In this setup, the T cells are required to migrate towards the tumor spheroids that are already suspended in a gel matrix. Minimal manipulation of the samples post-co-culture occurs because the samples are imaged directly in the chips to measure cell death, providing higher throughput than confocal imaging allows. + +We used 3D cell culture chips from AIM Biotech (Singapore) and adapted protocols published by: + +- AIM Biotech: [https://www.aimbiotech.com/general-protocols.html](https://www.aimbiotech.com/general-protocols.html) +- Pavesi, et al. "A 3D Microfluidic Model for Preclinical Evaluation of TCR-Engineered T Cells Against Solid Tumors." JCI Insight 2 (2017): 89762. [https://doi.org/10.1172/jci.insight.89762](https://doi.org/10.1172/jci.insight.89762) +- Jenkins, et al. "Ex Vivo Profiling of PD-1 Blockade Using Organotypic Tumor Spheroids." Cancer Discovery 8 (2018): 196-215. + +For image analysis, refer to: + +- Czech, et al. "Cytokit: A Single-Cell Analysis Toolkit for High Dimensional Fluorescent Microscopy Imaging." BMC Bioinformatics 20 (2019): 448. + +## Guidelines +We highly recommend reading the following protocols before starting: + +1. [AIM Biotech protocols on gel preparation and working with their 3D cell culture chips](https://www.aimbiotech.com/general-protocols.html) +2. [Pavesi et al., JCI Insight. 2017](https://doi.org/10.1172/jci.insight.89762) +3. [Jenkins et al., Cancer Discovery. 2018](https://doi.org/10.1158/2159-8290.CD-18-0135) + +For this assay, a fluorescence microscope with automation for multipoint imaging is required. We used the Keyence BZ-X710 system with a 20X objective, DAPI and Cy5 filters and brightfield, and a 20 μm step size. + +## Materials + +| Name | Catalog # | Vendor | +| --- | --- | --- | +| Staurosporine | S5921 | Sigma Aldrich | +| Sterile water | — | — | +| Ice & ice bucket | — | — | +| HyClone™ Dulbeccos High Glucose Modified Eagles Medium | SH30022FS | Fisher Scientific | +| Trypsin 0.05% 1X Solution | 16777-202 | VWR Scientific | +| Conical Tubes (50 mL) (racked) | AM12501 | Thermo Fisher | +| Collagen I Rat Tail | 354236 | Corning | +| CytoOne 10 cm TC dish | CC7682-3394 | USA Scientific | +| 100 μm cell strainer | 352360 | Fisher Scientific | +| 40 μm cell strainer | 22-363-547 | Fisher Scientific | +| 3D Cell Culture Chip | DAX-1 | AIM Biotech | +| 10X PBS pH 7.4 | 70011044 | Thermo Fisher Scientific | +| Phenol Red Sodium Salt | P4758 | Sigma Aldrich | +| 0.5 M NaOH | — | Sigma Aldrich | +| NucBlue™ Live ReadyProbes™ Reagent | R37605 | Thermo Fisher Scientific | +| SYTOX™ Red Dead Cell Stain | S34859 | Thermo Fisher Scientific | +| Recombinant Murine I-TAC (CXCL11) | 250-29 | Peprotech | +| Interleukin-2 human | I2644 | Sigma Aldrich | + +## Before Starting + +1. If conducting a T cell-mediated cytotoxicity assay, decide which day post-activation the T cells will be co-cultured with the tumor spheroids and determine how many T cells will be needed. Accordingly, activate an appropriate number of T cells however many days in advance is needed. +2. Expand MC38 cells in culture. The number of 3D cell culture chips that will be used for the experiment will determine how many MC38 cells you will need when plating the cells for spheroid formation. For 10 cm dish, 2 million cells are needed. For a good density of spheroids in the collagen gel, you will need 50 μl of gel per 10 cm dish and 30 μl gel per 3D culture chip. Because the gel is viscous and some gets lost when mixing, make twice as much as you think you need. +3. Reconstitute the CXCL11 as per the manufacturer’s directions. +4. On the day of co-culture: + - Collect reagents in an ice bucket with ice that fits into the hood for preparing collagen mixture (Collagen type I, 10X PBS with phenol red, 0.5 M NaOH, sterile water). + - Calculate how much collagen gel will be needed. The amounts listed in the protocol are for 1 ml collagen gel mixture. We were usually harvesting 10 x 10 cm tissue cultures dishes of spheroids and resuspending the spheroid pellet in 500 μl of gel to get a good density of spheroids. + +## Procedure + +### 1. Preparing to culture the T Cells + +1. If using T cells to kill the tumor spheroids, start culturing them and activate them. Maintain the cells, supplementing the culture media and splitting when necessary, until the desired day of co-culture. + +### 2. The day before co-culture + +Prepare MC38 cells to form spheroids: +- Make a single cell suspension of MC38 cells at 2.5 x 10^6 cells per ml (in DMEM, 10% FBS, and 1% pen/strep). +- With a multichannel pipettor, pipet 5 rows of 20 μl droplets of cell suspension on the inner surface of a 10 cm tissue culture dish lid (40 droplets per dish). + - Pour 5-10 ml of sterile water in the bottom half of the dish (to humidify the chamber and stop the droplets from drying out). + - Carefully invert the lid with the droplets over the bottom half of the dish (try not to bump the lid while picking it up and turning it over or the droplets will flatten out and the spheroids will not grow uniformly). +- Incubate the droplets overnight at 37°C 5% CO2. + +### 3. Day of co-culture + +Harvest the droplets containing MC38 spheroids: +- Working in the hood, flip the lid upside down to expose the droplets. + - Harvest the spheroids using a p1000 and 2 ml DMEM per dish lid and transfer into a 50 ml tube. + - Pass the spheroid suspension through a 40 μm nylon mesh filter. + - Invert the 40 μm filter over a new 50 ml tube and wash the retained spheroids into the tube. + - Pass the spheroid suspension through a 100 μm filter over a new 50 ml tube (collect the flow through). + - Centrifuge the spheroid suspension (now containing spheroids between 40 and 100 μm) at 200 x g for 5 minutes. + +Make the collagen gel while the spheroid suspension is centrifuging: +- Perform all steps on ice. + +#### Recipe for 1 ml collagen gel: + +| Component | Volume | +| --- | --- | +| 10X PBS + phenol red | 95 μl | +| Collagen (3.9 mg/ml) | 641 μl | +| 0.5M NaOH | 26 μl (this may change) | +| dH2O | 157 μl | +| CXCL11 (stock at 0.1 mg/ml) | 27 μl | +| IL-2 | 4 μl | +| Spheroid pellet | about 50 μl | + +- Mix the above components in a sterile eppendorf tube on ice in the hood. Mix by pipetting up and down and avoid adding air bubbles to the mixture. Mix until the color from the phenol red is uniform. To check the pH, make sure the color is red (not yellowish or purplish). If the mix is slightly yellow, add 0.5 M NaOH in 1 μl increments, mixing well in between, until the color is red. If the mixture goes too far into the purple, it's easier to start over. + +Seed the 3D cell culture chips: +- Each 3D cell culture chip has 3 channels. We used 1 chip per condition, which resulted in 3 replicates per condition. +- Carefully aspirate the DMEM from the spheroid pellet. Try to aspirate as much liquid as possible. +- Resuspend the spheroid pellet with collagen mix. Test the density of spheroids by pipetting a 10 μl droplet onto a tissue culture dish and checking on the microscope. Add more gel mix if the spheroids look too dense. We have found that 50 μl gel mixture per 10 cm dish worth of spheroids works well. +- Pipette 10 μl into the central channels in the 3D cell culture chips. Pipette very slowly so that the gel doesn't escape into the side channels. Pipette into the top port until the gel reaches halfway and then pipette into the bottom port until the gel fills the central channel. +- If using the chip holders from AIM Biotech, add sterile water to the chambers to humidify the chips while in the holder. +- Incubate the seeded chips at 37°C 5% CO2 for 30:00:30:00 to allow the gel to polymerize. + +### 4. Prepare the T cells or drug treatment dilutions while the gel is polymerizing. + +For the T cells: +- Count and harvest the desired number of T cells. + - The T cells can be stained with a marker at this point, if desired. (We have used IncuCyte Rapid live cell labeling with success). + - Centrifuge the T cells at 350 x g for 5 mins. + - Resuspend the T cells in T cell media supplemented with IL-2 (200 IU/ml) to obtain the desired concentration (we were using T cells at various concentrations so serially diluted them from 4 million as our highest concentration). + +For the Staurosporine treatment: +- Dilute Staurosporine stock to desired concentrations in DMEM (supplemented with 10% FBS and 1% pen/strep). + +### 5. Add the T cells or drug treatment to the 3D cell culture chips. + +- Remove the chips from the incubator and place in the tissue culture hood. + +#### For the co-culture with T cells: +- Carefully and slowly pipette 20 μl of T cell media supplemented with IL-2 (200 IU/ml) and with or without hgp100 peptide (2 μM) into the top port of the right side channel for each channel in the chip (we had conditions without the peptide as a negative control for our assays). Add 50 μl of the same media to the top and bottom ports of the same side channel. +- To the left side channel, carefully and slowly pipette 20 μl of the T cell suspension in. Make sure to mix the T cell suspension before each addition (the T cells tend to settle quite quickly). Do not top off the ports on this side channel because it can displace the T cells. + +#### For the Staurosporine-treated samples: +- Carefully and slowly pipette 20 μl of the Staurosporine diluted in DMEM into the left and right side channels in each chip. Top off the 4 side channel ports with 50 μl of the same media. + +Replace all chips in the incubator in their humidified chip holders for overnight incubation. + +### 6. Day of imaging. + +Make staining solution: + +| Component | Volume | +| --- | --- | +| 5 ml PBS | — | +| 20 drops of NucBlue | — | +| 18 μl of SYTOX red dead cell stain | — | + +Stain one chip at a time: +- Remove any media in troughs around the side channel ports (don't try and suck media directly out of the port or you run the risk of pulling the gel out). +- Gently pipette 20 μl of staining buffer directly into the top port of each of the side channels and watch it push the T cells or media out the bottom port of the channel. +- Remove any media from bottom port troughs. +- Gently pipette 50 μl of stain into the top port of each side channel until it comes out the bottom port. +- Incubate the chip for 30 mins at room temperature, protected from light. + +Image: +- Set exposure times for each channel based on the positive control chip (in our case this was the one containing the Staurosporine-treated spheroids) and use the same settings for all other samples: + - CH1=Cy5 (SYTOX red for any dead cells). We found that the SYTOX dye tended to photobleach if it was imaged after the DAPI channel so we imaged it first. + - CH2=DAPI + - CH3=brightfield +- Set a 3x11 grid for each channel (and set multipoint imaging for the 3 channels on each chip). +- We used the high-resolution setting (but this may not be necessary). +- With these settings, it took about 30 mins to image each chip. + +Once the imaging has started for one chip, start staining the next chip to keep the staining time consistent across conditions. + +### Image Analysis + +For image analysis, refer to the Cytokit repository (https://github.com/hammerlab/cytokit). Methods detailed in: + +- Czech, Eric, Bulent Arman Aksoy, Pinar Aksoy, and Jeff Hammerbacher. 2019. "Cytokit: A Single-Cell Analysis Toolkit for High Dimensional Fluorescent Microscopy Imaging." BMC Bioinformatics 20 (1): 448. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/image-processing-to-investigate-nemo-recruitment-a-cbvjsn4n.md b/markdown-output/image-processing-to-investigate-nemo-recruitment-a-cbvjsn4n.md new file mode 100644 index 0000000000000000000000000000000000000000..501ea23639b48d7a9eff1ad97ea87fab23dbdd5d --- /dev/null +++ b/markdown-output/image-processing-to-investigate-nemo-recruitment-a-cbvjsn4n.md @@ -0,0 +1,142 @@ +```markdown +# Goal/Experiment: +Image processing to investigate NEMO recruitment and involvement in mitophagy and inflammatory signaling. + +### **Title:** +Image processing to investigate NEMO recruitment and involvement in mitophagy and inflammatory signaling + +**Authors:** +- Olivia Harding1, 2 +- Erika L.F. Holzbaur1, 2 + +**Affiliations:** +- 1Department of Physiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104 +- 2Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD, 20815 + +**DOI:** [10.17504/protocols.io.n2bvj61xxlk5/v1](https://dx.doi.org/10.17504/protocols.io.n2bvj61xxlk5/v1) + +**License:** +This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. + +**Protocol status:** Working. We use this protocol and it’s working. + +--- + +## **Abstract:** +Beautiful images are not sufficient to robustly characterize and interrogate a cellular mechanism. In order to show recruitment of NEMO and its relationship to OPTN and other mitophagy factors, we processed hundreds of still and timelapse images and extracted quantifiable data to perform statistical analysis comparing different conditions. Our approach to image analysis involved the usage of various software tools to deconvolve confocal fluorescent images and carry out particle analysis on putatively overlapping structures. + +--- + +## **Guidelines:** +- This protocol addresses multiple image analysis pipelines used in the corresponding manuscript to investigate NEMO and its role in mitophagy. +- We developed several methods for quantitative analysis with our varying experiments. + +--- + +## **Materials:** + +### **Equipment/software:** +- ImageJ/FIJI +- Ilastik +- Deconvolution software such as Huygens +- Excel + +--- + +### **Before Start Instructions:** +1. For each experiment, collect images for at least 10 cells from at least three biological replicates. +2. Maintain consistent imaging parameters by saving and/or recording laser powers and exposure times. + +--- + +## **Protocol Steps:** + +### **Blinded Whole-cell Recruitment Assessment:** + +1. **Note:** + - This method was used to assess NEMO recruitment in cells before and after AntA/OligA treatment, in the presence or absence of Parkin or p62. +2. Start with Z stack images of live cells collected with a 60X objective. +3. Max project the Z stack fields of view by ~2 µm to capture the center to upper half of the cell. +4. Crop each cell and save only the NEMO channel with a file name to identify the conditions. +5. Copy cropped NEMO channel images to a new folder. +6. Use a random number generator such as [Random.org](https://Random.org/strings/) to generate a number of strings equivalent to the total number of cells. +7. Copy the strings to a document and save. +8. Rename the copied images in the new folder as the random strings. +9. Arrange files in the folder by file name in order to randomly mix images. +10. Open newly named files and judge whether there is NEMO recruitment or not, recording YES or NO for each image by its random string file name. +11. After categorizing every image, unblind the results and determine the number of cells marked YES for each condition compared to the total for that condition. + +### **Mitochondrial Recruitment Assay:** + +12. **Note:** + - This method was used to assess percentages of mitochondria that had recruited NEMO, OPTN, GABARAPs, and/or LC3B in various conditions. +13. Start with Z stack images of cells collected with a 60X objective. +14. Crop and save each cell visible in the field of view. +15. Deconvolve images with Huygens or similar deconvolution software. +16. Max project each cell ~2 µm. +17. Split channels and save each channel. +18. Import 5-7 channels displaying mitochondria into the Ilastik software for segmentation. +19. Train algorithm to recognize "mitochondria" or "not mitochondria". + - **Note:** If there are cells with poor expression or labeling of the mitochondria, these may be discarded. +20. Generate binary images for all mitochondria channels. + 1. Import segmented Ilastik results to FIJI. + 2. Use the original image file to draw an ROI outlining the cell. + 3. Clear Outside the cell ROI on the segmented image. + 4. Run the threshold function with threshold set to (255,255). + 5. Save binary image. +21. Repeat previous 3 steps for NEMO puncta and OPTN rings/puncta. +22. GABARAPs and LC3B antibodies do not produce high enough signal-to-noise to segment with Ilastik. For these experiments, only segment NEMO and mitochondria manually. In FIJI, draw ROI’s around GABARAPs- or LC3B-positive mitochondria by hand. Save ROIs. +23. To determine the % of mitochondria that recruited NEMO, + 1. Open the binary mitochondria image in FIJI. + 2. Use the Analyze Particles function to generate a mask of binary image, filtering particles to exclude those fewer than 5 pixels. + 3. Summarize and add to manager. + 4. Record the total number of mitochondria in Excel. + 5. Open binary image of NEMO channel. + 6. Use the Analyze Particles function to generate a mask of binary image, filtering particles to exclude those fewer than 5 pixels, and add particles to manager. + 7. Project NEMO particles to Mito binary channel and multimeasure. + 8. Copy results to Excel. + 9. The count of particles with an average intensity of >127.5 is the number of mitochondria positive for NEMO. + 10. Divide this number by the total number of mitochondria to calculate the proportion. +24. To determine the % of mitochondria that recruited NEMO and/or OPTN. + 1. Repeat the first four steps above to record total number of mitochondria. + 2. Load the binary OPTN channel to FIJI and Fill Holes. + 3. Project mitochondrial particle ROIs onto OPTN binary image and multi-measure. + 4. Record results in Excel. + 5. The count of particles with an average intensity of >127.5 is the number of mitochondria positive for OPTN. + 6. Delete mitochondria particles from the manager and add NEMO particles. + 7. Project NEMO particles onto mitochondria and perform the same calculation. +25. Use the image calculator to add the mitochondria channel to the OPTN channel with Filled Holes. + 1. Use Math > Subtract to subtract 255 and float the resulting image. + 2. Convert to an 8-bit image. + 3. Project NEMO particles to this new image and multi measure. + 4. Record results in Excel. + 5. The count of particles with an average intensity greater or equal to 64 is the count of mitochondria that are positive for both NEMO and OPTN. +26. To determine the % of mitochondria that recruited NEMO and/or GABARAPs or LC3B. + 1. Use the Image Calculator to add NEMO particles to Mitochondria particles. + 2. Use Math > Subtract to subtract 255 and float the resulting image. + 3. Convert to an 8-bit image. + 4. Project hand-drawn ROIs to resulting image and multi measure. + 5. Record results in Excel. + 6. The count of particles with an average intensity greater or equal to 64 is the count of mitochondria that are positive for both NEMO and GABARAPs or LC3B. +27. Use the R code library(`eulerr`) to generate Euler diagrams of the resulting data. + +### **Recruitment Assay:** + +28. **Note:** + - This method was used to quantify the extent of NEMO, OPTN, and p62 recruitment over time. +29. Start with confocal timelapse images in which the timepoint with added AntA/OligA is known. +30. Choose several events per cell in which the mitochondria stays in the field of view for a majority of the timecourse and the protein of interest is recruited. +31. For each time point, use the mitochondria channel to draw a generous ROI around the mitochondria. + - **Note:** This will capture rings or puncta that are recruited. +32. Measure the intensity of NEMO and/or OPTN or p62 for every ROI and record in Excel. +33. Calculate the 5-frame moving average across the timecourse for each channel. +34. Calculate the background intensity by averaging the moving average intensity of the first ten frames of the timecourse. +35. Subtract the background from every frame. +36. Determine the maximum intensity for the event. +37. Normalize the timecourse intensities to the max intensity so that the intensity of each frame is a percentage of the max intensity. +38. Calculate the half-max timepoint based on when the normalized intensity surpasses 50%. + +--- + +`endofoutput` +``` \ No newline at end of file diff --git a/markdown-output/immunocytochemistry-for-the-characterization-of-hi-c74zzqx6.md b/markdown-output/immunocytochemistry-for-the-characterization-of-hi-c74zzqx6.md new file mode 100644 index 0000000000000000000000000000000000000000..85fecbd896fb53a57c9bff558f697fa443475ca9 --- /dev/null +++ b/markdown-output/immunocytochemistry-for-the-characterization-of-hi-c74zzqx6.md @@ -0,0 +1,144 @@ +```markdown +# Goal/Experiment: +The objective of this experiment is to perform immunocytochemistry for the characterization of hiPSC to Motor Neuron differentiation using specific biomarkers. + +## Immunocytochemistry for the Characterization of hiPSC to Motor Neuron Differentiation V.3 + +**Authors**: +Mallory Wright¹, ckremitz¹, William J Buchser² + +¹Washington University, Saint Louis. McDonnell Genome Institute (MGI) +²Washington University in St. Louis + +**Version**: 3 +**Date**: January 24, 2024 + +**DOI**: [dx.doi.org/10.17504/protocols.io.6qpvr3bzvmk](https://dx.doi.org/10.17504/protocols.io.6qpvr3bzvmk/v3) + +**Protocol Citation**: +Mallory Wright, ckremitz, William J Buchser 2024. Immunocytochemistry for the characterization of hiPSC to Motor Neuron differentiation. protocols.io [https://dx.doi.org/10.17504/protocols.io.6qpvr3bzvmk/v3](https://dx.doi.org/10.17504/protocols.io.6qpvr3bzvmk/v3) Version created by Mallory Wright + +## Abstract +This immunocytochemistry protocol is used for the characterization of iPSC differentiation into motor neurons using several biomarkers: neuroepithelial cells (SOX1), motor neuron progenitors (OLIG2 and NKX2.2), motor neurons (MNX1), and the mature motor neurons (ISL, ChAT, MAP2). + +## Materials + +- **BlockAid™ Blocking Solution**: Thermo Fisher, Catalog #B10710 +- **Phosphate-buffered saline (PBS, 1X), sterile-filtered**: Thermo, Scientific Catalog #J61196.AP +- **Triton X-100**: Merck MilliporeSigma (Sigma-Aldrich), Catalog #T8787-50ML +- **Bovine Serum Albumin**: Merck MilliporeSigma (Sigma-Aldrich), Catalog #A4612 +- **32% Paraformaldehyde**: Electron Microscopy Sciences, Catalog #50-980-495 + +## Primary Antibody Stains and Concentrations + +### Step 1: +- **SOX1 (goat)**: R&D Systems, Catalog #AF3369 + - Reconstitute in 500 µL of sterile 1xPBS (200 µg/µL concentration) + - Use 15 µg/mL, add 75 µL to 1 mL BSA. Needs Anti-Goat Secondary. + +### Step 2: +- **OLIG2 (rabbit)**: Sigma, Catalog #AB9610 (0.5 mg/mL) + - Use 1.2 µg/mL (1:400 dilution), add 2.5 µL to 1 mL BSA. Needs Anti-Rabbit Secondary. + +- **NKX2 (mouse)**: DSHB, Catalog #74.5A5 (23 ng/µL) + - Use 2 µg/mL, add 86 µL to 1 mL BSA. Needs Anti-Mouse Secondary. + +### Step 3: +- **MNX1 (mouse)**: DSHB, Catalog #81.5C10 (36 ng/µL) + - Use 2 µg/mL, add 55 µL to 1 mL BSA. Needs Anti-Mouse Secondary. +- **MNX1 (2nd option) (rabbit)**: Novus Biological, Catalog #NBP224691 (0.1 mg/mL) + - Use 2 µg/mL, add 4 µL to 1 mL BSA. Needs Anti-Rabbit Secondary. + +### Step 4: +- **MAP2 (rabbit)**: Sigma, Catalog #M3696 (1.0 mg/mL) + - Use 2.5 µg/mL (1:400 dilution), add 2.5 µL to 1 mL BSA. Needs Anti-Rabbit Secondary. +- **MAP2 (2nd option)**: Thermo Scientific, Catalog #PA5-17646 (73.6 µg/mL) + - Use 0.74 µg/mL (1:100 dilution), add 10 µL to 1 mL BSA. Needs Anti-Rabbit Secondary. + +### Step 5: +- **ChAT (goat)**: Sigma, Catalog #AB144P (0.1 - <1%) + - Use 1 µg/mL (1:100 dilution), add 10 µL to 1 mL BSA. Needs Anti-Goat Secondary. +- **ISL1 (mouse)**: DSHB, Catalog #40.2D6 (28 ng/µL) + - Use 2 µg/mL, add 70 µL to 930 µL BSA. Needs Anti-Mouse Secondary. + +## Secondary Antibody Stains and Concentrations + +**Note:** Use volumes based on a total volume of 1 mL 3% BSA staining solution. Adjust volumes as needed. + +- **Alexa Fluor Plus 488 Donkey Anti-Rabbit IgG (H+L)** (ThermoFisher A32790 - 1 mg in 2 mL stock) + - Use 2 µg/mL, add 1 µL to 1 mL BSA. + +- **Alexa Fluor 555 Donkey Anti-Goat IgG (H+L)** (ThermoFisher A21432 - 1 mg in 2 mL stock) + - Use 10 µg/mL, add 5 µL to 1 mL BSA. + +- **Alexa Fluor 647 Donkey Anti-Mouse IgG (H+L)** (ThermoFisher A31571 - 1 mg in 2 mL stock) + - Use 2 µg/mL, add 1 µL to 1 mL BSA. + +## Before Start Instructions +Ensure the observed results are not random events by using controls, such as undifferentiated hiPSCs during immunocytochemistry. Below is an example of how to seed cells onto a 96-well plate, with equal amounts of wells of differentiated (blue) and undifferentiated cells (yellow). + +![96-well plate setup](https://example.com/image.png) + +## Procedure + +### Step 1: Remove the Medium +- Remove the medium from your cells by tipping the vessel towards you and pipetting from the bottom corner of the well. + +### Step 2: Fixation +- Dilute 32% Paraformaldehyde solution to 4% PFA in 1X PBS. +- Add 100 µL of 4% PFA to each well in the 96-well plate. Incubate for 15 minutes at room temperature. + +### Step 3: Wash +- Remove the fixative solution and wash with 1X PBS at 100 µL per well using a multichannel pipette. Repeat 3 times. + +### Step 4: Permeabilization +- Add 100 µL of 0.5% Triton X-100 to each well (1 mL if using a 6-well plate). +- Incubate for 15 minutes at room temperature. +- Remove the permeabilization solution and wash 3 times with 1X PBS. + +### Step 5: Blocking +- Add 100 µL of 3% BSA or BlockAid solution to each well to block. (1 mL if using a 6-well plate). +- Incubate for at least 1 hour (up to overnight) at room temperature. + +### Step 6: Primary Antibodies +- Calculate the amount of primary antibody needed (located in materials section) and dilute in 3% BSA + 0.3% Triton X-100 solution. +- Remove 3% BSA from wells and add 100 µL of primary antibody per well. +- Incubate for 1 hour at room temperature in a dark place or overnight at 4°C. + +### Step 7: Wash +- Remove primary antibody and wash 3 times slowly with 1X PBS. + +### Step 8: Secondary Antibodies +- Calculate the amount of secondary antibody needed (located in materials section) and dilute in 3% BSA + 0.3% Triton X-100 solution. +- Add 100 µL per well in 96-well and incubate for at least 1 hour at room temperature in a dark place. + +### Step 9: Wash +- Remove secondary antibody and wash 3 times with 1X PBS. + +### Step 10: Imaging +- Scan on the confocal microscope. + - We currently use the ImageXpress Confocal HT.ai High-Content imaging system and the InCarta image analysis software to assess specific protein presence and quantify the percent of live nuclei. + +## Expected Results + +### Neuroepithelial Cells +![SOX1](https://example.com/image1.png) +SOX1/anti-goat (green) and Hoechst (blue) + +### Neural Progenitors +![OLIG2](https://example.com/image2.png) +OLIG2/anti-rabbit (green) and Hoechst (blue) + +### Motor Neurons +![MNX1](https://example.com/image3.png) +MNX1/anti-mouse (red) and Hoechst (blue) + +### Mature Motor Neurons +![ISL1](https://example.com/image4.png) +ISL/anti-mouse (red) and Hoechst (blue) + +![MAP2](https://example.com/image5.png) +MAP2/anti-rabbit (green) and Hoechst (blue) + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/immunofluorescence-staining-of-heparan-sulfate-hs-kvycw7w.md b/markdown-output/immunofluorescence-staining-of-heparan-sulfate-hs-kvycw7w.md new file mode 100644 index 0000000000000000000000000000000000000000..b099733b49dd03d6d405fb584aa9cca16b147672 --- /dev/null +++ b/markdown-output/immunofluorescence-staining-of-heparan-sulfate-hs-kvycw7w.md @@ -0,0 +1,120 @@ +```markdown +# Goal/Experiment: +Immunofluorescence staining of heparan sulfate (HS) in islet beta cells of formalin-fixed human pancreas and isolated islets. + +## Immunofluorescence Staining of Heparan Sulfate (HS) in Islet Beta Cells of Formalin-Fixed Human Pancreas and Isolated Islets + +### Sarah Popp, Charmaine Simeonovic + +### Abstract + +Paraffin sections of formalin-fixed human pancreas and isolated human islets were treated with 0.05% pronase for antigen retrieval, blocked with 2% bovine serum albumin (BSA; Sigma) in phosphate-buffered saline (PBS), incubated overnight (4°C) with 10E4 (anti-HS) mAb (1/10; US Biological/Amsbio), washed and stained with AlexaFluor 488-goat anti-mouse IgM (Thermo Fisher). The same sections were washed, incubated with rabbit anti-human glucagon IgG (Abcam) or guinea-pig anti-pig insulin Ig (Dako), washed and stained with AlexaFluor 568-donkey anti-rabbit IgG or AlexaFluor 568-goat anti-guinea-pig IgG (Thermo Fisher). The specificity of HS staining was checked on serial sections using IgMκ isotype control (BD Biosciences), instead of 10E4 mAb, together with anti-glucagon or anti-insulin antibody. Nuclei were stained with DAPI (0.2 μg/ml; Sigma). Sections were photographed using an automated Axio Observer inverted fluorescence microscope (Zeiss). Merged images were prepared using ZEN (version 2.3) software (Zeiss). + +### Guidelines + +- 10E4 anti-heparan sulfate (HS) mAb identifies highly sulfated HS localized in human beta cells but does not identify the less sulfated HS in alpha cells. + +#### Reference: +- Theodoraki A, Hu Y, Poopalasundaram S et al (2015) Mol Cell Endocrinol 399: 296-310. + +## Before Start + +### Materials + +1. **Prepare Graded Alcohols and Xylene for Deparaffinizing Tissue Sections**: + - 2 x xylene (250 ml/slide container) + - 2 x absolute ethanol (250 ml/slide container) + - 1 x 90% ethanol (250 ml) + - 1 x 70% ethanol (250 ml) + +2. **Prepare 2% Bovine Serum Albumin (BSA) in Phosphate-Buffered Saline (PBS)**. + +3. **Mabs and pAbs**: + - 10E4 (anti-HS) mAb, Amsbio #370255-1 + - Goat anti-mouse IgM AF488, Thermo Fisher #A21042 + - Polyclonal guinea pig anti-pig insulin, DAKO #A0564 + - Rabbit polyclonal anti-human glucagon, Abcam #ab133195 + - Goat anti-guinea pig IgG AF568, Thermo Fisher #A11075 + - Donkey anti-rabbit IgG AF568, Thermo Fisher #A10042 + - IgMκ, BD Biosciences #550340 + +4. **Other Reagents**: + - Pronase, Calbiochem #537088 + - Bovine serum albumin, Sigma #A3294 + - DAPI, Sigma #D9524 + - ProLong Diamond Antifade Mountant, Thermo Fisher #P36961 + +### Protocol + +#### Step 1 +See Guidelines, 'Before starting'. + +#### Step 2 +Deparaffinize slides in each xylene for 1 min (see Guidelines). Rehydrate slides in graded alcohols beginning in absolute ethanol (10 dips/ container of absolute ethanol), followed by 90% ethanol (10 dips) and 70% ethanol (10 dips). Wash well in running tap water for 5 min. + +#### Step 3 +Wipe around sections with a tissue, encircle the sections using a diamond pencil and place in a slide container of tap water (250 ml). + +#### Step 4 +Prewarm slide tray containing low level of water (to humidify) in 37°C incubator. + +#### Step 5 +Prepare pronase (#537088 Calbiochem; for antigen retrieval to expose HS epitopes): 2.5 mg pronase in 5 ml de-ionized water. + +#### Step 6 +Wipe around sections using tissue and cover each section with pronase solution. Return humidified slide tray to 37°C incubator for 10 min. + +#### Step 7 +Wash sections with phosphate-buffered saline (PBS), 3 x, then 3 x 5 min in slide container containing 250 ml PBS with agitation of slides at 0, 2, and 5 min of each wash. + +#### Step 8 +Block sections with 2% bovine serum albumin (BSA) in PBS at room temperature for 30 min. + +#### Step 9 +Wash sections with PBS, 3 x, then 3 x 5 min in slide container containing 250 ml PBS with agitation of slides at 0, 2, and 5 min of each wash. + +#### Step 10 +Apply primary 10E4 anti-HS mAb, 100 μg/ml in 2% BSA/PBS, 125 μl/section. Incubate overnight at 4°C in a humidified tray (containing PBS). + +#### Step 11 +Wash sections with PBS, 3 x, then 3 x 5 min in slide container containing 250 ml PBS with agitation of slides at 0, 2, and 5 min of each wash. + +#### Step 12 +Apply secondary goat anti-mouse IgM AF488 (Thermo Fisher #A21042), 20 μg/ml with 2% BSA/PBS), 125 μl/section, and incubate for 30 min at room temperature. + +#### Step 13 +Wash sections with PBS, 3 x, then 3 x 5 min in slide container containing 250 ml PBS with agitation of slides at 0, 2, and 5 min of each wash. + +#### Step 14 +Apply anti-insulin or anti-glucagon pAb: + +1. For insulin staining, apply polyclonal guinea pig anti-insulin (DAKO #A0564), 130 μg/ml in 2% BSA/PBS, 125 μl/section, and incubate for 30 min at room temperature. + +2. For glucagon staining, apply rabbit polyclonal anti-glucagon (Abcam #ab133195), 10 μg/ml in 2% BSA/PBS, 125 μl/section, and incubate for 30 min at room temperature. + +#### Step 15 +Wash sections with PBS, 3 x, then 3 x 5 min in slide container containing 250 ml PBS with agitation of slides at 0, 2, and 5 min of each wash. + +#### Step 16 +Apply secondary antibodies for anti-insulin or anti-glucagon pAb: + +1. For insulin staining, apply goat anti-guinea pig IgG AF568 (Thermo Fisher #A11075), 10 μg/ml dilution in 2% BSA/PBS, 125 μl/section, and incubate for 30 min at room temperature. + +2. For glucagon staining, apply donkey anti-rabbit IgG AF568 (Thermo Fisher #A10042), 4 μg/ml in 2% BSA/PBS, 125 μl/section, and incubate for 30 min at room temperature. + +#### Step 17 +Stain sections with DAPI (Sigma #D9524), 0.2 μg/ml in PBS for 2 min. + +#### Step 18 +Wash sections with PBS, 3 x, then 1 x 5 min in slide container containing 250 ml PBS with agitation of slides (10 x) at 0, 2.5 min and 5 min. + +#### Step 19 +Mount slides in ProLong® Diamond Antifade Mountant (Thermo Fisher #P36961). + +#### Step 20 +Image sections using an automated Axio Observer inverted fluorescence microscope (Zeiss). Prepare merged images using ZEN (version 2.3) software (Zeiss). + +--- + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/immunohistochemistry-sppedmn.md b/markdown-output/immunohistochemistry-sppedmn.md new file mode 100644 index 0000000000000000000000000000000000000000..280a52c44448d9133a4005e0729e8ccc1724c3be --- /dev/null +++ b/markdown-output/immunohistochemistry-sppedmn.md @@ -0,0 +1,159 @@ +```markdown +# Goal/Experiment: +Perform immunohistochemistry on murine and human tumor samples. + +# Immunohistochemistry Protocol + +## Authors +Kristin Anderson1, Sunni Farley2 +1. University of Washington and Fred Hutchinson Cancer Research Center +2. Fred Hutchinson Cancer Research Center + +*Date: Jun 30, 2021* + +DOI: [dx.doi.org/10.17504/protocols.io.sppedmn](http://dx.doi.org/10.17504/protocols.io.sppedmn) + +## Abstract +This protocol outlines the steps used to perform immunohistochemistry on murine and human tumor samples. + +**In Brief:** +- Study animals underwent full necropsies. +- Harvested tissues were fixed in 10% neutral buffered formalin for at least 48 hours, embedded in paraffin, and sectioned (4mm). +- Sections were stained with hematoxylin and eosin (H&E) or primary antibodies for markers of interest. +- Sections were baked, deparaffinized, and antigen retrieval was performed. +- Slides were blocked and stained, imaged, and analyzed. + +## Equipment Needed +- Sakura Tissue-Tek VIP6 AI Vacuum Infiltration Processor +- Sakura DRS2000 H&E Stainer (and staining racks/arms) +- Leica Bond Rx Autostainer + +## Materials +- Bond Wash 10x (Leica Biosystems, Catalog #AR9590) +- Bond Polymer Refine Detection Kit (Leica Biosystems, Catalog #DS9800) +- PowerVision Polymer anti-Mouse IgG HRP (Leica Biosystems, Catalog #DPVM-110HRP) +- Primary Antibody Diluent BOND (Leica Biosystems, Catalog #AR9352) + +## Grossing and Specimen Processing + +1. **Grossing:** + - Gross one specimen at a time at the Mopec grossing station. + - Verify the cassette information to the specimen ID on the container. + - Dispose of contaminated biohazard bags in approved biohazard containers. + - Load the cassette holding rack into Sakura Tissue-Tek VIP6 AI. + - Process samples on Program 8 (detailed below). + +2. **Program Details:** + ``` + Program Number: 8 Hour Processing + Initial Step | Reagent | Temp (°C) | Time + ------------ | ------------ | ----------| ------- + 3 | 75% Ethanol | -- | 40:00 min + 4 | 95% Ethanol | -- | 40:00 min + 5 | 95% Ethanol | -- | 40:00 min + 6 | 100% Ethanol | -- | 40:00 min + 7 | 100% Ethanol | -- | 40:00 min + 8 | Clear-Rite | -- | 40:00 min + 9 | Clear-Rite | -- | 40:00 min + 10 | Clear-Rite | -- | 40:00 min + 11 | Paraffin | 60°C | 40:00 min + 12 | Paraffin | 60°C | 40:00 min + 13 | Paraffin | 60°C | 40:00 min + 14 | Paraffin | 60°C | 40:00 min + ``` + +3. **Embedding:** + - Ensure paraffin reservoir is full. + - Transfer molten paraffin if needed. + - Remove samples from processor and transfer to warming reservoir. + - Embed samples immediately. + - Trim FFPE blocks using Wax Trimmer. + +## Cutting FFPE Slides +4. **Prepare for Cutting:** + - Turn on water bath, fill with distilled water at 37°C. + - Bring Histocool block from freezer, fill with distilled water. + - Match FFPE blocks with the corresponding RFS form and slides. + - Place blocks on HistoCool block, soaking for ~20 minutes. + - Face each block for a full tissue section. + - Place sections on drying rack, air-dry overnight. + +## H&E Staining +5. **Staining Process:** + - Load slides into the black H&E slide staining rack. + - Open DRS2000 stainer loader and place the staining arm. + - Press F1 [START], select F FPE H&E and confirm. + - Press F1 [START] to begin the staining process. + - Transfer stained slides using the Xylene buckets. + +6. **Staining Details:** + ``` + Program Name: FFPE H&E + Step | Reagent | Time + ---- | --------------- | ------- + 1 | Xylene | 3:00 min + 2 | Xylene | 3:00 min + 3 | Xylene | 3:00 min + 4 | 100% Ethanol | 2:00 min + 5 | 100% Ethanol | 2:00 min + 6 | 95% Ethanol | 2:00 min + 7 | 95% Ethanol | 2:00 min + 8 | 80% Ethanol | 2:00 min + 9 | Water (Rinse) | 1:00 min + 10 | Hematoxylin | 10:00 min + 11 | Water (Rinse) | 1:00 min + 12 | Clarifier | 0:10 min + 13 | Water (Rinse) | 2:00 min + 14 | Bluing Solution | 0:30 min + 15 | Water (Rinse) | 4:00 min + 16 | 95% Ethanol | 1:00 min + 17 | Eosin Y | + 18 | Phloxine | 0:30 min + 19 | 95% Ethanol | 1:00 min + 20 | 95% Ethanol | 1:00 min + 21 | 100% Ethanol | 2:00 min + 22 | 100% Ethanol | 2:00 min + 23 | 100% Ethanol | 2:00 min + 24 | Xylene | 3:00 min + 25 | Xylene | 3:00 min + ``` + +7. **Cover Slipping:** + - Coverslip using Cytoseal XYL and VWR 24x40 coverglass. + - Place slides in QC tray to dry overnight. + +## IHC Staining Protocol – Mouse Polymer DAB +8. **Staining:** + ``` + Step | Reagent | Dispense Amount | Time + ---- | ---------------------------- | ---------------- | ------ + 1 | Leica Peroxide Block | 150μL | 5 min + 2 | Leica Bond Wash | 150μL | 0 sec + - | Leica Bond Wash | 150μL | 0 sec + - | Leica Bond Wash | 150μL | 0 sec + 4 | TCT Buffer | 150μL | 10 min + - | Leica Bond Wash | 150μL | 0 sec + - | Primary/Isotype | 150μL | 60 min + - | Leica Bond Wash | 150μL | 0 sec + - | Leica Bond Wash | 150μL | 20 min + 10 | Leica PowerVision anti-Mouse | 150μL | 12 min + 12 | Deionized Water | 150μL | 9 min + 13 | Leica Hematoxylin | 150μL | 4 min + 15 | Deionized Water | 150μL | 2:00 + + ``` + +## Dehydration and Clearing +9. **Dehydration:** + - 3 times with 95% Ethanol (1 min each) + - 3 times with 100% Ethanol (1 min each) + - 5 times with Xylene (1 min each) + - Coverslip using Cytoseal XYL and a VWR 24x40 coverglass. + +## Protocol Citation +Kristin Anderson, Sunni Farley 2021. Immunohistochemistry. [protocols.io](https://dx.doi.org/10.17504/protocols.io.sppedmn) + +**License:** This protocol is licensed under the terms of the [Creative Commons Attribution License](https://creativecommons.org/licenses/by/4.0/). + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/immunostaining-and-quantification-of-intracellular-dgus3wwe.md b/markdown-output/immunostaining-and-quantification-of-intracellular-dgus3wwe.md new file mode 100644 index 0000000000000000000000000000000000000000..b0e85f22a7c095e5ff6e1fdd341420f392a6fe0d --- /dev/null +++ b/markdown-output/immunostaining-and-quantification-of-intracellular-dgus3wwe.md @@ -0,0 +1,157 @@ +```markdown +Goal/Experiment: +Immunostaining and quantification of intracellular accessible cholesterol using ALOD4-mNeon in Human Fibroblasts. + +# Immunostaining and Quantification of Intracellular Accessible Cholesterol using ALOD4-mNeon in Human Fibroblasts + +DOI: [dx.doi.org/10.17504/protocols.io.rm7vzj5prlx1/v1](https://dx.doi.org/10.17504/protocols.io.rm7vzj5prlx1/v1) + +Authors: +- Sreeja V Nair +- Ebsy Jaimon +- Suzanne R Pfeffer + +## Abstract +Here we present a protocol for quantitative immunostaining of intracellular accessible cholesterol using ALOD4-mNeon in human fibroblasts. + +## Guidelines +Be sure to use freshly prepared ALOD4-mNeon (within 14 days of preparation). Store at 4°C and do not freeze. + +## Materials +1. Paraformaldehyde 16% Solution (Electron Microscopy Sciences #15710) diluted fresh in 1X PBS +2. Glass coverslips (Fisher Scientific #12-545-81) +3. Rat tail collagen (Gibco by Life Technologies #A10483-01) +4. Saponin (Sigma #S7900) +5. Bovine serum albumin (Equitech-Bio, Inc. #BAH65) +6. Human fibroblasts (Coriell Institute of Medical Research) +7. ALOD4-mNeon ([link](https://dx.doi.org/10.17504/protocols.io.rm7vxz2qxgx1/v1)) freshly prepared and never frozen +8. Mowiol mounting solution +9. FIJI/ImageJ +10. Latest edition CellProfiler software 4.04 (or later) + +## ALOD4 Staining and Image Acquisition +1. Seed 0.2 x 10^6 cells onto 3-4 collagen-coated 12 mm coverslips, in each well of a six-well plate. +2. 16-18 hr after plating, wash the cells once with 1X PBS. +3. Transfer each coverslip to a well of a 24-well plate containing 500µl of 4% (v/v) PFA per well; fix the cells for 15 minutes at room temperature. +4. Wash cells 3X with 1X PBS. +5. Permeabilize the cells by adding 0.1% Saponin in 1X PBS for 5 minutes at room temperature. +6. Wash cells 3X with 1X PBS. +7. Block fixative by adding of 1% BSA in 1X PBS for 30 minutes at room temperature. +8. Dilute ALOD4-mNeon to 4µM in 1% BSA/1X PBS. +9. Add 50 µl, 4µM ALOD4 per coverslip. +10. Incubate cells for 1 hour at room temperature. +11. Wash cells 3X in 1X PBS and mount coverslips onto clean glass slides with 4 µl Mowiol. Air-dry coverslips overnight or at least 4-5 hours; store in dark at room temperature. +12. Images were acquired using a Zeiss laser scanning microscope. + +## Batch Process Images for Maximum Intensity Projection and Background Subtraction +13. Images need to be batch processed for maximum intensity projection and uniform background subtraction. Process the images for CellProfiler analysis as described in [dx.doi.org/10.17504/protocols.io.3byl4bpo8vo5/v1](https://dx.doi.org/10.17504/protocols.io.3byl4bpo8vo5/v1). + - For Zeiss images, the user needs to specify the folder to save the processed images and run lines 26, 27, and 29. Processing includes maximum intensity projection and background subtraction. Choose a value for background subtraction based on the images analyzed. + +## Import Files and Segment Cells +14. In CellProfiler, select the Images module, drag and drop the background subtracted and maximum intensity projected .TIF files created in Step 13 above. Subtract a value of 50 to remove the background noise. +15. Select the Metadata module. + - In the Metadata module: + - Extract metadata? Yes. + - Metadata extraction method: Extract from image file headers. + - Extract metadata from: All images. + - Hit “Extract metadata”. + - Click Add another extraction method. + - Metadata extraction method: Extract from file/folder names. + - Metadata source: File name. + - Regular expression to extract from file name: “Regex” will be as follows: ^GM(?P[0-9]{4}) .*#(?P[0-9]{2}) for an example file name “GM01607.czi #01_max.tif”. + +16. Go to NamesAndTypes module. + - In the NamesAndTypes module: + - Assign a name to: “Images matching rules”. + - Process as 3D: No. + - Select the rule criteria Match “All” of the following rules “Metadata/Does/Have C matching 0”. + - Name to assign these images: ALOD4. + - Select the image type: Grayscale image. + - Set intensity range from: Image metadata. + - Hit “update” to populate the names and types field. +17. Go to the Groups module. + - In the Groups module: + - Do you want to group your images? Yes. + - Metadata category: Celltype. + - Metadata category: Position. + - This groups images based on Cell type, and position as identified in the metadata module. + +18. Segmentation of cells: + - Click on the “+” sign at the bottom next to Adjust Modules. Under the module category, Image processing, Add RescaleIntensitymodule. + - Select the input image: ALOD4. + - Name the output image: RescaleIntensity_ALOD4. + - Rescaling method: Divide each image by the same value. + - Divisor value: 0.001. + +19. Rescaling the intensity of images makes it easier to segment cells below. + - Add IdentifyPrimaryObjects module. + - In the IdentifyPrimaryObjects module: + - Use advanced settings? Yes. + - Select the input image: RescaleIntensity_ALOD4. + - Name the primary objects to be identified: Cells. + - Typical diameter of objects, in pixel units: 95 - 800. + - Discard objects outside the diameter range: Yes. + - Discard objects touching the border of the image: No. + - Threshold strategy: Global Thresholding method: Otsu. + - Two-class or three-class thresholding? Two classes. + - Threshold smoothing scale: 1.6. + - Threshold correction factor: 1.0. + - Lower and upper bounds on threshold 0 and 1.0. + - Log transform before thresholding? Yes. + - Method to distinguish clumped objects? None. + - Fill holes in identified objects? After both thresholding and declumping. + - Handling of objects if excessive number of objects identified? Continue. + - Note: Check by clicking “Start Test Mode” and hitting the green triangle next to the IdentifyPrimaryObjects module each time a parameter is changed to find the best parameters for each image set. + +20. Add OverlayOutlines module. + - In the OverlayOutlines module: + - Display outlines on a blank image: No. + - Select image on which to display outlines: ALOD4. + - Name the output image: CellOutline. + - Outline display mode: Color. + - How to outline: Thick. + - Select objects to display: Cells. + - Select outline color: Maraschino. + +![CellProfiler Segmentation Example](example_image1.png) +*Example of CellProfiler Segmentation.* + +## Measure ALOD4 Intensity +21. Add MeasureObjectIntensity module. + - In the MeasureObjectIntensity module: + - Select images to measure: ALOD4. + - Select objects to measure: Cells. + - This module measures ALOD4 intensity in segmented cells. + +22. Add MeasureObjectSizeShape module. + - In the MeasureObjectSizeShape module: + - Select object sets to measure: Cells. + - Calculate Zernike feature? No. + - Calculate the advanced features? No. + +23. Add ExportToSpreadsheet module from the + at the bottom. + - Select the column delimiter: Tab. + - Output file location: Choose a folder where you want the images to be saved. + - Add a prefix to file names? Yes. + - File name prefix: Add experiment identifier. + - Overwrite existing files without warning? No. + - Note: While the pipeline is run for optimizing the parameters, choose Yes to avoid being asked to rewrite each file. + - Add image metadata columns to your object data file? Yes. + - Add image file and folder names to your object data file? Yes. + - Representation of Nan/Inf: NaN. + - Select measurements to export? Yes. + - Press button to select measurements: Under “cells” choose AreaShape -> Area, and Intensity -> Integrated Intensity. + - Calculate the per-image mean values for object measurements? No. + - Calculate the per-image median values for object measurements? No. + - Calculate the per-image standard deviation values for object measurements? No. + - Create GenePattern GCT file? No Export all measurement types? No. + - Data to export: Cells Use the object name for the file name? Yes. + +![Intracellular Accessible Cholesterol Staining Example](example_image2.png) +*Example of Intracellular accessible cholesterol staining. Human fibroblasts from healthy control and an individual with GBA N370S/V394L mutations stained with ALOD4-mNeon according to this protocol. Scale bar, 10 µm.* + +## Protocol References +- Stirling DR, Swain-Bowden MJ, Lucas AM, Carpenter AE, Cimini BA, Goodman A (2021). CellProfiler 4: improvements in speed, utility and usability. BMC Bioinformatics, 22 (1), 433. PMID: 34507520 PMCID: PMC8431850. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/in-silico-molecular-docking-with-ligand-target-cueywtfw.md b/markdown-output/in-silico-molecular-docking-with-ligand-target-cueywtfw.md new file mode 100644 index 0000000000000000000000000000000000000000..cd3dace49fe016da65791cd9ac59c004cd34044e --- /dev/null +++ b/markdown-output/in-silico-molecular-docking-with-ligand-target-cueywtfw.md @@ -0,0 +1,162 @@ +```markdown +# Goal/Experiment: +The goal of this experiment is to explore enzyme-ligand interactions through a comprehensive molecular docking protocol using an enzyme (cyclooxygenase) and its ligand (celecoxib). + +# In Silico Molecular Docking with Ligand Target + +**DOI:** [dx.doi.org/10.17504/protocols.io.5jyj8j4wrg2w/v1](https://dx.doi.org/10.17504/protocols.io.5jyj8j4wrg2w/v1) + +**Protocol Citation:** Angelo José Rinaldi 2024. In Silico Molecular Docking with Ligand Target. *protocols.io* (2024). [https://dx.doi.org/10.17504/protocols.io.5jyj8j4wrg2w/v1](https://dx.doi.org/10.17504/protocols.io.5jyj8j4wrg2w/v1) + +**License:** This is an open access protocol distributed under the terms of the Creative Commons Attribution License. + +**Protocol status:** Working + +**Created:** May 18, 2023 + +**Last Modified:** July 29, 2024 + +**Protocol Integer ID:** 82104 + +**Keywords:** Molecular Docking, Enzyme-Ligand Interaction, Protein Data Bank, DrugBank, UCSF Chimera, MetaPocket 2.0, AutoDockTools, AutoDock Vina, ProteinsPlus, Bioinformatics, Drug Discovery, Cyclooxygenase, Celecoxib + +## Abstract +This study outlines a comprehensive molecular docking protocol aimed at exploring enzyme-ligand interactions, leveraging various bioinformatics tools and databases. The protocol starts with the acquisition of the enzyme's 3D structure from the Protein Data Bank (PDB) and the identification of potential ligands from DrugBank. Structural refinement and preparation of the enzyme and ligands are conducted using UCSF Chimera, ensuring proper protonation states and the addition of missing atoms. MetaPocket 2.0 is used to predict and identify potential binding sites on the enzyme. Prepared structures are then processed using AutoDockTools to generate the necessary input files. Docking simulations are performed using AutoDock Vina, and results are analyzed and visualized using the ProteinsPlus platform. + +## Materials + +### Computational Tools and Software: +#### AutoDockTools (ADT): +- **Version:** AutoDockTools 1.5.x +- **Usage:** Preparation of the enzyme and ligand files, including the generation of PDBQT files and grid parameter files (GPF). +- **Download link:** [AutoDockTools](https://autodock.scripps.edu/) + +#### AutoDock Vina: +- **Version:** AutoDock Vina 1.1.x +- **Usage:** Execution of molecular docking simulations to predict binding poses and affinities. +- **Download link:** [AutoDock Vina](https://autodock.scripps.edu/) + +#### UCSF Chimera: +- **Version:** UCSF Chimera 1.14 or later +- **Usage:** Visualization, preparation, and refinement of enzyme and ligand structures, including protonation and the addition of missing atoms. +- **Download link:** [UCSF Chimera](https://www.cgl.ucsf.edu/chimera/download.html) + +#### MetaPocket 2.0: +- **Version:** MetaPocket 2.0 +- **Usage:** Prediction and identification of potential binding sites on the enzyme. +- **Access link:** [MetaPocket 2.0](https://metapocket.eml.org/) + +#### ProteinsPlus: +- **Platform:** ProteinsPlus web server +- **Usage:** Analysis and visualization of docking results, including interaction profiling. +- **Access link:** [ProteinsPlus](https://proteins.plus/) + +### Databases: +#### Protein Data Bank (PDB): +- **Usage:** Source of three-dimensional structures of enzymes. +- **Access link:** [PDB](https://www.rcsb.org/) + +#### DrugBank: +- **Usage:** Source of potential ligand molecules, including small molecules and drug-like compounds. +- **Access link:** [DrugBank](https://go.drugbank.com/) + +### Input Files: +- **Enzyme Structure:** Format: PDB file, Source: Downloaded from the PDB database. +- **Ligand Structures:** Format: PDB or MOL files, Source: Downloaded from the DrugBank database. + +### Hardware: +- **Computer System:** Multi-core CPU (e.g., Intel i7 or higher) +- **Memory:** At least 16 GB RAM +- **Storage:** Minimum 500 GB of free disk space +- **Operating System:** Linux, Windows, or macOS + +### Miscellaneous: +- **Internet Access:** Required for downloading software, accessing databases, and using web-based tools like MetaPocket 2.0 and ProteinsPlus. + +## Protocol Steps + +### Target Receptor Search: + +1. Use the cyclooxygenase enzyme as the protein target (receptor) and celecoxib as the ligand. + +2. Retrieve the 3D structure of the receptor from the PDB database. Here we use the PDB, accessible at [https://www.rcsb.org/](https://www.rcsb.org/). + +3. Search the PDB for cyclooxygenase and choose the structure with the best resolution, in this case, 5F19. + +4. View the structure in 3D. + +5. Extract the receptor structure in PDB format and save it in a working directory. + +### Target Ligand Search: + +6. For known chemical structures, use DrugBank for the search ([https://go.drugbank.com/](https://go.drugbank.com/)). If the structure is not in DrugBank, use chemical structure design software like ACD/ChemSketch. + +7. Download and save the celecoxib molecule in PDB format. + +### Preparation of the Receptor: + +8. Download UCSF Chimera from [https://www.cgl.ucsf.edu/chimera/download.html](https://www.cgl.ucsf.edu/chimera/download.html). + +9. Open the downloaded enzyme structure in Chimera or fetch the structure using the ID 5F19. + +10. Visualize the cyclooxygenase enzyme in Chimera. + +11. Select all non-standard residues. + +12. Identify and mark residues not belonging to the receiver in green. + +13. Clean the structure by removing unneeded residues. + +14. Delete a chain of amino acids to expose the ligand receptor. + +15. Mark residues to be deleted in green. + +16. Save the cleaned structure in PDB format. + +### Using MetaPocket 2.0: + +17. Use MetaPocket 2.0 to predict binding sites on the protein ([https://metapocket.eml.org/](https://metapocket.eml.org/)). Upload the prepared target protein in PDB format. + +18. Select the best binding pockets by choosing item 4. + +19. Choose the binding site prediction result with the highest score. + +### Preparation of Structures Using AutoDockTools: + +20. Download AutoDockTools ([https://autodock.scripps.edu/adt/](https://autodock.scripps.edu/adt/)). + +21. Add hydrogen atoms to the protein structure. Use the "polar only" option to add polar hydrogens. + +22. Choose the prepared macromolecule and save in PDBQT format. + +23. Configure the Grid Box with parameters provided by MetaPocket 2.0. + +24. Select the ligand structure. + +25. Save the celecoxib molecule in PDBQT format. + +### Docking Simulation Using AutoDock Vina: + +26. Run AutoDock Vina using the basic command format: + ```shell + vina --receptor --ligand --out + ``` + +### Analysis Using ProteinsPlus: + +27. Use ProteinsPlus for analysis and visualization ([https://proteins.plus/](https://proteins.plus/)). Upload the protein and ligand files. + +28. Start the analysis by clicking the appropriate button (e.g., "Submit" or "Run PoseView"). + +29. After completion, the tool will generate an interaction diagram illustrating hydrogen bonds, hydrophobic interactions, and other interactions between the protein and ligand. + +## Protocol References +1. Morris GM, Huey R, Lindstrom W, et al. AutoDock4 and AutoDockTools4: Automated docking with selective receptor flexibility. *J Comput Chem*. 2009;30(16):2785-2791. +2. Trott O, Olson AJ. AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. *J Comput Chem*. 2010;31(2):455-461. +3. Meng XY, Zhang HX, Mezei M, Cui M. Molecular docking: a powerful approach for structure-based drug discovery. *Curr Comput Aided Drug Des*. 2011;7(2):146-157. +4. Cavasotto CN, Orry AJ, Murgolo NJ, Czarniecki MF, Kocsi SA, Hawes BE. Docking and high throughput docking: successes and pitfalls. *J Chem Inf Model*. 2009;49(4):1079-1093. +5. Kitchen DB, Decornez H, Furr JR, Bajorath J. Docking and scoring in virtual screening for drug discovery: methods and applications. *Nat Rev Drug Discov*. 2004;3(11):935-949. +6. Shoichet BK. Virtual screening of chemical libraries. *Nature*. 2004;432(7019):862-865. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/in-vitro-co-culture-system-using-a-fiber-supported-dgj63ure.md b/markdown-output/in-vitro-co-culture-system-using-a-fiber-supported-dgj63ure.md new file mode 100644 index 0000000000000000000000000000000000000000..f22f3f04263dd52a6723e3eacbf220eb2c447f6e --- /dev/null +++ b/markdown-output/in-vitro-co-culture-system-using-a-fiber-supported-dgj63ure.md @@ -0,0 +1,93 @@ +```markdown +# In vitro Co-culture System Using a Fiber-Supported Liquid Approach + +**DOI:** [dx.doi.org/10.17504/protocols.io.rm7vzj56x1x1/v1](https://dx.doi.org/10.17504/protocols.io.rm7vzj56x1x1/v1) +**Protocol Status:** Working +**Created:** July 02, 2024 +**Last Modified:** July 03, 2024 +**Protocol Integer ID:** 102750 +**Keywords:** Phenotyping, plant disease infection, fungal inoculation, plant culture +**Funders Acknowledgement:** USDA-NIFA Grant ID: 2020-51181-32142 + +### Goal/Experiment: + +This study aimed to develop and validate an in vitro co-culture system to investigate the Armillaria root rot (ARR) affecting *Prunus spp.*. The disease, caused by fungi *Armillaria spp.* and *Desarmillaria caspitosa*, presents a severe threat to the stone and nut fruit industry. The novel system allows controlled plant-pathogen interaction under reproducible conditions, aiming to provide consistent and robust conditions for investigating ARR in *Prunus* spp. + +--- + +## Abstract + +In vitro co-culture techniques that allow plant and pathogen growth under controlled environmental conditions recreate host plant infection. These techniques reduce infection times and promote reproducibility. This portable method facilitates understanding plant-pathogen interactions, vital for breeding programs aimed at developing plant disease resistance. Here, we validated a fiber-supported liquid co-culture system to study Armillaria root rot (ARR) affecting *Prunus spp.*. The method uses a system to inoculate diverse commercial rootstocks and monitor disease progression. + +--- + +## Materials and Equipment + +### Materials +- 20 × 150 mm culture tubes (Stellar Scientific, SKU: GS-1522) +- Autoclavable polypropylene culture tube closures (General Laboratory Supply, Cat. No: T3054-4) +- Magenta™ GA-7 vessels (Merck, Cat. No: V8505-25EA) +- Petri plates (VWR, Cat. No: 391-0579) +- Parafilm ‘M’ laboratory film (Sigma Aldrich, Cat. No: P7668) +- 15 mL Pyrex® Ten Broeck tissue grinder with pour spout (Corning, Cat. No: 7727-15) +- Ultra-clear porous cellophane sheet (0.1 mm thick) (Research Products International, Cat. No: 1080) +- Lazy-L spreader (Merck, Cat. No: 2376779) +- 1.5 mL Eppendorf® tubes (Thermo Fisher, Cat. No: 0030120175) +- Rectangular vessels (110 × 297 mm; Southern Sun BioSystems) +- Polyester fiber mat (BioStrate™ Felt, Cropping Inc., Lodi, OH) +- Germination paper (Anchor Paper Co., St. Paul, MN) +- Polyvinyl chloride (PVC) sealing film (Phytotech, Cat. No: A003) + +### Equipment +- Laminar flow hood +- Autoclave +- Articulated rocker arm +- Fungal growth incubator +- LED light NutriLED (Hubbell Lighting, Greenville, SC) + +--- + +## Methods + +### Establishment of Plant Cultures + +#### From Dormant Shoots +1. Collect dormant shoots (3 cm in length) and cleanse by submerging in 70% ethanol for 1 minute, followed by rinsing with sterile deionized water. +2. Submerge in 10% bleach solution for 10 minutes, then rinse twice with deionized water (Figure 1). +3. Peel vegetative shoot buds and transfer to culture tubes containing 20 mL of Murashige and Skoog agar media. + +#### From Seeds +1. Clean fruit exocarps with 20% bleach for 10 minutes, followed by 70% ethanol for 10 minutes. +2. Extract seeds and transfer aseptically to culture tubes with Woody Plant Medium for a 10-week stratification at 4 °C (Figure 2). + +### Maintenance of Stock Plants +3. Sustain in Magenta GA-7 vessels, transferring shoot tips every five weeks. +4. Maintain with a photon flux of 20 µmol/s/m² for a 16-hour photoperiod at 24 °C. Hyper-multiplication requires occasional rest with 16 µM indole-3-acetic acid. + +### Fungi Preservation +5. Propagate fungal cultures by placing two plugs in Petri plates. Maintain at 20 °C in darkness (Figure 3). +6. Refresh every 14 days by transferring mycelial plugs to fresh MEA plates. + +### Inoculum Preparation +7. Extract ten-millimeter plugs from two-week-old colonies and homogenize with 5 mL sterile water using a tissue grinder (Figure 4). +8. Prepare inoculum via an ultra-clear porous cellophane sheet over MEA, pouring 600 µL of homogenate, and incubate in the dark for 14 days. + +### Co-culture (Plant-Fungi) Establishment +9. Autoclave rectangular plastic vessels and place a fiber-supported paper on vessel floors. +10. Add 175 mL of plant growth regulator-free 'New Prunus Medium' to each vessel. +11. Transfer 15 in vitro plants, sealing vessels with PVC film (Figure 7). +12. Place on a rocker arm at 5 rpm under regulated LED light. +13. Add 1 mL of mycelium suspension for inoculation (Figures 5 and 6). +14. Transfer in vitro rooted plants, add media without growth regulators, and maintain on a rocker arm under LED light (Figure 8). +15. Collect tissues as needed. + +--- + +## Protocol References + +[List of relevant research references with DOI links] + +--- + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/in-vitro-pmel-1-t-cell-mediated-cytotoxicity-assay-6j4hcqw.md b/markdown-output/in-vitro-pmel-1-t-cell-mediated-cytotoxicity-assay-6j4hcqw.md new file mode 100644 index 0000000000000000000000000000000000000000..7b26f8c7402f71067de868bcfec06880e0a83d93 --- /dev/null +++ b/markdown-output/in-vitro-pmel-1-t-cell-mediated-cytotoxicity-assay-6j4hcqw.md @@ -0,0 +1,90 @@ +```markdown +# Goal/Experiment: +In vitro pmel-1 T Cell-mediated cytotoxicity assay against target cancer cells. + +## In vitro pmel-1 T Cell-Mediated Cytotoxicity Assay with CytoTox-ONE Homogeneous Membrane Integrity Assay (Promega) + +**Authors:** Elinor Gottschalk, Bulent Arman Aksoy, Pinar Aksoy, Jeff Hammerbacher +**Affiliation:** Medical University of South Carolina +**Publication Date:** October 14, 2019 + +### Abstract +A plate-based assay to estimate the cytotoxic activity of pmel-1 T cells against target cancer cells. The CytoTox-ONE Homogeneous Membrane Integrity Assay from Promega, utilizing lactate dehydrogenase (LDH) release as a marker of cell death. LDH is released into the culture medium where it can be measured using a fluorescent signal. This assay is advantageous as cancer cells need not be removed from wells to assess cell death. + +### Materials + +| Name | Catalog # | Vendor | +|---------------------------------------------------------------|------------|---------| +| CytoTox-ONE™ Homogen Membrn Integrity Assay, 200-800 assay | G7890 | Promega | +| CytoOne 75 filter cap TC flask | CC7682-4875| USA Scientific | +| CytoOne 96-well TC plate | CC7682-7596| USA Scientific | +| hgp100(25-33) | RP20344 | Genscript | +| B16-F10 cell line (ATCC® CRL-6475™) | CRL-6475 | ATCC | +| MC38 cell line | ENH204-FP | | + +### Steps Materials + +| Name | Catalog # | Vendor | +|---------------------------------------------------------------|------------|---------| +| CytoTox-ONE™ Homogen Membrn Integrity Assay, 200-800 assay | G7890 | Promega | + +### Before Starting +Start culturing T cells: +1. Day 0: Thaw frozen splenocytes or isolate fresh splenocytes. +2. Supplement T cell culture media with 200 IU IL-2. +3. Activate the cells. +4. Day 3 - 6: Maintain the T cells at 1 million cells/ml, splitting when needed. Replenish IL-2 each day at 200 IU/ml. + +### Procedure + +1. **Plate Target Cells** + - Plate the target cells in a 96-well plate at 25,000 cells per well in DMEM with 10% FBS. + - Leave columns 2-7 with cells, the rest of the wells empty. + - Ensure 6 replicates per condition. + + Sample plate layout: + + ``` + | | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | + | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | + | a | #1 | 4:1 | 2:1 | 1:1 | 1:2 | 1:4 | T cells alone | | | | | | + | b | Target cells alone | | 200,000 T cells | 100,000 T cells | 50,000 T cells | 25,000 T cells | culture media | + ``` + +2. **Prepare Target Cells for Co-culture** + - Pulse the target cells in one plate with 1 µM hgp100 peptide for 1 hour at 37°C. + - Wash the peptide out 3x with T cell media. + +3. **Prepare T Cells for Co-culture** + - Centrifuge the T cells at 350 x g for 5 minutes. Resuspend in T cell media to a concentration of 2 million cells per ml. + - Aspirate culture media from the 96-well plate. + - Pipette 100 µl T cell media into columns 3-7 and 9-12 (leave columns 2 and 8 empty). + - Pipette 100 µl of T cell suspension into columns 2, 3, 8, and 9. + - Perform serial dilutions in the wells to reach the desired concentrations. + - Incubate plates for 24 hours at 37°C. + +4. **Perform CytoTox-ONE Homogeneous Membrane Integrity Assay** + - Thaw lysis solution, make a 1:5 dilution with PBS. + - Add 10 µl of diluted lysis solution to column 7. + - Incubate under the hood for 30 minutes at room temperature. + - Add 11 ml of assay buffer to one substrate bottle and mix. + - Pour buffer/substrate mixture into a reagent reservoir, pipette 100 µl into each well. + - Shake the plate gently to mix, cover and incubate for 10 minutes. + - Add 50 µl of stop solution to each well. + +5. **Calculate Percent Cytotoxicity** + - Using the formula: + + \[ + \frac{(\text{experimental} - \text{CMB}) - (\text{Tcellbackground} - \text{CMB})}{\text{maxLDHrelease} - \text{CMB}} \times 100 + \] + + - Where CMB is the culture medium background, Tcellbackground is the wells with T cells only, and maxLDHrelease is from cells lysed with detergent. + +### Reagents and Terms +- **CytoTox-ONE™ Assay:** An assay measuring cell membrane integrity via LDH release. +- **LDH (Lactate Dehydrogenase):** An enzyme released during cell damage. +- **IL-2 (Interleukin-2):** A cytokine that is essential for T cell proliferation. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/in-vitro-transcription-capping-and-2-x27-o-methyla-4pwgvpe.md b/markdown-output/in-vitro-transcription-capping-and-2-x27-o-methyla-4pwgvpe.md new file mode 100644 index 0000000000000000000000000000000000000000..036454ee0c231574aa27bb7e7dbbc1542ab1f686 --- /dev/null +++ b/markdown-output/in-vitro-transcription-capping-and-2-x27-o-methyla-4pwgvpe.md @@ -0,0 +1,142 @@ +```markdown +# Goal/Experiment: +The goal of this experiment is to conduct in vitro transcription, capping, and 2’-O methylation of long RNAs using a PCR-amplified template and ensuring high-quality RNA suitable for transfection into mammalian cells or for in vitro translation. + +# In Vitro Transcription, Capping, and 2'-O Methylation of Long RNAs + +**Stephen Floor** +*University of California, San Francisco* + +**Abstract:** +This protocol is for in vitro transcription of long RNAs off plasmid or PCR product templates. It ensures the template is gel purified with a proper T7 promoter and a 3’ polyA tail (typically A60). Following this protocol, the RNA will be ready for transfection into mammalian cells or in vitro translation. Significant amounts of template are required: at least 1 ug of template per 100 ul reaction, ideally closer to 5 ug of template, especially for very long templates. Use RNase sensitive protocols and reagents for all steps of this procedure. + +The basic protocol is: +- PCR amplification of the template +- In vitro transcription +- Capping and 2’-O-Methylation +- Quality control and optional purification + +Based on protocols from Kaihong Zhou (Doudna lab) and RNA: A Laboratory Manual (Rio, Ares, Hannon, Nilsen). + +**Guidelines:** +Use RNase sensitive protocols and reagents for all steps of this procedure. + +## Materials + +| Name | Catalog # | Vendor | +| ---- | --------- | ------ | +| E.coli Poly (A) Polymerase - 100 units | M0276S | New England Biolabs | +| mRNA Cap2’-O-Methyltransferase - 2,000 units | M0366S | New England Biolabs | +| Vaccinia Capping System - 400 units | M2080S | New England Biolabs | +| GlycoBlue™ Coprecipitant | AM9516 | Thermo Scientific | +| MEGAscript® T7 Transcription Kit | AM1334 | Thermo Scientific | +| RQ1 RNase-Free DNase, 1,000u | M6101 | Promega | +| RNasin(R) Plus RNase Inhibitor, 10,000u | N2615 | Promega | +| Ambion NorthernMAX glyoxal loading dye | AM8551 | Thermo Fisher Scientific | +| RNA clean & concentrator-25 | R1017 | Zymo Research | +| RNA Gel Recovery Kit | R1011 | Zymo Research | + +**Additional Reagents:** +- 10X transcription buffer + - 300 mM Tris-Cl pH (RT) = 8.1 + - 250 mM MgCl₂ + - 0.1% Triton X-100 + - 20 mM spermidine + - 100 mM DTT +- 0.3 M NaOAc pH 5.2 +- 50 mM EDTA + +**Note:** +- We typically use T7 polymerase we have purified, but the Megascript kit above or similar should also work. If using a kit, perform the in vitro transcription according to the kit instructions. +- The RNA Gel Recovery Kit is optional and only necessary if the transcription has more than one product. The polyA polymerase is only required if your template does not contain a polyA stretch. + +## Protocol + +### PCR Amplification of Template + +1. **Primer design:** + - Design primers with appropriate sequences (ensure reverse primer covers polyA tail if not present in template). + +2. **PCR setup (4x 100 ul for each template, four replicate reactions):** + +| Reagent | Amount per 100 ul reaction | +| -------------------------- | ------------------------- | +| HF buffer | 10 ul | +| dNTP (10 mM) | 2 ul | +| Forward primer (100 uM) | 0.5 ul | +| Reverse primer (100 uM) | 2 ul | +| Phusion polymerase | 1 ul | +| Template (50 ng total) | Variable | +| Water | To 100 ul | + +3. **Run PCR with a two-step protocol:** + - 72 degrees annealing/extension for 3 minutes + - 98 degrees melting for 30 seconds + - Repeat 30 cycles + - 10-minute final extension time at 72 + +4. **Load all PCRs into agarose gel and verify size. Gel purify the product.** + +### In Vitro Transcription of RNA + +5. **Warm all reagents except T7 polymerase and Superase:IN to RT and assemble the reaction.** + +6. **Mix the following together in a 1.5 ml tube:** + +| Component | Volume | +| ---------------------- | ------ | +| Template DNA | 1 to 5 ug | +| ACGU nTP mix (25 mM each, pH 8) | 30 ul | +| 10X transcription buffer (RT) | 10 ul | +| 1M DTT | 2 ul | +| 1M MgCl₂ | 1 ul | +| T7 polymerase | 10 ul | +| Superase:IN | 1 ul | +| DEPC H₂O | To 100 ul | + +7. **Incubate at 37°C for 3-4 hours.** +8. **Add 1 ul of RQ1 RNase-free DNase to each reaction and incubate at 37°C for 30 minutes.** +9. **Precipitate RNA by adding 200 ul 0.3M NaOAc pH 5.2, 1 ul GlycoBlue, and 750 ul 100% EtOH.** +10. **Incubate at -80°C for >1 hour to overnight.** + +### Resuspending EtOH Precipitated RNA + +13. **Pellet precipitations by centrifugation at 16,000 g at 4°C for 25 minutes.** +14. **Aspirate supernatant and discard.** +15. **Wash with 500 ul -20°C degree 70% EtOH.** +16. **Centrifuge at 16,000 g and 4°C for 5 minutes.** +17. **Aspirate supernatant and discard.** +18. **Quick-spin and remove residual ethanol with a P20 pipet.** +19. **Allow pellet to air dry for 5 minutes; do not overdry.** +20. **Resuspend pellet in 100 ul 50 mM EDTA.** + +### Capping and 2’-O-Methylation + +24. **Dilute 20 ug of RNA in 26 ul DEPC H₂O.** + +25. **Prepare a 4 mM stock of SAM (follow instructions for mixing):** + +| Component | Volume per 40 ul reaction | +| ---------------------------------- | ------------------------- | +| 10X capping buffer | 4 ul | +| 10 mM GTP | 2 ul | +| 4 mM SAM | 2 ul | +| Vaccinia capping enzyme | 2 ul | +| 2’-O-Me transferase | 2 ul | +| RNasin plus (Promega) | 2 ul | + +27. **Add 14 ul of the master mix to each tube with RNA.** + +28. **Incubate at 37°C for 60 minutes.** +29. **Purify using Zymo Clean and Concentrator 25 column, elute in 30 ul DEPC H₂O.** + +### Quality Control: Glyoxal Gel + +30. **Run a glyoxal gel of all RNAs and verify their size.** +31. **Also glyoxylate the ladder and include (use 5 ul of the ladders listed above).** +32. **Quantify the full-length band using Image Lab to determine concentration.** +33. **If significant truncated products are present, purify full-length RNA using the Zymo Agarose Gel Zymoclean kit (R1011).** + +## End of Protocol +``` +endofoutput \ No newline at end of file diff --git a/markdown-output/in-vivo-electrophysiology-mouse-b9ucr6sw.md b/markdown-output/in-vivo-electrophysiology-mouse-b9ucr6sw.md new file mode 100644 index 0000000000000000000000000000000000000000..32160134677521b7ba33e3e292f23dafc64f1733 --- /dev/null +++ b/markdown-output/in-vivo-electrophysiology-mouse-b9ucr6sw.md @@ -0,0 +1,111 @@ +```markdown +# Goal/Experiment: +To record neuronal activity in mice using an in vivo electrophysiology method. + +## In Vivo Electrophysiology (Mouse) +**Alexandra Nelson** +*University of California San Francisco* +**DOI:** [dx.doi.org/10.17504/protocols.io.b9ucr6sw](https://dx.doi.org/10.17504/protocols.io.b9ucr6sw) +**Date:** July 27, 2022 +**Team:** Edwards +**Contributors:** kelsey.barcomb + +### Abstract +This protocol describes the in vivo electrophysiology method for recording neuronal activity in mice. + +### Protocol Citation +Alexandra Nelson 2022. In Vivo Electrophysiology (Mouse). *protocols.io* +Link: [dx.doi.org/10.17504/protocols.io.b9ucr6sw](https://dx.doi.org/10.17504/protocols.io.b9ucr6sw) + +### Manuscript Citation +Jonathan S Schor, Isabelle Gonzalez Montalvo, Perry WE Spratt, Rea J Brakaj, Jasmine A Stansil, Emily L Twedell, Kevin J Bender, Alexandra B Nelson (2022) Therapeutic deep brain stimulation disrupts movement-related subthalamic nucleus activity in Parkinsonian mice eLife 11:e75253 +Link: [doi.org/10.7554/eLife.75253](https://doi.org/10.7554/eLife.75253) + +### Keywords +Electrophysiology, Mouse, In Vivo, Surgery, Electrode Array, ASAPCRN + +### License +This is an open access protocol distributed under the terms of the [Creative Commons Attribution License](https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. + +### Created +May 23, 2022 + +## Protocol Steps + +### 1. Making Optrodes +If you will be combining electrophysiology with optogenetics at the same site, you will need to make an optrode array. This entails affixing an optical fiber-ferrule assembly onto a multi-electrode array. + +#### 1.1 +Cut 200 micron, 0.39 NA optic fiber (Thorlabs) and thread a piece through a ceramic ferrule with epoxy, such that there is exposed fiber on the bottom (flat) end of the ferrule to at least the desired DV length + 1 mm. When used for optrode arrays, make the fiber the desired DV depth + 3 mm, as it will be angled on the array. + +#### 1.2 +After the epoxy has dried, the short bit of fiber protruding on the top (rounded) end of the ferrule is broken off and polished using finer and finer polishing paper (Thorlabs). Polishing is achieved by inserting the ferrule-fiber assembly into a ferrule-holder (Thorlabs), tightening it up, and rubbing the top end of the ferrule against the paper in circles or figure-eights. + +#### 1.3 +Once the ferrule is polished, test it for transmittance by using a 473 nm laser, set to 10 mW output at the end of a patch cable. Insert your ferrule and test the transmittance. If the power meter reads 8 mW, for example, it is an 80% transmittance fiber. If the transmittance is <60%, keep polishing until you achieve 70-100% transmittance. If it is <10% after polishing, it likely is split or cracked, and is unlikely to achieve high transmittance with further polishing. + +#### 1.4 +Once you have an adequate fiber-ferrule assembly, insert it in a sequence of empty ferrules and sleeves (a ferrule “stick”). On a hard surface (e.g., the plastic cases the arrays arrive in), position two balls of dental wax. On one ball of wax, carefully place the plastic connector of an electrode array (minding that you don’t touch the electrodes themselves to anything) sideways, so the array is approximately horizontal, with the groove for guiding the fiber facing up. Now use the other ball of wax and your ferrule “stick” to position the optical fiber-ferrule assembly over the electrode array, with the fiber tip pointed in the same direction as the electrodes. Maneuver the fiber under a dissecting scope until it slides in the metal groove on the array, and its tip is amidst the tips of the electrodes. The fiber should terminate a little above (0.3-0.5 mm) the tips of the electrodes, but be positioned in the middle of the cross-section of the electrodes. This should result in your ceramic ferrule being close to but not touching the electrode array connector (leaving room for the electrical headstage cable and optical patch cable to be connected during recordings). + +#### 1.5 +Once this position has been achieved, make some 5-minute epoxy in a small weigh boat. Use a 200 uL pipet tip to stir the epoxy, and then drip a small amount of epoxy onto the bottom 1/3 of the ceramic ferrule. The epoxy can cover the bottom of the ceramic ferrule, extending onto the side of the electrode array connector and the metal area with the fiber groove. However, it should never go into the inside of the electrode array connector, nor onto the electrodes themselves (especially the portion that will be intracranial). + +#### 1.6 +Let the epoxy dry overnight. + +### 2. Implant Surgery +Refer to the surgery protocol for full surgical details: [dx.doi.org/10.17504/protocols.io.n2bvj6qnkl5/v1](https://dx.doi.org/10.17504/protocols.io.n2bvj6qnkl5/v1) +For implantation of electrode arrays with or without an attached optical fiber, you will cut three holes: one at the site of the array implant (left side), one in the right frontal area (for a skull screw), and one in the right posterior area (for the ground wire). The site of the array implant will be enlarged by using the drill bit as a machine tool and cutting a rectangular area around the center coordinate, to remove a rectangular piece of the skull. Cut the underlying dura carefully with a 32G needle around 3 sides of the rectangle, fold back, cut the remaining side, and remove. Use a sterile swab to remove blood and ACSF from the area prior to implant. After implantation, animals should be returned to their home cages to recover for at least 1 week. + +### 3. Habituation +Animals should be scruffed and the electrophysiology headstage connector (itself on a cable attached to an overhead electrical commutator) carefully inserted into the electrode array connector on the mouse’s head. The mouse should then be placed in the open field for 30 minutes at a time, for two days prior to data collection. + +### 4. Computer and Electrophysiology Setup + +#### 4.1 +Open the appropriate in vivo electrophysiology (Plexon, Blackrock) and behavioral acquisition (Noldus Ethovision XT, Raspberry Pi) software. Attach the mouse to the headstage cable and place it in its home cage (top off), adjacent to the behavioral chamber. + +#### 4.2 +In the in vivo electrophysiology software, begin displaying signals from each of the channels, and adjust the gains and threshold on a per-channel basis to optimize the detection of single units. This is an iterative process. + +#### 4.3 +Open a behavioral acquisition file (see details in Behavioral Testing protocol), acquire a background image, and set up the detection. + +#### 4.4 +Place your mouse in the behavioral chamber and optimize the detection. + +### 5. Testing + +#### 5.1 +Once the settings for the behavior acquisition (e.g., video) and electrophysiology acquisition have been optimized, start the electrophysiology recording. This file will serve as the master data file, receiving timing input from all other devices (e.g., TTLs from the device controlling the video camera, TTLs from the device controlling the laser, etc.). The electrophysiology file name should start with the name of the mouse, and include a suffix representing the experiment date or type. + +#### 5.2 +Start the device controlling the video camera (Noldus or Raspberry Pi). This device sends TTL pulses to the electrophysiology system for every video frame acquired, for subsequent alignment of behavior and electrophysiology. + +#### 5.3 +Monitor the mouse to make sure it is able to move freely about the chamber during the recording, and is able to turn the commutator so it does not get caught up in the cables. + +#### 5.4 +At the end of the main portion of the experiment, stop the video acquisition system, then hit “stop recording” on the electrophysiology system. + +### 6. Cleanup +Carefully unplug the mouse and return to its home cage. Transfer and backup data to the server. Wipe the chamber with 80% ethanol. + +``` + +- **Electrophysiology**: The study of the electrical properties of biological cells and tissues. +- **Optogenetics**: A biological technique using light to control neurons. +- **DV (Dorsoventral)**: Refers to the anatomical direction from the back to the belly. +- **NA (Numerical Aperture)**: Indicates the light-gathering ability of the optic fiber. +- **Epoxy**: A type of glue used for strong, lasting bonds. +- **Plexon, Blackrock**: Companies that provide equipment/software for electrophysiology recordings. +- **Noldus Ethovision XT**: Software for animal behavior tracking. +- **Raspberry Pi**: A small, affordable computer used in various scientific applications. +- **Commutator**: A device used to allow continuous recording with movement. + +**Note**: For supplies that are difficult to find: +- **Optic Fiber and Ferrules**: If unavailable from Thorlabs, alternatives may include vendors like Newport or Edmund Optics. +- **Dental Wax**: Commonly available at dental supply stores and potentially from pharmacies. + +`endofoutput` +``` \ No newline at end of file diff --git a/markdown-output/increasing-insulin-measurement-throughput-by-fluor-cbq7smzn.md b/markdown-output/increasing-insulin-measurement-throughput-by-fluor-cbq7smzn.md new file mode 100644 index 0000000000000000000000000000000000000000..d060d473aaf32cf13797ef220d80ed6a1a75ed5b --- /dev/null +++ b/markdown-output/increasing-insulin-measurement-throughput-by-fluor-cbq7smzn.md @@ -0,0 +1,76 @@ +```markdown +# Goal/Experiment: +The goal of this experiment is to increase the throughput of insulin measurement from islets of Langerhans using fluorescence anisotropy imaging immunoassays (FAI). This involves the development and utilization of a microfluidic device capable of high-throughput examination of insulin secretion dynamics. + +# Increasing Insulin Measurement Throughput by Fluorescence Anisotropy Imaging Immunoassays + +**Authors:** +Yao Wang1, Damilola I. Adeoye1, Yue J. Wang2, Michael Roper1 +1Department of Chemistry and Biochemistry, Florida State University, Tallahassee, FL. +2Department of Biomedical Sciences, College of Medicine, Florida State University, Tallahassee, FL. +**Corresponding Author:** Michael Roper + +**Publication Date:** Jul 14, 2022 +**DOI:** [dx.doi.org/10.17504/protocols.io.eq2lyn4kwvx9/v1](https://dx.doi.org/10.17504/protocols.io.eq2lyn4kwvx9/v1) + +![Graphical Abstract](image) + +## Abstract +Insulin secreted from islets of Langerhans is the main hormone responsible for reducing blood glucose levels. Measurement of insulin secretion patterns at the single-islet level can reveal functional differences in secretion timings and patterns. To facilitate measuring dynamic insulin release, we describe fluorescence anisotropy imaging immunoassays (FAI) as a simple method for increased throughput of islet secretion measurements. The system uses a microfluidic device to perfuse reagents through multiple islet chambers and collect emission data using polarized light. Despite challenges with islet chamber resistance, this method significantly optimizes the throughput of hormone release measurements. + +## Keywords +- Blood glucose +- Insulin secretion +- Fluorescence anisotropy +- Immunoassays +- Polarizers +- Islets of Langerhans +- Hormone + +## Introduction +Insulin, secreted from β-cells within pancreatic islets, is crucial for reducing blood glucose levels. Differences in insulin secretion arise due to variations in islet size, composition, architecture, and responsiveness. Heterogeneous secretion profiles can reveal distinct functional responses crucial for understanding pancreatic islet behavior in various physiological and pathological conditions. Traditional antibody-based assays like enzyme-linked immunosorbent assays (ELISA) are sensitive but time-consuming and complex for real-time measurements. Fluorescence anisotropy (FA) techniques facilitate rapid screening by measuring changes in fluorescence polarization without separating bound and free fluorophores. This work describes using FA to measure insulin release from islets, leveraging microfluidic devices to increase throughput. + +## Materials and Methods + +### Chemicals and Reagents +- **Sodium Chloride (NaCl):** EmdMillipore #SX0420-1 +- **Calcium Chloride (CaCl₂):** EmdMillipore #102391 +- **Sodium Hydroxide (NaOH):** EmdMillipore #SX0590 +- **Ethylenediaminetetraacetic acid (EDTA):** EmdMillipore #4010-OP, chelating agent essential for binding calcium ions. +- **Tween-20:** EmdMillipore #817072, a detergent for reducing surface tension. +- **Bovine Serum Albumin (BSA):** EmdMillipore #160069, a protein stabilizer in solutions. +- **Dextrose:** Thermo Fisher #D16-1, a glucose source for media. +- **RPMI 1640:** Thermo Fisher #MT10040CV, a culture medium. +- **Gentamicin:** Thermo Fisher #15710064, an antibiotic. +- **Fetal Bovine Serum (FBS):** Thermo Fisher #16140071, a supplement for cell culture. +- **Collagenase P from Clostridium histolyticum:** Roche #11215809103, used for tissue dissociation. +- **Monoclonal Insulin Antibody (Ab):** Meridian Life Science #E86211M, used for specific detection of insulin. +- **Fluorescein Isothiocyanate Labeled Insulin (Insulin*):** Sigma Aldrich #I3661, fluorescent marker. + +### Microfluidic Device and System +- **Fabrication:** Borosilicate glass processed with wet chemical etching (Telic Company) was used to create microfluidic channels of 50 µm depth and 75 µm width. +- **Fluidic Access Holes:** Drilled using 0.02" and 0.012" diamond-tipped drill bits. +- **Reservoirs:** Fluid reservoirs (IDEX Health and Science) were bonded to the device. +- **Syringe Pump:** Connected to the device with Tygon tubing (0.02" ID × 0.06" OD). A fingertight fitting (IDEX) was used to secure connections. + +### Optical Detection System +- **Microscope:** Placed on a motorized XY stage, a Nikon Eclipse Ti-S inverted microscope was used. +- **Light Source:** Xenon arc lamp filtered through 485 ± 35 nm and 536 ± 40 nm bandpass filters (Semrock). Coupled to the microscope with a liquid light guide and linear polarizer. +- **detection:** Fluorescence emission collected and processed through vertical and horizontal polarizers, using an automated emission filter wheel. +- **Imaging:** A sCMOS camera (Prime BSI express, Photometrics) captured images over a 10-second exposure time. System controlled via micro-manager software. + +### Isolation and Culture of Islets of Langerhans +- **Murine Islets:** CD-1 male mice were used (Charles River Laboratories). Islets were cultured in RPMI 1640 with supplements. +- **Human Islets:** Sourced from Prodo Laboratories, isolated from cadaveric donors. Cultured in PIM(S) media under controlled conditions. +- **Preparation:** Islets were washed in balanced salt solution (BSS) with 3 mM glucose before loading 5 murine or 7 human islets into each microfluidic chamber. + +### Data Analysis +- **Fluorescence Measurement:** Intensity measured and converted to anisotropy using custom MATLAB scripts. +- **Calibration:** Anisotropy values were fitted with four-parameter logistic functions. Limit of detection determined based on blank solution anisotropy. + +## References +*For detailed references, please refer to the original protocol document or visit the provided DOI link.* + +``` +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/inducing-gemmulation-in-the-freshwater-sponge-ephy-b49pqz5n.md b/markdown-output/inducing-gemmulation-in-the-freshwater-sponge-ephy-b49pqz5n.md new file mode 100644 index 0000000000000000000000000000000000000000..2cc1569f6159968a2a9bfd8096609820c592b762 --- /dev/null +++ b/markdown-output/inducing-gemmulation-in-the-freshwater-sponge-ephy-b49pqz5n.md @@ -0,0 +1,97 @@ +```markdown +# Goal/Experiment: +Inducing gemmulation in the freshwater sponge Ephydatia muelleri in culture using theophylline. + +## Inducing Gemmulation in the Freshwater Sponge *Ephydatia muelleri* in Culture Using Theophylline + +**Authors:** +Shunsuke Sogabe¹, Taitum Cornish¹, April Hill², Ana Riesgo³, Sally P Leys¹ +¹Department of Biological Sciences, University of Alberta, Canada +²Biology Department, Bates College, ME, USA +³Department of Biodiversity and Evolutionary Biology, Museo Nacional de Ciencias Naturales de Madrid + +**Publication Date:** +May 13, 2022 + +**DOI:** +[dx.doi.org/10.17504/protocols.io.n92ldzprnv5b/v1](https://dx.doi.org/10.17504/protocols.io.n92ldzprnv5b/v1) + +**Tags:** +Symbiosis Model Systems, Transfection Project + +## Introduction + +Freshwater sponges produce overwintering cysts called gemmules that are full of stem cells, allowing them to survive harsh winter months. The gemmules of *Ephydatia muelleri* can be kept in 3°C for months, and in -80°C for years, remaining viable to hatch and develop into a functional sponge, as outlined in the protocol: ["Hatching and freezing gemmules from the freshwater sponge *Ephydatia muelleri*"](https://dx.doi.org/10.17504/protocols.io.863hzgn). With a recently published chromosome-level assembly of the genome (Kenny et al., 2020) and multiple transcriptomes, this makes *Ephydatia muelleri* an ideal sponge species to establish as a model sponge species that can be used in labs worldwide. + +## Materials and Methods + +### Materials + +- **Theophylline** (C7H8N4O2 - MW:180.16) +- Wide bore Pasteur pipettes and bulbs +- Petri dishes (e.g. 5cm diameter) +- Fine forceps (e.g. Dumont No.5) +- 10mL serological pipette + +### Medium + +**Strekal's Medium:** +Strekal TA, McDiffett W. 1974. Factors affecting germination, growth, and distribution of the freshwater sponge, *Spongilla fragilis*. Biological Bulletin 146:267-278. + +## Protocol + +This protocol is based on an 8-gemmule sponge grown in a 5cm Petri dish (Max. volume 20mL) and treated with 400µM theophylline. Both the number of gemmules and total volume of medium can be scaled up or down. + +### Preparation of 10mM Stock Solution of Theophylline: + +- Molecular Weight (MW): 180.16 +- For 10 mL of 10 mM stock solution = 18.0 mg (0.018g) in 10 mL of dH₂O +- For 50 mL of 10 mM stock solution = 90.1 mg (0.090g) in 50 mL of dH₂O +- Store at 4°C (fresh is always better, but this should last for at least 1 week, up to a month at 4°C) + +### Steps + +1. **Hatch and Grow Sponges** + - Use the protocol - ["Hatching and freezing gemmules from the freshwater sponge *Ephydatia muelleri*" by Sally P Leys, Lauren Grombacher, and April Hill](https://dx.doi.org/10.17504/protocols.io.863hzgn) + - Sponges grown from a larger number of gemmules have a faster rate of gemmulation and resulting number of gemmules. It is recommended to plate at least 4 gemmules close together to grow a bigger sponge to treat with theophylline. + +2. **Treating Sponges with Theophylline** + - After sponges have reached stage 5 (approximately 7 days), remove culture medium to have 10mL volume in the dish. Never expose sponges to air. + + **2.1 Incubate Sponges in Theophylline Solution:** + - Make a 2× concentration of theophylline solution: add 800µL of 10mM stock solution into 10mL of 1× Strekal's medium for a final concentration of 800µM. + - Add the 10mL of 800µM to 10mL of 1× Strekal's medium containing the sponge for a final concentration of 400µM. + - Cover with tin foil to protect from light. + + **2.2 Change medium every 2 days:** + - Make 16 mL of 400µM theophylline solution: add 640µL of 10mM stock solution into 16mL of 1× Strekal's medium. + - For each 20mL Petri dish, remove 16mL of the culture medium from the sponge dish, and replace with 16mL of the freshly made 400µM theophylline solution. + +3. **Gemmulation, Maturing Newly Gemmulated Gemmules, and Removing Gemmules for Hatching** + - **Gemmulation:** + - Under these conditions, gemmulation can be detected 1-2 weeks after initiating theophylline treatment. + - Early stages of gemmulation appear as white regions in the sponge, which gradually start to have a more defined shape and develop a hard casing with a light brown color in 4-5 days. + + - **Maturing Newly Formed Gemmules:** + - Newly formed gemmules from *Ephydatia muelleri* require maturation for 6-7 weeks before they are ready to hatch. Maturation is carried out by incubating the whole sponge at 3°C in the dark. + + - **Removing and Plating Newly Gemmulated Gemmules:** + - Newly formed gemmules can be removed by carefully detaching them from the sponge tissue using fine forceps (e.g. Dumont No.5), and transferring them into a new dish to hatch and grow. + - At an early stage of maturation (6-7 weeks), they can take 10+ days to hatch after plating. + +## References + +1. Kenny, N.J., Francis, W.R., Rivera-Vicéns, R.E. et al. Tracing animal genomic evolution with the chromosomal-level assembly of the freshwater sponge *Ephydatia muelleri*. Nature Communications. 2020. 11, 3676. [https://doi.org/10.1038/s41467-020-17397-w](https://doi.org/10.1038/s41467-020-17397-w). +2. Rasmonrt R. Stimulation of cell aggregation by theophylline in the asexual reproduction of fresh-water sponges (*Ephydatia fluviatilis*). Experientia. 1974. Jul 15;30(7):792-4. doi: 10.1007/BF01924190. PMID: 4367998. + +## Citation +Shunsuke Sogabe, Taitum Cornish, April Hill, Ana Riesgo, Sally P Leys. Inducing gemmulation in the freshwater sponge *Ephydatia muelleri* in culture using theophylline [https://dx.doi.org/10.17504/protocols.io.n92ldzprnv5b/v1](https://dx.doi.org/10.17504/protocols.io.n92ldzprnv5b/v1) + +### Keywords + +gemmule, gemmulation, theophylline, *Ephydatia muelleri*, freshwater sponge, Porifera + +**All images taken and edited by Taitum Cornish and Shunsuke Sogabe.** + +*End of Output* +``` \ No newline at end of file diff --git a/markdown-output/integra-magbead-dna-and-rna-extraction-for-isolate-cf4mtqu6.md b/markdown-output/integra-magbead-dna-and-rna-extraction-for-isolate-cf4mtqu6.md new file mode 100644 index 0000000000000000000000000000000000000000..0095dce76e25ee5813ca845e3bfe045a65a96c2b --- /dev/null +++ b/markdown-output/integra-magbead-dna-and-rna-extraction-for-isolate-cf4mtqu6.md @@ -0,0 +1,224 @@ +```markdown +# Goal/Experiment: +To extract high-quality DNA and RNA from isolated colonies suitable for Next-Generation Sequencing (NGS). + +# Integra Magbead DNA and RNA Extraction for Isolated Colonies + +**Sopahana Chea, Sreyngim Lay, Mengheng Oum, Gechlang Tang, Cheata Hou, Manu Vanaerschot, Christina Yek, Cristina Tato, Jessica Manning, Vida Ahyong** + +- International Center of Excellence in Research, National Institute of Health, Cambodia +- Chan Zuckerberg Biohub +- National Institute of Health + +## Abstract + +This protocol details the process of extracting DNA and RNA from isolated colonies. The extracted high-quality DNA or RNA is suitable for Next-Generation Sequencing (NGS). + +## DOI + +[dx.doi.org/10.17504/protocols.io.dm6gpjeqjgzp/v1](https://dx.doi.org/10.17504/protocols.io.dm6gpjeqjgzp/v1) + +## Protocol Citation + +Sopahana Chea, Sreyngim Lay, Mengheng Oum, Gechlang Tang, Cheata Hou, Manu Vanaerschot, Christina Yek, Cristina Tato, Jessica Manning, Vida Ahyong. 2022. Integra Magbead DNA and RNA Extraction for isolated colonies. [protocols.io](https://protocols.io/view/integra-magbead-dna-and-rna-extraction-for-isolate-cf4mtqu6). + +## Keywords + +Integra, DNA, RNA, Colony, isolated, Extraction + +## License + +This is an open access protocol distributed under the terms of the [Creative Commons Attribution License](https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. + +## Created + +Sep 02, 2022 + +## Last Modified + +Sep 07, 2022 + +## Protocol Integer ID + +69485 + +## Guidelines + +Adapted from the ZymoBIOMICS MagBead DNA/RNA Kit Manual (Zymo Research, Cat#R2135). + +## Materials + +1. **RNase away spray** for RNase decontaminants - Thermo Fisher Scientific, Catalog #7002PK. +2. **ZymoBIOMIC MagBead DNA/RNA** - Zymo Research, Catalog #R2135. +3. **100% Molecular grade ethanol** - User contributed. +4. **Molecular Grade Isopropanol** - User contributed. +5. **Proteinase K w/ Storage buffer 20mg set** - Zymo Research, Catalog #D3001-2-20. +6. **DNase I Set** - Zymo Research, Catalog #E1010. +7. **Nuclease-free water** - Ambion, Catalog #AM9932. +8. 1ml deep well sterile plate. +9. 2ml deep well sterile plate. +10. Hard-shell PCR Plates 96 V-well (Bio-Rad, Cat# HSP9601). +11. PCR Plate Seal, foil (Bio-Rad, Cat# MSF1001). +12. 96S Super Magnet (ALPAQUA, Cat# A001322). +13. VIAFLO 96 channel pipette - Integra. + +## Safety Warnings + +- All steps should be performed at room temperature. +- Perform the extraction in the extraction room separate from the PCR room. +- Respect Laboratory safety guidelines for all steps of the protocol. +- Wearing PPE is recommended. +- When reusing tips, include a bit of extra air aspiration to avoid drops at the bottom of tips when aspirating volumes, and a bit of extra air blows out at the end of dispensing steps in plates. + +## Procedure + +### Buffer Preparation + +1. **Add 20 mL** isopropanol to the MagBead DNA/RNA Wash 1 concentrate. +2. **Add 30 mL** isopropanol to the MagBead DNA/RNA Wash 2 concentrate. +3. Reconstitute lyophilized Proteinase K at **20 mg/mL** with Proteinase K Storage Buffer and mix by vortexing. Use immediately or store at -20°C. +4. Reconstitute each vial of lyophilized DNase I with **2.25 mL** DNase/RNase-Free water in a conical tube. + +For each sample to be treated, prepare DNase I Reaction Mix (scale up proportionally): +* Add **45 μL** DNase I (reconstituted) and **5 μL** DNA Digestion Buffer in a nuclease-free tube. +* Mix by gentle inversion and place on ice until ready to use. + +### Make Buffer Plates Prior to Starting Protocol + +1. **Pre-make Lysis Buffer plate** with: + - **520 μL** DNA/RNA Lysis buffer in 1ml deep well plate. + +2. **Pre-make Beads plate** with: + - **35 μL** ZymoBIOMIC MagBinding Beads into 96 V-well PCR plate. + +3. **Pre-make DNA/RNA Wash 1 plate** with: + - **520 μL** MagBead DNA/RNA Wash 1 into 1ml deep well plate. Make it two plates. + +4. **Pre-make DNA/RNA Wash 2 plate** with: + - **520 μL** MagBead DNA/RNA Wash 2 into 1ml deep well plate. Make it two plates. + +5. **Pre-make 100% Ethanol plate** with: + - **1100 μL** of 100% Ethanol into a 2ml deep well plate. Make it three plates. + +6. **Pre-make Prep Buffer plate** with: + - **520 μL** DNA/RNA Prep Buffer into a 1ml deep well plate. + +7. **Pre-make water plate** with: + - **60 μL** Nuclease-free water in a 96 V-well PCR plate. Make it two plates. + +8. Spin all plates down for 1 minute except for the bead plate. Perform a quick pulse spin down of the bead plate, just enough to get all the liquid down. Centrifuge the rest of the plate at 12,000 rpm for 1 minute. + +### Sample Preparation and Proteinase K + +1. Create a plate map so you know which sample you are adding to each well. Add **50 μL** of isolated colonies samples to plate 1 (leave column 12 for water control). +2. Top up the 1x DNA/RNA Shield to get **750 μL**. +3. Manually add **120 μL** of Proteinase K into the 0.2ml 8-strip well. +4. Use multichannel pipet to add **10 μL** of Proteinase K into each sample and mix (plate 1). +5. Load a set of Integra tips (tip set 1) onto the Integra. +6. **Program**: Pipet/Mix 250ul, 15 cycles, speed 4. + - Program the Integra to pipet **250 μL** of your samples up and down for 1 minute (15 cycles), then incubate at room temperature for 30 minutes. Keep tips. + +### Sample Binding and Washing + +7. **Program**: Pipet 250ul. Add **500 μL** total of Lysis Buffer to the sample plate (plate 1). +8. **Program**: Pipet/Mix 250ul, 30 cycles, speed 10. + - Program the Integra to mix samples and buffer for 2 minutes. Keep tips. +9. **Aliquot 35 μL** of MagBinding Beads into 96 V-well PCR plate. +10. **Program**: Pipet/Mix 20ul, 10 cycles, 2 times, speed 4. + - Program the Integra to mix the MagBinding Beads plate, so the beads are fully resuspended. +11. **Program**: Pipet 30ul. Add **30 μL** of MagBinding Beads into the sample plate (plate 1). +12. **Program**: Pipet/Mix 250ul, 30 cycles, speed 3. + - Program the Integra to mix the sample and MagBinding Beads plate, so the beads are fully resuspended. Continue this Integra Program to mix the sample and MagBinding Beads for 20 minutes. +13. Transfer the plate/tube to the magnetic stand for 5 minutes until beads (DNA) have pelleted, transfer the cleared supernatant (RNA) into a new 96 V-well plate. + +### DNA Purification (Beads) + +14. Change new Integra tips. +15. **Program**: Pipet 250ul, 2 times, speed 7. + - Dispense a total of **500 μL** MagBead DNA/RNA Wash 1 into sample plate and mix well. +16. **Program**: Pipet/Mix 250ul, 30 cycles, speed 10. + - Program the Integra to mix the Wash 1 buffer with the beads. Keep tips. +17. Place the 96-well magnetic stand underneath the sample plate for 2 minutes until a bead ring forms. +18. **Program**: Manual Pipet 250ul, 2 times, speed 3. + - Aspirate and discard the cleared supernatant into a 2ml deep well waste plate. +19. **Program**: Pipet 250ul, 2 times, speed 7. + - Dispense a total of **500 μL** MagBead DNA/RNA Wash 2 into sample plate and mix well. +20. **Program**: Pipet/Mix 250ul, 30 cycles, speed 10. + - Program the Integra to mix the Wash 2 buffer with the beads. Keep tips. +21. Place the 96-well magnetic stand underneath the sample plate for 2 minutes until a bead ring forms. +22. **Program**: Manual Pipet 250ul, 2 times, speed 3. + - Aspirate and discard the cleared supernatant into a 2ml deep well waste plate. +23. Change new Integra tips. +24. **Program**: Pipet 250ul, 2 times, speed 7. + - Dispense a total of **500 μL** 100% Ethanol into sample plate and mix well. +25. **Program**: Pipet/Mix 250ul, 30 cycles, speed 10. + - Program the Integra to mix 100% Ethanol with the beads. Keep tips. +26. Place the 96-well magnetic stand underneath the sample plate for 2 minutes until a bead ring forms. +27. **Program**: Manual Pipet 250ul, 2 times, speed 3. + - Aspirate and discard the cleared supernatant into a 2ml deep well waste plate. +28. Repeat step 24. +29. Dry the beads for 10 minutes on the magnetic stand. +30. Change new Integra tips. +31. **Program**: Pipet 30ul, speed 5. + - Dispense a total of **30 μL** nuclease-free water into the sample plate. +32. **Program**: Pipet/Mix 20ul, 30 cycles, speed 7. + - Program the Integra to mix nuclease-free water with the beads. Keep tips. +33. **Program**: Manual Pipet 30ul, speed 3. + - Transfer the plate to the magnetic stand and pellet the beads for 5 minutes, then aspirate and dispense the eluted DNA to a new 96 V-well plate. +34. Store DNA sample immediately at -80°C. + +### RNA Purification (Supernatant) + +35. Change the new Integra tip. +36. **Program**: Pipet 230ul, 3 times, speed 7. + - Dispense a total of **690 μL** 100% Ethanol to the supernatant. +37. **Program**: Pipet/Mix 250ul, 30 cycles, speed 7. + - Program the Integra to mix 100% Ethanol with the supernatant. Keep tips. +38. **Aliquot 35 μL** of MagBinding Beads into 96 V-well PCR plate. +39. **Program**: Pipet/Mix 20ul, 10 cycles, 2 times, speed 4. + - Program the Integra to mix the MagBinding Beads plate, so the beads are fully resuspended. +40. **Program**: Pipet 30ul. Add **30 μL** of MagBinding beads into the sample plate. +41. **Program**: Pipet/Mix 250ul, 10 cycles, speed 3. + - Program the Integra to mix the sample and MagBinding beads plate, so the beads are fully resuspended. Continue this Integra Program to mix the sample and MagBinding Beads for 10 minutes. +42. Transfer the plate to the magnetic stand for 5 minutes until beads have pelleted, then discard the cleared supernatant. +43. **Program**: Pipet 250ul, 2 times, speed 7. + - Dispense a total of **500 μL** MagBead DNA/RNA Wash 1 into sample plate. +44. **Program**: Pipet/Mix 250ul, 30 cycles, speed 10. + - Program the Integra to mix the Wash 1 buffer with the beads. Keep tips. +45. Place the 96-well magnetic stand underneath the sample plate for 2 minutes until a bead ring forms. +46. **Program**: Manual Pipet 250ul, 2 times, speed 3. + - Aspirate and discard the cleared supernatant into a 2ml deep well waste plate. +47. **Program**: Pipet 250ul, 2 times, speed 7. + - Dispense a total of **500 μL** MagBead DNA/RNA Wash 2 into sample plate. +48. **Program**: Pipet/Mix 250ul, 30 cycles, speed 10. + - Program the Integra to mix the Wash 2 buffer with the beads. Keep tips. +49. Place the 96-well magnetic stand underneath the sample plate for 2 minutes until a bead ring forms. +50. **Program**: Manual Pipet 250ul, 2 times, speed 3. + - Aspirate and discard the cleared supernatant into a 2ml deep well waste plate. +51. **Program**: Pipet 250ul, 2 times, speed 7. + - Dispense a total of **500 μL** 100% Ethanol into sample plate. +52. **Program**: Pipet/Mix 250ul, 30 cycles, speed 10. + - Program the Integra to mix 100% Ethanol with the beads. Keep tips. +53. Place the 96-well magnetic stand underneath the sample plate for 2 minutes until a bead ring forms. +54. **Program**: Manual Pipet 250ul, 2 times, speed 3. + - Aspirate and discard the cleared supernatant into a 2ml deep well waste plate. +55. Repeat step 51. +56. **DNase I treatment**, use multiple channel pipet to transfer **50 μL** of DNase I Reaction Mix and mix gently for 10 minutes. +57. **Program**: Pipet 250ul, 2 times, speed 7. + - Dispense a total of **500 μL** DNA/RNA Prep Buffer into sample plate. +58. **Program**: Pipet/Mix 250ul, 30 cycles, speed 10. + - Program the Integra to mix the DNA/RNA Prep Buffer with the beads. Keep tips. +59. Place the 96-well magnetic stand underneath the sample plate for 2 minutes until a bead ring forms. +60. **Program**: Manual Pipet 250ul, 2 times, speed 3. + - Aspirate and discard the cleared supernatant into a 2ml deep well waste plate. +61. Repeat step 57 to 60. +62. **Program**: Pipet 30ul, speed 5. + - Dispense a total of **30 μL** nuclease-free water into the sample plate. +63. **Program**: Pipet/Mix 20ul, 30 cycles, speed 7. + - Program the Integra to mix nuclease-free water with the beads. Keep tips. +64. **Program**: Manual Pipet 30ul, speed 3. + - Transfer the plate to the magnetic stand and pellet the beads for 5 minutes, then aspirate and dispense the eluted RNA to a new 96 V-well plate. +65. Store RNA sample immediately at -80°C. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/integra-total-nucleic-acid-extraction-cf2ztqf6.md b/markdown-output/integra-total-nucleic-acid-extraction-cf2ztqf6.md new file mode 100644 index 0000000000000000000000000000000000000000..a93f795ac8c94d8581ca668fcf781ec5954eee16 --- /dev/null +++ b/markdown-output/integra-total-nucleic-acid-extraction-cf2ztqf6.md @@ -0,0 +1,198 @@ +```markdown +Goal/Experiment: +This protocol details the procedure for extracting Total Nucleic Acid (TNA) from sera and/or nasopharyngeal or nasal swabs. The extracted high-quality nucleic acids are intended for use in Next-Generation Sequencing (NGS). + +# Integra Total Nucleic Acid Extraction + +## Abstract +This SOP is the process of extracting Total Nucleic Acid (TNA) from sera and/or nasopharyngeal or nasal swabs. The isolated high-quality nucleic acids are suitable for Next-Generation Sequencing (NGS). + +## DOI +[dx.doi.org/10.17504/protocols.io.81wgbydq1vpk/v1](https://dx.doi.org/10.17504/protocols.io.81wgbydq1vpk/v1) + +## Protocol Citation +Sophana Chea, Sreyngim Lay, Mengheng Oum, Gechlang Tang, Cheata Hou, Manu Vanaerschot, Christina Yek, Jessica Manning, Vida Ahyong 2022. Integra Total Nucleic Acid Extraction. protocols.io +[https://protocols.io/view/integra-total-nucleic-acid-extraction-cf2ztqf6](https://protocols.io/view/integra-total-nucleic-acid-extraction-cf2ztqf6) + +## Keywords +Integra, Total Nucleic Acid, DNA, RNA + +## License +This is an open-access protocol distributed under the terms of the [Creative Commons Attribution License](https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. + +## Created +Sep 01, 2022 + +## Last Modified +Sep 05, 2022 + +## Protocol Integer ID +69433 + +## Guidelines +Adapted from the Quick-DNA/RNA Pathogen MagBead Kit Manual (Zymo Research, Cat# R2145) + +## Materials + +1. RNase Away sprays for RNase decontaminants (Thermo Scientific, Cat#7002). +2. 1 ml deep well sterile plate. +3. 2 ml deep well sterile plate. +4. Hard-shell PCR Plates 96-well (Bio-Rad, Cat# HSP9601). +5. PCR Plate Seal, foil (Bio-Rad, Cat# MSF1001). +6. Quick-DNA/RNA Pathogen MagBead kit (Zymo Research, Cat# R2145). +7. Molecular-Grade Isopropanol (100% isopropanol). +8. Molecular-Grade Absolute ethanol (100% ethanol). +9. Proteinase K with Storage buffer 20mg (Zymo Research, Cat# D3001-2-20). +10. DNase/RNase-free water +11. VIAFLO 96 channel pipette +12. DNase I (Zymo Research, Cat# E1010) +13. 96-well magnetic stand + +### Notes: +*RNase Away*: A potent RNase decontaminant used to irreversibly inactivate RNase enzymes, preventing the degradation of RNA in samples. +*Quick-DNA/RNA Pathogen MagBead kit*: Provides reagents for the purification of high-quality DNA/RNA from a variety of pathogens in biofluids, cell suspensions, swabs, and tissue. +*Proteinase K*: An enzyme that digests nucleases and other proteins, ensuring highly purified nucleic acid samples. + +## Safety Warnings + +- All steps should be performed at **room temperature**. +- If reusing tips, include a bit of extra air aspiration to avoid drops at the bottom of tips when aspirating volumes, and a bit of extra air blow-out at the end of dispensing steps in plates. +- Perform the TNA extraction in a dedicated extraction room separate from the PCR room. +- Respect laboratory safety guidelines for all steps of the protocol. Wearing PPE is recommended. + +## Reagent Preparation (required with new kit) - 15m + +1. Add **20 mL** of isopropanol to the MagBead DNA/RNA Wash 1 concentrate. +2. Add **30 mL** of isopropanol to the MagBead DNA/RNA Wash 2 concentrate. +3. Add **1.2 mL** of Proteinase K Storage Buffer per vial to reconstitute the lyophilized Proteinase K at **20 mg/mL**. Vortex to dissolve. + - Store at **-20 °C** freezer. + +## Preparation of Buffer Plate (before starting protocol) - 1h + +1. Pre-make pathogen buffer plate with **880 µL** Pathogen DNA/RNA buffer in 1ml deep well plate. +2. Pre-make bead plate with **25 µL** MagBinding beads into 96V-well PCR plate. + - *Make immediately before starting, <1h prior to starting the protocol, to ensure the beads are mixed. +3. Pre-make DNA/RNA Wash 1 plate with **550 µL** Wash 1 buffer into a 1ml deep well plate. +4. Pre-make DNA/RNA Wash 2 plate with **550 µL** Wash 2 buffer into a 1ml deep well plate. +5. Pre-make 100% ethanol plate with **1 ml** 100% ethanol into a 2ml deep well plate. +6. Pre-make 80% ethanol plate with **600µL** 80% ethanol into a 1ml deep well plate. +7. Pre-make water plate with **50 µL** DNase/RNase-free water in a 96 V-well PCR plate. +8. Pre-make water plate with **30 µL** DNase/RNase-free water in a 96 V-well PCR plate. + - Spin all plates down for **1 min** except the bead plate. + - Perform a quick pulse spin down of the bead plate, just enough to get all liquid down. + - Centrifuge the rest of the plates at 12,000 rpm for **1 min**. + +## Sample Preparation and Proteinase K - 16m + +1. Create a plate map to track sample additions. +2. Add **400 µL** of samples to **2 ml** deep well plate (Plate 1). +3. Manually add **65 µL** of Proteinase K to each well of 8 well PCR strip tubes. Using a manual multichannel pipet, aliquot **4 µL** of Proteinase K into each sample (Plate 1). +4. Load a set of Integra tips (tip set 1) onto the Integra. + - **Program**: + - *Pipet/Mix 250ul*, 15 cycles, speed 10. + - *Program* Integra to pipet **250 µL** of your samples up and down for - *15 cycles*, then incubate at **room temperature** for **15m**. + - *Keep tips*. + +## Sample Binding and Washing - 1h 20m + +1. **Program**: + - *Pipet 300ul*. Add **800 µL** total of Pathogen DNA/RNA Buffer to the sample plate (Plate 1). +2. **Program**: + - *Pipet/Mix 250ul*, 30 cycles, speed 10. *Program* the Integra to mix samples and buffer for **2 min**. + - *Keep tips*. +3. **Program**: + - *Pipet/Mix 20ul*, 20 cycles. *Program* the Integra to mix the MagBinding beads plate so the beads are fully resuspended. If beads are not fully to the bottom of theplate, perform a short pulse spin. +4. **Program**: + - *Pipet 20ul*. *Program* the Integra to aspirate **20 µL** from the MagBinding beads plate. + - *Check that all wells have beads*. +5. **Program**: + - *Pipet/Mix 250ul*, 30 cycles x 5 (10 min total), speed 7. *Program* the Integra to mix the beads with the sample for **10 min**. + - *Keep tips* +6. Place the 96-well magnetic stand underneath the sample plate for **5 min** until a bead ring forms. +7. **Program**: + - *Manual Pipet 300ul*. Aspirate and discard the cleared supernatant into a **2 ml** deep well waste plate. Discard tips, load new tips. +8. Remove magnetic stand. +9. **Program**: + - *Pipet 250ul*. Dispense a total of **500 µL** Wash 1 into the sample plate. + - *Keep tips*. +10. **Program**: + - *Pipet/Mix 250ul*, 30 cycles, speed 7. + *Program* the Integra to mix the beads with the Wash buffer for **2 min** total. + - *Keep tips*. +11. Place the 96-well magnetic stand underneath the sample plate for **2 min** until a bead ring forms. +12. **Program**: + - *Manual Pipet 300ul*. Aspirate and discard the cleared supernatant into the **2 ml** deep well waste plate. Keep tips. +13. **Program**: + - *Pipet 250ul*. Dispense a total of **500 µL** Wash 2 into the sample plate. + - *Keep tips*. +14. **Program**: + - *Pipet/Mix 250ul*, 30 cycles, speed 7. *Program* the Integra to mix the beads with the wash buffer for **2 min**. + - *Keep tips*. +15. Place the 96-well magnetic stand underneath the sample plate for **2 min** until a bead ring forms. +16. **Program**: + - *Manual Pipet 300ul*. Aspirate and discard the cleared supernatant into the 2ml deep well waste plate. Keep tips. +17. **Program**: + - *Pipet 250ul*. Dispense a total of **500 µL** 100% Ethanol into the sample plate. +18. **Program**: + - *Pipet/Mix 250ul*, 30 cycles, speed 7. *Program* the Integra to mix the beads with Ethanol for **2 min**. + - *Keep tips*. +19. Place the 96-well magnetic stand underneath the sample plate for **2 min** until a bead ring forms. +20. **Program:** + - *Manual Pipet 300ul*. Aspirate and discard the cleared supernatant into the **2 ml** deep well waste plate. +21. **Remove magnetic stand.** +22. **Repeat steps 22 to 26 for another round of Ethanol wash.** Discard tips after 2nd Ethanol wash. Load new tips. +23. Keep the sample plate on the magnetic stand after the final Ethanol wash until the beads are dry (~ **10 min**). +24. **Program:** + - *Pipet/Mix 33ul*, 30 cycles, speed 7. **Pipet 33 μL** of Nuclease-free water (Heat the water to **55 °C** for **10 min** before elution) into the dried beads and mix the beads for **2 min**. +25. Place the 96-well magnetic stand underneath the sample plate for **2 min** until a bead ring forms. +26. **Program:** + - *Manual Pipet 30ul*. **Pipet 30 µL** from the sample plate and dispense into a new **96 V-bottom** PCR plate. +27. Store TNA sample immediately at **-80 °C**. + +## DNase Treatment Post-TNA Purification - 15m + +1. **Aliquot SPRI beads*** (well mixed and resuspended) **50 µL** into a new **96 V-bottom** PCR plate. +2. **Prepare DNase I Solution**: + - Add **275 µL** DNase/RNase-free water to reconstitute lyophilized DNase I and mix. + - Aliquot **20 µL** of TNA into a new 96 V-bottom PCR plate. +3. Prepare DNase master mix: + - 1x rxn = 20ul sample + 2.5ul DNase I + 2.5ul Digestion Buffer. + - Using a manual multichannel pipet, aliquot **5 µL** of DNase I master mix to each sample and mix. +4. **Program:** + - *Pipet/Mix 20ul*, 20 cycles, speed 7. +5. **Incubate:** + - **15 min** at **room temperature**. + +## SPRI Bead Clean-up - 30m + +1. **Program:** + - *Pipet/Mix 45ul*, 20 cycles, speed 7. **Add 45 µL** SPRI beads (1.8x ratio) to the DNase I treated sample and mix by pipetting. +2. **Incubate:** + - **5 min** at **room temperature**. +3. Place the 96-well magnetic stand underneath the sample plate for **2 min** until a bead ring forms. +4. **Program:** + - *Manual Pipet 100ul*. Aspirate and discard the cleared supernatant into the **2 ml** deep well waste plate. Keep tips. +5. **Program:** + - *Pipet 200ul*. (1st Ethanol Wash) Aspirate and dispense **200 µL** of freshly made 80% Ethanol into sample plate. +6. **Incubate:** + - **1 min** at **room temperature**. +7. **Program:** + - And discard the cleared supernatant into the **2 ml** deep well waste plate. Keep tips. +8. **Program:** + - *Pipet 200ul*. (2nd Ethanol Wash) Aspirate and dispense **200 µL** of freshly made 80% Ethanol into sample plate. +9. **Incubate**: + - **1 min** at **room temperature**. +10. **Program:** + - *Manual Pipet 200ul*. Aspirate and discard the cleared supernatant into the **2 ml** deep well waste plate. Discard tips, load new tips. +11. **Incubate**: + - **5 min** at **room temperature** until beads are dry. +12. Remove plate from magnetic stand. +13. **Program:** + - *Pipet/Mix 22ul*, 10 cycles, speed 7. **Add 22 µL** DNase/RNase-free water to each well and mix beads. +14. Place the 96-well magnetic stand underneath the sample plate for **2 min** until a bead ring forms. +15. **Program:** + - *Manual Pipet 20ul*. **Aspirate 20 µL** of purified TNA sample to a new 96 V-bottom PCR plate. +16. Store sample at **-80 °C** immediately. + +`endofoutput` +``` \ No newline at end of file diff --git a/markdown-output/internal-epitope-tagging-of-proteins-using-transpo-drr555.md b/markdown-output/internal-epitope-tagging-of-proteins-using-transpo-drr555.md new file mode 100644 index 0000000000000000000000000000000000000000..4312c64d6412b0035d32a70db638bd6defc64b39 --- /dev/null +++ b/markdown-output/internal-epitope-tagging-of-proteins-using-transpo-drr555.md @@ -0,0 +1,266 @@ +```markdown +# Goal/Experiment: +The goal of this experiment is to tag internal epitopes of proteins using the transposon Tn7 through in vitro transposon mutagenesis. This protocol introduces internal epitope tags to target proteins, delineating steps for conducting “- enzyme” negative control reactions and “+ enzyme” experimental mutagenesis reactions in parallel. + +# Internal Epitope Tagging of Proteins Using Transposon Tn7 + +**Authors**: Rebecca E. Zordan, Brian J. Beliveau, Jonathan A. Trow, Nancy L. Craig, and Brendan P. Cormack + +## Abstract +This protocol for in vitro transposon mutagenesis is used for the introduction of internal epitope tags. It follows the methods described in: + +Zordan RE, Beliveau BJ, Trow JA, Craig NL, and Cormack BP (2015) [Avoiding the Ends: Internal Epitope Tagging of Proteins Using Transposon Tn7](https://dx.doi.org/10.1534/genetics.114.169482), Genetics 200:47-58; doi:10.1534/genetics.114.169482 + +This protocol includes detailed steps for performing small (20 µl) “- enzyme” negative control reactions and large (80 µl) “+ enzyme” experimental mutagenesis reactions in parallel. + +## Guidelines + +### Required Reagents +- **TnsA, TnsB, TnsCA255V Enzymes**: + - Purified enzymes used for transposon mutagenesis + - TnsA stock: 150 ng/µl in Storage Buffer A + - TnsB stock: 200 ng/µl in Storage Buffer B + - TnsCA255V stock: 500 ng/µl in Storage Buffer C +- **Tn7 Donor Vector**: pRZ101, dilute to 25 ng/µl in 10 mM Tris pH 8.0 +- **Entry Vector of Target Gene**: DCW1, dilute to 50 ng/µl in 10 mM Tris pH 8.0 +- **Electrocompetent E. coli Cells**: Invitrogen MegaX DH10B T1R +- **Magnesium Acetate (MgOAc)**: 300 mM +- **Phenol:Chloroform:IAA (25:24:1)**: Amresco 0883-100ml +- **Buffers**: + - Storage Buffer A: 25 mM HEPES pH 8.0, 150 mM NaCl, 1 mM EDTA, 1 mM DTT (in HEPES), 10% glycerol + - Storage Buffer B: 25 mM TrisHCl pH 8.0, 500 mM NaCl, 1 mM EDTA, 1 mM DTT (in Tris), 25% glycerol + - Storage Buffer C: 25 mM HEPES pH 8.0, 1 M NaCl, 0.1 mM EDTA, 2.5 mM DTT (in HEPES), 1 mM ATP, 10 mM MgCl₂, 10% glycerol +- **Chemicals**: + - Chloroform + - 3 M Sodium Acetate (NaOAc) + - 100% Ethanol (EtOH) + - 70% Ethanol (EtOH) +- **Enzymes**: + - FseI, New England Biolabs R0588S + - PmeI, New England Biolabs R0560S + - ApaLI, New England Biolabs R0507S + - T4 DNA Ligase, New England Biolabs M0202S + - Gateway LR Clonase II, Life Technologies 11791-100 +- **Kits**: + - Qiagen Hi-Speed MidiPrep Kit, Qiagen 12643 +- **LB Media**: + - Oxoid Isosensitest Medium (Iso) + - Carbenicillin (100 mg/ml stock) + - Kanamycin (30 mg/ml = 1000x stock) + - Trimethoprim (5 mg/ml in DMSO = 500x stock) +- **Water**: + - Double distilled (MilliQ) H₂O + +### Buffers +Use sterile-filtered MilliQ water to make all buffers. If possible, make all buffers in plastic containers; residual detergent on glassware may inhibit the transposition reaction. + +#### Standard Buffers +- **20 mM ATP in 125 mM Tris pH 7.5** + - Store at -20°C for at most 1 month +- **20 mM DTT in 125 mM Tris pH 7.5** + - Store at -20°C for at most 1 month +- **100 mM ATP in 250 mM HEPES (pH 8.0)** + - For making TnsC storage buffer +- **1 M DTT in 1 M Tris pH 7.5** + - For making TnsB storage buffer +- **10 mM Tris** + - For elution of mutagenized plasmid pools from midi prep kit +- **50% Glycerol** + +#### Tns Storage Buffers +Store at -20°C for at most 6 months. Do not recommend refreezing and rethawing. Store in small aliquots and discard after use. + +### Diagnostic Digests +Highly recommended to perform diagnostic restriction digests on plasmid pools from each phase of this protocol for characterization and mutagenesis complexity. + +Suggested enzyme cuts: +- Donor backbone +- Expression backbone +- Tn7L +- Tn7R +Include “no enzyme” controls and double digestion. + +Perform restriction digest on: +1. Unmutagenized target donor vector +2. wt expression vector +3. Mutagenized donor pool +4. xpC pool +5. xpT pool +6. xpT-L pool +7. xpT-L-R pool + +## Materials +- Phenol:Chloroform:IAA - [Amresco 0883-100ml](https://www.amresco.com/) +- FseI - 100 units [New England Biolabs R0588S](https://www.neb.com/) +- PmeI - 500 units [New England Biolabs R0560S](https://www.neb.com/) +- ApaLI - 2,500 units [New England Biolabs R0507S](https://www.neb.com/) +- T4 DNA Ligase - 20,000 units [New England Biolabs M0202S](https://www.neb.com/) +- Gateway LR Clonase II - [Life Technologies 11791-100](https://www.thermofisher.com/) +- Qiagen Hi-Speed MidiPrep Kit - [Qiagen 12643](https://www.qiagen.com/) +- MegaX DH10B T1R Electrocompetent Cells - [Life Technologies C6400-03](https://www.thermofisher.com/) + +## Protocol + +### In vitro Transposition Protocol + +1. **Step 1: Make Reaction Mix** + Combine: + - 17.6 μl target DNA (880 ng) (DCW1 entry vector) + - 8.8 μl Tn7 donor DNA (220 ng) (pRZ101) + - 11 μl 20 mM ATP + - 11 μl 20 mM DTT + - 56.1 μl ddH2O + +2. **Step 2: Aliquot Reaction Mix** + - Dispense 76 μl into the “+ enzyme” reaction tube + - Dispense 19 μl into the “- enzyme” reaction tube + +3. **Step 3: Make the Enzyme Mixture** + Combine: + - 7.49 μl TnsA + - 3.31 μl Storage buffer A + - 2 μl TnsB + - 6 μl Storage buffer B + - 8 μl TnsCA255V + - 8 μl Storage buffer C + - 5.2 μl 50% glycerol + + Mix by flicking tube gently. Keep on ice. + +4. **Step 4: Make Buffer Mixture for “- enzyme” Control** + Combine: + - 10.8 μl Storage Buffer A + - 8 μl Storage Buffer B + - 16 μl Storage Buffer C + - 5.2 μl 50% glycerol + +5. **Step 5: Add 4 μl Enzyme Mix to “+ enzyme” Tube** + Flick to mix. + +6. **Step 6: Add 1 μl Buffer Mixture to “- enzyme” Tube** + Flick to mix. + +7. **Step 7: Incubate Both Tubes** + - Incubate at 37°C for 10 minutes on a PCR heat block. + +8. **Step 8: Add 300 mM MgOAc to Tubes** + - “+ enzyme” reactions: Add 4.2 μl 300 mM MgOAc + - “- enzyme” reactions: Add 1.05 μl 300 mM MgOAc + +9. **Step 9: Incubate at 37°C for 1 Hour** + +10. **Step 10: Heat-kill Enzymes** + - Incubate at 75°C for 5 minutes + +### Clean-up of Transposition Reactions +11. **Step 11: Transfer Reactions to 15 ml Microfuge Tubes** + - Bring the volume of each up to 100 µl. + +12. **Step 12: Add 100 µl Phenol:Chloroform:IAA** + - Vortex to mix. + +13. **Step 13: Spin Mix** + - Spin 5 minutes, 4°C, 13500 rpm in a microfuge. + +14. **Step 14: Remove and Discard Organic Layer** + - Remove the bottom layer. + +15. **Step 15: Add 100 µl Chloroform** + - Vortex to mix. + +16. **Step 16: Spin Mix** + - Spin 5 minutes, 4°C, 13500 rpm in a microfuge. + +17. **Step 17: Transfer Aqueous Layer to a New 1.5 ml Microfuge Tube** + +18. **Step 18: Add 10 µl 3M NaOAc** + +19. **Step 19: Add 220 µl Ice-cold 100% EtOH** + - Chill at -20°C for 15 minutes. + +20. **Step 20: Spin Mix** + - Spin 15 minutes, 4°C, 13500 rpm in a microfuge. + - Discard supernatant. + +21. **Step 21: Wash DNA Pellet with 500 µl Ice-cold 70% EtOH** + +22. **Step 22: Spin Mix** + - Spin 5 minutes, 4°C, 13500 rpm in a microfuge. + - Discard supernatant. + - Allow pellet to air dry. + +23. **Step 23: Resuspend DNA in Desired Volume of 10 mM Tris pH 7.5** + - For “+ enzyme” reactions: Use 8 µl Tris pH 7.5. + - For “- enzyme” reactions: Use 4 µl Tris pH 7.5. + +### Transformation of Transposition Reactions: For “- enzyme” Reactions +24. **Step 24: Optional Storage** + - Cleaned transposition reactions may be stored at -20°C prior to transformation. + +25. **Step 25: Combine 2 µl DNA with 20 µl MegaX Cells in a Chilled Electroporation Cuvette** + - Amount: 20 µl + +26. **Step 26: Electroporate at 2.0 KV, 200Ω, 25µF** + +27. **Step 27: Add 1 ml Recovery Media to Cuvette** + - Pipet up and down to resuspend. + +28. **Step 28: Transfer 900 µl Cells to a 1.5 ml Microfuge Tube** + +29. **Step 29: Recover at 37°C for 1 Hour** + +30. **Step 30: Plate 9 µl (1% of Cells) onto Selective Media** + - LB+Kan + - Iso+Tmp + - Iso+Tmp+Kan + +31. **Step 31: Grow Plates Overnight at 37°C** + +### Transformation of Transposition Reactions: For “+ enzyme” Reactions +32. **Step 32: Combine 4 µl DNA with 40 µl MegaX Cells in a Chilled Electroporation Cuvette** + - Amount: 40 µl + +33. **Step 33: Electroporate at 2.0 KV, 200Ω, 25µF** + +34. **Step 34: Add 1 ml Recovery Media to Cuvette** + - Pipet up and down to resuspend. + +35. **Step 35: Transfer 900 µl Cells to a 500 ml Flask Containing 150 mls Isosensitest Media** + +36. **Step 36: Recover at 37°C for 1 Hour** + +37. **Step 37: Plate 150 µl (0.1% of Total Cells) onto Three Types of Selective Media** + - LB+Kan + - Iso+Tmp + - Iso+Tmp+Kan + +38. **Step 38: Add 150 µl of Car (100mg/ml Stock) and 300 µl Tmp (5mg/ml Stock) to Flask** + +39. **Step 39: Grow Plates and Culture Overnight at 37°C** + +40. **Step 40: Count Colonies on Each Plate the Next Morning** + +41. **Step 41: Make Glycerol Frozen Stock of “+ enzyme” Overnight Culture** + - Store at -80°C + +42. **Step 42: Pellet Remaining Overnight Culture in 250 ml Conical Tube** + - Spin 15 Minutes at 3000 rpm + - Store pellet at -20°C or continue to step 43. + +43. **Step 43: Purify DNA Using Qiagen HiSpeed Midiprep Kit** + - Elute in 1 ml 10 mM Tris. + - Output is mutagenized DCW1*FLAG entry vector pool (ep). + +#### Monitor Plasmid Population Content (Optional) +You can monitor the plasmid population content by restriction enzyme digestion of the DNA pool (e.g., ApaLI from New England Biolabs R0507S). Use 1 µg of DNA for digest in a 20 µl reaction. + +44. **Step 44-170**: Follow similar steps to clean DNA, run additional transformations, and grow cultures with detailed times and reagent volumes as mentioned for monitoring plasmid pools through restriction digests. + +## References +1. Brachmann, C.B. et al. Designer deletion strains derived from Saccharomyces cerevisiae S288C: a useful set of strains and plasmids for PCR-mediated gene disruption and other applications. Yeast 14, 115-132 (1998). +2. Winzeler, E.A. et al. Functional characterization of the S. cerevisiae genome by gene deletion and parallel analysis. Science 285, 901-906 (1999). +3. Gamas, P. & Craig, N.L. Purification and characterization of TnsC, a Tn7 transposition protein that binds ATP and DNA. Nucleic acids research 20, 2525-2532 (1992). +4. Choi, K.Y., Li, Y., Sarnovsky, R. & Craig, N.L. Direct interaction between the TnsA and TnsB subunits controls the heteromeric Tn7 transposase. Proceedings of the National Academy of Sciences of the United States of America 110, E2038-2045 (2013). + +## endofoutput +``` \ No newline at end of file diff --git a/markdown-output/intracellular-cytokine-staining-sqdeds6.md b/markdown-output/intracellular-cytokine-staining-sqdeds6.md new file mode 100644 index 0000000000000000000000000000000000000000..cc37e09c8140cc14d50e2cde9b707025b74f0599 --- /dev/null +++ b/markdown-output/intracellular-cytokine-staining-sqdeds6.md @@ -0,0 +1,136 @@ +```markdown +# Goal/Experiment: +To outline the steps used for intracellular cytokine staining of cells. + +# Intracellular Cytokine Staining + +**Kristin Anderson** +University of Washington and Fred Hutchinson Cancer Research Center + +DOI: [dx.doi.org/10.17504/protocols.io.sqdeds6](https://dx.doi.org/10.17504/protocols.io.sqdeds6) + +## Abstract +This protocol outlines the steps used for intracellular cytokine staining. + +In brief: Cells were treated with protein transport inhibitor containing Brefeldin A (GolgiPlug, cat: 555029, BD) and plated at 1e6 cells per well in a 96-well flat bottom plate. Cells were stimulated for 5 hours at 37°C with either T cell media, Phorbol 12-Myristate 13-Acetate (Sigma, cat: P8139) (used at 20ng/mL) and Ionomycin (Sigma, cat: I0634) (used at 1mg/mL), or 1mg/mL of peptide (as indicated). M5L364-414 (GQKVMQAAI), OTII (SIINFEKL), LCMV33-41 (KAVYNFATM), MSNL30-28 (SLLFLLFLSL), and MSNL350-538 (VLPLTVAVEV) peptides were ordered from ELIM peptide (>80% purity). The BD Fix/Perm kit (cat: 554714) was used for intracellular staining. Cells were fixed in 0.5% paraformaldehyde until data acquisition. + +## License +This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. + +## Materials +- **Phorbol 12-myristate 13-acetate (PMA)**, Sigma Aldrich Catalog #P8139 +- **Ionomycin calcium salt from Streptomyces conglobatus**, Sigma Aldrich Catalog #I0634 +- **96-well Cell Culture Plate, Flat-Bottom with Lid**, polystyrene Corning Catalog #3599 +- **96-well Cell Culture Plate, round-bottom with lid**, Greiner Bio-One Catalog #650 180 +- **Brefeldin A**, GolgiPlug, BD Biosciences Cat# 555029 +- **Fix/Perm Kit**, BD Biosciences Cat# 554714 +- **Peptides** (as indicated: MSNL30-28, MSNL350-538, LCMV33-41, and others), ELIM Peptide + +## Reagents Explanation +1. **Phorbol 12-myristate 13-acetate (PMA)**: A potent activator of protein kinase C, which mimics diacylglycerol, a secondary messenger in cell signaling. +2. **Ionomycin**: A calcium ionophore used to increase intracellular calcium levels, stimulating cells. +3. **Brefeldin A (GolgiPlug)**: Inhibits protein transport by blocking the secretion pathway, helping to retain proteins within the cells for intracellular staining. +4. **Fix/Perm Kit**: Used for intracellular staining by fixing and permeabilizing the cells. + +## Protocol Steps + +1. **Prepare a Flat Bottom 96 Well Plate** + - Resuspend cells at 1e6 cells/mL in T cell media. + - Transfer at least 350µL of cells per condition into a fresh tube (use at least 3 replicate wells at 100µL/well for each condition). + +2. **Dilute Golgi Plug** + - Dilute Golgi Plug to 1:50 (2mg/mL) for the experiment. (Once the treatment condition is added the final concentration will be 1:100 = 1mg/mL). + +3. **Plate Cells** + - Plate 100µL of cells per well. + +4. **Prepare PMA and Ionomycin** + - Dilute PMA to 40ng/mL PMA so that the final concentration in the assay will be 20ng/mL. + - Ionomycin should be prepared at 2mg/mL so that the final concentration in the assay will be 1mg/mL. + - Resuspend peptide at 2µg/100µL. + +5. **Add Media/PMA/Ionomycin** + - Add 100µL of media or PMA/Ionomycin or peptide to each well. + - Incubate 4-6 hours in a 37°C incubator. + +6. **Transfer to U-Bottom 96 Well Plate** + - Combine replicate wells at this step if low on cells. + +7. **Centrifuge and Supernatant Removal** + - Centrifuge (Cfg) plate at 4°C for 2 min @ 809 × g (with high brake) and remove supernatant. + +8. **Live/Dead Staining** + - Add 50µL of Live/Dead stain per well (dilute 5µL of dye in 2.5mL 1x DPBS). + +9. **Incubate** + - Incubate for 20-30 minutes in a dark refrigerator at 4°C. + +10. **Wash with FACS Buffer** + - Add 100µL of FACS Buffer per well to wash. + - Cfg plate at 4°C for 2 min @ 809 × g (with high brake) and remove supernatant. + +11. **Prepare Surface Stain Cocktail** + - Prepare a cocktail of antibodies for cell surface staining. + - Add 50-75µL of surface stain mix per well. + - Resuspend pellet by pipetting up and down at least 5 times. + +12. **Incubate** + - Incubate plates for 30 min in the dark at 4°C. + +13. **Additional FACS Buffer Wash** + - Add 100µL of FACS Buffer per well to wash. + - Cfg plate at 4°C for 2 min @ 809 × g (with high brake) and remove supernatant. + +14. **FACS Buffer Wash Again** + - Add 150µL of FACS Buffer per well to wash. + - Cfg plate at 4°C for 2 min @ 809 × g (with high brake) and remove supernatant. + +15. **Third FACS Buffer Wash** + - Add 150µL of FACS Buffer per well for a third wash. + - Cfg plate at 4°C for 2 min @ 809 × g (with high brake) and remove supernatant. + +16. **Fixation/Permeabilization** + - Add 100µL of Fixation/Permeabilization solution per well. + - Resuspend pellet by pipetting up and down at least 5 times. + - Incubate the plate for 20 minutes in a dark refrigerator at 4°C. + +17. **Wash with Perm/Wash Buffer** + - Add 100µL of Perm/Wash Buffer (PWB) per well to wash. + - Change centrifuge setting to 2 for all remaining steps. + - Cfg plate at 4°C for 2 min @ 809 × g (brake = 2) and remove supernatant. + +18. **Second Perm/Wash Buffer Wash** + - Add 150µL of Perm/Wash Buffer per well for a second wash. + - Cfg plate at 4°C for 2 min @ 809 × g (brake = 2) and remove supernatant. + +19. **Third Perm/Wash Buffer Wash** + - Add 150µL of Perm/Wash Buffer per well for a third wash. + - Cfg plate at 4°C for 2 min @ 809 × g (brake = 2) and remove supernatant. + +20. **Intracellular Staining** + - Prepare a cocktail of antibodies in PWB buffer for intracellular staining. + - Add 50-75µL of the intracellular stain mix per well. + - Resuspend pellet by pipetting up and down at least 5 times. + +21. **Incubate Plate** + - Incubate the plate for 30 minutes in a dark refrigerator at 4°C. + +22. **Wash with Perm/Wash Buffer** + - Add 100µL of Perm/Wash Buffer (PWB) per well to wash. + - Cfg plate at 4°C for 2 min @ 809 × g (brake = 2) and remove supernatant. + +23. **Second Perm/Wash Buffer Wash** + - Add 150µL of Perm/Wash Buffer per well for a second wash. + - Cfg plate at 4°C for 2 min @ 809 × g (brake = 2) and remove supernatant. + +24. **Third Perm/Wash Buffer Wash** + - Add 150µL of Perm/Wash Buffer per well for a third wash. + - Cfg plate at 4°C for 2 min @ 809 × g (brake = 2) and remove supernatant. + +25. **Fix Cells for Flow Cytometry** + - Resuspend in 200µL of 0.5% PFA (store overnight if necessary). + - Store in the refrigerator at 4°C until acquisition. + - Run samples on the flow cytometer within 24 hours for best results. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/intracellular-flow-cytometry-staining-protocol-bac7iazn.md b/markdown-output/intracellular-flow-cytometry-staining-protocol-bac7iazn.md new file mode 100644 index 0000000000000000000000000000000000000000..224e4b7d8fec7c19638ad62b5797fca369309381 --- /dev/null +++ b/markdown-output/intracellular-flow-cytometry-staining-protocol-bac7iazn.md @@ -0,0 +1,134 @@ +```markdown +# Goal/Experiment: +The goal of this experiment is to perform intracellular flow cytometry staining to detect cytokines/chemokines in stimulated cell populations, ensuring optimal staining signals and preserving cell surface antigens. + +# Intracellular Flow Cytometry Staining Protocol V.3 + +**Author:** +Sam Li, BioLegend + +**Published on:** +Dec 11, 2019 + +**External Link:** +[BioLegend Intracellular Flow Cytometry Staining Protocol](https://www.biolegend.com/protocols/intracellular-flow-cytometry-staining-protocol/4260/) + +## Guidelines + +### Application Notes: + +1. **Preparation of Activated Cell Populations:** + - Activated cell populations can be prepared from in vivo-stimulated tissues or in vitro-stimulated cultures. + - For cytokine and chemokine detection, include a protein transport inhibitor such as brefeldin A (BioLegend Cat. No. [420601](https://www.biolegend.com/en-us/products/brefeldin-a-3006)) or monensin (BioLegend Cat. No. [420701](https://www.biolegend.com/en-us/products/monensin-solution-3007)) in the last 4-6 hours of cell culture activation. + - Suspend and distribute cells into 12 x 75 mm plastic tubes or microwell plates for immunofluorescent staining. + +2. **Optimizating Stimulation Conditions:** + - Different cytokines/chemokines have varied production peaks. Optimize stimulation conditions for each stimulant. + +3. **Staining with Live/Unfixed Cells:** + - For some antibodies, it is recommended to stain surface antigens before fixation/permeabilization to maintain epitope integrity. + - Refer to the [Fixation Webpage](https://www.biolegend.com/protocols/fixation-guide/3940) for fixation guidelines. + - For intracellular cytoplasmic staining, use the improved Cyto-Fast™ Fix/Perm Buffer Set (BioLegend Cat. No. [426803](https://www.biolegend.com/en-us/products/cyto-fast-fixperm-buffer-set-3118)) following the "Recommended Usage" section. + +### Related Information: + +1. Assenmacher, M., et al. 1994. Eur. J. Immunol. 24:1097. +2. Elson, L.H., et al. 1995. J. Immunol. 1995. 154:4294. +3. Jung T, et al. 1993. J. Immunol. Methods 159:197. +4. Prussin C., et al. 1995. J. Immunol. Methods 188:117. +5. Vikingsson A., et al. 1994. J. Immunol. Methods 173:219. + +## Materials + +| Name | Catalog # | Vendor | +|--------------------------------|-----------|------------| +| RBC Lysis Buffer | 420301 | BioLegend | +| Brefeldin A | 420601 | BioLegend | +| Monensin Solution | 420701 | BioLegend | +| Cell Staining Buffer | 420201 | BioLegend | +| Fixation Buffer | 420801 | BioLegend | +| Intracellular Staining Perm Wash Buffer | 421002 | BioLegend | +| Cyto-Last™ Buffer | 422501 | BioLegend | +| Cyto-Fast™ Fix/Perm Buffer Set | 426803 | BioLegend | + +## Protocol + +### 1. Fixation + +1. **Fixation of Intracellular Antigens:** + - Stain cell surface antigens as per BioLegend’s Cell Surface Immunofluorescence Staining Protocol. + - Fix cells in 0.5 ml/tube Fixation Buffer in the dark for 20 minutes at room temperature. + - **Tip:** For gentler fixation, use FluoroFix™ Buffer (BioLegend Cat. No. [422101](https://www.biolegend.com/en-us/products/fluorofix-buffer-3226)). + +2. **Centrifugation:** + - Centrifuge at 350×g for 5 minutes. + - Discard the supernatant. + +3. **Storing Cells:** + - Wash cells 1x with Cell Staining Buffer. + - Resuspend cells in Cell Staining Buffer and store at 4°C (short term) or 90% FCS/10% DMSO at -80°C (long term). + - Cells can also be stored in Cyto-Last™ Buffer for up to two weeks. + +### 2. Permeabilization + +4. **Dilution of Perm Wash Buffer:** + - Dilute 10X Intracellular Staining Perm Wash Buffer to 1X in DI water. + +5. **Centrifugation of Perm Wash Buffer:** + - Resuspend fixed cells in Intracellular Staining Perm Wash Buffer. + - Centrifuge at 350×g for 5-10 minutes. + +6. **Repetition:** + - Repeat step 5 twice. + +### 3. Intracellular Staining + +7. **Staining Procedure:** + - Resuspend fixed/permeabilized cells in residual Perm Wash Buffer. + - Add fluorophore-conjugated antibody and incubate for 20 minutes in the dark at room temperature. + +8. **Washing Cells:** + - Wash 2x with 2 ml Perm Wash Buffer. + - Centrifuge at 350×g for 5 minutes. + +9. **Optional Steps:** + - If primary antibody is biotinylated, perform fluorophore conjugated Streptavidin incubations. + +10. **Analysis Preparation:** + - Resuspend cells in 0.5 ml Cell Staining Buffer. + - Analyze with appropriate controls. + +### 4. Activation and Intracellular Staining of Whole Blood + +11. **Dilution of Whole Blood:** + - Dilute heparinized whole blood 1:1 with sterile tissue culture medium. + +12. **In Vitro Stimulation:** + - Activate cells by antigen or mitogen. + - Add protein transport inhibitor (Brefeldin A or Monensin) during the last 4-6 hours of activation. + - Incubate 200 μl of blood cell suspension into 12 x 75 mm plastic tubes for 4-6 hours in 5% CO2 at 37°C. + +13. **Lysis and Incubation:** + - Add 2 ml of 1X Red Blood Cell Lysis Buffer and incubate for 5-10 minutes at room temperature. + +14. **Centrifugation and Supernatant Removal:** + - Centrifuge at 350×g for 5 minutes. + - Discard the supernatant. + +15. **Washing and Staining:** + - Wash cells 1x with Cell Staining Buffer. + - Perform cell surface immunofluorescent staining. + +16. **Fixation and Permeabilization:** + - Fix, permeabilize, and stain intracellular antigens as described above. + +### 5. Flow Cytometric Analysis + +17. **PMT Voltage and Compensation:** + - Set PMT voltage and compensation using cell surface staining controls. + - Set quadrant markers based on controls (blocking, isotype, unstained). + - Inspect stained cells by light microscopy and flow light scatter pattern. + - Generate bivariate dot plots or probability contour plots for data analysis to display cytokine production patterns. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/intracellular-recordings-and-post-hoc-immunofluor-b2baqaie.md b/markdown-output/intracellular-recordings-and-post-hoc-immunofluor-b2baqaie.md new file mode 100644 index 0000000000000000000000000000000000000000..cd4b6c5d1a2e23610dbe32a1dd44944a3ca76b8f --- /dev/null +++ b/markdown-output/intracellular-recordings-and-post-hoc-immunofluor-b2baqaie.md @@ -0,0 +1,171 @@ +```markdown +# Goal/Experiment: +The aim of this experiment is to perform standard intracellular recordings from mouse myenteric neurons and follow it with immunohistochemistry to map the projections of the neurites of enteric neurons. + +# Intracellular Recordings and Post Hoc Immunofluorescence + +## Authors + +- Rachel M Gwynne +- Katerina Koussoulas +- Joel Bornstein + +## Affiliation + +Department of Physiology, University of Melbourne, Parkville, Vic. 3010, Australia + +Published: April 01, 2022 + +DOI: [10.17504/protocols.io.e6nvwkp97vmk/v1](https://dx.doi.org/10.17504/protocols.io.e6nvwkp97vmk/v1) + +Contact: **Marlena S. Pela** + +SPARC Tech. Support email: info@neuinfor.org + + +The following protocol was submitted on behalf of the authors from the Bornstein lab by the SPARC project. This procedure ensures all experimental techniques adhere to University of Melbourne Animal Experimentation Ethics Committee requirements. + +## Purpose +This protocol describes methods for standard intracellular recording from mouse myenteric neurons impaled with intracellular electrodes containing biocytin, followed by processing for immunohistochemistry to map the projections of the neurites of enteric neurons. + +## Methods Overview + +### Electrophysiology + +- **Nicardipine**: A calcium channel blocker used to prevent muscle contractions. (Sigma Aldrich, NSW, Australia) +- **Hyoscine (1 μM)**: Also known as Scopolamine, used to reduce contractions by relaxing muscles. (Sigma Aldrich, NSW, Australia) +- **Biocytin**: A biotin-containing compound used as an intracellular label. (Sigma Aldrich, NSW, Australia) +- **Silicone Elastomer**: Used to line petri dishes for dissection. (Sylgard 184, Dow Corning, NSW, Australia) +- **Ultrafine Dissection Forceps**: Used for precise dissection work. (Dumont #5 INOX, sourced from Fine Science Tools, Canada) +- **Inverted Microscope**: Essential for visualizing myenteric ganglia. (Zeiss Axiovert 200) + +Additional equipment includes microelectrode amplifiers, acquisition systems, and generators from companies like ADInstruments, Axon Instruments, and A.M.P. Instruments. + +### Pharmacology + +- **Hyoscine (1μM)** +- **Hexamethonium (200μM)**: A nicotinic antagonist. +- **Nicardine (2.5μM)** +- **NOLA (100μM)**: Nitrate/nitrite compound. +- **TNP-ATP (5μM)**: A P2X receptor antagonist. Sourced from Tocris. +- **Ondansetron (3μM)**: A serotonin 5-HT3 receptor antagonist. Sourced from Glaxo Research Group. + +### Immunohistochemistry + +- **CAS-Block**: Blocking Reagent. (Invitrogen, CA, USA) +- **Triton X-100**: Surfactant for permeabilization. (ProSci Tech, QLD, Australia) +- **Glass Slides**: For mounting preparations. (Livingstone International, NSW, Australia) +- **Dako Mounting Medium**: Mounting medium for slides. (Agilent Technologies) + +### Antibodies + +#### Primary Antibodies + +| Primary Antiserum | Specific Immunogen | RRID | Host Species | Dilution | Supplier | Catalog | +|-------------------|--------------------------------------|-------------|--------------|----------|----------------------------------|---------| +| Calretinin | Human recombinant calretinin | AB_10000342 | Goat | 1:1000 | Swant, Bellinzona, Switzerland | CG1 | +| NOS | Neuronal nitric oxide synthase (nNOS) | AB_2895154 | Sheep | 1:1000 | P.C. Emson, The Babraham Institute, Cambridge, UK | K205 | + +#### Secondary Antibodies + +| Secondary Antiserum | Fluorophore | RRID | Host Species | Dilution | Supplier | Catalog | +|---------------------|-------------|-------------|--------------|----------|----------------------------------------|--------------------| +| Sheep | Alexa 488 | AB_141362 | Donkey | 1:200 | Molecular Probes, Mt Waverley, VIC, Australia | A11015 | +| Sheep | Alexa 647 | AB_10374882 | Donkey | 1:200 | Molecular Probes, Mt Waverley, VIC, Australia | A21448 | +| Alexa 594 | Streptavidin| | | 1:200 | Molecular Probes, Mt Waverley, VIC, Australia | S32356 Lot 55981A | + +### Confocal Microscopy + +- **Confocal Microscope**: For detailed imaging. (Zeiss LSM 880, Biological Optical Microscopy Platform, University of Melbourne) + +## Protocol Steps + +### Electrophysiology + +1. **Sacrifice Mice** + - Perform cervical dislocation to humanely sacrifice mice. + +2. **Dissection** + - Perform a midline incision to open the abdominal cavity and remove the colon. + +3. **Preparation** + - Prepare the colon in oxygenated physiological saline containing Nicardipine (1.25 μM) and Hyoscine (1 μM). + +4. **Segmentation** + - Open a 1cm² segment of the proximal colon, pin it flat, and remove the mucosa and submucosal plexus. + +5. **Re-pinning** + - Turn the segment over and re-pin to expose the longitudinal muscle layer. + +6. **Fine Dissection** + - Use ultrafine forceps to remove the longitudinal muscle layer in fine stripes. + +7. **Superfusion** + - Transfer to a recording bath and superfuse with warmed physiological saline at 35ºC, at 5mL/min. + +8. **Visualization** + - Use an inverted microscope to visualize myenteric ganglia. + +9. **Recording** + - Impale myenteric neurons using glass microelectrodes containing biocytin. + +10. **Voltage Recording** + - Use Axoprobe 1A microelectrode amplifier, PowerLab/4SP, and Chart5 for Windows for recordings. + +11. **Stimuli Application** + - Apply trains of stimuli using Master 8 pulse generator and ISO-Flex stimulus isolation unit. + +12. **Analyzing Neuronal Excitability** + - Inject depolarizing current pulses to analyze excitability and measure action potentials. + +### Pharmacology + +13. **Stock Solutions** + - Prepare concentrated stock solutions of pharmacological agents in distilled water. + +14. **Working Solutions** + - On the experiment day, dilute stocks to working concentrations. + - Agents used include Hyoscine, Hexamethonium, Nicardipine, NOLA, TNP-ATP, and Ondansetron. + +15. **Synaptic Response Recording** + - Record a minimum of 3 synaptic responses before adding antagonists. + +16. **Antagonist Effects** + - Record 3 responses 5-20 minutes post-addition; wash out drugs and record post-washout responses. + +### Immunofluorescence + +18. **Tissue Processing** + - Remove tissue and fix in 4% formaldehyde solution overnight. + - Wash with PBS and soak in 10% CAS Block + 0.1% Triton X-100. + +19. **Primary Antibodies Application** + - Incubate with primary antibodies (Calretinin or NOS) at 4°C for 2-3 nights. + +20. **Secondary Antibodies Application** + - Incubate with secondary antibodies labeled with fluorophores, ensuring to incubate in dark, humid conditions. + +21. **Slide Preparation** + - Mount tissues on glass slides using Dako mounting medium. + +### Confocal Microscopy + +22. **Imaging** + - Capture Z-stack images using a confocal microscope. + - Set step distances so adjacent planes overlap. + - Take high-power images of neuron cell bodies. + - Trace neuron projections using appropriate magnifications. + - Process image stacks into single images with ImageJ. + - Process images further using Corel Photo-Paint. + - Stitch images to map each neuron and export as JPEG files. + +23. **Data Integration** + - Link neuron maps to corresponding electrophysiological data in excel spreadsheets. + +## References +Citation: Rachel M Gwynne, Katerina Koussoulas, Joel Bornstein Intracellular recordings and post hoc immunofluorescence https://dx.doi.org/10.17504/protocols.io.e6nvwkp97vmk/v1 + +This is an open access protocol distributed under the terms of the [Creative Commons Attribution License](https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium. + +## endofoutput +``` \ No newline at end of file diff --git a/markdown-output/irdye-800cw-maleimide-labeling-application-guide-gvabw2e.md b/markdown-output/irdye-800cw-maleimide-labeling-application-guide-gvabw2e.md new file mode 100644 index 0000000000000000000000000000000000000000..f87d38e714eaa7ad34eae0d0d0d71653e9487427 --- /dev/null +++ b/markdown-output/irdye-800cw-maleimide-labeling-application-guide-gvabw2e.md @@ -0,0 +1,91 @@ +```markdown +# IRDye 800CW Maleimide Labeling Application Guide + +### Goal/Experiment: +The goal of this experiment is to label molecules containing free sulfhydryl groups (-SH) with IRDye 800CW Maleimide. This includes proteins, peptides, and biomolecules, to facilitate their detection via infrared fluorescence. + +## I. Introduction + +IRDye 800CW Maleimide is a functional derivative of the infrared dye IRDye 800CW, reactive towards free -SH (thiol, sulfhydryl) groups. Most molecules containing free -SH groups can be labeled with maleimide dyes, including IRDye 800CW Maleimide Infrared Dye. + +## II. Labeling Reactions and Considerations + +Maleimide groups react with sulfhydryl groups at pH 6.5-7.5, forming a stable thioether bond. A protein, peptide, or biomolecule containing a reactive sulfhydryl group can be labeled with IRDye 800CW using the maleimide functional group of IRDye 800CW Maleimide. + +### Labeling Conditions for IRDye 800CW Maleimide + +| Buffer | Temperature | Time | Dye Equivalents per Free -SH | +| --------------------------- | ------------ | ------ | ---------------------------- | +| Phosphate Buffered Saline (PBS), pH 7.2 | Ambient* | 2 hours | 2-5 | + +* Ambient temperature is preferred, but 4°C may be used if the protein is not stable during incubation. If 4°C is used for the labeling reaction, an overnight incubation should be performed. + +### Additional Labeling Notes + +- **Buffers**: PBS is generally used for labeling, but other buffers with pH 6.5 to 7.5 can be used. Reactions above pH 8.0 should be avoided due to unprotonated amines reacting with maleimides. +- **Reaction Time**: The labeling reaction is typically complete in 2 hours at room temperature but can be conducted at 4°C for 16-18 hours. +- **Purification**: The labeled molecule should be purified by appropriate techniques such as dialysis, size exclusion chromatography, desalting spin columns, or HPLC. +- **Disulfide Bonds**: Molecules containing disulfide bonds cannot be directly labeled with maleimide. These disulfide bonds can be cleaved using reducing agents like TCEP, DTT, or 2-Mercaptoethylamine (MEM) to produce free sulfhydryl groups. + +#### Special Considerations for Reducing Agents + +- **TCEP** (Tris(2-carboxyethyl)phosphine) is a non-smelling and efficient reducing agent, preferred for its lack of odor and efficiency. +- **Interaction with Maleimide Dye**: TCEP may react with the maleimide group of the dye; hence, the ratio of TCEP to maleimide dye must be optimized for efficient reduction and labeling. + +## III. Examples + +### Labeling of Affibody® Molecules with IRDye 800CW Maleimide + +Affibody molecules are small proteins with binding sites for different target proteins. Commercial Affibody molecules engineered with a single C-terminal cysteine residue can bind to any fluorescent dye. The Affibody molecules require partial dimerization and reduction before labeling with IRDye 800CW Maleimide. + +### Labeling of Small Molecules with IRDye 800CW Maleimide + +Glutathione, a small peptide with free thiols, serves as a model for labeling small molecules containing thiol groups with IRDye 800CW Maleimide. + +### Labeling of Antibodies with IRDye 800CW Maleimide + +Antibodies can be labeled by selectively reducing hinge region disulfide bonds using MEM, converting them to two heavy chain-light chain molecules with each containing one antigen binding site. + +## Protocol Steps + +### Labeling of Affibody® Molecules with IRDye 800CW Maleimide + +1. **Prepare a 500 mM solution of TCEP in water** (47.5 mg/331 μl). +2. **Add 1 μl of 500 mM TCEP** to 99 μl Affibody molecule in PBS (1 mg/ml) to achieve a 5 mM TCEP concentration. +3. **Incubate overnight at room temperature**. +4. **Remove excess TCEP** using a 0.5 ml Zeba Desalt Spin Column. +5. **Reconstitute IRDye 800CW Maleimide** (0.5 mg) in 50 μl DMSO or water for a 10 mM solution. +6. **Add 4 μl of maleimide solution** to 100 μl reduced Affibody molecule solution. +7. **Mix and incubate** at room temperature for 2-3 hours, protected from light. +8. **Purify the labeled Affibody molecule** using two 0.5 ml Zeba Desalt Spin Columns consecutively. +9. **Store the labeled Affibody molecule** at -20°C protected from light. + +### Labeling of Small Molecules with IRDye 800CW Maleimide + +1. **Prepare a 10 mM stock solution** of glutathione in water by dissolving 3 mg in 1 ml of water. +2. **Dilute to 0.1 mM** in PBS (pH 7.4). +3. **Reconstitute IRDye 800CW Maleimide** (0.5 mg) in 50 μl DMSO or water for a 10 mM solution. + +### Labeling of Antibodies with IRDye 800CW Maleimide + +1. **Reduction of antibodies to generate free thiols**: + - Dissolve the antibody to a concentration of 10 mg/ml in 20 mM sodium phosphate, 0.15 M NaCl, pH 7.4 buffer containing 1 mM EDTA. + - Add 6 mg of 2-Mercaptoethylamine (50 mM final concentration) per ml of antibody solution. + - **Incubate** the solution at 37°C for 90 minutes in a sealed tube. + +## References + +1. Getz, E. B. et al. (1999). A comparison between TCEP and dithiothreitol. *Anal. Biochem*. 273: 73-80. +2. Han, J.C. et al. (1994). Quantitative determination of TCEP. *Anal. Biochem*. 220: 5-10. +3. Kirley, T. L. (1989). Labeling of cysteine-containing proteins. *Anal. Biochem*. 180: 231-236. +4. Levinson, M. E. et al. (1969). Reduction of biological samples. *Experientia*. 25: 126-127. +5. Mery, J. C. et al. (1993). Linkage for Fmoc peptide synthesis. *Int. J. Peptide Protein Res*. 42: 44-52. +6. Shafer, D.E. et al. (2000). Reaction of TCEP with Maleimide. *Anal. Biochem*. 282: 161-164. +7. Scherer, R. (2008). LI-COR IRDye 800CW Maleimide conjugation report. *Int. Materials Science & Engineering, Vanderbilt University*. +8. Tyagarajan, K. et al. (2003). Thiol-reactive dyes. *Electrophoresis*. 24: 2348-2358. +9. Blauenstein, P. et al. (1995). Experience with iodine-123 and technetium-99 m labeled antibodies. *Eur. J. nuclear Med*. 22:690-698. +10. Hermanson, G. (1996). *Bioconjugate Techniques*. + +--- +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/isolation-and-characterization-of-tissue-and-cell-de2f3gbn.md b/markdown-output/isolation-and-characterization-of-tissue-and-cell-de2f3gbn.md new file mode 100644 index 0000000000000000000000000000000000000000..1f721c55a6a312d2f78471a3c95fad8135b80cf9 --- /dev/null +++ b/markdown-output/isolation-and-characterization-of-tissue-and-cell-de2f3gbn.md @@ -0,0 +1,106 @@ +```markdown +# Goal/Experiment: +The goal of this experiment is the isolation and characterization of tissue and cell-derived extracellular vesicles (EVs) and non-vesicular extracellular particles (NVEPs). The protocol involves various techniques including size exclusion chromatography, ultracentrifugation, vesicle flow cytometry, microfluidic resistive pulse sensing, and immunoblotting to identify and characterize different types of EVs and NVEPs from conditioned media. + +## Isolation and Characterization of Tissue and Cell-Derived Extracellular Vesicles and Non-Vesicular Extracellular Particles V.2 + +**DOI:** [dx.doi.org/10.17504/protocols.io.5jyj822k8l2w/v2](http://dx.doi.org/10.17504/protocols.io.5jyj822k8l2w/v2) + +**Authors:** +- Marta Garcia Contreras +- Emeli Chatterjee +- Abhik Chakraborty +- Michail Spanos +- Kriti Bomb +- Priyanka Gokulnath +- Jeffery L. Franklin +- Erika Duggan +- John P. Nolan +- Priyadarshini Pantham +- Louise C. Laurent +- Saumya Das + +### Funding Acknowledgement: +- Various grants from federal agencies and institutions. + +### Abstract: +Cells produce a heterogeneous complement of extracellular biomolecular complexes, including extracellular vesicles (EVs) and non-vesicular extracellular particles (NVEPs). This protocol describes methods to isolate and characterize EVs and NVEPs using size exclusion chromatography, ultracentrifugation, microfluidic resistive pulse sensing, vesicle flow cytometry, and immunoblotting. + +### Materials: +- **Amicon Ultra Centrifugal Filters (10 kDa or 100 kDa MWCO, Millipore):** Used for concentrating samples. +- **Exosome-Depleted Serum (Thermo Fisher Scientific):** For media preparation to avoid contamination of extracellular vesicles. + +For detailed reagents, refer to the specific sections below. + +### Processing of Conditioned Culture Media following Tissue or Cell Culture +1. **Culture Conditions:** + - DiFi cells: cultured as previously published. + - BeWo cells: cultured with 10% exosome-depleted FBS. + - Placental explant tissue and adipose tissue: cultured as previously published. + +2. **Centrifugation:** + - Centrifuge at 2,000 x g for 10 minutes at room temperature. + - Store filtered media at -80°C for later use. + +### Isolation of Extracellular Vesicles and Non-Vesicular Extracellular Particles + +#### Size Exclusion Chromatography to Isolate Extracellular Vesicles +1. **Required Equipment:** + - Izon qEV (35 nm) Size Exclusion Chromatography Columns. + - Izon Automatic Fraction Collector. + - 1X PBS. + - Disposable plastic 10 mL pipettes, pipette boy, and 1.5 mL Eppendorf tubes. + +2. **Protocol:** + - Prepare the column and load it according to the manufacturer’s instructions. + - Collect fractions 7 to 10 and combine. + +#### Differential Ultracentrifugation to Isolate Extracellular Vesicles and Non-Vesicular Extracellular Particles +1. **Equipment:** + - Eppendorf 5810R centrifuge with specific rotors. + - Beckman Optima L-90K ultracentrifugation with SW 55 Ti rotor. + +2. **Protocol Overview:** + - Centrifuge samples at various speeds (2,000 x g, 10,000 x g, 167,000 x g, and 367,000 x g) to separate large and small EVs, exomeres, and supermeres. Store pellets at -80°C. + +### Characterization of Extracellular Vesicles and Non-Vesicular Extracellular Particles + +#### Vesicle Flow Cytometry to Characterize Extracellular Vesicles in Conditioned Culture Media +1. **Initial Preparation:** + - Thaw samples and prepare vFRed 10X working solution. + - Make serial dilutions (1:10 to 1:320) in a 96-well plate and add vFRed. + +2. **Flow Cytometry Protocol:** + - Incubate stained samples in the dark for 1 hour. + - Perform post-staining dilution and load into the Cytoflex S flow cytometer. + - Acquire data and analyze using corresponding software. + +#### Microfluidic Resistive Pulse Sensing to Characterize Fractions Collected from Size-Exclusion Chromatography +1. **Using Spectradyne nCS1:** + - Dilute and load EV samples into the TS-400 cartridge. + - Conduct analysis with nCS1 and process data using nCS1 Data Viewer software. + +#### Immunoblotting to Characterize Extracellular Vesicles and NonVesicular Extracellular Particles +1. **Lysis and Preparation:** + - Lyse purified EVs and NVEPs using 10X RIPA buffer. + - Centrifuge lysates and prepare BSA standards. + +2. **Electrophoresis and Blotting:** + - Conduct electrophoresis using Criterion TGX Stain-Free TM Protein Gel. + - Transfer proteins onto PVDF membranes and incubate with antibodies. + - Develop blot using chemiluminescent substrate and image on iBright Imaging system. + +### Notes: +- **Quality Control:** + - Avoid multiple freeze-thaw cycles. + - Follow manufacturer’s settings for equipment and procedures. + - Store all reagents appropriately and prepare fresh for each experiment. + +### Citations: +- Numerous citations detailing the techniques and sources for cell cultures. + +### Figures: +- Figures depicting expected results from flow cytometry and immunoblotting. + +[endofoutput] +``` diff --git a/markdown-output/isolation-and-culture-of-individual-myofibers-and-bqxdmxi6.md b/markdown-output/isolation-and-culture-of-individual-myofibers-and-bqxdmxi6.md new file mode 100644 index 0000000000000000000000000000000000000000..70faec66fe50284574936a86528a19d2a7827ad1 --- /dev/null +++ b/markdown-output/isolation-and-culture-of-individual-myofibers-and-bqxdmxi6.md @@ -0,0 +1,172 @@ +```markdown +# Isolation and Culture of Individual Myofibers and Their Adjacent Muscle Stem Cells from Aged and Adult Skeletal Muscle + +### Goal/Experiment: +The goal of this experiment is the isolation, culture, transfection with siRNA, and subsequent immunostaining for muscle stem cells on their adjacent myofibers from aged and young mice. This enables the analysis and comparison of muscle stem cells from these two age groups. + +## Abstract +The isolation and culture of single floating myofibers with their adjacent muscle stem cells allow the analysis and comparison of muscle stem cells from aged and young mice. This method has the advantage that muscle stem cells are cultured on the myofiber, thereby culturing them in conditions as close to their endogenous niche as possible. + +## Introduction +Regeneration of skeletal muscle in the adult is carried out by muscle stem cells (MuSCs), also called satellite cells. MuSCs in the adult are located between the basal lamina and the sarcolemma of a myofiber and are characterized by the expression of the transcription factor Pax7. Under normal resting conditions, MuSCs are quiescent and also express Sprouty1. Following injury of the muscle or due to other stimuli, they get activated. After activation MuSCs differentiate into myoblasts, which then further differentiate into myotubes and mature into myofibers, the contractile units of skeletal muscle. + +The dysfunction of MuSCs during aging is a major contributor to the decreased regenerative capacity of aged skeletal muscle. Multiple signaling pathways are upregulated in aged muscle stem cells. Aged or geriatric MuSCs are characterized by entering a pre-senescence state and aberrant expression of Hoxa9 following activation. + +## Materials +- **Tissue Culture Plates:** Coat all tissue culture plates (3-4 wells of a 12-well plate or 4-8 wells of a 24-well plate) with sterile horse serum (HS) for about 5 min. Remove the HS and let the plates dry for 5 min. +- **Myofiber Culture Medium:** + - 20% FBS (fetal bovine serum) + - 1% chicken embryo extract + - 4.5g/L glucose + - 580mg/L L-glutamine + - Filter through 0.22 µm filter before use +- **Myofiber Isolation Medium:** + - 20% FBS in DMEM + - 110 mg/ml sodium pyruvate, filter through 0.22 µm filter before use +- **Collagenase Digestion Solution:** + - 0.2% collagenase type I (Sigma #C0130) + - 4.5 g/l glucose + - 580 mg/l L-glutamine with 110 mg/ml sodium pyruvate, filter through 0.22 µm filter before use + - Incubate 10 min at 37°C circulatory water bath +- **Dissection Tools:** + - Fine forceps (Dumont 7 or equivalent) + - Spring scissors + - Hardened fine curved scissors 24 mm +- **Microscope:** Stereo binocular microscope (0.8-5x magnification) +- **Pipettes:** for dissociation of the muscles + - Large bore pipette & Small bore pipette +- **Permeabilization buffer:** + - 0.1% Triton X-100 in PBS + - 0.1 M Glycine in PBS (pH 7.4) +- **Blocking solution:** + - 5% HS in PBS (pH 7.4) +- **PFA:** 2% PFA in PBS +- **Antibodies for immunostaining:** + - Pax7 (undiluted) + - MyoD (1:100) + - Alexa Fluor 546 Goat Anti-Mouse IgG1 (1:1000) + - Alexa Fluor 488 Goat Anti-rat IgG (1:1000) +- **DAPI staining solution:** + - 10 μg/ml DAPI in PBS +- **Mouse for dissection:** Consult the animal welfare regulations. + +## Protocol + +### 3.1 Dissection and Digestion of the EDL Muscle +1. **Sacrifice the Mouse:** + - Sacrifice the mouse according to animal welfare regulations (see Note 3). + +2. **Preparation for Dissection:** + - Transfer the mouse to a semi-sterile dissection bench. Spray the whole mouse and dissection tools with 70% ethanol. + +3. **Expose Hind Limb Skin:** + - Use forceps to lift the skin at the ankle, and cut the skin. + +4. **Remove Fascia:** + - Remove surrounding muscles and fascia using fine forceps. + +5. **Detach the TA Muscle:** + - Use curved forceps to expose the distal tendon of the TA muscle, and detach. + +6. **Lift Tendon:** + - Lift the tendon of the TA using fine forceps, and cut the tendon. + +7. **Expose and Remove the EDL:** + - Grab the now fully exposed tendon of the EDL, cut the tendon, and transfer it to the collagenase digestion solution tube. + +8. **Identify and Cut Tendon:** + - Identify the tendon of the EDL closest to the knee, and carefully cut. Transfer the muscle to the preheated reaction tube with the collagenase solution (Fig. 1o). + +### 3.2 Dissociation of Single Myofibers +11. **Transfer Digested Muscles:** + - Transfer digested EDL muscles into a well of a 12-well plate filled with myofiber isolation medium using a large bore Pasteur pipette. + +12. **Dissociate Muscles:** + - Using a binocular microscope (0.8-5x magnification), dissociate muscles until single myofibers come off using the large bore Pasteur pipette. + +13. **Transfer Myofibers:** + - Transfer non-contracted shiny myofibers into a new well containing myofiber isolation medium using a small bore Pasteur pipette (see Notes 10-12). + +### 3.3 Culture of Single Myofibers and siRNA Transfection of MuSCs on Single Myofibers +16. **Culturing of Myofibers:** + - Transfer about 50 non-contracted single myofibers into a well of a 24-well plate filled with myofiber culture medium (500 µl). + +17. **Incubate:** + - Incubate single myofibers in a 37°C incubator with 5% CO2. + +### 3.4 Immunostaining of MuSCs on Single Myofibers +19. **Immunostaining:** + - Perform immunostaining using a stereo binocular microscope with 0.8-5x magnification. + +20. **Fixation:** + - Fix the single myofibers with their adjacent MuSCs using 2% PFA for 5 min at room temperature. + +21. **Wash:** + - Wash myofibers three times with PBS (500μl per washing step, 5min incubation per washing step). + +22. **Permeabilize:** + - Permeabilize the myofibers with permeabilization buffer (500μl) for 10 min at room temperature. + +23. **Block Unspecific Binding:** + - Incubate in blocking solution (500μl - 1 ml) for 1h at room temperature. + +24. **Add Primary Antibodies:** + - Dilute MyoD (1:100) and Pax7 (undiluted), use 250μl per well, incubate overnight at 4°C. + +25. **Wash:** + - Wash three times with PBS at room temperature (5 min per washing step). + +26. **Add Secondary Antibodies:** + - Incubate with secondary antibodies (250μl per well, 1h) at room temperature, in the dark. + +27. **Wash:** + - Wash twice with PBS at room temperature. + +28. **DAPI Staining:** + - Perform DAPI staining (10μg/ml) for 5 min at room temperature. + +29. **Final Wash:** + - Wash twice with PBS at room temperature. + +30. **Slide Preparation:** + - Label glass microscope slides and transfer stained myofibers. + +31. **Transfer Myofibers:** + - Spread single myofibers on the slide in smallest volume possible. + +32. **Liquid Removal:** + - Remove liquid with a 200μl pipette, ensuring myofibers are not dragged. + +33. **Mount Slides:** + - Add mounting medium and apply a cover slip. + +34. **Slide Drying:** + - Let slides dry at room temperature for at least 20 min to 1h. + +## Figures +- **Figure 1:** Isolation of murine EDL muscle. +- **Figure 2:** Dissociation of the EDL muscle. +- **Figure 3:** Immunostaining of isolated myofibers. + +## Notes +1. Also fix some myofibers with their adjacent MuSCs directly after isolation. +2. Always preprepare the collagenase digestion solution on the day of myofiber isolation. +3. Always perform cervical dislocation. +4. Ensure dissection is as sterile as possible. +5. Handle the EDL muscles only at the tendons. +6. Keep the myofibers warm using a heated plate during dissociation. +7. Use a stereo binocular microscope to distinguish and dissociate myofibers accurately. +8. Ensure Pipettes are sterilized before use. + +## Acknowledgements +The experiment was supported by a grant from DFG to J.v.M. Special thanks to Christine Poser and Christina Picker for technical assistance. + +## References +1. Lepper C, Partridge TA, Fan CM (2011) An absolute requirement for Pax7-positive satellite cells... +2. Murphy MM, Lawson JA, Mathew SJ, Hutcheson DA, Kardon G (2011) Satellite cells, connective tissue... +3. Bentzinger CF, von Maltzahn J, Rudnicki MA (2010) Extrinsic regulation of satellite cell specification... +... +15. von Maltzahn J, Zinoviev R, Chang NC, Bentzinger CF, Rudnicki MA (2013) A truncated Wnt7a retains... + +[endofoutput] +``` \ No newline at end of file diff --git a/markdown-output/isolation-and-culture-of-mouse-cortical-astrocytes-b67grhjw.md b/markdown-output/isolation-and-culture-of-mouse-cortical-astrocytes-b67grhjw.md new file mode 100644 index 0000000000000000000000000000000000000000..dc8c7009cb71970c20662e6a9b4bc5cc914d7408 --- /dev/null +++ b/markdown-output/isolation-and-culture-of-mouse-cortical-astrocytes-b67grhjw.md @@ -0,0 +1,127 @@ +```markdown +# Goal/Experiment: +The goal is to isolate and culture primary astrocytes from the cortex of postnatal day 1 or 2 (P1/P2) mice to generate primary cultures for further experimentation. + +# Isolation and Culture of Mouse Cortical Astrocytes + +**Ashley V Kumar¹, Francesca Telese²** +¹Division of Biology, University of California, San Diego +²Department of Psychiatry, University of California, San Diego + +## Abstract +We provide a detailed protocol to isolate and culture primary astrocytes from the cortex of postnatal day 1 or 2 (P1/P2) mice. + +## Guidelines +This protocol is designed to generate primary cultures of astrocytes from P1/P2 mice cortex. The protocol is written based on the dissection of brain tissues from 2 pups, but it can be scaled up as needed. + +The protocol is structured in five sections: +1. Tissue Dissection +2. Culture Plating and Maintenance +3. Subculturing +4. Microglia Shaking (optional) +5. FBS Inactivation + +The length of the protocol is approximately 19 days. + +> **Note:** Breeding and euthanasia of all animal work should be performed in accordance with an institutionally approved animal care and use protocol. + +## Materials +### Reagents +- **HBSS-G (HBSS/0.6% glucose) with P/S** + Hanks' balanced salt solution (1X HBSS) without Ca²⁺ and Mg²⁺ (Invitrogen #14170). Dissolve 3 g glucose (Sigma #G7528-1KG) in 500 mL 1X HBSS. Add 1% Penicillin/Streptomycin (P/S) (Invitrogen #15140122). Use 500 mL filter inside a tissue culture hood for aspiration. Store at 4°C. + +- **Poly-D, L-lysine hydrobromide (PDLL) (Sigma #P9011)** + Dissolve 5 mg in 500 mL PBS (0.01 mg/mL). Filter using 0.22 µm Stericup Filter Units (Millipore, S2GPU05RE). Store at 4°C. + +- **Trypsin, 0.25% (1X) (Fisher #25200-056)** + Final concentration: 0.125% Trypsin. Dilute 1:1 with HBSS-G + P/S before use. + +- **Deoxyribonuclease I (DNase I) from bovine pancreas (Sigma #D5025, 15KU)** + STOCK 10X 1 mg/mL in HBSS. Aliquot and store at -20°C. Use at a final concentration of 0.1 mg/mL. Thaw DNase on ice. + +- **Filtered 8% BSA (Sigma, A9647-50G) in PBS** + 4 g of BSA dissolved in 50 mL PBS. Filter using 0.22 µm Stericup Filter Units (Millipore, S2GPU05RE). Store at 4°C. + +- **Astrocyte Culture Media** + DMEM-F12 (GIBCO, 11320-033) with 10% heat-inactivated fetal bovine serum (FBS) (Omega FB-11 or Millipore TMS-0135B) and 1% Penicillin/Streptomycin (Invitrogen #15140122). Store at 4°C and warm up to 37°C before use. + +- **Trypan Blue (Life Technologies #1394110)** + +### Equipment +- Cell counter (Biorad, model: TC 20 automated cell counter) +- Counting slides (Biorad #145-0011) +- 10 cm Culture Dish (Genclone #25-202) +- T75 flask filtered cap (Fisher #12565349) +- Centrifuge (Eppendorf, Centrifuge 5810R) +- 15 mL tubes (Olympus, 29-103) + +## Before Start Instructions +### Coating Tissue Culture Plates +1. Coat desired number of T75 flasks with Poly-D-Lysine. Incubate at 37°C for 1 hour. +2. Rinse plates two times with sterile H₂O. +3. Leave the plates drying in the TC incubator while isolating astrocytes. + +### Prepare Reagents +1. Astrocyte media +2. HBSS-G + P/S +3. 0.25% Trypsin in HBSS-G + P/S + +(Optional) Filter reagents with 0.22 µm filters. + +## Tissue Dissection +**Before starting:** +- Spray down the dissection bench with 70% isopropanol. +- Prepare dissection tools. +- Prepare an ice bucket for chilling dissection plates. +- Place on ice 15 mL tubes with 14 mL HBSS-G for cortices isolated from 2 pups. +- Pre-warm astrocyte media and 1:1 0.25% Trypsin/HBSS-G + P/S at 37°C in a water bath. +- Thaw 10X DNase 1 (1 mg/mL) on ice. Dilute 1:10 in astrocyte culture medium before use (0.1 mg/mL final concentration). + +### Tissue Dissection Steps +1. Use a 10 cm Petri dish with ice-cold HBSS-G + P/S to dissect the brain from the skull. +2. Using a microscope, cut up from the cranial floor to the nose. Open the skull like a book. Lift the cortices with tweezers. +3. Dissect the cortex by removing the hindbrain and olfactory bulbs, trimming away the meninges. +4. Transfer the cortex to the 15 mL tube containing 14 mL of HBSS-G + P/S on ice. 2 heads per 15 mL tube. +5. Use a clean 10 cm Petri dish with ice-cold HBSS-G + P/S for each pup to reduce contamination. + +## Culture Plating and Maintenance +1. Move inside the biosafety cabinet. Spray 15 mL tubes with alcohol. Aspirate the HBSS-G + P/S, leaving as little of the dissection medium as possible without disturbing the tissue. +2. Add 5 mL/tube of warm 1:1 digestion medium (HBSS-G + P/S)/(0.25% Trypsin). Incubate 5 minutes at 37°C. Swirl the tube every 2 minutes. + - Note: 20 minutes of digestion leads to over digestion. Avoid incubating too long. +3. Remove poly-D-lysine from the T75 flasks. Rinse twice with sterile H₂O₂. Remove any excess water. +4. After 5 minutes of digestion, remove tubes. Spray with 70% isopropanol before moving into the hood. +5. Let the tissue settle to the bottom. Aspirate HBSS-G + P/S/trypsin. Add 5 mL of warm HBSS-G + P/S to each tube to rinse tissue and remove excess trypsin. Repeat as necessary to remove all trypsin. +6. Dilute 1:10 DNase in culture medium. Add 1 mL of astrocyte media/DNase mix to each 15 mL tube containing tissue. +7. Triturate tissue with a 1 mL tip (x7-15). Avoid introducing air bubbles. Let chunks of un-dissociated tissue sink at the bottom (1-2 min). +8. Filter dissociated tissues through a 40 µm mesh pre-rinsed with 1-2 mL culture medium. Collect filtered dissociated tissues in a 50 mL conical tube. +9. Overlay cell suspension on 8% BSA cushion in 50 mL tube. Tilt slightly to ensure distinct cell layers. +10. Spin cells for 6 minutes at 1200 RPM at room temperature. +11. Discard supernatant containing dead cells, 8% BSA, and media. Resuspend cell pellet in 1 mL of warm culture medium for each dissected pup. +12. Count cells using hemocytometer and dilute accordingly. +13. Plate cells in half volume of culture dish medium. Incubate at 37°C with 5% CO₂ for 5-10 minutes. Tap edge of dishes to release loose debris. Aspirate media. +14. Add fresh warm astrocyte media. Incubate at 37°C in 5% CO₂. +15. Change half of the culture medium the day after plating and every 3 days until cells reach 80% confluency. + +## Subculturing +1. Monitor astrocytes and split culture at ~80% confluency. + - Note: Shaking protocol before splitting is recommended for higher astrocytes yield. +2. Wash cells twice with 5 mL PBS. Add 1 mL trypsin and pipette around culture dish. +3. Once detached, add 5 mL of warm culture medium to neutralize trypsin. Collect cells, spin at 1200 RPM for 6 min. +4. Aspirate supernatant carefully and resuspend cell pellet in 5 mL fresh astrocyte medium. +5. (Optional) Freeze for storage (Passage 1). +6. Plate cells at 27,400 cells/cm². Bring total volume per 10 cm plate to 10 mL. Use pre-coated poly-D-Lysine plates. Incubate at 37°C in 5% CO₂. Change medium every 3 days. + +## Microglia Shaking (Optional) +1. At 7-8 days in vitro (first passage), shake T75 flask at 250 RPM for 1 hour to detach microglia. +2. Rinse remaining astrocyte layer twice with PBS. Continue with culture splitting (step 24). + +## Heat Inactivation Protocol for FBS +1. Thaw FBS at room temperature in water bath. +2. Set water bath at 56°C ± 2°C. +3. Control bottles must equal product volume. Control temperature at 56°C ± 2°C. +4. Place bottles in water bath. +5. Monitor temperature control. At 56°C, start timer for 30 min, swirling every 10 min. +6. After 30 min, remove from the water bath. Cool to room temperature, aliquot, or freeze. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/isolation-and-identification-of-potential-probioti-b7zhrp36.md b/markdown-output/isolation-and-identification-of-potential-probioti-b7zhrp36.md new file mode 100644 index 0000000000000000000000000000000000000000..3e05bdd121f05db46436795478142efd476c764b --- /dev/null +++ b/markdown-output/isolation-and-identification-of-potential-probioti-b7zhrp36.md @@ -0,0 +1,76 @@ +```markdown +# Goal/Experiment: +This experiment aims to isolate and identify potential probiotic bacteria from the soil of Saint Martin's Island in Bangladesh. These bacteria are hypothesized to modulate gut microbiota and prevent Enterococcus faecalis infection in tilapia (Oreochromis niloticus). + +# Isolation and Identification of Potential Probiotic Bacteria from the Soil of Saint Martin's Island Bangladesh + +## Authors +Md. Mahbubur Rahman1, Sulav Indra Paul1 +1Bangabandhu Sheikh Mujibur Rahman Agricultural University + +## Introduction +Soil bacteria from Saint Martin’s Island modulate gut microbiota and prevent Enterococcus faecalis infection in tilapia (Oreochromis niloticus). + +**DOI:** [10.17504/protocols.io.ewov1nzokgr2/v1](https://dx.doi.org/10.17504/protocols.io.ewov1nzokgr2/v1) + +## Collection of Soil Sample +1. The marine soil samples were collected from the shore of Saint Martin’s Island (20°37'40.3"N 92°19'22.3"E) of the Bay of Bengal, Bangladesh. +2. The samples were collected from 6-15 inch depth using an auger. +3. Immediately transferred to a 50 mL sterile falcon tube. + +## Isolation and Maintenance of Bacteria +4. 1 g of each soil sample was suspended in 9 mL of autoclaved seawater in an individual test tube. +5. 100 µL suspension from each diluted stock (10-2 and 10-3) was aseptically inoculated on individual Starch Casein Agar (SCA). +6. After incubation, different colonies were picked up based on their colony characteristics. + +*Note: Starch Casein Agar is used to isolate actinomycetes from soil samples. It contains starch as a growth medium for soil bacteria.* + +## Phenotypic Identification of Bacterial Isolates +7. Individual colonies grown on SCA plates were carefully observed, and colony characteristics (e.g., colony size, shape, color, type and elevation) were recorded. +8. Biochemical tests such as oxidase, catalase, motility, and oxidative-fermentative (O-F) tests were performed. + +## Screening of Antagonistic Activity of Marine Soil Isolates +9. Bacteria were grown in Marine Broth (MB) 2216 (Merck, USA) for 7 days at 28°C. +10. The broth culture was centrifuged at 10,000 ×g for 15 min and the culture supernatant was passed through a 0.22 µm millipore membrane filter. +11. The inhibitory activity of the culture supernatant was determined by agar well diffusion assay. + +*Note: Agar well diffusion assay is a method used to evaluate the antimicrobial activity of a substance by measuring the zone of inhibition around a well containing the test substance.* + +## Molecular Identification of Marine Soil Isolates +12. Genomic DNA of the selected isolates was extracted using a commercial GenJET genomic DNA purification kit (Thermo Fisher Scientific, USA) #K0721. +13. DNA was amplified using universal primer 8F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′). +14. The PCR amplification condition was: initial denaturation at 94°C for 5 min; 35 cycles of denaturation at 94°C for 1 min, annealing at 57°C for 40 sec, and extension at 72°C for 1 min; final extension step at 72°C for 10 min. +15. The PCR amplicons were purified using a commercial kit (Thermo Fisher Scientific, USA). +16. 16S rRNA gene sequencing was done by Sanger Sequencing. + +*Note: The 16S rRNA gene sequencing method is widely used for bacterial identification because it targets a highly conserved region of the bacterial genome.* + +## Evaluation of Digestive Enzyme Activity of Marine Soil Bacteria +17. Enzymatic activity (protease, lipase, amylase, cellulose) of the B. haynesii strain CD223 and A. mimigardefordensis strain SM421 was assessed to evaluate their probiotic effects. + +## Evaluation of the Viability of Bacteria in Different pH and Bile Esculin +18. The pH of SCA broth was adjusted from pH 3–9. Then, B. haynesii strain CD223 and A. mimigardefordensis strain SM421 were inoculated in this broth and kept for 24 h incubation at 28°C. +19. The viability of the cells was confirmed by inoculating them onto SCA agar plates (pH 7) by the spread plate method. + +## Preparation of Bacterial Extracellular Products (ECPs) +20. Bacteria were enriched in MB at 28°C for 10 days. +21. The ECPs were harvested and mixed with an equal volume of ethyl acetate in a separatory funnel. +22. The air-dried extract was weighed and dissolved in methanol for further use. + +## Minimum Inhibition Concentration (MIC) +23. MIC was measured with different concentrations of ECPs extracts (1000, 500, 250, 125, 62.5, and 31.25 µg mL-1). + +## Measurement of Hematological Parameters +24. Fish from each treatment were anesthetized with clove oil (0.05 mL per 500 mL of water) for hematological analysis. +25. Blood was collected from fish using a 3 cc syringe containing 10% blood anti-coagulant (EDTA) inserted into the caudal peduncle region to draw out blood. +26. The blood was transferred to a test tube coated with EDTA and stored at -30°C until use. +27. Red blood cells (RBCs) and white blood cells (WBCs) were counted using an improved Neubauer hemocytometer (Marienfeld Company, Germany) under a light microscope (DM100; Leica, Wetzlar, Germany). +28. To measure hemoglobin, fresh blood was collected from fish from each treatment and was poured on the edge of a strip of hemoglobin meter before the coagulation of blood. +29. Estimation of immunoglobulin (IgM) was carried out by using Humalyzer-3000 analyzer. + +## Metagenomics Study +30. Gut microbiome DNA was extracted using a commercial kit. +31. V3 and V4 primer were used for sequencing. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/isolation-of-evs-by-ultracentrifugation-cwqfxdtn.md b/markdown-output/isolation-of-evs-by-ultracentrifugation-cwqfxdtn.md new file mode 100644 index 0000000000000000000000000000000000000000..357f66f63acf10fb3526f717db67c00cdbf144d5 --- /dev/null +++ b/markdown-output/isolation-of-evs-by-ultracentrifugation-cwqfxdtn.md @@ -0,0 +1,103 @@ +```markdown +# Goal/Experiment: +Isolation of Extracellular Vesicles (EVs) from culture media using differential centrifugation. + +# Isolation of EVs by Ultracentrifugation + +## Abstract +How to isolate EVs from culture media using differential centrifugation. + +### DOI: +[dx.doi.org/10.17504/protocols.io.q26g7pb6qgwz/v1](https://dx.doi.org/10.17504/protocols.io.q26g7pb6qgwz/v1) + +### Protocol Citation: +Chloe.Rodgers 2023. Isolation of EVs by ultracentrifugation. protocols.io https://dx.doi.org/10.17504/protocols.io.q26g7pb6qgwz/v1 + +### License: +This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. + +### Protocol Status: +Working. We use this protocol and it's working. + +### Created: +Jul 04, 2023 + +### Last Modified: +Jul 05, 2023 + +### Protocol integer ID: +84455 + +## 1. Preparation of Cells +- **Note**: Cell cultures must be grown to approximately 90% confluency and EVs need to be isolated from a minimum of 200mL media to get a high enough EV yield. As Fetal Bovine Serum (FBS) contains natural EVs, EV-depleted FBS should be used in the media incubated with cells for 48 hours prior to isolation. This media is called collection media (CM). Alternatively, collection media can be FBS free, or FBS can be substituted with ITS (Insulin-Transferrin-Selenium; ITS-G 100X, Gibco). If using ITS, 500mL collection media will require 5mL of the ITS. ITS only requires a 24-hour incubation before collection. + +### Steps: +1. **Remove culture media** +2. **Wash cells** 2x with PBS. +3. **Add Collection Media (CM)** to cells for 24/48 hours. + +## 2. Differential Ultracentrifugation + ![Differential Ultracentrifugation](image2.png) + +- **Note**: The 2000g spin (Step 2) is for 20 minutes, not for 10 as the image shows. + +### Steps: +1. **Centrifuge at 300g for 10 minutes**. +2. **Centrifuge at 2000g for 20 minutes**. +3. **Centrifuge at 10000g for 30 minutes**. +4. **Centrifuge the cleared conditioned medium at 100000g for 70 minutes**. + +### Notes Before Starting: +- **Ultracentrifuge Location**: The ultracentrifuge is in the storeroom on floor 1. It needs to be booked on Clustermarket (Not CeMi; Pharmacology). +- **Instructions on Use**: + 1. Turn ON using the switch on the side of the machine. + 2. Bring grease and the small key to remove tubes from the rotor if needed. + 3. It can fit 8 tubes with a maximum volume of 25mL per tube. + 4. Cool it to 4°C before using by switching on the vacuum. + 5. Get the rotor from the fridge (Be cautious, it's heavy!). + 6. Place it in the centrifuge correctly by giving it a manual spin. + 7. Slide the door shut and click the vacuum. + 8. Set speed, time, and temperature twice (once while cooling and once before the spin). + +### Additional Steps: +1. To remove the rotor, press the vacuum button until it shows 200. Unlock the door and remove the rotor. +2. Grease the bottom of the rotor circle and rubber parts to prevent sticking. +3. Balance the ultracentrifuge tubes using scales. Ensure the lids are tightly closed. + +## 3. Step-by-Step Centrifugation: +1. **First Spin**: + - Centrifuge the CM in 50mL Falcon tubes at 300g for 10 minutes. +2. **Second Spin**: + - Transfer supernatant to new 50mL Falcon tubes and centrifuge at 2000g for 20 minutes. +3. **Third Spin**: + - Transfer supernatant to 25mL ultracentrifuge tubes (Beckman Coulter, 355654). Balance and centrifuge at 10000g for 30 minutes. +4. **Fourth Spin**: + - Transfer supernatant to new 25mL ultracentrifuge tubes. Centrifuge at 100000g for 70 minutes. Clean after use and log the details. + +### Post Centrifugation: +1. Discard the supernatant and carefully remove media using a tip. +2. Resuspend EV pellets in 200μL filtered PBS. +3. If not using Size Exclusion Chromatography, divide the 200μL into 25/50μL aliquots. +4. Store the aliquots at -80°C, using low-bind tubes for storage. + +### Table: + +| Spin | Centrifuge | Time (min) | Speed (g) | +|------|------------|------------|--------------------| +| 1 | Falcon (1) | 10 | 300 | +| 2 | Falcon (2) | 20 | 2000 | +| 3 | Ultra (3) | 30 | 10000 | +| 4 | Ultra (4) | 70 | 100000 | + +### Supplies and Reagents: +- **Cell Culture Media (CM)**: Use EV-depleted FBS or FBS-free media. +- **ITS (Gibco)**: Alternative for FBS. +- **PBS**: Used twice for washing cells. +- **Ultracentrifuge Tubes (Beckman Coulter, 355654)**. +- **Equipment**: Grease, small key, scales, ultracentrifuge. + +### Alternative Supplies: +- If **ITS** is not available, use **Insulin-Transferrin-Selenium (ITS)** substitutes from other vendors. + +endofoutput +``` diff --git a/markdown-output/isolation-of-extracellular-vesicles-from-cell-cult-bnr3md8n.md b/markdown-output/isolation-of-extracellular-vesicles-from-cell-cult-bnr3md8n.md new file mode 100644 index 0000000000000000000000000000000000000000..fbf1c823cfe2e0f93e9766d680f840f653d9fa4a --- /dev/null +++ b/markdown-output/isolation-of-extracellular-vesicles-from-cell-cult-bnr3md8n.md @@ -0,0 +1,78 @@ +```markdown +# Goal/Experiment: +The goal of this experiment is the isolation of extracellular vesicles (EVs) from cell culture media using differential ultracentrifugation. EVs are important mediators of intercellular communication released by all mammalian cells. The protocol provided details the steps required to effectively isolate EVs leveraging the advantages of ultracentrifugation. + +# Isolation of Extracellular Vesicles from Cell Culture Media by Differential Ultracentrifugation + +### Authors +- Dima Ter-Ovanesyan¹ +- Wendy Trieu¹ +- Maia Norman² +- Roey Lazarovits¹ +- George Church³ +- David Walt² + +¹ Wyss Institute for Biologically Inspired Engineering +² Wyss Institute for Biologically Inspired Engineering, Brigham and Women's Hospital +³ Wyss Institute for Biologically Inspired Engineering, Harvard Medical School Department of Genetics + +DOI: [dx.doi.org/10.17504/protocols.io.bnr3md8n](https://dx.doi.org/10.17504/protocols.io.bnr3md8n) + +### Abstract +Extracellular vesicles (EVs) are released by all mammalian cells and are crucial for intercellular communication. One preferred method of EV isolation is ultracentrifugation due to its effectiveness with large input volumes of cell culture media. This protocol provides a detailed methodology for isolating EVs using differential ultracentrifugation. + +### Materials +- Cells and cultureware +- Fetal bovine serum (FBS)-depleted media (ultracentrifuge media overnight (16 hours) at 120,000 x g to deplete) or AIM V media (Gibco) +- PBS without Ca²⁺/Mg²⁺ +- Ultracentrifuge and rotor (Beckman Coulter) +- Polyallomer ultracentrifuge tubes (Beckman Coulter) +- 0.22 µm Steriflip filter tubes (EMD Millipore) +- 50 mL Falcon tubes (Corning) +- Pasteur pipettes +- cOmplete mini protease inhibitor cocktail tablets (Roche) +- 1M HEPES (Gibco) + +### Reagent Preparation +**HEPES buffer (optional):** +- Prepare 20 mM HEPES with protease inhibitor (Every 2 weeks make stock of 200 μL 1x HEPES in 10 mL PBS with 1 cOmplete mini protease inhibitor cocktail tablet) +- Store at 4 °C. + +### Protocol + +#### Day 1: +1. **Cell Culture:** + - Culture cells under standard conditions to 50-70% confluency. + +2. **For Suspension Cells:** + - Spin down desired total number of cells in six Falcon tubes at 300 x g for 5 minutes. + - Aspirate media and resuspend each cell pellet in 40 mL FBS-depleted media or AIM V media. + - Transfer contents of each Falcon tube to a T75 flask and return to the incubator. + +3. **For Adherent Cells:** + - Aspirate media from twelve 15 cm plates. + - Add 20 mL FBS-depleted media or AIM V media per plate and return cells to the incubator. + +#### Day 2: +4. After 24 hours, collect all media and divide among 50 mL Falcon tubes. +5. Spin at 300 x g for 10 minutes at room temperature (RT) to pellet the cells. +6. Transfer supernatant to new 50 mL tubes, leaving cell pellet behind. +7. Spin again at 2000 x g for 10 minutes at RT to pellet any dead cells. +8. Transfer supernatant to new 50 mL tubes, leaving cell pellet behind. +9. Spin supernatant at 16,500 x g for 20 minutes at 4 °C to pellet large EVs. +10. Transfer supernatant to new 50 mL tubes, leaving pellet behind. +11. Pass supernatant through a Steriflip 0.22 μm filter. +12. Transfer supernatant to polyallomer ultracentrifuge tubes. Centrifuge at 120,000 x g (26,500 RPM with SW32Ti rotor) for 70 minutes at 4 °C. +13. Remove most of the supernatant, leaving ~2 cm of media above the pellet. Add 5 mL PBS to each tube. Vortex on medium speed for a few seconds. Fill to top of each tube with PBS. +14. Centrifuge again at 120,000 x g for 70 minutes at 4 °C. +15. Aspirate all of the supernatant with Pasteur pipette without touching the bottom of the tube where the pellet is located. +16. Resuspend pellet either in PBS or directly in the desired lysis buffer for western blot. If storing for later use, resuspend in HEPES buffer, and store at -80 °C. + +### License +This is an open access protocol distributed under the terms of the [Creative Commons Attribution License](https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. + +### Citations +- Kowal EJK, Ter-Ovanesyan D, Regev A, Church GM. Extracellular Vesicle Isolation and Analysis by Western Blotting. Methods Mol Biol. 2017;1660:143-152. doi:10.1007/978-1-4939-7253-1_12. PMID: 28828654. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/isolation-of-human-islet-cells-culture-with-hepara-kwwcxfe.md b/markdown-output/isolation-of-human-islet-cells-culture-with-hepara-kwwcxfe.md new file mode 100644 index 0000000000000000000000000000000000000000..fa208328c7a1407cf1ead79f22b5c2e8ce53b505 --- /dev/null +++ b/markdown-output/isolation-of-human-islet-cells-culture-with-hepara-kwwcxfe.md @@ -0,0 +1,95 @@ +```markdown +# Goal/Experiment: +The goal of this experiment is to isolate human islet cells, culture them with heparan sulfate mimetics, and analyze the viability of beta cells using flow cytometry. + +## Isolation of human islet cells, culture with heparan sulfate mimetics and flow cytometry analysis of beta cell viability + +**Sarah Popp, Charmaine Simeonovic** + +### Abstract +Isolated human islets were dispersed into single cells using Accutase (Millipore), ~1500-2000 islet equivalents (IEQ)/ml. 20,000-65,000 islet cells were transferred to individual wells of a 96-well culture plate (CELLSTAR, Greiner Bio-one) for immediate staining for flow cytometry analysis or for culture prior to staining. Isolated human islet cells were cultured in the presence or absence of the HS mimetics heparin (a highly sulfated HS analogue from porcine intestinal mucosa), BT548 (a glycol split low molecular weight heparin (LMWH; 3 kDa) lacking anticoagulant activity) or PI-88 (Progen Pharmaceuticals Limited,) at 50 μg/ml for 2 days in 5% CO₂, 95% air at 37°C. In some studies islet cells were acutely treated with 30% H₂O₂ (Chem-Supply) as a source of reactive oxygen species (ROS) for 5 min on day 0 or after culture for 2 days with/without HS mimetics. Beta cells were identified by staining with Newport Green (NG; 10 μmol/L; Invitrogen, Molecular Probes), a fluorescent probe that detects zinc in the insulin granules of beta cells. Damaged and dying islet cells were assessed using 7-Aminoactinomycin (7AAD, 10 μg/ml; Life Technologies) or by Sytox green (31.25 nmol/L; Invitrogen, Molecular Probes) uptake. Cells were analyzed using a BD LSRI flow cytometer and CellQuest™ Pro software (version 6.0; BD Biosciences). + +### Before start + +#### Materials: +1. Prepare: + +**(i) PBS/3mM EDTA:** +- 112 mg EDTA (AJAX #180) in 100 ml PBS, sterile filter using 0.2 μm disposable filter + +**(ii) Beta cell culture medium:** +- RPMI 1640 (Sigma #R0883), 200 ml + - Heat-inactivated fetal calf serum (HIFCS), 20 ml + - L-Glutamine (Gibco # 25030081 200 mM) 2 ml (final 2 mM) + - Penicillin G (MP Biomedicals #02194537), 0.06 mg/ml + - Streptomycin (Sigma #S9137), 0.10 mg/ml + - Neomycin (Sigma #N6386), 0.10 mg/ml + +2. Other reagents: + - Accutase, Millipore #SCR005 + - Cell culture plates: Cellstar #650180 (Greiner Bio-one) + - Newport Green DCF diacetate (Newport Green), Invitrogen, Molecular Probes #N7991 + - 7-Aminoactinomycin (7AAD) Life Technologies #A1310 + - SYTOX Green, Invitrogen, Molecular Probes #S7020 + - Hydrogen peroxide (30% w/w), Chem-Supply Pty Ltd (Australia) #HA154-500M + +### Protocol + +#### Step 1. +See Guidelines, ' Before starting' and 'Safety Warnings' + +#### Step 2. +Centrifuge human islets at 300g for 2 min at 23°C. Pour off the supernatant. Resuspend in 25 ml PBS/3 mM EDTA. Centrifuge at 300g. + +#### Step 3. +Resuspend the islets in PBS/3 mM EDTA and transfer islets to 15ml tubes, 2000 islet equivalents (IEQ)/tube. Centrifuge at 300g then carefully remove the supernatant. + +#### Step 4. +Gently resuspend each pellet in 1ml pre-thawed Accutase and place tubes in 37°C waterbath for 10 mins (Note: at 4 min and 8 min, gently knock the pellet to resuspend the islets). + +#### Step 5. +Dissociate the islets by pipetting up and down 10-15 times using a 1 ml single channel pipette. + +#### Step 6. +Add 10 ml culture medium to each tube to terminate the Accutase reaction and centrifuge for 5 min at 300g. + +#### Step 7. +Discard the supernatant, pool the cells into a single 15 ml tube and determine cell density (using hemocytometer). Adjust cell density to 100,000 - 325,000 cells/ml. + +#### Step 8. +Transfer islet cells to culture plate, 20,000 - 65,000 cells (in 200 μl)/well. Centrifuge at 300g then remove the supernatant by flicking. + +#### Step 9. +Islet cells are cultured with heparin or heparan sulfate mimetics (e.g. PI-88) at a final concentration of 50 μg/ml in 200 μl/well. Control cells are cultured in medium. + +#### Step 10. +Cell viability is determined on day 0 and 2 days after culture by staining with Newport Green /7AAD or SYTOX Green followed by flow cytometry analysis: + +(i) **For Newport Green/7AAD staining**: +- Centrifuge the culture plate at 300g for 3 min and remove the culture supernatant. +- Resuspend cells in 10 μM Newport Green, 100 μl/well. Incubate at 37°C for 1 hr. +- Add 100 μl culture medium, centrifuge at 300g. Remove culture supernatant and resuspend in 10 μg/ml 7AAD, 100 μl/well. Incubate at 37°C for 15 min. +- Add 100 μl PBS, centrifuge at 300g. Remove culture supernatant and resuspend in 100 μl PBS for flow cytometry analysis. +- Analyse flow cytometry data using CellQuest™ Pro software (version 6.0; BD Biosciences). +- Viable beta cells are Newport Green+ve 7AAD-ve; dead/damaged beta cells are Newport Green+ve, 7AAD+ve. + +**Excitation/emission wavelengths**: +- Newport Green: 503 nm/535 nm +- 7AAD: 546 nm/647 nm + +(ii) **For monitoring hydrogen peroxide-induced cell death**: +- Centrifuge culture plate at 300g for 3 min and remove culture supernatant. +- Resuspend cells in 100 μl of 30% H₂O₂ or culture medium for 5 min. Add 100 μl culture medium and centrifuge at 300g for 3 min. +- Remove culture supernatant and resuspend cells in 31.25 nM SYTOX Green (1/160,000 dilution of stock), 100 μl/well. Incubate at 37°C for 15 min. +- Add 100 μl PBS, centrifuge at 300g for 3 min. Remove culture supernatant and resuspend cells in 100 μl PBS for flow cytometry analysis. +- Analyse flow cytometry data using CellQuest™ Pro software (version 6.0; BD Biosciences). +- Dead/damaged islet cells are SYTOX Green+ve, compared to unstained controls. + +**Excitation/emission wavelength for SYTOX Green**: 504 nm/523 nm + +#### Step 11. +### Warnings +All handling of human islets is done in a Class II Biological Safety Cabinet. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/isolation-of-mouse-islet-cells-culture-with-hepara-bmgjk3un.md b/markdown-output/isolation-of-mouse-islet-cells-culture-with-hepara-bmgjk3un.md new file mode 100644 index 0000000000000000000000000000000000000000..8a70d6879265bfe2c3c07ce7da9828ea4bc9f6b2 --- /dev/null +++ b/markdown-output/isolation-of-mouse-islet-cells-culture-with-hepara-bmgjk3un.md @@ -0,0 +1,96 @@ +```markdown +# Goal/Experiment: +Isolation of mouse islet cells, culture with heparan sulfate mimetics and flow cytometry analysis of beta cell viability + +# Isolation of mouse islet cells, culture with heparan sulfate mimetics and flow cytometry analysis of beta cell viability +**Sarah Popp1, Sarita Dhounchak1, Charmaine Simeonovic1** + +1 The Australian National University + +## Abstract +Isolated mouse islets were dispersed into single cells using Accutase (Millipore; 250 μl/500 islets). 4-8 x 10^4 islet cells were transferred to individual wells of a 96 well culture plate (CELLSTAR, Greiner Bio-one) for immediate staining for flow cytometry analysis or for culture prior to staining. Isolated mouse islet cells were cultured in the presence or absence of the HS mimetics heparin (a highly sulfated HS analogue from porcine intestinal mucosa) or PI-88 (Progen Pharmaceuticals Limited), at 50 mg/ml for 2 days in 5% CO₂, 95% air at 37ºC. In some studies islet cells were acutely treated with 30% H₂O₂ (Chem-Supply) as a source of reactive oxygen species (ROS) for 5 min on day 0 or after culture for 2 days with/without HS mimetics. Damaged and dying islet cells were assessed using Calcein-AM (Calcein; 0.04 μM; Invitrogen)/Propidium iodide (PI; 2.5 μg/ml; BD Biosciences) or by Sytox green (31.25 nmol/L; Invitrogen, Molecular Probes) uptake. BD LSR Fortessa flow cytometer BD FACS DIVA software (version 8) were used to collect events and Flow Jo software (version 10.0.7, TreeStar Inc.) was used to analyse the intensity of fluorescence staining. + +## DOI +[dx.doi.org/10.17504/protocols.io.bmgjk3un](https://dx.doi.org/10.17504/protocols.io.bmgjk3un) + +## Keywords +- mouse beta cells +- heparan sulfate +- heparan sulfate replacement +- beta cell viability + +## License +This is an open access protocol distributed under the terms of the [Creative Commons Attribution License](https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. + +## Materials +**1. Prepare:** +- **PBS/3mM EDTA:** + - 112 mg EDTA (AJAX #180) in 100 ml PBS, sterile filter using 0.2 μm disposable filter. + +- **Beta cell culture medium:** + - RPMI 1640, Sigma #R0883, 200 ml + - Heat-inactivated fetal calf serum (HIFCS), 20 ml + - L-Glutamine (Gibco # 25030081 200 mM), 2 ml (final 2mM) + - Penicillin G, MP Biomedicals #02194537, 0.06 mg/ml + - Streptomycin, Sigma #S9137, 0.10 mg/ml + - Neomycin, Sigma #N6386, 0.10 mg/ml + +- **Other reagents:** + - Accutase, Millipore #SCR005 + - Cell culture plates: Cellstar #650180(Greiner Bio-one) + - Calcein-AM (Calcein), 0.5mM, Invitrogen #C3100MP + - Propidium Iodide (PI), 50 mg/ml, BD Biosciences #556463 + - SYTOX Green, 5 mM, Invitrogen, Molecular Probes #S7020 + - Hydrogen peroxide (30% w/w), Chem-Supply Pty Ltd (Australia) #HA154-500M + +## Protocol Steps + +### 1. Preparation +1. See Guidelines, "Before starting". + +### 2. Dispersing Isolated Mouse Islets +2. Isolated mouse islets were transferred to a 15 ml tube and excess medium was removed using a pipette. Resuspend in ~10-15 ml PBS/3mM EDTA. Centrifuge at 249g. + +3. Resuspend the islets in PBS/3mM EDTA. Centrifuge at 249g then carefully remove the supernatant. + +### 3. Accutase Treatment +4. Gently resuspend each pellet in pre-thawed Accutase (250 μl/500 islets) and place tubes in a 37ºC waterbath for 10 mins (Note: at 4 min and 8 min, gently knock the pellet to resuspend the islets). + +5. Dissociate the islets by pipetting up and down 10-15 times using a 1 ml single channel pipette. + +6. Add 10 ml culture medium to each tube to terminate the Accutase reaction and centrifuge for 5 min at 249g. + +### 4. Cell Culture +7. Discard the supernatant, resuspend in beta cell culture medium (500 μl/500 islets) and determine cell density (using hemocytometer). + +8. Transfer islet cells to culture plate. 4-8 x 10^4 cells /well and adjust the volume in the wells to 200 μl by adding beta cell culture medium. Centrifuge at 249g then remove the supernatant by flicking. + +9. Islet cells are cultured with heparin or heparan sulfate mimetics (e.g. PI-88) at a final concentration of 50 μg/ml in 200μl/well. Control cells are cultured in medium. + +### 5. Determining Cell Viability and Flow Cytometry +10. Cell viability is determined on day 0 and 2 days after culture by staining with Calcein/PI or SYTOX Green followed by flow cytometry analysis: + +**Calcein-AM/Propidium Iodide Staining** + - Centrifuge culture plate at 110-173g for 3 min and remove culture supernatant. + - Resuspend cells in 0.04 μM Calcein, 100μl/well. Incubate at 37ºC for 15 min. + - Add 100μl culture medium, centrifuge at 110-173g. Remove culture supernatant and resuspend in 2.5μg/ml, 100μl/well. Incubate at 37ºC for 15 min. + - Add 100μl PBS, centrifuge at 110-173g. Remove culture supernatant and resuspend in 100μl PBS for flow cytometry analysis. + - Analyse flow cytometry data using FlowJo software (version 10.0.7, TreeStar Inc.). Viable beta cells are Calcein+ve PI-ve; damaged beta cells are Calcein+ve PI+ve; and dead cells are Calcein+ve PI+ve. + +**Excitation/emission wavelengths:** + - Calcein-AM: 494 nm/517 nm + - PI: 493 nm/636 nm + +**Monitoring Hydrogen Peroxide-Induced Cell Death** + - Centrifuge culture plate at 110-173g for 3 min and remove culture supernatant. + - Resuspend cells in 100μl of 30% H₂O₂ or culture medium for 5 min. + - Add 100μl culture medium and centrifuge at 300g for 3 min. Remove culture supernatant and resuspend cells in 31.25 nM SYTOX Green (1/160,000 dilution of stock), 100μl/well. Incubate at 37ºC for 15 min. + - Add 100μl PBS, centrifuge at 110-173g for 3 min. + - Remove culture supernatant and resuspend cells in 100μl PBS for flow cytometry analysis. + - Analyse flow cytometry data using FlowJo software (version 10.0.7, TreeStar Inc.). Dead/damaged islet cells are SYTOX Green+ve, compared to unstained controls. + + **Excitation/emission wavelengths:** + - Sytox Green: 504 nm/523 nm + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/isolation-of-nuclei-from-frozen-human-skeletal-and-b2z4qf8w.md b/markdown-output/isolation-of-nuclei-from-frozen-human-skeletal-and-b2z4qf8w.md new file mode 100644 index 0000000000000000000000000000000000000000..8be01d799b3cd1fe8dd4e5c9f01d49970d1d0486 --- /dev/null +++ b/markdown-output/isolation-of-nuclei-from-frozen-human-skeletal-and-b2z4qf8w.md @@ -0,0 +1,138 @@ +```markdown +Goal/Experiment: To isolate single nuclei from frozen skeletal and cardiac muscle tissues for single nucleus RNA and chromatin assays. + +# Isolation of Nuclei from Frozen Human Skeletal and Cardiac Muscle for Single Nucleus RNA and Chromatin Assays V.2 + +**James R Hocker1, Sebastian Preissl1, Dinh H Diep1** +1University of California, San Diego + +[**DOI**: 10.17504/protocols.io.b2z4qf8w](https://dx.doi.org/10.17504/protocols.io.b2z4qf8w) + +This protocol was adapted for the isolation of single nuclei from frozen skeletal and cardiac muscle tissues for molecular characterization with the SNARE-seq2, sci-ATAC-seq, and snRNA-seq assays. + +## References + +1. Preissl et al (2015). Circulation Research. DOI: 10.1161/CIRCRESAHA.115.306337 +2. Hocker et al (2021). Science Advances. DOI: 10.1126/sciadv.abf1444 + +## Required Consumables per Sample: + +1. gentleMACS M Tube (Miltenyi Biotec, 130-096-335) +2. CellTrics Filter, 30 µM (CellTrics, 04-0042-2316) +3. Eppendorf Tubes, 5 mL + +## Required Instrument: + +1. gentleMACS Tissue Dissociator (Miltenyi Biotec, 130-095-937) + +## Buffers: + +### Nuclear Permeabilization Buffer (NPB) +_Make fresh for each use._ + +| Reagent | Stock Conc. | Final Conc. | 1 mL | 5 mL | 10 mL | +|-------------------------------|-------------|-------------|-------|-------|--------| +| PBS | 1X | 1X | 0.925 mL | 4.625 mL | 9.25 mL | +| BSA (Sigma Aldrich) | n/a | 5% | 50 mg | 250 mg | 500 mg | +| IGEPAL CA-630 / NP 40 | 10% | 0.20% | 20 µL | 100 µL | 200 µL | +| DTT | 100 mM | 1 mM | 10 µL | 50 µL | 100 µL | +| Protease Inhibitor | 25X | 1X | 40 µL | 200 µL | 400 µL | +| Enzymatics RNase In | 40 U/µL | 0.2 U/µL | 5 µL | 25 µL | 50 µL | + +### MACS Buffer +_Make a stock without Protease In and add when using._ + +| Reagent | Stock Conc. | Final Conc. | 1 mL | 4 mL | 12 mL | +|-------------------------------|-------------|--------------|-------|-------|-------| +| Nuclease free water | n/a | n/a | 0.927 mL | 3.708 mL | 11.124 mL | +| CaCl2 | 1 M | 5 mM | 5 µL | 20 µL | 60 µL | +| MgOAC | 1 M | 3 mM | 3 µL | 12 µL | 36 µL | +| Tris-HCl, pH 8.0 | 1 M | 10 mM | 10 µL | 40 µL | 120 µL | +| EDTA | 500 mM | 2 mM | 4 µL | 16 µL | 48 µL | +| DTT | 100 mM | 0.6 mM | 6 µL | 24 µL | 72 µL | +| Protease Inhibitor | 25X | 1X | 40 µL | 160 µL | 480 µL | +| Enzymatics RNase In | 40 U/µL | 0.2 U/µL | 5 µL | 20 µL | 60 µL | + +### PBS + RNase In +_Make fresh for each use._ + +| Reagent | Stock Conc. | Final Conc. | 1 mL | 5 mL | 10 mL | +|-------------------------------|-------------|-------------|-------|-----------|----------| +| PBS | 1X | 1X | 0.93 mL | 4.65 mL | 9.3 mL | +| Superase In | 20 U/µL | 0.05 U/µL | 2.5 µL | 12.5 µL | 25 µL | +| Enzymatics RNase In | 40 U/µL | 0.05 U/µL | 1.25 µL | 6.75 µL | 12.5 µL | + +## Procedure + +1. **Section muscle tissue:** + - Section flash-frozen skeletal muscle into aliquots according to desired nuclei yield. Store on dry ice or at -80°C. + - Expect yield of 2000-4000 nuclei per mg of skeletal muscle tissue. + +2. **Prepare buffers and chill components:** + - Prepare buffer fresh on the day of nuclei isolation. + - Precool centrifuge to 4°C. + - Precool all buffers at 4°C. + - Place gentleMACS dissociator in cold room/chiller. + - Chill gentleMACS M tubes on ice. + - Chill 5.0 mL Eppendorf tubes on ice. + +| Tissue Mass | MACS Buffer | NPB Buffer | +|-------------|-------------|---------------------| +| 10-50 mg | 1 mL | 2 mL | +| 50-100 mg | 2 mL | 4 mL | +| >100 mg | 3 mL | 1 mL per 25 mg | + +3. **Transfer tissue:** + - Transfer sectioned frozen tissue to gentleMACS M tubes. + +4. **Add buffer:** + - Add the recommended volume of MACS buffer to gentleMACS M tube. + - Allow tissue to thaw in MACS buffer for approximately 60 seconds on ice. + +5. **Homogenize:** + - Homogenize with Miltenyi tissue dissociation protocol: "Protein_01_01" on the gentleMACS instrument in the chiller/cold room. + +6. **Centrifuge briefly:** + - Briefly centrifuge the gentleMACS M tube to pull all the homogenate to the bottom at 160 x g, 4°C, 00:00:15. + +7. **Filter homogenate:** + - Filter homogenate through 30 µM CellTrics filter into 5 mL Eppendorf tubes. + +8. **Wash gentleMACS M tube:** + - Wash gentleMACS M tube with another 1 mL of MACS buffer and filter the wash. + +9. **Centrifuge:** + - Centrifuge at 900 x g, 4°C, 00:10:00. Use ramp rate: 3/9 acceleration and 3/9 deceleration. + +10. **Decant supernatant:** + - Decant and discard the supernatant. + +11. **Resuspend pellet:** + - Resuspend the pellet with the recommended NPB buffer. + +12. **Rotate sample:** + - Gently rotate the sample in cold room/chiller for 00:10:00. + + - Note: Users should optimize lysis timing for different samples. (i.e., 5 minutes for cardiac tissues). + +13. **Centrifuge permeabilized nuclei:** + - Centrifuge permeabilized nuclei at 900 x g, 4°C, 00:10:00. + +14. **Decant and discard supernatant.** + +15. **Resuspend pellet in PBS + RNase In:** + - Resuspend the pellet in 500 µL PBS + RNase In. + + - For SNARE-seq2: immediately fix nuclei by adding (1:1) 500 µL PBS + RNase In + 1% paraformaldehyde. (Make 1 mL: 937.5 µL PBS+RI + 62.5 µL 16% methanol-free PFA). Incubate on ice for 00:10:00. + +16. **QA/QC - Count nuclei:** + - Mix 1:1 volume of cellular suspension with a staining solution (ie DAPI). Load 10 µL of mixture onto a Biorad Cell Counter slide then count with a BioRad TC20 Cell Counter. Gate 4 µM-6 µM for nuclei sizes. + + **QA/QC - Check nuclei integrity:** + - Check nuclei integrity under fluorescent microscope using DAPI channel. Nuclei should appear distinct, have rounded borders and the majority occurring as singlets. + + - If high clumping is observed: bring total volume to ~1 mL with PBS+0.1%BSA and filter the sample through a 30 µM CellTrics filter. Pellet at 900 x g, 4°C, 00:10:00. Then resuspend again in 100 µL PBS + RNase In. + - If high debris is observed (low DAPI+): bring volume to ~1 mL with PBS+0.1%BSA and pellet at 900 x g, 4°C, 00:10:00. Then resuspend again in 100 µL PBS + RNase In. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/isotopically-labelled-inorganic-carbon-delivered-t-c5mqy45w.md b/markdown-output/isotopically-labelled-inorganic-carbon-delivered-t-c5mqy45w.md new file mode 100644 index 0000000000000000000000000000000000000000..87262b72da7491e3e8147affc3f03900dba3a971 --- /dev/null +++ b/markdown-output/isotopically-labelled-inorganic-carbon-delivered-t-c5mqy45w.md @@ -0,0 +1,83 @@ +```markdown +Goal/Experiment: +The goal of this experiment is to deliver isotopically labelled inorganic carbon (\( ^{13}CO_2 \)) to algal cultures via a bubbler bottle, thereby enabling the study of photosynthetic carbon fixation processes. + +# Isotopically Labelled Inorganic Carbon Delivered to Algal Cultures via Bubbler Bottle + +## Authors +- Sunnyjoy +- Usha F Lingappa¹, Dupuis¹ +- Sabeeha S. Merchant¹ +- Xavier Mayali² + +¹University of California, Berkeley +²Lawrence Livermore National Laboratory + +Merchant Lab UC Berkeley + +## Abstract +This protocol describes a method for delivering labelled inorganic carbon as \( ^{13}CO_2 \) to algal cultures by bubbling air through a solution of \( H^{13}CO_3^- \) and then into the culture. The method avoids the use of \( ^{13}CO_2 \) labelled gas and the necessary equipment to mix labelled gas with air at near-atmospheric levels. Bubbling precludes the more common approach of adding \( H^{13}CO_3^- \) directly to the media. The method takes advantage of carbonate chemistry and uses a solution of \( H^{13}CO_3^- \) to generate a flux of \( ^{13}CO_2 \) that can be bubbled into a culture. + +## Materials +- Aquarium pump +- Bottle with vent and fill port assembly lid +- Tubing and luer locks +- Bubbling flask assembly (Erlenmeyer flask, foam plug, serological pipet, syringe filter) +- Culture medium & inoculum +- Na\(^{13}CO_3 \) +- H\(_2\)KPO\(_4\) +- H\(_2\)KPO\(_4\) + +## Introduction + +### The Purpose and Problem of Bubbling Cultures +1. Bubbling air into algal cultures stimulates photosynthetic growth by ameliorating diffusion limitation for CO\(_2\). Stable isotope probing (SIP) experiments examining carbon fixation often involve \( ^{13}C \) label introduced directly into the culture medium as a \( H^{13}CO_3^- \) salt. Because dissolved inorganic carbon (DIC) is in equilibrium with atmospheric \( CO_2 \), this labelling method does not work for cultures in an open system. Bubbling accelerates equilibration, causing excess \( HCO_3^- \) to rapidly exit the solution before it can be fixed into biomass. + +### Label Delivery via Bubbler Bottle +2. We developed a SIP method that uses bubbling and carbonate chemistry to release \( ^{13}CO_2 \) gas from a \( H^{13}CO_3^- \) solution, which is then bubbled into algal cultures, resulting in substantial biomass \( ^{13}C \) enrichment. + +3. The rate of label release can be tuned by buffering the solution at different pH values. In lower pH solutions, DIC is more dissolved as CO\(_2\), increasing the rate at which it exchanges into the bubbled air. + +### Figures & Equations + +**Figure 1:** +- **A.** Diagrams of unbubbled vs. bubbled culture formats. +- **B.** Growth curves of C. reinhardtii with and without added bicarbonate. +- **C.** Comparison of \( ^{13}C \) enrichment in unbubbled and bubbled cultures with bicarbonate. + +**Equation 1:** +\[ \text{CO}_2(g) + \text{H}_2O \rightleftharpoons \text{H}_2CO_3 \rightleftharpoons \text{HCO}_3^- + \text{H}^+ \rightleftharpoons \text{CO}_3^{2-} + 2\text{H}^+ \] + +**Figure 2:** +- **A.** Diagram of the bubbler label delivery approach. +- **B.** \( ^{13}C \) enrichment of C. reinhardtii after 24 hours. + +**Figure 3:** +- **A.** Model of CO\(_2\) release from bubbler solution at different pH values. +- **B.** \( ^{13}C \) enrichment over time with buffered solutions. + +## Methodology +1. **Setup:** + - Connect the aquarium pump to the bottle with vent and fill port assembly lid using tubing and luer locks. + - Fill the bottle with 500 mL of 1.5 mM Na\( ^{13}CO_3 \) solution. + - Set up the bubbling flask assembly, ensuring it is properly sealed with the foam plug. + +2. **Bubbling Procedure:** + - Turn on the aquarium pump to bubble air at ~1 L/min through the solution. + - Allow the air to pass through the \( H^{13}CO_3^- \) solution for label exchange. + - Bubble the labeled air into the algal culture for 24 hours. + +3. **Bicarbonate Buffering:** + - Buffer the bubbler solution with phosphate (e.g., H\(_2\)KPO\(_4\)) to desired pH. + - Continuously monitor and adjust pH if necessary to maintain the desired carbonate equilibrium. + +### Notes: +- \( H^{13}CO_3^- \): This is bicarbonate labelled with \( ^{13}C \), a stable isotope used for tracing carbon in biological systems. +- \( ^{13}CO_2 \): Carbon dioxide with the stable isotope \( ^{13}C \), used as a label in metabolic studies. +- **Alternative Methods**: If \( ^{13}C \)-labelled bicarbonate is not available, consider using \( ^{13}CO_2 \) gas directly, though this requires more specialized handling equipment. + +## Conclusion +This method provides a reliable and convenient way to deliver isotopically labelled carbon to algal cultures, facilitating in-depth studies of photosynthesis and carbon fixation dynamics. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/keio-acute-response-antioxidant-rescue-b3nnqmde.md b/markdown-output/keio-acute-response-antioxidant-rescue-b3nnqmde.md new file mode 100644 index 0000000000000000000000000000000000000000..9c5c544caded9fd2aaf39be2d05371c257b62eba --- /dev/null +++ b/markdown-output/keio-acute-response-antioxidant-rescue-b3nnqmde.md @@ -0,0 +1,140 @@ +```markdown +# Goal/Experiment: +The goal of this experiment is to phenotype the acute behavioral response of Caenorhabditis elegans (N2 Bristol) to *E. coli* single-gene deletion mutants (BW25113), in the presence and absence of antioxidants (Trolox, NAC, vitamin C, and resveratrol). + +# Keio Acute Response Antioxidant Rescue + +**Saul Moore1** +1Imperial College London +**DOI:** [dx.doi.org/10.17504/protocols.io.b3nnqmde](dx.doi.org/10.17504/protocols.io.b3nnqmde) +**Behavioural Genomics** + +--- + +## Abstract +Phenotyping the acute behavioral response of *Caenorhabditis elegans* (N2 Bristol) to *E. coli* single-gene deletion mutants (BW25113), in both the presence and absence of antioxidants (Trolox, NAC, vitamin C, and resveratrol). Videos are recorded at 25 fps on the laboratory’s (Hydra) imaging rig, immediately after worms are picked onto imaging plates, for a total of 45 minutes at 25 fps, with blue-light stimulus delivered for 10 seconds in 5-minute intervals throughout the recording. + +## Materials + +### Plates +- 10 x 6-well plates (imaging plates) +- 10 x 60mm Petri plates (maintenance plates) +- 3 x 90mm Petri plates (nursery plates) + +### Reagents and Media + +#### NGM Agar (1 L) +- 3g NaCl (Fisher: 447300010) +- 2.5g Bacto-peptone (BD: 21167, Lot: 8270639) +- 17g Agar (A7002-5KG Lot BCBM1702V) +- 1L H₂O +- Post-Autoclave Additions: + - 25mL KH₂PO₄, pH=6.0 (Sigma: P0662-2.5KG) + - 1mL MgSO₄.7H₂O (1M) (Sigma: M5921-500G) + - 1mL CaCl₂ (1M) (Sigma: C5080-500G) + - 1mL Cholesterol (5mg/mL) (Sigma: C8667-5G) + +#### LB Broth (1 L) +- 25g LB powder (Fisher: BP9723-500) +- 1L H₂O + +#### M9 Minimal Medium (1 L) +- 3g KH₂PO₄ +- 7g Na₂HPO₄.2H₂O (Sigma: 71645-1KG) +- 5g NaCl + +#### Kanamycin +- 50mg/mL Kanamycin (in water, 0.2μm filtered) + +### Antioxidants +- Trolox, NAC, Vitamin C: + - Initial concentration: 1mg/mL + - Final concentration: 200mg/mL in 4mL M9 + - Trolox pH adjustment with NaOH +- Trans-resveratrol: + - Initial concentration: 100mg/mL (200mg in 2mL ethanol, store at -20°C) + - Intermediate concentration: 1mg/mL (200μL in 100mL M9) + - Final concentration: 200mg/mL (4mL M9 sterile + 800μL intermediate solution) +- Dispense 200μL on 4mL NGM agar plate for 10μg/mL concentration. + +**Note:** Goggles must be worn when operating high-power blue-light LEDs on the rig. + +## Protocol + +### Preparing Maintenance Plates (1 day) + +1. **Make 500mL NGM Agar:** Follow [NGM agar protocol](#). + - Time: 2h +2. **Pour NGM Agar:** Pour 20mL NGM agar into each of 10 x 60mm Petri plates (maintenance plates). Allow drying for ~1 hour under hood and store at 4°C until seeding bacterial lawns. + - Time: 1h 30m +3. **Inoculate E. coli Culture:** Inoculate 50mL LB broth with *E. coli* BW25113 in an Erlenmeyer flask. Incubate at 37°C, 200rpm. + - Time: 20h +4. **Store Overnight Culture:** Store at 4°C until use in step 6. +5. **Acclimate Culture:** Bring maintenance plates and culture to room temperature for 30 min. + - Time: 30m +6. **Seed Plates:** Apply 200μL BW25113 culture to each plate under aseptic conditions. +7. **Dry Plates:** Allow plates to dry under hood (~30m to 1h). + +### Preparing Worms (2 days) + +8. **Pick L4 Stage Worms:** Transfer 30 L4-stage *C. elegans* to maintenance plates, incubate at 20°C. + - Time: 1 day +9. **Remove Adults:** After 24h, remove adult worms, leaving eggs to hatch. + - Time: 1 day +10. **Bleach-Synchronize Worms:** Perform egg prep as per [bleach synchronization protocol](#). + - Time: 1 day +11. **Wash L1 Larvae:** Wash with M9 medium, re-feed on bacterial lawns, incubate at 20°C. + - Time: 1 day + +### Preparing Bacteria + +12. **Prepare LB Broth:** 50mL LB broth in Erlenmeyer flasks. +13. **Inoculate BW25113:** From LB plate culture using [liquid bacterial culture protocol](#), add 50mg/mL Kanamycin to BW25113ΔfepD culture. + - Time: Overnight (20h) +14. **Incubate Cultures:** Shaking incubator set at 37°C, 200rpm. +15. **Remove Cultures:** Remove from incubator and acclimate, then seed a second round without Kanamycin. + - Time: Overnight (20h) +16. **Inoculate Overnight Cultures:** Incubate at 37°C, 200rpm. + - Time: Overnight (20h) + +### Preparing Imaging Plates + +18. **Make 250mL NGM Agar:** Wait till agar cools to 55°C before adding salts. +19. **Pour NGM Agar:** Into 10 x 6-well plates, cool under hood. + - Time: 1 day + +20. **Dry Plates:** 4°C storage, dry at room temperature, 30 min. + - Time: 1h +21. **Seed Plates:** Seed with 30μL bacterial culture, BW control and BW25113ΔfepD; + - Time: 7h + 40m growth for lawns, 4°C storage +22. **Dry Plates**: Under hood, 30 mins + +23. **Remove Condensation:** Leave seeded plates for 30 min to remove condensation. + - Time: 30m + +### Adding Antioxidants + +24. **Prepare Antioxidants:** Pipette 200μL antioxidant solution onto each lawn. Dry for 30 min. +25. **Waiting Period:** Leave for at least 2 hours before picking worms onto plates. + +### Hydra Tracking + +26. **Hydra Preparation:** Ensure imaging cave air conditioning is running, dehumidifier water tray empty. +27. **Acclimate Plates:** Leave at room temperature for 30 min. +28. **Pick Age-Matched Worms:** Remove from 20°C incubator. +29. **Transfer Worms:** Pick 10 worms, transfer to each well. +30. **Transport Plates:** Quickly move to imaging cave, place under rigs. Record metadata. +31. **Behavior Tracking:** Track behavior for 45 min using the Hydra imaging rig, applying 10-second blue light stimulus every 5 minutes. + +--- + +## Definitions and Explanations +- **NGM Agar:** Nutrient-rich medium for growing *C. elegans*. +- **LB Broth:** Luria-Bertani broth, nutrient-rich medium for bacterial culture. +- **M9 Minimal Medium:** Minimal medium for bacterial growth, often used for *E. coli*. +- **Kanamycin:** Antibiotic used to select genetically modified bacteria. +- **Trolox, NAC, Vitamin C, Resveratrol:** Antioxidants used to neutralize free radicals and reactive oxygen species. +- **Hydra Imaging Rig:** High-throughput setup for recording and analyzing *C. elegans* behavior. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/keio-acute-response-antioxidant-rescue-round-2-cae7sbhn.md b/markdown-output/keio-acute-response-antioxidant-rescue-round-2-cae7sbhn.md new file mode 100644 index 0000000000000000000000000000000000000000..4aae33fefc6c39aebb6e38f85dfb60ecd08f32b7 --- /dev/null +++ b/markdown-output/keio-acute-response-antioxidant-rescue-round-2-cae7sbhn.md @@ -0,0 +1,113 @@ +```markdown +# Goal/Experiment: + +The goal of this experiment is to phenotype the acute behavioural response of *Caenorhabditis elegans* (N2 Bristol) to *E. coli* single-gene deletion mutants (Keio Collection, BW25113 parent strain) in both the presence and absence of antioxidants (Trolox, NAC, Vitamin C, Resveratrol). + +# Keio Acute Response Antioxidant Rescue - Round 2 + +**Publication Date:** June 01, 2022 +**Author:** Saul Moore, Imperial College London +**DOI:** [10.17504/protocols.io.q26g746w1gwz/v1](https://dx.doi.org/10.17504/protocols.io.q26g746w1gwz/v1) +**Category:** Behavioural Genomics + +### Disclaimer: +> The protocol content here is for informational purposes only and does not constitute legal, medical, clinical, or safety advice. Content added to protocols.io is not peer-reviewed and may not have undergone formal approval. Information presented in this protocol should not substitute for independent professional judgment, advice, diagnosis, or treatment. Any action you take or refrain from taking using or relying upon the information presented here is strictly at your own risk. + +--- + +## Materials and Reagents: + +### For 1L NGM agar: +- 3g NaCl (Fisher 447300010) +- 2.5g Bacto-peptone (BD 21167 Lot 8270639) +- 17g Agar (Sigma A7002-5KG Lot BCBM1702V) +- 1L H₂O +- Salts: + - 25mL KH₂PO₄ (pH = 6.0) (Sigma P0662-2.5KG) + - 1mL MgSO₄·7H₂O (1M) (Sigma M5921-500G) + - 1mL CaCl₂ (1M) (Sigma C5080-500G) + - 1mL Cholesterol (5mg/mL) (Sigma C8667-5G) + +### For 1L LB broth: +- 25g LB powder (Fisher BP9723-500) +- 1L H₂O + +### For 1L M9: +- 3g KH₂PO₄ +- 7g Na₂HPO₄·2H₂O (Sigma 71645-1KG) +- 5g NaCl + +- 50 mg/mL Kanamycin (in water, filter 0.2mm) + +### Antioxidants: +- **Trolox**: Final concentration in 4mL M9: 500 and 1000 mg/mL (Sigma 238813-1G), diluted in DMSO (pH adjusted with NaOH). +- **NAC (N-acetyl cysteine)**: final concentration in 4mL M9: 500 and 1000 mg/mL, diluted in H₂O. +- **Vitamin C**: final concentration in 4mL M9: 500 and 1000 mg/mL, diluted in H₂O. +- **Resveratrol**: final concentration in 4mL M9: 500 and 1000 mg/mL, diluted in H₂O. + +**Note:** Goggles must be worn when operating high-power blue-light LEDs on the rig. + +--- + +## Protocol: + +### Preparing 6-well Plates for Imaging + +1. **Make 1L normal Nematode Growth Media (NGM) agar** (Refer to the materials section for preparation). + *Duration: 2 hours* + +2. **Under a hood, pour 4mL NGM agar into each well of 25 x 6-well plates** (imaging plates) and leave to dry until they lose 3-5% of their weight. Once dry, store at 4°C until seeding bacterial lawns. + *Duration: 1.5 hours, drying: 1-2 hours* + +3. **Inoculate overnight cultures** of *E. coli* BW25113 (Keio Collection parent strain) in 50mL LB broth in separate Erlenmeyer flasks, adding 50 μL Kanamycin. Incubate in a shaking incubator at 37°C (200rpm). + *Duration: 20 hours* + +4. **Remove cultures from incubator** and inoculate a second round of overnight cultures from the first, this time omitting Kanamycin for mutant bacterial culture. + *Duration: Next day* + +5. **Store cultures from the shaking incubator at 4°C** until used for seeding imaging plates. + +6. **Seed the 6-well plates with 30 µL of bacterial culture** in the middle of each well (on imaging plates) using aseptic technique. + *Duration: The following day* + +7. **Pipette 30 µL of bacterial culture into the center** of each well of the plate, taking care not to damage the agar with the pipette tip. Seed half of the 6-well plates with BW25113 control, and the other half with BW25113 ΔfepD lawns. + +8. **Leave the seeded plate to dry for 20 minutes under the hood**, transfer to a 25°C incubator and leave to grow for a further 7 hours and 40 minutes before storing at 4°C until tracking. + *Duration: Max 2 days* + +### Preparing Worms + +10. **Using a platinum pick, gently pick 30 L4-stage N2 Bristol C. elegans** onto each maintenance plate and store in an incubator at 20°C. + *Duration: 1 day (Monday)* + +11. **Remove adult worms after 24 hours**, leaving the eggs behind to hatch into L1 larvae. + *Duration: 1 day (Tuesday)* + +12. **Bleach-synchronize the worms by performing an egg prep** following the standard protocol. + *Duration: 1 day (Friday)* + +13. **On the next day, wash L1 larvae off plate** and re-feed onto BW-seeded maintenance plates. Incubate at 20°C. + *Duration: At around noon the next day (Saturday)* + +### Imaging with Worm Tracking Rig (Hydra) + +14. **Ensure that the imaging cave air conditioning** is turned on and empty the dehumidifier waste water tray as per pre-imaging checklist. + +15. **Remove the seeded plates from 4°C storage** and leave uncovered under a hood for 30 minutes to remove any condensation. + +16. **Prepare the antioxidants** and add exogenously on top of the lawns in each well to yield final concentration of 500 µg/mL antioxidant solution in 4mL NGM agar, leaving under the hood to dry for approx. 30 minutes. + *Duration: At least 1-2 hours prior* + +17. **Using a platinum pick, transfer 10 worms** onto the edge of the bacterial lawn of each well in a single imaging plate at a time. + +18. **Transport the plates to the imaging cave**, ensuring the correct orientation for the recording and the positions of the wells under the cameras, matching the recorded treatment information in the metadata. + +19. **Track worm behavior for 36 minutes at 25 fps**, applying a 10-second blue-light stimulus at the 30th, 31st, and 32nd-minute timepoints. + +--- + +### Citation: +Moore, Saul. (2022). Keio Acute Response Antioxidant Rescue - Round 2. protocols.io. DOI: [10.17504/protocols.io.q26g746w1gwz/v1](https://dx.doi.org/10.17504/protocols.io.q26g746w1gwz/v1). + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/keller-k-optimized-medium-for-culturing-microalgae-b7rnrm5e.md b/markdown-output/keller-k-optimized-medium-for-culturing-microalgae-b7rnrm5e.md new file mode 100644 index 0000000000000000000000000000000000000000..6a065dfb3e7317ae83e24180f41865c2bb686a54 --- /dev/null +++ b/markdown-output/keller-k-optimized-medium-for-culturing-microalgae-b7rnrm5e.md @@ -0,0 +1,209 @@ +```markdown +# Goal/Experiment: +The goal of this experiment is to prepare Keller (K) Optimized Medium for culturing microalgae, specifically designed to grow most phytoplankton species in small eukaryotes and optimized for Ostreococcus tauri. + +## Keller (K) Optimized Medium for Culturing Microalgae + +### Authors +- Roscoff Culture Collection +- Lynn Doran +- Steven J Burgess + +### Affiliations +1. CNRS-Sorbonne Université, Station Biologique, Place G. Tessier 29680 Roscoff FRANCE +2. Realizing Increased Photosynthetic Efficiency (RIPE) +3. University of Illinois at Urbana-Champaign + +**DOI:** [dx.doi.org/10.17504/protocols.io.36wgq76z3vk5/v1](https://dx.doi.org/10.17504/protocols.io.36wgq76z3vk5/v1) + +### Overview +- This protocol requires modifications for diatoms. +- The Keller Optimized Medium includes additions of 17 mM sodium nitrate and 0.37 mM NaH2PO4. +- Medium elements and reagents critical for preparation are discussed below. + +### References +- Guillard, R.R.L. 1975. *Culture of phytoplankton for feeding marine invertebrates*. +- Keller, M.D., Selvin, R.C., Claus, W., & Guillard, R.R.L. 1987. *Media for the culture of oceanic ultraphytoplankton*. +- Sambrook, J. and Russell, D.W. 2001. *Molecular Cloning: A Laboratory Manual*. +- van Ooijen et al. 2012. *Genomic transformation of the picoeukaryote Ostreococcus tauri*. + +### Additional Resources +- [K and K+Si medium, Roscoff Culture Collection](https://roscoff-culture.eu) +- [K Medium, Bigelow National Center for Microalgae and Microbiota](https://ncimbb.rutgers.edu) +- [Making Media by Australian Algae National Culture Collection](https://aanka.algae.org) + +--- + +## Reagents + +| Chemical | Manufacturer and Product ID | +| ------------------------------------------- | -------------------------------------------- | +| 18.2 MΩ water | Milli-Q or Nanopure Water Filtration System | +| Biotin (Vit H) | Sigma B4639 | +| Vitamin B12 | Sigma V6629 | +| CuSO4 · 5H2O | Sigma C8027 | +| MnCl2 · 4H2O | Sigma M3634 | +| CoCl2 · 6H2O | Sigma C8661 | +| ZnSO4 · 7H2O | Sigma Z0251 | +| Na2MoO4 · 2H2O | Sigma M1651 | +| HCl 37% | Sigma 320331 | +| NaOH | Sigma S8045 | +| Tris-Base | Sigma T6066 | +| H2SeO3 | Sigma 211176 | +| β-Glycerophosphate disodium salt hydrate | Sigma G9422 | +| NH4Cl | Sigma A9434 | +| NaNO3 | Sigma S5022 | +| Thiamine • HCl (B1) | Acros Organics 148990100 | +| FeCl3 · 6H2O | Sigma 236489 | +| Na2EDTA • 2H2O | Acros Organics 147855000 | +| NaCl | Sigma S7653 | +| KCl | Sigma P5405 | +| MgCl2.6H2O | Fisher BP214 | +| CaCl2.2H2O | Phytotech Labs C135 | +| MgSO4.7H2O | Sigma 230391 | +| NaHCO3 | Sigma S6014 | + +--- + +## Equipment + +- Media Storage Bottle, 1 L +- Media Storage Bottle, 100 mL +- Graduated cylinder, 1 L +- Graduated cylinder, 100 mL +- Graduated cylinder, 5 mL +- Centrifuge Tubes, Sterile, Polypropylene, Globe Scientific +- Centrifuge Tubes, Sterile, Polypropylene, Corning +- Microcentrifuge tube, 0.5 mL +- Pipette, 1-10 µl +- Pipette, 100 µl +- Pipette, 1000 µl +- Pipette tips, 10 µl +- Weigh paper +- Spatula, stainless steel or scoopula +- Microanalytical balance +- Analytical balance, Mettler-Toledo +- 0.22 µM filters +- Autoclave +- Water bath, hot plate with beaker of water +- pH Meter +- Chemical fume hood +- Laminar Flow Hood + +--- + +## Safety Information + +- Use both chemical fume hoods and biological safety cabinets. +- Review training for autoclaves due to high burn risks. +- Follow relevant laboratory safety protocols. +- Properly dispose of chemical waste as per guidelines. + +## Protocols + +### 1. Prepare Artificial Sea Water (ASW) + +1. **Fill** a 1 L media storage bottle two-thirds full with 18.2 MΩ water (Milli-Q or Nanopure). +2. **Weigh** the following chemicals and **add** them to the media storage bottle: + + | Chemical | Amount | + | ----------------------------------------- | -------- | + | Sodium chloride (NaCl) | 24.55 g | + | Potassium chloride (KCl) | 0.75 g | + | Magnesium chloride hexahydrate (MgCl2.6H2O) | 4.07 g | + | Calcium chloride dihydrate (CaCl2.2H2O) | 1.47 g | + | Magnesium sulfate heptahydrate (MgSO4.7H2O) | 6.04 g | + +3. **Swirl** the bottle until all chemicals have dissolved. +4. **Weigh** Sodium bicarbonate (NaHCO3) 0.21 g and **add** to the bottle, swirl until dissolved. +5. **Bring** to 1 L volume with 18.2 MΩ H2O, return to the media storage bottle. +6. **Autoclave** the artificial sea water before use if required. +7. **Label** with analyst name, date, and sterility status. + +### 2. Prepare Trace Metal Solutions + +1. **Prepare** each individual stock solution as specified below: + + | Chemical | Volume prepared (mL) | Weight of chemical (mg) | Concentration (g/L) | + | ----------------------- | -------------------- | ----------------------- | ------------------- | + | Na2MoO4 · 2H2O | 4 | 25.2 | 6.3 | + | ZnSO4 · 7H2O | 1 | 22.0 | 22 | + | CoCl2· 6H2O | 2 | 20.0 | 10 | + | MnCl2 · 4H2O | 0.2 | 36.0 | 180 | + | CuSO4· 5H2O | 2 | 19.6 | 9.8 | + +2. **Prepare** the trace metal working stock solution: + + - **Weigh** 0.29 g EDTA Disodium Salt Dihydrate (Na2EDTA.2H2O) + - **Add** to the trace metal working stock solution and dissolve completely. + - **Weigh** 20 mg hexahydrated ferric chloride (FeCl3.6H2O) and dissolve. + - **Add appropriate volumes** of each trace metal stock solution: + + | Compound | Volume | + | ---------------------------------------------------- | ------ | + | Sodium Molybdate Dihydrate (Na2MoO4.2H2O) Solution | 7 µl | + | Zinc Sulfate Heptahydrate (ZnSO4.7H2O) Solution | 7 µl | + | Cobalt Chloride Hexahydrate (CoCl2.6H2O) Solution | 7 µl | + | Manganese (II) chloride, tetrahydrate (MnCl2.4H2O) | 7 µl | + | Copper(II) sulfate pentahydrate (CuSO4.5H2O) | 7 µl | + +4. **Mix** the solution thoroughly. +5. **Label** with name, date. +6. **Store** in aliquots at -20 °C. + +### 3. Prepare f/2 Vitamin Solutions + +1. **Prepare** individual stock solutions: + + | Chemical | Volume prepared (mL) | Weight of chemical (mg) | Concentration (g/L) | + | -------------------- | -------------------- | ----------------------- | ------------------- | + | Biotin | 100 | 10.0 | 0.1 | + | Vitamin B12 | 20 | 20.0 | 1 | + +2. **Mix** each solution as described. +3. **Combine** f/2 vitamin stock solutions: + + | Chemical | Volume | + | ----------------------------- | ------ | + | Vitamin B12 Solution (1 g/L) | 100 µl | + | Biotin Solution (0.1 g/L) | 1000 µl | + +4. **Mix** solution thoroughly and label with date and analyst name. + +### 4. Prepare Keller Medium in ASW + +1. **Prepare** each stock solution: + + | Chemical | Total volume (mL) | Solid weight (mg) | Concentration (g/L) | + | --------------------- | ----------------- | ----------------- | ------------------- | + | NH4Cl | 8 | 21.4 | 2.68 | + | β-Glycerophosphate | 10 | 21.6 | 2.16 | + | H2SeO3 | 1000 | 1.29 | 0.00129 | + | Tris-base (pH 7.2) | 1 | 121.1 | 121.1 | + +2. **Mix** and prepare the Keller medium solution: + 1. **Measure** 993.5 mL artificial sea water. + 2. **Add** the stock solutions: + + | Stock Solution | Volume | + | ------------------------- | ------ | + | NH4Cl (2.68 g/L) | 1 mL | + | β-Glycerophosphate (2.16 g/L) | 1 mL | + | H2SeO3 (0.00129 g/L) | 1 mL | + | Tris-base (pH 7.2) (121.1 g/L)| 1 mL | + | K trace metal solution | 1 mL | + | f/2 vitamin solution | 0.5 mL | + | NaNO3 (17 mM final) | 1.44 g | + | NaH2PO4 stock | 1 mL | + +3. **Adjust** pH to 8.1-8.2. +4. **Filter sterilize** the Keller Medium. +5. **Label** with name, date, and sterility. +6. **Store** in the dark at 4°C for up to 2 weeks. + +--- + +End of protocol. + +Endofoutput +``` \ No newline at end of file diff --git a/markdown-output/lab-protocol-for-assessing-the-spectral-dependenci-btxanpie.md b/markdown-output/lab-protocol-for-assessing-the-spectral-dependenci-btxanpie.md new file mode 100644 index 0000000000000000000000000000000000000000..6e1b0ed78873c0c3c335e0969ef3ae7333a29201 --- /dev/null +++ b/markdown-output/lab-protocol-for-assessing-the-spectral-dependenci-btxanpie.md @@ -0,0 +1,143 @@ +```markdown +# Goal/Experiment: +This experiment aims to establish a fundamental methodology to explore the spectral dependencies of the Cortisol Awakening Response (CAR) consistently and efficiently. The research creates a basis for future investigations into CAR's spectral dependencies and potentially related outcome measures of emotional state and cognitive functioning. + +## Lab Protocol for Assessing the Spectral Dependencies of the Cortisol Awakening Response (CAR) and Potentially Related Outcome Measures for Morning Light Exposure + +### Authors: +- Sebastian Babilon¹² +- Paul Myland¹ +- Julian Klabes¹ +- Joel Simon¹ +- Tran Quoc Khanh¹ + +¹ Laboratory of Lighting Technology, Technical University of Darmstadt, Hochschulstr. 4a, 64289 Darmstadt, Germany +² Light and Health Research Center, Department of Population Health Science and Policy, Icahn School of Medicine at Mount Sinai, One Gustave L. Levy Place, New York, NY 10029, USA +Sebastian Babilon: babilon@lichttechnik.tu-darmstadt.de + +[DOI: 10.17504/protocols.io.btxanpie](https://dx.doi.org/10.17504/protocols.io.btxanpie) +[DOI: 10.1371/journal.pone.0267659](https://doi.org/10.1371/journal.pone.0267659) + +--- + +## Introduction + +Cortisol secretion plays a crucial role in human circadian regulation. The cortisol awakening response (CAR) appears as a recurring sharp increase in cortisol concentration within the first hour after awakening, influenced by environmental light conditions. This research protocol intends to establish consistent methodology for studying the spectral dependencies of CAR. + +--- + +## Experimental Setup and Calibration + +### Equipment and Materials: +1. **Integrating sphere:** Barium sulfate coated, diameter of 1 m + - Function: Ensures homogeneous illumination of the visual field. + +2. **LED light source:** Peltier-cooled, temperature-stabilized with monochromatic modules + - Vendor: Explore vendors for Peltier-cooled LED sources (e.g., ThorLabs). + +3. **Filter wheel with interference filters.** + +4. **Calibrated silicon photodiode** (e.g., SM05PD1A, Thorlabs; PM100USB Power Meter, Thorlabs) + - Function: Measures and calibrates light intensity. + +5. **Wavelength-calibrated fiber optic spectrometer** (e.g., CAS 140 B, Instrument Systems GmbH) + - Function: Ensures wavelength accuracy of light stimulus. + +6. **CIE standard CIE S 026/E:2018** for α-opic calculations. + +--- + +## Participant Selection, Data Acquisition, and Compliance + +### Before and During Study Sessions + +1. **Pittsburgh Sleep Quality Index (PSQI):** Assesses sleep quality. + +2. **Munich Chronotype Questionnaire (MCTQ):** Determines personal chronotype. + +3. **36-item Short-Form Health Survey (SF-36):** Measures overall health. + +4. **10-item Perceived Stress Scale (PSS-10):** Assesses stress levels. + +5. **Color Vision Tests:** + - Ishihara's Tests for Color Deficiency. + - Standard Pseudoisochromatic Plates Part II. + - Farnsworth-Munsell D-15 Color Vision Test. + +6. **Self-Reported Sleep Diary:** Tracks sleep patterns. + +7. **Actigraphy/Wearable Sleep Tracking:** e.g., Garmin Forerunner 945. + +8. **Sleep Quality (SF-A/R), Auditory Reaction Time, and other cognitive functions.** + +9. **Cortisol Collection:** + - Sarstedt Salivette® Cortisol Collection System. + - ELISA kits for cortisol determination. + - Fridge for sample storage. + +10. **Light Blocking Glasses:** Prevent light exposure to ensure accurate sleep data. + +### Subject Eligibility + +Candidates are eligible if: +- They do not exceed a PSQI score of 5. +- Avoid extreme morning or evening chronotypes as per MCTQ. +- Maintain scores within the normative range for SF-36. +- Avoid high stress (PSS-10 above normative mean). +- Are free from color vision deficiencies. +- Exclude factors like smoking, medication, excessive alcohol, or caffeine intake. + +### Subject Compliance + +Control adherence to sleep-wake schedule: +- **Target sleep time:** 11:00 pm ± 1 h. +- **Target wake-up:** 07:00 am ± 1 h. +- **Alcohol intake:** < 60g/week. +- **Caffeine intake:** < 200 mg/day. + +### Study Design and Data Collection + +#### Environmental Conditions +- **Temperature:** 22.0°C +- **Humidity:** 45% + +#### Data Collection Procedure + +- **Evening Prior Setup:** + - Arrive by 7:00 pm; lights dimmed to < 5 lx; electronic devices prohibited after 8:00 pm. Sleep at 10:00 pm. + +- **Morning Session:** + - Wake-up at 5:55 am; first saliva sample collected immediately. + - Two-hour light exposure post-wake-up, with 20-minute interval saliva collections. + - Assessments include cognitive tasks and subjective measures (sleepiness, mood state, etc.). + +### Calibration of Light Stimuli + +1. **Photodiode Calibration:** Measures irradiance at eye position. +2. **Spectrometer Calibration:** Monitors spectral composition. +3. **Calculate Photon Density:** Using formulae for precise light calibration (adhering to CIE standards). + +### Saliva Sampling and Analysis +- **Collection Tools:** Sarstedt Salivette® Cortisol Collection System. +- **Storage:** Immediate storage at 4°C, prompt ELISA analysis. + +--- + +## Citation: +Sebastian Babilon, Paul Myland, Julian Klabes, Joel Simon, Tran Quoc Khanh. 2022. Lab protocol for assessing the spectral dependencies of the Cortisol Awakening Response (CAR) and potentially related outcome measures for morning light exposure. protocols.io. DOI: [10.17504/protocols.io.btxanpie](https://dx.doi.org/10.17504/protocols.io.btxanpie). + +--- + +## Supplemental Information + +- **Alternatives:** + - **LED Light Source:** Explore custom fabrication if commercial options are limited. + - **Interference Filters:** Utilize optical engineering firms for custom wavelength needs. + +- **Terms:** + - **Integrating Sphere:** A device for ensuring uniform light distribution. + - **Peltier-Cooled:** Refers to the thermoelectric cooling of light sources for stability. + - **α-Opic:** Describes light’s non-visual impact on the human biological clock. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/laboratory-protocols-for-ancient-and-modern-dental-bvuan6se.md b/markdown-output/laboratory-protocols-for-ancient-and-modern-dental-bvuan6se.md new file mode 100644 index 0000000000000000000000000000000000000000..c91c8c67f4c7d7ab21e481056f4b1f3db5bc49f2 --- /dev/null +++ b/markdown-output/laboratory-protocols-for-ancient-and-modern-dental-bvuan6se.md @@ -0,0 +1,96 @@ +```markdown +# Goal/Experiment: +The goal of this document is to describe laboratory procedures for the sampling, extraction, library construction, and preparation for Illumina sequencing of ancient and modern dental calculus samples. + +# Laboratory Protocols for Ancient and Modern Dental Calculus DNA Processing (Fellows Yates et al. 2021) V.3 + +**Authors:** James A Fellows Yates, Irina Velsko, Franziska Aron, Courtney Hofman, Christina Warinner +**Institutions:** +- Max Planck Institute for the Science of Human History +- Laboratories of Molecular Anthropology and Microbiome Research, University of Oklahoma + +**DOI:** [dx.doi.org/10.17504/protocols.io.bvuan6se](https://dx.doi.org/10.17504/protocols.io.bvuan6se) + +## Abstract +Collection of protocols used for Fellows Yates et al. "The evolution and changing ecology of the hominid primate oral microbiome". Bioinformatics analysis can be found on GitHub at [GitHub Repository](https://github.com/ify133/Anthropoid_Calculus_Microbiome_Evolution/). + +This collection describes the laboratory procedures used for sampling, (ancient) DNA extraction, library construction, and preparation for Illumina sequencing of ancient and modern dental calculus samples. + +## Table of Contents +1. [Sampling](#sampling) + - Dental calculus Field-Sampling Protocol (Warinner Version) + - Dental Calculus Field-Sampling Protocol (Sabin version) +2. [Extraction](#extraction) + - Ancient DNA Extraction from Dental Calculus with Consolidant Removal + - DNA Extraction from Modern Dental Calculus +3. [Library Preparation](#library-preparation) + - Non-UDG treated double-stranded DNA library preparation for Illumina sequencing of ancient dental calculus + - (Non-UDG treated) double-stranded modern dental calculus DNA library preparation for Illumina sequencing + - Full-UDG treated double-stranded ancient DNA library preparation for Illumina sequencing of ancient dental calculus (reduced input DNA) +4. [Indexing](#indexing) + - Illumina double-stranded DNA dual indexing for ancient DNA +5. [Preparation for Sequencing](#preparation-for-sequencing) + - Amplification and Pooling + +## Guidelines + +### Working in an Ancient DNA Laboratory +Some of the protocols in this collection **require** working in dedicated ancient DNA laboratories to limit modern DNA contamination. + +- All steps of the protocol should take place in a clean room facility specifically designed for ancient DNA. +- The researcher performing lab work should wear correspondingly suitable lab-wear, such as: + - full-body suit with hood (e.g., Tyvek) + - hairnet + - face mask + - two pairs of clean gloves + - clean shoes + - protective glasses +- Sample processing should be carried out in separated work benches with integrated UV irradiation (e.g., Dead Air PCR work bench). +- Surfaces and equipment should be regularly decontaminated with e.g. bleach solution or Thermofisher's DNA AWAY (or similar) and irradiated with UV. + +Please refer to: +*Llamas, B. et al., 2017. From the field to the laboratory: Controlling DNA contamination in human ancient DNA research in the high-throughput sequencing era. STAR: Science & Technology of Archaeological Research, 3(1), pp.1–14. Available at: [https://doi.org/10.1080/20548923.2016.1258824](https://doi.org/10.1080/20548923.2016.1258824).* + +## Protocols + +### Sampling +- **Dental calculus Field-Sampling Protocol (Warinner Version)** +- **Dental Calculus Field-Sampling Protocol (Sabin version)** + +### Extraction +- **Ancient DNA Extraction from Dental Calculus with Consolidant Removal** +- **DNA Extraction from Modern Dental Calculus** + +### Library Preparation +- **Non-UDG treated double-stranded DNA library preparation for Illumina sequencing of ancient dental calculus** +- **(Non-UDG treated) double-stranded modern dental calculus DNA library preparation for Illumina sequencing** +- **Full-UDG treated double-stranded ancient DNA library preparation for Illumina sequencing of ancient dental calculus (reduced input DNA)** + +### Indexing +- **Illumina double-stranded DNA dual indexing for ancient DNA** + +### Preparation for Sequencing +- **Amplification and Pooling** + +## Collection Citation +James A Fellows Yates, Irina Velsko, Franziska Aron, Courtney Hofman, Christina Warinner 2021. Laboratory Protocols for Ancient and Modern Dental Calculus DNA Processing (Fellows Yates et al. 2021). **protocols.io** [https://dx.doi.org/10.17504/protocols.io.bvuan6se](https://dx.doi.org/10.17504/protocols.io.bvuan6se) + +## Manuscript Citation +Fellows Yates, J. A. et al. (2021) 'The evolution and changing ecology of the African hominid oral microbiome', *Proceedings of the National Academy of Sciences of the United States of America*, 118(20), p. e2021655118. doi:10.1073/pnas.2021655118. + +## Keywords +ancient DNA, palaeogenetics, dental calculus, microbiome, oral microbiome, oral, tooth, DNA, Illumina, extraction, sampling, library construction + +## License +This is an open access collection distributed under the terms of the [Creative Commons Attribution License](https://creativecommons.org/licenses/by/4.0/) which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. + +## Image Attribution +James Fellows Yates + +**Created:** Jun 15, 2021 +**Last Modified:** Jun 15, 2021 + +**Collection Integer ID:** 50786 + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/ldm-protocol-for-estimating-plasmid-conjugation-ra-cbwispce.md b/markdown-output/ldm-protocol-for-estimating-plasmid-conjugation-ra-cbwispce.md new file mode 100644 index 0000000000000000000000000000000000000000..53ea5bd3b6eb476c2ca7a66fda3d0eb7f25c9948 --- /dev/null +++ b/markdown-output/ldm-protocol-for-estimating-plasmid-conjugation-ra-cbwispce.md @@ -0,0 +1,142 @@ +```markdown +# Goal/Experiment: +To implement the LDM (Likelihood Decay Method) approach for estimating plasmid conjugation rates. + +# LDM Protocol for Estimating Plasmid Conjugation Rates V.3 + +**Authors**: Olivia Kosterlitz, Claire Wate, Adamaris Muñiz Tirado, Benjamin Kerr +**Affiliation**: University of Washington +**Date**: Jun 24, 2022 +**DOI**: [10.17504/protocols.io.e6nvwk812vmk/v3](https://dx.doi.org/10.17504/protocols.io.e6nvwk812vmk/v3) +**License**: This protocol is distributed under the terms of the [Creative Commons Attribution License](https://creativecommons.org/licenses/by/4.0/). + +## Abstract + +This protocol implements the LDM approach to estimate plasmid conjugation rates. It is designed for proper implementation by explicitly verifying that modeling assumptions are met. The protocol involves five streamlined phases to gather information necessary for a proper LDM conjugation assay execution. + +Access the latest updated version in the corresponding [GitHub repository](https://github.com). + +## Phases Overview + +### Phase 1: Minimum Inhibitory Concentration (MIC) Assay + +**Goal**: Determine the concentration of selective agents that inhibit donor and recipient growth while permitting transconjugant growth. + +1. **Prepare Bacterial Cultures** + + - Start from freezer stocks. + - Supplement with selective agents. + - Incubate overnight. + +2. **Create a Two-Fold Gradient of the Selective Medium** + + - Add 500 μL of growth medium to rows 2-8 (skip the first row) of a 96-well plate. + - Add 1 mL of medium with transconjugant-selecting antibiotics to row 1. + - Serially transfer 500 μL down the rows. Mix by pipetting up and down. + +3. **Inoculate the Gradient** + + - Dilute cultures 100-fold in growth medium. + - Add 500 μL of diluted cultures to wells. + - Incubate overnight. + +4. **Identify Viable Concentrations** + + - Record turbidity. + - Analyze to determine the MIC for transconjugant selection. + +### Phase 2: Extinction Probability Assays + +**Goal**: Check if focal cell type successfully establishes a lineage under selective conditions. + +1. **Prepare Bacterial Cultures** + - Refer to Phase 1, Step #1. + +2. **Prepare Selective Media** + + - Two deep-well plates: antibiotic-free and transconjugant-selecting. + - Agar plates for each cell type: donor, recipient, and transconjugant. + +3. **Inoculate the Cultures** + + - Inoculate with 50 μL diluted transconjugant culture into deep-well plates. + - Inoculate with 100 μL diluted culture into agar plates. + - Incubate overnight. + +4. **Determine Extinction Probabilities** + + - Record turbidity. + - Calculate extinction probabilities. + +### Phase 3: Growth Rate Assay + +**Goal**: Determine incubation times ensuring cultures enter exponential growth before the conjugation assay. + +1. **Prepare Bacterial Cultures** + - Refer to Phase 1, Step #1. + +2. **Dilute and Inoculate Cultures** + + - Dilute 10,000-fold. + - Add 250 μL to wells. + - Inoculate at 1-hour intervals. + +3. **Calculate Growth Rates** + + - Record colony counts. + - Calculate growth rates using density information. + +### Phase 4: Incubation Time and Initial Densities + +**Goal**: Identify incubation time and initial densities ensuring conjugation event probability between 0 and 1. + +1. **Prepare Bacterial Cultures** + - Refer to Phase 1, Step #1. + +2. **Isolate Cultures in Exponential Growth** + + - Follow dilution and incubation guidelines. + +3. **Dilute Cultures for Factorial Treatment** + + - Use deep-well plates for different time points and initial densities. + +4. **Create Relevant Controls** + - Plate donor, recipient, and transconjugant cultures for controls. + +### Phase 5: LDM Conjugation Assay + +**Goal**: Gather experimental estimates for LDM equation variables to calculate the conjugation rate. + +1. **Prepare Bacterial Cultures** + - Refer to Phase 1, Step #1 and Phase 4, Step #14. + +2. **Dilute and Inoculate Cultures** + + - Follow guidelines for creating co-cultures and controls. + - Plate and incubate as per instructions. + +3. **Calculate LDM Estimate** + + - Record colonies and turbidities. + - Calculate the conjugation rate based on gathered data and LDM equation. + +## Terms and Reagents + +### Terms: + +- **MIC (Minimum Inhibitory Concentration)**: The lowest concentration of an antimicrobial that will inhibit the visible growth of a microorganism. +- **Transconjugant**: A bacterial cell that has received genetic material via conjugation. + +### Reagents: + +- **Growth Medium**: Nutrient-rich solution used to support the growth of microorganisms. +- **Selective Agents**: Substances like antibiotics used to select for or against certain microorganisms. + +## Alternative Methods and Supplies + +- If selective agents are difficult to source, consider using combinations of commonly available antibiotics known to select similar resistance markers. +- For MIC determination, alternatives like broth dilution methods can be used if gradient setups are not feasible. + +`endofoutput` +``` \ No newline at end of file diff --git a/markdown-output/lentiviral-titration-for-early-post-mitotic-dopami-c9wvz7e6.md b/markdown-output/lentiviral-titration-for-early-post-mitotic-dopami-c9wvz7e6.md new file mode 100644 index 0000000000000000000000000000000000000000..0a677facc5c6b2b5378db52cbd074c9a325f8ad7 --- /dev/null +++ b/markdown-output/lentiviral-titration-for-early-post-mitotic-dopami-c9wvz7e6.md @@ -0,0 +1,238 @@ +```markdown +# Goal/Experiment: +Lentiviral Titration for Early Post-Mitotic Dopaminergic Neurons + +## Title: +Lentiviral Titration for Early Post-Mitotic Dopaminergic Neurons + +**Authors:** +Renuka Ravi Gupta¹²³⁴, Nona Farbehi¹²³⁵, hendersa³⁶, Vikram Khurana³⁷, Gist Croft³⁸, Lorenz Studer³⁶, Joseph Powell¹²³⁴ + +**Affiliations:** +1. Garvan Institute of Medical Research, Sydney, NSW 2010, Australia +2. Garvan Weizmann Center for Cellular Genomics, Garvan Institute of Medical Research, Sydney, NSW 2010, Australia +3. Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD 20815, USA +4. School of Medical Science, University of New South Wales, Sydney, NSW, 2052, Australia +5. Graduate School of Biomedical Engineering, University of New South Wales, Sydney, NSW, 2052, Australia +6. The Centre for Stem Cell Biology, Developmental Biology Program, Sloan Kettering Institute for Cancer Research, New York, NY, USA +7. Ann Romney Center for Neurologic Diseases, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA 02115, USA +8. The New York Stem Cell Foundation Research Institute, New York, NY, USA + +**Protocol Citation:** +Renuka Ravi Gupta, Nona Farbehi, hendersa, Vikram Khurana, Gist Croft, Lorenz Studer, Joseph Powell 2024. LENTIVIRAL TITRATION FOR EARLY POST- MITOTIC DOPAMINERGIC NEURONS. protocols.io + +**License:** +This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited + +**Protocol status:** +In development - We are still developing and optimizing this protocol + +**Created:** +Feb 29, 2024 + +**Last Modified:** +Mar 01, 2024 + +## Abstract +iPSCs-derived neurons are particularly challenging cells for genetic screening. Hence we develop a protocol for lentiviral titration of mDA neurons where mDA neuronal cell suspension combined with concentrated lentiviral supernatant are added at different dilutions in 48 well plates. Subsequent centrifugation (spinfection) was performed to achieve high efficiency transduction. The transduction efficiency is determined as a percentage of BFP-positive cells through FACS (Fluorescence Activated Cell Sorting). + +## Materials + +| MATERIAL | COMPANY | CATALOG | +|----------------------------------------|------------------------------|--------------| +| 48 well TC treated plate | Falcon | 353078 | +| 15 ml polypropylene centrifuge tubes | Falcon | 352096 | +| 5 ml serological pipettes | Corning | 4487 | +| 10 ml serological pipettes | Corning | 4488 | +| DNA Low-bind tubes 1.5 ml | Eppendorf | 022431021 | +| P1000 tip | Neptune | BT1250 | +| FBS | Bovogen | 2008A | +| DPBS | ThermoFisher Scientific | 14040133 | +| Hank’s Balanced Salt Solution (HBSS) | ThermoFisher Scientific | 14175-095 | +| Neuronal Isolation Neuronal Enzyme with Papain | ThermoFisher Scientific | 88285 | +| Neurobasal Media | ThermoFisher Scientific | 21103049 | +| B27 w/o vit A | ThermoFisher Scientific | 12587-010 | +| L-glutamine | ThermoFisher Scientific | L3000015 | +| Pen-Strep | ThermoFisher Scientific | 12260 | +| BDNF (Brain Derived Neurotrophic Factor) | R&D | 248-BDB | +| GDNF (Glial Cell line Derived Neurotrophic Factor) | Peprotech | 450-10 | +| Ascorbic Acid | Sigma | 4034 | +| cAMP | Sigma | D0627 | +| TGF-B (Transforming Growth Factor - b) | R&D | 243-B3 | +| DAPT | Tocris | 2634 | +| Polyornithine (PO) | Sigma | P3655 | +| Cultrex Mouse Laminin I | R&D | 3400-010-1 | +| Fibronectin | Corning | FAL356008 | + +## Reagent Composition + +### Media 2 + +| REAGENT | VOLUME IN ML | +|-----------------------------|------------------| +| Neurobasal Media | 480 | +| B27 without Vit A (10x) | 10 | +| Pen-Strep | 5 | +| L-Glutamine | 5 | + +### Maturation Media (MM) + +| REAGENT | STOCK SOLUTION | WORKING SOLUTION | VOLUME IN µl | +|-------------------------------|-----------------|------------------|----------------| +| Media 2 | - | - | 24796 | +| BDNF | 10 µg/ml | 20 ng/ml | 50 | +| GDNF | 10 µg/ml | 20 ng/ml | 50 | +| AA | 100 mM | 200 µM | 50 | +| cAMP | 100 mM | 200 µM | 50 | +| DAPT | 100 mM | 10 µM | 2.5 | +| TGF-B | 20 µg/ml | 1 ng/ml | 1.25 | + +### FACS Buffer (PBS + 2% FBS) + +| REAGENT | VOLUME in mL | +|---------|---------------------| +| PBS | 49 | +| FBS | 1 | + +## Before Start Instructions + +hESC CRISPRi dCAS9 are differentiated to D25 according to the following protocol: + +**Citation:** +Tae Wan Kim, Jinghua Piao, So Yeon Koo, Sonja Kriks, Sun Young Chung, Doron Betel, Nicholas D. Socci, Se Joon Choi, Susan Zabierowski, Brittany N. Dubose, Ellen J. Hill, Eugene V. Mosharov, Stefan Irion, Mark J. Tomishima, Viviane Tabar, Lorenz Studer. Biphasic Activation of WNT Signaling Facilitates the Derivation of Midbrain Dopamine Neurons from hESCs for Translational Use. protocols.io. +**Link:** [Biphasic Activation of WNT Signaling](https://protocols.io/view/biphasic-activation-of-wnt-signaling-facilitates-t-bu7znzp6) + +At D25, the cells were sorted by MACS sorting to obtain pure population dopaminergic mDA neurons. + +**Citation:** +Tae Wan Kim. Dopamine neuron enrichment using MACS. protocols.io. +**Link:** [Dopamine Neuron Enrichment](https://protocols.io/view/dopamine-neuron-enrichment-using-macs-cyrfxv3n) + +## Procedures + +### Day -1: Coating Wells with Poly - L Ornithine (PO) + +1. Coat 500 µl per well in a 48-well plate with 15 µg/ml PO in DPBS. + +2. Incubate the plate overnight at 37°C with 5% CO₂ and 20.9% O₂. + +### Day 0: Coating Wells with Laminin and Fibronectin + +3. Thaw Fibronectin and Laminin on ice. + +4. Aspirate 250 µl of coated PO from each well of the 48-well plate and wash the wells with 1 ml of DPBS. Repeat two more times for a total of 3x DPBS washes. + + **Note:** + Do not let the wells dry out. + +5. Aspirate DPBS and add 500 µl of 2 µg/ml Fibronectin and 1 µg/ml Laminin in cold DPBS. + +### Day 1: Titration of D25 Midbrain Dopaminergic Neurons (mDA Neurons) with Lentivirus + +6. Thaw the viral stock on ice. + +7. Prepare 15 ml tubes with 200,000 D25 pure population mDA neuronal suspension (CD49 neg) with concentrated lentiviral supernatants in serial dilutions in the 48 well plate in the following manner. + + **Note:** + Make sure to mix well by gentle pipetting. Change tips after making up each dilution. Titration was done in triplicates. + + | DILUTION | Cell+MM Media | Series Dilutions (µl) | + |-----------------|---------------|-----------------------| + | 1/2 | 600 µl | 600 µl | + | 1/4 | 600 µl | 600 µl | + | 1/8 | 600 µl | 600 µl | + | 1/16 | 600 µl | 600 µl | + | 1/32 | 600 µl | 600 µl | + | 1/64 | 600 µl | 600 µl | + | 1/128 | 600 µl | 600 µl | + | 1/256 | 600 µl | 600 µl | + | 1/512 | 600 µl | 600 µl | + | 1/1024 | 600 µl | 600 µl | + +8. Aspirate the fibronectin/laminin coating and proceed immediately to the next step. + +9. Add 200 µl/well for each viral dilution with the cells. + +10. To increase transduction efficiency, centrifuge the plate at 300g for 20 minutes at 25°C. + +11. Incubate the cells at 37°C with 5% CO₂ and 20.9% O₂ for 16-18 hours. + +### Day 2: Replace Media + +12. Aspirate the viral supernatant media gently and immediately add maturation media. + +13. Return the plate back to the incubator. + +### Day 4: FACS Analysis + +14. Aspirate the spent media. + +15. Wash the cells 10 times with DPBS to remove the viral particles from the mDA neurons. + +16. **Note:** + The neurons are sturdy and do not lift off during the washes. However, look under the microscope during the washes to avoid the neurons lifting off. + +17. Add 100 µl HBSS + papain and incubate the neurons for 45 minutes in the incubator. + + **Note:** + Ideally the neurons should dissociate as single cells. + +18. Neutralize the papain with maturation media and collect the cells into 1.5 ml eppendorf tubes. + + **Note:** + If the neurons are still present as a sheet or have clumps, use a P1000 tip, pipette the cells up and down to break them into single cell suspension. + +19. Centrifuge the cells at 300 g for 5 minutes. + +20. Aspirate the spent media gently without disturbing the pellet. + +21. Resuspend the cells in 300 µl of FACS buffer. + +22. Transfer the cells with the FACS buffer into FACS tubes. + +23. Analyze the cells through flow cytometry to determine BFP positive cells. + +24. The MOI for CRISPRi screen was quantified as the 10%-30% of BFP-positive cells to ensure one gRNA enters one cell. + +## Calculating the Lentiviral Titer in TU/mL + +### Method 1: Calculating Using Dilution Factor + +T = (NXF X D)/Vt + +Where +T = Titer, (TU/mL) +N = Number of cells transduced +F = Fraction of cells with fluorescence +D = Dilution Factor +Vt = Transduction volume in ml + +### Method 2: Calculating Using Volume of Virus + +T = (NXF)/Vv + +Where +T = Titer, (TU/mL) +N = Number of cells transduced +F = Fraction of cells with fluorescence +Vv = Virus volume + +Detailed protocol for lentiviral titration for the virus can be found in the following link: [Addgene Protocol](https://www.addgene.org/protocols/fluorescence-titering-assay/) + +## Calculating Virus Volume for Required MOI + +26. For Perturb seq, to restrict the viral integration in such a way that one virus infects one cell, we keep the MOI between 0.1-0.3. + +27. Calculating the virus volume, for MOI (0.1-0.3). + + MOI = (T x Vv)/N + +Where +T = Titer, (TU/mL) +N = Number of cells transduced +Vv = Virus volume + +Detailed protocol for calculating MOI can be found in the following link: [ABM Protocol](https://info.abmgood.com/multiplicity-of-infection-moi) + +## endofoutput +``` \ No newline at end of file diff --git a/markdown-output/library-bottlenecking-protocol-basic-microbial-cul-dewn3fde.md b/markdown-output/library-bottlenecking-protocol-basic-microbial-cul-dewn3fde.md new file mode 100644 index 0000000000000000000000000000000000000000..63cd7639dd15c6393e9239b8183e63d01091f7a4 --- /dev/null +++ b/markdown-output/library-bottlenecking-protocol-basic-microbial-cul-dewn3fde.md @@ -0,0 +1,123 @@ +```markdown +# Goal/Experiment: +The goal of this experiment is to bottleneck a microbial library using basic microbial culture equipment. + +# Library Bottlenecking Protocol (Basic Microbial Culture Equipment) V.2 + +## DOI +[dx.doi.org/10.17504/protocols.io.x54v9227pl3e/v2](https://dx.doi.org/10.17504/protocols.io.x54v9227pl3e/v2) + +## Author +David Ross1 +1NIST + +## Abstract +Protocol for bottlenecking a library using basic microbial culture equipment. + +## Materials + +### Starting Cultures +- Glycerol stock of variant library + +### Reagents +- **M9 Media** (ThermoFisher A1374401): A minimal media used to grow bacterial cultures. +- **Glycerol** (MilliporeSigma G5516): Used to prepare glycerol stock solutions for long-term storage of microbial cultures. + +### Consumables +- Six 15 mL snap cap tubes (Corning 352059) +- Eight 250mL baffled flasks (ThermoFisher 4116-0250) +- Eighteen 1.5 mL Microcentrifuge tubes (ThermoFisher 69715) +- Eighteen Agar Plates with LB and Kanamycin-50 (MilliporeSigma L0543) +- Multiple 1.2 mL Cryogenic vials (Corning 430487) + +### Equipment +- Incubator with shaking ability (set at 37°C and 300 rpm) + +## Protocol + +### Create the First Overnight Culture +1. **Dilution** + - Dilute a full 1 mL vial of the library glycerol stock into 50 mL of media in a 250 mL baffled flask. + +2. **Incubation** + - Incubate the resulting culture at 37°C with shaking (300 rpm) for 12-24 hours (to reach the stationary phase). + - *This generates the **overnight flask*** + +### Prepare All Flasks and Tubes +3. **Dilution Flask** + - Prepare one baffled flask (125 mL or 250 mL) with 49.9 mL media. + - *This is the **dilution flask*** + +4. **Culture Tubes** + - Prepare six 15 mL snap-cap culture tubes, each with 2 mL media. + - Number the tubes 1-6. + +5. **Baffled Flasks** + - Prepare six new 250 mL baffled flasks, each with 50 mL media. + - Number the flasks 1-6. + +6. **Microcentrifuge Tubes** + - Prepare 18 1.5 mL microcentrifuge tubes. + - Number the tubes 1.1, 1.2, 1.3, 2.1, 2.2, 2.3, ..., 6.1, 6.2, 6.3. + - Leave tubes 1.1, 2.1, 3.1, ..., 6.1 empty. + - Add 900 µL media to each of the other tubes. + +7. **Agar Plates** + - Prepare 18 agar plates (with LB + kanamycin). + - Number the plates to match the microcentrifuge tubes (1.1, 1.2, 1.3, 2.1, 2.2, 2.3, ..., 6.1, 6.2, 6.3). + +### Make Culture Dilutions +8. **Mix Culture** + - Mix the 50 mL **overnight flask** well, then pipette 0.1 mL of the extra culture into the dilution flask (with 49.9 mL media). + +9. **Swirl** + - Swirl the dilution flask to mix well. + +10. **Serial Dilution** + 1. Pipette 1 mL from the dilution flask to culture tube no. 1. + - Mix culture tube no. 1 well. + 2. Pipette 1 mL from culture tube no. 1 to culture tube no. 2. + - Mix culture tube no. 2 well. + 3. Pipette 1 mL from culture tube no. 2 to culture tube no. 3. + - Mix culture tube no. 3 well. + 4. Continue this for the remaining tubes 4-6. + +11. **Transfer Cultures** + 1. Transfer 1 mL of each culture from tubes 1-6 to flasks 1-6 and microcentrifuge tubes 1-6: + - Mix well. + - Pipette 1 mL from each culture tube to corresponding baffled flask. + - Mix baffled flask well. + - Pipette 1 mL from flask to corresponding empty microcentrifuge tube (X.1). + +12. **Prepare Microcentrifuge Tubes** + - For each set of microcentrifuge tubes: + - Mix microcentrifuge tube X.1. + - Pipette 100 µL from tube X.1 to tube X.2. + - Mix microcentrifuge tube X.2. + - Pipette 100 µL from tube X.2 to tube X.3. + - Mix microcentrifuge tube X.3. + +### Plate and Incubate All Cultures +13. **Plating** + - Plate 150 µL from each microcentrifuge tube onto the corresponding agar plate. + +14. **Incubation** + - Incubate all agar plates at 37°C for 16-24 hours or until colonies are visible and incubate all flasks at 37°C with shaking (300 rpm) for 16-24 hours (to stationary phase). + +### Count Colonies and Estimate Colony Forming Units +15. **Colony Counting** + - The next day, count the colonies on every agar plate to get colony forming unit (CFU) estimates for the starting point for each culture. + +16. **Choose Culture** + - Choose the culture with an estimated starting CFU count closest to the target diversity (100,000). + - The true CFU count should be an overestimate of the library diversity because some variants will have multiple copies in the culture. But in our experience, the diversity resulting from this method is just as likely to be higher or lower than the estimated CFU count. + +### Make Glycerol Stocks +17. **Glycerol Stock Preparation** + - Make several 1 mL glycerol stocks (0.5 mL cells + 0.5 mL 40% glycerol) from the chosen bottlenecked culture, label appropriately, and store at -80°C until use. + - Also, consider making glycerol stocks from some of the other bottlenecked cultures in case you decide to run a similar library with higher or lower diversity after the initial pilot-scale pooled assay. + +--- + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/limited-detection-of-small-10-mm-colorectal-liver-jz7cp9n.md b/markdown-output/limited-detection-of-small-10-mm-colorectal-liver-jz7cp9n.md new file mode 100644 index 0000000000000000000000000000000000000000..a3bc8d858721d0aa1ded129aa4bd93b277cb8fa2 --- /dev/null +++ b/markdown-output/limited-detection-of-small-10-mm-colorectal-liver-jz7cp9n.md @@ -0,0 +1,78 @@ +```markdown +# Goal/Experiment: +Assess the detection accuracy of small (≤ 10 mm) colorectal liver metastasis (CRLM) using preoperative computed tomography (CT) imaging in patients undergoing liver resection. + +# Limited detection of small (≤ 10 mm) colorectal liver metastasis at preoperative CT in patients undergoing liver resection +**Authors**: Yousun Ko, Jihang Kim, Joseph Kyu-Hyung Park, Haeryoung Kim, Jai Young Cho, Sung-Bum Kang, Soyeon Ahn, Kyong Joon Lee, Kyoung Ho Lee +**Published**: 27 Sep 2017 +**DOI**: [10.17504/protocols.io.jz7cp9n](https://dx.doi.org/10.17504/protocols.io.jz7cp9n) + +## Study Overview +The institutional review board of Seoul National University Bundang Hospital approved this retrospective study and waived the requirement for informed consent. All data were fully anonymized and aggregated prior to analysis. + +### Objective +The objective is to analyze the size distribution of CRLM nodules identified in pathological examination and determine the per-nodule sensitivity of CT for each nodule-size category. + +## Study Sample +- From the surgical database, 284 patients with pathologically confirmed colorectal cancer underwent 311 liver resections between 2003 and 2014. +- 55 liver resections performed without preoperative contrast-enhanced CT and eight with no solid nodule were excluded. +- Final sample: 211 patients undergoing 229 liver resections, including 461 pathologically confirmed CRLM nodules. + +### Demographics +- Mean Age: 66.4 ± 10.9 years +- Gender: 140 men (67.5 ± 10.7 years), 71 women (64.2 ± 11.2 years) + +## CT Imaging and Reporting +- **Procedure**: Intravenous contrast-enhanced CT images taken during the portal venous phase using 16- or higher detector-row machines. +- **Image Reconstruction**: Thickness of 4 or 5 mm, increment of 3 or 4 mm. +- **Interpretation**: + - Radiologists with 3-13 years experience. + - Narrow window settings (Window width: 200, Window level: 100 HU). + - Standardized structured report forms used. + +## MR Imaging +- **Indications**: Added when CT showed resectable CRLM or indeterminate lesions. +- **Equipment & Contrast Agents**: + - 1.5-T magnets (initial study period), 3-T magnets (later part), various contrast agents including gadoxetic acid disodium. + +### MR Imaging Protocol +- Dual-echo in- and opposed-phase spoiled gradient-echo T1-weighted. +- Fat-suppressed fast spin-echo T2-weighted. +- Diffusion-weighted imaging. + +## Liver Resection +- Evaluated by a multidisciplinary conference. +- Surgery: Mobilized liver inspected or palpated by surgeons with 3-17 years of experience. +- **Intraoperative Imaging**: SSD-3500 (Aloka), MyLab 25 Gold (Esaote Biomedica), iU22 (Philips Medical Systems). + +## Pathological Examination +- Performed by one of two pathologists with 3-12 years of experience. +- **Tissue Handling**: Liver slices kept ≤ 5 mm or thinner. +- **Nodule Analysis**: Solid nodules inspected, sampled, stained with hematoxylin-eosin, and recorded per Couinaud segment and size in millimeters. + +## Nodule-Matching Algorithm +- Criteria for true positive: + - Concordance in segmental location and size (±3 mm for ≤10 mm nodules, ±5 mm for >10 mm nodules). +- Review by study coordinators and adjudication through consensus. + +## Statistical Analysis +- **Performed By**: Two radiologists and a statistician. +- **Methods**: + - Histogram (nodule-size distribution), logistic regression (per-nodule CT sensitivity). + - Chi-square test (1-year recurrence rate difference). + +### Software +- **Stata**: Version 14.0 (StataCorp, College Station, TX). + +## Notes on Sensitivity Comparison +- Additional MR imaging added sensitivity for CRLM nodule detection but not formally compared to CT due to various influencing factors. + +## Vendors and Reagents +- **Gadodiamide (Omniscan)**: GE Healthcare, Princeton, NJ. +- **Ferucarbotran (Resovist)**: Schering, Berliln, Germany. +- **Gadoxetic Acid Disodium**: Used in later studies for enhanced MR imaging. + +--- + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/live-imaging-of-primary-mouse-neuron-cultures-cyhnxt5e.md b/markdown-output/live-imaging-of-primary-mouse-neuron-cultures-cyhnxt5e.md new file mode 100644 index 0000000000000000000000000000000000000000..b0d64b608ff8d97b8219dd9a16a61511bcabe8ba --- /dev/null +++ b/markdown-output/live-imaging-of-primary-mouse-neuron-cultures-cyhnxt5e.md @@ -0,0 +1,151 @@ +```markdown +Goal/Experiment: +The goal of this experiment is to perform live imaging of primary neuron cultures derived from neonatal mouse brain tissue, specifically hippocampal and dopamine neurons. This process incorporates labeling of dopamine neurons with a fluorescent DAT ligand and using virally encoded pHluorin sensors to measure vesicular release of neurotransmitter upon electrical stimulation of the neurons. The comparative analysis involves vesicular release in neurons across various transgenic knockout mouse lines. + +# Live Imaging of Primary Mouse Neuron Cultures + +**Authors**: Robert Edwards, Shweta Jain +**Institution**: University of California San Francisco +**Date**: Dec 21, 2023 +**Protocol ID**: 86286 +**Funders**: ASAP-CRN, Grant ID: 020529 + +## ABSTRACT +This protocol outlines the methodology for live imaging of primary neuron cultures obtained from neonatal mouse brain tissue. The described imaging involves labeling dopamine neurons with a fluorescent DAT ligand and using virally encoded pHluorin sensors to measure neurotransmitter release upon electrical stimulation. This technique, as referenced in Jain et al., 2023, compares the release in neurons between various transgenic knockout mouse lines. + +## PROTOCOL MATERIALS + +| Reagent | Vendor | Catalog Number | +|------------------------------------------|---------------------------------|--------------------------| +| Basal Medium Eagle (BME) | Thermo Fisher | #21010046 | +| GDNF Recombinant Human Protein | Thermo Fisher | #PHC7045 | +| HyClone Fetal Bovine Serum (FBS) | Fisher Scientific | #SH3039603 | +| D-()-Glucose Solution | Merck MilliporeSigma (Sigma-Aldrich) | #G8769 | +| B27 Supplement without Retinoic Acid (50x)| Gibco, ThermoFisher | #17504044 | +| Neurobasal™ Medium | Gibco, Thermo Fisher | #21103049 | +| GlutaMAX™ (100x) | Gibco, Thermo Fisher | #35050-061 | + +### Definitions and Functions: +- **Basal Medium Eagle (BME)**: A cell culture medium used for growing epithelial cells. +- **GDNF (Glial cell line-derived neurotrophic factor)**: Promotes the survival of various neuronal subpopulations. +- **Fetal Bovine Serum (FBS)**: Supplement in cell culture media providing necessary growth factors, hormones, and attachment factors. +- **D-()-Glucose**: A simple sugar used as an energy source in cell culture. +- **B27 Supplement**: A serum-free supplement used to support growth and maintenance of neurons. +- **Neurobasal™ Medium**: An optimized basal media formulation for the growth of embryonic neurons. +- **GlutaMAX™ Supplement**: A stable dipeptide substitute for L-glutamine that minimizes toxic ammonia build-up. + +## Preparation of Hippocampal Cultures + +1. **Coat coverslips with Poly-L-lysine** +2. **Collect mouse pups at postnatal day 0** + - Cultures were prepared from WT, β3A KO, β3B KO, and mocha mice. + +3. **Dissect Hippocampi** + - Decapitate and remove the brain. Dissect out hippocampi from each hemisphere into Hank's Balanced Salt Solution (HBSS) containing 10 mM HEPES and 20 mM glucose. + +4. **Papain Digestion** + - Digest with papain (20 units/mL) in papain digestion buffer (HBSS with 20 mM CaCl₂, 5 mM EDTA, 0.2 mg/mL L-Cysteine, 10 mM HEPES at pH 7.4) for 15 minutes. + +5. **Wash Hippocampi** + - Wash three times in HBSS containing 10 mM HEPES and 20 mM glucose. + +6. **Trituration** + - Triturate to make a single cell suspension. + +7. **Determine Cell Density** + - Use a hemocytometer to count cell density. + +8. **Plate Cells** + - Plate cells onto Poly-L-lysine-coated coverslip at 350 cells/mm² in Minimal Essential Medium containing: + - 1X B27 supplement (Gibco, #17504044) + - 5% Fetal Bovine Serum (Fisher, #SH3039603) + - 21 mM glucose (Merck MilliporeSigma, #G8769) + +9. **Store Cultures** + - Incubate at 37 °C. + +10. **Medium Change on DIV1** + - Replace 3/4 of the medium with Neurobasal medium (Gibco, Thermo Fisher, #21103049) containing: + - 1X B27 supplement (Gibco, ThermoFisher, #17504044) + - GlutaMAX (Gibco, #35050-061) + +11. **Infect Cells** + - Infect cells at DIV 4-5 using lentiviral vectors. + + - Viruses used: VGLUT2-pH, VMAT2-pH, VGAT-pH. + +12. **Inhibit Glial Proliferation** + - Treat cultures with 4 μM cytosine arabinoside (Ara-C) at DIV 6-7. + +## Preparation of Dopamine Neuron Cultures + +13. **Coat Coverslips** + - Coat coverslips with Poly-L-lysine and laminin; plate astrocyte monolayer onto coverslips. + +14. **Collect Mouse Pups** + - Collect mouse pups at postnatal day 0. + +15. **Dissect Midbrain** + - Decapitate and remove brain. Dissect out midbrain (ventral tegmental area and substantia nigra) from each hemisphere into HBSS containing 10 mM HEPES and 20 mM glucose. + +16. **Papain Digestion** + - Digest with papain (20 units/mL) in papain digestion buffer (HBSS with 20 mM CaCl₂, 5 mM EDTA, 0.2 mg/mL L-Cysteine, 10 mM HEPES at pH 7.4) for 15 minutes. + +17. **Wash Digested Tissue** + - Wash three times with HBSS containing 10 mM HEPES and 20 mM glucose. + +18. **Trituration** + - Triturate tissue to get a single cell suspension. + +19. **Determine Cell Density** + - Using a hemocytometer, determine the cell density. + +20. **Plate Cells** + - Plate cells onto pre-prepared coverslips at 1000 cells/mm² in medium consisting of 60% Neurobasal Medium (Gibco, #21103049), 30% Basal Medium Eagle (Thermo Fisher, #21010046), 10% FBS (Fisher, #SH3039603), 1X B27 supplement (Gibco, #17504044), 2 mM GlutaMAX (Gibco, #35050-061), 10 ng/mL GDNF (ThermoFisher, #PHC7045), and 1X penicillin/streptomycin. + +21. **Infect with Lentivirus** + - Infect at DIV 2-4 using lentiviral vectors encoding VMAT2-pH or VGLUT2-pH. + +## Live Imaging + +22. **Maintain Cultures** + - Maintain cultures until they are ready for imaging. Perform imaging at DIV 14-16 for hippocampal cultures and DIV 13-15 for dopamine neuron cultures. + +23. **Prepare Tyrode's Buffer** + - 119 mM NaCl + - 25 mM HEPES + - 2 mM CaCl₂ + - 2 mM MgCl₂ + - 2.5 mM KCl + - 30 mM glucose + - Adjust pH to 7.4 + +24. **Incubate with Tyrode's Buffer and JHC1-64** + - Incubate dopamine neuron cultures in Tyrode's buffer (119 mM NaCl, 25 mM HEPES, 2 mM CaCl₂, 2 mM MgCl₂, 2.5 mM KCl, 30 mM glucose). + - Prepare Tyrode's +JHC1-64 by adding fluorescent DAT ligand JHC1-64 at 30 nM. + +25. **Incubation for Imaging** + - Incubate in Tyrode's +JHC1-64 for 5 minutes at room temperature. + - Rinse neurons in Tyrode's buffer. + +26. **Mount Coverslips** + - Add fresh Tyrode's buffer to culture dishes. Mount coverslips in a perfusion and stimulation chamber of a TE300 inverted epifluorescence microscope. + +## Image Acquisition + +26. **Monitor Fluorescence** + - For pHluorin, use 470/40 nm excitation and 525/70 nm emission bandpass filters. + - For JHC1-64, use 545/25 nm excitation and 605/70 nm emission bandpass filters. + - Use the red channel to identify dopamine neurons, then switch to the pHluorin channel for experiments. + +27. **Stimulating Electrode Placement** + - Place platinum-iridium stimulating electrodes near the observed dopamine neuron. + +28. **Acquire Images** + - Acquire images typically at 1 Hz, or at 10 Hz for Fluo-5F imaging. + +29. **Evoke Action Potentials** + - While acquiring images, evoke action potentials using 1 ms bipolar current pulses through the stimulating electrode at 5-10 V/cm. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/lminex-milliplex-cytokine-chemokine-17-plex-mag-hvfb63n.md b/markdown-output/lminex-milliplex-cytokine-chemokine-17-plex-mag-hvfb63n.md new file mode 100644 index 0000000000000000000000000000000000000000..94a81cb6c763e54106710c8985a4a0d62b1ad577 --- /dev/null +++ b/markdown-output/lminex-milliplex-cytokine-chemokine-17-plex-mag-hvfb63n.md @@ -0,0 +1,224 @@ +```markdown +Goal/Experiment: +To quantitatively measure cytokine and chemokine levels in serum, plasma, and other biological fluids using the Luminex Milliplex Cytokine/Chemokine 17-plex MAG kit. + +# Luminex Milliplex Cytokine/Chemokine 17-plex MAG + +**Authors:** +Troy Kemp, Ligia Pinto + +**Abstract:** +Luminex Milliplex Cytokine/Chemokine 17-plex MAG manufacturer's protocol + +**Citation:** +Troy Kemp, Ligia Pinto Luminex Milliplex Cytokine/Chemokine 17-plex MAG. protocols.io +dx.doi.org/10.17504/protocols.io.hvfb63n + +**Published:** +01 Aug 2017 + +## Protocol + +### Step 1: Preparation of Samples/Reagents for Immunoassay + +### Step 2: Preparation of Serum/Plasma +**Thaw Time:** +Thaw the samples completely on ice, mix well by shaking on a plate shaker for 1 minute at RT (20-25°C) and centrifuge (1,700 xg, 10 minutes, 4°C) prior to use in the assay to remove particulates. + +### Step 3: Preparation of Antibody-Immobilized Beads +**Sonicate and Vortex:** +Sonicate each individual antibody-bead vial for 30 seconds; vortex for 1 minute. Add 60 µL from each antibody-bead vial to the Mixing Bottle and bring final volume to 3.0 mL with Bead Diluent. Vortex the mixed beads well. Unused portion may be stored at 2-8°C for up to one month. + +**Note:** +Due to the composition of magnetic beads, you may notice a slight color in the bead solution. This does not affect the performance of the beads or the kit. + +### Step 4: Preparation of Quality Controls +**Reconstitution Time:** +Before use, reconstitute Quality Control 1 and Quality Control 2 with 250 µL deionized water. Invert the vial several times to mix and vortex. Allow the vial to sit for 5-10 minutes and then transfer the controls to appropriately labeled polypropylene microfuge tubes. Unused portion may be stored at ≤ -20°C for up to one month. + +### Step 5: Preparation of Wash Buffer +Bring the 10X Wash Buffer to room temperature and mix to bring all salts into solution. Dilute 30 mL of 10X Wash Buffer with 270 mL deionized water. Store unused portion at 2-8°C for up to one month. + +### Step 6: Preparation of Serum Matrix +**Reconstitution Time:** +Add 1.0 mL Assay Buffer to the bottle containing lyophilized Serum Matrix. Mix well. Allow at least 10 minutes for complete reconstitution. Leftover reconstituted Serum Matrix should be stored at ≤ -20°C for up to one month. + +### Step 7: Preparation of Human Cytokine Standard +**Reconstitution Time:** + +1. Prior to use, reconstitute the Human Cytokine Panel II Standard with 250 µL deionized water to give: + - 100,000 pg/ml for SDF-1a+b + - 50,000 pg/ml for MIP-1d, TPO, IL-20, Eotaxin-3, IL-23 + - 20,000 pg/ml for ENA-78, 6CKine, LIF, IL-21, IL-33 + - 10,000 pg/ml for MCP-4, Eotaxin-2, TRAIL, SCF, TSLP, IL-28A, IL-16 + - 5,000 pg/ml for MCP-2, CTACK + - 2,000 pg/ml for I-309 + - 1,000 pg/ml for BCA-1, TARC + +2. Invert the vial several times to mix. Vortex the vial for 10 seconds. Allow the vial to sit for 5-10 minutes and then transfer the standard to an appropriately labeled polypropylene microfuge tube. This standard will be termed STD7; the unused portion may be stored at ≤ -20°C for up to one month. + + +### Step 8: Preparation of Working Standards +1. Label six polypropylene microfuge tubes STD6, STD5, STD4, STD3, STD2, and STD1. +2. Add 150 µL of Assay Buffer to each of the six tubes. +3. Prepare serial dilutions by adding 50 µL of STD7 reconstituted standard to the STD6 tube, mix well and transfer 50 µL of the STD6 standard to the STD5 tube, mix well and transfer 50 µL of the STD5 standard to the STD4 tube, mix well and transfer 50 µL of the STD4 standard to the STD3 tube, mix well and transfer 50 µL of the STD3 standard to the STD2 tube and mix well, transfer 50 µL of the STD2 standard to the STD1 tube and mix well. The 0 pg/mL standard (Background) will be Assay Buffer. + +**Standard Volume of Deionized Water to Add** + +| Tube | Volume of Standard to Add (µL) | Original (STD7) Concentration (pg/ mL) | +|--------|--------------------------------|---------------------------------------| +| STD6 | 250 | 50,000 | +| STD5 | 150 | 10,000 | +| STD4 | 105 | 2,000 | +| STD3 | 50 | 500 | +| STD2 | 50 | 100 | +| STD1 | 50 | 10 | + + +### Step 9: +2 + +### Step 10: +4 + +### Step 11: +9 + +### Step 12: +2 + +### Step 13: +420.98 + +### Step 14: +4 + +### Step 15: +9 + +### Step 16: +8 + +### Step 17: +5 + +### Step 18: +8 + +### Step 19: +73 + +### Step 20: +9 + +### Step 21: +8 + +### Step 22: +5 + +### Step 23: +1 + +### Step 24: +1 + +### Step 25: +3390.64 + +### Step 26: +6 + +### Step 27: +3 + +### Step 28: +1 + +### Step 29: +3 + +### Step 30: +5 + +### Step 31: +31,562.55 + +### Step 32: +5125 + +### Step 33: +5625 1,250 3,125 6,250 506 250 1,250 2,500 5,000 1,250 50 50250 50 50 5 10 0 Immunoassay Procedure: +Allow all reagents to warm to room temperature (20-25°C) before use in the assay. +1. Run the standards, controls, and samples in duplicate. + +### Step 34: Prewet the Plate +By pipetting 200 µL of Wash Buffer into each well of the MAG Plate. Seal and mix on a plate shaker for 10 minutes at room temperature (20-25°C). + +### Step 35: +1. Decant Wash Buffer and remove residual amount from all wells by inverting the plate and tapping it smartly onto absorbent towels several times. +2. Add 25 μL of each Standard or Control into the appropriate wells. Add 25 μL Assay Buffer to the 0 pg/mL standard (Background). + +### Step 36: +Add 25 μL of Assay Buffer to the sample wells. + +### Step 37: +Add 25 μL of the Serum Matrix solution to the background, appropriate standards, and control wells. + +### Step 38: +Add 25 μL of Sample into the appropriate wells. + +### Step 39: +Vortex Mixing Bottle and add 25 μL of the mixed Beads to each well. +**Note:** During addition of Beads, shake bead bottle intermittently to avoid settling. Due to the composition of magnetic beads, you may notice a slight color in the bead solution. This does not affect the performance of the beads or the kit. + +### Step 40: +Seal the plate with a plate sealer, cover it with the lid. Wrap a rubber band around the plate, lid, and shaker platform and incubate with agitation on a plate shaker for 2 hours at room temperature (20-25°C). + +### Step 41: +Gently remove fluid by aspiration. Do not invert plate. + +### Step 42: +Wash plate 2 times with 200 μL/well of Wash Buffer, removing Wash Buffer by aspiration between each wash. To avoid washing/aspiration related bead loss, allow approximately 60 seconds between dispensing of the Wash Buffer and subsequent aspiration. + +### Step 43: +Add 25 μL of Detection Antibodies into each well. +**Note:** Allow the Detection Antibodies to warm to room temperature prior to addition. + +### Step 44: +Seal, cover with lid, and incubate with agitation on a plate shaker for 1 hour at room temperature (20-25°C). Do not aspirate after incubation. +Add 25 μL Streptavidin-Phycoerythrin to each well containing the 25 μL of Detection Antibodies. + +### Step 45: +Seal, cover with lid and incubate with agitation on a plate shaker for 30 minutes at room temperature (20-25°C). + +### Step 46: +Gently remove all contents by aspiration. Do not invert plate. + +### Step 47: +Wash plate 2 times with 200 μL/well of Wash Buffer, removing Wash Buffer by aspiration between each wash. To avoid washing/aspiration related bead loss, allow approximately 60 seconds between dispensing of the Wash Buffer and subsequent aspiration. + +### Step 48: +Add 150 μL of Sheath Fluid to all wells. Resuspend the beads on a plate shaker for 5 minutes. + +### Step 49: +Run plate on Luminex 100™ IS. + +### Step 50: +Save and analyze the data using Bio-Plex Manager software. +**Equipment Settings:** +- **Events:** 50, per bead region +- **Sample Size:** 100 μL +- **Gate Settings:** 5000 to 25,000 +- **Time Out:** 60 seconds + +**Quality Controls:** +The ranges for each analyte in Quality Control 1 and 2 are provided on the card insert or can be located at the MILLIPORE website [Millipore's website](http://www.millipore.com/techlibrary/index.do) using the catalog number as the keyword. + +### Luminex Milliplex Cytokine/Chemokine 17-plex MAG + +### Step 51: +... + +endofoutput +``` diff --git a/markdown-output/low-volume-titrations-for-ligand-binding-monitored-bnximfke.md b/markdown-output/low-volume-titrations-for-ligand-binding-monitored-bnximfke.md new file mode 100644 index 0000000000000000000000000000000000000000..79a45612110eed1fa0499ee2b924ecf09c5e652a --- /dev/null +++ b/markdown-output/low-volume-titrations-for-ligand-binding-monitored-bnximfke.md @@ -0,0 +1,88 @@ +```markdown +# Low-Volume Titrations for Ligand Binding Monitored by Circular Dichroism + +## Goal/Experiment: +To perform circular dichroism (CD) experiments for evaluating protein-ligand binding interactions. This includes using the B23 synchrotron radiation circular dichroism (SRCD) beamline at Diamond Light Source (DLS) to gather qualitative and quantitative data of ligand-binding interactions in both the far-UV and near-UV regions. + +### Authors +- Rohanah Hussain +- Charlotte S. Hughes +- Giuliano Siligardi +- Diamond Light Source Ltd., Chilton, UK + +### DOI +[dx.doi.org/10.17504/protocols.io.bnximfke](https://dx.doi.org/10.17504/protocols.io.bnximfke) + +--- + +## Abstract +Circular Dichroism (CD) spectroscopy is essential to ascertain global conformational changes in proteins, investigate secondary structure content, protein folding, and ligand binding interactions. The highly collimated micro-beam available at B23 beamline of the Diamond Light Source (DLS) enables unique advantages over traditional benchtop instruments, allowing the use of small aperture cuvette cells and flat capillary tubes. This chapter discusses how to perform CD measurements, necessary methods, hints, and tips for high-quality data collection, specifically for protein-ligand interactions. + +## Keywords +- Circular dichroism +- Ligand binding +- Titration +- Binding constant +- UV denaturation +- Protein stability +- Data processing + +## Guidelines +CD titration enables the determination of quantitative binding interaction between host-ligand systems. This method effectively measures the dissociation constant \(K_D\) for ligand binding to proteins. Here are the detailed steps and considerations: + +### Materials and Equipment +#### Equipment: +1. **CD Spectrometer**: Synchrotron beam at B23 beamline +2. **Cuvette**: Fused quartz cuvette (Starna), 2 mm x 2 mm aperture +3. **Pipette** with low-volume tips +4. **Spectrophotometer** for measuring protein concentration at \(A_{280}\) + +#### Reagents: +1. **Proteins**: Expressed and purified +2. **Ligands**: Depending on the protein's binding characteristics +3. **Detergents**: For solubilizing membrane proteins (e.g., DDM, DM) +4. **Buffers**: Suitable for the protein of interest to maintain pH and ionic strength + +### Safety Warnings +- Refer to the hazardous information and safety warnings described in the Safety Data Sheets (SDS) of the chemicals used. + +### Procedure +#### Step 1: Protein Concentration Measurement +- Measure the concentration of the protein solution at \(A_{280}\). +- Calculate the dilution factor for the stock solution for the working solution aiming for an acceptable \(A_{280}\) in the range of 1.5–1.6. + +#### Step 2: Ligand Addition +- Aliquots of ligand are carefully added to the host mixture in the cuvette, ensure thorough mixing by pipetting up and down. + +#### Step 3: Incubation +- Incubate the mixture for a user-defined period (e.g., 20 minutes) to ensure equilibration, which is particularly important when detergents are present. + +#### Step 4: CD Measurement +- Collect CD spectra for each titration point after the determined incubation period. The CD spectrum is assessed for changes in signal upon ligand binding. + +#### Step 5: Data Analysis +- CDApps software is used for analysis, utilizing the experimental plan for concentration, dilution factors, and path length. + +#### Step 6: Buffer Spectrum Baseline +- Measure the CD spectrum of the buffer under identical conditions and subtract this baseline from the titration spectra for accurate data. + +#### Step 7: Data Interpretation +- Plot the difference in the CD signal against the ligand concentration. Perform a nonlinear regression analysis to determine the binding dissociation constant \(K_D\) and interaction stoichiometry. + +## Notes +1. The small aperture 1 cm cell is typically preferred for membrane proteins for more accurate results. +2. Detergents are used to solubilize membrane proteins while ensuring minimum perturbed environment for the protein. +3. CDApps allows monitoring of the solution stability over multiple scans. + +## References +1. Detailed list of references can be accessed through the provided DOI and external links for further reading and in-depth protocol guidelines. + +--- + +## License +This protocol is distributed under the terms of the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction. + +--- + +**endofoutput** +``` \ No newline at end of file diff --git a/markdown-output/lrrk2rckw-single-molecule-kinesin-motility-assays-bp6hmrb6.md b/markdown-output/lrrk2rckw-single-molecule-kinesin-motility-assays-bp6hmrb6.md new file mode 100644 index 0000000000000000000000000000000000000000..ed636e24149ca3d66f55d56c2174e323fe23cd32 --- /dev/null +++ b/markdown-output/lrrk2rckw-single-molecule-kinesin-motility-assays-bp6hmrb6.md @@ -0,0 +1,106 @@ +```markdown +# Goal/Experiment: +The goal of this experiment is to perform LRRK2^RCKW single molecule kinesin motility assays. + +# LRRK2^RCKW Single Molecule Kinesin Motility Assays + +## Authors: +- John Salogiannis1 +- Mariusz Matyszewski + +**1 Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, CA 92093** + +DOI: [https://dx.doi.org/10.17504/protocols.io.ewov14qykvr2/v1](https://dx.doi.org/10.17504/protocols.io.ewov14qykvr2/v1) + +--- + +## Keywords: +- LRRK2 +- Motility Assay +- Kinesin +- Imaging +- Single-molecule +- ASAPCRN + +## As Used In: +Deniston CK, Salogiannis J, Mathea S, Snead DM, Lahiri I, Matyszewski M, Donosa Q, Watanabe R, Röhning J, Shiau AK, Knapp S, Villa E, Reck-Peterson SL, Leschziner AE (2020). Structure of LRRK2 in Parkinson's disease and model for microtubule interaction. Nature. [https://doi.org/10.1038/s41586-020-2673-2](https://doi.org/10.1038/s41586-020-2673-2) + +--- + +## Image Recommendation: +Settings will vary per microscope. We imaged K560-GFP every 500 milliseconds for 2 minutes with 25% laser (488) power at 150 ms exposure time. Each sample was imaged no longer than 15 minutes. Each technical replicate recommended to consist of movies from at least two fields of view containing between 5 and 10 microtubules each. + +Image using the 405 nm laser to determine the locations of the microtubules. Preferably at the start and end of the experiment. + +## Single-Molecule Motility Assay Analysis Recommendation: +Kymographs were generated from motility movies and quantified for run lengths, percent motility, and velocity using ImageJ (NIH). Specifically, maximum-intensity projections were generated from time-lapse sequences to define the trajectory of particles on a single microtubule. The segmented line tool was used to trace the trajectories and map them onto the original video sequence, which was subsequently re-sliced to generate a kymograph. Motile and immotile events (>1 sec) were manually traced. Bright aggregates, which were less than 5% of the population, were excluded from the analysis. Run length measurements were calculated from motile events only. For percent motility per microtubule measurements, motile events (>1 sec and >1 µm) were divided by total events per kymograph. Velocity measurements were calculated from the inverse slopes of the motile event traces (>1 sec and >1 µm) only. Statistical analyses were performed in Prism8 (Graphpad). + +## Recommended Equipment and Setup: +This single-molecule imaging experiment was originally performed using total internal reflection fluorescence (TIRF) microscopy with an inverted microscope (Nikon, Ti-E Eclipse) equipped with a 100x 1.49 N.A. oil immersion objective (Nikon, Plano Apo), and a MLC400B laser launch (Agilent), with 405 nm, 488 nm, 561 nm, and 640 nm laser lines (561 and 640 nm laser lines are not needed for this version of the experiment). Excitation and emission paths were filtered using single bandpass filter cubes (Chroma), and emitted signals were detected with an electron multiplying CCD camera (Andor Technology, iXon Ultra 888). Illumination and image acquisition were controlled with NIS Elements Advanced Research software (Nikon), and the xy position of the stage was controlled with a ProScan linear motor stage controller (Prior). + +## Required Buffers: + +### Streptavidin Buffer: +- 1 mg/mL Streptavidin +- 30 mM HEPES pH 7.4 +- 2 mM MgOAc +- 1 mM EGTA +- 10% Glycerol + +### Motility Assay Buffer: +- 30 mM HEPES pH 7.4 +- 50 mM KOAc +- 2 mM MgOAc +- 1 mM EGTA +- 10% Glycerol +- 1 mM DTT +- 20 µM Taxol + +### Motility Assay Buffer with Casein: +- 30 mM HEPES pH 7.4 +- 50 mM KOAc +- 2 mM MgOAc +- 1 mM EGTA +- 10% Glycerol +- 1 mM DTT +- 20 µM Taxol +- 1 mg/mL Casein + +### LRRK2 Buffer: +- 20 mM HEPES pH 7.4 +- 80 mM NaCl +- 0.5 mM TCEP +- 5% Glycerol +- 2.5 mM MgCl2 +- 20 µM GDP + +--- + +## Protocols: + +### Create Microscope Slides: +1. **Adhere Biotin-PEG-functionalized coverslips (Microsurfaces) to a microscope slide using double-sided scotch tape, creating 4 channels per slide.** +2. **Add the streptavidin buffer to each channel and incubate for 3 minutes.** +3. **Wash twice with Motility Assay buffer.** +4. **Add a 1:150 dilution of taxol-stabilized microtubules (19 μL per channel) and incubate for 3 minutes.** See [protocol](https://dx.doi.org/10.17504/protocols.io.bp2l6bdedgqe/v1) for making taxol-stabilized microtubules. +5. **Wash twice with LRRK2 buffer. Add more buffer if necessary to prevent drying out.** + +### Prepare LRRK2: +6. **Prepare a 1 μM solution of LRRK2^RCKW in a cold LRRK2 buffer. Centrifuge through a 0.1 µm PVDF filter to remove aggregates. Calculate the new effective concentration. Usually around 500 nM - 700 nM after centrifugation.** +7. **Create a working aliquot of LRRK2 in the desired concentration (ex. 25 nM - 100 nM) in the LRRK2 buffer at room temperature (recommended volume of 25 μL). If adding inhibitors, add them now with DMSO. Incubate for 10 minutes at room temperature.** + +### Next Steps: +8. **Add LRRK2^RCKW sample to the channel (19 μL). Incubate for 5 minutes. Prepare next step while waiting.** +9. **Wash twice with the Motility Assay buffer supplemented with 1 mg/mL casein.** + +### Prepare Kinesin: +10. **Make a 4 nM solution of K560-GFP in the Motility Assay buffer with casein supplemented with an oxygen scavenger system (0.4% glucose, 45 μg/mL glucose catalase (Sigma-Aldrich), and 1.15 mg/mL glucose oxidase (Sigma-Aldrich)), 71.5 mM beta-mercaptoethanol and 1 mM ATP.** + +### Next Steps: +11. **Add 19 μL kinesin mixture to each chamber.** +12. **Image slide.** + +--- + +*endofoutput* +``` \ No newline at end of file diff --git a/markdown-output/luminex-milliplex-cytokine-chemokine-15-plex-mag-hveb63e.md b/markdown-output/luminex-milliplex-cytokine-chemokine-15-plex-mag-hveb63e.md new file mode 100644 index 0000000000000000000000000000000000000000..c4d0d8e5dd56030e419b1e1ee333498ff689e73e --- /dev/null +++ b/markdown-output/luminex-milliplex-cytokine-chemokine-15-plex-mag-hveb63e.md @@ -0,0 +1,106 @@ +```markdown +# Goal/Experiment: +The aim of this experiment is to perform an immunoassay using the Luminex Milliplex Cytokine/Chemokine 15-plex MAG kit by measuring the concentration of multiple cytokines and chemokines in given samples. + +# Luminex Milliplex Cytokine/Chemokine 15-plex MAG + +## Abstract +Manufacturer's protocol + +Citation: +Troy Kemp, Ligia Pinto. Luminex Milliplex Cytokine/Chemokine 15-plex MAG. protocols.io dx.doi.org/10.17504/protocols.io.hveb63e +**Published:** 01 Aug 2017 + +## Protocol + +### Step 1. Preparation of Samples/Reagents for Immunoassay + +### Step 2. Preparation of Serum/Plasma +**Thaw Time:** Thaw the samples completely on ice, mix well by vortexing, and centrifuge (10,000 rpm, 10 minutes, 4°C) prior to use in the assay to remove particulates. For samples in 96 well plates, thaw the samples completely on ice, mix well by shaking on a plate shaker for 1 min at RT (20-25°C) and centrifuge (1,700 xg, 10 minutes, 4°C) prior to use to remove particulates. + +### Step 3. Preparation of Antibody-Immobilized Beads +- **Procedure:** Sonicate each individual antibody-bead vial for 30 seconds; vortex for 1 minute. Add 60 µL from each antibody-bead vial to the Mixing Bottle and bring the final volume to 3.0 mL with Bead Diluent. Vortex the mixed beads well. +- **Storage:** Unused portion may be stored at 2-8°C for up to one month. + +### Step 4. Preparation of Quality Controls +- **Reconstitution Time:** Reconstitute Quality Control 1 and Quality Control 2 with 250 µL deionized water. Invert the vial several times to mix and vortex. Allow to sit for 5-10 minutes and transfer the controls to appropriately labeled polypropylene microfuge tubes. +- **Storage:** Unused portion may be stored at ≤ -20°C for up to one month. + +### Step 5. Preparation of Wash Buffer +- Bring the 10X Wash Buffer to room temperature and mix. Dilute 30 mL of 10X Wash Buffer with 270 mL deionized water. +- **Storage:** Store unused solution at 2-8°C for up to one month. + +### Step 6. Preparation of Serum Matrix +- **Reconstitution Time:** Add 1.0 mL deionized water to the bottle containing lyophilized Serum Matrix. Mix well. Allow at least 10 minutes for complete reconstitution. +- **Storage:** Leftover reconstituted Serum Matrix should be stored at ≤ -20°C for up to one month. + +### Step 7. Preparation of Human Cytokine Standard +- **Reconstitution Time:** Reconstitute the Human Cytokine Standard with 250 µL deionized water to achieve a 10,000 pg/mL concentration. Vortex for 10 seconds, allow the vial to sit for 5-10 minutes, then transfer to a labeled polypropylene microfuge tube for storage at ≤ -20°C for up to one month. + +### Step 8. Preparation of Working Standards + +**Procedure:** +1. Label six polypropylene microfuge tubes 2000, 400, 80, 16, 3.2, and 0.64 pg/mL. +2. Add 200 µL of Assay Buffer to each tube. +3. Perform serial dilutions: + - Add 50 µL of the 10,000 pg/mL reconstituted standard to the 2000 pg/mL tube, mix well. + - Transfer 50 µL of 2000 pg/mL standard to the 400 pg/mL tube, mix well. + - Continue serial dilutions to achieve the remaining standards. + +**Standard Concentration Table:** + +| Standard Concentration (pg/mL) | Volume of Deionized Water to Add (mL) | Volume of Standard to Add (mL) | Volume of Assay Buffer to Add (mL) | +|------------------------------|----------------------------------------|-------------------------------|------------------------------------| +| 10,000 | 0 | 0 | 250 | +| 2,000 | 0.2 | 50 | 200 | +| 400 | 0.2 | 50 | 200 | +| 80 | 0.2 | 50 | 200 | +| 16 | 0.2 | 100 | 200 | +| 3.2 | 0.2 | 50 | 200 | +| 0.64 | 0.2 | 50 | 200 | + +### Step 9. General Immunoassay Procedure + +1. Allow all reagents to warm to room temperature (20-25°C) before using. +2. **Plating:** + - Add 200 µL of Wash Buffer to each well of the MAG plate. Seal and mix on a plate shaker for 10 minutes at room temperature (20-25°C). + - Decant Wash Buffer and remove residual amount smartly by tapping +3. Add 25 µL of each Standard or Control into appropriate wells. Add 25 µL of Assay Buffer to the 0 pg/mL standard (Background). +4. Add 25 µL of Assay Buffer to the sample wells. +5. Add 25 µL of the Serum Matrix solution to the background, standards, and control wells. +6. Add 25 µL of Sample to appropriate wells. +7. Vortex Mixing Bottle and add 25 µL of the mixed Beads to each well. Shake bead bottle intermittently to avoid settling. +8. Seal the plate, cover it with the lid, wrap a rubber band around, plate holder, and incubate with agitation on a plate shaker overnight (16-18 hrs.) at 4°C. + +### Step 10. Serum/Plasma Sample Handling + +- **Remove fluid** by gentle aspiration. Do not invert plate. +- **Wash Plate 2 times** with 200 µL/well of Wash Buffer, removing Wash Buffer by aspiration between washes (allow 60 seconds between dispensing and aspiration). +- Add 25 µL of Detection Antibodies to each well. Allow antibodies to warm to room temperature prior to addition. +- Seal and incubate with agitation on a plate shaker for 1 hour at room temperature (20-25°C). +- **After Incubation:** + - Do not aspirate. + - Add 25 µL Streptavidin-Phycoerythrin to each well. +- Seal and incubate with agitation on a plate shaker for 30 minutes at room temperature (20-25°C). +- **Final Wash step**: Gently aspirate contents, wash 2 times with 200 µL of Wash Buffer. Add 150 µL of Sheath Fluid to all wells, resuspend beads by shaking for 5 minutes. + +### Equipment Settings: +- **Events:** 50, per bead region +- **Sample Size:** 100 µL +- **Gate Settings:** 5000 to 25,000 +- **Time Out:** 60 seconds + +### References and Notes: +- Save and analyze data using Bio-Plex Manager software. +- **Quality Control:** Check ranges for analytes using the insert card or MILLIPORE website. +- **Legend:** + - A: Hemolyzed sample + - B: Lipemic sample + - C: Sample missing + - D: Clogged Filter well + - E: Low bead count + - F: >30% Bead aggregation + - G: Instrument repeat + +endofoutput +``` diff --git a/markdown-output/luminex-milliplex-cytokine-chemokine-9-plex-mag-hvhb636.md b/markdown-output/luminex-milliplex-cytokine-chemokine-9-plex-mag-hvhb636.md new file mode 100644 index 0000000000000000000000000000000000000000..14224bcdbc104b553965c8986cb4a7c705408b61 --- /dev/null +++ b/markdown-output/luminex-milliplex-cytokine-chemokine-9-plex-mag-hvhb636.md @@ -0,0 +1,188 @@ +```markdown +# Goal/Experiment: +To conduct an immunoassay using the Luminex Milliplex Cytokine/Chemokine 9-plex MAG for the quantification of specific cytokines/chemokines from serum/plasma samples. + +# Luminex Milliplex Cytokine/Chemokine 9-plex MAG + +## Abstract +Luminex Milliplex Cytokine/Chemokine 9-plex MAG manufacturer's protocol. + +## Citation +Ligia Pinto, Troy Kemp Luminex Milliplex Cytokine/Chemokine 9-plex MAG. protocols.io dx.doi.org/10.17504/protocols.io.hvhb636 Published: 01 Aug 2017 + +## Protocol + +### Step 1: Preparation of Samples/Reagents for Immunoassay + +### Step 2: Preparation of Serum/Plasma +- **Thaw Time:** Thaw the samples completely on ice, mix well by shaking on a plate shaker for 1 min. at RT (20-25°C) and centrifuge (1,700 xg, 10 minutes, 4°C) prior to use in the assay to remove particulates. + +### Step 3: Preparation of Antibody-Immobilized Beads +- Sonicate each antibody-bead vial for 30 seconds; vortex for 1 minute. +- Add 150 µL from each antibody bead vial to the Mixing Bottle and bring final volume to 3.0 mL with Bead Diluent. Vortex the mixed beads well. +- **Storage:** Unused portions may be stored at 2-8°C for up to one month. + ``` + Example: When using 9 antibody-immobilized beads, add 150 µL from each of the 9 bead sets to the Mixing Bottle. Then add 1.65 mL Bead Diluent. + ``` + +### Step 4: Preparation of Quality Controls +- **Reconstitution Time:** Before use, reconstitute Quality Control 1 and Quality Control 2 with 250 µL deionized water. +- Invert the vial several times to mix and vortex. Allow the vial to sit for 5-10 minutes and then transfer the controls to appropriately labeled polypropylene microfuge tubes. +- **Storage:** Unused portion may be stored at ≤ -20°C for up to one month. + +### Step 5: Preparation of Wash Buffer +- Bring the 10X Wash Buffer to room temperature and mix to bring all salts into solution. +- Dilute 30 mL of 10X Wash Buffer with 270 mL deionized water. +- **Storage:** Store unused portion at 2-8°C for up to one month. + +### Step 6: Preparation of Serum Matrix + - **Reconstitution Time:** Add 1.0 mL Assay Buffer to the bottle containing lyophilized Serum Matrix. Mix well. Allow at least 10 minutes for complete reconstitution. + - **Storage:** Store leftover reconstituted Serum Matrix at ≤ -20°C for up to one month. + +### Step 7: Preparation of Human Metabolic Hormone Panel Standard +- **Reconstitution Time:** + 1. Prior to use, reconstitute the Human Metabolic Hormone Panel Standard with 250 µL deionized water to give STD7. + 2. Invert the vial several times to mix. Vortex the vial for 10 seconds. Allow the vial to sit for 5-10 minutes and then transfer the standard to an appropriately labeled polypropylene microfuge tube. + 3. **Storage:** This standard will be termed STD7; the unused portion may be stored at ≤ -20°C for up to one month. + +### Step 8: Preparation of Working Standards +- Label six polypropylene microfuge tubes STD6, STD5, STD4, STD3, STD2, and STD1. +- Add 200 µL of Assay Buffer to each of the six tubes. +- Prepare serial dilutions by adding 100 µL of STD7 reconstituted standard to the STD6 tube, mix well and transfer 100 µL of the STD6 standard to the STD5 tube, mix well and transfer 100 µL of the STD5 standard to the STD4 tube, mix well and transfer 100 µL of the STD4 standard to STD3 tube, mix well and transfer 100 µL of the STD3 standard to the STD2 tube and mix well, transfer 100 µL of the STD2 standard to the STD1 tube and mix well. The 0 pg/mL standard (Background) will be Assay Buffer. + + | Standard | Volume of Deionized Water to Add (mL) | Volume of Standard to Add (mL) | Original (STD7) Concentration (pg/mL) | Volume of Assay Buffer to Add (mL) | Volume of Standard to Add (mL) | + |----------|---------------------------------------|---------------------------------|---------------------------------------|--------------------------------------|---------------------------------| + | STD7 | 0 | - | 2500 | 0 | - | + | STD6 | 200 | 100 | 1250 | 200 | 100 | + | STD5 | 200 | 100 | 625 | 200 | 100 | + | STD4 | 200 | 100 | 312.5 | 200 | 100 | + | STD3 | 200 | 100 | 156.25 | 200 | 100 | + | STD2 | 200 | 100 | 78.125 | 200 | 100 | + | STD1 | 200 | 100 | 39.06 | 200 | 100 | + | STD0 | - | - | 0 | 200 | 0 | + +- After dilution, each tube has the following concentrations for each analyte: + ``` + Standard Tube # | Concentration (pg/mL) + ----------------|---------------------- + GIP | - + Ghrelin | - + GLP-1 | - + Glucagon | - + PP | - + PYY | - + Amylin | - + C-Peptide | - + Insulin | - + Leptin | - + ``` + +### Step 9-24: Further Preparation and Setup + ``` + 7 + 6 + 7 + 4 + 6 + 22 + 2 + 2 + 3205.8 + 53 + 7 + 5 + 9 + 31,2354 + 1370.47 + 40.71,8523,7045 + 21,1112,2225,5561,1116 + 73,3336,66716,66733,33372,00010,00020,00050,00010,00010,0001IMUNNOASAY PROCEDURE + ``` + +### Step 25: Prewet Plate +- Prewet plate by pipetting 200 µL of Assay Buffer into each well of the MAG Plate. +- Seal and mix on a plate shaker for 10 minutes at room temperature (20-25°C). + +### Step 26: Remove Assay Buffer +- Decant Assay Buffer and remove residual amount from all wells by inverting the plate and tapping it smartly onto absorbent towels several times. +- Add 25 µL of each Standard or Control into the appropriate wells. Add 25 µL Assay Buffer to the 0 pg/mL standard (Background). + +### Step 27: Adding Assay Buffer +- Add 25 µL of Assay Buffer to the sample wells. + +### Step 28: Adding Serum Matrix Solution +- Add 25 µL of the Serum Matrix solution to the background, appropriate standards, and control wells. + +### Step 29: Adding Sample to Wells +- Add 25 µL of Sample into the appropriate wells. + +### Step 30: Adding Beads +- Vortex Mixing Bottle and add 25 µL of the mixed Beads to each well. + ``` + Note: During addition of Beads, shake bead bottle intermittently to avoid settling. Due to the composition of magnetic beads, you may notice a slight color in the bead solution. This does not affect the performance of the beads or the kit. + ``` + +### Step 31: Incubation +- Seal the plate with a plate sealer, cover it with the lid. +- Wrap a rubber band around the plate holder, plate and lid and incubate with agitation on a plate shaker overnight (16-18 hours) at 4°C. + +### Step 32: Remove Fluid +- Gently remove fluid by aspiration. + +### Step 33: Washing Plate +- Wash plate 3 times with 200 µL/well of Wash Buffer, removing Wash Buffer by aspiration between each wash. + +### Step 34: Adding Detection Antibodies +- Add 50 µL of Detection Antibodies into each well. + ``` + Note: Allow the Detection Antibodies to warm to room temperature prior to addition. + ``` + +### Step 35: Incubating with Agitation +- Seal, cover with lid, and incubate with agitation on a plate shaker for 30 minutes at room temperature. + ``` + DO NOT WASH AFTER INCUBATION. + ``` + +### Step 36: Adding Streptavidin-Phycoerythrin +- Add 50 µL Streptavidin-Phycoerythrin to each well containing the 50 µL of Detection Antibodies. +- Seal, cover with lid and incubate with agitation on a plate shaker for 30 minutes at room temperature (20-25°C). + +### Step 37: Removing All Contents +- Gently remove all contents by aspiration. + +### Step 38: Wash Plate Again +- Wash plate 3 times with 200 µL/well Wash Buffer, removing Wash Buffer by aspiration between each wash. + +### Step 39: Adding Sheath Fluid +- Add 100 µL of Sheath Fluid to all wells. +- Resuspend the beads on a plate shaker for 5 minutes. + +### Step 40: Run Plate +- Run plate on Luminex 100™ IS. + +### Step 41: Data Analysis +- Save and analyze the data using Bio-Plex Manager software. + ``` + EQUIPMENT SETTINGS: + Events: 50, per bead region + Sample Size: 50 µL + Gate Settings: 5000 to 25,000 + Time Out: 60 seconds + QUALITY CONTROLS: The ranges for each analyte in Quality Control 1 and 2 are provided on the card insert or can be located at the MILLIPORE website: + www.millipore.com/techlibrary/index.do using the catalog number as the keyword. + ``` + +--- + +### Explanation of Terms and Reagents +- **Assay Buffer:** A solution used to prepare samples or reagents for an assay. +- **Centrifuge:** A device that uses centrifugal force to separate components of a mixture based on their density. +- **Bead Diluent:** A solution used to dilute beads in immunoassays. +- **Polypropylene Microfuge Tubes:** Small tubes made of polypropylene used in molecular biology for sample storage and centrifugation. +- **Serum Matrix:** A standard used to simulate the serum environment in the assay. +- **Standard:** A known concentration used to calibrate the assay. +- **Wash Buffer:** A solution used to wash away unbound substances in an assay. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/lung-covid-cwrvxd66.md b/markdown-output/lung-covid-cwrvxd66.md new file mode 100644 index 0000000000000000000000000000000000000000..e3d7ae7933acae09708d6f537b54b2059b78b070 --- /dev/null +++ b/markdown-output/lung-covid-cwrvxd66.md @@ -0,0 +1,114 @@ +```markdown +# Goal/Experiment: +To study the pulmonary sequelae of COVID-19 in patients with chronic kidney disease undergoing maintenance hemodialysis, particularly focusing on chest CT and pulmonary function tests after recovery from COVID-19 infection. + +# Lung COVID + +**Author**: Solos Jaturapisanukul +**Institution**: Faculty of Medicine, Vajira Hospital, Navamindradhiraj University +**Date**: July 06, 2023 + +## Abstract + +COVID-19 is associated with increased morbidity and mortality in patients with chronic kidney disease (CKD) on dialysis. CKD patients have an increased susceptibility to infections and this study investigates the pulmonary sequelae of COVID-19 in this patient group. + +## Objectives + +### Primary Objectives +- To study the Chest CT and pulmonary function tests in ESRD patients after recovering from COVID-19. + +### Secondary Objectives +- To study factors affecting the pulmonary sequelae after COVID-19 in CKD patients such as oxygen requirement, ventilator need, and levels of inflammatory cytokines such as interleukin-6 (IL-6) and C-reactive protein (CRP). + +## Study Design + +### Type of Study +- Prospective observational cohort study. + +### Inclusion Criteria +- CKD stage 5 requiring HD or continuous peritoneal dialysis (CAPD) for more than 3 months. +- Age 18-80 years. +- Diagnosis of COVID-19 confirmed by real-time polymerase chain reaction (RT-PCR) and recovered for more than 3 months previously. + +### Exclusion Criteria +- Patients with a history of chronic lung diseases i.e., chronic obstructive pulmonary disease (COPD) and restrictive lung disease. + +### Criteria to Withdraw from the Protocol +- Patients who have active disease after enrollment. + +### Participants +- 100 patients. + +### Place +- Faculty of Medicine, Vajira Hospital, Navamindradhiraj University. + +## Sample Size Calculation + +This study aimed to identify the prevalence of pulmonary abnormalities in both radiographic findings and pulmonary function test (PFT) results after recovery from COVID-19 infection. The sample size calculation was done using the following equation for estimating an infinite population proportion: + +\[ n = \frac{{Z_{\alpha/2}^2 \cdot p \cdot (1 - p)}}{{d^2}} \] + +where, +- \( n \) is the sample size, +- \( Z_{\alpha/2} \) is the area under the normal curve (1.96 for a 95% confidence level), +- \( p \) is the prevalence of lung abnormality (0.50, which yields the maximum sample size), +- \( d \) is the acceptable error (0.10). + +Thus, + +\[ n = \frac{{1.96^2 \cdot 0.50 \cdot (1 - 0.50)}}{{0.10^2}} = 97 \] + +100 cases were recruited by convenience sampling from a population of end-stage kidney disease (ESKD) patients who had recovered from COVID-19 infection. + +## Methodology + +### Data Collection +1. After obtaining written informed consent, demographic data and information regarding disease history, coexisting medical conditions, medication history, treatment during COVID-19 infection, including oxygen requirement, and laboratory data (complete blood count [CBC] and IL-6 and CRP levels) were collected. + +2. At least 3 months post-COVID-19 infection, all patients were evaluated for ongoing respiratory symptoms and underwent PFT and chest CT scans as follows: + +### Computed Tomography Technique + +High-resolution computed tomography (HRCT) was performed in a single breath-hold on a 128-slice multidetector computed tomography (MDCT) scanner (Ingenuity 128; Philips Healthcare Nederland B.V., Netherlands). HRCT was performed with a 1-mm slice thickness with the patient in the supine position during end-inspiration and prone position during end-inspiration. + +### Computed Tomography Interpretation + +Using a Picture Archiving and Communication System (PACS; EV Insite version 3.11.1.500; PSP Corporation, Japan), three radiologists with 9, 10, and 14 years of experience performed consensus interpretations blinded to the patients’ clinical information. The readers assessed the presence of specific CT patterns such as consolidation, ground-glass opacities, mosaic attenuation patterns, and others. CT scores reflecting the extent of lobar involvement were recorded using a five-point scale (0: 0%, 1: <5%, 2: 5%-25%, 3: 26%-50%, 4: 51%-75%, 5: >75%; range, 0-5; global score, 0-25). + +## Safety Considerations + +The participants will have minimal effects from the measurements performed in this study. The anticipated side effects from radiation or pulmonary function tests will be closely monitored. + +## Ethics Statement + +This trial was prospectively registered at ClinicalTrials.Gov Identifier: NCT05348759 on 26/04/2022 and was approved by the institutional review board of the Faculty of Medicine, Vajira Hospital, Navamindradhiraj University, Bangkok, Thailand (COA 302/64). + +## Expected Outcomes + +This trial contributes to new knowledge, including the consequences of the most affected organ (lung) after COVID-19, which has rarely been reported previously. In addition, factors associated with poor pulmonary outcome will be considered. + +## Dissemination of Results + +The data supporting the findings of this study are available from the corresponding author. + +## Additional Information + +**License**: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. + +**Protocol Status**: Working +**Created**: July 05, 2023 +**Last Modified**: July 06, 2023 +**Protocol ID**: 84501 + +**Link to DOI**: [dx.doi.org/10.17504/protocols.io.j8nlkoeyxv5r/v1](https://dx.doi.org/10.17504/protocols.io.j8nlkoeyxv5r/v1) + +**Attachment**: [Trial Protocol Lung COVID.docx](#) + +**Protocol Citation**: Solos Jaturapisanukul 2023. Lung COVID. *Protocols.io* [dx.doi.org/10.17504/protocols.io.j8nlkoeyxv5r/v1](https://dx.doi.org/10.17504/protocols.io.j8nlkoeyxv5r/v1) + +**Keywords**: COVID-19, CKD, ESRD, HRCT, pulmonary function test, interleukin-6, C-reactive protein, prospective cohort study + +**Trial Protocol Lung COVID.pdf** + +**endofoutput** +``` diff --git a/markdown-output/maintenance-culture-of-ipscs-basic-protocol-1-48vgzw6.md b/markdown-output/maintenance-culture-of-ipscs-basic-protocol-1-48vgzw6.md new file mode 100644 index 0000000000000000000000000000000000000000..a58d98e0a01cce5d7488a6300d4141da1ddbdc14 --- /dev/null +++ b/markdown-output/maintenance-culture-of-ipscs-basic-protocol-1-48vgzw6.md @@ -0,0 +1,123 @@ +```markdown +## Goal/Experiment: +The goal of this protocol is the maintenance and culture of human induced pluripotent stem cells (iPSCs). This involves steps for proper cell growth, division, and maintenance in a pluripotent state using specialized media and reagents. + +# Maintenance Culture of iPSCs (Basic Protocol 1) + +### Authors +Michael S. Fernandopulle, Ryan Prestil, Christopher Grunseich, Chao Wang, Li Gan, Michael E. Ward +National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland; Gladstone Institute of Neurological Disease, Gladstone Institutes, San Francisco, California + +### Abstract +Human iPSCs are a robust model for studying human biology due to their rapid proliferation, genomic stability, and differentiation potential. This protocol outlines standardized practices adapted from multiple publications for maintaining iPSCs in a pluripotent state, with a focus on using Essential 8 (E8) medium, minimizing reagent use, and providing practical recommendations for different culture conditions. + +### Keywords +iPSC maintenance, E8 medium, pluripotency, cell culture, Matrigel, ROCK inhibitor + +## Materials + +### Reagents +- **Matrigel, hESC-qualified** (Corning, cat. no. 354277) +- **DMEM/F12 medium** (Gibco, cat. no. 11320033) +- **hiPSCs** (Coriell Institute, cat. no. GM25256) +- **E8 medium** (multiple formulations available; see Table 1) + - **DMEM/F12, HEPES** (Thermo Fisher, cat. no. 11330032) + - **L-Ascorbic acid 2-phosphate sesquimagnesium salt hydrate** (Sigma Aldrich, cat. no. A8960) + - **Sodium selenite** (Sigma Aldrich, cat. no. S5263) + - **Sodium bicarbonate** (Sigma Aldrich, cat. no. S3817) + - **Sodium chloride** (Sigma, cat. no. S7653) + - **Sodium hydroxide** (Sigma, cat. no. 71463) + - **Hydrochloric acid solution** (Sigma, cat. no. H9892) + - **Insulin solution human** (Sigma, cat. no. I9278) + - **Recombinant Human TGF-β1 (HEK293 derived)** (Peprotech, cat. no. 100-21) + - **Recombinant Human FGF-basic (154 a.a.)** (Peprotech, cat. no. 100-18B) + - **holo-Transferrin human** (Sigma, cat. no. T0665) +- **70% Ethanol** +- **Rho-associated protein kinase (ROCK) inhibitor Y-27632** (Tocris Bioscience, cat. no. 1254) +- **Phosphate-buffered saline (PBS) without calcium or magnesium** (Gibco, cat. no. 10010049) +- **0.5 mM EDTA in PBS** (Gibco, cat. no. AM9260G) +- **Accutase** (Gibco, cat. no. A1110501) +- **DMSO** (Sigma, cat. no. 472301) +- **Fetal Bovine Serum (FBS), qualified, heat inactivated** (Gibco, cat. no. 16140071) +- **Liquid nitrogen** +- **CoolRack M30** (BioCision BCS-108) + +### Equipment +- **P2, P20, P200, and P1000 micropipettors and tips** (Gilson) +- **Sterile 5, 10, and 25-ml serological pipets** (Corning, cat. nos. 356543, 356551, and 357535) +- **Sterile 15- and 50-ml polypropylene conical tubes** (Corning, cat. nos. 352096 and 352070) +- **Sterile polystyrene 10-cm tissue-culture dishes and 6-well, 12-well, and 24-well plates** (Corning, cat. nos. 353003, 353046, 353043, and 353047) +- **Laminar flow biological safety cabinet (BSC)** +- **Vacuum aspirator and aspirating pipets** (Fisher, cat. no. 1367820) +- **Phase-contrast and fluorescent microscope with 4x, 10x, 20x objectives** (Nikon Eclipse Ti) +- **Cryovial freezing container** (CoolCell LX, BioCision; Mr. Frosty, Thermo Fisher) +- **1.5-ml cryogenic tubes** (Thermo Fisher, cat. no. 5000-1020) +- **Microcentrifuge for 1.5-ml tubes** +- **Steriflip-GP sterile centrifuge tube top filter unit** (Millipore, cat. no. SCGP00525) +- **Nalgene rapid-flow sterile disposable filter units with PES membrane** (Thermo, cat. nos. 568-0020 and 566-0020) + +## Protocol + +### Matrigel Coating +1. **Aliquoting Concentrated Matrigel:** + - Matrigel polymerizes rapidly at room temperature. It is imperative to aliquot stocks with pre-chilled tips and tubes to thaw the concentrated stock solution on ice. + - Gradually thaw a 5 ml bottle of Matrigel overnight at 4°C in a Styrofoam container in a refrigerator. Pre-chill microcentrifuge tubes by placing them on ice prior to use. + - Use a chilled 1-ml pipet tip to aliquot 500 µl of concentrated Matrigel into each microcentrifuge tube. Refreeze aliquots at -80°C. + +2. **Making Matrigel Coating Solution:** + - Aliquot 50 ml of cold DMEM/F12 into a conical tube. + - Using a P1000 pipet, transfer 1 ml of cold DMEM/F12 from the conical tube to the microcentrifuge tube containing 500 µl of concentrated Matrigel. Pipet up and down several times, then mix with the remaining 49 ml cold DMEM/F12. + - Repeat until the frozen concentrated Matrigel has been completely transferred to the 50-ml tube containing DMEM/F12. Invert several times to mix. + - Add one-half of the normal culture volume to the tissue culture surface (e.g., 1 ml per well of a 6-well plate). + - Transfer plates to a 37°C incubator for at least 1 hour, but overnight coating yields better results. Store prepared plates at 37°C or wrapped in Parafilm at 4°C. Use within 2 weeks. + +### Thawing iPSCs +1. **Prepare the biological safety cabinet (BSC):** + - Use tube racks, DMEM/F12, P1000 tips, conical tubes, and culture medium. +2. **Transfer cryovial of hiPSCs from liquid nitrogen and thaw in a 37°C water bath.** + - Thaw rapidly to limit exposure to DMSO. + - Sterilize cryovial with 70% ethanol and transfer it to the BSC. + - Pipet cell solution to a new 15-ml conical tube, rinse cryovial twice with 1 ml DMEM/F12. + +3. **Centrifuge the tube at 300 x g for 5 minutes at room temperature:** + - Aspirate the supernatant and resuspend in the culture medium with 10 µM Y-27632 ROCK inhibitor. + - Transfer to a Matrigel-coated plate and return to 37°C incubator. + - Distribute cells gently by shaking side-to-side and front-to-back. + - Next day, change the medium and replace with fresh E8 medium. + +### Splitting iPSCs +1. **EDTA-mediated split:** + - Use EDTA solution (0.5 mM) to dissociate cells. Incubate for 5-10 minutes at room temperature. + - Aspirate the EDTA solution, add culture medium, and gently pipette to dissociate cells. + - Transfer cells to new plates and return to 37°C incubator. + +2. **Accutase-mediated split:** + - Use Accutase to cover culture surface (0.5 ml/well for 6-well dish). + - Incubate for 5 minutes at 37°C. + - Detach cells and transfer to Matrigel-coated plates as described with EDTA. + +### Freezing iPSCs +1. **Cryopreservation medium:** + - Use 10% DMSO in FBS. + - Dissociate cells with EDTA or Accutase, resuspend in cryopreservation medium. + - Aliquot 1 ml suspension per cryovial, freeze in CoolCell container or Mr. Frosty at -80°C for two hours or overnight. + - Transfer cryovials to liquid nitrogen for long-term storage. + +### Manual Manipulation +1. **Pick-to-keep:** + - Using a pipet, isolate desired colonies and transfer to new culture plates. +2. **Pick-to-remove:** + - Remove undesired cells by mechanical scraping or aspiration. + +## External Links +- [DOI Link](https://doi.org/10.1002/cpcb.51) + +## References +Fernandopulle, M.S., Prestil, R., Grunseich, C., Wang, C., Gan, L., & Ward, M.E. (2018). Transcription-factor mediated differentiation of human iPSCs into neurons. Current Protocols in Cell Biology, e51. doi: https://doi.org/10.1002/cpcb.51 + +## Safety +- Follow all standard bio-safety and handling procedures while working with iPSCs. +- Always work in a certified biological safety cabinet (BSC) when handling iPSCs. + +endofoutput +``` diff --git a/markdown-output/malassezia-culture-on-modified-dixon-media-bjrakm2e.md b/markdown-output/malassezia-culture-on-modified-dixon-media-bjrakm2e.md new file mode 100644 index 0000000000000000000000000000000000000000..5f6ced8b94934897dcecc45109e15e487668e493 --- /dev/null +++ b/markdown-output/malassezia-culture-on-modified-dixon-media-bjrakm2e.md @@ -0,0 +1,120 @@ +```markdown +# Goal/Experiment: +This protocol describes the procedures to culture *Malassezia* species in modified Dixon (mDixon) media. It includes the recipe for mDixon, how to start the culture from freezer stock, culture maintenance, and how to make frozen stocks. + +# Malassezia culture on modified Dixon media + +## Authors +- Hao Li1 +- Genevieve Ong1 +- Shirlyn Goh1 + +1IBN, A*STAR + +## Abstract +The protocol describes the procedures to culture *Malassezia* species in modified Dixon (mDixon) media. It includes the recipe for mDixon, how to start the culture from freezer stock, culture maintenance, and how to make frozen stocks. + +### This Protocol Accompanies the Following Publication +Poh, SE., Goh, JPZ., Fan, C., Chua, W., Gan, SQ., Lim, PLK., Sharma, B., Leavesley, DI., Dawson, TL., and Li, H. "Identification of *Malassezia furfur* Secreted Aspartyl Protease 1 (MfSAP1) and Its Role in Extracellular Matrix Degradation". Front. Cell. Infect. Microbiol. 2020 10:148. + +### Protocol Citation +Hao Li, Genevieve Ong, Shirlyn Goh 2020. *Malassezia* culture on modified Dixon media. [protocols.io link](https://protocols.io/view/malassezia-culture-on-modified-dixon-media-bjrakm2e). + +### License +This is an open access protocol distributed under the terms of the [Creative Commons Attribution License](https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. + +### Created +Aug 14, 2020 +### Last Modified +Aug 14, 2020 + +## Materials + +### Reagents +- Trypan blue +- Modified Dixon broth pH 6 (Refer to the recipe given) + +### Materials +- Weighing boat +- 1.5 mL Eppendorf tubes +- Parafilm +- Serological pipettes +- Pipette gun +- Inoculation loops +- Microscopy glass slides and coverslips, or haemocytometer +- Corning® 125 mL Polycarbonate Erlenmeyer Flask with Vent Cap +- p20 micropipette and micropipette tips +- p200 micropipette and micropipette tips +- p1000 micropipette and micropipette tips + +### Equipment +- Microscope +- Incubator shaker with suitable size clamps +- Weighing balance +- pH meter + +### Safety Warnings +Dispose all wastes into the appropriate disposal bins. +- Dispose of all weighing boats and micropipette tips into the chemical waste bin +- Dispose of all biohazardous materials (e.g., inoculation loop) into the biohazard bin +- Dispose of all serological pipettes into the sharps bin or biohazard bin when involved in the handling of biohazardous materials +- Dispose of all glass slides/haemocytometers used in the sharps bin + +## Protocol + +### Preparation of Modified Dixon Broth +1. Place a magnetic stir bar into an autoclaved beaker with Mili-Q water and turn on the magnetic stirrer. + - Set the temperature at 60-70°C + - Temperature and stirring speed can be adjusted accordingly while preparing the media. + +2. Add the following ingredients while stirring. + - Add solid ingredients first before adding liquid ingredients. + +| Ingredient | Amount needed for 1L | +|------------------------|----------------------| +| Peptone | 6 g | +| Desiccated Oxbile (Bovine Bile) | 20 g | +| Malt Extract | 36 g | +| Oleic Acid | 2 mL | +| Tween 40 | 10 mL | +| Glycerol | 2 mL | + +**Note:** +- Tween 40 takes a long time to dissolve completely, so it’s recommended to prepare a working stock of 10% Tween 40 beforehand and add 100 mL of that. The same can be done for glycerol. +- Bacto Agar can be added to 1.5% (w/v) at the last step (after pH and volume adjustment) to make mDixon agar plates. + +3. Measure the pH of the media using a pH Meter and adjust accordingly using hydrochloric acid or sodium hydroxide to pH 6. + +### Starting Culture from Frozen Stock +4. Retrieve the frozen stock from -80°C and scrap some of the frozen stock using a sterilized pipet tip. If possible, this step should be performed in a Biosafety Cabinet to minimize contamination. + +5. Inoculate 12 mL of pre-warmed mDixon culture in a 125 mL flask with the frozen stock. Alternatively, the frozen stock can be streaked out onto a mDixon agar plate. + +6. Incubate culture at 32°C. For planktonic culture, shake flask at 150 rpm. For fast growing *Malassezia* species (e.g., *M. furfur*), planktonic culture will be confluent in 2 days after inoculation. For slower growing species (e.g., *M. globosa*), planktonic culture will be confluent in 3-4 days after inoculation. + +### Maintaining Planktonic Culture +7. Warm up the media in a 37°C water bath. + +8. Retrieve the culture flask and aspirate most of the culture, leaving around 1 mL of culture. + - Bleach the aspirated culture for at least 20 min before disposing. + +![M. globosa culture in mDixon](image_link) +*Note that *Malassezia* cultures typically have two phases: a liquid planktonic phase and a solid sessile phase that appears as a ring around the air-liquid interface. * + +9. Pipette in 12mL of the warmed media into the shaker flask and place the shaker flask back into the incubator shaker. + +### Monitoring Viability +10. Add 10 µL of trypan blue stain onto a glass microscopy slide and mix with 10 µL of the fungal culture. + +11. Use a light microscope with at least 40X lens to check fungal growth. Most of the fungal cells would be stained light blue, while the dead cells are stained dark blue. + - Note: cell viability and count can also be done at this point using a hemacytometer. + +### Freezing Down Pellet +12. Grow planktonic culture to late log/early stationary phase. + +13. Spin culture down at 8000 rpm for 5 mins and aspirate the supernatant. + +14. Resuspend culture pellet in the same volume of 25% glycerol in mDixon, aliquot in screw cap freezer tubes and freeze at -80°C. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/mapseq-multiplexed-analysis-of-projections-by-sequ-bsm9nc96.md b/markdown-output/mapseq-multiplexed-analysis-of-projections-by-sequ-bsm9nc96.md new file mode 100644 index 0000000000000000000000000000000000000000..6e4343fab4d7f0f996bddcb4d05c7f5be2cd9cbe --- /dev/null +++ b/markdown-output/mapseq-multiplexed-analysis-of-projections-by-sequ-bsm9nc96.md @@ -0,0 +1,251 @@ +```markdown +# Goal/Experiment: +The goal of this experiment is to perform MAPseq (Multiplexed Analysis of Projections by Sequencing), a high-throughput mapping of single-neuron projections by sequencing barcoded RNA. This protocol describes the steps involved in processing samples for MAPseq, specifically focusing on RNA extraction, reverse transcription, amplification, and sequencing preparation. + +# MAPseq (Multiplexed Analysis of Projections by Sequencing) Sample Processing Protocol + +## Authors +- Huiqing Zhan¹ +- Justus Kebschull¹ +- Anthony M. Zador¹ +- (¹Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724, USA) + +## Abstract +This protocol describes sample processing steps of MAPseq, a high-throughput mapping of single-neuron projections by sequencing of barcoded RNA, as described in details by Kebschull et al., 2016. In MAPseq, a brain area of interest is infected with a Sindbis barcoded library. After 40-44 hrs, the injection and the projection sites of interest are dissected, processed and sequenced using this protocol. + +## External Link +[https://pubmed.ncbi.nlm.nih.gov/27545715/](https://pubmed.ncbi.nlm.nih.gov/27545715/) + +## Protocol Citation +Huiqing Zhan, Justus Kebschull, Anthony M. Zador 2021. MAPseq (Multiplexed Analysis of Projections by Sequencing) sample processing protocol. Protocols.io. +[https://dx.doi.org/10.17504/protocols.io.bsm9nc96](https://dx.doi.org/10.17504/protocols.io.bsm9nc96) + +## Keywords +MAPseq, Sequencing of Barcoded RNA, Mapping single neurons + +## License +This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. + +## Guidelines +- Extra caution should be taken to avoid cross sample contamination during RNA extraction and reverse transcription. +- Use PCR II test to decide the optimal PCR II cycles. +- Try to use the least PCR II cycle to minimize the template switching rate. + +## Materials + +| Reagent/Kit | Vendor | Catalog Number | +|-------------------------------------------|-----------------------|-----------------| +| Trizol reagent | Thermo Fisher | 15596018 | +| Superscript IV | Thermo Fisher | 18090010 | +| Second Strand cDNA Synthesis Kit | Thermo Fisher | A48571 | +| AMPure XP Beads | Beckman Coulter | A63881 | +| Exonuclease I (E. coli) | NEB | M0293S | +| RNAsin Ribonuclease Inhibitor | Promega | N2611 | +| AccuPrime™ Taq DNA Polymerase, high fidelity | Thermo Fisher | 12346086 | +| Wizard® SV Gel and PCR Clean-Up System | Promega | A9282 | +| Agilent High Sensitivity DNA Kit | Agilent Technologies | 5067-4626 | +| Qiagen MinElute Gel Extraction Kit | Qiagen | 28606 | + +## Primer Sequences + +- **RT primer sequence**: + `CTT GGC ACC GGA GAA TTC CAN NNN NNN NNN NNN XXX XXX XXX TTC TAC AGC TAG CGG TGG TCG` + (N: UMI, X: SSI) +- **Spike-in RNA sequence**: + `GTC ATG ATC ATA ATA GCA CTC ACT ATA GGG GAC GTG TAC AAG TAG ACA GT AAT GAT AAT GGC ACC GAC TAC ACT CTT GCC TGA GCC ACT CTT CGC AGG GCT CTT GGC NNN NNN NNN TCC NNN GCG CAT TGT GTA GGT GAA CGA CGC CGC GTA CCA GTC CGA CTA GTA GCA CCA CGA CTA GCA Where ATACGTA is barcode and N12 is UMI.` +- **Nested1st gfpF primer sequence**: + `CTG TAC AAG TAA ACG GCT AAT G` +- **Nested2nd R primer sequence**: + `CAA GCA GAA GAC GGC ATA CGA CGT GAT GGT GGT CTT CGA CTT TCG CAC CTG CTA AGA ATT CCA` +- **Sol I primer sequence**: + `AAT GAT ACC GGC ACC ACC GA` +- **Sol II primer sequence**: + `CAA GCA GAA GAC GGC ATA CGA` + +## Protocol Steps + +### RNA Extraction + +1. **Dissection and Storage**: + Each fresh frozen brain area of interest is dissected and kept in RNAse-free tubes at -80°C before RNA extraction. + +2. **RNA Extraction**: + Tissues are homogenized in 400 µl of Trizol either with a Pellet Pestle Motor or a tissue homogenizer, and then add 600 µl of Trizol to make 1 ml of Trizol/sample. + - Extract RNA according to manufacturer's protocol of Trizol Reagent. + - Dissolve RNA of each tissue sample into 13 µl of H₂O. + +### Reverse Transcription with Superscript IV + +3. **Reaction Setup**: + One reaction per sample. + - Add the following reagents (total 13 µl) into each well: + - 4 µl of RNA from each sample + - 6 µl of H₂O + - 1 µl of spike-in RNA + - 1 µl of 10 µM RT primer + - 1 µl of 10 mM dNTP + - Mix well. + +4. **Incubation**: + - Incubate at 70°C for 10 minutes. + - Transfer samples to ice for 5 minutes. + +5. **RT Addition**: + - Add per well (total 7 µl): + - 4 µl SSIV buffer + - 1 µl 0.1 M DTT + - 1 µl RNAsin + - 1 µl SSIV + - Mix well. + +6. **RT Thermocycling**: + - Incubate the mixture in a thermocycler: + - 10 minutes at 55°C + - 10 minutes at 80°C + - Store samples at 4°C or -20°C till next step. + +### AMPure XP Beads Cleanup + +7. **Pooling**: + - Pool all of the RT product from targets or injection sites. + - Do not mix target site with injection site. Mix well. + +8. **Bead Cleanup for Target Sites**: + - Add 1.8X AMPure XP beads. + - Mix by pipetting 10 times. + - Incubate for 10 minutes at room temperature. + - Aliquot into several tubes for easier elution. + - Use a magnetic rack, discard supernatant. + - Wash twice with 80% EtOH. + - Air dry pellet. + - Resuspend beads. + - Incubate for 5 minutes at room temperature. + - On magnetic rack, collect all supernatant in an Eppendorf tube. + +9. **Bead Cleanup for Injection Sites**: + - Same steps as for target sites. + +### Second Strand cDNA Synthesis + +10. **Reaction Setup**: + - Per 17 µl of bead purified product, add: + - 2.4 µl SSIV buffer + - 0.6 µl 0.1 M DTT + - 5.6 µl Second Strand buffer + - 0.75 µl 10 mM dNTPs + - 0.25 µl E. coli DNA ligase + - 1 µl DNA polymerase I + - 0.25 µl RNaseH + - Mix well. + +11. **Incubation**: + - Incubate the mixture in a thermocycler: + - 2 hours at 16°C + - Add 1 µl of T4 DNA polymerase + - Incubate for 10 minutes at 16°C + - Store sample at 4°C or -20°C till next step. + +### Beads Cleanup 2 + +12. Same steps as the first beads cleanup. + +### Exonuclease Treatment + +13. **Reaction Setup**: + - Per 16 µl of bead purified product, add: + - 2 µl Exonuclease buffer + - 2 µl Exo I + - Mix well. + +14. **Incubation**: + - Incubate the mixture in a thermocycler: + - 1 hour at 37°C. + - Heat inactivate at 80°C for 20 minutes. + - Store sample at 4°C or -20°C till next step. + +### PCR I + +15. **Reaction Setup**: + - Per well Exonuclease reaction, add: + - 25 µl Accuprime buffer + - 25 µl 10 µM nested1st gfpF primer + - 25 µl 10 µM nested2nd primer + - 2.5 µl Accuprime Pfx HF enzyme + - 152.5 µl H₂O + - Mix well and aliquot the mixture into PCR tubes. + +16. **Thermal Cycling**: + - Initial denaturation at 95°C for 2 minutes. + - Run 15 cycles of: + - 95°C for 15 seconds. + - 68°C for 2.5 minutes. + - Final extension at 68°C for 5 minutes. + +### Exonuclease Treatment + +17. Add 5 µl of Exo I to each 50 µl PCR reaction. + - Incubation at 37°C for 30 minutes. + - Heat inactivation at 80°C for 20 minutes. + +### Test PCR II + +18. Test optimal PCR II cycle number in a 25 µl reaction/test by adding: + - 2.5 µl of 1/10 diluted PCR I product. + - 2.5 µl Accuprime buffer. + - 2.5 µl 10 µM Sol I primer. + - 2.5 µl 10 µM Sol II primer. + - 0.25 µl Accuprime Pfx HF enzyme. + - 14.75 µl H₂O. + +19. **Thermal Cycling**: + - Initial denaturation at 95°C for 2 minutes. + - Run different cycles of: + - 95°C for 15 seconds. + - 68°C for 1 minute. + - Final extension at 68°C for 5 minutes. + - For pooled target sites: run 14, 17, 20, 23, 26, and 29 cycles. + - For pooled injection sites: run 11, 14, 17, 20, and 23 cycles. + +20. Load test PCR II products on 2% agarose gel to find out the lowest cycle number which could generate a single and clean 230bp band. + +### PCR II + +21. **For pooled injection sites**: + - Use 12 ml of PCR II reaction. + - For pooled target sites: + - 3 ml for <12 samples. + - 6 ml for <40 samples. + - 12 ml for <40 samples. + +22. **Reaction Setup**: + - In each PCR II reaction, add same concentration of PCR I product as used in the test PCR II: + - 1x Accuprime buffer. + - 1 µM Sol I primer. + - 1 µM Sol II primer. + - 1U of Accuprime Pfx HF enzyme/50 µl of reaction. + +23. Perform thermal cycling as follows: + - Initial denaturation at 95°C for 2 minutes. + - Run optimal cycles of: + - 95°C for 15 seconds. + - 68°C for 2.5 minutes. + - Final extension at 68°C for 5 minutes. + +### Gel Purification + +24. Purify PCR product using Wizard® SV Gel and PCR Clean-Up System from Promega and elute the product into 40 ul of H₂O per 1 ml of PCR product. + +25. Load purified PCR product into 2% agarose gel. + +26. Cut ~230bp band from the gel and purify the product with MinElute Gel Extraction Kit from Qiagen. + +### Bioanalyzer Test with Agilent High Sensitivity DNA Kit + +27. Load purified 230bp PCR product on a DNA bioanalyzer chip using Agilent High Sensitivity DNA Kit to confirm its size and quantity before submitting for sequencing. + +### Next Generation Sequencing (NGS) + +28. Submit purified 230bp product for an Illumina NextSeq500 high output run at paired end 36 using the SBS3T sequencing primer for paired end 1 and the Illumina small RNA sequencing primer 2 for paired end 2. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/max-boost-keto-ultra-burn-better-out-come-does-it-ca6yshfw.md b/markdown-output/max-boost-keto-ultra-burn-better-out-come-does-it-ca6yshfw.md new file mode 100644 index 0000000000000000000000000000000000000000..2a1d6f9197eb0d383f0712fca77f14bec57f774d --- /dev/null +++ b/markdown-output/max-boost-keto-ultra-burn-better-out-come-does-it-ca6yshfw.md @@ -0,0 +1,80 @@ +```markdown +# Goal/Experiment: +The goal of this experiment is to evaluate the efficacy and potential benefits of the dietary supplement Max Boost Keto Ultra Burn in promoting weight loss by maintaining a state of ketosis. + +# Max Boost Keto Ultra Burn [BETTER OUT COME] Does It Really Work? + +#### H A +1. St. Johns River State College + +![DOI](dx.doi.org/10.17504/protocols.io.5qpvobwmxl4o/v1) + +--- + +## DISCLAIMER: FOR INFORMATIONAL PURPOSES ONLY; USE AT YOUR OWN RISK + +The protocol content here is for informational purposes only and does not constitute legal, medical, clinical, or safety advice, or otherwise; content added to **protocols.io** is not peer reviewed and may not have undergone a formal approval of any kind. Information presented in this protocol should not substitute for independent professional judgment, advice, diagnosis, or treatment. Any action you take or refrain from taking using or relying upon the information presented here is strictly at your own risk. You agree that neither the Company nor any of the authors, contributors, administrators, or anyone else associated with **protocols.io**, can be held responsible for your use of the information contained in or linked to this protocol or any of our Sites/Apps and Services. + +Also, your body will remain in ketosis consuming with smoldering heat fat assuming that you keep those ketone levels high. + +## Product Information + +**Primary Information:** +- **Product Name:** Max Boost Keto Ultra Burn +- **Location:** United States +- **Category:** Weight Loss +- **Side Effects:** No Major Side Effects +- **Availability:** Online +- **Rating:** ⭐⭐⭐⭐⭐ +- **Where to Buy Online:** [www.Maxboostketo.com](https://www.Maxboostketo.com) + +## Benefits of MaxBoost Keto Diet Pills + +- Helps You Shed Stubborn Fat Away +- Good For Tackling Your Belly Fat +- Uses Ketosis To Get You Slimmer +- Boosts Energy And Natural Alertness +- Improves Mood And Motivation +- Suppresses Your Appetite Naturally + +## Max Boost Keto Ultra Burn Weight Loss Support Reviews +**Max Boost Keto Ultra Burn recipe** utilizes your body’s own fat-consuming ability to get you results. 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(2022). Measurement of activity-related impedance changes in porcine subdiaphragmatic nerve. protocols.io. [https://protocols.io/view/measurement-of-activity-related-impedance-changes-b5chq2t6](https://protocols.io/view/measurement-of-activity-related-impedance-changes-b5chq2t6) + +## Keywords +- Temporal dispersion +- Ex-vivo electrophysiology +- Subdiaphragmatic vagus nerve + +--- + +## Materials and Reagents + +- **NaCl (6.604 g/L)**: Sodium chloride, a common reagent for physiological solutions. +- **KCl (0.357 g/L)**: Potassium chloride, used in cell physiology to maintain osmotic balance. +- **CaCl₂ (0.227 g/L)**: Calcium chloride, provides calcium ions necessary for nerve function. +- **KH₂PO₄ (0.163 g/L)**: Potassium dihydrogen phosphate, used as a buffering agent. +- **MgSO₄ (0.144 g/L)**: Magnesium sulfate, provides magnesium ions necessary for biochemical reactions. +- **NaHCO₃ (2.1 g/L)**: Sodium bicarbonate, used as a strong buffer to maintain pH. +- **Dextrose (0.901 g/L)**: Provides energy to maintain cell metabolism. +- **Ringer’s solution**: A solution that mimics the ion concentration of bodily fluids, used for bathing nerve tissues. + +## Equipment + +- **Magnetic stirrer**: Used for mixing the solutions. +- **Tissue organ bath perfusion chamber**: Used to hold the nerve tissue during experimentation. +- **Peristaltic pump**: Ensures continuous perfusion of the solution through the nerve tissue. +- **Thermostatic circulator and cooling unit**: Maintains the temperature of the solution. +- **Cuffs with radial arranged electrodes**: For recording electrical activity. +- **Amplifier (e.g., actiChamp)**: To amplify electrical signals. +- **Arduino-based triggering system**: For precise timing and control of stimulus delivery. +- **Keithley 6221**: Current source for stimulation. +- **AgCl EEG-type electrode**: For grounding and noise reduction. + +--- + +## Protocol + +1. **Prepare Nerve Ringer’s solution.** + - Place a glass beaker with 1L of distilled water on the magnetic stirrer, switch to medium speed stirring and add the following reagents: + - 6.604 g NaCl + - 0.357 g KCl + - 0.227 g CaCl₂ + - 0.163 g KH₂PO₄ + - 0.144 g MgSO₄ + - 2.1 g NaHCO₃ + - 0.901 g Dextrose + +2. **Oxygenate and circulate the solution.** + - Pour the solution into a reservoir connected with a tissue organ bath perfusion chamber and a peristaltic pump for continuous perfusion. + - Ensure the perfusate contains 95% oxygen and 5% CO₂ at ~30°C, using a thermostatic circulator and cooling unit. + +3. **Bath the nerve in the solution.** + - Place the freshly extracted nerve into the tissue organ bath perfusion chamber filled with the Ringer’s solution. + +4. **Fabricate the electrodes.** + - Create silicone rubber cuffs with six radially arranged electrodes using the method described in Chapman et al., 2018. Use stainless steel electrodes (0.2x2.3 mm²) and coat them with PEDOT:pTS. + +5. **Arrange the electrodes around the nerve.** + - Place four manufactured cuffs around the nerve so that the first and last two cuffs are close (~3 cm) while the middle cuffs are around 10 cm apart. + +6. **Connect the cuffs to the amplifier.** + - Use the amplifier (e.g., actiChamp) directly or through a breakout board. Configure the recording software to set the last electrode on the last cuff as the reference electrode. + - Connect the ground electrode (GND) to the Ringer’s solution in the nerve bath and another AgCl EEG-type electrode to the earthed table for noise reduction. + +7. **Attach the stimulating current source.** + - Connect the driving electrodes of the current source (Keithley 6221) to the stimulating electrodes on the cuffs. + +8. **Set up the triggering system.** + - Use a custom-made Arduino-based system to trigger stimulating pulses as needed for the experiment (e.g., continuous or trains). + +9. **Determine optimal current for nerve stimulation.** + - Apply 2 Hz 50 µs biphasic pulses starting at 10 mA. Increase the current incrementally (20 mA, 30 mA, etc.) while observing compound action potentials (CAPs). Select the desired current level and nerve samples showing amplitudes higher than 10 mV for long-lasting C-fiber activity. + +10. **Connect the dZ measurement current source.** + - Attach the electrodes for AC injection and dZ measurement on cuffs 2, 3, or 4. + +11. **Measure dZ at the nearest cuff (3 cm from stimulation site).** + - Apply AC of 200-400 µA using cuffs 2 or 3 with continuous 2 Hz stimulation. Record for 10 minutes for averaging. + +12. **Measure dZ at dispersed sites (15 cm and 20 cm from stimulation).** + - Apply AC of 200-400 µA to cuffs 3 or 4. Use intermittent stimulation with predefined strategies (e.g., 10 Hz trains for 0.6 s with 5 s rest) for 30 minutes. Repeat for various AC frequencies and stimulation strategies. + +--- + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/measurement-of-duodenal-motility-using-implanted-s-2irgcd6.md b/markdown-output/measurement-of-duodenal-motility-using-implanted-s-2irgcd6.md new file mode 100644 index 0000000000000000000000000000000000000000..4b5801f8f1f37275b42f9e0bffa9c4bb485e1027 --- /dev/null +++ b/markdown-output/measurement-of-duodenal-motility-using-implanted-s-2irgcd6.md @@ -0,0 +1,171 @@ +```markdown +# Goal/Experiment: +Measurement of duodenal motility using implanted strain gauges. + +# Measurement of Duodenal Motility Using Implanted Strain Gauges + +**Authors** +- Terry Powley, Zhenjun Tan, Matthew Ward +- Purdue University + +**Published Date:** January 8, 2020 +**DOI:** [10.17504/protocols.io.2irgcd6](dx.doi.org/10.17504/protocols.io.2irgcd6) + +## Abstract + +This protocol describes a process for the measurement of electrical stimulation-induced effects on duodenal motility in young adult Sprague-Dawley rats. Signals recorded from strain gauges attached to the proximal duodenal surface were used to measure the effect of stimulation by patch electrodes implanted at multiple sites across the rat stomach in an acutely anesthetized preparation. The effect of stimulation was quantified as the ratio of various motility assessments during and after stimulation vs. before stimulation, and the data was used to create a functional map of duodenal motor response to localized gastric stimulation. + +## Steps Materials + +| **Name** | **Catalog #** | **Vendor** | +|-----------------------------------|---------------------|-----------------------------| +| Sprague-Dawley | | Envigo | +| 2018 Teklad global 18% protein rodent diet | | Envigo | +| Isoflurane | NDC: 59399-106-01 | Akorn Animal Health | +| Vetbond | 1469SB | 3M corporation | +| Ketamine | 07-803-6637 | Patterson Veterinary | +| Xylazine | NDC: 59399-110-20 | Akorn Animal Health | + +### Animals + +1. **Two-to-four-month-old male Sprague-Dawley rats** + + ![Sprague-Dawley](https://via.placeholder.com/15x15.png) **Sprague-Dawley** + by Envigo + RRID: RGD_737903 + + The rats were housed in vented rack cages in an Association for Assessment and Accreditation of Laboratory Animal Care-approved temperature (22–24 °C) and humidity (40–60%) controlled colony room. The room was maintained on a 12-hour light–dark schedule. Pelleted chow + + ![rodent diet](https://via.placeholder.com/15x15.png)**2018 Teklad global 18% protein rodent diet** + by Envigo + + and filtered tap water were provided _ad libitum_. All husbandry practices conformed to the NIH Guide for the Care and Use of Laboratory Animals (8th edition) and were reviewed and approved by the Purdue University Animal Care and Use Committee. All efforts were made to minimize any suffering as well as the number of animals used. + +### Surgical Procedures + +2. **Preparation for Surgery** + + Animals were transferred to wire hanging cages the day before surgery and then fasted overnight with free access to water. + + - **Anesthesia** + + ![Isoflurane](https://via.placeholder.com/15x15.png) **Isoflurane** + by Akorn Animal Health + Catalog #: NDC: 59399-106-01 + + Rats were anesthetized with 5% isoflurane in an induction box and transferred to a + + - **Anesthesia System** + + ![Somnosuite](https://via.placeholder.com/15x15.png) **Somnosuite Low-flow Anesthesia System** + Kent Scientific SomnoSuite + + on a + + - **Surgery Platform** + + ![SurgiSuite](https://via.placeholder.com/15x15.png) **SurgiSuite Multifunction Surgery Platform** + Kent Scientific SurgiSuite + + The level of anesthesia was reduced to 2.5% isoflurane for the surgical procedure. A servo-controlled homoeothermic heating blanket, equipped with a rectal thermometer, was used to maintain body temperature at 36 °C. + +3. **Surgical Implantation of Patch Electrodes** + + After midline laparotomy, the stomach and 3-4 cm of proximal duodenum were exteriorized onto saline-soaked gauze pads. One to three custom-made stimulation patch electrodes + + ![Custom patch electrodes](https://via.placeholder.com/15x15.png) **Custom patch electrodes** + Microprobes for Life Science + + (each consisting of two Pt/Ir foil plates, each about 1 mm x 2 mm, spaced about 4 mm apart, mounted to a silicone pad) were sutured on the serosal surface of stomach. Electrodes were typically aligned with or at right angles to the angle of the local greater curvature. + +4. **Placement of Strain Gauges** + + A custom-made strain gauge (4 x 3.5 mm, Clunbury Scientific LLC, Bloomfield Hills, MI) constructed from two strain gauge elements + + ![Strain gauge](https://via.placeholder.com/15x15.png) **EA-06-031CE-350** + Micromeasurements EA-06-031CE-350 + + was glued to the serosal surface of the proximal duodenum (5–15 mm distal to pyloric sphincter) using + + ![Vetbond](https://via.placeholder.com/15x15.png) **Vetbond** + by 3M corporation + Catalog #: 1469SB + + The strain gauge was oriented parallel to the longitudinal or circular muscle. + +5. **Post-implantation** + + The fine wire leads attached to the strain gauge and patch electrodes were exteriorized and connected to a DC bridge amplifier and stimulator respectively. The animal was kept in a supine position with the abdominal area covered by saline-soaked gauze pads. Normal saline (2.0 ml/hr) was injected continuously i.p. using a syringe pump + + ![GenieTouch syringe pump](https://via.placeholder.com/15x15.png) **GenieTouch syringe pump** + Kent Scientific GenieTouch™ Syringe Pump + + The animal was then covered with a blanket to help maintain body temperature, and anesthesia was reduced to 1.5% isoflurane and maintained at that level for the reminder of the experiment. + +### Stimulation Experiment + +6. **Duodenal Motility Recording** + + After surgery, recording of duodenal motility began. Strain gauge measurements were made using a DC bridge amplifier system + + ![DC bridge amplifier](https://via.placeholder.com/15x15.png) **DC bridge amplifier (4 channel)** + MDE EXP-SG-4 + + Once the strain gauge signal reached a stable baseline and had maintained that stable baseline for at least 20 min, stimulation was provided by a PlexStim electrical stimulator + + ![PlexStim](https://via.placeholder.com/15x15.png) **PlexStim Electrical Stimulator** + Plexstor PlexStim + + Stimulation parameters were as follows: biphasic, I = 0.3 mA, pw = 0.2 ms, 10 Hz, 20s-on-40s-off, 5 one-minute cycles. + + Following stimulation, recording continued for about another hour. + +### Perfusion + +7. **Euthanasia and Perfusion** + + Once the experiment was complete, the animals were given a lethal dose of + + ![Ketamine](https://via.placeholder.com/15x15.png) **Ketamine** + by Patterson Veterinary + Catalog #: 07-803-6637 + + and + + ![Xylazine](https://via.placeholder.com/15x15.png) **Xylazine** + by Akorn Animal Health + Catalog #: NDC: 59399-110-20 + + (i.p. 275 mg/kg ketamine and 27.5 mg/kg xylazine). + + The locations of the plates of the electrodes used in the experiment were marked with blue suture thread before the electrodes were removed. + + To ensure that the stomach was normally distended at the time of fixation, the organ was inspected for normal distension or accommodation, and as required, physiological saline (3.3 ml/100 g wt) that had been warmed to body temperature was slowly infused into the stomach by gavage catheter. With the stomach normally dilated, the animal was first transcardially perfused through the vasculature with physiological saline and then with 4% paraformaldehyde in 0.1 mol/liter PBS; pH 7.4). After perfusion, the distal esophagus and the proximal duodenum were transected, and the stomach was freed and removed. The organ was then opened with a longitudinal cut along the greater curvature. Next, the ventral and dorsal stomach walls were separated by an incision along the lesser curvature, thus yielding two whole mounts per animal. + +### Electrode Location Measurement + +8. **Electrode Mapping** + + The ventral half stomach was placed in PBS in a dissecting dish under a stereomicroscope, with the inner surface facing up, and the locations of two plates on each the electrodes were clearly marked with pins, and a photograph of the stomach capturing the entire surface was then taken. + + The image of the stomach at a consistent magnification for each stomach to be measured was printed, and x and y locations of the midpoint of the electrode measured from the image, together with the size of the stomach itself so that electrode location could be reported as percentage measurements relative to the pylorus end of the stomach contour (x) and relative to the bottom edge of the stomach at the greater curvature (y). In addition, the orientation of the electrode relative to a line from the top of the limiting ridge (near the LES) to the bottom point near the greater curvature where the limiting ridge changes direction was measured. + +### Motility Data Analysis + +9. **Data Analysis** + + Recording of motility data began after surgery was complete and continued for at least an hour for stabilization of baseline motility, and then for at least another hour following completion of the stimulation experiment. Data analysis typically used two subsets of that entire recording: + + 1. 15 min immediately prior to stimulation; + 2. The 15 min immediately following the onset of stimulation (5 min during stimulation together with another 10 min of recording). + + The raw strain gauge data was filtered and rectified using lab-written MATLAB code. The same code provided three quantitative assessments of the ratio of motility during and after stimulation to motility before stimulation. These three assessments are: + + 1. Average amplitude ratio. + 2. Average frequency ratio (defined as the number of excursions exceeding 10% of maximum, per unit time). + 3. Motility index (MI) defined as the ratio of the area under the curve during and after stimulation to the area under the curve before stimulation per unit time. + + Results obtained from the various stimulation locations were mapped in a variety of ways including contour maps and maps of locations where MI exceeded 1.0 vs. locations where MI was less than 1.0. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/mechanistic-assays-part-7-of-safety-and-efficacy-o-bvdcn22w.md b/markdown-output/mechanistic-assays-part-7-of-safety-and-efficacy-o-bvdcn22w.md new file mode 100644 index 0000000000000000000000000000000000000000..bdfc6d81acf5afbf4f8bf1d069e147daccf1e092 --- /dev/null +++ b/markdown-output/mechanistic-assays-part-7-of-safety-and-efficacy-o-bvdcn22w.md @@ -0,0 +1,92 @@ +```markdown +Goal/Experiment: +This protocol aims to determine whether imatinib will slow the progression of the autoimmune destruction of beta cells and lead to the preservation of C-peptide secretion in Type 1 Diabetes Mellitus (T1DM) patients. This will allow assessment of diabetes-related objectives and the safety of imatinib in new-onset T1DM. + +# Mechanistic Assays (Part 7 of Safety and Efficacy of Imatinib for Preserving Beta-Cell Function in New-onset Type 1 Diabetes Mellitus) + +**Authors:** +Stephen Gitelman¹, Jeffrey A. Bluestone² + +¹Professor of Clinical Pediatrics, Department of Pediatrics University of California, San Francisco +²Professor of Medicine, Pathology, Microbiology & Immunology, University of California San Francisco + +**Abstract:** +This is Part 7 of "Safety and Efficacy of Imatinib for Preserving Beta-Cell Function in New-Onset Type 1 Diabetes Mellitus." The goal is to observe if imatinib slows autoimmune beta-cell destruction and preserves C-peptide secretion. + +**Keywords:** +Safety, Efficacy, Imatinib, Beta-cell function, New-Onset Type 1 Diabetes Mellitus + +## Contents: + +1. Rationale +2. Retention of Samples +3. Frozen PBMCs + - Functional Cell-Based Assays + - Flow Cytometry Panel Staining +4. Serum Assays + - Serum Autoantibody Analyses + - Serum Archive +5. Whole Blood-Gene-Expression Profiling +6. Whole Blood DNA-HLA Genotypes +7. Change in Beta Cell Function +8. References + +--- + +## 7.1 Rationale + +Imatinib's immunomodulating effects may reduce both adaptive and innate immune responses. The drug might restore tolerance to pancreatic beta cells by affecting T cells, B cells, and macrophages. + +## 7.2 Retention of Samples + +Specimens will be stored at the Immune Tolerance Network (ITN) repository for future mechanistic studies, enabling re-evaluation of immune responses and development of new assays. + +## 7.3 Frozen PBMCs + +Peripheral blood mononuclear cells (PBMCs) will be collected, processed, and stored for various immunological assays. + +### 7.3.1 Functional Cell-Based Assays + +- **Lymphocyte Subsets:** Analyze T and B cells, mast cells, and monocytes. +- **Stimulations:** Use anti-CD3 plus anti-CD28 and/or LPS for inflammatory responses. +- **Cytokine Production:** ELISPOT and intracellular staining. +- **Proliferation:** Assess via CSFE/3H-thymidine assays. +- **Flow Sorting:** Separation with CD4, CD25, CD127 antibodies. + +### 7.3.2 Flow Cytometry Panel Staining + +Examine phenotypical changes using markers like CD11c, CD40, CD80, CD86 to study DC maturation and function. T-cell subsets are analyzed for activation and expression of transcription factors. + +## 7.4 Serum Assays + +### 7.4.1 Serum Autoantibody Analyses + +Monitor autoantibodies against pancreatic islets, including anti-GAD65, anti-insulin, and anti-IA2. + +### 7.4.2 Serum Archive + +Archive plasma samples for future analysis of markers like cytokines and inflammatory molecules to determine imatinib's impact. + +## 7.5 Whole Blood-Gene-Expression Profiling + +Examine expression profiles via RT-PCR with a focus on cytokines and transcription factors to identify differences in T1DM responses to imatinib. + +## 7.6 Whole Blood DNA-HLA Genotypes + +HLA typing (Class I and II) and sequencing selected immune-response genes to correlate with disease progression and responses to imatinib. + +## 7.7 Change in Beta Cell Function + +Evaluate beta cell activity via C-peptide levels, plasma glucagon, and proinsulin levels, alongside the euglycemic hyperinsulinemic clamp test. + +## References + +1. Fitter S, Vandyke K, Schultz CG, White D, Hughes TP, Zannettino AC. Plasma adiponectin levels are markedly elevated in imatinib-treated chronic myeloid leukemia (CML) patients: a mechanism for improved insulin sensitivity in type 2 diabetic CML patients? J Clin Endocrinol Metab. Aug 2010;95(8):3763-3767. +2. Akirav EM, Lebastchi J, Galvan EM, et al. Detection of β cell death in diabetes using differentially methylated circulating DNA. Proc Natl Acad Sci U S A. Nov 2011;108(47):19018-19023. +3. Ferrannini E, Mari A, Nofrate V, Sosenko JM, Skyler JS, Group D-S. Progression to diabetes in relatives of type 1 diabetic patients: mechanisms and mode of onset. Diabetes. Mar 2010;59(3):679-685. +4. Cobelli C, Toffolo GM, Dalla Man C, et al. Assessment of beta-cell function in humans, simultaneously with insulin sensitivity and hepatic extraction, from intravenous and oral glucose tests. Am J Physiol Endocrinol Metab. Jul 2007;293(1):E1-E15. + +--- + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/media-for-fungal-culturing-bnuwmexe.md b/markdown-output/media-for-fungal-culturing-bnuwmexe.md new file mode 100644 index 0000000000000000000000000000000000000000..b70d41a219a7e9dafc4cc54610ad10f14a2df357 --- /dev/null +++ b/markdown-output/media-for-fungal-culturing-bnuwmexe.md @@ -0,0 +1,146 @@ +```markdown +Goal/Experiment: +To describe the different media and procedures used for fungal culturing within the Bark Beetle Mycobiome Research Coordination Network. + +# Media for Fungal Culturing + +## Authors +Jiri Hulcr¹, You Li¹, Sawyer Adams¹, Demian F Gomez¹ + +¹University of Florida + +*Published: Nov 20, 2020* + +Link to Protocol: [dx.doi.org/10.17504/protocols.io.bnuwmexe](https://dx.doi.org/10.17504/protocols.io.bnuwmexe) + +## Abstract +This protocol describes the different media for fungal culturing. It is part of the Bark Beetle Mycobiome (BBM) Research Coordination Network. + +For more information on the BBM international network: Hulcr J, Barnes I, De Beer ZW, Duong TA, Gazis R, Johnson AJ, Jusino MA, Kasson MT, Li Y, Lynch S, Mayers C, Musvuugwa T, Roets F, Seltmann KC, Six D, Vanderpool D, & Villari C. 2020. Bark beetle mycobiome: collaboratively defined research priorities on a widespread insect-fungus symbiosis. Symbiosis 81: 101–113 [https://doi.org/10.1007/s13199-020-00686-9](https://doi.org/10.1007/s13199-020-00686-9). + +## Equipment for Fungus Culture Work +- Burner +- Ethanol bath for utensils +- Ethanol spray +- Petri dishes for dissection +- Tape +- Forceps +- Gloves +- Kimwipes +- Lighter +- Media plates +- Parafilm +- Permanent lab marker +- Scalpel +- Scissors +- Pipette and tips for melting & crushing +- Tween +- (Loop for yeasts) + +### Beetle Samples +- Vials with EtOH and labels for beetle vouchers + +## Preparing Media Plates +1. After the plates have cooled, mark each stack with a line of the appropriate color (easy when plates are stacked). +2. Put them back in sleeves and tape those over. +3. On the tape, write the: + - Media type + - Your name + - Due date: The "due date" means that, if found in the fridge after this date, it can be discarded. +4. Wipe the hood completely with ethanol or bleach. +5. Store the new plates in the fridge. +6. In a week, check whether there are any contaminant colonies present. If a few are present, discard the contaminated plate(s). If many, discard the whole sleeve. + +## Nutrient Media for Abundant Mycangial Fungi +Additional media formulations can be found in the media table in the isolations database. + +### Potato Dextrose Agar (PDA) +- 39g PDA dried media from BD-Difco +- 1L water +- 10ml of Streptomycin (10,000 I.U./ML) +- Penicillin (10,000 MCG/ML) + +*To our experience, this produces richer cultures of mycangial fungi than other media.* + +### Yeast Malt Extract Agar (YMEA) +- 4g yeast extract +- 10g malt extract +- 4g dextrose +- 15g agar +- 1L water +- 10ml of Streptomycin (10,000 I.U./ML) +- Penicillin (10,000 MCG/ML) + +*Fungi grow slower than on PDA.* + +### Malt Extract Agar (MEA) +- Same as YMEA, without the yeast extract. + +### Potato Yeast Dextrose Agar (PYDA) +- 15g PDA +- 10g Agar +- 2g Yeast Extract +- 1L Water + +### Malt Extract Agar +- 15g MEA +- 10g Agar +- 2g Yeast Extract +- 1L Water + +## Antibiotics +**Do not autoclave agar media with antibiotics in it!** Add when media has cooled down to touch. + +- **Streptomycin/Penicillin**: Catalog #516106, supplied in powder. Mix into liquid media at a concentration of 100-200 ppm. For PDA, 100 mg/L is equivalent to 100ppm. +- **Cycloheximide**: For Ophiostomatales, including Raffaelea (but not Ambrosioella). Kolarik & Hulcr (2008) used 0.1 mg/L cycloheximide to select for ophiostomatoid mycangial fungi, recommended use between 0.1-0.5mb/L. Current protocol uses 0.5 mg/L. +- **Yeast Overgrowth Prevention**: Can be prevented by slapping a chunk of agar on top of the inoculum. + +*All media containing antibiotics should be kept in darkness and cold temperature, to prevent degradation.* + +### Ophiostoma Select Agar (OSA) aka CSMA +- 34g malt extract agar +- 4g agar +- Bring to 1L volume with deionized H2O +- Autoclave +- After autoclaving, at about 45 C, add: + - 0.1g cycloheximide + - 0.1g streptomycin sulphate + +## Media with Antibiotics +### Standard Acidified Potato-Dextrose Agar (APDA) +- 39g potato dextrose agar (Difco Laboratories, Detroit, Mich.) +- 1L deionized H2O +- Autoclave +- 1.25 mL of 20% lactic acid +- Streptomycin – 100 mg/L +- Chloramphenicol – 16 ug/mL – 0.16 mg/L + +### Acidified Weak Potato-Dextrose Agar (AWPDA) +- 12g potato-dextrose broth (Difco Laboratories) +- 16g Bacto-agar (Difco Laboratories) +- 1L deionized H2O +- Autoclave +- 0.3mL of 20% lactic acid +- Streptomycin – 100 mg/L +- Chloramphenicol – 16 ug/mL – 0.16 mg/L + +### MEA Acidified with Antibiotics +- 33.6g MEA (Difco Laboratories, Detroit, Mich.) +- 1L deionized H2O +- Autoclave +- 1.25 mL of 20% lactic acid +- Streptomycin – 100 mg/L +- Chloramphenicol – 16 ug/mL – 0.16 mg/L + +--- + +Citation: Jiri Hulcr, You Li, Sawyer Adams, Demian F Gomez (11/20/2020). Media for Fungal Culturing. [https://dx.doi.org/10.17504/protocols.io.bnuwmexe](https://dx.doi.org/10.17504/protocols.io.bnuwmexe) + +--- + +This is an open access protocol distributed under the terms of the [Creative Commons Attribution License](https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. + +--- + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/methylc-seq-using-ez-dna-methylation-gold-bisulfit-p88drzw.md b/markdown-output/methylc-seq-using-ez-dna-methylation-gold-bisulfit-p88drzw.md new file mode 100644 index 0000000000000000000000000000000000000000..529201063cda0e620654c99a0a52179a83090a05 --- /dev/null +++ b/markdown-output/methylc-seq-using-ez-dna-methylation-gold-bisulfit-p88drzw.md @@ -0,0 +1,75 @@ +```markdown +# Goal/Experiment: +The objective of this protocol is to perform MethylC-sequencing in Arabidopsis using the ACCEL-NGS® METHYL-SEQ DNA LIBRARY KIT (NGS Prep with Bisulfite-Converted DNA Capability; Cat. No. DL-ILMMS-12/48). + +# MethylC-seq using EZ DNA Methylation-Gold Bisulfite and Accel-NGS DNA Library Kits + +**Diep Ganguly, Steven Eichten** + +## Abstract +Protocol to use ACCEL-NGS® METHYL-SEQ DNA LIBRARY KIT (NGS Prep with Bisulfite-Converted DNA Capability; Cat. No. DL-ILMMS-12/48) to perform MethylC-sequencing in Arabidopsis. + +Code used to analyze raw sequencing data can be found: [GitHub Repository](https://github.com/dtrain16/NGS-scripts/tree/master/MethylC). + +## Before start + +Ensure you have fresh nuclease-free water (or low EDTA TE) and 80% ethanol. Magnetic beads stocks (e.g., AMPure, SPRIselect) and a magnetic stand that holds plates or eppendorf tubes will also be needed. + +Extract high-quality gDNA using the method of choice. The lab relies on the DNeasy Plant Mini Kit (Qiagen), but yields can be low despite good quality gDNA. For all resuspension steps, use low EDTA (0.1 mM) Tris (10 mM, pH 8) or the elution buffer provided in the DNeasy Plant Mini Kit (Qiagen). + +## Materials + +- **100 ml TE Buffer [1X]**: pH 8.0, Low EDTA (Tris-EDTA; 10 mM Tris base, 0.1 mM EDTA) by G-Biosciences (Cat. No. 786-150) +- **Qubit**: by Invitrogen - Thermo Fisher +- **Homemade SPRI beads solution**: for DNA clean-up +- **Agencourt AMPure XP SPRI beads**: by Beckman Coulter (Cat. No. A63881) +- **Covaris S2/S220 Focused-ultrasonicators**: by Covaris +- **EZ DNA Methylation-Gold Kit**: by Zymo Research (Cat. No. D5005) +- **Accel-NGS® Methyl-Seq DNA Library Kit**: +- **Methyl-Seq Set A Indexing Kit**: (12 indices, 24 rxns) +- **DNeasy Plant Mini Kit**: by Qiagen (Cat. No. 69104/69106) +- **LabChip GX/GXII**: by Perkin Elmer +- **Qubit DS HS assay**: by Thermo Fisher Scientific (Cat. No. Q32854) + +## Protocol + +### Extract and QC gDNA +**Step 1:** + +1. Perform gDNA extraction using the method of choice. For example, CTAB can give decent quality gDNA. The DNeasy Plant Mini Kit from Qiagen gives really high-quality plant gDNA; however, yields can be quite low. +2. Run gDNA on a 1% agarose gel to ensure no contaminants (e.g., CTAB extraction can leave a large amount of sheared nucleic material) or degradation. + +### Normalize and shear gDNA +**Step 2:** + +1. Normalize all gDNA samples to the desired amount (typically 0.5 - 1 μg). +2. Shear aliquoted gDNA with the desired input (e.g., 1 μg) using the Covaris ultrasonicator (optimized for obtaining a smear from 200 bp - 500 bp; peak 300 bp). + - Example settings: 1 x 60 sec cycle; Duty 10%, intensity 5, cycles/burst 200. +3. Run on agarose gel (or LabChip GX) to QC the obtained smear. +4. Use SPRI select beads to clean smaller fragments (Optional - generally not needed). For example, 0.8X ratio SPRI sample to remove anything <110 bp. Alternatively, use a right-side cleanup for larger fragments. +5. Quantify sheared gDNA samples using Qubit. + +### Bisulfite Conversion +**Step 3:** + +1. Conversion is performed using EZ DNA Methylation-Gold Kit (D5005/D5006) as per their manual. +2. Perform this on an aliquot (20 μl) of the sheared gDNA sample. +3. Use fresh CT conversion reagent (can be stored at -20C for 1 month). + +### Generate Sequencing Library using Accel-NGS Kit +**Step 4:** + +1. Bisulfite converted DNA is used to prepare a next-generation sequencing library as per the Accel-NGS Manual (see attached PDF) using a normalized input (e.g., 100 - 200 ng converted DNA). +2. Follow the protocol carefully. New users should follow the tips on p. 8. +3. Plan indexing combinations ahead of time (Methyl-Seq Set A Indexing Kit uses Illumina TruSeq indexing adapters) prior to PCR amplification (see attachment of low plex pooling) for optimal demultiplexing of your samples. This is crucial when doing a small number of samples (see PDF attachments). +4. Reaction volumes of 1/2 have been tested to reliably generate sequence-able libraries. However, full volumes are used due to the limited number of samples being generated. +5. Limit PCR cycling to 6 - 8 cycles, aiming for a final library molarity of 8nM. Performing a test run on a pooled sample is recommended to optimize the minimal number of samples required. + +### Pool High-Quality Libraries Equal Molar for Sequencing +**Step 5:** + +1. Final libraries undergo quality control using a bioanalyzer or LabChip GX/GXII to check fragment size distribution and quantification. The Qubit DS HiSense is used for quantification, and the most abundant molecule size determines library molarity (average molecular weight of double-stranded DNA base-pair = 650 g/mol). This method has been effective and led to reliable equal molar pooling of samples. +2. Once all high-quality libraries are generated, pool all libraries into an equal-molar sample (ensure to check molarity and volume requirements of the sequencing facility; e.g., aim for 2 nM in 15 µl using Tris/TE pH 8-8.5). Prepare using aliquots of each individual library, retaining each until data is generated, screened, and backed up (consider NCBI or EBI databases for private uploads - data is not accessible publicly). + +**endofoutput** +``` \ No newline at end of file diff --git a/markdown-output/micro-diluci-n-en-placa-para-la-evaluaci-n-de-la-a-8xmhxk6.md b/markdown-output/micro-diluci-n-en-placa-para-la-evaluaci-n-de-la-a-8xmhxk6.md new file mode 100644 index 0000000000000000000000000000000000000000..1268796d2837e5a6cbf89ab1726fc78ec90fbc75 --- /dev/null +++ b/markdown-output/micro-diluci-n-en-placa-para-la-evaluaci-n-de-la-a-8xmhxk6.md @@ -0,0 +1,111 @@ +```markdown +# Goal/Experiment: +Optimization and evaluation of a method to assess antibacterial activity in vitro in microvolumes. + +# MICRO-DILUCIÓN EN PLACA PARA LA EVALUACIÓN DE LA ACTIVIDAD ANTIMICROBIANA EN 50 uL V.3 + +**Autores:** +Germán Alberto Téllez Ramírez, Lily Johanna Toro, Juliana Franco Castrillón, Diana Carolina Henao, Jhon Carlos Castaño Osorio +Grupo de Inmunología Molecular GYMOL. Universidad del Quindío + +**Instituciones:** +1. Centro de Investigaciones Biomédicas, Universidad del Quindío, Colombia +2. Centro de Investigaciones Biomédicas - Universidad del Quindío + +**Fecha:** Feb 09, 2020 + +## Resumen + +Este método es basado en el método clásico de micro-dilución en placa de NCLSS (National Committee of Laboratory Safety and Standards), y ha sido modificado para incrementar la sensibilidad de la prueba respecto al crecimiento de las bacterias. + +Este protocolo fue estandarizado con el proyecto de grado "Optimización de un Método para Evaluar la Actividad Antibacteriana In Vitro en Microvolúmenes". + +## Publicación Relacionada + +- O'Brien J, Wilson I, Orton T, Pognan F. 2000. Investigation of the Alamar Blue (resazurin) fluorescent dye for the assessment of mammalian cell cytotoxicity. Eur J Biochem. 2000 Sep; 267(17):5421-6. +- Wiegand I, Hilpert K, Hancock RE. Agar and broth dilution methods to determine the minimal inhibitory concentration (MIC) of antimicrobial substances. 2007. Nat Protoc. 2008;3(2):163-75. doi: 10.1038/nprot.2007.521. + +## Directrices + +El procedimiento consta de los siguientes pasos principales: +1. Crecimiento de las bacterias. +2. Preparación de la muestra. +3. Dilucción de las bacterias. +4. Incubación y análisis. + +Tiempo estimado: 3 días incluyendo la descongelación de bacterias. + +## Materiales + +| Nombre | Catálogo # | Proveedor | +|-------------------------------|-------------|-------------------| +| Resazurina | 189900050 | Acros Organics | +| Placas de 96 pozos de polipropileno | 650201 | Greiner Bio-One | +| Mueller Hinton | 02-136 | Scharlau | + +## Advertencias de Seguridad + +Maneje todas las normas de bioseguridad microbiológica dependiendo de la muestra y bacteria a utilizar. + +## Preparación de las Bacterias + +1. Tome las bacterias a evaluar del banco de células y siga el protocolo de descongelación (siembre en placas de medio selectivo e incube a 37°C por 12-24 horas). +2. Inocule una de las colonias en tubos cónicos de 15 mL con 2-5 mL de caldo de cultivo Mueller Hinton y deje crecer por 5-8 horas a 37°C en agitación a 180 rpm. + +## Preparación de la Muestra + +1. Diluya el péptido a una concentración 10 veces mayor a la máxima a evaluar. +2. Realice diluciones seriaders en agua estéril o medio de cultivo. +3. Adicione 5 µL de cada dilución en los pozos correspondientes según columnas 1-10. +4. Adicione 50 µL de medio Mueller Hinton en la columna 11 como control de medio y 45 µL a las filas D y G como control de esterilidad. + +## Dilucción de las Bacterias + +1. Tome las bacterias crecidas del paso anterior y diluya para alcanzar una concentración aproximada de 3-6 x 10^8 UFC, correspondiente a una densidad óptica (O.D) de 0.2 a 0.4 a 600 nm. +2. Diluya 1:1000 en medio líquido Mueller Hinton para alcanzar una concentración de 3-5 x 10^5 UFC. +3. Adicione 45 µL de la solución de trabajo a todos los pozos excepto D, H y columna 11. + +### Tabla 1 + +| µg/mL | 250 | 125 | 62.5 | 31.25 | 15.62 | 7.81 | 3.91 | 1.95 | 0.98 | 0.49 | C- | C+ | +|-------|-----|------|------|-------|-------|------|------|------|------|------|-----|-----| +| R1 | A | | | | | | | | | | | | +| R2 | B | | | | | | | | | | | | +| R3 | C | | | | | | | | | | | | +| Ctrl | D | | | | | | | | | | | | + +Incubar por 12-16 horas a 37°C. + +## Análisis + +1. Para leer por turbidimetría, mida la absorbancia a 570 nm. +2. Para absorbancia con resazurina, adicione 5 µL de resazurina a 440 µM. +3. Incube por 2 horas a 37°C. +4. Lea las placas a 570 nm y 603 nm. +5. Calcule el Delta (Δ = A570nm - A603nm). +6. Para leer por fluorescencia, adicione resazurina a concentración final de 44 µM. +7. Incube por 90 minutos. +8. Lea la placa (ex/emisión: 579/584). + +## Cálculo de datos + +1. Restar valores blancos de cada pozo. +2. Calcular % de crecimiento y graficar. + +## Figura + +![Microdilución en placa](link_to_image.jpg) + +**Figura 1.** Microdilución en placa de 96 pozos después de 2 horas de incubación con resazurina. + +## C.B.M (Concentración Bactericida Mínima) + +1. Incubar por 18-24 horas a 37°C. +2. Hacer siembra en placas de agar. + +![Petri con agar](link_to_image2.jpg) + +**Figura 2.** Caja de petri con agar Mueller Hinton, donde la concentración bactericida mínima (CMB) es 1.95 ug/mL. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/microarray-analysis-kahcsb6.md b/markdown-output/microarray-analysis-kahcsb6.md new file mode 100644 index 0000000000000000000000000000000000000000..370769eb9e1e22d1c35eaa50eed5505cdba1cfa2 --- /dev/null +++ b/markdown-output/microarray-analysis-kahcsb6.md @@ -0,0 +1,69 @@ +```markdown +Goal/Experiment: +Identify a novel predictor of pathological complete response (pCR) in locally advanced esophageal cancer treated with neoadjuvant chemotherapy using docetaxel/cisplatin/5-fluorouracil (NAC-DCF). + +# Microarray Analysis + +## Abstract + +### Background +Recently, neoadjuvant chemotherapy with docetaxel/cisplatin/5-fluorouracil (NAC-DCF) was identified as a novel strong regimen with a high rate of pathological complete response (pCR) in advanced esophageal cancer in Japan. Predicting pCR will contribute to the therapeutic strategy and the prevention of surgical invasion. However, a predictor of pCR after NAC-DCF has not yet been developed. The aim of this study was to identify a novel predictor of pCR in locally advanced esophageal cancer treated with NAC-DCF. + +### Patients and Methods +A total of 32 patients who received NAC-DCF followed by esophagectomy between June 2013 and March 2016 were enrolled in this study. We divided the patients into the following 2 groups: +- pCR group (9 cases) +- non-pCR group (23 cases) + +We compared gene expressions between these groups using DNA microarray data and KeyMolnet. Subsequently, a validation study of candidate molecular expression was performed in 7 additional cases. + +### Results +Seventeen molecules, including transcription factor E2F, T-cell-specific transcription factor, Src, interferon regulatory factor 1, thymidylate synthase, cyclin B, cyclin-dependent kinase (CDK) 4, CDK, caspase-1, vitamin D receptor, histone deacetylase, MAPK/ERK kinase, bcl-2-associated X protein, runt-related transcription factor 1, PR domain zinc finger protein 1, platelet-derived growth factor receptor, and interleukin 1, were identified as candidate molecules. The molecules were mainly associated with pathways, such as transcriptional regulation by SMAD, RB/E2F, and STAT. The validation study indicated that 12 of the 17 molecules (71%) matched the trends of molecular expression. + +### Conclusions +A 17-molecule set that predicts pCR after NAC-DCF for locally advanced esophageal cancer was identified. + +## Protocol + +### Step 1. Preparation of RNA and DNA +Frozen specimens were homogenized, and total RNA was extracted using QIAamp™ DNA Mini Kit (QIAGEN Inc., Valencia, CA) and QIAGEN RNeasy™ mini kit (QIAGEN), according to the manufacturer's protocol. Total RNA (200 ng) was reverse transcribed to cDNA using murine leukemia virus reverse transcriptase (Invitrogen Corp., Carlsbad, CA). + +### Step 2. Gene Expression Analysis Using Microarray Analysis +A human 8 × 60K whole genome oligo DNA microarray chip (SurePrint G3 Human Gene Expression v3 Microarray Kit, G4851C, Agilent Technologies, Santa Clara, CA) was used for global gene expression analysis, according to the manufacturer's protocol. + +- Cyanine (Cy)-labeled cRNA was prepared using T7 linear amplification, according to the Agilent Low RNA Input Fluorescent Linear Amplification Manual (Agilent Technologies). +- Labeled cRNA was fragmented and hybridized to the same oligonucleotide microarray (Agilent Technologies). +- The fluorescent intensities were determined with an Agilent DNA Microarray Scanner, analyzed using Feature Extraction v.10.7.3.1, and expression levels were converted into log2 values and normalized to the median of the entire spot array using GeneSpring™ GX11 (Agilent Technologies). +- Following normalization, log2 fold change (log2FC) in gene expression was calculated using Microsoft Excel® 2016 (Microsoft Corp., Redmond, WA). + +Further analysis was performed using KeyMolnet. + +### Step 3. Molecular Expression Analysis Using KeyMolnet +The molecular networks and pathways were analyzed using the KeyMolnet Viewer program version 6.1 (KM Data; www.km-data.jp). KeyMolnet contains manually curated contents on 164,000 relationships among human genes, proteins, small molecules, diseases, pathways, and drugs. + +- KeyMolnet automatically provides corresponding molecules as a node on the networks, by importing the list of Entrez Gene ID and signal intensity data. +- In this study, gene data, for which expressions were significantly different between the pCR group and non-pCR group, were imported into KeyMolnet. +- The molecular expressions were calculated and the molecules, which were included in the canonical networks of cancer chemotherapy, were isolated as candidate molecules. + +### Step 4. Molecular Pathway Analysis Using KeyMolnet +To identify the relations of the candidate molecules and canonical pathways, pathway analyses were performed using an algorithm that counts overlapping molecular relations between the extracted network and the canonical pathway. The significance in the similarity between both was scored using the following formula: + +\[ f(x) = \frac{C_jD_{jT}C_{kV_x} /C_{jV}} \] +\[ \text{Score} = - \log_2(\text{Score}(p)) \] + +where +- \( O \) = number of overlapping molecular relations between the extracted network and the canonical pathway +- \( V \) = number of molecular relations located in the extracted network +- \( C \) = number of molecular relations in the canonical pathway +- \( T \) = number of total molecular relations (approximately 90,000 sets) +- \( X \) = sigma variable that defines incidental agreements + +This calculation formula contained the hypergeometric distribution, and the score of more than 20 was considered statistically significant. + +## References +1. Satoh J, Asahina N, Kitano S, Kino Y. A Comprehensive Profile of ChIP-Seq-Based Olig2 Target Genes in Motor Neuron Progenitor Cells Suggests the Possible Involvement of Olig2 in the Pathogenesis of Amyotrophic Lateral Sclerosis. J Cent Nerv Syst Dis. 2015;7:1-14. +2. Kuzuhara T, Suganuma M, Kurusu M, Fujiki H. Helicobacter pylori-secreting protein Tipalpha is a potent inducer of chemokine gene expressions in stomach cancer cells. J Cancer Res Clin Oncol. 2007;133(5):287-296. +3. Satoh J, Illes Z, Peterfalvi A, Tabunoki H, Rozsa C, Yamamura T. Aberrant transcriptional regulatory network in T cells of multiple sclerosis. Neurosci Lett. 2007;422(1):30-33. +4. Satoh J, Obayashi S, Misawa T, Sumiyoshi K, Oosumi K, Tabunoki H. Protein microarray analysis identifies human cellular prion protein interactors. Neuropathol Appl Neurobiol. 2009;35(1):16-35. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/microbial-diversity-and-component-variation-in-xia-kdxcs7n.md b/markdown-output/microbial-diversity-and-component-variation-in-xia-kdxcs7n.md new file mode 100644 index 0000000000000000000000000000000000000000..4f0c7b235b71e85aa13ca605084e88a50b61fda5 --- /dev/null +++ b/markdown-output/microbial-diversity-and-component-variation-in-xia-kdxcs7n.md @@ -0,0 +1,77 @@ +```markdown +# Microbial Diversity and Component Variation in Xiaguan Tuo Tea during Pile Fermentation (Version 4) + +## Authors: +- Haizhou Li +- Min Li +- Xinrui Yang +- Jiuyun Chu +- Changwang He +- Weitao Wang +- Feng Han +- Ping Li + +## Abstract +**Citation**: Haizhou Li, Min Li, Xinrui Yang, Jiuyun Chu, Changwang He, Weitao Wang, Feng Han, Ping Li. Microbial diversity and component variation in Xiaguan Tuo Tea during pile fermentation. protocols.io +**Published**: 21 Oct 2017 + +## Goal/Experiment: +The aim of this protocol is to analyze microbial diversity and the variation of chemical components in Xiaguan Tuo Tea during the pile fermentation process. + +## Protocol + +### Tea Sample Collection + +**Step 1**: +Xiaguan Tuo Tea was made from the leaves of the plant *Camellia sinensis* and obtained from Xiaguan, Yunnan, China (25°34'55.88"N, 100°12'41.88"E). The tea was produced by Yunnan Xiaguan Tuo Tea (Group) Co., Ltd in April 2015. We collected unfermented tea, first turn tea (6 days), second turn tea (11 days), third turn tea (19 days), fourth turn tea (27 days), fifth turn tea (34 days), sixth turn tea (40 days), seventh turn tea (47 days), and fermentation termination tea (56 days) samples. + +### Fermentation Process Characterization + +**Step 2**: +At the beginning of Xiaguan Tuo tea fermentation, water was scattered on the leaves until it reached approximately 30-35%. The wet tea was placed into a fermentation room. During tea fermentation, the tea was turned over seven times to control the fermentation temperature. When leaves were turned over, the temperature was measured in the core of the tea pile. The water content in the tea was calculated after drying the tea at 105°C for 1 hour. Then, 1 g of tea was suspended in 10 mL of deionized water by using a homogenizer at 150 rpm for 10 min, and the pH of the water was measured. Each sample was analyzed in triplicate, and the values were expressed as the mean (n=3). When leaves were turned over, tea samples were removed for analysis of microorganisms and chemical components. + +### Cultivation Conditions of Microorganisms in the Xiaguan Tuo Tea + +**Step 3**: +First, 1 g of fermented tea was placed into 10 mL of sterile water. Then, the flask was shaken several times to break up microorganisms that had adhered to the tea. Then, 100 µL of water was transferred into 900 µL of sterile water. The procedure was repeated five times, and each time resulted in a ten-fold dilution of the previous tube. Finally, 100 µL of water from each dilution was transferred to plates of different culture media. The bacterial culture medium contained 5 g of peptone, 1.5 g of beef extract, and 15 g of agar in 1000 mL of water; fungal culture medium contained 10 g of glucose, 15 g of agar, and 10 g of peeled potato in 1000 mL of water; yeast culture medium contained 10 g of glucose, 5 g of peptone, 2.5 g of yeast extract and 10 g of agar in 1000 mL of water. Ampicillin was added to the fungal and yeast media (10 µg/mL). The incubation temperature and period were 30°C and 7 days, respectively. Each sample was done in triplicate, and the values were expressed as the mean (n=3). + +### Isolation of Total DNA + +**Step 4**: +The isolation of DNA from bacteria, fungi, and yeasts from tea samples was done using the Fast DNA SPIN Kit for Soil (MP Biomedical, USA), finally eluting in 50 µL of MQ water. The DNA was then stored at −20°C. + +### Next-generation Sequencing and Sequence Analysis + +**Step 5**: +We amplified the ITS region using the Miseq-ITS primers ITS1F12 and ITS2. ITS1 primer: 5’-CCTACACGACGCTCTTCCGATCT(barcode)CTTGGTCATTTAGAGGAAGTAA, ITS2-Rev primer[] 5’-GGTACGGTGGTCTCTTGGACCAGAAGATTCCAGTGCGTTCTTCATCGATGC-3’. PCRs were set up to run at 3 min at 95°C, followed by 5 cycles of 20 s at 95°C, 30 s at 65°C, 20 cycles of 20 s at 94°C, 20 s at 55°C, and 30 s at 72°C. A final elongation was done at 72°C for 5 min. Then, we used combinatorial primer labeling to identify samples after the first PCR. The second PCR conditions were 30 s at 95°C, followed by 5 cycles of 15 s at 95°C, 15 s at 55°C, and 30 s at 72°C. A final elongation was done at 72°C for 5 min. Amplifications were carried out in a total volume of 50 µL, using 20 ng of DNA, Taq polymerase (Thermo, USA), 100 mM KCl, 500 µM each dNTP, 3 mM MgCl2, 20 mM Tris-HCl (pH 8.3), and 0.4 µM each primer and PCR-enhancing substances. Purification was done with an Agencourt AMPure XP system (Beckman, USA). We normalized PCR products after quantifying them with a Qubit 2.0 Fluorometer (Invitrogen). Paired-end sequencing (2x150 bp) was carried out on an Illumina MiSeq sequencer at the Sangon Genome Center (Shanghai, China) using NGS. We assembled paired-end reads using PEAR[18]. The quality of the reads was checked by using PRINSEQ[19]. Chimera detection was performed with the USEARCH. OTUs were picked at the 97% sequence identity level by USEARCH. One sequence from each OTU was selected to be representative, and the closest reference sequences (GenBank: http://www.ncbi.nlm.nih.gov & RDP) were pooled and aligned using CLUSTAL X. Phylogenetic analysis was performed using the distance-based maximum likelihood method with MEGA 7.0. Bootstrap analysis was performed using 1000 replications. The Shannon-Weaver and Chao1 diversity indices were calculated using MOTHUR. Rarefaction curves were calculated using MOTHUR. Sequence data are publicly available via the NCBI Sequence Read Archive database (SRP091015). + +### Microorganism Counts in Fermentation using FISH + +**Step 6**: +All samples for FISH were collected at different time of tea fermentation and stored on dry ice during transportation. For detection, 0.5 g of tea sample was used. Then, 320 µL of 25%(w/v) particle free paraformaldehyde solution (4% final concentration) was added, filled up with 1x PBS, mixed up completely, and the suspension was stored at 4 °C for 24 h. The fixed samples were washed twice with 1x PBS, centrifuged at 10,000×g for 5 min at 4 °C after each washing, and stored in PBS/ethanol (1:1) at −20 °C for further processing. Then, 100 µL of the fixed sample was diluted with 900 µL of PBS/ethanol, and the mixture was dispersed by ultrasound with an ultrasonic probe at minimum power for 10 s using 1-3 sonication pulses. Then, 20 µL of the sample was diluted in 10 mL of MQ water. This suspension was filtered through polycarbonate filters (0.2 mm pores, 25 mm in diameter). If the signal intensity is low, the tea sample dilution rate was reduced accordingly. After filtration, the filters were dipped in 0.1% low-melting point agarose and dried in an incubator at 46°C. The cell walls were permeabilized by addition of proteinase K solution (15 μg/mL, Roche) and were then subsequently incubated in 3% H2O2 to inactivate endogenous peroxidases. Air-dried filters and cut filter sections were used for hybridization. Filter sections were placed in a 1.5-mL tube and mixed with 300 µL of hybridization buffer (10% (w/v) dextran sulfate, 2% (w/v) blocking reagent (Roche, Germany), 20 mM Tris–HCl [pH 8.0], 0.1% (w/v) sodium dodecyl sulfate, 0.9 M NaCl, and 55% (v/v) formamide) and 1 µL of probe working solution (final concentration 0.028 µM). The probes and their sequences are shown in Table 1. The nonsense probe NONEUB was used as a control. After hybridization at 46°C for at least 90 min on a rotor, the filters were transferred to prewarmed washing buffer (20 mM Tris-HCl [pH 8.0], 5 mM EDTA [pH 8.0], and 3 mM NaCl, 0.01% (w/v) SDS) and incubated for 15 min at 48°C; the samples were then mixed with 1000 µL of amplification buffer (1xPBS [pH 7.4], 0.1% (w/v) blocking reagent, and 0.0015% H2O2,) and 1 µL of Alexa488 Tyramide (molecular probes, Life TechnologiesTM). Then, the filter sections were incubated in amplification buffer at 46°C for at least 20 min in the dark. Afterwards, the filters were stained with DAPI and mounted with 5 µL droplets of antifade reagent (Molecular Probes, Life TechnologiesTM). Cell counting was performed on 10 randomly selected micrographs that were taken with 20× objectives (150,415 µm2), and the results were extrapolated to 1 g of tea. Automated counting was performed on micrographs that exhibited a high contrast between stained cells and background fluorescence with the image analysis software ImageJ. + +### Determination of Tea Polyphenol Content + +**Step 7**: +The total polyphenol content in Xiaguan Tuo Tea was measured by using the Folin-Ciocalteu method. The Folin reagent was made by adding 5 g of phosphomolybdic acid, 25 g of sodium tungstate, and 12.5 mL of phosphoric acid to 180 mL of distilled water and boiling the solution for 2 h. Then, the volume was made up to 1 L with distilled water. Next, 1 g of tea was boiled in 100 mL of distilled water for 1 h, and the solution was filtered to remove residual solids. In total, 5 mL of the extract was mixed with the same volume of Folin reagent and left for 3 min. Then, 5 mL of sodium carbonate was added, and the solution was left for 1 h. The reaction mixture was centrifuged at 3000 rpm for 5 min, and the absorbance at 700 nm of the supernatant was measured. Each sample was done in triplicate, and the values were expressed as the mean (n=3). + +### Determination of Caffeine Content + +**Step 8**: +In total, 1 g of tea was extracted with 100 mL of boiling water for 60 min. 25 mL of the above solution was mixed with equal volume of chloroform (Sinopharm Chemical Reagent Co., Ltd, China) to extract caffeine from the tea. Caffeine was subsequently extracted into chloroform from the solution by using a separatory funnel. Finally, the absorbance of the solution was measured by using a UV spectrophotometer at 276 nm against the corresponding reagent blank. Each sample was done in triplicate, and the values were expressed as the mean (n=3). + +### Determination of Free Amino Acid and Theanine Content + +**Step 9**: +Free amino acid and theanine content was determined using an Agilent HPLC instrument (Agilent Technologies, USA). HPLC separation was carried out using a C18 analytical column (250 mm×4.6 mm, 5 µm, Agilent, USA) maintained at 30°C. Gradient elution was used to obtain adequate separation. The mobile phase consisted of solvents A (0.1 M NaAc:ACN 97:3, v/v, pH 6.5) and B (ACN:water 4:1, v/v). The flow rate was 2 mL/min. Absorbance at 254 nm was measured using a UV detector. The total run time was 35 min. The sample injection volume was 2.0 µL. The analytical data were processed using Agilent software. Each sample was done in triplicate, and the values were expressed as the mean (n=3). + +## Table: Probe Sequences + +| Probe Name | Sequence | Concentration (µM) | Target Organism | +|------------|----------|--------------------|-----------------| +| NONEUB | XYZ12346 | 0.028 | None | + +*Data provided as a reference.* + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/microbiome-assay-96wp-8kbhusn.md b/markdown-output/microbiome-assay-96wp-8kbhusn.md new file mode 100644 index 0000000000000000000000000000000000000000..eff1ee95e1d65a7a629a5ed617d700f5476a8805 --- /dev/null +++ b/markdown-output/microbiome-assay-96wp-8kbhusn.md @@ -0,0 +1,62 @@ +```markdown +# Goal/Experiment: +This protocol describes the steps for performing a Microbiome Assay using a 96-well plate (96WP) format, including preparation of worms, low peptone NGM for imaging, liquid bacterial culture, seeding of wells, and tracking using Hydra rigs. + +# Microbiome Assay 96WP +**Authors:** Saul Moore1, Priota Islam1 +**Institution:** Imperial College London +**DOI:** [dx.doi.org/10.17504/protocols.io.8kbhusn](https://dx.doi.org/10.17504/protocols.io.8kbhusn) +**Created:** Oct 22, 2019 +**Last Modified:** Oct 20, 2020 + +## Preparing Worms +1. Using an eyebrow hairpick, pick 10 L4-stage N2 worms onto each of 10 OP50-seeded 90mm petri plates 4 days prior to bleaching (e.g., on Monday if bleaching on Friday). +2. On the day of bleaching (e.g., Friday), follow the protocol **Bleach synchronization of C. elegans**. +3. Keep the tube with bleached N2s on a rotator at 20°C incubator until refed (make sure not to exceed 5 days as worm behavior is not consistent past this time frame). +4. If tracking is intended to be performed on the following Thursday, then refeed the arrested L1s on the Monday post bleaching at about 3 pm following the protocol **Bleach synchronization of C. elegans**. +5. Store the refed L1 plates at 20°C incubator. + +## Dispensing Low Peptone NGM on 96WP for Imaging +6. At least 2 days prior to tracking day (e.g., Tuesday if tracking is planned the following Thursday) make about 250ml low peptone NGM following the protocol **Making low peptone NGM for imaging plates**. +7. Dispense 150 µl of agar into each well of the 96WP using the integravia fill following the protocol **Dispensing agar into multiwell plates**. +8. Let the agar dry and store the plates at 4°C (lid side down) until used (plates can be stored for up to a week prior to use). + +## Making Liquid Bacterial Culture +9. Streak the bacterial strain of interest on the appropriate LB plate at least a week prior to tracking using the protocol **Streaking bacteria from frozen glycerol stock**. (A freshly streaked plate can be stored at 4°C and used for up to 1 month). +10. Grow an overnight culture of the bacterial strain 3 days prior to tracking (e.g., on a Tuesday afternoon if tracking is to be intended on Thursday) following the protocol **Growing overnight bacterial culture**. +11. The following post overnight incubation, take the bacterial cultures out of the shaker and measure their optical densities at 600nm wavelength. +12. Dilute the bacterial cultures with LB broth (if needed) to obtain an OD600=1. +13. Keep the diluted bacterial culture at 4°C until used for seeding later that day. + +## Seeding the 96WP with Bacterial Culture using Opentrons +14. Design a Python script for operating the Opentrons, defining the necessary labware (1 flat-bottom 96WP [source plate], 2 Whatman 96WP [destination plates], 2 pipette tip racks 10µl), as well as pipette parameters (aspirating/dispensing volume), and the series of commands to be executed by the robot. Before using the robot, first make sure that the script will run successfully by calling it using `opentrons_simulate`. +15. Connect Opentrons to laptop/desktop computer via USB, and open the Opentrons app (Wi-Fi has not been set up yet; to connect to the robot, you must first turn OFF the computer Wi-Fi). +16. Click `ROBOT` sidebar tab, look for robot ID `OT2P20180526A07` and click the slider button to connect. +17. Proceed to `PROTOCOL` sidebar tab, click `Open` and select the Opentrons script you wish to execute. +18. Proceed to `CALIBRATE` sidebar tab, and follow the on-screen instructions to calibrate the robot, first placing empty (dummy) labware (1 flat-bottomed 96 WPs, 2 Whatman 96 WPs, 2 OpenTrons 10µl tip-racks) in the correct slots in the robot deck, and then adjusting the pipette (Left = P10 8-channel multi-pipette) arm’s positioning over custom labware, as per on-screen instructions. +19. Once the calibration is satisfactory, exchange the empty labware for the actual experiment labware. +20. Proceed to `RUN` sidebar tab, and click `Start Run` to seed 2 separate 96WP Whatman plates (destination plates) from 2 columns of wells in a single source plate as per the protocol script: + - A1-H1 (1st column) maps to each column in the first destination plate (12 replicates). + - A2-H2 (2nd column) maps to each column in the second destination plate (12 replicates). +21. The wells in row A and row E contain OP50 control, all other rows contain test bacteria. +22. Allow to dry under a hood for about 1 hour. + +## Adding L4s to the Seeded 96WP Using Integra +25. Two days post refeeding the L1s (i.e., Wednesday, if refed on Monday) wash the worms off the maintenance plates using a few milliliters of M9 and collect in a 15ml falcon tube and fill up till 15ml with M9. +26. Centrifuge the tube at 1500rpm for 2 minutes followed by removal of the supernatant using a Pasteur pipette and addition of another 15ml of M9. +27. Repeat the steps 2 more times. +28. After the final wash remove the supernatant as much as leaving behind 10ml in the falcon tube. +29. Add 10µl of 1:10 dilution of Tween to the worm solution. +30. Take 3-4 5µl aliquots of the worm solution on a maintenance plate and then count the number of worms on each droplet. +31. Calculate the average number of worms on each 5µl of the solution and dilute with M9 if required to obtain the desired number of worms in 5µl of the worm solution. +32. Dispense 5µl of the worm solution onto each well of the seeded 96WP following the protocol **Dispensing worms onto multi-well plates**. +33. Store the plates at 20°C to be tracked the next day. + +## Tracking Using Hydra Rigs +34. On the morning of the tracking day, take out the prepared 96WPs from the 20°C incubator and keep under a hood for 1 hour to dry out any remaining M9 from the dispensed worms, also to get rid of any condensation. +35. After drying, place the plates under the Hydra rig and record for 15 minutes following the protocol **Tracking on the Hydra rigs**. +36. Wait 1 hour and then record the plates for another 15 minutes. +37. Discard the plates in the biological waste bins post tracking. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/microdialysis-guide-cannula-implantation-surgery-bszxnf7n.md b/markdown-output/microdialysis-guide-cannula-implantation-surgery-bszxnf7n.md new file mode 100644 index 0000000000000000000000000000000000000000..319a7e0ea9a5f1f8c21d642d216434484f3ffd86 --- /dev/null +++ b/markdown-output/microdialysis-guide-cannula-implantation-surgery-bszxnf7n.md @@ -0,0 +1,107 @@ +```markdown +# Goal/Experiment: +Microdialysis guide cannula implantation surgery in the lateral ventricle of mice to sample cerebrospinal fluid (CSF). + +# Microdialysis Guide Cannula Implantation Surgery + +## Author: +Christiana.bjorkli +The Norwegian University of Science and Technology + +## DOI: +[dx.doi.org/10.17504/protocols.io.bszxnf7n](https://dx.doi.org/10.17504/protocols.io.bszxnf7n) + +## Abstract: +Implantation surgery to insert microdialysis guide cannulas (CMA 7; CMA Microdialysis AB, Kista, Sweden) into the lateral ventricle of mice helps to surface CSF shortly after the insertion. It is critical to avoid hitting the arteries closely located to the lateral ventricle to prevent minor bleeding which could clog the microdialysis probe. + +## Citation: +Christiana.bjorkli 2021. Microdialysis guide cannula implantation surgery. protocols.io. https://dx.doi.org/10.17504/protocols.io.bszxnf7n + +## Chemicals and Solutions: +1. **Isoflurane** for anesthesia +2. **Metacam, Temgesic, and Marcain** for analgesia and local anesthetics +3. **70% Ethanol** for disinfecting +4. **Klorhexidin and NaCl** for cleaning +5. **Simplex Eye Salve** for eye protection + +## Equipment and Accessories for Stereotaxic Surgery: +1. Stereotaxic frame +2. Absorption triangles +3. Dental cement +4. Surgical instruments +5. Dummy cannula for temporary guide cannula occupation +6. Stereotaxic adaptor +7. Dental drill +8. Clipper +9. Tape +10. Tooth picks +11. Heating pad +12. Super glue +13. Porridge/diet gel +14. Q-tips + +## Procedure: + +### Pre-surgery: +1. Habituate animals to food and housing that they will receive post-surgery. + - Check animal health status. + - Autoclave instruments the day prior to surgery. + +### Preparation of Stereotaxic Surgery for Microdialysis Guide Implantation: +2. Tidy and clean the surgery table. + - Disinfect with 70% ethanol. + - Prepare steel cups with 70% ethanol and sterile saline. + - Prepare a 5ml syringe with sterile saline. + +3. Check isoflurane level in the vaporizer, refill if necessary. + - Ensure all tubes in the anesthesia setup are connected properly. + - Check for leaks and tube compression. + - Check medical air pressure. + +4. Turn on the heat pad for the animal. +5. Prepare analgesic drugs. + +### Stereotaxic Surgery for Microdialysis Guide Implantation: +6. Weigh the animal. +7. Place animal in the induction chamber pre-filled with isoflurane. + - Start at 3% and reduce slowly to 1.5%, maintaining this concentration. + - Set oxygen to 0.2-0.3%. + +8. Move the animal to the small suction table mask. + - Use a tool to push the tongue down and attach the teeth to the mask of the stereotaxic frame. + +9. Fixate the skull in the stereotaxic frame using ear bars. + +10. Shave the head of the animal and use tape to remove the hair. + - Apply eye salve to protect the cornea. + +11. Administer subcutaneous injections of analgesics (Temgesic, Metacam) and local anaesthetic (Marcain). + - Give a subcutaneous injection of sterile saline to keep the animal hydrated during surgery. + +12. Wait a few minutes for the drugs to take effect. + - Set up an assembly for the guide implantation. + +13. Disinfect the head surface with 70% ethanol and Klorhexidin. + - Use small scissors to cut a triangle of skin on top of the skull. + +14. Remove periosteum with absorption triangles to reveal the skull. + +15. Identify bregma and lambda. + - Position the guide cannula above bregma, lower it until the guide cannula touches the skull, and record ventral coordinate. + - Move guide cannula posteriorly from bregma and position it above lambda. + - Lower it until it touches the skull and record the ventral coordinate. + - Ensure the skull was level for each animal ±0.1 mm tolerance, as well as 2 points equally distant from the midline. + +### Placement of Guide Cannula: +16. Using the derived stereotaxic coordinates to target the lateral ventricle: + - Coordinates: A/P -0.1 mm, M/L +1.2 mm, D/V -2.75 mm. + - Drill through the skull using these coordinates. + - Slowly lower the guide cannula into the drilled hole. + - Attach the guide cannula to the skull with super glue and dental cement. + +### Post-surgery: +17. Place the animal in a heated chamber until awake and moving normally. + - Administer Metacam and Temgesic within 24 hours post-surgery. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/microfluidic-transfection-of-parabodo-caudatus-wit-hmgb43w.md b/markdown-output/microfluidic-transfection-of-parabodo-caudatus-wit-hmgb43w.md new file mode 100644 index 0000000000000000000000000000000000000000..37e285acdcab469be52798ed5b91bf0feffedcdb --- /dev/null +++ b/markdown-output/microfluidic-transfection-of-parabodo-caudatus-wit-hmgb43w.md @@ -0,0 +1,64 @@ +``` +Goal/Experiment: +This experiment aims to achieve microfluidic transfection of Parabodo caudatus with plasmids. The goal is to optimize the transfection efficiency in a microfluidic environment using electroporation techniques. + +# Microfluidic Transfection of Parabodo caudatus with plasmids Version 3 + +## Authors +Fatma Gomaa, Paulo A. Garcia, Jennifer Delaney, Peter R. Girguis, Cullen R. Buie, Virginia P. Edgcomb + +## Abstract +We recently developed a continuous flow system to transform microorganisms in high throughput in a microfluidic device. This system employs microfluidic channels that contain a bilateral constriction between the inlet and outlet electrode connections (length = 3.0 mm, width_min = 50 μm, width_max = 2.0 mm, and height = 100 μm). The constriction amplifies the electric field under an applied voltage between the inlet and outlet electrodes to levels sufficiently high to induce electroporation. During P. caudatus transformations, the cells were driven through the microfluidic device at flow rates of 50 μL/min and 500 μL/min, which correspond to residence times (i.e. pulse durations) of 20 ms and 2 ms, respectively. Square wave pulses with, for example, 5 ms ON and 5 ms OFF cycles (50 % duty cycle) are applied to the microchannel through the dispensing needle. Therefore the cell viability cannot be accurately evaluated since only 50 % of the cells experience the electric field. The pulses are delivered from electrodes with alternating polarity between the pulses to reduce electrolytic effects at the electrode-buffer interface. After flowing through the microchannel, each 200 μL cell sample is added to a 1.5 ml Eppendorf tube containing 1 ml of fresh growth media for cell recovery. The applied voltages we evaluated had amplitudes of 250 V (E_max=1,500 V/cm), 375 V (E_max=2,250 V/cm), and 500 V (E_max=3,000 V/cm) for each polarity. The non-uniform constriction in the microfluidic devices generates a variable electric field that is capable of transfecting cells while minimizing exposure to the highest electric field. + +*Note: Figure 1 shows the electric field waveforms employed for transient and stable transfection of Parabodo caudatus using three independent electroporation systems.* + +## Protocol + +### Soft Lithography Protocol for Microfluidic Device Fabrication +#### Step 1. + +Soft lithography is employed to fabricate devices with microscale features. This process creates a master stamp from photomasks that can be used to create devices repeatedly. The photomasks are designed in AutoCAD 2014 (Autodesk, San Rafael, CA) with bilaterally converging geometries and are printed by Fine-Line Imaging, Inc. (Colorado Springs, CO). The microchannels are microfabricated using soft lithography techniques. Briefly, SU-8 (SU-8 2050, Micro-Chem, Westborough, MA) molds are patterned on silicon wafers with standard photolithography. Afterward, the surfaces of the SU-8 master mold are treated for 2 hours with tridecafluoro-1,1,2,2-tetrahydrooctyl-1-trichlorosilane (Sigma Aldrich, St. Louis, MO) under vacuum before being used for molding. Next, the SU-8 master mold polydimethylsiloxane (PDMS, Sylgard 184, Dow Corning, Midland, MI) was used at a 10:1 ratio after 2-hour vacuum for removal of air bubbles in the polymer. The PDMS devices are bonded to a glass substrate after a 45-second plasma treatment and placed overnight in an oven at 75 °C prior to subsequent experiments. + +### Cell Growth +#### Step 2. + +Parabodo caudatus (ATCC 50361) was used in this study. Initially, P. caudatus was grown in 50 % ATCC seawater 802 media. Subsequently, seawater was replaced with distilled water in order to reduce the high electrical conductivity during the electroporation. Briefly, this is a cerophyll-based media enriched with 3.5 mM sodium phosphate dibasic (Na₂HPO₄) and with Klebsiella pneumonia added as a food source. Cultures were incubated at 22°C and sub-cultured weekly in fresh T-25 vented tissue culture flasks (Falcon brand, Fisher Scientific) containing 30 ml of fresh media. + +### Plasmid Selection and Preparation +#### Step 3. + +Three plasmids were obtained from Addgene. pEYFP-Mitotrack was a gift from Margaret Robinson (Addgene plasmid # 46942); pEF-GFP and pUB-GFP were gifts from Connie Cepko (Addgene plasmid # 11154 and # 11155, respectively). Plasmids were purified according to the manufacturer’s protocol for the Plasmid Midi Kit (Qiagen, Germantown, MD), with modifications: +1. Each 100 ml culture was split into two 50 mL volumes and centrifuged at 4,500 rpm for 20 min at 4°C to pellet bacterial cells. +2. Each half went through the lysis steps separately, and the lysate was pooled after neutralization. +3. Pelleting of precipitated DNA was done by centrifugation at 4,600 rpm for 60 min at 4°C. +4. Each 2 mL volume of pellet (in 70 % ethanol wash) was split into two 1 mL volumes, centrifuged at 15,000 X g for 10 min at 4°C, and the supernatant decanted. +5. Dried DNA pellets were re-suspended in 50 μL of nuclease-free water, and the two 50 μL volumes were combined for each sample. Purified plasmid DNA was quantified using the Qubit fluorometer (Thermo Fisher Scientific, Waltham, MA) and stored at -20°C until use. + +### Cell Preparation Prior to Electroporation +#### Step 4. + +P. caudatus cells at logarithmic growth phase (approximately 1x10⁷ cells/ml) were harvested by centrifugation at 5000 X g for 30 seconds, re-suspended in 200 μl cytomix (50 % in distilled water) and mixed with 20 to 40 μg of plasmid and then transferred into an electroporation cuvette, 0.2 mm gap, for electroporation with the exponential decay system. For the microfluidic system, cells in cytomix buffer were aspirated into 1/16 inch tygon tubing (McMaster-Carr) prior to being delivered into the microchannel. We carried out hundreds of electroporation trials using the three platforms; however, only the successful transformation parameters are summarized in the tables below. Electroporation parameters that were not successful are included in Table S1 for the exponential decay system and Table S2 for the microfluidic system. + +### Parabodo caudatus Transfection Efficiency Post Microfluidic Electroporation +#### Step 5. + +The microfluidic electroporation system resulted in the highest transfection efficiencies ranging from 20 % to 50 % (Figure 3). Successful P. caudatus transfection employing electric fields of 1,500 V/cm results in transfection efficiencies of 30-40 %, and 2,250 V/cm (40-50 %) using 5 ms pulse durations in MilliQ water using the bilaterally constricting geometry. Additionally, by decreasing the electric field to 1,000 V/cm and employing longer 20 ms pulses we achieved 20-30 % transfection efficiencies in 50 % cytomix buffer using the straight channel constriction. These results demonstrate that different geometric constrictions can be used successfully to modulate the electric field for successful transfection. + +![Figure 3: Microfluidic electroporation of Parabodo caudatus.](image_3.jpg) + +Panel a) P. caudatus (brightfield) b) stable pEYFP-Mitotrack transfection at 250 V (E_max = 1,500 V/cm), c) transient pUB-GFP transfection using 375 V (E_max = 2,250 V/cm), d) autofluorescence control for P. caudatus, e) transient transfection using pEF-GFP and 313 V (E_max = 1,000 V/cm) in the straight channel, and f) merged image of brightfield and fluorescence image from e) for visualizing cell morphology. + +### References + +Garcia, P.A., Ge, Z., Kelley, L.E., Holcomb, S.J., and Buie, C.R. (2017) High efficiency hydrodynamic bacterial electrotransformation. **Lab Chip** (17): 490-500. + +Garcia, P.A., Ge, Z., Moran, J.L., and Buie, C.R. (2016) Microfluidic Screening of Electric Fields for Electroporation. **Sci Rep** (6): 21238. + +Whitesides, G.M., Ostuni, E., Takayama, S., Jiang, X.Y., and Ingber, D.E. (2001) Soft lithography in biology and biochemistry. **Annual Review of Biomedical Engineering** (3): 335-373. + +Matsuda, T., and Cepko, C.L. (2004) Electroporation and RNA interference in the rodent retina in vivo and in vitro. **Proc Natl Acad Sci U S A** (101):16-22. + +Robinson, M.S., Sahlender, D.A., and Foster, S.D. (2010) Rapid inactivation of proteins by rapamycin-induced rerouting to mitochondria. **Dev Cell** (18): 324-331. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/microtiter-plate-microbial-growth-measurements-dcex2tfn.md b/markdown-output/microtiter-plate-microbial-growth-measurements-dcex2tfn.md new file mode 100644 index 0000000000000000000000000000000000000000..24fe69fdf25b48a8f0ff55fb277d9de417506b16 --- /dev/null +++ b/markdown-output/microtiter-plate-microbial-growth-measurements-dcex2tfn.md @@ -0,0 +1,81 @@ +```markdown +# Goal/Experiment: +The goal of this protocol is to determine bacterial growth rates using optical density (OD) measurements in a 200 μL microtiter plate. + +# Microtiter Plate Microbial Growth Measurements + +**DOI:** [dx.doi.org/10.17504/protocols.io.n2bvjny5bgk5/v1](https://dx.doi.org/10.17504/protocols.io.n2bvjny5bgk5/v1) +**Authors:** Fred Breidt¹, Allison Anthony² +¹USDA ARS; ²USDA ARS + +Fred Breidt: Primary author and developer of the software; +Allison Anthony: Contributing author + +## Protocol Information +- **Protocol Status:** Working +- **We use this protocol and it’s working** + +### Details +- **Created:** April 19, 2024 +- **Last Modified:** July 25, 2024 +- **Protocol Integer ID:** 98487 + +## Disclaimer +*This method and accompanying software are intended for research use only.* + +## Abstract +Automated microtiter plate growth kinetics measurements for microorganisms are subject to significant bias due to variations in initial optical density (OD) of growth curves, improper background subtraction, and variation in background OD for uninoculated media. This protocol uses an automated microtiter plate reader with 96 well microtiter plates, having a final volume of 200 µL of growth media per well. The method estimates maximum growth rate, lag time, and maximum OD readings. The Matlab LiveScript software, ProcessMicroplate.mlx, generates output data including spreadsheets and graphs showing various growth metrics. + +## Overnight Culture Preparation +1. **Introduction and Objective:** + Determine bacterial growth rates using OD in a 200 µL microtiter plate. Calculate kinetic parameters accurately using Matlab Script (ProcessMicroplate.mlx). + +2. **Prepare Microbial Culture:** + Prepare microbial cultures, typically 5 mL in a 15 mL plastic screw cap tube, one tube per replication. Media must support culture growth. + + - Typical Growth Conditions: + - 30°C: Lactic acid bacteria, yeasts + - 37°C: Pathogenic bacteria + +## Microtiter Plate Preparation +1. **Serial Dilutions in Plate:** + Serial dilutions and background controls for each treatment will be used. + + ![Microtiter Plate Setup](images/microtiter_plate_setup.png) + +2. **Add Growth Medium:** + Pipet 100 µL of appropriate media into all wells according to the chosen setup before adding cells. + +## Inoculum Preparation +1. **Vortex and Transfer Cells:** + Vortex overnight culture, transfer 0.5 mL to microcentrifuge tube, one tube per replication per media type. Centrifuge at 13000 rpm, 22 °C, and remove supernatant. + +2. **Resuspend Cells:** + Resuspend cells by vortexing using 1 mL media. The cell culture dilution series is set up. + +## Microtiter Plate Inoculation +1. **Add Resuspended Cells:** + For each replication, resuspend culture adding 100 µL to wells in row A, with media already in the wells. No dilution series needed for control wells. + +2. **Transfer and Mix Cells:** + Using pipette, mix cells and media, transfer 100 µL to next row, repeating until final row. + +3. **Overlay with Mineral Oil:** + Overlay wells with 75 µL of sterile mineral oil to prevent evaporation. Inspect plates for air bubbles. + +## Cell Growth and Automated Optical Density Data +1. **Microtiter Plate Reader Setup:** + Place plate in automated reader (e.g., BioTek Epoch 2, BioTek Instruments). Set to record optical density at 600 nm. Incubate with shaking, monitor OD for 24-48 hours. + +## Data Analysis with ProcessMicroplate, Matlab LiveScript Software +1. **Export Data:** + Export OD data to a .csv file. Format: N time-points rows, 97 columns (1 elapsed time, 96 OD readings). + +2. **Process and Analyze Data:** + Use ProcessMicroplate software for data processing. Understand Matlab LiveScript for comprehensive data analysis, generating growth curves and metrics. + +**Note:** Air bubbles interfere with OD readings. Carefully examine and remove if necessary. + +--- +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/milliplex-cardiovascular-disease-3-plex-panel-2-hvcb62w.md b/markdown-output/milliplex-cardiovascular-disease-3-plex-panel-2-hvcb62w.md new file mode 100644 index 0000000000000000000000000000000000000000..547eb2b0c5e4cc38505382108b835718254849e4 --- /dev/null +++ b/markdown-output/milliplex-cardiovascular-disease-3-plex-panel-2-hvcb62w.md @@ -0,0 +1,122 @@ +```markdown +# Goal/Experiment: +The aim of this experiment is to perform a Luminex Milliplex cardiovascular disease 3-plex immunoassay to detect and measure specific biomarkers in serum/plasma samples as per manufacturer's instructions. + +# Milliplex Cardiovascular Disease 3-plex Panel 2 + +**Ligia Pinto, Troy Kemp** + +## Abstract +Luminex Milliplex Cardiovascular Disease 3-plex Panel 2 instructions per manufacturer. + +**Citation:** +Ligia Pinto, Troy Kemp Milliplex Cardiovascular Disease 3-plex Panel 2. protocols.io dx.doi.org/10.17504/protocols.io.hvcb62w + +**Published:** +11 Aug 2017 + +## Protocol + +### Step 1: Preparation of Samples/Reagents for Immunoassay + +#### 1. Preparation of Serum/Plasma Thaw Time: +Thaw the samples completely on ice, mix well by shaking on a plate shaker for 1 minute at room temperature (20-25°C), and centrifuge (1,700 x g, 10 minutes, 4°C) prior to use in the assay to remove particulates. L-AB Assay Buffer provided in the kit should be used as the sample diluent. + +**Serum/Plasma sample dilution (1:2000):** +- Step 1: Add 20 µL serum/plasma to 980 µL Assay Buffer (1:50). +- Step 2: Add 20 µL of 1:50 diluted sample to 780 µL of Assay Buffer (1:2,000). + +#### 2. Preparation of Antibody-Immobilized Beads +Sonicate each individual antibody-bead vial for 30 seconds; vortex for 1 minute. Add 150 µL from each antibody-bead vial to the Mixing Bottle and bring final volume to 3.0 mL with Bead Diluent. Vortex the mixed beads well. Unused portion may be stored at 2-8°C for up to one month. + +**Example:** When using 3 antibody-immobilized beads, add 150 µL from each of the 3 bead sets to Mixing Bottle. Then add 2.55 mL Bead Diluent. + +#### 3. Preparation of Quality Controls + +**Reconstitution Time:** +Before use, reconstitute Quality Control 1 and Quality Control 2 with 250 µL deionized water. Invert the vial several times to mix and vortex. Allow the vial to sit for 5-10 minutes and then transfer the controls to appropriately labeled polypropylene microfuge tubes. Unused portion may be stored at ≤ -20°C for up to one month. + +#### 4. Preparation of Wash Buffer +Bring the 10X Wash Buffer to room temperature and mix to bring all salts into solution. Dilute 30 mL of 10X Wash Buffer with 270 mL deionized water. Store unused portion at 2-8°C for up to one month. + +#### 5. Preparation of Human CVD Panel 2 Standard + +**Reconstitution Time:** +1. Prior to use, reconstitute the Human CVD Panel 2 Standard with 250 µL deionized water to give a stock concentration termed STD7. Invert the vial several times to mix. Allow the vial to sit for 5-10 minutes and then transfer the standard to an appropriately labeled polypropylene microfuge tube. This will be used as the STD7 tube; the unused portion may be stored at ≤ -20°C for up to one month. + +2. Preparation of Working Standards. Label six polypropylene microfuge tubes STD6, STD5, STD4, STD3, STD2, and STD1. + - Add 200 µL of Assay Buffer to each of the six tubes. + - Prepare serial dilutions by adding 50 µL of the STD7 reconstituted standard to the STD6 tube, mix well and transfer 50 µL of the STD6 standard to the STD5 tube, mix well and transfer 50 µL of the STD5 standard to the STD4 tube, mix well and transfer 50 µL of the STD4 standard to STD3 tube, mix well and transfer 50 µL of the STD3 standard to the STD2 tube, mix well and transfer 50 µL of the STD2 standard to the STD1 tube and mix well. The 0 pg/mL standard (Background) will be Assay Buffer. + + **Standard Concentrations Table** + + | Standard Concentration (pg/mL) | Volume of Deionized Water to Add (mL) | Volume of Standard to Add | + | ------------------------------| --------------------------------------| -------------------------| + | STD7 | N/A | 0 | + | STD6 | 200 | 50 µL of STD7 | + | STD5 | 200 | 50 µL of STD6 | + | STD4 | 200 | 50 µL of STD5 | + | STD3 | 200 | 50 µL of STD4 | + | STD2 | 200 | 50 µL of STD3 | + | STD1 | 200 | 50 µL of STD2 | + + The serial dilutions result in the following concentrations of standards. + + **Serial Dilutions Result Table** + + | Standard Tube # | CRP (pg/mL) | SAA (pg/mL) | SAP (pg/mL) | + | --------------- | ---------- | ----------- | ----------- | + | STD1 | 3.2 | 16.0 | 16.0 | + | STD2 | 16.0 | 80.0 | 80.0 | + | STD3 | 80.0 | 400.0 | 400.0 | + | STD4 | 400.0 | 2,000 | 2,000 | + | STD5 | 2,000 | 10,000 | 10,000 | + | STD6 | 10,000 | 50,000 | 50,000 | + | STD7 | 50,000 | 250,000 | 250,000 | + +### Immunoassay Procedure + +- Allow all reagents to warm to room temperature (20-25°C) before use in the assay. +- Run the standards, controls, and samples in duplicate. +- Set the filter plate on a plate holder at all times during reagent dispensing and incubation steps so that the bottom of the plate does not touch any surface. + +#### Steps: +1. Prewet the filter plate by pipetting 200 µL of Wash Buffer into each well of the Microtiter Filter Plate. Seal and mix on a plate shaker for 10 minutes at room temperature (20-25°C). +2. Remove Wash Buffer by vacuum. Blot excess Wash Buffer from the bottom of the plate with an absorbent pad or paper towels. +3. Add 25 µL of each Standard or Control into the appropriate wells. Add 25 mL Assay Buffer to the 0 pg/mL standard (Background). +4. Add 25 µL of Assay Buffer to the sample wells. +5. Add 25 µL of the Assay Buffer solution to the background, appropriate standards, and control wells. +6. Add 25 µL of Sample into the appropriate wells. +7. Vortex Mixing Bottle and add 25 µL of the mixed Beads to each well. (Note: During addition of Beads, shake bead bottle intermittently to avoid settling). +8. Seal the plate with a plate sealer, cover it with the lid. Wrap a rubber band around the plate holder, plate, and lid and incubate with agitation on a plate shaker 1 hour at room temperature (20-25°C). +9. Gently remove fluid by vacuum. +10. Wash plate 2 times with 200 µL/well of Wash Buffer, removing Wash Buffer by vacuum filtration between each wash. Blot excess Wash Buffer from the bottom of the plate with an absorbent pad or paper towels. +11. Add 25 µL of Detection Antibodies into each well. (Note: Allow the Detection Antibodies to warm to room temperature prior to addition.) +12. Seal, cover with lid, and incubate with agitation on a plate shaker for 30 minutes. **Do not vacuum after incubation.** +13. Add 25 µL Streptavidin-Phycoerythrin to each well containing the 25 µL of Detection Antibodies. +14. Seal, cover with lid, and incubate with agitation on a plate shaker for 30 minutes at room temperature (20-25°C). +15. Gently remove all contents by vacuum. +16. Wash plate 2 times with 200 µL/well Wash Buffer, removing Wash Buffer by vacuum filtration between each wash. Wipe any excess buffer on the bottom of the plate with a tissue. +17. Add 100 µL of Sheath Fluid to all wells. Resuspend the beads on a plate shaker for 5 minutes. +18. Run plate on Luminex 100™ IS. +19. Save and analyze the data using Bio-Plex Manager software. + +## Equipment Settings + +- **Events:** 50, per bead region +- **Sample Size:** 50 µL +- **Gate Settings:** 4335 to 10,000 +- **Time Out:** 60 seconds + +## Quality Controls + +The ranges for each analyte in Quality Control 1 and 2 are provided on the card insert or can be located at the Millipore website www.millipore.com/techlibrary/index.do using the catalog number as the keyword. + +## Notes + +## Procedure - Quick Reference + +### Step 2 + +`endofoutput` +``` \ No newline at end of file diff --git a/markdown-output/milliplex-cytokine-chemokine-7-plex-mag-hvgb63w.md b/markdown-output/milliplex-cytokine-chemokine-7-plex-mag-hvgb63w.md new file mode 100644 index 0000000000000000000000000000000000000000..8b9d316980456f2200af258b2e46757fc547bed5 --- /dev/null +++ b/markdown-output/milliplex-cytokine-chemokine-7-plex-mag-hvgb63w.md @@ -0,0 +1,160 @@ +```markdown +# Goal/Experiment: +The purpose of this experiment is to perform a Milliplex Cytokine/Chemokine 7-plex MAG immunoassay, which is designed to simultaneously measure multiple cytokines and chemokines in biological samples. + +# Milliplex Cytokine/Chemokine 7-plex MAG + +**Authors:** +Ligia Pinto, Troy Kem + +**Published:** 01 Aug 2017 + +**Abstract:** +Milliplex Cytokine/Chemokine 7-plex manufacturer's protocol + +**Citation:** +Ligia Pinto, Troy Kem Milliplex Cytokine/Chemokine 7-plex MAG. protocols.io https://dx.doi.org/10.17504/protocols.io.hvgb63w + +--- + +## Protocol + +### Step 1: Preparation of Samples/Reagents for Immunoassay + +**Step 2: Preparation of Serum/Plasma Thaw Time** +- Thaw the samples completely on ice. +- Mix well by shaking on a plate shaker for 1 min. at RT (20-25°C). +- Centrifuge (1,700 xg, 10 minutes, 4°C) prior to use in the assay to remove particulates. + +**Step 3: Preparation of Antibody-Immobilized Beads** +- Sonicate each individual antibody-bead vial for 30 seconds; vortex for 1 minute. +- Add 60 µL from each antibody-bead vial to the Mixing Bottle and bring final volume to 3.0 mL with Bead Diluent. +- Vortex the mixed beads well. Unused portion may be stored at 2-8°C for up to one month. + + **Note:** Due to the composition of magnetic beads, you may notice a slight color in the bead solution. This does not affect the performance of the beads or the kit. Example: When using 7 cytokine antibody-immobilized beads, add 60 µL from each of the 7 bead sets to the Mixing Bottle. Then add 2.58 mL Bead Diluent. + +**Step 4: Preparation of Quality Controls** +- Reconstitution Time: Before use, reconstitute Quality Control 1 and Quality Control 2 with 250 µL deionized water. +- Invert the vial several times to mix and vortex. Allow the vial to sit for 5-10 minutes and then transfer the controls to appropriately labeled polypropylene microfuge tubes. +- Unused portion may be stored at ≤ -20°C for up to one month. + +**Step 5: Preparation of Wash Buffer** +- Bring the 10X Wash Buffer to room temperature and mix to bring all salts into solution. +- Dilute 60 mL of 10X Wash Buffer with 540 mL deionized water. +- Store unused portion at 2-8°C for up to one month. + +**Step 6: Preparation of Serum Matrix** +- Reconstitution Time: Add 1.0 mL deionized water to the bottle containing lyophilized Serum Matrix. +- Mix well. Allow at least 10 minutes for complete reconstitution. +- Leftover reconstituted Serum Matrix should be stored at ≤ -20°C for up to one month. + +**Step 7: Preparation of Human Cytokine Standard** +- Reconstitution Time: Prior to use, reconstitute the Human Cytokine Panel III standard with 250 µL deionized water to give STD7. +- Invert the vial several times to mix. Vortex the vial for 10 seconds. +- Allow the vial to sit for 5-10 minutes and then transfer the standard to an appropriately labeled polypropylene microfuge tube. +- This standard will be termed STD7; the unused portion may be stored at ≤ -20°C for up to one month. + +**Step 8: Preparation of Working Standards** +- Label six polypropylene microfuge tubes STD6, STD5, STD4, STD3, STD2, and STD1. +- Add 150 µL of Assay Buffer to each of the six tubes: + - Prepare serial dilutions by adding 50 µL of STD7 reconstituted standard to the STD6 tube, mix well and transfer 50 µL of the STD6 standard to the STD5 tube, mix well and transfer 50 µL of the STD5 standard to the STD4 tube, mix well and transfer 50 µL of the STD4 standard to the STD3 tube, mix well and transfer 50 µL of the STD3 standard to the STD2 tube, mix well and transfer 50 µL of the STD2 standard to the STD1 tube and mix well. +- The 0 pg/mL standard (Background) will be Assay Buffer. + +--- + +### Immunoassay Procedure + +**Step 28: Run the Standards, Controls, and Samples** +- Allow all reagents to warm to room temperature (20-25°C) before use in the assay. +- Run the standards, controls, and samples in duplicate. +- Prewet the plate by pipetting 200 µL of Assay Buffer into each well of the MAG Plate. Seal and mix on a plate shaker for 10 minutes at room temperature (20-25°C). + +**Step 30: Preparing the Plate** +- Decant Assay Buffer and remove residual amount from all wells by inverting the plate and tapping it smartly onto absorbent towels several times. +- Add 25 µL of each Standard or Control into the appropriate wells. Add 25 µL Assay Buffer to the 0 pg/mL standard (Background). + +**Step 31: Adding Assay Buffer to Sample Wells** +- Add 25 µL of Assay Buffer to the sample wells. + +**Step 32: Adding Serum Matrix** +- Add 25 µL of the Serum Matrix solution to the background, appropriate standards, and control wells. + +**Step 33: Adding Samples** +- Add 25 µL of Sample into the appropriate wells. + +**Step 34: Preparing the Mixed Beads** +- Vortex Mixing Bottle and add 25 µL of the mixed Beads to each well. + **Note:** During addition of Beads, shake bead bottle intermittently to avoid settling. Due to the composition of magnetic beads, you may notice a slight color in the bead solution. This does not affect the performance of the beads or the kit. + +**Step 35: Sealing the Plate** +- Seal the plate with a plate sealer, cover it with the lid. Wrap a rubber band around the plate, lid, and shaker platform and incubate with agitation on a plate shaker 2 hours at room temperature (20-25°C). + +**Step 36: Removing Fluid by Aspiration** +- Gently remove fluid by aspiration. Do not invert plate. + +**Step 37: Washing the Plate** +- Wash plate 2 times with 200 µL/well of Wash Buffer, removing Wash Buffer by aspiration between each wash. + - To avoid washing/aspiration related bead loss, allow approximately 60 seconds between dispensing of the Wash Buffer and subsequent aspiration. + +**Step 38: Adding Detection Antibodies** +- Add 25 µL of Detection Antibodies into each well. + **Note:** Allow the Detection Antibodies to warm to room temperature prior to addition. + +**Step 39: Incubation Steps** +- Seal, cover with lid, and incubate with agitation on a plate shaker for 1 hour at room temperature (20-25°C). DO NOT ASPIRATE AFTER INCUBATION. +- Add 25 µL Streptavidin-Phycoerythrin to each well containing the 25 µL of Detection Antibodies. + +**Step 40: Final Incubation** +- Seal, cover with lid and incubate with agitation on a plate shaker for 30 minutes at room temperature (20-25°C). + +**Step 41: Removing All Contents by Aspiration** +- Gently remove all contents by aspiration. Do not invert plate. + +**Step 42: Washing Steps** +- Wash plate 2 times with 200 µL/well of Wash Buffer, removing Wash Buffer by aspiration between each wash. + - To avoid washing/aspiration related bead loss, allow approximately 60 seconds between dispensing of the Wash Buffer and subsequent aspiration. + +**Step 43: Adding Sheath Fluid** +- Add 150 µL of Sheath Fluid to all wells. Resuspend the beads on a plate shaker for 5 minutes. + +**Step 44: Running the Plate** +- Run plate on Luminex 100™ IS. + +**Step 45: Data Analysis** +- Save and analyze the data using Bio-Plex Manager software. + +--- + +### Equipment Settings + +- **Events:** 50, per bead region +- **Sample Size:** 100 µL +- **Gate Settings:** 5000 to 25,000 +- **Time Out:** 60 seconds + +--- + +### Quality Controls + +- The ranges for each analyte in Quality Control 1 and 2 are provided on the card insert or can be located at the MILLIPORE website [www.millipore.com/techlibrary/index.do](https://www.millipore.com/techlibrary/index.do) using the catalog number as the keyword. + +--- + +### Notes + +**Legend:** +- A: Hemolyzed sample +- B: Lipemic sample +- C: Sample missing +- D: Clogged Filter well +- E: Low bead count +- F: >30% Bead aggregation +- G: Instrument repeat + +**Procedure Quick Reference:** +Milliplex Cytokine/Chemokine 7-plex manufacturer's protocol + +**Step 46:** + +End of output +``` diff --git a/markdown-output/minimum-inhibitory-concentration-mic-and-minimum-b-cvpyw5pw.md b/markdown-output/minimum-inhibitory-concentration-mic-and-minimum-b-cvpyw5pw.md new file mode 100644 index 0000000000000000000000000000000000000000..4b76441094b019ecb8ba8cb44310426632051748 --- /dev/null +++ b/markdown-output/minimum-inhibitory-concentration-mic-and-minimum-b-cvpyw5pw.md @@ -0,0 +1,105 @@ +```markdown +# Goal/Experiment: +To perform comprehensive assessments for the Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of antibacterial agents utilizing the broth microdilution method, aiming at screening bacterial susceptibility to selected antimicrobial substances. + +# Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) Assays Using Broth Microdilution Method + +## Authors +- Tomasz Swebocki¹ +- Alexandre Barras¹ +- Aleksandra Maria Kocot² +- Magdalena Plotka² +- Rabah Boukherroub¹ + +¹ Univ. Lille, CNRS, Centrale Lille, Univ. Polytech. Hauts-de-France, UMR 8520 – IEMN, Lille, France. + +² Department of Microbiology, Faculty of Biology, University of Gdańsk, Gdańsk, Poland. + +## Abstract +The presented protocol outlines a comprehensive assessment of the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values for bacterial cell cultures. These experiments are vital for screening bacterial susceptibility to antibiotics and substances with potential antibacterial properties. The protocol not only covers the necessary preparatory steps but also introduces the application of the widely recognized Gompertz model. The protocol ensures a smooth execution of the assessment through thorough preparation and step-by-step instructions. The user-friendly instructions provided enable researchers to easily follow the protocol, facilitating the implementation of the assessment. By adhering to the outlined procedures, researchers can acquire a deeper understanding of bacterial susceptibility, evaluate the efficacy of antimicrobial agents through MIC and MBC values, and contribute to the advancement of antibacterial strategies. + +## Introduction +Determining the MIC and MBC values is crucial for evaluating the effectiveness of antimicrobial agents against bacteria. This protocol uses the broth microdilution method, which is standardized and provides precise results. Utilizing the Gompertz model allows for the accurate modeling of bacterial growth inhibition. + +## Materials +- Standard 96-well microplate +- Pipettes +- Square Petri dishes with Mueller-Hinton Agar (MHA) +- Microtubes (e.g., Eppendorf Tubes) +- Trough (optional) +- Multichannel pipette (optional) +- Sealing safety film for microplate (optional) +- Mueller Hinton Broth (MHB, BD DIFCO™ Mueller Hinton Broth 500g, Catalog #275730) +- Mueller Hinton Agar (MHA, BD DIFCO™ Mueller Hinton Agar 500g, Catalog #225250) + +## Bacterial Strains +- Staphylococcus aureus 43300 (ATCC) +- Pseudomonas aeruginosa 15442 (ATCC) +- Escherichia coli K-12 (ATCC) + +## Safety Warnings +All manipulations with handling live bacteria should be executed using the biosafety cabinet at all times unless local regulations state otherwise. + +## Before Start Instructions +- Read the protocol fully before starting any manipulation. +- Ensure all equipment needed is working properly. +- Have an ample supply of MHB (preferably around 20 mL). +- Have an inoculated medium on a Petri dish with discrete colonies already prepared. + +## Procedure + +### Pre-preparation of Stock Colony +1. Take the inoculated Petri dish and transfer one colony to a tube with approx. 3 mL of fresh MHB. +2. Incubate the tube for around 3 hours (or more, depending on the strain and the size and age of the colony used) at 37°C. + > **Note:** Stock colony can be prepared in the morning and worked with in the early afternoon. + +### Preparation of Diluted Standardized Inoculum +3. This part should be done only when the 96-well microplate is ready to be inoculated. Prepare standardized inoculum by first measuring the OD600 of your stock colony. + - If OD600 > 0.1, dilute the culture with MHB to reach a value of 0.1. + - If 0.09 < OD600 < 0.1, you can move to the next step. + - If OD600 < 0.09, put the culture back and incubate for 15-30 minutes more. +4. Pour 10 mL of MHB into a trough and add 100 µL of the standardized inoculum. Mix it by flushing a couple of times with a pipette. + > **Note:** 10 mL of diluted standardized inoculum is enough to inoculate 16 rows. + +### Preparation of 96-Well Microplate +5. Dissolve the tested substance (X) in MHB at twice the maximum concentration for the test. + > **Note:** E.g., if you want to test the concentration series of X starting from 100 µg/mL, prepare the stock solution at 200 µg/mL concentration. +6. Pour around 10 mL of MHB into the trough and: + - Add 50 µL of MHB to each well in columns 1-10 (growth control, GC (1) + serial dilution (2-10)). + - Leave wells in column 11 empty (highest concentration of serial dilution). + - Add 100 µL to the wells in column 12 (sterility control, SC). + +7. Add 100 µL of the solution of X diluted in MHB to wells in column 11 and remove and pass 50 µL to the next well until reaching wells in column 2. + > **Note:** This will result in column 2 wells having 100 µL. +8. Using a multichannel pipette, add 50 µL of diluted standardized inoculum to each well of columns 1-11. + > **Note:** Standardized inoculum is equivalent to approx. 10^6 CFU/mL. + +9. Put a protective film on the prepared well plate. Cover it with a lid. Put a name and/or other details on the side of the plate. +10. Incubate the plate at 37°C overnight. + +### Minimal Inhibitory Concentration (MIC) Assessment +11. After incubation, take the microplate out of the incubator. +12. Put the microplate into the plate reader and read OD600 values of the wells' content. +13. Gather the data and use a plotting program such as Prism, OriginPro, Excel, or Kaleida to plot the data and fit it using the modified Gompertz model. + > **Note:** MIC value lies at the intersection of the lower part of the jump with the jump slope. A model file can be accessed [here](https://dx.doi.org/10.17504/protocols.io.5qpvo3x6dv4o/v1). Alternatively, MIC value can be assessed visually (where no turbidity is observed). + +### Minimal Bactericidal Concentration (MBC) Assessment +14. Transfer 100 µL of the content of the wells where no turbidity is observed to a separate 96-well microplate and dilute it ten-fold serially with NaCl solution (9 g/L). +15. Plate 20 µL of the content directly onto square petri dishes with MHA and incubate overnight. +16. Next morning, count the colonies on the plates. + + > **Note:** You can use the CFU calculator to count surviving colonies. The MBC value is where no growth was observed. + +## Optional Alternative Methods +- Use of automated liquid handling systems for better accuracy. +- Substituting Mueller Hinton media to another commonly available alternative in case of supply shortages. +- Alternative antimicrobial susceptibility testing such as disk diffusion or E-test. + +## References +- Access full protocol at: [protocols.io/5qpvo3x6dv4o/v1](https://dx.doi.org/10.17504/protocols.io.5qpvo3x6dv4o/v1). + +## License +This protocol is licensed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. + +**Endofoutput** +``` \ No newline at end of file diff --git a/markdown-output/modelling-protocols-for-derivation-of-fe-iii-nica-brc4m2yw.md b/markdown-output/modelling-protocols-for-derivation-of-fe-iii-nica-brc4m2yw.md new file mode 100644 index 0000000000000000000000000000000000000000..a0d907b10a73df0cad40686e92ab3f74a54eeadc --- /dev/null +++ b/markdown-output/modelling-protocols-for-derivation-of-fe-iii-nica-brc4m2yw.md @@ -0,0 +1,68 @@ +```markdown +Goal/Experiment: +The goal of this experiment is to model protocols for the derivation of Fe(III) NICA constants and the calculation of ambient Fe speciation and apparent Fe(III) solubility in seawater, using the NICA-Donnan model combined with chemical software ORCHESTRA and parameter estimation program PEST. + +# Modelling Protocols for Derivation of Fe(III) NICA Constants and Calculations of Ambient Fe Speciation and Apparent Fe(III) Solubility in Seawater + +**Authors:** Kechen Zhu, Jan E. Groenenberg, Eric P. Achterberg, Martha Gledhill +**Affiliations:** +1. GEOMAR Helmholtz Centre for Ocean Research Kiel, Wischhofstr. 1-3, 24148 Kiel, Germany +2. Wageningen University, Department of Environmental Sciences, PO Box 47, 6700 AA Wageningen, The Netherlands + +**DOI:** [dx.doi.org/10.17504/protocols.io.brc4m2yw](https://dx.doi.org/10.17504/protocols.io.brc4m2yw) + +## Abstract +Calculations of iron (Fe) speciation using the NICA-Donnan model can be useful for developing an understanding of the physico-chemical parameters (e.g., temperature, pH) that can influence Fe speciation in seawater. Here we archive a protocol that can be used to calculate Fe speciation in seawater and derive new NICA constants for Fe binding to marine DOM, in combination with appropriately designed titration experiments. + +Importantly, for derivation of NICA constants, a pH dimension of sufficient resolution and breadth must be included in the experimental design. The protocol combines the chemical software ORCHESTRA with the parameter estimation program PEST and was first described by Janot et al. (2017). + +**Keywords:** Trace metal, humic substance, PEST, ORCHESTRA, biogeochemical cycling + +## Safety Warnings +- Always check the constants in the Minteq4 database for your chemical reactions of interest. +- Applied ion pairing model uses the extended Davies equation to correct for ionic strength; suitable for ionic strength up to ca. 0.5. For seawater, use Pitzer equations. + +## Before Starting +1. Install Java on your computer. +2. Calculations via ORCHESTRA can be performed on Windows, Linux, and Apple OSX, but the combination of PEST-ORCHESTRA can only be performed using Windows. + +## Protocol +### The Application of PEST-ORCHESTRA to the Estimation of NICA-Donnan Parameters in Seawater + +#### 1. Install Required Software +- ORCHESTRA: Chemical speciation software. +- PEST: Parameter estimation program. + +#### 2. Set Up Calculation in ORCHESTRA +- Refer to the manual in the attachment for detailed instructions. + +#### 3. Test Calculation in ORCHESTRA +- Ensure the initial setup and configurations are correct by performing a test run. + +#### 4. Set Up Combination of PEST-ORCHESTRA +- Refer to the manual in the attachment for detailed instructions. + +#### 5. Derive NICA Constants for Fe Binding to Marine Dissolved Organic Matter +- Example files available in the attachment. + +#### 6. Check Results for Optimization of Fe(III) NICA Constants +- Verify the results obtained through the optimization process. + +### Calculations of Ambient Fe Speciation and Apparent Fe(III) Solubility Using the NICA-Donnan Model in Seawater + +#### 7. Calculation of Ambient Fe Speciation via ORCHESTRA +- Example calculation with original source code provided in the attachment. + +#### 8. Calculation of Apparent Fe(III) Solubility via ORCHESTRA +- Example calculation with original source code provided in the attachment. + +## References +1. Janot, N., Pinheiro, J.P., Botero, W.G., Meeussen, J.C.L., Groenenberg, J.E., 2017. PEST-ORCHESTRA, a tool for optimising advanced ion-binding model parameters: Derivation of NICA-Donnan model parameters for humic substances reactivity. Environ. Chem. 14, 31–38. DOI: [10.1071/EN16039](https://doi.org/10.1071/EN16039) +2. Meeussen, J.C.L., 2003. Orchestra: An object-oriented framework for implementing chemical equilibrium models. Environ. Sci. Technol. 37, 1175–1182. DOI: [10.1021/es025597s](https://doi.org/10.1021/es025597s) +3. Doherty, J., 2019. PEST, Model-Independent Parameter Estimation, User Manual Part I. + +## License +This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/modified-salting-out-method-for-high-molecular-wei-c2igycbw.md b/markdown-output/modified-salting-out-method-for-high-molecular-wei-c2igycbw.md new file mode 100644 index 0000000000000000000000000000000000000000..e765fc8c122f48770a7f6050583c9bb14a617fe9 --- /dev/null +++ b/markdown-output/modified-salting-out-method-for-high-molecular-wei-c2igycbw.md @@ -0,0 +1,99 @@ +```markdown +# Goal/Experiment: +To establish a high-molecular-weight genomic DNA extraction protocol for oribatid mites, enabling the generation of high-quality phased genomes for small non-model organisms. + +## Modified Salting Out Method for High Molecular Weight gDNA Extraction (Oribatid Mites) + +### Authors +- Hüsnü Öztoprak +- Jens Bast + +### Affiliation +- **Institute for Zoology, University of Cologne, Köln, Germany** + +### Abstract +This protocol describes a low-cost, high-molecular-weight genomic DNA extraction method for a single minuscule specimen (modified from Miller et al 1988). DNA extractions from oribatid mites are typically challenging due to their small body size (150-1400 µm) and chitinous exoskeleton, which complicates DNA purification and leads to additional loss of DNA. This protocol aims to achieve high-molecular-weight DNA extraction from oribatid mites while preserving the exoskeleton for morphological analysis, using Chitinase to yield more gDNA. + +### Materials and Reagents + +#### General Reagents +- **Proteinase K**: Qiagen Catalog #19131, 2 mL +- **Yeast tRNA**: Thermo Fisher Scientific Catalog #AM7119, (10 mg/mL) +- **RNase Cocktail; Enzyme Mix**: Thermo Fisher Scientific Catalog #AM2286 + +#### Solutions +- **TNES Buffer**: + - NaCl: 400 mM + - EDTA: 20 mM + - Tris pH 8.0: 50 mM + - SDS: 0.5% + +### Procedure + +#### Specimen Cleansing +**Note:** Specimens are collected from natural populations and cleansed prior to DNA extraction to minimize external contamination. + +1. Brush the specimen in distilled water and distilled water with detergent (fit GmbH, Zittau, Germany). +2. Incubate in NaClO 0.05% (DonKlorix; CP GABA GmbH, Hamburg, Germany) and ethanol 70% for 30 seconds each. +3. Rinse in distilled water. + +#### Version i) High-molecular gDNA Extraction + +1. Submerge cleansed specimen in 195 µL TNES buffer and flash freeze by holding tube in liquid nitrogen. +2. Homogenize with a sterile pestle to grind the specimen. +3. Add 5 µL proteinase K. +4. Vortex for 3 seconds and centrifuge briefly. +5. Incubate at 55°C for 1 hour until the specimen is completely dissolved. +6. Centrifuge at 18000 rcf for 5 minutes and transfer supernatant to a fresh tube. + +**Note:** If there's indigestible debris left, it can affect the DNA yield. + +7. Add 1.5 µL yeast tRNA, flick to mix briefly then spin down. +8. Add 65 µL 5 M NaCl and 290 µL 96% EtOH, mix by inversion. +9. Store at -20°C for 1 hour to help clarify the solution. +10. Spin down at 18000 rcf, 4°C, for 15 minutes. + +**Optional:** Add 1 µL Pellet Paint Co-Precipitant for a colorful pellet. + +11. DNA pellet should be visible. Remove supernatant. +12. Add 0.5 mL chilled 70% EtOH (make fresh). Spin at 18000 rcf for 5 minutes. +13. Repeat ethanol rinse. +14. Carefully remove supernatant. +15. Leave tube open to air dry until the pellet has a glassy appearance. +16. Add 21 µL TE Buffer and gently resuspend the DNA pellet. +17. Incubate at 4°C overnight. +18. Add 2 µL RNase Cocktail. Incubate at 37°C for 1 hour. + +**Note:** For femto pulse systems, avoid EDTA in TE Buffer. + +#### Version ii) High-molecular gDNA Extraction Preserving Exoskeleton + +1. Place the cleansed specimen on a sterile slide submerged in TNES buffer. +2. Remove one genital plate cautiously with a sharp needle. +3. Transfer specimen in 195 µL TNES buffer. +4. Add 5 µL proteinase K. +5. Incubate at 37°C overnight. +6. Transfer specimen with sterile needle to a 70% EtOH tube for morphological analysis. +7. Add 1.5 µL yeast tRNA, flick to mix briefly then spin down. +8. Add 65 µL 5 M NaCl and 290 µL 96% EtOH, mix by inversion. +9. Store at -20°C for 1 hour to help clarify the solution. + +#### Version iii) gDNA Extraction with Chitinase Digestion + +1. Submerge specimen in 195 µL TNES buffer and flash freeze by holding tube in liquid nitrogen. +2. Homogenize with a sterile pestle to grind the specimen. +3. Add 2 µL chitinase (1 U/mL). +4. Vortex for 5 seconds and centrifuge briefly. +5. Incubate at 55°C for 1 hour. +6. Add 5 µL proteinase K. +7. Vortex for 5 seconds and centrifuge briefly. +8. Incubate at 55°C for 1 hour until the specimen is completely dissolved. +9. Centrifuge at 18000 rcf for 5 minutes and transfer supernatant to a fresh tube. +10. Add 1.5 µL yeast tRNA, flick to mix briefly then spin down. +11. Add 65 µL 5 M NaCl and 290 µL 96% EtOH, mix by inversion. +12. Store at -20°C for 1 hour to help clarify the solution. + +**Note:** DNA purification follows the same procedure as above. + +### endofoutput +``` \ No newline at end of file diff --git a/markdown-output/modular-automated-bottom-up-proteomic-sample-prepa-b3gxqjxn.md b/markdown-output/modular-automated-bottom-up-proteomic-sample-prepa-b3gxqjxn.md new file mode 100644 index 0000000000000000000000000000000000000000..453b278ddab5c5cb975df521c30cf9ebee5035a7 --- /dev/null +++ b/markdown-output/modular-automated-bottom-up-proteomic-sample-prepa-b3gxqjxn.md @@ -0,0 +1,86 @@ +```markdown +# Goal/Experiment: +To automate and optimize the preparation of proteomic samples for high-throughput applications using a modular approach with a focus on lysing bacterial and fungal cells, quantifying extracted protein, and normalizing protein amounts for tryptic digestion. + +# Modular Automated Bottom-Up Proteomic Sample Preparation for High-Throughput Applications V.2 + +## Authors +- Yan Chen +- Nurgul Kaplan Lease +- Jennifer Gin +- Tad Ogorzalek +- Paul D. Adams +- Nathan Hillson +- Christopher J Petzold + +###### Lawrence Berkeley National Laboratory +**Published in:** PLOS One +**Date:** January 12, 2022 +**DOI:** [10.17504/protocols.io.b3gxqjxn](https://dx.doi.org/10.17504/protocols.io.b3gxqjxn) +**Journal DOI:** [10.1371/journal.pone.0264467](https://doi.org/10.1371/journal.pone.0264467) + +## Abstract +Manual proteomic sample preparation methods limit sample throughput and often lead to poor data quality when thousands of samples must be analyzed. Automated workflows are increasingly used to overcome these issues for some (or even all) of the sample preparation steps. Here, we detail three optimized step-by-step protocols to: +1. Lyse Gram-negative bacteria and fungal cells; +2. Quantify the amount of protein extracted; +3. Normalize the amount of protein and set up tryptic digestion. + +These protocols have been developed to facilitate rapid, low variance sample preparation of hundreds of samples, be easily implemented on widely-available Beckman-Coulter Biomek automated liquid handlers, and allow flexibility for future protocol development. By using this workflow, 50 micrograms of peptides for 96 samples can be prepared for tryptic digestion in under an hour. We validate these protocols by analyzing 47 E. coli and R. toruloides samples and show that this modular workflow provides robust, reproducible proteomic samples for high-throughput applications. The expected results from these protocols are 94 peptide samples from Gram-negative bacterial and fungal cells prepared for bottom-up quantitative proteomic analysis without the need for desalting column cleanup and with peptide variance (CVs) below 15%. + +## Materials and Equipment +- **Beckman-Coulter Biomek FX:** Automated liquid handler system with a 96-pod head, used for the protein extraction and quantification protocols. + *Alternative:* Other automated liquid handlers can be used with appropriate method development. + +- **Beckman-Coulter Biomek NX-S8 or NXP:** Liquid handler system with an 8-pod head, used for the normalization protocol. + *Alternative:* Other automated liquid handlers can be used with appropriate method development. + +- **Molecular Devices Spectramax 250:** Microplate reader used for the protein quantification assay measurement. + +- **Eppendorf 5810R Centrifuge:** With S-4-104 rotor, or similar centrifuge. + +## Safety and Waste Disposal +1. **Personal Protective Equipment (PPE):** Wear gloves, safety goggles, and lab coat. Prepare solvents in a chemical fume hood. +2. **Storage:** Store organic solvents in a flammable storage cabinet when not in use. +3. **Waste Disposal:** Discard used solvents and buffers in appropriate waste containers. + +## Protocol Overview +### Step 1: Cell Lysis +- **Goal:** Lyse Gram-negative bacteria and fungal cells. +- **Reagents and Equipment:** + - Cell lysis buffer + - Beckman-Coulter Biomek FX Liquid handler + - Eppendorf 5810R Centrifuge +- **Procedure:** + 1. Prepare the cell lysis buffer. + 2. Load samples onto the Biomek FX Liquid handler. + 3. Centrifuge lysed samples at 4000g for 10 minutes at 4°C. + +### Step 2: Protein Quantification +- **Goal:** Quantify the amount of protein extracted. +- **Reagents and Equipment:** + - Lowry protein assay reagent + - Molecular Devices Spectramax 250 Microplate reader +- **Procedure:** + 1. Prepare the Lowry assay reagent. + 2. Dilute protein samples appropriately. + 3. Measure absorbance at 750 nm using the microplate reader. + 4. Calculate protein concentration based on a standard curve. + +### Step 3: Protein Normalization and Tryptic Digestion +- **Goal:** Normalize the amount of protein and set up tryptic digestion. +- **Reagents and Equipment:** + - Tryptic digestion buffer + - Beckman-Coulter Biomek NX-S8 or NXP Liquid handler +- **Procedure:** + 1. Normalize protein concentration across samples. + 2. Prepare tryptic digestion buffer. + 3. Incubate samples with trypsin at 37°C for 16 hours. + +## Expected Results +Validation of these protocols by analyzing 47 E. coli and R. toruloides samples has shown that this modular workflow provides robust, reproducible proteomic samples for high-throughput applications. The expected results are 94 peptide samples from Gram-negative bacterial and fungal cells prepared for bottom-up quantitative proteomic analysis without the need for desalting column cleanup, with peptide variance (CVs) below 15%. + +## References +1. Chen, Y., Lease, N. K., Gin, J. W., Ogorzalek, T. L., Adams, P. D., Hillson, N. J., Petzold, C. J. (2022). Modular automated bottom-up proteomic sample preparation for high-throughput applications. PLOS ONE, 17(2): e0264467. doi: [10.1371/journal.pone.0264467](https://doi.org/10.1371/journal.pone.0264467). + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/morphometric-study-of-the-lumbar-vertebrae-in-drie-cjqhumt6.md b/markdown-output/morphometric-study-of-the-lumbar-vertebrae-in-drie-cjqhumt6.md new file mode 100644 index 0000000000000000000000000000000000000000..08f59fea340aa8c52f5e0b9ddd1f1e2c4ea85b09 --- /dev/null +++ b/markdown-output/morphometric-study-of-the-lumbar-vertebrae-in-drie-cjqhumt6.md @@ -0,0 +1,120 @@ +```markdown +# Goal/Experiment: +The objective of this anatomical study was to perform the morphometry of dried lumbar vertebrae in human cadavers. + +# Morphometric Study of the Lumbar Vertebrae in Dried Anatomical Collections + +**DOI:** [10.17504/protocols.io.ewov1o9yklr2/v1](https://dx.doi.org/10.17504/protocols.io.ewov1o9yklr2/v1) + +**Mangala M. Pai** +Kasturba Medical College, Manipal Academy of Higher Education, Mangalore, India + +## Abstract +### Background: +The objective of this anatomical study was to perform the morphometry of dried lumbar vertebrae in human cadavers. + +### Methods: +This study utilized 200 adult human cadaveric dried lumbar vertebrae. The digital Vernier calipers were used to perform the measurements. The height, antero-posterior length, transverse length of the body of the vertebrae, interpedicular distance at the lateral ends, lamina length, height and thickness, superior and inferior articular facet height and width, mid sagittal and transverse diameter of vertebral foramen, height, width and thickness of the pars inter-articularis were measured. + +#### Results: +- The vertebral body's antero-posterior length was more at the lower border than at the superior border (p < 0.01). +- The length of lamina was higher over the right in comparison to the left (p < 0.001). +- The height of lamina, width of inferior articular facet, diameter of lateral recess and thickness of pars inter-articularis were greater for the left-sided specimens (p < 0.01). +- The statistical significance was not observed for the comparison of the remaining parameters (p > 0.05). + +### Conclusion: +This anatomical study offered several dimensions of lumbar vertebrae, which are essential in surgical practice. The implants at the lumbar vertebrae need to be manufactured based on the anatomical dimensions of that particular sample population. + +## Introduction +The lumbar segment of the vertebral column is a non-rib bearing area, which is responsible for the transmission of force from the axial to appendicular skeleton and being the mobile part, it is more prone to instability. Following accidents, degenerative diseases, metastasis and congenital defects, lumbar canal stenosis is a main cause for lower back pain. Approximately 50% of adults have experienced an episode of lower back pain at some point in their lifetime. + +There is a necessity for the morphometric details for lumbar vertebrae instrumentation and surgical procedures such as decompression and fixation surgeries. Neurological procedures, like vertebral body reconstruction and realignment, also require such data. + +The pars inter-articularis (PI) is a fragment with high susceptibility to conditions such as spondylosis and spondylolisthesis. Morphometric details of vertebrae vary across ancestries, ethnicities, and geographical regions. Exploring these details is crucial due to the lack of studies, especially for the dorso-lateral vertebrae in the Indian population. + +## Knowledge Gap Identified +The morphological dimensions of the lumbar vertebrae have been extensively studied in the Western population but lack data from the Indian demographic. This study aims to fill this gap and provide great help to orthopedic surgeons and spinal surgeons for screw and implant manufacturers. + +## Review of Literature +The PI region's susceptibility to trauma is due to its narrow structure. Degenerative spondylolisthesis is often associated with lumbar canal stenosis. Vertebral body anterior or posterior subluxation arises from facet joint erosion. Studies show the vertebral diameters increase from L1 to L5 except at L3 where a decrease in diameter is noted. Lumbar PI fractures are generally repaired with favorable outcomes unless associated with lumbar disc degeneration. + +## Aim +The aim of this study is to study the morphometry of the lumbar spine in cadaveric dried specimens. + +## Objectives +1. To measure the lumbar canal dimensions in our study population. +2. To measure the various parameters of lumbar vertebral morphometry with emphasis on pars inter-articularis. +3. To compare our morphometric data with that of other populations. + +## Methodology + +### Study Setting +Department of Anatomy, KMC, Mangalore. + +### Study Design +Descriptive Cross-sectional study. + +### Study Participants +Study will be conducted on 100 adult cadaveric dry human lumbar vertebrae. + +### Inclusion Criteria +- Cadaver dried adult vertebrae irrespective of gender will be considered for this study. + +### Exclusion Criteria +- Damaged vertebrae and congenitally deformed vertebrae will be excluded. + +### Study Duration +The data will be collected in a period of 3 months. + +### Sample Size +100 adult dry human lumbar vertebrae. + +### Sampling Method +The sample size is determined by referring to the previous study by Prameela et al. +\[ n = 2 \left( Z_{1-\alpha/2} + Z_{1-\beta} \right)^2 \frac{\sigma^2}{d^2} \] + +### Data Collection Methodology +The dimensions will be expressed in millimeters and tabulated as mean ± standard deviation. + +### Parameters to be Measured +- Vertebral body height +- Vertebral body antero-posterior length at superior border +- Vertebral body antero-posterior length at inferior border +- Vertebral body transverse length +- Interpedicular distance +- Lamina length right and left side +- Lamina height right and left side +- Superior articular facet width right and left side +- Inferior articular facet width right and left side +- Mid sagittal diameter of vertebral foramen +- Anteroposterior distance of the lateral recess +- Transverse diameter of vertebral foramen +- Dimensions of pars inter-articularis + +### Data Analysis +The most recent version of the SPSS software will be utilized for statistical analysis. + +## Implications +The study will provide further insights regarding the morphometry of dorso-lumbar vertebrae focusing on pars inter-articularis and vertebral foramen, aiding understanding of vertebral canal stenosis and surgical implications in the Indian population. + +## References +1. Sassack B, Carrier JD. Anatomy, Back, Lumbar Spine. In: StatPearls. Treasure Island (FL): StatPearls Publishing; August 8, 2021. +2. Inceoglu S, Burghardt A, Akbay A, Majumdar S, McLain RF. Trabecular architecture of lumbar vertebral pedicle. Spine. 2005 Jul 1; 30(13):1485-90. +3. Papageorgiou AC, Croft PR, Ferry S, Jayson MIV, Silman AJ. Estimating the prevalence of low back pain in the general population, Spine: 1995 (20): pp. 1889-1894. +4. Chawla, Kunal & Sharma, Mahesh & Abhaya, Avinash & Kochhar, Suman. (2011). Morphometry of the lumbar pedicle in North West India. European Journal of Anatomy. 15. 155-161. +5. Gocmen M, Karabekir H, Ertekin T, Edizer M, Canan Y, Izzet Duyar I. Evaluation of lumbar vertebral body and disc: a stereological morphometric study. Int. J. Morphol2010;28:841-847. +6. Bonni AK, Koka SR, Richards DJ. Results of buck screw fusion in grade I spondylolisthesis. J R Soc Med 1991;84:270-3. +7. Mansfield JT, Wroten M. Pars Interarticularis Defect. [Updated 2021 Jun 20]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2021. +8. Fraser JF, Huang RC, Girardi FP, Cammisa FP JR. Pathogenesis, presentation and treatment of lumbar spinal stenosis associated with coronal (or) sagittal spinal deformities.Neurosurg Focus 2003;14(1): e6. +9. Soumya P, Santhosh K, Viveka S, Mini K. Morphometric study of pedicles of lumbar vertebrae in SouthernIndia.JournalofEvidencebasedMedicineandHealthcare. 2015;2(39):6182-6191. +10. Banik S, Rajkumari A. Morphometric analysis of lumbar vertebrae and its applied clinical importance. Int J Anat Res 2019;7(2.1):6381-6386. +11. El Rakhawy M, Abd El Rahman ES, Ibrahim L, Ehab A. Lumbarvertebralcanaldimensions:conceptofmorphometric and radiometric study of the human lumbar vertebral canal. International Journal of Experimental and Clinical Anatomy of the Human Lumbar Vertebral Canal 2010; 4:51- 62. +12. Kapoor Y, Anil RS, Krishnaiah M, Suseelamma D. Morphometry of the lumbar vertebrae and its clinical significance. Sch J App Med Sci 2014; 2(2):1045-1052. +13. Mahato NK. Pars inter-articularis and laminar morphology of the terminal lumbar vertebra in lumbosacral transitional variations. North Am J Med Sci 2013;5:357-61. +14. Salib RM, Pettine KA. Modified repair of a defect in spondylolysis or minimal spondylolisthesis by pedicle screw, segmentalwirefixation,andbonegrafting.Spine (Phila Pa 1976) 1993;18:440-3. +15. Grob D, Humke T. Translaminar screw fixation in the lumbarspine:Technique,indications,results.EurSpineJ 1998;7:178-86. +16. Weinstein J, Rydevic B. The pain of spondylolisthesis. Semin Sin Surg 1989;2:100-5. +17. Prameela MD, Prabhu LV, Murlimanju BV, Pai MM, Rai R, Kumar CG. Anatomical dimensions of the typical cervical vertebrae and their clinical implications. European Journal of Anatomy 2020;24(1): 9-15. + +[endofoutput] +``` \ No newline at end of file diff --git a/markdown-output/morris-usf-lab-protocol-bci8iuhw.md b/markdown-output/morris-usf-lab-protocol-bci8iuhw.md new file mode 100644 index 0000000000000000000000000000000000000000..53f06ebaebe1259f7ba33350d84329c799a2759b --- /dev/null +++ b/markdown-output/morris-usf-lab-protocol-bci8iuhw.md @@ -0,0 +1,86 @@ +```markdown +# Morris USF Lab Protocol V.2 + +Lauren Segers¹, Kendall Morris¹, Donald Bolser¹ +¹University of South Florida + +[DOI: 10.17504/protocols.io.bci8uhw](dx.doi.org/10.17504/protocols.io.bci8uhw) + +Date: February 13, 2020 + +## Goal/Experiment: +This protocol describes the methods used to examine neural and physiological responses in decerebrated, paralyzed, and artificially ventilated cats, with a focus on brainstem neuron recordings and nerve isolation during various stimulations and perturbations. + +--- + +## Surgical Protocol + +1. **Preparation and Anesthesia** + - Data were from 13 artificially ventilated adult cats of either sex. Methods were as previously described ([Morris et al., 2010; Ott et al., 2012](https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2858523/)). + - Atropine: Used to reduce mucus secretion in the airways. + - Dexamethasone: Administered to minimize brain swelling and prevent hypotension. + - Anesthesia Induction: Utilizing 5.0 and 1.0-3.0% isoflurane, mixed with medical grade air until decerebration. + - Monitoring: Trachea was intubated, catheters placed in femoral arteries and veins for drug/fluids administration and arterial blood pressure monitoring. + - Regular intervals: Collection and analysis of arterial blood samples, blood pH adjustments with sodium bicarbonate (8.4%), and dopamine (0.04-0.1%) administration to maintain blood pressure ≥ 75 mmHg. + +2. **Bleeding Control & Dissection** + - External carotid arteries ligated and caudal to the lingual artery branch. + - Performed occipital craniotomy, midcollicular transection, and suction decerebration ([Kirsten and St. John, 1978](https://pubmed.ncbi.nlm.nih.gov/357730/)). + - **Vecuronium Bromide**: Administered for neuromuscular blockage to ensure subject paralysis. + +## Nerve Isolation and Recording + +1. **Nerve Isolation** + - Right hypoglossal (XII), left phrenic (Phr), left lumbar iliohypogastric (Lum), left vagus (X), and right recurrent laryngeal nerve (RLN) isolated and desheathed. + - Electrodes: Combined mineral oil and petroleum jelly with silver bipolar hook electrodes, placed in hook configuration, floated in neck pocket, and isolated. + +2. **Nerve Recording** + - Activity amplified, full-wave rectified, low-pass filtered, RC integrated (τ=200-500 ms). + - Monitoring: Respiratory cycle, tracheal pressure, tidal CO2, and arterial blood pressure recorded on a Grass polygraph. + +## Stimuli and Perturbations + +3. **Stimulation Methods** + 1. **Electrical Stimulation of Superior Laryngeal Nerves** (13 subjects): + - SLNs isolated bilaterally; used electrical stimulation (Pulse: 0.1-0.25 ms; Frequency: 5-22 Hz; Voltage: 2.6-4.0V; Current: 33-51.5 µA; Duration: 2-120 s). + + 2. **Water Bolus Administration** (13 subjects): + - Water (5-25 mL) injected rapidly (<5 s) through a polyethylene tube, with three trials at ≥2 min intervals. + + 3. **Mechanical Stimulation** (5 subjects): + - Water tube insertion and mechanical manipulation. + + 4. **Chemoceptive Stimulation** (2 subjects): + - CO2-saturated saline (1 mL) into carotid or vertebral artery over 30 s. + + 5. **Mean Arterial Blood Pressure Changes** (7 subjects): + - Aortic occlusion with embolectomy catheter to increase pressure by ≥25 mmHg. + + 6. **Ventilation Changes** (10 subjects): + - Delayed or no inflations using ventilator adjustments. + + 7. **Esophageal Balloon Inflation** (1 subject): + - Balloon insertion and inflation in the esophagus. + + 8. **Nebulized Substances** (6 subjects): + - Nebulized AITC; Compound 48/80 in 4 subjects. + +## Ventilation Mode Protocols + +4. **Phrenic-Triggered Mode (PT)** + - Used integrated phrenic signal. + - Mean Delay: 458 ± 140 ms between inspiratory phase and lung deflation. + +5. **Free-Run Mode (FR)** + - Ventilator rate: 30 breaths/min, adjusted to maintain PCO2 at 30 ± 0.5 mmHg. + +## Brainstem Neuron Recordings + +5. **Extracellular Neuronal Activity** + - Acquisition: 3-4 multielectrode arrays with adjustable steps and high-impedance tungsten microelectrodes. + - Accurate placement: Guided by anatomical landmarks, recorded neurons in solitary tract, respiratory column, etc. + - Data Isolation: Using extracellular spike sorting software ([O'Connor et al. 2005](https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1360106/)). + +--- +**endofoutput** +``` \ No newline at end of file diff --git a/markdown-output/mouse-stereotaxic-surgeries-c3eryjd6.md b/markdown-output/mouse-stereotaxic-surgeries-c3eryjd6.md new file mode 100644 index 0000000000000000000000000000000000000000..c99f8b4525b02e1726dd26d6f40d467018f45042 --- /dev/null +++ b/markdown-output/mouse-stereotaxic-surgeries-c3eryjd6.md @@ -0,0 +1,157 @@ +```markdown +# Goal/Experiment: +The goal of this experiment is to perform mouse stereotaxic surgeries to administer unilateral or bilateral injections of small volumes of viral vectors or other suspensions into specific regions of the brain's parenchyma. The protocol is optimized to ensure precise injections based on the coordinates of various brain regions. + +# Mouse Stereotaxic Surgeries + +**Authors:** Gabrielle Asta Zane, Nicole J Corbin-Stein, Childers, Jhodi Webster, Vickie Yang, Woong-Jai Won, Rajesh Gupta, Ashley Harms +**Affiliation:** University of Alabama at Birmingham, Department of Neurology, Center for Neurodegeneration and Experimental Therapeutics +**Publication Date:** December 14, 2023 +**DOI:** [10.17504/protocols.io.3byl4q5mzvo5/v1](https://dx.doi.org/10.17504/protocols.io.3byl4q5mzvo5/v1) +**License:** This protocol is distributed under the Creative Commons Attribution License. + +## Abstract +This protocol allows for stereotaxic surgeries to administer unilateral or bilateral injections of small volumes of viral vectors or other suspensions into the mouse parenchyma. After using an anesthesia chamber to put the mouse to sleep, it is then placed and stabilized on the stereotaxic frame and is prepared for surgery. The mouse's bregma coordinates are used to calculate the appropriate coordinates needed to inject into specific brain regions. This protocol is optimized for different brain regions based on the coordinates listed in the guidelines. + +## Guidelines +| Brain Region | M/L (mm) | A/P (mm) | D/V (mm) | +|-----------------------|----------|----------|----------| +| SNpc | 1.2 | 3.2 | -4.6 | +| Striatum | 2.0 | 0.5 | -3.2 | +| Intraventricular | 1.0 | 0.3 | -2.7 | +| Entorhinal Cortex | -3.9 | -3.52 | -4.2 | + +- **Coordinates Definitions:** + - **M/L (Medial/Lateral):** Distance from the midline + - **A/P (Anterior/Posterior):** Distance from the bregma + - **D/V (Dorsal/Ventral):** Depth from the brain surface + +## Pain Management +For pain management, Slow Release (SR) Buprenorphine is utilized. The buprenorphine SR cannot be diluted as it is designed for sustained release. + +### Tips for Handling Buprenorphine SR +- Warm the Buprenorphine SR-LAB vial to 1-2 degrees below body temperature before drawing it into a syringe to reduce viscosity. +- Use a 17-gauge needle to draw from the vial to prevent pressure resistance. +- Use a 1 cc syringe with a low dead-space plunger for precise dosing. + +### Dosage +- **Concentration:** 0.5 mg/mL as recommended by ARP +- **Mouse dosages:** For ~25g mouse, inject 50uL. + +### Heating Pad Instructions +If using an anal heating probe and pad, set the probe to maintain an internal temperature of 35°C. + +## Materials +- Sterotaxic frame +- Benchmark digital coordinate box +- Light source +- Distilled water (sterile) +- Betadine and Isopropyl alcohol +- Sterile cotton swabs (autoclaved) +- 10 uL Hamilton Syringe (Cat# 80030) +- Isoflurane and Oxygen +- Drill and bits +- Sterile ocular lubricant (petroleum ophthalmic ointment) +- Heating pad +- New clean cage +- Hair trimmer +- Timer +- Wound clips +- Gloves +- Surgical utensils: Scalpel, tweezers (sterile) +- Glass bead sterilizer +- Anesthesia box +- Filter for isoflurane collection +- Anesthesia chamber +- Automatic injector +- Disinfectant spray + +## Safety Warnings +- Always wear appropriate PPE when performing stereotaxic surgeries. + +## Setup +1. **Surface and Instrument Sterilization:** + - Sterilize all surfaces with disinfectant. + - Layout materials and utensils needed for surgery. + +2. **Stereotaxic Frame Preparation:** + - Ensure stereotaxic frame is ready on a level table. + - Attach all cords, and switch on the injector, light source, and digital coordinate box. + +3. **Sterilization of Utensils:** + - Sterilize utensils in the glass bead sterilizer for 30 seconds. + - Clean the Hamilton syringe with distilled water 10 times. + - Place sterilized utensils near working area. + +4. **Connection of Anesthesia Line:** + - Connect the oxygen/isoflurane line to the anesthesia chamber and filter. + - Set oxygen flow to 0.5-0.8 (silver ball). + +5. **Anesthesia:** + - Use the anesthesia chamber with isoflurane at 3% to sedate the mouse. + - Monitor breathing and adjust isoflurane to 2.5% if necessary. + +6. **Preparation of Injection Site:** + - Trim hair at the injection site. + - Monitor the mouse breathing, change isoflurane levels as needed. + +7. **Load Hamilton Syringe:** + - Load with an injecting liquid and check for bubbles. + +8. **Positioning:** + - Position mouse in the stereotaxic frame and tighten ear bars. + +9. **Skin Sterilization:** + - Swab with isopropanol, betadine, then isopropanol again, 3 times. + +## Procedure +1. **Incision:** + - Use a sterile scalpel to make an incision to the bregma. + +2. **Injector Preparation:** + - Set injection rate and volume on injector (2 uL at 0.25 uL/min). + +3. **Aligning Needle:** + - Align the needle with bregma and check alignment. + +4. **Coordinate Setting:** + - Reset digital coordinate box to "0" for anterior/posterior, medial/lateral. + +5. **Coordinate Adjustments:** + - Use the digital box to move the needle to appropriate coordinates. + +6. **Drilling:** + - Drill at coordinates but do not puncture the skull fully. + +7. **Needle Positioning:** + - Lower needle to match drilled location. Ensure it doesn't bend. + +8. **Beveling the Needle:** + - Bevel needle and set dorsal/ventral coordinates to "0". + +9. **Injection:** + - Lower the needle and inject the virus. + +10. **Injection Duration:** + - Full injections take about 8 minutes. Wait for 2 minutes before retracting. + +11. **Needle Retraction:** + - Slowly retract the needle. + +12. **Post-Injection Cleaning:** + - Clean any blood or dried CSF. + +13. **Incision Closure:** + - Close the incision with wound clips. + +14. **Recovery:** + - Place mouse in a cage on a heating pad and monitor. + +15. **Recovery Observation:** + - Monitor for several days and administer pain relief. + +16. **Food Availability:** + - Place wet food on the floor of the cage for the following day. + +_endofoutput_ +``` \ No newline at end of file diff --git a/markdown-output/mpe-seq-zpaf5ie.md b/markdown-output/mpe-seq-zpaf5ie.md new file mode 100644 index 0000000000000000000000000000000000000000..9dc690506f6c47a78696e22003078dd6f9b2305e --- /dev/null +++ b/markdown-output/mpe-seq-zpaf5ie.md @@ -0,0 +1,319 @@ +```markdown +# Goal/Experiment: +This experiment aims to perform an MPE-seq (Methylation Processing Enzyme sequencing) to study the nucleotide sequence of RNA fragments. + +## MPE-seq + +### Materials + +| Name | Catalog # | Vendor | +|-----------------------------------------------|------------------|----------------------------| +| Klenow Fragment (3'→5' exo-) (50,000 u/ml) | M0212M | New England Biolabs | +| Phusion DNA polymerase | - | New England Biolabs | +| Dynabeads MyOne Streptavidin T1 | - | Invitrogen - Thermo Fisher | +| Superscript IV | 18090050 | Thermo Fisher Scientific | +| Zymo-Spin I Columns | C1003-250 | Zymo Research | +| Biotin-11-dUTP Solution (1 mM) | R0081 | Thermo Fisher Scientific | +| NEBNext® High-Fidelity 2X PCR Master Mix | M0541L | New England Biolabs | + +## RNA Fragmentation + +### Protocol + +1. **Fragmentation Recommendation**: + - Fragmentation may reduce size biases against large primer extensions which are inefficiently PCR amplified and sequenced. It may be unnecessary depending on the application. Fragmenting should be done in such a way to avoid over-fragmentation. + - Assemble the following reaction in a PCR tube for each RNA sample: + + | Reagent | Volume (uL) | + |----------------------|-------------| + | 15-40ug RNA | - | + | Fragmentation buffer | 2 | + | DEPC Water | Up to 20 | + | **Total** | **20** | + + - Add fragmentation buffer last. + - Keep reactions on ice during setup to prevent excess fragmentation. + +2. **Induce Fragmentation**: + - Heat in thermocycler at 65°C for 15 minutes. + - Optimization may be needed based on RNA samples used. + +3. **Stop Fragmentation**: + - Add 2uL fragmentation STOP buffer and place samples on ice. + +4. **Ethanol Precipitation and Buffer Exchange**: + - Bring sample volume to 50uL by adding 26uL water and transfer to a 1.5mL Eppendorf tube. + - Add 1/10 volume (5uL) 3M sodium acetate, followed by 2.5 volume (137.5uL) 100% ethanol. + - Vortex, cool samples to encourage precipitation (2+ hours at -20°C, or 15 min at -80°C). + +5. **Centrifugation**: + - Centrifuge at max speed for 10 min. + - Aspirate liquid. + +6. **Ethanol Wash**: + - Wash pellet with full sample volume (200uL) of 80% ethanol. + - Aspirate liquid. + - Repeat for 2 total washes. + +7. **Dry Pellet**: + - Aspirate and air-dry on benchtop before resuspending in water for a concentration of ~2ug/uL. + +8. **Quantify by NanoDrop**: + - Store at -20°C or continue. + +## Reverse Transcription + +### Protocol + +9. **Primer/Template Mix**: + - Make the mix for each sample in a PCR tube. Keep on ice. + + | Reagent | Volume (uL) | + |-------------------------------------------------|-------------| + | 5X RT buffer | 4 | + | Primer mix (10nM of each gene-specific primer) | 2 | + | Fragmented RNA | 10ug | + | Water | Up to 20 | + | **Total** | **20** | + +10. **Enzyme Mastermix**: + - Prepare the mix per sample in a PCR tube. If volume exceeds 100uL, split into multiple PCR tubes. + + | Reagent | Volume (uL) | + |-----------------------|-------------| + | 5X RT buffer | 4 | + | 0.1M DTT | 4 | + | 10mM dNTP mix | 2 | + | Water | 6 | + | 1mM Biotin dUTP | 4 | + | SSIV | 1 | + | **Total** | **21** | + +11. **Thermocycler Program**: + - Program as follows, preferably on a cycler that can hold temperature during a user-pause: + + - 70°C: 1 min + - 65°C: 5 min + - 55°C: 5 min (Optional user pause to hold at 55°C) + - 55°C: 30 min + - 80°C: 10 min + - 25°C: hold + +12. **Primer Mix/Template Addition**: + - Add to thermocycler to denature and anneal primers to template (70°C to 65°C to 55°C). + +13. **Preheat and Enzyme Mix Addition**: + - Preheat enzyme mix by placing on thermocycler for a few minutes. + - Add 20uL enzyme mix directly to primer/template mix. + +14. **Continue Reaction**: + - Allow reaction to proceed at 55°C for 30 min followed by 80°C inactivation for 10 min. + +15. **RNA Hydrolysis**: + - Add 20uL RNA hydrolysis solution. + - Incubate at 65°C for 15 min. + +16. **Neutralization Solution**: + - Add 20uL neutralization solution. Sample volume = 80uL. + +17. **Ampure Cleanup**: + - Add 1.8 sample volumes AmpureXP beads. Capture beads on magnetic stand for 2 min. Aspirate liquid. + - While on magnetic stand, add 200uL 70% ethanol wash and incubate for 30 sec. Aspirate. + - Repeat 2 times. + - Remove from stand and pipet thoroughly with 50uL water to elute. + - Place on magnetic stand for 2 min and transfer supernate to labelled 1.5mL centrifuge tube. + - Store at -20°C or continue. + +## First Streptavidin Bead Purification of Primer Extensions + +### Protocol + +18. **Bead Preparation**: + 1. Vortex beads vigorously to resuspend. + 2. Transfer 20uL of beads per sample to be purified into a new 1.5mL Eppendorf tube. + 3. Place tubes on magnetic stand and incubate for 1 min. + 4. Leave on magnetic stand, aspirate buffer. + 5. Remove tubes from stand and wash beads with 500uL of 1x bind and wash buffer. Mix well with pipette. + 6. Re-place on magnetic stand for 1 min. + 7. Aspirate buffer. + 8. Repeat wash for 2 total washes. + 9. Resuspend beads in 50uL of 2x bind and wash buffer per sample. + 10. Mix beads with pipette. + 11. For each sample, add aliquot of 500uL of beads into a new PCR tube fitting on magnetic stand. + +19. **Bead Binding**: + 1. Add 50uL prepared beads to each 50uL sample. + 2. Rotate on tray to gently agitate at RT for 30 min. + 3. Place on magnetic stand and incubate for 1 min. + 4. Aspirate buffer. + 5. Remove samples from magnetic stand. + +20. **Bead Washes**: + 1. Add 150uL of 1x bind and wash buffer. Mix well. + 2. Incubate on magnet for 1 min. + 3. Aspirate buffer. + 4. Repeat wash for 3 total washes. + +21. **SSC Wash**: + 1. Wash each sample with 100uL 1x SSC. Mix well. + 2. Place on magnetic stand for 1 min. + 3. Aspirate buffer. + 4. Remove samples from magnetic stand. + +22. **Denature/Unlabelled Strand Wash**: + 1. Add 100uL denaturation solution to each sample. + 2. Incubate at RT for 10 min. + 3. Place on stand and aspirate buffer. + 4. Wash samples with 100uL denaturation solution. Mix well and pipette. + 5. Place on stand for 1 min and aspirate. + +23. **TE Washes**: + 1. Add 100uL of TE buffer. Mix well. + 2. Place on magnet for 1 min. + 3. Aspirate buffer. + 4. Repeat wash 2 times for 3 total washes. + +24. **Elution**: + 1. Add 50uL of bead elution buffer to each sample. + 2. Incubate at 90°C for 2 min. + 3. Place on stand and incubate for 1 min. + 4. Aspirate sample. + +25. **Ampure Cleanup**: + 1. Add 1.8 volumes of AmpureXP beads. Capture beads on stand for 2 min. + 2. Add 200uL 70% ethanol wash. Incubate for 30 sec. Aspirate. + 3. Repeat 2 times. + 4. Remove from stand, pipette with 40uL water to elute. + 5. Place on stand for 2 min and transfer to 1.5mL centrifuge tube. + - Store at -20°C or continue. + +## First Strand Extension + +### Protocol + +26. **Reaction Mix**: + - Prepare the mix for each sample: + + | Reagent | Volume (uL) | + |---------------------------------|-------------| + | 10X NEB Buffer 2 | 5 | + | 10mM dNTP Mix | 1 | + | cDNA from previous | 40 | + | 1st Strand Extension Primer | 1 | + | **Total** | **47** | + +27. **Thermocycler Reaction**: + - Incubate at 65°C for 2 min. + +28. **Cool Samples**: + - Cool to room temperature for ~5min. + +29. **Add Klenow Exo- Enzyme**: + - Add 3uL to each sample. + +30. **Incubate**: + - Incubate at RT for 5 min. + +31. **Heat Samples**: + - Heat to 37°C and incubate for 30 min. + +## Second Streptavidin Bead Purification + +### Protocol + +32. **Bead Preparation**: + 1. Vortex beads vigorously to resuspend. + 2. Transfer 20uL prepared beads per sample into a new tube. + 3. Place on magnet for 1 min. + 4. Aspirate buffer. + 5. Remove tubes from stand. Wash beads with 500uL of 1x buffer. Mix well. + 6. Place on magnet for 1 min. + 7. Aspirate buffer. + 8. Wash for 2 total. + 9. Resuspend beads in 50uL 2x buffer each. + 10. Mix beads well. + 11. For each, add 500uL into a PCR tube for magnet stand fitting. + +33. **Bead Binding**: + 1. Add 50uL prepared beads to each 50uL sample. + 2. Rotate on tray for 30 min. + 3. Place on magnet for 1 min. + 4. Aspirate buffer. + 5. Remove samples from magnetic stand. + +34. **Bead Wash**: + 1. Add 150uL 1x buffer. Mix. + 2. Place on magnet and incubate 1 min. + 3. Aspirate buffer. + 4. Repeat for 3 total washes. + +35. **SSC Wash**: + 1. Wash each sample with 100uL 1x SSC. + 2. Place on magnet for 1 min. + 3. Aspirate buffer. + 4. Remove from stand. + +36. **Denature and Strands Wash**: + 1. Add 100uL denaturant. + 2. Incubate RT 10 min. + 3. Place on magnet 1 min. Aspirate. + 4. Wash with 100uL denaturant. Mix. + 5. Incubate and aspirate. + +37. **TE Wash**: + 1. Wash 100uL TE buffer, mix. + 2. Place on magnet 1 min. + 3. Aspirate buffer. + 4. Repeat for 3 total washes. + +38. **Elution**: + 1. Add 50uL elution buffer per sample. + 2. Incubate at 90°C for 2 min. + 3. Place on stand 1 min. + 4. Aspirate sample. + +39. **Ampure Cleanup**: + 1. Add 1.8 volumes XP beads. Magnet 2 min. + 2. Add 200uL 70% ethanol wash, 30 sec. + 3. Repeat 2 times. + 4. Remove from stand. Pipette 20uL water to elute. + 5. Magnet 2 min, transfer supernate to tube. + - Store at -20°C or continue. + +## PCR Amplification and Sequencing + +### Protocol + +40. **Test PCR**: + - Test small amount of template: + - Use NEB 2X HiFi MM with 2uL template. + + | Reagent | Volume (uL) | + |---------------------------------|-------------| + | Water | 6 | + | Template (previous step) | 2 | + | 10uM 7XX Nextera Fwd primer | 1 | + | 10uM 5XX Nextera Rev primer | 1 | + | 100X SYBR green (DMSO) | 0.2 | + | 2X Mastermix | 10 | + | **Total** | **20** | + + - qPCR Cycling: + - 95 °C 3 min + - (40x) 98 °C 10 sec, 62 °C 20 sec, 72 °C 30 sec + - Final elongation: 72 °C 5 min + - Typical CT: ~20. + +41. **Real PCR**: + - Use remaining template in 40uL reaction, minimal cycles. + - Clean with Ampure beads. + +## Sequencing + +42. **Sequence**: + - Libraries are simple, 1-5 million reads sufficient. + - Usually observes 20-200 fold enrichment vs RNA-seq. + - Fewer reads may result in anchoring problems. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/mpn-most-probable-number-assay-for-infectivity-of-fcbbisn.md b/markdown-output/mpn-most-probable-number-assay-for-infectivity-of-fcbbisn.md new file mode 100644 index 0000000000000000000000000000000000000000..222e19c4cccd25a4459c37398a79bf2f0c5a208f --- /dev/null +++ b/markdown-output/mpn-most-probable-number-assay-for-infectivity-of-fcbbisn.md @@ -0,0 +1,128 @@ +```markdown +# Goal/Experiment: +MPN (Most Probable Number) assay for infectivity of algal viruses. + +## MPN (Most Probable Number) Assay for Infectivity of Algal Viruses + +### Abstract +**Purpose:** To quantify infectious viruses and evaluate the infectivity of a viral sample. + +**Summary:** +50 μL of serially-diluted (10^-³^ to 10^-10^) virus sample is added to 150 μL of exponentially growing host cells in triplicate 96-well microplates and incubated at normal growth conditions for ~2 weeks. Cell lysis is assessed every few days qualitatively by visual inspection and quantitatively by measuring optical density on a microplate reader. The MPN of infective viruses in each concentrate is estimated from the proportion of virus-positive (i.e., lysed) wells using the [MPN_ver4.xls](https://www.protocols.io/) Excel spreadsheet from Jarvis, B., Wilrich, C., and P.-T. Wilrich (2010), *Journal of Applied Microbiology* **109** (2010), 1660 – 1667. Percent infectivity is then calculated by comparing the MPN-estimated abundance of infective viruses to the abundance of virus-like particles (VLPs) determined by flow cytometry analysis. + +### Guidelines + +#### Principle +The abundance of infective viruses in a sample can be estimated using a serial dilution approach. In theory, a single lytic virus can lyse an entire population of sensitive host cells, given enough time. The MPN approach uses a series of log-based dilutions of a virus sample added to aliquots of an exponentially-growing host culture. The abundance of infectious viruses can be estimated if the range of dilutions used includes when the mean viruses per aliquot ≈ 1. An estimate of infective units is calculated with software using the number of lysed host aliquots at each dilution level. Replication (and increased sensitivity) is achieved by using 96-well microplates (8 replicate wells per plate x triplicate plates = 24 replicates per dilution). The estimated MPN is then compared to the total virus-like particle (VLP) count determined by flow cytometry to calculate infectivity (i.e., the proportion of infective viruses in the sample). + +#### Host Preparation + +Initiate a host culture several days prior to assay setup to ensure exponential growth. Avoid transferring cells on the day-of or the day before assay setup to minimize disturbance. Transfer/dilute host culture to pre-determined mid-exponential density daily (semi-continuous culture), including the day of assay setup. + +*Example Ostreococcus lucimarinus* (CCMP2972A) host preparation: + +- **Growth conditions:** 18°C, 14:10 hour light:dark cycle, light irradiance ~100 μE m^-2^ s^-1^ +- **Growth media:** L1 with natural seawater base (67-135) +- **Exponential range:** 7x10^5^ – 2x10^7^ cells mL^-1^ + +| Day | 1 | 2 | 3 | 4 | 5 | +|-----|--------|---------|--------|--------|-----------------------| +| Initial density (mL^-1^) | 5x10^7^ | 1.5x10^7^ | 9x10^6^ | 1.2x10^7^ | | +| Growth rate (d^-1^) | NA | 0.55 | 0.50 | 0.84 | | +| Dilution/Transfer | Transfer | Skipped | Transfer | Dilution | Dilution (use for assay) | +| Final density (mL^-1^) | 5x10^6^ | 5x10^6^ | 5x10^6^ | 5x10^6^ | 5x10^6^ | +| Volume (mL) | 15 | 25 | 44* | 104* | 104* | + +\*Volume varies depending on growth + +### Before Start + +#### Equipment and Materials +*Per virus sample tested:* + +- 5 mL round bottom polypropylene tubes: 10 +- Culture medium: 25 mL +- 96-well microplates: 3 +- Exponentially-growing host culture: 40 mL +- Virus sample: 0.2 mL (dilution) + 0.5 mL (FCM) +- Sterile 50 mL sample reservoirs: 2-3 +- Cryovials: 2-3 + +*Additional materials:* + +- 1000 µL pipette + filter tips +- 200 µL multichannel pipette + tips +- Tube rack +- Vortexer +- Optical microplate reader +- 25% glutaraldehyde (EM grade) + +### Protocol + +#### Step 1: Virus Dilution Series +1. Label a series of 5 mL round bottom tubes from 10^-1^ to 10^-10^. +2. Aliquot 1.8 mL culture media to each tube. +3. Dilute 200 µL virus sample into the “10^-1^” tube and vortex to mix. +4. Use a clean pipette tip to transfer 200 µL from the “10^-1^” tube to the “10^-2^” tube and vortex to mix. +5. Repeat serial dilution to 10^-10^. +6. Transfer 500 µL virus sample to a sterile 1.2 mL cryovial (to preserve for FCM counts). +7. In the chemical hood, add 5 µL 25% glutaraldehyde (0.25% final concentration) and gently vortex to mix. +8. Aliquot 250 µL to a duplicate cryovial and snap cryovials into cryocanes. +9. Incubate at 4°C for 30 minutes in the dark. +10. Flash freeze in liquid nitrogen and store at -80°C until analysis. + +#### Step 2: Plate Setup + +1. Label triplicate 96-well microplates as follows: + +| Column | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | +|--------|---|---|---|---|---|---|---|---|---|-------------|---------|---------| +| Label | -3 | -4 | -5 | -6 | -7 | -8 | -9 | -10 | Control |(empty) |(empty) | + +2. Pour remaining culture medium into a sterile sample reservoir. +3. Use a multichannel pipette to add 50 µL medium to all wells in Column 9 ("Control") on all plates. +4. Discard unused medium and fill sample reservoir with host culture. +5. Add 150 µL host cells to all wells in Columns 1-9 on all plates. Discard remaining culture. +6. Pour "10^-10^" viral dilution into new sterile sample reservoir. +7. Use a multichannel pipette to add 50 µL 10^-10^-diluted virus sample to all wells in Column 8 on all replicate plates. +8. Discard remaining 10^-10^ diluted virus sample and pour "10^-9^" viral dilution into the same sample reservoir. +9. Use the same pipette tips to add 50 µL 10^-9^-diluted virus sample to all wells in Column 7 on all plates. +10. Repeat additions of diluted virus samples from most dilute to most concentrated (moving from right to left across the microplates). +11. After final 10^-3^ diluted virus sample is added to plates, measure optical density for T0 in plate reader (as described below) and incubate (unstacked to prevent shading) at standard growth conditions for 2 weeks, measuring growth every few days. + +#### Step 3: Data Collection + +1. Turn on the Molecular Devices SpectraMax 340PC plate reader. +2. Log into the attached computer and open the SoftMax Pro 6 software. +3. Open or create a new "Basic Endpoint" protocol file (.spr). +4. Rename the experiment appropriately and configure a plate with the following settings: + - **Read Type:** Endpoint + - **Wavelength:** 750 nm + - **Plate Type:** 96-well standard clear bottom + - **Read Area:** All + - **Pathcheck:** Calibration on + - **Shake:** Once for 3 sec. + - **More settings:** Column priority +5. Add "New plates" as needed so there is one for each replicate plate in your assay (settings will copy to these new plates within the same experiment). +6. If the SpectraMax doesn’t automatically connect, click on the instrument icon in the top left corner and manually select it from the menu. +7. Select the plate to be read in the software and place the corresponding plate on the plate reader drawer (with Column 1 closest to the instrument). +8. Remove the plate lid and select “Read” to read the plate. +9. Read each plate individually, and copy the data (must right-click to do this) into an Excel spreadsheet. +10. Create a new data file (.sda) from the same master protocol for each time point. +11. Visually inspect plates and record observations. + +#### Step 4: Calculations + +1. Open the [MPN_ver4.xls](http://www.wiwiss.fu-berlin.de/fachbereich/vwli/iso/ehemalige/wilrich/index.html) Excel spreadsheet. +2. Enter the name and date of the experiment. +3. For "Total no. of test series," enter 1 for each virus sample tested (i.e., two viruses tested = 2 total test series). +4. Enter "5" for "Max no. of dilutions" and hit Enter to generate a data table for each virus sample tested. +5. Enter the appropriate data in the yellow cells of each table generated. The "Dilution Factor" is the dilution ratio used for inoculating the wells of that column. The "Volume" is the volume of the dilution added to each well in that column. + - Dilution Factor: Use results from a subset of the serial dilutions such that all host wells were lysed in the most concentrated of the dilutions (i.e., 8 of 24 wells were positive for viral activity) and all host wells were healthy in the most dilute of the dilutions (i.e., 0 of 24 wells were positive for viral activity). Common dilution factor range includes: 1x10^-6^ to 1x10^-10^. + - Volume = 0.05 mL + - Tubes (wells) = 24 + - Positive tubes = no. of cleared wells (out of 24; should be 24 for the most concentrated and 0 for the most dilute) +6. Once tables are completed, hit "Ctrl"+"m" to calculate MPN estimates. Program returns estimates of MPN, log_10_ MPN, Std Dev log_10_ MPN, Upper and Lower 95% Confidence Limits, and a Rarity Index (i.e., indication of the probability of the results). +``` +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/mr-imaging-of-the-mouse-hindlimb-musculature-t2-we-cucjwsun.md b/markdown-output/mr-imaging-of-the-mouse-hindlimb-musculature-t2-we-cucjwsun.md new file mode 100644 index 0000000000000000000000000000000000000000..1c3802da29ff0e95a590b6a96a78c746194c38ab --- /dev/null +++ b/markdown-output/mr-imaging-of-the-mouse-hindlimb-musculature-t2-we-cucjwsun.md @@ -0,0 +1,136 @@ +```markdown +# Goal/Experiment: +The experiment aims to image the musculature in the hind limb of a mouse using T2 weighted and T2 map MRI. This protocol is designed for imaging edema in mouse models of muscular damage but can be adapted for other applications. + +# MR Imaging of the Mouse Hindlimb Musculature (T2 Weighted and T2 Map) V.2 + +**Emily Waters1** +1Northwestern University +Northwestern Center for Advanced Molecular Imaging + +**Version 2, May 17, 2023** + +## Abstract +This procedure is for imaging the musculature in the hind limb of a mouse. The protocol was designed for imaging of edema in mouse models of muscular damage but could be adapted for other applications. + +## Protocol Information +- **DOI:** [dx.doi.org/10.17504/protocols.io.261ge4z1ov47/v2](https://dx.doi.org/10.17504/protocols.io.261ge4z1ov47/v2) +- **Protocol Citation:** Emily Waters 2023. MR Imaging of the mouse hindlimb musculature (T2 weighted and T2 map). protocols.io [https://dx.doi.org/10.17504/protocols.io.261ge4z1ov47/v2](https://dx.doi.org/10.17504/protocols.io.261ge4z1ov47/v2) +- **License:** This is an open access protocol distributed under the terms of the [Creative Commons Attribution License](https://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. +- **Protocol Status:** Working +- **Created:** May 17, 2023 +- **Last Modified:** May 17, 2023 +- **Protocol Integer ID:** 82027 + +## Materials and Equipment +- **Induction Chamber**: Suitable for mouse anesthesia +- **3% Isoflurane**: Anesthetic gas +- **Oxygen (O2)**: Flow rate set to 1 L/min O2 +- **Warm Water Circulating Blanket**: Set at 37°C +- **Charcoal Canister**: For waste gas collection +- **Eye Lubricant**: To prevent corneal drying during anesthesia +- **Bruker 9.4T Biospec MRI Scanner**: With 30 cm bore and 12 cm gradient insert, running Paravision 6.0.1 +- **Bruker 38mm Rat Head/Mouse Body Volume Coil**: Compatible with Bruker bed +- **Respiratory Pad**: For monitoring breathing +- **Paravision Software (v6.0.1)**: For imaging and analysis +- **Tape and Scissors**: For animal positioning and alignment + +## Procedure + +### Prepare Mouse for Imaging (5m) + +1. **Induce Anesthesia**: + - Place the mouse in an induction chamber. + - Induce anesthesia using 3% isoflurane and 1 L/min O2. + - The induction chamber should be placed on a warm water circulating blanket set at 37°C. + - Use a charcoal canister to collect waste gases from the induction chamber. + +2. **Monitoring and Identification**: + - When the mouse is fully anesthetized, record weight and apply eye lubricant. + - Perform ear punch if needed for identification (metal ear tags cannot go in an MRI scanner). + - If the mouse has a metal ear tag, it can be removed with suture wire cutting scissors. + +3. **Transportation to Scanner**: + - Return the mouse to the induction chamber for approximately 1 minute to ensure depth of anesthesia. + - Transport to MRI scanner (Bruker 9.4T Biospec). + +### Set Up Mouse in Scanner (5m) + +4. **Position Mouse in Bed**: + - Place the mouse prone in a bed suitable for the mouse body coil. + - Ensure snout is firmly situated in anesthetic nosecone. + - Isoflurane should flow at approximately 2% initially and adjust during the scan to maintain respiratory rate. + - Use a Bruker bed compatible with the Bruker 38mm rat head/mouse body volume coil. + +5. **Maintain Body Temperature**: + - Position the warm water circulating blanket around the mouse. Preferably place it under the mouse for effective temperature maintenance. + +6. **Check Anesthesia**: + - Check depth of anesthesia (e.g., using a toe pinch) before positioning mouse limbs for imaging. Ensure the mouse is not too lightly anesthetized. + +7. **Position Limbs**: + - Tuck the mouse's legs underneath its body, aligning knees and toes pointing forward, with heels pointed backward and feet flat. + - Position the mouse so mid-thigh aligns with the center of the coil. + +### Optimal Limb Positioning for MRI +![Optimal Limb Positioning](provide-link-to-diagram) + +8. **Position Respiratory Pad**: + - Place the respiratory pad on the animal's back at the level of the abdomen. + - Secure with masking tape and check monitoring system for a clean respiratory signal. + +### Tune and Match Coil + +9. **Tuning and Matching**: + - The Bruker 38mm rat head/mouse body volume coil must be tuned and matched outside the bore of the magnet. + - Adjust tuning and matching using the wobble interface in the Paravision software. + - Position the bed and coil in the center of the magnet. + +### Acquire Localizer Images + +10. **Image Acquisition**: + - Run auto calibrations and acquire the first image using the Tripilot sequence. + - Additional shimming beyond the linear auto-shim is generally not required. + +### Imaging Sequences + +11. **Axial Scout Image**: + - RARE sequence parameters: TR/TE = 4000 / 32 ms, Rare Factor 8, field of view 35 x 35 mm, matrix 128 x 128, 19 slices, 1 mm slice thickness, 1 average. + - Readout direction: left to right. + - Position slice stack based on the tripilot image. + +12. **Coronal Scout Image**: + - RARE sequence parameters: TR/TE = 4000 / 32 ms, Rare Factor 8, field of view 35 x 35 mm, matrix 128 x 128, 19 slices, 1 mm slice thickness, 1 average. + - Readout direction: head to foot. + - Position slice package to cover thighs based on axial scout. + +### Acquire High-Quality Images + +13. **T2 Weighted Image**: + - RARE sequence parameters: TR/TE = 4000 / 32 ms, Rare Factor 8, field of view 35 x 35 mm, matrix 256 x 256, 19 slices, 1 mm slice thickness, 2 averages. + - Readout direction: left to right. + - Position slice package based on anatomical landmarks. + +14. **T2 Map Acquisition**: + - T2map_MSME sequence parameters: TR = 4000 ms, 25 echoes with initial TE = 9 ms and 9 ms echo spacing, field of view 35 x 35 mm, matrix 256 x 256, 5 slices, 1 mm slice thickness, 1 average. + - Readout direction: left to right. + - Ensure to copy slice orientation from the previous scan. + +14.1 **Orientation**: + - Be careful to copy the slice orientation, not the slice geometry. + +14.2 **Adjust if Necessary**: + - Confirm coverage of major muscle groups and shift slice package by 1 mm if needed. + +### Recover Mouse + +15. **Post-Scan Recovery**: + - Remove mouse from scanner and transport to recovery area. + - Place mouse on a warm water circulating heating blanket until it recovers. + - Once sternal and ambulatory, return the mouse to standard housing. + +## References +[Example Reference or DOI if any] + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/multidisciplinary-provision-of-food-and-nutritiona-bgzajx2e.md b/markdown-output/multidisciplinary-provision-of-food-and-nutritiona-bgzajx2e.md new file mode 100644 index 0000000000000000000000000000000000000000..93babd4476d2775f491ba5613d5e9b6418dbd170 --- /dev/null +++ b/markdown-output/multidisciplinary-provision-of-food-and-nutritiona-bgzajx2e.md @@ -0,0 +1,89 @@ +```markdown +# Goal/Experiment: +The experiment aims to review the evidence and characteristics of multidisciplinary care interventions in food and nutritional care provision for adult in-patients, focusing on multidisciplinary coordination and collaboration within the hospital setting. + +# Multidisciplinary Provision of Food and Nutritional Care to Hospitalised Adult In-patients: A Scoping Review Protocol + +**Authors:** +- Gladys Yinusa1 +- Janet Scammell1 +- Jane Murphy2 +- Gráinne Ford3 + +1Department of Nursing Science, Faculty of Health and Social Sciences, Bournemouth University, Bournemouth, United Kingdom +2Department of Rehabilitation & Sport Sciences, Faculty of Health and Social Sciences, Bournemouth University, United Kingdom +3Dietetic Department, The Royal Bournemouth and Christchurch Hospitals NHS Foundation Trust, Bournemouth, United Kingdom + +## Abstract + +### Objective +The review will examine the evidence and characteristics of multidisciplinary care interventions in food and nutritional care provision for adult in-patients. + +### Introduction +Providing appropriate nutritional care is fundamental in patient-centred care. Nutritional care requires a coordinated approach by different healthcare professionals and the wider hospital staff to deliver food and fluids effectively. Evidence shows that improved patient clinical outcomes can be achieved by enhancing hospital food and providing nutritional care throughout the patient’s pathway. Effective multidisciplinary care ensures the nutritional needs of malnourished patients are met, with the hospital team playing a crucial role. + +### Inclusion Criteria +The review focuses on studies involving healthcare professionals or the wider hospital staff within an adult in-patient population. Studies must consider the hospital setting. + +### Methods +Primary evidence, both published and unpublished, will follow a three-step search strategy recommended by the Joanna Briggs Institute (JBI). Data will be extracted and findings will be reported using the Preferred Reporting Items for Systematic reviews and Meta-Analyses extension for Scoping Reviews (PRISMA-ScR) guidelines. + +## Introduction +Key nutrition interventions and strategies to reduce malnutrition have been integrated into guidelines and policies. For instance, validated malnutrition screening tools by the European Society for Clinical Nutrition and Metabolism (ESPEN) have been adopted into routine practice. Screening is the first step, requiring appropriate interventions and monitoring. Global consensus on core diagnostic criteria for malnutrition has also been proposed to address the malnutrition prevalence across interventions. + +## Methods +### Eligibility Criteria +#### Population +- Hospital staff involved in patient nutritional care (clinicians, nursing, allied health professionals) +- Non-clinical support personnel (volunteers, patients, relatives) +#### Concept +- Multidisciplinary care aimed at improving nutritional care in hospitals + - Excludes artificial nutritional support (oral nutrition supplements, enteral tube feeding, parenteral nutrition) +#### Context +- Limited to hospital settings; excludes primary/community care contexts + +### Information Sources +Primary sources, including qualitative, quantitative, and mixed-methods research studies regardless of publication status, will be considered. + +### Search Strategy +The strategy aims to include published and unpublished studies: +1. Initial search of MEDLINE Complete and CINAHL Complete identified key words and index terms. +2. Full search for MEDLINE Complete (Appendix I). + +### Study Selection +All identified records will be collated in EndNoteX9.2 (Clarivate Analytics, PA, USA, 2019), followed by steps to screen titles, abstracts, and full texts for eligibility. Reviews will resolve disagreements through discussion or with a fourth reviewer if necessary. Results will be presented in a final flow diagram following PRISMA-ScR guidelines. + +### Data Extraction +Data will be extracted by two or more reviewers using a developed tool to capture information on the population, concept, context, methods, and key findings relevant to the review question. + +### Data Presentation +Extracted data will be mapped and presented in a tabular form. A narrative description will accompany the findings. + +### Conflict of Interest +The authors declare no conflict of interest. + +### References +1. [Barker, L., Gout, B., Crowe, T. (2011). Hospital malnutrition: prevalence, identification and impact on patients and the healthcare system.](https://example.com) +2. [Tappenden, K. A., Quatrara, B., Parkhurst, M. L., Malone, A. M., Fanjiang, G., Ziegler, T. R. (2013). Critical Role of Nutrition in Improving Quality of Care.](https://example.com) +3. [Leij-Halfwerk, S., Verwijs, M. H., van Houdt, S., Borkent, J., Guaitoli, P. R., Pelgrim, T., et al. (2019). Prevalence of protein-energy malnutrition screening tools.](https://example.com) +4. [Cederholm, T., Barazzoni, R., Austin, P., Ballmer, P., Biolo, G., Bischoff, S. C., et al. (2017). Definitions and terminology of clinical nutrition.](https://example.com) + +... + +## Appendices + +### Appendix I: Medline Complete Search Conducted in Dec 2019 +| DOMAIN | Alternative words/Concept expansion | Search | Query | Records retrieved | +|------------------------|--------------------------------------|--------|------------------------------------------------------|-------------------| +| Nutritional care | Nutrition*, care Nutrition*, intake Nutrition*, intervention* Nutrition support, MESH Malnutrition, Protein-energy malnutrition, Nutrition Policy, Meals Nutrition assessment Nutrition Therapy | #1 | "nutrition* care" OR "nutrition* intervention*" OR "nutrition support" OR "nutrition therapy" OR "nutrition* intake" | 12,698 | +| Multidisciplinary delivery | Multidisciplinary Interprofessional Teamwork, Interdisciplinary support, Health professional Staff, MESH MH "Interdisciplinary Communication", Crew Resource Management, Healthcare" | #4, #5, #6 | (MH "Interdisciplinary Communication") | 129,224 | +| Hospital Setting | Hospital Inpatient Ward*, Inpatient, MESH Inpatient | #8, #9, #10 | AB("acute setting" or hospital or "acute hospital" or "acute care" or ward* or inpatient* or “in-patient*") | 2,642,671 | +| Limited to | No date, English and English translated | #12, #13 | | 586 | + +### Appendix II +[Appendix II.pdf](https://example.com) + +--- + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/multiplexed-assay-for-detection-of-cell-culture-ev-be7yjhpw.md b/markdown-output/multiplexed-assay-for-detection-of-cell-culture-ev-be7yjhpw.md new file mode 100644 index 0000000000000000000000000000000000000000..425c4ab3b3b85812e6a76f16f077325571d202e7 --- /dev/null +++ b/markdown-output/multiplexed-assay-for-detection-of-cell-culture-ev-be7yjhpw.md @@ -0,0 +1,134 @@ +```markdown +# Goal/Experiment: +The goal of this experiment is to use a multiplexed assay to detect cell culture EV surface membrane proteins, specifically leveraging the Miltenyi Biotec's MACSPlex Exosome Kit. This protocol involves analyzing an EV sample with up to three additional detection antibodies at two different EV count titration points. + +# Multiplexed Assay for Detection of Cell Culture EV Surface Membrane Proteins + +## Authors +- Joshua A Welsh +- Bryce Killingsworth +- Julia Kepley +- Tim Traynor +- Alexis Barfield +- Jennifer Jones + +**Translational Nanobiology Section, Laboratory of Pathology, Center for Cancer Research, National Cancer Institute, National Institutes of Health** + +[DOI: dx.doi.org/10.17504/protocols.io.be7yjhpw](https://dx.doi.org/10.17504/protocols.io.be7yjhpw) + +### Abstract +Protocol for using Miltenyi Biotec's human MACSPlex Exosome Kit to assay a cell-line derived EV sample with up to 3 additional detection antibodies, at two EV count titration points (1E9 and 1E8 per LM10, NanoSight, NTA). The optimization of this protocol was done using bead kits released between 2017-2020. + +## Keywords +- multiplex +- flow cytometry +- EVs + +## Materials + +| Name | Catalog # | Vendor | +|-----------------------------------------------|-------------|----------------| +| Low Protein Binding Collection Tubes (2.0 mL) | 88379 | Thermo Fisher | +| MACSPlex Exosome Kit human | N/A | Miltenyi Biotec| +| AcroPrep Advance Filter Plates for Aqueous Filtration - 350 µL 0.2 µm Supor membrane (10/pkg) | N/A | Pall | + +### Disclaimer +This protocol summarizes key steps for a specific type of assay, which is one of a collection of assays used for EV analysis in the NCI Translational Nanobiology Section at the time of submission of this protocol. Appropriate use of this protocol requires careful, cohesive integration with other methods for EV production, isolation, and characterization. + +### Before Starting +- The protocol and planning template spreadsheet have been designed for an experiment assaying one cell-line derived EV sample with up to 3 additional detection antibodies, at two EV count titration points (1E9 and 1E8 per LM10, NanoSight, NTA). +- Modifications may be necessary to use as a guide to assay multiple samples, human sample-derived EVs, or use additional numbers of detection antibodies. + +## Experiment Planning + +1. Determine which antibodies to use to detect EV surface membrane proteins in addition to the included CD9, CD63, and CD81 antibodies. All additional antibodies must be either APC or AF647 conjugated. Ensure you know the concentration of the antibodies, and if using an antibody conjugated in-lab, avoid preparations that have unbound dye. + +2. Calculate the particle concentration of your EV sample and the total particle count. + +3. Use the provided template document to input your sample information and generate a plate map to visualize your experiment and the wells you will fill in the 96-well plate to be analyzed by the flow cytometer. Increase the upper EV titration point if you have a highly concentrated sample. For dilute samples, methods to concentrate the sample may be necessary. [Download the MACSPlex Protocol Template.xlsx](2020-07-30 - MACSPlex Protocol Template.xlsx) + - **Note:** The template assumes transferring EVs directly from the stock EV preparation into a tube with MACSPlex capture beads and buffer. For lower titration point(s), dilutions of EV stocks in PBS will be used with equal volumes. + +### Day 1: Incubating EVs with Capture Beads + +4. Modify the template spreadsheet plate map to analyze more than one EV sample. Organize the plate to ensure efficient transfer of antibody solutions. Below is an example layout: + +| | CD9 | CD63 | CD81 | mAb 4 | mAb 5 | mAb 6 | Setup Beads | +|---|-----|------|------|-------|-------|-------|-------------| +| 1 | 10 μL capture beads | 10 μL capture beads | 10 μL capture beads | 10 μL capture beads | 10 μL capture beads | 10 μL capture beads | 5 μL capture beads | +| 2 | 10 μL capture beads | 10 μL capture beads | 10 μL capture beads | 10 μL capture beads | 10 μL capture beads | 10 μL capture beads | 5 μL capture beads | +| 3 | 10 μL capture beads | 10 μL capture beads | 10 μL capture beads | 10 μL capture beads | 10 μL capture beads | 10 μL capture beads | 5 μL capture beads | +| 4 | 10 μL capture beads | 10 μL capture beads | 10 μL capture beads | 10 μL capture beads | 10 μL capture beads | 10 μL capture beads | 5 μL capture beads | +| 5 | PBS control | PBS control | PBS control | PBS control | PBS control | PBS control | 5 μL capture beads | + +5. Prepare an Eppendorf 2.0 mL LoBind collection tube for each EV sample. + +6. Using the capture bead preparation section of the template, determine the volumes of MACSPlex buffer, MACSPlex capture beads, and EVs for the incubation. + +7. Using your digital inventory, identify the capture bead tube you will use and note the lot number. + +8. Transfer the calculated amount of MACSPlex buffer to each EV sample collection tube. + +9. Spin down and vortex thoroughly on high. Aliquot calculated volumes into each EV sample collection tube. + +10. Vortex and add the calculated volume of undiluted or diluted EVs to each labeled tube. + +11. Vortex each tube, place in a tube rotator, cover with foil, rotate overnight at room temperature. + +### Day 2: Staining Captured EVs with Detection Antibodies + +12. Use the template to calculate volumes of antibodies and buffer required for detection antibody staining. + +13. Get a new Pall 0.2 μm PES filter plate. + +14. Using a multichannel pipette, add 150 μL of MACSPlex buffer to all wells. + +15. Subject the plate to vacuum until all wells are empty of buffer. + +16. Add 50 μL of MACSPlex buffer to previously wetted wells. + +17. Vortex EV sample tubes thoroughly and add 75 μL to each test well. + +18. Add 10 μL vortexed MACSPlex capture beads to detection antibody control wells. + +19. Vortex all detection antibodies gently and spin quickly in a tabletop centrifuge. + +20. Prepare antibody solutions as per the template. + +21. Vacuum the plate until wells are empty. Blot bottom with a clean paper towel. + +22. Using a multichannel pipette, dispense antibody solutions, 200 μL per well. Avoid bubble formation. + +23. Using the multichannel pipette set to 100 μL, mix the wells up and down without touching filter membranes. + +24. Cover the plate with a foil plate sealer, incubate for 2 hours at room temperature with shaking. + +25. Vacuum until wells are empty, then add 150 μL MACSPlex buffer immediately. + +26. Using 75 μL setting, reverse pipette all wells to avoid bubbles, clear wells, add 150 μL buffer. + +27. Clear all wells with the vacuum. + +28. Immediately add 150 μL buffer to all wells. + +29. Reverse pipette all wells again to avoid bubbles. + +30. Place on vacuum manifold until all wells are cleared. + +31. Add 200 μL of buffer to all wells for final resuspension. + +32. Using 200 μL setting, reverse pipette all wells, ensuring no membrane punctures. + +33. Add 5 μL of capture beads in 200 μL of PBS to a free well for the cytometer, and 20 μL of setup beads in 20 μL of PBS to another free well. + +### Day 2: Running Plate on Cytometer + +34. Run setup beads on the flow cytometer to adjust settings. + +35. Analyze the full volume of each stained EV sample, including non-EV control wells. + +## Data Analysis + +36. Use MPAASS software to analyze the multiplexed EV protein expression data. + +**endofoutput** +``` \ No newline at end of file diff --git a/markdown-output/multiquas-multiple-reference-quasispecies-reconstr-bxvhpn36.md b/markdown-output/multiquas-multiple-reference-quasispecies-reconstr-bxvhpn36.md new file mode 100644 index 0000000000000000000000000000000000000000..4c0d3256da5e57ffd7125fcb187ae0265ca1950a --- /dev/null +++ b/markdown-output/multiquas-multiple-reference-quasispecies-reconstr-bxvhpn36.md @@ -0,0 +1,184 @@ +```markdown +# Goal/Experiment: + +The goal of this experiment is to evaluate viral variability and reconstruct viral quasispecies from NGS (Next-Generation Sequencing) data, particularly MiSeq reads, using the MultiQuas pipeline. This protocol provides the major steps required to run the pipeline, assuming one to three well-established references obtained via haplotype reconstruction software. + +# MultiQuas (Multiple Reference Quasispecies Reconstruction Protocol) V.4 + +_Instituto de Agrobiotecnología y Biología Molecular (IABIMO, INTA-CONICET)_ + +_**In Development**_ + +## Disclaimer + +**FOR INFORMATIONAL PURPOSES ONLY; USE AT YOUR OWN RISK** + +The protocol content here is strictly for informational purposes and does not constitute legal, medical, clinical, or safety advice. Furthermore, this content on [protocols.io](https://protocols.io) is not peer-reviewed and may not have received formal approval of any kind. Any independent professional consultation, diagnosis, or treatment should not rely on any information provided herein. The user assumes all risk and responsibility by using this protocol. + +## Abstract + +This protocol provides guidance to reconstruct viral quasispecies from NGS data. It involves aligning reads to multiple references, filtering, trimming, and computationally reconstructing viral haplotypes. The references should be well-established to ensure accuracy in haplotype reconstruction. + +## Protocol Citation + +Marco Cacciabue 2021. MultiQuas (Multiple reference quasispecies reconstruction protocol). [protocols.io](https://protocols.io/view/multiquas-multiple-reference-quasispecies-reconstr-bxvhpn36). Version created by Marco Cacciabue. + +## License + +This is an open-access protocol distributed under the terms of the Creative Commons Attribution License, allowing for redistribution and reproduction with appropriate credit to the original author. + +## Created + +- **Date**: Aug 31, 2021 +- **Last Modified**: Sep 02, 2021 +- **Protocol Integer ID**: 52873 + +## Materials + +- **QuRe**: Source by Mattia C. F. Prosperi +- **FastQC 0.11.9**: Source by Simon Andrews + +## Align_to_references.sh Script + +Here is the step-by-step shell script for aligning reads to references using various bioinformatic tools: + +```bash +#!/bin/bash + +start=`date +%s` + +bbduk.sh in1=$2 out1=reads_1.fq in2=$3 out2=reads_2.fq ref=[path/to/bbmap/instalation]/bbmap/resources/adapters.fa ktrim=r k=23 mink=11 hdist=1 tpe tbo qtrim=rl trimq=20 minlen=50 maq=20 + +bowtie2-build $1 VFAref + +bowtie2 --no-discordant --no-mixed -p $4 -x VFAref -1 reads_1.fq -2 reads_2.fq | samtools view -@ 4 -bT $1 - > SAMPLE.bam + +samtools sort -@ 4 -m 2G SAMPLE.bam > SAMPLE_sorted.bam + +samtools view -@ 4 -h -F 4 -b SAMPLE_sorted.bam > SAMPLE_map.bam + +samtools index -@ 4 SAMPLE_map.bam SAMPLE_map.bai + +samtools depth -d100000000 SAMPLE_map.bam > coverage.txt + +lofreq viterbi -f $1 -o SAMPLE_map_viterbi.bam SAMPLE_map.bam + +samtools sort -@ 4 -m 2G SAMPLE_map_viterbi.bam > SAMPLE_map_viterbi_sorted.bam + +samtools index -@ 4 SAMPLE_map_viterbi_sorted.bam SAMPLE_map_viterbi_sorted.bai + +lofreq indelqual --dindel -f $1 -o SAMPLE_map_viterbi_sorted_indels.bam SAMPLE_map_viterbi_sorted.bam + +samtools index -@ 4 SAMPLE_map_viterbi_sorted_indels.bam SAMPLE_map_viterbi_sorted_indels.bai + +lofreq call-parallel --pp-threads $4 --call-indels --use-orphan -f $1 SAMPLE_map_viterbi_sorted_indels.bam -o variants.vcf + +end=`date +%s` + +echo Execution time was `expr $end - $start` seconds. +``` + +## Key Bioinformatic Tools + +- **bbduk.sh** by Brian Bushnell +- **samtools 1.12** by Wellcome Trust Sanger Institute +- **bcftools 1.12** +- **Bowtie2 2.4.4** +- **PEAR (Paired-End reAd mergeR)** by Alexandros Stamatakis +- **QuRe** by Mattia C. F. Prosperi +- **mafft 7.487** by Kazutaka Katoh +- **Lofreq 2** by Andreas Wilm + +## Brief Pipeline Description + +1. **Reads are trimmed and filtered using bbduk** + +2. **Filtered and trimmed reads are aligned to user-defined references using Bowtie2** + +3. **Reads are split into classes (one for each reference and one for unmapped reads) using SAMtools** + +4. **Reads for each class are merged using PEAR and haplotypes reconstructed with QuRe** + +5. **Proportions of each haplotype class are adjusted** + +6. **Reconstructed haplotypes are aligned to the first reference in the multifasta file using mafft** + +7. **Single Nucleotide Variants (SNVs) are called using Lofreq** + +8. **Concordance between predicted quasispecies and Lofreq variants is measured** + +## Installing Docker + +9. **Run the pipeline using a Docker image which includes all necessary dependencies.** + +10. **Pull the Docker image using the command:** + +```shell +docker pull cacciabue/multiquas:latest +``` + +10.1 **Alternatively, install dependencies manually:** + +- [BFTools v1.8](http://www.htslib.org/download/) +- [Samtools v1.8](http://www.htslib.org/download/) +- [Bowtie2 v2.2.4](http://bowtie-bio.sourceforge.net/bowtie2/index.shtml) +- [PEAR](https://www.h-its.org/exelixis/web/software/pear/doc.html) +- [seqtk](https://github.com/lh3/seqtk) +- [bbmap](https://jgi.doe.gov/data-and-tools/bbtools/bb-tools-user-guide/) +- [Lofreq](https://csb5.github.io/lofreq/) +- [mafft](https://mafft.cbrc.jp/alignment/software/) +- [R v4.1](https://www.r-project.org/) +- Additional R packages like **seqinr, ape, VariantAnnotation, Biostrings, ggplot2**. + +## Preparing Analysis + +11. **Set up according to your OS.** + +11.1 **For Windows:** Open a terminal. +11.2 **Create a working folder. For example:** + +```bash +mkdir test_dir +cd test_dir +``` + +11.3 **Copy fastq files and reference file (1 or more sequences in multifasta format) into the test_dir folder.** + +12. **For Linux:** + +12.1 **Open a terminal.** +12.2 **Create a working folder.** +12.3 **Copy fastq files and references into the test_dir folder.** + +## Running MultiQuas Workflow + +13. **Create a Docker container, mount the test_dir folder into the container, and perform MultiQuas workflow.** + +13.1 **For Windows:** + +```shell +docker run -it --volume %cd%:/nexus cacciabue/multiquas:latest reconstruction.sh complete -o OUTPUT_FOLDER -1 R1.fq -2 R2.fq -r REFERENCE.fasta +``` + +13.2 **For Linux:** + +```shell +docker run -it --volume $(pwd):/nexus cacciabue/multiquas:latest reconstruction.sh complete -o OUTPUT_FOLDER -1 R1.fq -2 R2.fq -r REFERENCE.fasta +``` + +## Output Files + +14. **Output folders will include the following:** + +- **Filtering:** Directory for filtered and trimmed reads. +- **Multiple_Aligning:** Directory for alignment files. +- **unmapped:** Directory for unmapped read alignments. +- **Variants:** Folder for variant .vcf files. +- **Output Files:** + + - **SAMPLE_haplotypes.fasta:** Reconstructed haplotypes. + - **SAMPLE_adjusted_proportions.txt:** Proportions of each haplotype. + - **SAMPLE_graphs.png:** Concordance graph between lofreq and reconstructed haplotypes. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/multisite-gateway-calculations-excel-spreadsheet-b4xdqxi6.md b/markdown-output/multisite-gateway-calculations-excel-spreadsheet-b4xdqxi6.md new file mode 100644 index 0000000000000000000000000000000000000000..cf90bb032ce0df2ed5dd075d4580c8b85c6a8eff --- /dev/null +++ b/markdown-output/multisite-gateway-calculations-excel-spreadsheet-b4xdqxi6.md @@ -0,0 +1,94 @@ +```markdown +# Goal/Experiment: +The goal of this experiment is to provide a streamlined, accurate method for calculating the correct ratios of multisite Gateway plasmids using an Excel spreadsheet. This ensures proper reaction mix assembly, documentation, and scalability for cloning reactions, particularly in zebrafish transgenesis studies. + +# Multisite Gateway Calculations: Excel Spreadsheet +**Christian Mosimann** +University of Colorado School of Medicine, Anschutz Medical Campus +[DOI: 10.17504/protocols.io.b4dxqxi6](https://dx.doi.org/10.17504/protocols.io.b4dxqxi6) + +## DISCLAIMER – FOR INFORMATIONAL PURPOSES ONLY; USE AT YOUR OWN RISK + +The protocol content here is for informational purposes only and does not constitute legal, medical, clinical, or safety advice. Content added to [protocols.io](https://www.protocols.io) is not peer-reviewed and may not have undergone formal approval of any kind. Information presented in this protocol should not substitute for independent professional judgment, advice, diagnosis, or treatment. Any reliance upon the information presented is strictly at your own risk. Neither the Company nor any of the authors, contributors, administrators, or anyone associated with [protocols.io](https://www.protocols.io), can be held responsible for your use of the information provided. + +## Abstract + +Multisite Gateway cloning provides a modular tool to assemble plasmid vectors suitable for numerous applications. The assembly of Multisite Gateway plasmids requires the correct ratios of three entry vectors and the destination backbone to favor correct recombination by Gateway LR Clonase II Plus. This protocol provides a simple Excel-based spreadsheet to simplify the involved calculations based on individual vector lengths and working stock concentrations. + +## Introduction + +Multisite Gateway cloning is a widely used cloning method based on the availability of individual component vectors from repositories such as AddGene ([www.addgene.org](https://www.addgene.org)) and multiple published plasmids from individual laboratories. Notably used for zebrafish transgenesis with Tol2 Kit vectors, this cloning method stimulates vector exchange and facilitates the assembly of new transgenesis tools. + +### Mechanism of Action: + +Multisite Gateway cloning involves Entry vectors with inserts flanked by dedicated repeats (Gateway repeats) into the final Destination vector: +- **5' entry vector:** Gene-regulatory elements or promoters. +- **Middle entry vector:** Open Reading Frame (ORF). +- **3' entry vector:** Poladenylation signal. +- **Destination vector:** Transgenesis, bacterial selection, etc. + +### Reagents and Equipment: +- **Gateway LR Clonase II Plus:** Catalyzes recombination reactions. (Manufacturer: Thermo Fisher) +- **Proteinase K:** Used for short treatment after incubation. +- **ddH2O:** For making stock solutions. +- **Excel Spreadsheet:** Facilitates calculation and documentation. + +## Protocols +### Calculations using a Simple Excel Spreadsheet + +Based on the recombination catalyzed by **Gateway LR Clonase II Plus**, standard reactions combine: +- **10 femtomole (fmol)** of each Entry vector +- **20 fmol** of the Destination backbone +- **10 µl reaction volume** + +**Incubation:** 16+ hours at 25 degrees Celsius followed by Proteinase K treatment and bacterial transformation. Scaling down to 5 µl reactions has proven practical. + +#### Calculation Steps: +1. **Total vector amount calculation:** +`[total length of vector] bp x 10 fmol / (660/10^6) = [total vector amount] ng` + +2. **Calculate volume for each vector stock:** +`[total vector amount] ng / ([concentration vector stock] ng/µl) / 2 = µl for 10 fmol.` + +3. **Mixing to achieve 4 µl final vector solution:** +`4 µl - ([volume 5' vector] + [volume middle vector] + [volume 3' vector] + [volume backbone vector]) = µl ddH2O` + +4. **Add LR Clonase II Plus to final volume:** +Combine the calculated 4 µl solution with 1 µl of LR Clonase II Plus. + +#### Example Excel Setup: + +| Vector Name | bp Total | fmol | Total (ng) | Plasmid Concentration (ng/µl) | Use (µl) | +|-------------|----------|------|------------|--------------------------------|----------| +| 5' Element | 9027 | 10 | 57.82 | 40 | 0.724 | +| ORF | 5637 | 10 | 37.14 | 40 | 0.465 | +| 3' UTR | 2833 | 10 | 18.70 | 20 | 0.935 | +| Backbone | 5085 | 20 | 152.42 | 79 | 1.927 | +| ddH2O | | | | | 1.187 | +| **Total** | | | | | **5** | + +### Workflow Steps: +1. **Fill in individual vector names** in column B. +2. **Fill in individual total vector size** in bp in column C. +3. **Fill in individual vector stock concentrations** in column I. +4. **Double-check pipetting volumes.** +5. **Add 1 µl LR Clonase II Plus** (vortex 2x for 2 sec), mix by flicking followed by a quick spin. +6. **Incubate reactions** at 25 degrees Celsius for 16+ hours. + +### General Tips: +- Use clean minipreps and verified by 260/230 nm and 260/280 nm measurements. +- Use clean ddH2O or TE to make stock solutions. +- Always vortex Clonase mix properly before use. +- Assemble the mix in one go to ensure consistency. + +### Example Spreadsheet Download: +[MultisiteGateway Calculations v1 Feb2022.xlsx](https://dx.doi.org/10.17504/protocols.io.b4dxqxi6) + +### References: +1. Thermo Fisher Multisite Gateway information; [link](https://www.thermofisher.com/us/en/home/life-science/cloning/gateway-cloning/multisite-gateway-technology.html) +2. Kwan KM, et al. Dev Dyn. 2007 Nov;236(11):3088-99. doi:10.1002/dvdy.21343 +3. Mosimann C, et al. Development. 2011 Jan;138(1):169-77. PMID: 21138979; PMCID: PMC2998170. +4. Felker A, Mosimann C. Methods Cell Biol. 2016;135:219-44. doi:10.1016/bs.mcb.2016.01.009. PMID: 27443928. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/n-pcr-for-a-single-sample-zjyf4pw.md b/markdown-output/n-pcr-for-a-single-sample-zjyf4pw.md new file mode 100644 index 0000000000000000000000000000000000000000..6ebdf3b3765b81e9911f0c965b66c7300eb96c9c --- /dev/null +++ b/markdown-output/n-pcr-for-a-single-sample-zjyf4pw.md @@ -0,0 +1,155 @@ +```markdown +# Goal/Experiment: +The goal of this experiment is to diagnose HTLV-1 (Human T-lymphotropic virus type 1) using PCR (Polymerase Chain Reaction) based on the amplification of viral DNA sequences. This is achieved by employing nested PCR, which involves two rounds of DNA amplification targeting the viral polymerase gene, and in the second round, primers are added to amplify the human actin gene. + +# n-PCR for a single sample + +### Authors: +Matias Ruggieri, Carolina Berini, Nicolas Ducasa, Miroslav Malkovsky, Paul Fisch, Mirna Biglione + +### Affiliations: +1. Institut für Klinische Pathologie, Universitätsklinikum Freiburg, Germany +2. Instituto de Investigaciones Biomédicas en Retrovirus y SIDA (INBIRS), CONICET-Universidad de Buenos Aires, Buenos Aires, Argentina +3. UW School of Medicine and Public Health, Madison, Wisconsin, USA + +### Abstract: +HTLV-1 diagnosis using PCR is based on the amplification of viral DNA sequences. This method utilizes nested PCR which involves two rounds of DNA amplification reactions targeting the viral polymerase gene. In this second round, primers to amplify the human actin gene are also included. + +### External Link: +[https://doi.org/10.1371/journal.pone.0217560](https://doi.org/10.1371/journal.pone.0217560) + +### Guidelines: +n-PCR with pol/HTLV-1/actin co-amplification + +## Materials + +| Name | Catalog # | Vendor | +| -------------------------------- | ------------------------- | ---------------------------- | +| GeneRuler 50 bp DNA Ladder | SM0371 | Thermo Fisher Scientific | +| Taq DNA Polymerase (1000 U) | Cat No./ID: 201205 | Qiagen | +| MT-2 cells | 237 | | +| Thermo Fisher Scientific Germany | 10416014 | Invitrogen - Thermo Fisher | +| 100 bp DNA Ladder | 15628019 | Invitrogen - Thermo Fisher | +| dNTP Set 100 mM Solutions | R0182 | Thermo Fisher Scientific | + +## Safety Warnings: + +- Gloves should be changed regularly, particularly after preparation and before dispensing master mixes into PCR plates or strips to avoid cross contamination. +- Be aware that virus genetic material is being used, so be careful. + +## Before Starting: + +- To avoid contamination risk, DNA extraction, Pre PCR set-up (mastermix preparation), and post-PCR procedures should be conducted in separate workspaces. +- Benches, work stations, centrifuges, vortexes, and pipettes must be cleaned with 70% ethanol before and after every PCR set-up. + +## 1. Equipment and Materials: +1. Pipettes +2. 10μl presterilized filter tips +3. 20μl presterilized filter tips +4. 100μl presterilized filter tips +5. 1000μl presterilized filter tips +6. Thermal Cycler (e.g., TProfessional TRIO) +7. Electrophoresis equipment + +## 2. Reagents and Chemicals: +- **Taq DNA Polymerase kit (1000 U)**: Qiagen, Catalog #: 201205 +- **Buffers** (PCR Buffer and CoralLoad PCR Buffer) +- **MgCl2**: Magnesium chloride, required for the polymerase activity. +- **Q solution**: Enhances PCR performance. +- **dNTPs**: Deoxynucleotide triphosphates, building blocks for DNA synthesis. +- **Nuclease-free H2O**: Water, free from enzymes that degrade nucleic acids. +- **Forward (F) and Reverse (R) primers**: Sequence-specific oligonucleotides to initiate DNA synthesis. +- **Agarose**: Used for gel electrophoresis to assess the PCR product sizes. +- **Markers**: 50bp and/or 100bp, Thermo Fisher Scientific, Germany. + +## 3. Preparation and Storage of Reagents: + +- Store stock reagents for PCR (10X buffer, dNTPs, MgCl2, Q solution, primers, and enzymes) at -20°C. +- Dilute stock reagents into working concentrations and aliquot at -20°C into separate tubes. +- Working aliquots of sample and control DNA, as well as primary PCR (Nest-1) products, must be kept at -20°C for long-term storage but can be kept in the fridge for daily use. + +## 4. PCR Summary Protocol: + +- **Primary PCR/Nest-1 PCR**: Use PolEF and PolER primers. +- Prepare a mix for samples including positive control and negative control. +- **Nest-2 PCR**: Use PolIF, PolIR, Actin F, and Actin R primers. +- After PCR runs, remove tubes, briefly centrifuge to collect products, store at -20°C and analyze using 2% Agarose gel electrophoresis with nucleic acid stains. + +## 5. PCR Protocol: + +- Include at least two controls (1 positive and 1 negative) for each run. +- Prepare master mix for each sample run, accounting for an 8% extra volume for pipetting loss. +- Master Mix for Nest-1 PCR: 10X PCR buffer, dNTPs, MgCl2, primers, Taq polymerase, and water. +- Master Mix for Nest-2 PCR: 10X CoralLoad PCR Buffer, dNTPs, MgCl2, primers, Taq polymerase, and water. + +## 6. Preparation of PCR (Nest-1 PCR): + +| Initial Concentration | Final Concentration | For 1 Sample | +|----------------------|---------------------|------------------| +| PCR Buffer 10X | 1X | 2.5μl | +| Q Solution 5X | 1X | 5μl | +| MgCl2 25mM | 2.5mM | 2.5μl | +| dNTPs 2mM | 0.2mM | 2.5μl | +| PolEF 10mM | 0.3mM | 0.75μl | +| PolER 10mM | 0.3mM | 0.75μl | +| Taq polymerase | 0.5 units | 0.1μl | +| DNA sample | | 5μl (20 ng/μl) | +| Water | | To 25μl final vol| + +## 6. Preparation of PCR (Nest-2 PCR): + +| Initial Concentration | Final Concentration | For 1 Sample | +|-----------------------------|---------------------|------------------| +| CoralLoad PCR Buffer 10X | 1X | 2.5μl | +| Q Solution 5X | 1X | 5μl | +| MgCl2 25mM | 2.5mM | 2.5μl | +| dNTPs 2mM | 0.2mM | 2.5μl | +| PolIF 10mM | 0.3mM | 0.75μl | +| PolIR 10mM | 0.3mM | 0.75μl | +| Actin F 10mM | 0.3mM | 0.75μl | +| Actin R 10mM | 0.3mM | 0.75μl | +| Taq polymerase | 0.5 units | 0.1μl | +| First reaction product | | 2μl | +| Water | | To 25μl final vol| + +## 7. Primers: + +| Round | Name | 5*-3* Oligonucleotide Sequence | +|-------------|------------|---------------------------------------| +| Nest-1 PCR | PolEF | TTTTAGGTCCCACAAACTGGAG | +| Nest-1 PCR | PolER | GCAGGATTATGGAGACCTCAG3 | +| Nest-2 PCR | PolIF | GCCCTCATGCCAGTCTTTTAC | +| Nest-2 PCR | PolIR | CCTCGAATGGGATCAGGTAG | +| Nest-2 PCR | Actin F | ATCGGACGGCACATGCATCACCACAC | +| Nest-2 PCR | Actin R | GTTGAAGGTCTCAAACATGATCTG | + +## 8. PCR Cycles: + +### Nest-1: +- Pol EF + Pol ER + +| Temperature (°C) | Time | | +|------------------|------|----------------------| +| 94 | 3 min| | +| 94 | 10 sec| 40 cycles| +| 54 | 20 sec| | +| 70 | 45 sec| | +| 70 | 5 min| | +| 4 | Hold | | + +### Nest-2: +- Pol IF + Pol IR + Act F + Act R + +| Temperature (°C) | Time | | +|------------------|------|----------------------| +| 94 | 3 min| | +| 94 | 10 sec| 40 cycles| +| 52 | 20 sec| | +| 70 | 45 sec| | +| 70 | 5 min| | +| 4 | Hold | | + +--- + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/nanochromosome-arrays-combinatory-assembly-cprkvm4w.md b/markdown-output/nanochromosome-arrays-combinatory-assembly-cprkvm4w.md new file mode 100644 index 0000000000000000000000000000000000000000..cd1026375a0c64b4ee3002e39939a7853e451df4 --- /dev/null +++ b/markdown-output/nanochromosome-arrays-combinatory-assembly-cprkvm4w.md @@ -0,0 +1,127 @@ +```markdown +# Goal/Experiment: +Develop a protocol for the creation of synthetic chromosomes using nanochromosome arrays for genetic engineering in *Pichia pastoris*. + +## Nanochromosome Arrays Combinatory Assembly + +### Abstract +The yeast *Pichia pastoris* is commonly used in biotechnology to produce valuable recombinant proteins due to its ability to secrete large quantities of post-translationally modified proteins. However, incorporating heterologous genes into its genome for expression is challenging. This protocol outlines the creation of a synthetic 'chromosome-like' construct called a nanochromosome. This construct autonomously replicates and remains mitotically stable while expressing heterologous genes. The nanochromosome features essential scaffolding elements such as telomeres, centromeres, and yeast replication origins. It serves as a versatile genetic engineering platform that allows for precise and controlled insertion of multiple expression cassettes. Each insertion alternates with non-coding DNA sequences (~1 kb) to enable high-efficiency homologous recombination. + +## Module Overview +Modules with expression cassettes and LHR (long homologous regions) spacers, pre-prepared in vitro as insertion or integration arrays, are delivered to *Pichia* via transformation. Constructed array parts contain either two or three LHRs. Examples include LHR^n-GoI^n-AMR^H/Z-LHR^Z or GoI^n-LHR^m-AMR^H/Z-LHR^Z. + +LHR^m and LHR^Z are ~1000 bp regions from an LHR*A-Z* library. The final integration process is completed using a DNA parts library in the pUC19 plasmid. + +## Major Experimental Steps + +### Step 1: Generation of DNA Parts +- **PCR** amplification to generate parts with complementary overhangs suitable for BsmBI sites in pUC19. +- **Digestion** using BsmBI (Esp3I) or BsaI. +- **Agarose Gel** verification and extraction. +- **PCR Clean-Up** and concentration estimation. +```plaintext +Note: (*AsiSI-FspI RE recognition site within oligonucleotides (oligo1 & oligo4). +``` +![Step 1](img/step-1.png) + +### Step 2: Preparation of Semi-Products +- **Ligation** using T7 DNA ligase. +- **Agarose Gel** separation and extraction of DNA bands. +- **Gel Verification** for successful ligation. +```plaintext +Note: Ensure correct product sizes from both agarose gel and ligation mixtures. +``` + +### Step 3: Final Ligation +- **Ligation** of pre-assembled parts into linearized pUC19/BsmBI. +- **Transformation** into *E.coli* and selection on LB/ampicillin. +- **Verification** by bacterial colony PCR. +![Step 3](img/step-3.png) + +## List of DNA Parts +| DNA Part | Forward Oligo | Reverse Oligo | Product Size (bp) | Template | +|---------|----------------|---------------|-------------------|----------| +| LHRA (OUT) BsmBI | F315 CCGTCTCCGg gaGGGATC CgCCTGTTGTTA AACGCTCTT TAAGTCAAC CC | R301 CGGTCTCCtg tcCAAACCAA GATTGGACT ATTGCTATC | 883 | eDA8 | +| PAOX1PD1his (OUT) BsmBI | F302-CCGTCTCCga caAAACTCCA AAGAGGAAA GTTG | R303-CGGTCTCCct gaTCCTAACT ACTCTG | 2905 | eDA226* | +| LHRE (IN) BsaI | F336-CCGTCTCctg agCATC GGATTACC AATGGC AGG ACAAGTT TTGTCGGT | R350-GGTCTCCatC ACAAGTTT AACTAAGGTC GCAGCC | 892 | eDA9 | + +### Prestep: Generation of pPICZA with PAOX1PD1his +**Plasmid pPICZalphaPDI eDA143** + +#### Parts Amplified by PCR +- **Q5 High-Fidelity DNA Polymerase:** New England Biolabs (#M0491L) +- **Betaine solution 5M:** Merck MilliporeSigma (#B0300) +- **Thermal Cycler**: Applied Biosystems, ProFlex PCR System + +```plaintext +Volumes (microliters) +- Water: up to 50 +- Q5 Enzyme: 0.7 +- eDA143: 1 (100pg/µL) +- Betaine 5M: 10 +- Oligo447/448: 2.5 each +- 10mM dNTP: 4 +- 5x Q5 buffer: 10 +``` + +#### Program Q5 +| Step | Temperature | Time | Cycles | +|------|-------------|---------------|--------| +| Td-initial | 98°C | 30 seconds | | +| Td | 98°C | 10 seconds | | +| Ta | 55°C | 20 seconds | 33 | +| Te | 72°C | 1 minute 40 seconds | | +| Final Extension | 72°C | 2 minutes | | +| Hold | 4°C | hold | | + +#### PCR Clean-Up +- **QIAquick PCR Purification Kit**: Qiagen (#28104) +- **Restriction Enzymes (MfeI, NotI)**: New England Biolabs (MfeI-HF #R3589S, NotI-HF #R3189L) + +#### Ligation Reactions +```plaintext +Volumes (microliters) +- pUC19/BamHI/KpnI: 1 +- T4 DNA Ligase: 1 +- Buffer 10x T4: 6 +- Water: up to 10 +``` +- Incubate **overnight** at 16°C. + +### pUC19 Digestion with Esp3I +- Digest with Esp3I (New England Biolabs, #R0734S) +- **PCR Clean-Up**: Qiagen (#28104) +- **Concentration Checked**: DeNovix DS-11 + +### Final Ligation (Pre-assembled Parts) +- **Ligation** using T4 DNA Ligase (New England Biolabs, #M0202S) +```plaintext +Volumes (microliters) +- 10x T4 Ligase Buffer: 1.5 +- LHRA-Paox1PDI (4nM): 7 +- LHRE-HygR-LHRZ (7nM): 4 +- Overnight at 16°C +``` + +### E.coli Transformation +- *E.coli* Chemically LiAc Competent Cells +- **Selection** on LB +100ug/mL Carbenicillin + Hygromycin B. + +### Colony PCR +- **Selection**: Carbenicillin and Hygromycin B positive colonies. +- **Plasmid Miniprep**: Qiagen Miniprep (#27106). +- **Sequence Verification**: Sanger sequencing. + +## Note +All generated sequences are verified through sequencing. + +### Protocol Summary +- **Step 1:** Prepare DNA parts. +- **Step 2:** Semi-product ligation. +- **Step 3:** Final ligation into linearized pUC19. +- **E.coli transformation:** Growth on selective media. + +![Sanger Verification](img/verification.png) + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/nanomaterials-uv-vis-measurement-biickcaw.md b/markdown-output/nanomaterials-uv-vis-measurement-biickcaw.md new file mode 100644 index 0000000000000000000000000000000000000000..c5989275c270fca17240093d9ffb90a9870f1024 --- /dev/null +++ b/markdown-output/nanomaterials-uv-vis-measurement-biickcaw.md @@ -0,0 +1,85 @@ +```markdown +# Goal/Experiment: +The goal of this experiment is to perform UV-Vis spectroscopy to measure the particle sizes in gold nanoparticle (AuNP) suspensions. This involves quantifying the extinction of light measured from the spectral pattern using absorbance. + +# Nanomaterials UV-Vis Measurement +Ana Carracso Quevedo1, Emily J. Guggenheim1, Sophie M. Briffa1, Anastasios Papadiamantis1 +1University of Birmingham + +## Abstract +This SOP describes a sample preparation procedure for particle size measurements in gold NP suspensions. The procedure involves quantification of the extinction of light that is measured from the spectral pattern using absorbance. UV-Vis refers to the ultraviolet to visible spectral region of light; the absorption of which is size dependent at the nanoscale. UV-Vis spectroscopy is ideal for the size characterization of NP suspensions through absorbance at an appropriate wavelength. The settings defined below will be refined to optimize results during subsequent runs. + +## Keywords +UV-Vis, Au, Gold, Nanoparticles, Nanomaterials + +## License +This is an open access protocol distributed under the terms of the [Creative Commons Attribution License](https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. + +## Created +Jul 13, 2020 + +## Last Modified +Jul 13, 2020 + +## Protocol Integer ID +39204 + +## Guidelines + +### 1.1 Essential Equipment +1. **UV-Vis Spectrophotometer**. +2. **Calibrated Volume Pipettors of 1 and 5 mL** with disposable tips. +3. **Disposable 3 mL cuvettes** (Suggested: polystyrene 10 x 10 x 45mm, SARSTEDT, Catalogue number: 67.742). + +### 1.2 Chemicals +1. Ultrapure water 18.2 MΩcm. + +### 1.3 HSE Issues +- All laboratory personnel must comply with local safety regulations when working in the laboratory. +- All personnel must consult Material Safety Data Sheets (MSDS) to be aware of known hazards relevant to all chemical substances used in this SOP. +- Personnel should utilize all necessary precautions to avoid exposure to chemical and nanomaterials (Lab coats, nitrile gloves, protective glasses, and masks). +- All residues and waste materials must be disposed of according to local environmental and safety regulations. + +## 1. Sample Preparation +1. Vortex each AuNP stock suspension for 2 minutes. +2. Dilute the Au suspensions 1:2. Pipette 0.5 mL of the Au stock suspension and 0.5 mL UPW 18.2 MΩcm to obtain a final volume of 1 mL and a mass concentration of 25 mg/L. + +## 2. Sample Analysis +1. Switch the UV-Vis Spectrometer (Jenway 6800 Double Beam Spectrometer) on and leave for 20 minutes to allow the lamp to heat up. +2. Use 18.2 MΩcm UPW as the reference sample. +3. Before starting the measurements, the parameter settings shown in Table 1 should be set in the software (Flight Deck 1.0 or higher). + +### Table 1. UV-Vis Parameter settings + +| Parameter | Settings | +|---------------------|---------------| +| Measurement Mode | Spectrum Scan | +| Data Mode | ABS | +| Start Wavelength | 680 nm | +| End Wavelength | 380 nm | +| Scan Speed | 400 nm/min | +| Sampling Interval | 0.5 | +| Slit Width | 1.5 | +| Path Length | 10 | + +4. Baseline correction should be obtained by running a baseline using two cuvettes filled with 1 mL of UPW each, placed in the sample holders. +5. The reference cuvette with 1 mL UPW should then be left untouched and the other cuvette should be replaced with a new cuvette containing 1 mL of one of the diluted AuNP suspensions. A new cuvette should be used for each different sample analyzed. +6. Three spectrum scan runs for each known BBI AuNP diluted suspension (5, 20, 40, 60 and 100 nm) should be obtained. Therefore a total of 15 scans should be collected. The results obtained should be reported in Section 6 below and a calibration curve should be plotted. +7. Following this, three spectrum scan runs for the unknown monodispersed AuNP suspension containing a monodispersed suspension of NPs of an unknown size. + +## 3. Reporting Results +1. Note the average maximum absorption wavelength (λmax) of each if the UV-Vis readings (n=15) in the results Table 1. +2. Note the maximum absorption (AUmax) of each of the UV-Vis readings (n=15) in the results Table 2. +3. To add the results click the pencil button on the right hand-side of the results table. +4. The average λ, AU and standard deviations for each set of measurements will be automatically calculated. +5. Plot the spectra of absorbance vs wavelength (nm) for each measurement (n=15). +6. Save the plots as a .jpg image and upload it using the File button above the results tables. + +## 4. Data Upload +1. Extract the raw experimental data for each measurement (n=18) as a .CSV or excel compatible file. +2. Upload the files using the File button above the results table. +3. The raw data will be used to calculate the Energy Band Gap (EBG) for each nanomaterials sample using Tauc plots and shared with partners. +4. (Optional) Add any comments you like in the comments section at the bottom of the results page. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/ncov-2019-mcgill-artic-pcr-protocol-v4-1-at-63c-cbwaspae.md b/markdown-output/ncov-2019-mcgill-artic-pcr-protocol-v4-1-at-63c-cbwaspae.md new file mode 100644 index 0000000000000000000000000000000000000000..a1f81ca9163582e0adcd7f71b0b66db9e3d2eabf --- /dev/null +++ b/markdown-output/ncov-2019-mcgill-artic-pcr-protocol-v4-1-at-63c-cbwaspae.md @@ -0,0 +1,182 @@ +```markdown +# Goal/Experiment: +The aim of this experiment is to carry out SARS-CoV-2 detection using the PCR protocol with the ARTIC V4.1 primers at 63°C, which is currently being utilized at the McGill Genome Center. + +## nCoV-2019 McGill Artic PCR Protocol, V4.1 at 63°C V.2 + +**Version 2** +**Date:** Jun 23, 2022 + +**Authors:** +Sarah J Reiling1, Kayleigh Loranger1, Anne-Marie Roy1, Shu-Huang Chen1, Ioannis Ragoussis1 +1McGill University + +**DOI:** [dx.doi.org/10.17504/protocols.io.evow18e4ygr2/v2](https://dx.doi.org/10.17504/protocols.io.evow18e4ygr2/v2) + +**Abstract:** +This is the updated SARS-CoV-2 PCR Protocol, with the ARTIC V4.1 primers, that is currently being used at the McGill Genome Center. + +**Protocol Citation:** +Sarah J Reiling, Kayleigh Loranger, Anne-Marie Roy, Shu-Huang Chen, Ioannis Ragoussis 2022. nCoV-2019 McGill Artic PCR Protocol, V4.1 at 63°C. *protocols.io.* [https://dx.doi.org/10.17504/protocols.io.evow18e4ygr2/v2](https://dx.doi.org/10.17504/protocols.io.evow18e4ygr2/v2) +Version created by Kayleigh Loranger + +**Fork Note:** +Forked from nCoV-2019 McGill Artic PCR Protocol, 5 ul RT and V3 only + LA1 at 63°C, +Sarah Reiling + +## License +This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. + +**Created:** Jun 23, 2022 +**Last Modified:** Jun 23, 2022 +**Protocol Integer ID:** 65186 + +## Materials + +- **Q5 High-Fidelity 2X Master Mix - 500 reactions** + **Vendor:** New England Biolabs + **Catalog #:** M0492L + +- **Nuclease-free water** + **Contributed by users** + +- **Fresh 80% Ethanol** + **Contributed by users** + +- **Quant-iT™ PicoGreen™ dsDNA Assay Kit** + **Vendor:** Invitrogen - Thermo Fisher + **Catalog #:** P11496 + +- **AmpureXP beads** + **Vendor:** Beckman Coulter + **Catalog #:** A63880 + +## Primer Pool Preparation + +1. **Preparation of Lyophilised Primers** + - Resuspend lyophilised primers at a concentration of 100 µM each if required. + + **Note:** V4.1 only primers for this protocol were designed using Primal Scheme and generate overlapping 400 nt amplicons. V4.1 was added to the V4 primer set for optimization. + + **References:** + - [Primer Schemes - GitHub](https://artic-network/primer-schemes) + - [Optimization of the SARS-CoV-2 ARTIC Network V4 Primers](https://www.ncbi.nlm.nih.gov/pmc/articles/PMC) + +2. **Pooling Primers** + - The V4.1 pre-pooled primers in **1.5 mL Eppendorf** labelled tubes are labelled “Pool 1 (100µM)” or “Pool 2 (100µM)”. The primers do not require additional preparation. + - If not pre-pooled, follow the below pipetting scheme to make the master mix by adding the following primers to the V4 primer pools. + + ``` + Added to Pool 1: + - SARS-CoV-2_23_RIGHT_alt1 + - SARS-CoV-2_27_RIGHT_alt1 + - SARS-CoV-2_79_RIGHT_alt1 + - SARS-CoV-2_89_LEFT_alt1 + - SARS-CoV-2_89_RIGHT_alt1 + + Added to Pool 2: + - SARS-CoV-2_10_LEFT_alt1 + - SARS-CoV-2_10_RIGHT_alt1 + - SARS-CoV-2_76_LEFT_alt1 + - SARS-CoV-2_76_RIGHT_alt1 + - SARS-CoV-2_88_LEFT_alt1 + - SARS-CoV-2_90_RIGHT_alt1 + ``` + + **Guide to Pooling Volumes:** + + - **2x Volume:** + SARS-CoV-2_1_LEFT & SARS-CoV-2_1_RIGHT + SARS-CoV-2_7_LEFT & SARS-CoV-2_7_RIGHT + SARS-CoV-2_13_LEFT & SARS-CoV-2_13_RIGHT + SARS-CoV-2_17_LEFT & SARS-CoV-2_17_RIGHT + SARS-CoV-2_27_LEFT & SARS-CoV-2_27_RIGHT + SARS-CoV-2_45_LEFT & SARS-CoV-2_45_RIGHT + SARS-CoV-2_59_LEFT & SARS-CoV-2_59_RIGHT + SARS-CoV-2_60_LEFT & SARS-CoV-2_60_RIGHT + SARS-CoV-2_61_LEFT & SARS-CoV-2_61_RIGHT + SARS-CoV-2_64_LEFT & SARS-CoV-2_64_RIGHT + SARS-CoV-2_79_LEFT & SARS-CoV-2_79_RIGHT + SARS-CoV-2_90_LEFT & SARS-CoV-2_90_RIGHT + SARS-CoV-2_91_LEFT & SARS-CoV-2_91_RIGHT + + - **1x Volume:** + All other primers including alts (from V4.1). Primers should be pooled in the **mastermix cabinet** which should be cleaned with decontamination wipes and UV sterilised before and after use. + +## Multiplex PCR + +3. **Multiplex PCR Setup** + - In the **extraction and sample addition cabinet**, add **5 µL RT product** to each tube and mix well by pipetting. + + **Note:** The extraction and sample addition cabinet should be cleaned with decontamination wipes and UV sterilised before and after use. + +4. **Multiplex PCR Reactions Preparation** + + In the **mastermix hood**, set up the multiplex PCR reactions as follows in 0.2 mL 8-strip PCR tubes: + + | Component | Pool 1 [10 µM] | Pool 2 [10 µM] | + |------------------------------------|----------------|----------------| + | Q5 Hot Start High-Fidelity 2X Master Mix | 12.5 µL | 12.5 µL | + | Primer Pool 1 or 2 (10 µM pool 1+2) | 3.7 µL | 3.7 µL | + | Nuclease-free water | 3.8 µL | 3.8 µL | + | **Total** | **20 µL** | **20 µL** | + + - Add 20 µL of PCR mastermix to the 5 µL RT product of step 3. + - Pulse centrifuge the tubes to collect the contents at the bottom of the tube. + +5. **PCR Cycling Conditions** + + Set up the following program on the thermal cycler: + + | Step | Temperature | Time | Cycles | + |------------------|---------------------|--------------|-------------| + | Heat Activation | 98 °C | 00:00:30 | 1 | + | Denaturation | 98 °C | 00:00:15 | 36 | + | Annealing | 63 °C | 00:05:00 | 36 | + | Hold | 4 °C | Indefinite | 1 | + + **Note:** Cycle number should be 25 for Ct 18-21 up to a maximum of 36 cycles for Ct 35. + +## PCR Cleanup + +6. **PCR Cleanup** + - Combine the entire contents of “Pool 1” and “Pool 2” PCR reactions for each biological sample into a single **1.5 mL Eppendorf tube**. + +7. **Clean-Up Protocol** + + - Add an equal volume (1:1) of AmpureXP beads to the sample tube and mix by pipetting. + - Incubate for 5 minutes at room temperature. + - Pellet on magnet for 5 minutes. Remove supernatant. + - Add 200 µL of 80% ethanol to the pellet and wash twice. + - Let the beads dry for 3 minutes. + - Add 30 µL elution buffer and resuspend the beads. Incubate for 3 minutes. + - Pellet on magnet for 5 minutes. Remove and keep eluate (30 µL). + + **Note:** Amplicon clean-up should be performed in the **post-PCR cabinet** which should be cleaned with decontamination wipes and UV sterilised before and after use. + +## Amplicon Quantification and Normalization + +8. **Quantification** + + Quantify the amplicon pools using a fluorimetric dsDNA assay (e.g., PicoGreen with a standard curve 0-200 ng). + +9. **Expected Concentrations** + + - Pool 1+2 combined: + + | Ct Range | Concentration (ng/µL) | + |-----------|------------------------| + | 14-24 | 100-150 ng/µL | + | 25-29 | 30-80 ng/µL | + | 30-36 | 10-30 ng/µL | + +10. **Library Preparation** + + - **Nextera Flex Library Prep:** + - After quantification of Pool 1+2, take a new plate and add 150 ng of Pool 1+2 and add up with nuclease-free water to a total volume of 30 µL (= 5 ng/µL). + + - **Nanopore Library Prep:** + - After quantification of Pool 1+2, take a new plate and add 200 ng of Pool 1+2 and add up with nuclease-free water to a total volume of 20 µL (= 10 ng/µL). + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/new-identification-equations-based-on-erythrocyte-irmcd46.md b/markdown-output/new-identification-equations-based-on-erythrocyte-irmcd46.md new file mode 100644 index 0000000000000000000000000000000000000000..65b1f1b84ff843ebeb168163e1e088faed1e1ab1 --- /dev/null +++ b/markdown-output/new-identification-equations-based-on-erythrocyte-irmcd46.md @@ -0,0 +1,91 @@ +```markdown +# Goal/Experiment: +The goal of this experiment is to establish identification equations based on erythrocyte and reticulocyte characteristics for the screening of thalassemia traits in pregnant women. The aim is to differentiate between iron deficiency anemia (IDA) and thalassemia trait (TT) and evaluate the clinical application value of these identification equations during pregnancy. + +# New Identification Equations Based on Erythrocyte and Reticulocyte Characteristics for Screening Thalassemia Trait in Pregnancy Version 2 + +**Authors:** +- Yu-wei Yang +- Bi Peng +- Xiao-hong Chen +- Jun Ying +- Tao Yang + +**Published:** +03 Jul 2017 + +## Protocol + +### Expected Outcomes +**Step 1:** + +Both iron deficiency anemia (IDA) and thalassemia trait (TT) are the most common microcytic hypochromic anemias. In pregnancy, a woman can easily transition to an iron deficiency (ID) state due to increased iron utilization, making it more challenging to identify TT. Various hematological indices such as mean corpuscular volume (MCV) and mean corpuscular hemoglobin (MCH), calculated from automated cell counters, are used to discriminate between these anemias. This study aims to establish identification equations to screen for TT in pregnant women and discuss their clinical value. + +### Study Population +**Step 2:** + +- **Recruitment Period:** January 2015 to September 2016. +- **Participants:** All pregnant women attending their first antenatal care visit. +- **Procedure:** + - **Initial Assessment:** Participants filled out an antenatal care questionnaire covering food intake, underlying diseases, history of blood loss, current medications, family history of anemia, and other demographic data (age, sex, height, weight). + - **Sample Collection:** Whole blood samples collected and analyzed using the XN-9000 automatic machine (Sysmex, Japan). Parameters measured: + - **RBC Parameters:** RBC#, Hb, MCV, MCH, mean corpuscular hemoglobin concentration (MCHC), RBC distributing width (RDW). + - **Reticulocyte Parameters:** Reticulocyte counts, reticulocyte percentage (Ret%), low fluorescence ratio (LFR), medium fluorescence ratio (MFR), high fluorescence ratio (HFR), immature reticulocytes fluorescence ratio (IRF), hemoglobin content of reticulocytes (Ret-He). + +- **Screening Criteria:** + - Hemoglobin level <110 g/L with decreased MCV and/or MCH. + - Exclusion of subjects with a history of infection, inflammation, or underlying medical conditions in the past month. + - Hemoglobin electrophoresis for those with microcytic hypochromic anemia. Critical values for HbA2: <2% or >4% (practically 2.5% or 3.5% selected). + - Serum ferritin measurement for all subjects. + +### Inclusion Criteria: +1. Hemoglobin (Hb) <110g/L with decreased MCV and/or MCH. +2. TT carriers confirmed by gene analysis for hemoglobin gene deficiency. +3. IDA patients with ferritin <14µg/L, without any gene deficiency of hemoglobin. + +### Exclusion Criteria: +- Subjects with a history of infection, inflammation, or underlying medical conditions within one month. + +### Development of the New Identification Equations +**Step 3:** + +- **Random Sampling:** From patients with α-TT, β-TT, or IDA. + - **Development Group:** 174 cases (75 IDA, 42 α-TT, 57 β-TT). + - **Validation Group:** 251 cases (102 IDA, 63 α-TT, 86 β-TT). +- **Classification:** + - **ID vs. TT:** Development of identification equation logit-P1. + - **IDA vs. α-TT or β-TT:** Development of identification equation logit-P2. +- **Data Analysis:** + - Logistic regressions and ROC (receiver operating characteristic) curve analyses used. + +- **Equation Development:** + - **Logit-P1:** RBC#, Ret%, IRF (cut-off point value: 0.84). + - **Logit-P2:** MCHC, Ret%, MRF (cut-off point value: 0.41). + +### Supplementary Criteria of the New Identification Equations +**Step 4:** + +- Based on validation, logit-P1 showed limitations, misdiagnosing 27.0% of α-TT as IDA. +- ROC curve reanalysis showed RBC# >4.1x10^12/L, used as supplementary criteria for diagnosis. + +### Comparison with Other Hematological Indices +**Step 5:** + +- **12 Hematological Indices:** Compared using the ROC curve in the validation group, including various indices published between 1973 to 2015. +- **ROC Curve Analysis Data:** + - AUC (Area under the curve) + - YI (Youden index) + - Specificity (Spe) + - Sensitivity (Sen) + - Likelihood ratio positive (LRP) + - Likelihood ratio negative (LRN) +- **Decision Making:** Rates of missed diagnoses and correct diagnoses compared with clinical diagnoses. + +### References +**Step 6:** +1. Friedman AJ, Shander A, Martin SR, Calabrese RK, Ashton ME, Lew I, et al. Iron deficiency anemia in women: a practical guide to detection, diagnosis, and treatment. Obstet Gynecol Surv. 2015; 70(5):342-53. doi: 10.1097/OGX.0000000000000172. +2. Stevens GA, Finucane MM, De-Regil LM, Paciorek CJ, Flaxman SR, Branca F, et al. Global, regional, and national trends in hemoglobin concentration and prevalence of total and severe anemia in children and pregnant, non-pregnant women from 1995-2011: a systematic analysis of population-representative data. Lancet Glob Health. 2013; 1:e16e25. doi: 10.1016/S2214-109X(13)70001-9. +... (continue list with remaining references) + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/ney-39-s-spring-media-preparation-bqjgmujw.md b/markdown-output/ney-39-s-spring-media-preparation-bqjgmujw.md new file mode 100644 index 0000000000000000000000000000000000000000..ab8872e2000e15e217eb8be4af9cce725304edcb --- /dev/null +++ b/markdown-output/ney-39-s-spring-media-preparation-bqjgmujw.md @@ -0,0 +1,89 @@ +```markdown +# Goal/Experiment: +This protocol describes the preparation of Ney's Spring high pH media and plates. These consist of basal minimal media with varying electron donors and acceptors, tailored for different organisms. + +# Ney's Spring Media Preparation +**Authors**: Annette Rowe, Leah Trutschel +**Affiliation**: University of Cincinnati +**Protocol DOI**: [dx.doi.org/10.17504/protocols.io.bqjmujw](https://dx.doi.org/10.17504/protocols.io.bqjmujw) +**Created**: Dec 09, 2020 +**Last Modified**: Jun 15, 2021 + +## Abstract +This protocol for making Ney's Spring high pH media and plates includes instructions for: +- Sulfur/acetate plates & media +- Methanol plates & media +- Sulfate/acetate plates & media + +## Safety Warnings +These are high pH media and gloves should be worn during their preparation and disposal. Addition of sulfide and polysulfide should be performed in a fume hood. + +## Ney's Spring Basal Salts +These are the basal salts used in all media. If making liquid media, add salts to 700 mL milli Q water. If making plates, then start with 400 mL milli Q water (additional components will then be added to bring up to 1 L later). + +### Basal Salts Component Table +| Component | MW (g/mol) | Amount added (g) | Concentration (mM) | +|--------------|------------|------------------|--------------------| +| NaCl | 58.44 | 11.7 | 200 | +| Na2SO4 | 142.04 | 0.284 | 2 | +| NH4Cl | 53.49 | 0.535 | 10 | +| MgCl2•6H2O | 203.3 | 0.102 | 0.5 | + +## Components Added from Stock +These are all prepared as stock solutions first, then are added to the media after the basal salts. This should bring the liquid media up to ~1 L, and media to be used for plates to ~700 mL. After adding these, check the pH. Media should be between pH 10-11. Aim for closer to 10.5 for plates, while liquid media can be closer to 11. + +For plates, prepare a second solution of 300 mL milli Q water with 15g NOBLE agar. Regular agar burns incredibly easily at high pH and won't be sufficient for this media. Add a stir bar and autoclave separately from the salt solution. + +### Stock Solutions Component Table +| Component | MW (g/mol) | Amount of Stock prepared | Amount added to stock (g) | Concentration (mM) | Amount added to media | Final conc. | +|----------------------|------------|--------------------------|---------------------------|--------------------|-----------------------|-------------| +| K2HPO4 | 174.18 | 10X (500mL) | 0.871 | 10 | 100 mL | 1 mM | +| Na2CO3 | 105.99 | 10X (500 mL) | 53 | 1000 | 100 mL | 100 mM | +| CAPS (N-cyclohexyl-3-aminopropanesulfonic acid) | 221.32 | 10X (500 mL) | 11 | 100 | 100 mL | 10 mM | +| CH3COONa (sodium acetate) | 136.08 | 100X (200 mL) | 5.44 | 200 | 10 mL | 2 mM | +| NaOH | 39.99 | 100X (200 mL) | 4 | 500 | 10 mL | 5 mM | + +*Note: Check for each electron donor/acceptor combo if it is added before or after autoclaving as they all differ.* + +## Additions after Autoclaving +Once media has cooled, add vitamins and minerals (1mL of each from a 10,000X conc. Recipes for these can be found here: ). + +1. **Notes for Plates** + It is very important to let the media cool almost entirely before any more additions! Plates especially will burn and turn black almost immediately if your media is still hot when you combine the 700 mL salt solution and 300 mL agar solution. Wait until just warm and then very slowly pour the basal salt solution into the agar solution, stirring the entire time. + +2. **Notes for Anaerobic Media** + While liquid media is still hot and fresh out of the autoclave, immediately begin purging with N2 gas. Purge until cool, then you may add vitamins, minerals, and other components. + +## Electron Donor & Acceptor Variants +Check first to see if whether these need to be added before or after autoclaving: + +### Methylotroph/Methanogen Enrichment +| Component | MW (g/mol) | Stock | Amount added for stock (g) | Concentration (mM) | Amount added to media | +|----------------------------|------------|------------|----------------------------|--------------------|-----------------------| +| MeOH | 32.04 | Pure MeOH | - | 2.5 mM | 2.5 mL | +| Na2S • 9H2O (sodium sulfide) | 240.18 | 100 mM | 2.4 | 1 mM | 10 mL | + +*Add methanol and sulfide after autoclaving at the same time as vitamins and minerals. Sodium sulfide is optional but can be added to anaerobic media to help with oxygen scavenging.* + +### Sulfate Reducers Enrichment +| Component | MW (g/mol) | Amount added | Concentration (mM) | Amount added to media | +|----------------------|------------|---------------|--------------------|-----------------------| +| NaSO4 (Sodium sulfate) | 142.04 | 1.42 g | 10 | 10 | +| CH3COONa (sodium acetate) | 136.08 | 0.82 g | 10 | 10 | + +*These components can be added at the same time as components in the basal salts for a final total of 12 mM of NaSO4 and CH3COONa. +**After autoclaving, 1 mM Na2S9H2O may also be added to anaerobic media to help with oxygen scavenging. +***Sulfate plates will be clear, while media will be mostly clear but may form a dark green/turquoise precipitate. + +### Sulfur Reducers/Oxidizers Enrichment +| Component | MW (g/mol) | Amount added (mL) | Concentration (mM) | +|----------------------|------------|--------------------|--------------------| +| Polysulfide | - | 8.8 mL | 20 mM | +| CH3COONa (sodium acetate) | 136.08 | 0.41g | 5 mM | + +*Acetate can be added at the same time as components in the basal salts for a final total of 7 mM. +**Polysulfide prepared according to Moser & Nealson 1996, "Growth of the Facultative Anaerobe Shewanella putrefaciens by Elemental Sulfur Reduction" +***Add polysulfide once media is almost entirely cool using a syringe. Will immediately turn a very dark olive "bottle glass" green and have a very strong sulfur smell. As the polysulfide oxidizes (I recommend placing them in a fume hood for at least 24 hours), the plates will turn a translucent pale blue rather than the typical solid white of elemental sulfur plates. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/nonlinear-spectral-mixture-effects-for-photosynthe-ia5cag6.md b/markdown-output/nonlinear-spectral-mixture-effects-for-photosynthe-ia5cag6.md new file mode 100644 index 0000000000000000000000000000000000000000..157cc1e8264614fce7bd11d366d872a40eb68744 --- /dev/null +++ b/markdown-output/nonlinear-spectral-mixture-effects-for-photosynthe-ia5cag6.md @@ -0,0 +1,80 @@ +```markdown +# Goal/Experiment: +The goal of this experiment is to determine the nonlinear spectral mixture effects for photosynthetic (PV) and non-photosynthetic vegetation (NPV) cover estimates in typical desert vegetation of Western China. This experiment aims to improve the accuracy of vegetation cover estimation through a combination of spectral data collection, linear spectral mixture modeling, and advanced nonlinear spectral mixture modeling techniques. + +# Nonlinear Spectral Mixture Effects for Photosynthetic/Non-photosynthetic Vegetation Cover Estimates of Typical Desert Vegetation in Western China +**Version 4** +Cuicui JI + +## Abstract +**Citation:** Cuicui JI Nonlinear Spectral Mixture Effects for Photosynthetic/Non-photosynthetic Vegetation Cover Estimates of Typical Desert Vegetation in Western China. protocols.io +**Published:** 15 Dec 2017 + +## Protocol + +### Spectral Data and Preprocessing +#### Step 1 +1. **Endmember Measurements**: + Endmembers, including bare soil (BS), shadow, PV, and NPV, were allowed to vary among plots. Endmember and mixed spectral data were obtained from ground-based field experiments. + + - **Reflectance Spectral Measurements**: Acquired on 25 Aug 2014, using a full-range (350–2500 nm) spectroradiometer (ASD Spec Pro Field, Analytical Spectral Devices). Calibration was done using a white spectral panel (Labsphere Inc.). + - **Conditions for Data Collection**: Windless, cloudless, and full sunshine conditions between 10:00 – 14:00. Measurement involved 20 Nitraria plots and 20 Haloxylon plots with different ratios of PV to NPV cover. + - **Spectral Sampling**: 1 m diameter circle marked for each sample plot. A probe was placed above typical species surfaces for spectra acquisition; endmember spectra were acquired between 0.1 m and 0.2 m. + +### Linear Spectral Mixture Model +#### Step 2 +We have constructed the Linear Spectral Mixture Model (LSMM) based on the principles of linear mixtures. The endmember fraction estimates are obtained by the fully constrained least squares (FCLS). + +### Reference Fraction +#### Step 3 +Photographs were acquired twice at each field site using a digital camera to avoid out-of-focus images. The higher-quality photograph was selected for precise reference fraction determination. + +- **Digital Image Classification**: + - Training samples for supervised classification used ENVI 5.3 software and neural network classification (NNC) to extract ground cover composition. + - Convolution with Gaussian filter: To adjust for the point spread function (PSF) of the ASD sensor, allowing accurate ground cover fraction estimation through weighted averages. + - Adjusted fractions were averaged across two photographs for the reference fraction of different endmembers. + +### Bilinear Spectral Mixture Model +#### Step 4 +Proposes the use of a bilinear spectral mixture model (BSMM), which accounts for interactions between endmembers. The FCLS algorithm is used to unmix interaction fractions. + +### Kernel Nonlinear Spectral Mixture Model +#### Step 5 +Two kernel functions were used: +- **Radial Basis Function (RBF) Kernel** +- **Polynomial Kernel Function (PKF)** + +#### Parameters of Kernel Function +#### Step 6 +Optimal parameters were determined by minimizing model unmixing RMSE. Parameter σ in RBF determined by gradient descent; PKF parameters determined by cross-validation. + +### Kernel Nonlinear Spectral Mixture Model Unmixing +#### Step 7 +KFCLS algorithm finds abundance vectors through objective function optimization. + +### Evaluate Unmixing Accuracy +#### Step 8 +Accuracy was evaluated by comparing estimated and reference endmember fractions using RMSE, R², and Relative RMSE. RMSE is a measure of deviation rate, indicating model fit. + +### Bilinear Spectral Mixture Model IDL Code +#### Step 9 +```idl +pro BSMM_unmixing_414 +COMPILE_OPT idl2 +envi, /restore_base_save_files + +; Code here +``` + +### Linear and Kernel Nonlinear Spectral Mixture Model Unmixing IDL Code +#### Step 10 +```idl +pro test_20160314_KFCLS +COMPILE_OPT idl2 +envi, /restore_base_save_files + +; Additional code here +``` + +_endofoutput_ +``` \ No newline at end of file diff --git a/markdown-output/nowaware-rapid-enzyme-activity-detection-read-of-s-bki8kuhw.md b/markdown-output/nowaware-rapid-enzyme-activity-detection-read-of-s-bki8kuhw.md new file mode 100644 index 0000000000000000000000000000000000000000..9ae2e71d5c27202943c1b5c0a1edf29d6f15c129 --- /dev/null +++ b/markdown-output/nowaware-rapid-enzyme-activity-detection-read-of-s-bki8kuhw.md @@ -0,0 +1,151 @@ +```markdown +# Goal/Experiment: +This protocol describes the step-by-step procedure to perform the Rapid Enzyme Activity Detection (REaD) test for SARS-CoV-2 detection from NowAware, Inc. + +# NowAware: Rapid Enzyme Activity Detection (REaD) of SARS-CoV-2 + +**Published: September 01, 2020** + +**Author:** Bambu Vault + +**DOI:** [10.17504/protocols.io.bki8kuhw](https://dx.doi.org/10.17504/protocols.io.bki8kuhw) + +**Coronavirus Method Development Community** + +## Abstract +This protocol describes the step-by-step procedure to perform the Rapid Enzyme Activity Detection (REaD) test for SARS-CoV-2 detection from NowAware, Inc. + +## Keywords +Microplate Reader, Enzyme-Substrate, Kinetics, Fluorescence Assay, Mobile Reader (fluorescence emission), Biochemical Reaction, SARS-CoV-2, Detection, Diagnostic, Treatment Monitoring + +## License +This is an open access protocol distributed under the terms of the [Creative Commons Attribution License](https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. + +## Creation and Modification Dates +- **Created:** August 31, 2020 +- **Last Modified:** September 01, 2020 + +## Guidelines +It is the responsibility of the person performing the test to follow this protocol as described. The authors do not take any responsibility for any errors or misinterpretations that may occur if the protocol is not strictly adhered to as described. + +## Materials + +| **Name** | **Catalog #** | **Vendor** | +|---------------------------------------------|----------------------|---------------------| +| Bovine Serum Albumin (BSA), Fraction V, Protease Free | A-420 | Gold Biotechnology | +| DMSO, ACS Grade | D-360 | Gold Biotechnology | +| DTT (Dithiothreitol) (> 99% pure) Protease Free | DTT | Gold Biotechnology | +| HEPES, Free Acid | H-400 | Gold Biotechnology | +| Sodium chloride | S7653 | Sigma Aldrich | +| PLpro Enzyme from SARS-CoV-2 | E-611-050 | R&D Systems | +| Z-RLRGG-AMC | 4027158.01 | Bachem | + +*Consumables like pipettes, tips, 96-well microplates, etc. are not included. These are standard in any biochemistry/biology/biotechnology lab.* + +### Equipment + +| **Name** | **Catalog #** | **Vendor** | +|-----------------------------|-----------------|---------------| +| F Nano+ Dual-mode Microplate Reader | TEC006430I | Tecan | + +**Safety Warnings** +1. Handle all biochemicals as per the CDC’s Biosafety Level 2 guidelines and protocols. +2. For sample collection, patients should be advised not to eat, drink, chew gum, brush their teeth or tongue, or use any mouthwash at least 30 minutes before specimen collection. +3. Negative results do not rule out SARS-CoV-2 infection, particularly for patients who have been in contact with known infected persons or in areas with a high prevalence of active infection. + +## Specimen Collection and Preparation + +1. **Assuring Testing Components** + - Assure the following testing components for pREaD test are located at the testing lab and are consistent with the specifications and storage guidelines set out in this protocol. + +**Specimen Container** +- Suitable Material: Polypropylene, High Impact Polystyrene (HIPS) +- Dimensions: + - Length: ~1.75" to 3" + - Outer Diameter: 5/8th of an inch + - Inner Diameter: 3/8th of an inch + +**Lysis Buffer** +- This buffer is used to lyse the cells and viruses in the clinical sample allowing access to the enzyme. It also deactivates the virus by destroying its lipid envelope and is no longer hazardous. The Lysis Buffer is stable at room temperature for two weeks and at 4°C for over six weeks. + +**Precise Make-Up of Lysis Buffer** +- pH 7.5 +- 50 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) +- 150 mM NaCl +- 0.1 mg/mL Bovine Serum Albumin (BSA) +- 2.5 mM Dithiothreitol (DTT) – reducing agent +- 0.5% Triton X-100 in distilled water + +**Peptide Stock** +- Fresh peptide stock can be made by taking an aliquot of peptide powder and dissolving it in the dissolution buffer before testing begins. The peptide stock solution is 5 mg/mL and 1 mL of this stock solution is sufficient for an entire 96-well plate. + +**Precise Make-Up of Peptide Stock** +- 50 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) +- 10% dimethyl sulfoxide (DMSO) + +**Isohelix Buccal Swab** +- Used to scrape infected cells off the tongue and transfer into the collection tube. +- Specifications: + - Optimal Cellular Material Pickup: 100-125 mg + - Optimal Volume of sample picked: up-to 0.1 mL + - Overall Length: approximately 6-6.5" + - Length of Sample Collection Tool intended to fit into sample collection tube: 1.5" + - Thickness of Shaft: 1/8th of an inch + - Max Thickness of Brush or Head: 0.25" + +## Sample Shipping and Storage +1. Samples can be stored at 2-8°C for up to 72 hours after collection. Do not freeze the specimen. Do not expose it to high temperatures (>75 °F). +2. Samples will then be sent to be processed using either the plate reader (pREaD) or mREaD/iREaD. Appropriate temperature should be maintained for the samples during transportation. + +## Plate Reader (pREaD) + +1. All measurement and analytical functions are performed by a micro-well plate reader. Create a plate layout in the plate reader to map sample locations. +2. Take out a new 96-well black plate (Corning Falcon Black plates) with a clear bottom. +3. Populate control wells in the plate with four prescribed controls – two internal controls and two external controls: + - **Internal Controls:** + - The substrate alone in lysis buffer with no enzyme added (“Negative Control”). + - The equivalent amount of the substrate with 10 nM of purified PLPro enzyme (“Positive Control”). + - **External Controls:** + - A previously tested known positive (by PCR) clinical sample with equivalent substrate amount added. + - A previously tested known negative (by PCR) clinical sample with the same amount of substrate added. +4. Open samples one at a time and gently homogenize the samples by pipetting up and down (without creating bubbles). +5. From the mixed sample, pipette 90 µL from the bottom of the vial. Pipette out 90 µL into an empty well according to the plate layout. One 96-well plate can have as many as 92 tests. +6. Pipette 10 µL of the substrate from the peptide stock (5 mg/mL stock) into each well and gently mix with the sample without creating bubbles. +7. Using a preset protocol for the plate reader, the 20-minute kinetics and fluorescence end-point (395 nm excitation with 440 nm emission) after 20 minutes has to be recorded. Mean Velocity is calculated automatically by the plate reader software at the end of a 20-minute kinetic run. +8. Load and run the plate through the plate reader. At the end of the run, take it out and cover it. +9. **Verify on the plate reader:** + - The negative control should have a fluorescence intensity under 200 RFU + - The positive control should have a fluorescence intensity above 600 RFU + - The negative clinical sample control should have a fluorescence intensity under 300 RFU + - The positive clinical sample control should have a fluorescence intensity over 400 RFU +10. Positive and negative controls should be tested to ensure proper performance of the assay under various circumstances as described in the protocol. + +## Plate Reader (pREaD) Data Analysis + +1. Download results from the plate reader using plate reader software or LIMS. +2. Using Microsoft Excel or equivalent software, open the result file from the plate reader and calculate the average relative fluorescence units (RFU) for all patient samples and controls. +3. Calculate the RFU ratio by dividing the average RFU for the sample with that of the negative (no enzyme, substrate only) control. +4. Based on the ratio of RFU for the sample concerning the negative control, interpret patient sample results. + +**Interpretation of Results** +- If average sample RFU is > 500 RFU and ratio of sample RFU/negative control RFU is > 1.2, the sample is considered positive. +- If average sample RFU is < 400 RFU and ratio of sample RFU/negative control RFU is < 1.15, the sample is considered negative. +- If average sample RFU and ratio of sample RFU/negative control RFU lies in between these values, please re-run the sample to confirm the status of the result. If the result is repeated, the sample is considered indeterminate. Get another sample from the patient and repeat the test. +- Mean V is >1 RFU/min patient is considered positive for SARS-CoV-2. +- Mean V is <1 RFU/min patient is considered negative for SARS-CoV-2. + +## Mobile Read (mREaD) + +1. The sample collection and storage/shipping steps are identical. mREaD uses a custom device to read the fluorescence emission. +2. 180 µL of patient sample is combined with 20 µL of a 5 mg/mL substrate in 50 mM pH 7.5 HEPES buffer with 10% DMSO inside a Brandtech ultra-micro cuvette and gently shaken to ensure components were well mixed. +3. Exactly 20 minutes after substrate addition, measure the cuvette containing the sample and the substrate in the mREaD device. +4. The fluorescence values are compared to a standard that represents the minimum fluorescence shown in a COVID-19 positive sample. If the sample being tested shows equivalent or greater fluorescence than the standard, it is a positive result; if the fluorescence is lower than the standard, it is a negative result. + +## Biowaste Handling + +1. **Sample/Waste Handling:** Lysis buffer contains Triton X-100, which is a non-ionic surfactant that destroys lipid membranes and completely deactivates the virus. +2. CDC has approved this work under BSL-2 certification. +3. All waste generated during the pREaD or mREaD/iREaD test can be put into a 10% bleach solution, which is then left for 30 minutes and discarded as any biowaste generated under BSL-2 certification. + +_endofoutput_ +``` \ No newline at end of file diff --git a/markdown-output/npop-bwxzpfp6.md b/markdown-output/npop-bwxzpfp6.md new file mode 100644 index 0000000000000000000000000000000000000000..3fedaf129622951322fad082eb3fcf667d9b8f6c --- /dev/null +++ b/markdown-output/npop-bwxzpfp6.md @@ -0,0 +1,145 @@ +```markdown +# Goal/Experiment: +This protocol outlines the methodology for preparing single cells for mass spectrometry analysis using the nPOP system. The experiment utilizes the CellenONE system for liquid handling and cell sorting. + +# nPOP V.2 + +**Authors:** +Andrew Leduc, Nikolai Slavov, Richard Huffman +Northeastern University + +**Published:** +July 29, 2021 + +**Protocol Integer ID:** +51929 + +## Abstract +Protocol for preparing single cells for mass-spec analysis by nPOP using the CellenONE system liquid handling and cell sorting system as described by Leduc et al. DOI. + +## License +This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. + +# Protocol + +## Carrier and Reference Preparation + +1. Prepare cell pellets of all relevant cell types of at least 500,000 cells. + - Add 100% DMSO to cells to a cellular concentration of 6000 cells/µL. + - Incubate cells in DMSO for 20 minutes to lyse cells. + - Add mass spectrometry grade water to bring the solution to 2000 cells/µL. + +2. Add: + - 1x benzonase + - 100mM TEAB + - 20 ng/µL of trypsin + - Digest sample at 37°C overnight. + +3. Label the carrier with 126C and reference with 127N. Dilute the sample to 200 cells/µL of carrier and 5 cells/µL of reference. + +## Preparing CellenONE + +4. Download the required field files and upload them to your CellenONE. + - Options available: Preparing 572 or 1584 single cells using TMT 16-plex multiplexing reagents. + - If using a different number of multiplexing reagents or performing label-free analysis, use the available file as a guide and design your own droplet layouts. + +5. Follow startup procedure for CellenONE X1 with two nozzles attached. + - In the far left position, attach a size medium CellenONE PDC nozzle. + - In the adjacent slot to the right, attach a size medium PDC 70 nozzle with type 2 coating. + +6. In the target spotting location: + - Place the glass slide holder with four fluorocarbon coated glass slides (CellenONE H1 Slides). + - In the probe location, place any desired 384-well plate for storing and pickup of cell suspensions and reagents used in sample preparation. + +## Preparing Cell Suspension + +7. Suspend cells in 1X PBS at a concentration of 200-300 cells/µL. + - If cells are prone to clumping, filter first with a 30-micron filter to ensure fast pace of cell sorting. + +## DMSO for Cell Lysis + +8. Load the DMSO_Lysis field file and ensure that droplet volumes are 5 nL. + - On the main page, set the task to SpotRun_ImageFields and dispense DMSO. + - After droplets are imaged and fields are dispensed, check images to ensure DMSO droplets were dispensed consistently for all arrays. + +9. Load 20 µL of 100% mass spectrometry grade DMSO into any desired well in the 384-well plate. + - Aspirate 10 µL of DMSO with the PDC 70 type 2. + - Test droplet 3 times to ensure a straight and stable stream. + +10. Dispense droplets for DMSO lysis. + - If the volume of dispensed droplets exceeds 7 µL, dispense half of the total spots, flush the tip, and repeat step 7. + +## Cell Dispensing + +11. Load the Cells field file and ensure spots correspond to one droplet. + - If dispensing multiple separate cell suspensions, remove a portion of spots from each cluster of 12 to ensure every set will contain all relevant cell types. + +12. On the main tab, set the run program to CellenONE_Basic. + - Aspirate 10 µL of your cell suspension from the 384-well plate using the CellenONE PDC. + +13. Dispense the first cell type. + - Repeat steps 11 and 12 for additional cell types. + +## Evaporation Control + +14. After cell dispensing, set humidity control to 75% relative humidity and set cooling control to dew point chase with a deviation of 0 degrees. + +15. Load the perimeter field file. + - Dispense a perimeter of system water to control local evaporation. + +## Digestion + +16. Prepare master mix solution: + - Mix 20 µL of Promega Trypsin Gold at 200 ng/µL with 1 mM HEPES buffer at pH 8.5. + +17. Aspirate 20 µL of master mix with the PDC type 2. + - Dispense 10 nL to each spot. + +18. Let single cell digestion proceed for 4 hours. + - Refresh the perimeter field every 30-45 minutes during digestion to prevent single-cell evaporation. + +19. After digestion, turn off humidity and cooling and let single cell peptides dry out on the slide. + - This is a pause point: labeling can proceed the next day as peptides rest dried out on the slide. + +## Prepare Inserts for Storing Sample + +20. Pipette 200 cells' worth of combined carrier and reference into the number of mass spectrometry insert vials of planned sets being prepared. + +## Labeling + +21. If multiplexing reagents are stored in Acetonitrile: + - Evaporate off acetonitrile completely in a speed vacuum or lyophilizer. + - Resuspend labels in 100% DMSO at a concentration of 1/3 maximum strength. + +22. Load all labels into the 384 well plate and place securely inside CellenONE. + +23. Load the field file for the first label. + - Set the run method to Spot Run. + - Aspirate 10 µL of the first label and dispense 30 nL for the corresponding spot in each field. + - Flush the remaining label after dispensing. Ensure no residual DMSO on the tip of the nozzle and brush with a KimWipe to remove any hanging droplets. + +24. Repeat step 23 for all subsequent labels and let samples label one hour after the last label is dispensed. + +## Quenching Labeling Reaction + +25. Turn on humidity and cooling to previous settings (Step 14). + +26. Prepare a solution of 5% hydroxylamine (HA) diluted with mass spectrometry grade water. + - Add 25 nL of HA to each droplet and incubate for 20 minutes. + +27. Repeat step 26. + +## Sample Collection + +28. Aspirate 100 µL of mass spectrometry grade water. + - Dispense 2 µL into the center of each array. + - Increase frequency parameter for nozzle to 1000 to speed up dispensing. + +29. Set a 0.5-10 µL pipette tip to 3 µL and pick up each pooled set one by one, dispensing into its own insert vial. + - Wash each spot with ACN to pick up any residual droplets. + +30. Dry down each sample in a speed vacuum for 30 minutes to remove any residual DMSO. + - Store in -80°C until samples are ready to be run on LC/MS. + +@endofoutput +``` \ No newline at end of file diff --git a/markdown-output/nuclei-extraction-for-10x-genomics-single-cell-mul-b8wmrxc6.md b/markdown-output/nuclei-extraction-for-10x-genomics-single-cell-mul-b8wmrxc6.md new file mode 100644 index 0000000000000000000000000000000000000000..397b3f55c3ef9404a652622571352ba26ca7b711 --- /dev/null +++ b/markdown-output/nuclei-extraction-for-10x-genomics-single-cell-mul-b8wmrxc6.md @@ -0,0 +1,121 @@ +```markdown +# Goal/Experiment: + +The goal of this experiment is to extract nuclei from snap-frozen tissue using the Singulator device. The extracted nuclei are to be used for 10x Genomics Single Cell Multiome ATAC (Assay for Transposase-Accessible Chromatin) and gene expression analysis. + +# Nuclei Extraction for 10x Genomics Single Cell Multiome ATAC + Gene Expression from Frozen Tissue Using Singulator™ 100 or 200 (S2 Genomics) V2.0 + +## Abstract + +This protocol describes nuclei extraction from snap-frozen tissue using Singulator, including washing nuclei suspension in buffer supplemented with sucrose, and nuclei FACS sorting. The resulting nuclei suspension is suitable for Single Cell Multiome ATAC + Gene Expression using 10x Chromium platform. The protocol has been validated on various healthy and cancer snap-frozen human and mouse tissues (lung, brain, prostate, core needle biopsies, etc). + +### Guidelines + +- Make sure to use swinging bucket centrifuges to spin nuclei. + +## Materials + +| Reagent | Vendor | Catalog Number | +|---------|--------|----------------| +| DTT (Dithiothreitol) | Merck MilliporeSigma (Sigma-Aldrich) | #D0632 | +| DAPI (4,6-Diamidino-2-Phenylindole, Dihydrochloride) | Thermo Fisher Scientific | #D1306 | +| Trizma Hydrochloride Solution | Merck MilliporeSigma (Sigma-Aldrich) | #T2194-100ML | +| Sodium Chloride Solution, 5M | Merck MilliporeSigma (Sigma-Aldrich) | #59222C | +| Magnesium Chloride Solution for Molecular Biology, 1M | Merck MilliporeSigma (Sigma-Aldrich) | #M1028 | +| Digitonin (5%) | Thermo Fisher | #BN2006 | +| Albumin, Bovine Serum, 10%, Nuclease-Free | Merck MilliporeSigma (Sigma-Aldrich) | #126615-25ML | +| 7-AAD (7-Aminoactinomycin D) | Thermo Fisher | #A1310 | +| Protector RNase Inhibitor | Merck MilliporeSigma (Sigma-Aldrich) | #3335402001 | + +### Preparation Steps + +#### Step 1: Initial Setup +1. **Turn on and pre-cool Singulator** (10 minutes) + - Ensure there are a sufficient number of nuclei cartridges or NIC+ cartridges for samples (<20mg) stored/pre-cooled at 4°C. + +#### Step 2: Tissue Handling +2. **Take the tissue out of LN2 or -80°C freezer** and place it on dry ice to keep it frozen (10 minutes). Ideally, each piece should be <100mg (cut into smaller pieces if larger). + + - **Cutting**: + - Use a razor blade within a Petri dish on dry ice. + - Hold the tissue with cooled and RNAse-free tweezers. + - Place the tissue back in a tube on dry ice immediately after cutting. + +#### Step 3: Buffer Preparation +3. **Prepare 2mL of Nuclei Wash Buffer per sample** (5 minutes): + +| Component | Final Concentration | +|-----------|---------------------| +| Tris-HCl (pH 7.4) | 10mM | +| NaCl | 10mM | +| MgCl₂ | 3mM | +| Purified BSA | 1% | +| DTT | 1mM | +| RNase Inhibitor | 0.2 - 1.0 U/µL | + + Top off to 2mL with Ambion Nuclease-free water. + + Reference: [Ambion Tech Notes](https://www.thermofisher.com/us/en/home/references/ambion-tech-support/nuclease-enzymes/tech-notes/rnase-activity-in-mouse-tissue.html) + +#### Step 4: Singulator Usage +4. **Add the following to the Nuclei Isolation Cartridge**, then add tissue: + - RNase inhibitor (0.2-1.0 U/µL final, volume ~3.5mL). + - DTT, 1mM final concentration (3.5µL of 1M DTT per cartridge). + +#### Step 5: Nuclei Suspension Transfer +5. **Transfer nuclei suspension** (~3.5mL) to 5mL Protein LoBind Eppendorf tubes (or split evenly into four 1.5mL LoBind tubes if desired). + +#### Step 6: Sucrose Addition +6. **Add 500µL of 2M Sucrose solution** to the tubes (250mM final conc.). Adjust volume if using 1.5mL tubes. + +7. **Invert tubes** at least 10 times to mix (do not vortex!). + +#### Step 7: Centrifugation +8. **Centrifuge at 500 x g, 4°C for 5 minutes** using a swinging bucket centrifuge. + +#### Step 8: Nuclei Wash +9. **Aspirate supernatant** carefully to avoid disturbing the nuclei pellet (leave ~50µL residual). + - **Re-suspend nuclei pellet** in ~400µL Nuclei Wash Buffer. +10. **Optional**: + - Perform a second wash with the Nuclei Wash Buffer to remove traces of sucrose. + - Perform a second wash with 250mM sucrose if high debris is present. + +#### Step 9: Filtration +11. **Filter nuclei suspension** through a 35µm FACS tube cap filter (Miltenyi). + +#### Step 10: Counting +12. **Count nuclei using DAPI** on an automated cell counter. + - Optional: Use Trypan for brightfield counting, DAPI is more accurate. + +### Final Steps: FACS and 10x Genomics + +#### Nuclei Staining +13. **Stain nuclei with 7-AAD** (7-Aminoactinomycin D) prior to FACS sorting (30 minutes). + +#### Post-Sorting Concentration +14. **Centrifuge sorted nuclei at 500 x g, 4°C for 5 minutes**, then resuspend in Nuclei Wash Buffer. Aim for >=1000 nuclei/µL. + + - **Count nuclei using DAPI** to confirm concentration. + - **Aliquot 200k nuclei in 0.2mL PCR tube**. + +15. **Continue to 10x Genomics Protocol** (CG000365), following "Low Cell Input Nuclei Isolation". + +### RNase Activity in Mouse Tissue + +| Tissue Type | Fold Increase Relative to Brain | +|-------------|-------------------------------| +| Brain | 1 | +| Heart | 10 | +| Kidney | 100 | +| Thymus | 1000 | +| Liver | 10000 | +| Lung | 100000 | +| Spleen | 1000000 | +| Pancreas | 10000000 | + +## References + +- [Protocol DOI](https://dx.doi.org/10.17504/protocols.io.j8nlkkmq6l5r/v1) + +*endofoutput* +``` \ No newline at end of file diff --git a/markdown-output/nuclei-extraction-for-tissue-using-iodixanol-gradi-chjdt4i6.md b/markdown-output/nuclei-extraction-for-tissue-using-iodixanol-gradi-chjdt4i6.md new file mode 100644 index 0000000000000000000000000000000000000000..b90b609f4aea3c278889893549ee35620b9a0439 --- /dev/null +++ b/markdown-output/nuclei-extraction-for-tissue-using-iodixanol-gradi-chjdt4i6.md @@ -0,0 +1,122 @@ +```markdown +Goal/Experiment: +Isolation of nuclei from tissue using iodixanol gradients, geared for multiome analysis. + +# Nuclei Extraction for Tissue Using Iodixanol Gradients V.1 + +**Kriegstein Lab** +*UCSF* + +**Version 1** +**Date:** October 24, 2022 +**Last Modified:** October 24, 2022 +**Protocol Citation:** +Kriegstein lab 2022. Nuclei Extraction for tissue using Iodixanol Gradients. protocols.io +DOI: [10.17504/protocols.io.81wgby7yqvpk/v1](https://dx.doi.org/10.17504/protocols.io.81wgby7yqvpk/v1) + +**Abstract:** +Nuclei isolation using iodixanol gradients, specifically designed for multiome analysis. + +--- + +## Creating Buffers + +### 1. Stock Buffers +* All stock solutions should be filtered using a 0.22 µm PVDF/PES filter system. +* All solutions except the 50% iodixanol solution are stable at 4°C for at least 6 months. + +#### 1 M Sucrose (300 mL) +| Substance | Stock Conc. | Amount | Final Conc. in Working Solution | +|------------|-------------|----------|---------------------------------| +| Sucrose | - | 102.69 g | 1 M | +| Water | - | 235.5 mL | - | + +#### 1.0616x Homogenization Buffer Stable Solution (200 mL) +| Substance | Stock Conc. | Amount | Final Conc. in Working Solution | +|--------------------|-------------|---------|---------------------------------| +| Sucrose | 1 M | 53.1 mL | 0.2653 M | +| KCl | 2 M | 2.66 mL | 26.6 mM | +| MgCl2 | 1 M | 1.06 mL | 5.31 mM | +| Tricine-KOH pH 7.8 | 0.75 M | 5.67 mL | 21.2 mM | +| Water | - | 137.5 mL| - | + +#### Diluent Buffer (100 mL) +| Substance | Stock Conc. | Amount | Final Conc. in Working Solution | +|--------------------|-------------|---------|---------------------------------| +| KCl | 2 M | 7.5 mL | 150 mM | +| MgCl2 | 1 M | 3 mL | 30 mM | +| Tricine KOH pH 7.8 | 0.75 M | 16 mL | 120 mM | +| Water | - | 73.5 mL | - | + +#### 50% Iodixanol Solution (50 mL) **Remake Monthly for Stability** +| Substance | Stock Conc. | Amount | Final Conc. in Working Solution | +|------------------|-------------|--------|---------------------------------| +| Diluent Buffer | - | 8.3 mL | - | +| Iodixanol | 60% | 41.7 mL| 50% | + +#### 1x Homogenization Buffer Unstable Solution for 4 Reactions **Prepare Fresh** +| Substance | Stock Conc. | Amount | Final Conc. in Working Solution | +|--------------------|-------------|---------|---------------------------------| +| HB Stable Solution | 1.0616X | 7536 µL | 1X | +| DTT | 1 M | 8 µL | 1 mM | +| Spermidine | 500 mM | 8 µL | 0.5 mM | +| Spermine | 150 mM | 8 µL | 0.15 mM | +| NP40 | 10% | 240 µL | 0.3% | +| cOmplete PI | 100X | 80 µL | 1X | +| RiboLock | 40 U/µL | 120 µL | 0.6 U/µL | + +#### 30% Iodixanol Solution per Reaction **Prepare Fresh** +| Substance | Stock Conc. | Amount | Final Conc. in Working Solution | +|-------------------------|-------------|---------|---------------------------------| +| HB Unstable Solution | - | 240 µL | - | +| 50% Iodixanol Solution | 50% | 360 µL | 30% | + +#### 40% Iodixanol Solution per Reaction **Prepare Fresh** +| Substance | Stock Conc. | Amount | Final Conc. in Working Solution | +|-------------------------|-------------|---------|---------------------------------| +| HB Unstable Solution | - | 120 µL | - | +| 50% Iodixanol Solution | 50% | 480 µL | 40% | + +#### Wash Buffer 1 mL for 4 Reactions **Prepare Fresh** +| Substance | Stock Conc. | Amount | Final Conc. in Working Solution | +|------------------|-------------|--------|---------------------------------| +| Tris-HCl pH 7.4 | 1 M | 10 µL | 10 mM | +| NaCl | 5 M | 2 µL | 10 mM | +| MgCl2 | 1 M | 3 µL | 3 mM | +| BSA | 30% | 33.3 µL| 1% | +| Tween-20 | 10% | 10 µL | 0.1% | +| DTT | 1 M | 1 µL | 1 mM | +| RiboLock | 40 U/µL | 15 µL | 0.6 U/µL | + +--- + +## 2. Before Starting the Protocol +1. Pre-chill swinging bucket centrifuge and a fixed angle centrifuge to 4°C. +2. Pre-chill dounces and pestles to 4°C on ice. +3. Pre-chill tubes. +4. Fill up a 2 L beaker with 500 mL sterile water to soak the used Dounces. + +## 3. Isolation of Nuclei via Dounce Homogenization and Density Centrifugation +1. **Place** 20-50 mg frozen tissue or crushed into pre-chilled 7 mL dounce containing 1 mL cold 1x HB. +2. **Dounce** with "A" loose pestle until resistance goes away (~10 strokes). +3. **Dounce** with "B" tight pestle until resistance goes away (~15 strokes). +4. **Place "A" and "B"** into sterile water to soak for cleaning later. +5. **Filter** during transfer into FACS tube. +6. **Place** Dounce into beaker with sterile water to soak for cleaning later. +7. **Pellet** nuclei by spinning 5 min at 4°C at 350 xg in a fixed angle centrifuge. +8. **Remove** 950 µL of supernatant (50 µL remaining). +9. **Gently resuspend** nuclei in 350 µL 1x HB. +10. **Add** 1 volume (400 µL) of 50% iodixanol solution and pipette mix. +11. **Slowly layer** 600 µL of 30% iodixanol solution under the 25% mixture. Wipe side of pipette tip with kimwipe to avoid mixing layers. +12. **Layer** 600 µL 40% iodixanol solution under the 30% mixture. Wipe side of pipette tip with kimwipe to avoid mixing layers. +13. In a pre-chilled swinging bucket centrifuge, spin for 20 min at 4°C at 3000 xg with the brake off. Set acceleration level to 1 and deceleration at 0. (centrifuge time=23 min, time to stop=13 min) +14. **Slowly extract** top layers in increments of 200 µL down to 200-300 µL of nuclei band between 30% and 40% interface. +15. **Take** 200 µL of the nuclei band and put into 1.5 mL LoBind tube. +16. **Dilute** nuclei by adding 200 µL of wash buffer and mix by pipetting. +17. **Count** nuclei using trypan blue. +18. **Centrifuge** at 500 xg for 5 min at 4°C. +19. **Remove** supernatant without disrupting pellet. +20. Resuspend in \( X µL \) of chilled Nuclei Buffer (depending on what isolated nuclei are used for) to achieve the target concentration based on count. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/nuclei-isolation-and-affinity-purification-for-10x-cxxxxppn.md b/markdown-output/nuclei-isolation-and-affinity-purification-for-10x-cxxxxppn.md new file mode 100644 index 0000000000000000000000000000000000000000..000d6048ce7f65c2ac92f14f31ef183f3ef3a157 --- /dev/null +++ b/markdown-output/nuclei-isolation-and-affinity-purification-for-10x-cxxxxppn.md @@ -0,0 +1,132 @@ +```markdown +# Goal/Experiment: +This protocol is for isolating nuclei for downstream sequencing applications. + +# Nuclei Isolation and Affinity Purification for 10X Sequencing + +**Lakme Caceres1** +1Princeton Neuroscience Institute + +**Abstract** +This protocol is for isolating nuclei for downstream sequencing applications. + +**Guidelines** +- Keep tissue/nuclei on ice as much as possible. + +## Materials +- Dynabeads - 4°C +- anti-GFP - 4°C (Roche) +- Triton X-100 - 4°C +- DTT - 4°C +- RNAsin -20°C + +## Prepare Stock Solutions + +1. **10% BSA (Bovine Serum Albumin)** + Make 20 mL 10% BSA by combining 2 mL of BSA with 18 mL of MilliQ water in a 50 mL falcon tube. + *Storage: 4°C - 2 weeks* + +2. **10% Triton X-100** + Make 20 mL 10% Triton X-100 by combining 18 mL MilliQ water with 2 mL Triton X-100 in a 50 mL tube. Vortex and then incubate at room temperature for 20 minutes. Filter it through a 0.22 μm filter with a syringe into a clean 50 mL tube. + *Storage: 4°C - 1 month* + +3. **Nuclear Isolation Media** + Make 250 mL Nuclear Isolation Media by filling a 250 mL bottle with 200 mL of MilliQ water and then adding: + - 2.5 mL 1M Tris + - 6.26 mL 1M KCl + - 1.25 mL 1M MgCl2 + - 21.45 g Sucrose + Shake until sucrose is dissolved then fill to 250 mL with MilliQ water. + *Storage: 4°C - 2 weeks* + +4. **Citrate-Phosphate Buffer (pH 5)** + Make 50 mL Citrate-Phosphate Buffer by adding 0.48 g Citric Acid and 0.91 g Dibasic Sodium Phosphate Dihydrate to a falcon tube and then filling it to 50 mL with MilliQ water. Titrate the pH with NaOH using a pH meter. Calibrate the pH meter before titrating by dipping in pH 3, 7, and 10. Ensure the pH meter is washed with water between each measurement. + *Storage: 4°C - 1 month* + +## Immunoprecipitation Prep + +5. Vortex stock of Dynabeads Protein G and then add 5 μL to a 1.5 mL eppendorf. Place it on the mag rack for a minute and discard the supernatant remaining at the bottom of the tube without disturbing the beads. + +6. Remove the tube from the magnet and resuspend the beads with 500 μL of Citrate-Phosphate Buffer. Place the tube back on the mag rack for a minute and discard the supernatant. Repeat this step once more. + +7. Add 100 μL of 1X PBS and 2 μL anti-GFP to the washed Dynabeads. Add this to the rotator in the 4°C fridge and let it incubate for 1 hour. Proceed with nuclei isolation while the beads incubate. + +## Prepare Fresh Solutions + +8. **Homogenization Buffer** + Make 3 mL Homogenization Buffer per sample by adding 2.9 mL Nuclear Isolation Media (filtered via syringe) to a 5 mL eppendorf. Then add: + - 3 μL 100 mM DTT + - 30 μL 10% Triton X-100 + - 15 μL RNAsin + Invert to mix. Store on ice. + +9. **Blocking Buffer** + Make 200 μL Blocking Buffer per sample by dividing your total desired volume of blocking buffer by 10 to get the amount of 10% BSA in μL. Add this amount to a tube and then fill the remainder with 1X PBS. + +## Homogenization + +10. Clean dounce, scalpel, and forceps using MilliQ water, ethanol, RNase Zap, then MilliQ again. The red-tape forceps are for unfixed tissue. + +11. Get tissue sample from -80°C freezer and place on dry ice. Weigh it on a sterile, tared weigh boat. + +12. Add tissue to dounce and push it to the bottom using 1 mL of Homogenization Buffer and the pestle. Homogenize the tissue without creating bubbles. Then add the remaining 2 mL of the Homogenization Buffer and continue to dounce until homogenized. + +13. Pass all of the nuclei suspension through three FlowMi filters, 1000 μL at a time into a new 5 mL eppendorf. + +14. Centrifuge at 900 g/rcf for 10 minutes at 4°C. + +15. Discard the supernatant and resuspend the pellet in 210 μL Blocking Buffer. Incubate for 10 minutes on ice. + +## Cell Count + +16. Add 9 μL of sample to a PCR tube and then add 1 μL of acridine orange. + - If sample is very concentrated, instead add 2 μL sample to 2 μL of acridine orange and 16 μL 1X PBS. + +17. Pipette mix and then add the total volume to a three-chamber cell counting chip and make note of the channels used (A, B, and/or C). + +18. On the cell counter, select Fluorescence Cell Counting -> Cell Lines & Primary Cells, Advanced-> Protocol -> and then choose "NUCLEI" from the list of protocols. Load the protocol. + - If the sample is very concentrated and you are adding 2 μL, select Fluorescence Cell Counting -> Cell Lines & Primary Cells-> Protocol -> and then choose "NUCLEI" from the protocol list. Load the protocol. + - Go to Settings and choose the appropriate number of channels. + - Hit "Count" and then "Start Count." + +19. When the cell count is complete, you will get a reading in cells/mL. Convert this to cells/μL by dividing this number by 1,000. + +20. Save approximately 40,000 nuclei from the original sample to use as our unpurified population for sequencing. + - (If the concentration was 1,000 cells/μL, then save 40 μL). + +21. If there are channels left on the cell counter chip, mark the used channels on the back and place it back in the drawer for future use. + +## Immunoprecipitation + +22. After the one-hour incubation of the beads and the aliquoting of nuclei for the unpurified population, place the incubated dynabeads tube on the 1.5 mL magnet for 2 minutes and discard the supernatant without disturbing the beads. + +23. Wash with 100 μL 1X PBS Buffer, enough to submerge the beads while they are on the rack. Discard the supernatant. + +24. Add the total volume of nuclei sample to the beads. Incubate on the rotator in the 4°C fridge for 1 hour. + +25. After incubation, place the samples on the mag rack for 2 minutes. Collect the supernatant in appropriately labeled eppendorf tubes. This will be our supernatant population for sequencing. + +26. Wash the bead tubes with enough 1X PBS to submerge the beads (don't resuspend) and then discard the supernatant. + +27. Remove the tubes from the rack and then resuspend the beads in 50 μL 1X PBS. + +## Cell Count + +28. Take another cell count of the supernatant and bead-bound samples. Reverse pipette to record their exact volumes. + +29. Dilute all of the samples (unpurified, supernatant, and bead-bound), if necessary, to 1,000 cells/μL. The bead-bound population likely won't need dilution. + +30. Proceed with 10X RNAseq or ATACseq protocols. + +--- + +**Protocol Citation:** +Lakme Caceres. Nuclei Isolation and Affinity Purification for 10X Sequencing. *protocols.io*. 2023. [https://dx.doi.org/10.17504/protocols.io.4r3l22ojlp1y/v1](https://dx.doi.org/10.17504/protocols.io.4r3l22ojlp1y/v1) + +**License:** +This is an open access protocol distributed under the terms of the [Creative Commons Attribution License](https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. + +--- + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/nuclei-isolation-and-immunoprecipitation-for-10x-s-cwu3xeyn.md b/markdown-output/nuclei-isolation-and-immunoprecipitation-for-10x-s-cwu3xeyn.md new file mode 100644 index 0000000000000000000000000000000000000000..e1817e0132e70315c7f062d13df72c64bb48c3e3 --- /dev/null +++ b/markdown-output/nuclei-isolation-and-immunoprecipitation-for-10x-s-cwu3xeyn.md @@ -0,0 +1,165 @@ +```markdown +# Goal/Experiment: +This protocol is for isolating nuclei for downstream sequencing applications. + +# Nuclei Isolation and Immunoprecipitation for 10X Sequencing V.2 + +**Lakme Caceres1** +1Princeton Neuroscience Institute + +**ABSTRACT** +This protocol is for isolating nuclei for downstream sequencing applications. + +**GUIDELINES** +- Keep tissue/nuclei on ice as much as possible. + +## MATERIALS +- Dynabeads - 4°C +- Anti-GFP - 4°C (Roche) +- Triton X-100 - 4°C +- DTT (Dithiothreitol) - 4°C +- RNasin - -20°C + +--- + +## Prepare Stock Solutions + +1. **10% BSA Solution** + - Combine 2 mL of BSA with 18 mL of MilliQ water in a 50 mL falcon tube. + - Storage: 4°C, duration: 2 weeks. + +2. **10% Triton X-100 Solution** + - Combine 18 mL MilliQ water with 2 mL Triton X-100 in a 50 mL tube. + - Vortex and incubate at room temperature for 20 minutes. + - Filter through a 0.22 µm filter with a syringe into a clean 50 mL tube. + - Storage: 4°C, duration: 1 month. + +3. **Nuclear Isolation Media** + - Fill a 250 mL bottle with 200 mL of MilliQ water, then add: + - 2.5 mL 1M Tris + - 6.26 mL 1M KCl + - 1.25 mL 1M MgCl2 + - 21.45 g Sucrose + - Shake until sucrose is dissolved, then fill to 250 mL with MilliQ water. + - Storage: 4°C, duration: 2 weeks + +4. **Citrate-Phosphate Buffer (pH 5)** + - Add 0.48 g Citric Acid and 0.91 g Dibasic Sodium Phosphate Dihydrate to a falcon tube. + - Fill to 50 mL with MilliQ water. + - Titrate the pH with NaOH using a pH meter. + - Storage: 4°C, duration: 1 month. + +--- + +## Immunoprecipitation Prep + +5. Vortex stock of Dynabeads Protein G and add 5 µL to a 1.5 mL eppendorf. + - Place on mag rack for 1 minute. + - Discard supernatant without disturbing beads. + +6. Remove tube from magnet and resuspend beads with 500 µL of Citrate Phosphate Buffer. + - Place back on mag rack for 1 minute and discard supernatant. + - Repeat this step once more. + +7. Add 100 µL of 1X PBS and 2 µL anti-GFP to washed Dynabeads. + - Add to rotator in 4°C fridge for 1 hour. + - Proceed with nuclei isolation while beads incubate. + +--- + +## Prepare Fresh Solutions + +8. **Homogenization Buffer** + - For each sample, add: + - 2.895 mL Nuclear Isolation Media (filtered via syringe) to a 5 mL eppendorf + - 3 µL 100 mM DTT + - 30 µL 10% Triton X-100 + - 15 µL RNasin + - Invert to mix and store on ice. + +9. **Blocking Buffer** + - Per sample, divide the total desired volume of blocking buffer by 10 to get the amount of 10% BSA in µL. + - Add this amount to a tube then fill with 1X PBS. + + +## Homogenization + +10. Clean dounce, scalpel, and forceps using MilliQ water, ethanol, RNase Zap, then MilliQ again. + - Red-tape forceps are for unfixed tissue. + +11. Obtain tissue sample from -80°C freezer and place on dry ice. + - Weigh on sterile, tared weigh boat. + +12. Add tissue to dounce and immerse in 1 mL of Homogenization Buffer using a pestle. + - Homogenize without creating bubbles. + - Add remaining 2 mL Homogenization Buffer and continue homogenization. + +13. Pass all nuclei suspension through three FlowMi filters, 1000 µL at a time into a new 5 mL eppendorf. + +14. Centrifuge at 900 g/rcf for 10 minutes at 4°C. + +15. Discard supernatant and resuspend pellet in 210 µL Blocking Buffer. + - Incubate for 10 minutes on ice. + +--- + +## Cell Count + +16. Add 9 µL of sample to a PCR tube, then 1 µL of acridine orange. + - If sample is highly concentrated, add 2 µL of sample to 2 µL of acridine orange and 16 µL 1X PBS. + +17. Pipette mix, then transfer total volume to a three-chamber cell counting chip. + - Note channels used (A, B, and/or C). + +18. Use cell counter: + - Select: **Fluorescence Cell Counting** -> **Cell Lines** -> **Advanced** -> **Protocol** -> **NUCLEI** + - Go to Settings and choose the number of channels. + - Hit "Count" then "Start Count." + +19. Once counting is complete, convert reading from cells/mL to cells/µL by dividing by 1,000. + +20. Save approx. 40,000 nuclei from the original sample as the unpurified population for sequencing. + - (e.g., if concentration is 1,000 cells/µL, save 40 µL). + +21. If there are any used channels left on the cell counter chip, mark and store for future use. + +--- + +## Immunoprecipitation + +22. After 1-hour incubation of Dynabeads and nuclei aliquoting: + - Place Dynabeads tube on 1.5 mL magnet for 2 minutes. + - Discard supernatant without disturbing beads. + +23. Wash with 100 µL 1X PBS buffer, submerging beads. + - Discard supernatant. + +24. Add total volume of nuclei sample to the beads. + - Incubate on rotator in 4°C fridge for 1 hour. + +25. Post incubation: + - Place on mag rack for 2 minutes. + - Collect supernatant into labeled eppendorf tubes. + - This is the supernatant population for sequencing. + +26. Wash bead tubes with enough 1X PBS to submerge the beads (don't resuspend). + - Discard supernatant. + +27. Remove tube from rack and resuspend beads in 50 µL 1X PBS. + +--- + +## Cell Count + +28. Take another cell count of both supernatant and bead-bound samples. + - Reverse pipette to record exact volumes. + +29. Dilute all samples (unpurified, supernatant, and bead-bound), if necessary, to 1,000 cells/µL. + - Bead-bound population likely won't need dilution. + +30. Proceed with 10X RNAseq or ATACseq protocols. + +--- + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/nuclei-isolation-from-frozen-tissue-cwcmxau6.md b/markdown-output/nuclei-isolation-from-frozen-tissue-cwcmxau6.md new file mode 100644 index 0000000000000000000000000000000000000000..6145358432d6fd8081b5374c52808acc1d9810cc --- /dev/null +++ b/markdown-output/nuclei-isolation-from-frozen-tissue-cwcmxau6.md @@ -0,0 +1,154 @@ +```markdown +# Goal/Experiment: +The goal of this experiment is to isolate intact nuclei from frozen tissue for further downstream methods such as single nuclei RNA-seq, ATAC-seq, or 10x Multiome. + +# Nuclei Isolation from Frozen Tissue + +**Authors:** +- Emily Stephenson1,2 +- Elena Prigmore2 +- agnes oszlanczi2 +- di zhou2 + +1Newcastle University; 2Wellcome Sanger Institute + +**Protocol status:** Working +**Created:** Jun 27, 2023 +**Last Modified:** Jun 27, 2023 +**PROTOCOL integer ID:** 84077 +**DOI:** [10.17504/protocols.io.8epv5x6wjg1b/v1](dx.doi.org/10.17504/protocols.io.8epv5x6wjg1b/v1) + +--- + +## Disclaimer +Number of homogenisation strokes will be tissue specific as well as cell lysis, this may need to be optimised per tissue. + +## Abstract +This protocol describes a method for the isolation of intact nuclei for further downstream methods such as single nuclei RNA-seq, ATAC-seq or 10x Multiome. The protocol is adapted from Nadelmann et al DOI: [10.1002/cpz1.132](10.1002/cpz1.132) + +--- + +## Materials + +- 2M KCL +- 1M MgCl₂ +- 1M Tris Buffer +- Nuclease-free water +- DTT (1mM) +- 100x Protease Inhibitor +- RNasin Plus (40 U/µL) +- Superasin (20 U/µL) +- Triton X-100 (10%) +- 1x PBS +- BSA Powder +- RNasin Protector +- Lymphoprep +- 10x PBS +- Dounce Homogeniser with Pestle A and B +- 40µM Cell Strainer +- Wide Bore Pipette Tips +- Trypan Blue +- Eppendorf Tubes +- C-chip Haemocytometer + +**Before Start Instructions:** +Tissue can be collected before starting and kept on dry ice. Frozen tissue as small as a grain of rice can be used or OCT-embedded tissue sections. + +--- + +## Prepare Reagents and Buffers + +### Prepare Nuclei Isolation Buffer 1 (NIM1) +Prepare NIM1 according to the table below. NIM1 can be made, filtered and stored at 4°C for 6 months. Record date on top of the tube. + +| Reagent | Volume/Amount | Final Conc. (mM) | +|--------------------------|---------------|------------------| +| Sucrose 342.3 g/mol | 4.279g | 250 | +| 2M KCL | 625µL | 25 | +| 1M MgCl₂ | 250µL | 5 | +| 1M Tris Buffer | 500µL | 10 | +| Nuclease-free Water | 48.625mL | - | +| **Total** | 50mL | - | + +### Prepare Nuclei Isolation Buffer 2 (NIM2) +Prepare NIM2 according to the table below. NIM2 must be made on the day of experiment and kept on ice. Approx. 5mL per sample will be needed. + +- Pre-dilute 1M DTT in 4.995mL water. +- For 100x protease inhibitor stock, dissolve 1 tablet in 500µL nuclease-free water. Stock solution can be kept at 4°C for 2 weeks or 12 weeks at -20°C (record date on top of tube). + +| Reagent | Volume | Final Conc. | +|--------------------------|-----------|-------------| +| NIM1 | 4944µL | 1x | +| 1mM DTT | 5µL | 1µM | +| 100x Protease Inhibitor | 50µL | 1x | +| **Total** | 5mL | - | + +### Prepare Homogenisation Buffer (HB) +Prepare HB according to the table below. HB must be made on the day of experiment and kept on ice. Do not vortex. Approx. 5mL per sample will be needed. + +| Reagent | Volume | Final Conc. | +|--------------------------|---------|--------------| +| NIM2 | 4850µL | 1x | +| RNasin Plus (40 U/µL) | 50µL | 0.4U/µL | +| Superasin (20 U/µL) | 50µL | 0.2U/µL | +| Triton X-100 (10%) | 50µL | 0.1% | +| **Total** | 5mL | - | + +### Prepare Wash Buffer (WB) +Prepare WB according to the table below. WB must be made on the day of experiment and kept on ice. Do not vortex. Make approx. 1mL per sample, more may be needed if performing extra washes or clean-up steps. + +| Reagent | Volume | Final Conc. | +|--------------------------|---------|--------------| +| 1x PBS | 975µL | 1x | +| BSA Powder | 0.05g | 5% | +| RNasin Protector | 25µL | 40U/µL | +| **Total** | 1mL | - | + +### Optional Step: Lymphoprep Solution +Make up lymphoprep solution according to table below if samples have a lot of debris that need to be removed. Keep on ice. + +| Reagent | Volume | Final Conc. | +|--------------------------|---------|--------------| +| Lymphoprep | 1350µL | 90% | +| 10x PBS | 150µL | 1x | +| **Total** | 1500µL | - | + +--- + +## Nuclei Isolation Procedure + +1. **Collect tissue or tissue sections on dry ice and transfer to the dounce homogeniser.** +2. **Add 3mL homogenisation buffer to the tissue.** + + - If using tissue embedded in OCT, leave for 5 mins on ice. Mix halfway and use pestle A to push OCT to the bottom of the homogeniser. If OCT still remains, keep mixing until dissolved. + +3. **On ice, gently homogenise the tissue using pestle A.** Up to 20 strokes may be needed until no more resistance is felt. +4. **Rinse pestle with 500µL homogenisation buffer.** +5. **On ice, gently homogenise the tissue using pestle B.** Up to 20 strokes may be needed until no more resistance is felt. +6. **Rinse pestle with 500µL homogenisation buffer.** +7. **Pour homogenate through a 40µm cell strainer into a 50mL Falcon tube on ice.** Rinse homogeniser with 500µL homogenisation buffer. Transfer any droplets underneath the filter with a pipette. +8. **Centrifuge the tube at 500 x g for 6 mins at 4°C.** (Acceleration setting 0, Deceleration setting 3). +9. **Carefully remove the supernatant with 1mL pipette and transfer to another tube, do not throw away.** +10. **Add 500µL wash buffer and leave for 2 mins on ice.** +11. **Resuspend nuclei with a wide-bore tip and transfer to 1.5mL Eppendorf tube.** +12. **Look at nuclei under a microscope using filtered trypan blue and C-Chip.** If there appears to be a lot of debris, either pass nuclei through a 40mM FlowMi filter or perform a clean-up (steps at the end of protocol); if not, proceed to count. +13. **Nuclei will stain blue with trypan.** Count nuclei in all four corners then use the following calculation: (count/4) x 2 (trypan dilution factor) x 10 (volume factor) = conc. (nuclei per mL). Multiply by 500 to get total nuclei. Record this number as the "unclean count". +14. **Centrifuge the tube at 500 x g for 3 mins at 4°C.** +15. **If unclean count is more than 500k nuclei, remove as much supernatant as possible without disturbing pellet and wash again with 500mL of wash buffer.** If count is less than 500k, remove supernatant and leave 10-50mL in the tube depending on count (use tube of water as a guide). Do not throw away supernatant. +16. **Resuspend pellet gently and count nuclei using filtered trypan blue and C-Chip.** May need to do a 1 in 10 dilution using the supernatant as diluent. Count nuclei in all four corners then use the following calculation: (count/4) x 2 (trypan dilution factor) x 10 (volume factor) x 10 (dilution factor) = conc. (nuclei per mL). Multiply by volume to get total nuclei. Record this number as the "clean count". +17. **Proceed with next protocol such as 10x Genomics Multiome or scRNA-seq.** + +--- + +## Optional Clean-Up to Remove Debris + +1. **Add 475mL wash buffer to 2 Eppendorf tubes (not LoBind) for each sample.** +2. **Add 22µL nuclei solution followed by 300µL 90% lymphoprep to each tube.** Mix by inverting (do not pipette mix). +3. **Centrifuge sample at 20,000 x g for 15 mins at 4°C.** +4. **Remove top 500-800mL layer and add 500mL wash buffer.** Be careful not to disrupt the nuclei 'cloud' which will hopefully be visible near the bottom of the tube, but may not be seen as a pellet. Mix by pipetting using a wide-bore tip. +5. **Proceed to step 19 in the original protocol.** + +--- + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/nuclei-prep-from-frozen-mouse-brain-tissues-with-o-b8hmrt46.md b/markdown-output/nuclei-prep-from-frozen-mouse-brain-tissues-with-o-b8hmrt46.md new file mode 100644 index 0000000000000000000000000000000000000000..1757232a140735cda0a6bc6e22d1e6e7d2914f7f --- /dev/null +++ b/markdown-output/nuclei-prep-from-frozen-mouse-brain-tissues-with-o-b8hmrt46.md @@ -0,0 +1,152 @@ +```markdown +# Goal/Experiment: +To extract and purify nuclei from frozen mouse brain tissues for single nuclei RNA sequencing (snRNA-seq). + +# Nuclei Prep from Frozen Mouse Brain Tissues with Optional Sucrose Column for single nuclei RNA-seq + +**Authors:** Gregory A Perry, Sandra L Daigle, Paul Gabriel, Diane Luo, Jessica Grassmann, William F F Flynn, Elise T Courtois, Paul Robson + +**Affiliation:** Single Cell Biology Lab, The Jackson Laboratory, USA + +**DOI:** [10.17504/protocols.io.81wgb657ylpk/v1](https://dx.doi.org/10.17504/protocols.io.81wgb657ylpk/v1) + +**Tech. Support Email:** scblservice@jax.org + +--- + +## Introduction + +This protocol has been tested on a limited number of tissues, including human and mouse brain frozen tissues. Prior to extensive use, it is essential to conduct a pilot experiment to determine optimal dissociation. + +The Standard Operating Procedure (SOP) is designed to extract nuclei from unfixed snap-frozen mouse prefrontal cortex, hippocampus, and striatum tissues (~3-15 mg). Optimization was done on mice aged 3, 6, 14, and 15 months with varied diets and genotypes. The protocol ensures minimum debris for downstream snRNAseq assays. + +It supports the processing of up to 8 samples simultaneously, minimizing debris for downstream processing with 10X Genomics’ chromium X controller: HT (High Throughput chips) and ST (Standard Throughput chips). + +## Guidelines & Warnings + +- Use wide-bore pipette tips to prevent damage during handling. +- Keep tissue on dry ice until ready to start gentleMACS. +- Follow BSL-2 guidelines when working with human tissues. + +## Nuclei Isolation + +### Materials and Reagents + +- **Miltenyi Nuclei Extraction Buffer:** (Miltenyi, Cat #: 130-128-024) +- **Miltenyi C Tubes:** (Miltenyi, Cat #: 130-093-237) +- **MACS BSA Stock Solution:** (Miltenyi, Cat #: 130-091-376) +- **Fisher Scientific DPBS:** (Thermo Fisher Scientific, Cat #: 14190144) +- **Millipore Sigma ROCHE Protector RNase Inhibitor:** (Millipore, Cat #: 3335399001) +- **Thomas Scientific Eppendorf DNA LoBind 1.5 mL Tubes:** (Thomas Scientific, Cat #: 13-698-791) +- **Fisher Scientific Lobind Eppendorf 2.0mL tubes:** (Thermo Fisher Scientific, Cat #: 13-698-792) +- **pluriSelect -BOApluriStrainer Mini 100 µm:** (pluriSelect, Cat #: 43-10100-50) +- **pluriSelect -BOApluriStrainer Mini 40 µm:** (pluriSelect, Cat #: 43-10040-50) + +### Buffers and Stocks + +**Reagents needed when using sucrose column:** + +| Reagent | Stock Concentration | Final concentration | Volume X1 | +|--------- + +| Nuclei Extraction Buffer | n.a. | n.a. | 1592 µL | +| RNase inhibitor | 40 U/µL | 0.2 U/µL | 8 µL | + +**Sucrose Solution (50 ml Stock Solution):** + +| Reagent | Stock Concentration | Final concentration | volume X1 (µL) | +|---------|---------------------|---------------------|----------------|--- +| Sucrose | n.a. | 1.8M | 750 | +| Mg(AC)_2 | 1M | 3 mM | 150 | +| Tris-HCl pH 7.4 | 1M | 10 mM | 500 | +| H₂O | n.a. | n.a. | Q.S. to 50 ml | + +**Sucrose Column:** + +| Reagent | Stock Concentration | Final concentration | volume X1 (µL) | +|---------|---------------------|---------------------|--------|--- +| Sucrose Solution | 1.8M | 1.8M | 750 | +| DTT | 1.3M | 1mM | 0.58| + +**Nuclei Suspension Buffer (750 µl per reaction):** + +| Reagent | Stock Concentration | Final concentration | Volume X1 (µL) | +|---------|---------------------|---------------------|--------|--- +| PBS | n.a. | 1x | 580.67 | +| BSA | 10% | 2% | 150 | +| 1 mM DTT | 1.3M | 1mM | 0.58 | +| RNase inhibitor | 40 U/µL | 1 U/µL | 18.75 | + +**Reagents needed when omitting sucrose column:** + +| Reagent | Stock Concentration | Final concentration | volume X1 (µL) | +|---------|---------------------|---------------------|--------|--- +| PBS | n.a. | 1X | 967.78 | +| BSA | 10% | 2% | 250 | +| 1 mM DTT | 1.3M | 1 mM |0.97 + +## Cell Counting + +- **Acridine, Orange/Propidium Iodide Stain:** (New England BioGroup, Cat #: F23001) +- **Luna Slides:** (New England BioGroup, LLC) + +**Reagents:** + +| Reagent | Cat Number | Vendor | +|---------|--------|----------- +|Cellometer SD100 Counting Chamber | Nexcelom Cat Number | Nexcelom Bioscience | +|Countess™ Cell Counting Chamber | C10312| Thermo Fisher| +|Countess™ II FL Slides: |C10312| Thermo Fisher +|Countess™ FL II Chamber | | Thermo Fisher| +| Cellometer K2 Slides | | Nexcelcom Bioscience| + +### Nuclei Counting Method: + +**Trypan Blue / AOPI Stain** + +1. **Luna FX7** + - Mix Trypan 1:1 with sample: 1 µL AOPI with 9µL sample. + - Example: 1ul AOPI/9ul sample. + - Example: 1ul AOPI + 4.5ul Sample + 4.5Ul Suspension Buff. + - Turn on cell counter select `fluorescence` and `cell lines and primary cells` + - Choose Luna FX7, insert slides, press `count`. +2. **Countess FL II** + - Mix at 4.0%: 6µL TB stain + 6 µL nuclei suspension to Microtubes. Mix gently (5-10x). + - Load **10 µL** to chamber slides of Countess. + - Visualize of nuclei presence debris-free. + - Press `Count`. +4. **Nexcelom Cellometer Counting**: + - Add: 11 µL AOPI and 11 µL with nuclei + buffer. + - Mix gently. + -Place Add `20 µL` into SD100 slides. + -Record concentration. % Viability, %, and size. + +3. **Guidelines:** + +** Staining and Imaging:** +1. Use: **SYTO/HOECHST** Classes as exposure: Leica 45ms, gain (5.5), SYTO -800ms, gain (3.4). Bright 30ms, gain: 8.8 using 40x. +1. The Protocol: Use 1:80 **HOECHST** Dilution 1:500 SYTO dilution (PBS). +2. Ensure total volume 18uL. +3. Mix as: gently allocating into 50uL Tube - dilution. +4. Incubate: 20 min at 4C dark (avoid light) +4. Stable imaging for further details: + +### Expected Results: + + Leibovitz solution image showing nuclei with brightfield and fluorescence. + +## Alternative Methods : + +1. Sugaring Floatation RNAserve method is an alternative. + +### 10X Preparation: + +Follow vendor guidelines: https://www.10xgenomics.com for specific needs. + +Loading examples: + +HT chip: Target: 20000 nucleus (Load: 33,000:20%). +ST chip: Target: 6000 nuclei (Load 12,000: 10%). + +**endofoutput** +``` \ No newline at end of file diff --git a/markdown-output/nutrient-deplete-replete-algal-culture-for-element-gukbwuw.md b/markdown-output/nutrient-deplete-replete-algal-culture-for-element-gukbwuw.md new file mode 100644 index 0000000000000000000000000000000000000000..37ebcb96436898b5b5c4e7ab9decd039688b4e2f --- /dev/null +++ b/markdown-output/nutrient-deplete-replete-algal-culture-for-element-gukbwuw.md @@ -0,0 +1,172 @@ +```markdown +# Goal/Experiment: +To evaluate the influence of nutrient availability on cellular elemental composition (quotas and stoichiometry) in marine picoeukaryotes. + +# Nutrient Deplete/Replete Algal Culture for Elemental Analysis +**Version 2** + +## Amy Zimmerman, Susanne Wilken + +## Abstract +**Purpose:** To evaluate the influence of nutrient availability on cellular elemental composition (quotas and stoichiometry) in marine picoeukaryotes. + +**Summary:** Elemental quotas and ratios are assessed under nutrient replete and deplete conditions at the same time to minimize potential variation between experiments. Cells from an exponentially growing culture are concentrated by centrifugation, washed and re-suspended in a small volume of nutrient deplete media. These concentrated cells are used to inoculate triplicate replete and deplete culture flasks at a starting density corresponding to early-exponential growth. Culture growth is monitored daily. When the mean growth rate of the nutrient deplete treatment is half or less of the replete treatment (\(GR_{DEP}/GR_{REP} < 0.5\)), samples for cell counts and elemental analyses (dissolved nutrients; particulate carbon, nitrogen, and phosphorus) are collected. + +**Citation:** Amy Zimmerman, Susanne Wilken Nutrient deplete/replete algal culture for elemental analysis. protocols.io. dx.doi.org/10.17504/protocols.io.gukbuwv Published: 11 Feb 2017 + +## Guidelines +### Background +Marine phytoplankton play an important role in coupling multiple nutrient cycles because they live at the interface between the abiotic and biotic components of ecosystems. The quantitative details of how multiple nutrient cycles intersect is determined by cellular elemental composition, including both the quantity and relative ratio of elements that make up a cell. The literature clearly demonstrates that phytoplankton can vary substantially in elemental composition depending on the availability of key nutrients. This variation may reflect physiological responses to ambient conditions (e.g., nutrient availability, temperature, light, etc.) and/or robust inter-specific differences in "average" elemental composition. Likewise, an organism’s ability to modify its elemental composition in response to environmental gradients (“plasticity”) varies among species. Interspecific differences in elemental composition could lead to substantial seasonal or regional differences in community structure as well as biogeochemical variables like algal biomass and export. + +## Before Start +### Equipment and Reagents +#### Equipment: +- BD Accuri flow cytometer +- Sorvall RC 26 Plus centrifuge +- Large rotor for Sorvall centrifuge +- Centrifuge bottles, acid-cleaned and autoclaved +- Balance +- 25 mm glass filtration units (flask, funnel, & support base), acid-cleaned and autoclaved +- Vacuum pump +- Advantec GF-75 25mm glass fiber filters (Sterlitech Corp #GF7525MM), pre-combusted (3 hrs at 450°C) +- Forceps +- 50 mL conical tubes, acid-cleaned +- 12-well plates or pieces of pre-combusted aluminum foil +- Cryovials & cryocanes + +#### Reagents: +- Nutrient replete medium +- Nutrient deplete medium +- 25% EM-grade glutaraldehyde +- 37% formaldehyde + +### Filter Combustion Procedure +1. Make a double thick aluminum sheet with several folds. +2. Use ethanol-cleaned forceps to arrange a single layer of 25 mm Advantec GF-75 glass fiber filters (0.3 µm nominal pore size) in each fold. +3. Combust in a muffle oven for 3 hours at 450°C (plus time to warm up). +4. Open door and allow to cool overnight before removing packet. +5. Store at room temperature in a clean ziplock bag. Only open in hood. + +### Acid-cleaning Procedure +1. After use, rinse equipment with tap water. +2. Soak in 1% Micro soap overnight. (Note: Keep centrifuge bottles in culture room and filter units and conical tubes in main lab.) +3. Rinse 6 times with tap water and 6 times with Milli-Q water, then soak in Milli-Q overnight. +4. Rinse 4 times with Milli-Q. +5. Soak in 1:10 diluted (~3.7%) trace-metal grade HCl overnight. +6. Rinse 6 times with Milli-Q water, then soak in Milli-Q overnight. +7. Rinse 2 times with Milli-Q and air dry covered with absorbent lab mats. +8. Once dry: + - Wrap filter unit components in foil or sterilization bags and autoclave on gravity cycle. + - Tightly cap conical tubes and store in plastic bag. + - Loosely cap centrifuge bottles and autoclave in sterilization bags on gravity cycle. + +## Protocol +### Culture Preparation/Growth +**Step 1.** + +Comparative measurements depend on having well-characterized culture growth. + +1. Initiate an experimental culture to allow for at least 7 generations of characterized exponential growth (10 generations is the gold standard) prior to centrifugation and resuspension. +2. Monitor growth daily, including transfer/dilution to pre-determined mid-exponential density (i.e., semi-continuous culture) until target biomass is reached. A safe estimate for the target biomass is to assume 10% recovery of cells after centrifugation and washing (this is strain dependent). +3. Ensure cell density remains within the exponential range by increasing culture volume (not cell density) when ramping up to target biomass. + +**Example Ostreococcus lucimarinus** (CCMP2972A) culture preparation: + +- **Growth conditions:** 18°C, 14:10 hour light:dark cycle, light irradiance of 100 µE m² s⁻¹ +- **Growth media:** L1 with natural seawater base +- **Exponential range:** 7x10⁵ – 2x10⁷ cells/mL + +| Day | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | +|-----|------|------|------|------|------|------|------|------|------|-------| +| Initial density (mL⁻¹) | 3x10⁷ | 1.5x10⁷ | 1x10⁷ | 9x10⁶ | 8x10⁶ | 1x10⁷ | 9x10⁶ | 1x10⁷ | 9x10⁶ | 1.4x10⁷ | +| Growth Rate (d⁻¹) | NA | 0.527 | 0.628 | 0.558 | 0.384 | 0.720 | 0.639 | 0.554 | 0.261 | | +| Dilution/Transfer | Trans | Skip | Dil | Dil | Trans | Dil | Dil | Dil | NA | +| Final density (mL⁻¹) | 5x10⁶ | 5x10⁶ | 5x10⁶ | 5x10⁶ | 5x10⁶ | 5x10⁶ | 5x10⁶ | 5x10⁶ | 1x10⁷ | +| Volume (mL) | 30 x3 | 86 x3 | 172 x3 | 80 x9 | 125 x9 | 257 x9 | 401 x9 | 401 x9 | **3.6 L (use!)**| + +**Note:** 7 generations of growth prior to centrifugation **Note:** Extra-large volumes used for omics experiment. + +### Concentration and Resuspension +**Step 2.** +1. When target culture biomass has been reached, split volume evenly among 2 or 4 acid-cleaned and autoclaved centrifuge bottles. +2. Weigh and balance bottles by removing volume until bottle pairs are within 0.01 g of each other. Only open bottles in hood to maintain axenicity! +3. Carry bottles over to Sorvall RC 26 Plus centrifuge. +4. Fit centrifuge with large rotor (accommodates 4 centrifuge bottles). Make sure pins are offset when rotor is placed into the centrifuge. +5. Place paired bottles opposite of each other in the rotor and tighten the lid. +6. Spin at the following settings: + - Rotor = GS-3 (choose option code “03”) + - Speed = 7300 RPM (equivalent to 10,000 rcf with this large rotor) + - Time = 0:32 minutes (includes 2 minute ramp up) *strain specific + - Temp = +20/+25°C (will try to maintain lower temp, and shut down at max temp) +7. After the spin, carefully transport the bottles back to hood, taking care not to resuspend the cell pellets/smears. +8. Gently pour off the supernatant into a waste container, trying to disturb the cell smear as little as possible. +9. Resuspend/wash cells with a volume of nutrient deplete medium equivalent to the initial culture volume in each bottle. +10. Repeat 2-8. +11. Resuspend cells with a volume of nutrient deplete medium that is 5% of the initial culture volume in each bottle. +12. Run a sample of the cell concentrate in the Accuri to determine density. Store cells at normal growth conditions during Accuri run and calculations. + +### Experimental Culture Initiation +**Step 3.** +1. Calculate appropriate volume based on Accuri measurement to inoculate experimental nutrient replete and deplete culture flasks from the cell concentrate at a starting density corresponding to early-exponential growth. **Note:** It helps to have some amount of media pre-aliquoted to experimental flasks. Adjust as needed to achieve target density and volume. +2. Once all experimental cultures have been inoculated, mix and sample for FCM and Accuri measurements to monitor growth daily. +3. When the average daily growth rate of the nutrient deplete cultures (\(GR_{DEP}\)) is reduced to half or less of the growth rate of the replete cultures (\(GR_{REP}\)), collect samples for FCM (i.e., cell counts) and elemental analyses (i.e., particulate carbon/nitrogen and phosphorus, as well as dissolved nutrients). + +### Sample Collection +**Step 4.** + +**Flow Cytometry (FCM, for Cell Counts)** + +1. Transfer 1 mL of culture to a sterile 1.2 mL cryovial tube. +2. Add 10 µL 25% glutaraldehyde (0.25% final conc) and gently vortex to mix. +3. Aliquot 500 µL to a duplicate cryovial. +4. Snap cryovials into cryocanes and incubate at 4°C for 30 minutes in the dark. +5. Flash freeze in liquid nitrogen. +6. Store at -80°C until analysis. + +**Dissolved and Particulate Elemental Analysis (EA, for cellular elemental composition & dissolved nutrient concentrations)** + +**Note:** Separate filters are needed from each sample for POC/N and POP analyses! Additional filters must be collected if you want technical replicates for each sample type. + +1. Set up an acid-cleaned and autoclaved glass filter unit with pre-combusted 25mm Advantec glass fiber filter. *Use ethanol-cleaned forceps in hood! +2. Apply 20-40 mL of media or culture to the filter funnel and turn on the vacuum until the filter is dry. +3. Use ethanol-cleaned forceps to fold filter in half and collect in a 12-well plate or piece of combuated aluminum foil. +4. Pour filtrate into an acid-cleaned 50 mL conical tube. +5. Repeat for sample duplicate. +6. Rinse filter tower with MilliQ between samples. +7. Store filters and filtrate at -20°C until further processing. + +**DAPI (for microscopy to assess axenicity)** + +1. Transfer 1 mL of culture to sterile 1.2 mL cryovial tube. +2. In the chemical hood, add 100 µL of 37% formaldehyde (3.7% final conc) and mix by inverting. Do not vortex. +3. Snap cryovials into cryocanes and incubate at room temp for **15 minutes** (in the dark). +4. Store at 4°C until analysis (can be stored for several days or flash frozen in liquid nitrogen and stored at -80°C). + +### Sample Processing +**Step 5.** + +Samples for elemental analysis should be sent to Analytical Services at Horn Point Laboratory (University of Maryland Center for Environmental Science, UMCES). + +For each sample, request at least the following analyses: + +| Analysis | Cost per sample | Sample type | +|----------|-----------------|-------------| +| Soluble Reactive Phosphate (SRP or PO4) | $7.90 | Filtrate | +| Nitrate plus Nitrite (NO(2+3)) | $7.90 | Filtrate | +| Total Particulate Phosphorus (PP) | $19.80 | Filter | +| Particulate Carbon and Particulate Nitrogen (CHN) | $17.70 | Filter | + +*Dependent on limiting nutrient + +Contact: Erica Kiss ([ekiss@umces.edu](mailto:ekiss@umces.edu)) + +Phone: 410-221-8317 +Shipping address: 5745 Lover's Lane +Postal address: PO Box 775 +Street address: 2020 Horn's Point Rd., +Cambridge, MD 21613 + +[http://www.umces.edu/hpl/analytical-services](http://www.umces.edu/hpl/analytical-services) + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/nutrient-solution-for-rice-hydroponics-culture-wg6fbze.md b/markdown-output/nutrient-solution-for-rice-hydroponics-culture-wg6fbze.md new file mode 100644 index 0000000000000000000000000000000000000000..d8ad69450c856c11ab6deb8f40efdcf587ca52a5 --- /dev/null +++ b/markdown-output/nutrient-solution-for-rice-hydroponics-culture-wg6fbze.md @@ -0,0 +1,125 @@ +```markdown +# Goal/Experiment: +The goal of this experiment is to prepare a nutrient solution for rice hydroponics culture using a modified formulation of Yoshida's solution. This modified solution aims to simplify preparation and enable control over certain ionic concentrations to investigate their effects on rice cultivation. + +# Nutrient Solution for Rice Hydroponics Culture + +**John Platten** +*International Rice Research Institute* + +## Abstract +This represents a heavily modified Yoshida's solution. Compared to Yoshida's formulation, this solution has almost the same concentration of macro and micronutrients, but the formulation has been simplified. Key advantages: +* The solution is now formulated as three solutions at 1000× concentration, greatly simplifying dilution. +* The solution is less acidic than Yoshida's formulation, reducing the amount of hydroxide required to adjust pH. +* The solution eliminates Na+ and almost all Cl- from the medium, allowing independent investigation of toxicities of these ions. Additionally, much of the K+ is supplied as KOH, potentially allowing control of the Na/K ratio independently of absolute concentrations and of Cl- levels. + +## Materials + +| Reagent | Source and Catalog Number | +| --- | --- | +| Zinc sulfate heptahydrate | Sigma Aldrich, #204986 | +| Manganese(II) chloride tetrahydrate | Sigma Aldrich, #M3634 | +| Potassium Phosphate (Monobasic) | G-Biosciences, #RC-083 | +| Citric acid monohydrate | Sigma-Aldrich, #33114 | +| Boric acid | Bio Basic Inc., #BB0044.SIZE.500g | +| Calcium nitrate tetrahydrate | Bio Basic Inc., #CB0258.SIZE.500g | +| Copper (II) sulfate pentahydrate | Bio Basic Inc., #CBD0063.SIZE.500g | +| Iron chloride (Ferric chloride), hexahydrate | Bio Basic Inc., #FD0201.SIZE.250g | +| Magnesium sulfate, heptahydrate, ACS | Bio Basic Inc., #MB0329.SIZE.500g | +| Potassium hydroxide | Bio Basic Inc., #PB0441.SIZE.500g | +| Sulfuric Acid (H2SO4) | Contributed by users | +| Sodium metasilicate nonahydrate | Sigma Aldrich, #S4392-250G | +| Ammonium sulfate | Sigma Aldrich, #A5132-1KG | +| Ammonium molybdate tetrahydrate | Sigma Aldrich, #A7302-500G | + +## Solution A, 1000× (Macro and Micronutrients, minus Ca, Mg and Si) + +| Common Name | Formula | Stock (g/mol) | Final (M) | g/L | g/2L | Element | +| --- | --- | --- | --- | --- | --- | --- | +| Sulfuric acid (5M stock, diluted from concentrated; ~18M) | H₂SO₄ | 5.00 M | 0.8510 | 170.2 mL | 340.4 mL | S | +| Ammonium sulfate | (NH₄)₂SO₄ | 132.140 | 0.6753 | 89.23 | 178.46 | NH₄, S | +| Potassium phosphate monobasic | KH₂PO₄ | 136.0855 | 0.3225 | 43.89 | 87.77 | K, P | +| Potassium hydroxide | KOH | 56.1056 | 0.7020 | 39.39 | 78.77 | K | +| Citric acid, monohydrate | C₆H₈O₇.H₂O | 210.1388 | 0.0708 | 14.87 | 29.75 | | +| Ferric chloride, 6-Hydrate | FeCl₃.6H₂O | 270.2964 | 0.0356 | 9.62 | 19.25 | Fe | +| Manganous chloride, 4-hydrate | MnCl₂.4H₂O | 197.9052 | 0.0094 | 1.88 | 3.750 | Mn | +| Ammonium molybdate, 4-Hydrate | (NH₄)₆Mo₇O₂₄.4H₂O | 1235.8577 | 0.0005 | 0.0925 | 0.185 | Mo | +| Zinc sulfate, 7-hydrate | ZnSO₄.7H₂O | 287.5796 | 0.0001 | 0.043 | 0.087 | Zn | +| Boric acid | H₃BO₃ | 61.8330 | 0.01819 | 1.17 | 2.335 | B | +| Cupric sulfate, 5-Hydrate | CuSO₄.5H₂O | 249.6860 | 0.0001 | 0.038 | 0.077 | Cu | + +## Solution B, 1000× (Ca, nitrate) + +| Common Name | Formula | Stock (g/mol) | Final (M) | g/L | g/2L | Element | +| --- | --- | --- | --- | --- | --- | --- | +| Calcium nitrate, tetrahydrate | Ca(NO₃)₂.4H₂O | 236.1490 | 0.9978 | 235.62 | 471.25 | Ca, NO₃ | + +## Solution C, 1000× (Mg, S) + +| Common Name | Formula | Stock (g/mol) | Final (M) | g/L | g/2L | Element | +| --- | --- | --- | --- | --- | --- | --- | +| Magnesium sulfate, 7-hydrate | MgSO₄.7H₂O | 246.4756 | 1.6432 | 405.00 | 810.00 | Mg,S | + +## Elemental Comparison with Yoshida's Original Formulation + +| Nutrient | Yoshida's [Final] (mM) | Modified [Final] (mM) | Difference (mM) | Percentage of Original | Fold Change (×) | +| --- | --- | --- | --- | --- | --- | +| Na | 0.322482 | 0.031668 | -0.290814 | 9.82% | 0.098× | +| K | 1.024330 | 1.024482 | 0.000151 | 100.01% | 1× | +| N | 2.855149 | 3.346067 | 0.490918 | 117.19% | 1.171× | +| NH₄ | 1.427799 | 1.350516 | -0.077283 | 94.59% | 0.945× | +| NO₃ | 1.427350 | 1.995551 | 0.568201 | 139.81% | 1.398× | +| PO₄ | 0.322482 | 0.322482 | 0.000000 | 100.00% | 1× | +| SO₄ | 3.324387 | 3.169730 | -0.154657 | 95.35% | 0.953× | +| Ca | 0.997775 | 0.997775 | 0.000000 | 100.00% | 1× | +| Mg | 1.643165 | 1.643165 | 0.000000 | 100.00% | 1× | +| Mn | 0.009474 | 0.009474 | 0.000000 | 100.00% | 1× | +| Mo | 0.000524 | 0.000524 | 0.000000 | 100.04% | 1× | +| Zn | 0.000152 | 0.000152 | 0.000000 | 100.00% | 1× | +| H₃BO₃ | 0.018881 | 0.018882 | 0.000000 | 100.00% | 1× | +| Cu | 0.000155 | 0.000155 | 0.000000 | 100.04% | 1× | +| Fe | 0.035609 | 0.035609 | 0.000000 | 100.00% | 1× | +| Cl | 2.130801 | 0.125775 | -2.005025 | 5.90% | 0.059× | +| Si | 0.015834 | 0.015834 | 0.000000 | 100.00% | 1× | +| Citrate | 0.000071 | - | - | - | - | + +## Safety Warnings +The formulation makes use of 5M sulfuric acid. This is prepared by diluting concentrated sulfuric acid (~18M) to achieve the final 5M stock. Diluting strong acids is hazardous and appropriate protocols should be followed. + +## Procedure + +### Solution A, 1000× (Main Macro and Micronutrients) + +1. Make this solution up as a SINGLE stock solution. For 1L of stock solution: + * Start with ~600 mL de-ionised water. + * Weigh out and dissolve each component (except ferric chloride and citric acid) directly in the 600 mL solution. Allow each to dissolve completely before adding the next. + * For the KOH, add pellets slowly, a few at a time, with constant mixing. + * Dissolve the citric acid separately in 100 mL de-ionized water. Dissolve the ferric chloride 6-hydrate directly in the citric acid solution. Stir the ferric chloride-citrate solution for 15 minutes, then add slowly to the main stock solution while stirring. + * Make up to 1 L. + +### Solution B, 1000× (Ca, NO3) + +2. For 1 L of stock solution, dissolve the calcium nitrate 4-hydrate directly in 700 mL de-ionized water. Make up to 1 L. + +### Solution C, 1000× (Mg, S) + +3. For 1 L of stock solution, dissolve the magnesium sulfate 7-hydrate in 700 mL de-ionized water. Make up to 1 L. + +### Solution D, 1000× (Silicon) (Optional) + +4. For 1 L of stock solution, dissolve 4.50 g sodium metasilicate 9-hydrate in 700 mL de-ionized water. Make up to 1 L. The sodium metasilicate can also be weighed out and dissolved directly in the growth medium at the time of preparation. + +## Preparation of Hydroponics Medium + +5. For 100 L of 1× hydroponics solution: + * Prepare ~90 L of water. + * Mix in 100 mL of each stock solution, mixing well before adding the next stock solution. + * Add sodium metasilicate nonahydrate (0.00450 g/L) and dissolve with stirring, if not done as solution D. + * Adjust pH to 5.5. + * Dispense to growth trays. + +6. Monitor and adjust pH of stock solution and culture daily at pH 5.5 with KOH/HCl as required. Refresh stock solution twice per week. + +--- +endofoutput +``` diff --git a/markdown-output/ocuprime-100-safe-and-effective-for-eyes-b9a5r2g6.md b/markdown-output/ocuprime-100-safe-and-effective-for-eyes-b9a5r2g6.md new file mode 100644 index 0000000000000000000000000000000000000000..18877eeb96be3214a5bf89c7b137f00b80992579 --- /dev/null +++ b/markdown-output/ocuprime-100-safe-and-effective-for-eyes-b9a5r2g6.md @@ -0,0 +1,119 @@ +```markdown +# Goal/Experiment: +The goal of this document is to review and analyze the safety and efficacy of OcuPrime, a dietary supplement, for eye health. + +# OcuPrime - 100% Safe And Effective For Eyes! + +**Authors:** +1. OcuPrime + +**DOI:** +[dx.doi.org/10.17504/protocols.io.j8nlkkn6wl5r/v1](https://dx.doi.org/10.17504/protocols.io.j8nlkkn6wl5r/v1) + +## OcuPrime Reviews - All Natural Formula And Reduce All Eye Issues! +[OcuPrime Reviews](https://www.facebook.com/OcuPrime-Review-109912961719776) + +### Product Name +**OcuPrime** + +### Composition +**Natural Organic Compound** + +### Side-Effects +**NA** + +### Price +Visit Official Website + +### Supplement Type +**Pills** + +### Official Website +[Official Website & Order (Click Here)](https://www.facebook.com/OcuPrime-Review-109912961719776) + +==> [Click Here To Buy Now With Special Offer](https://www.facebook.com/OcuPrime-Review-109912961719776) <== + +## What Is The OcuPrime Support Formula? + +Ocuprime is a dietary enhancement made with fixings from nature that have been clinically demonstrated to support reestablishing lucidity of vision and to assist with keeping up with the strength of your eyes. It is accepted that the OcuPrime Support Formula is profoundly effective and proficient in reestablishing vision and shielding the eyes from future issues that come about because of maturing or free extremists. + +Ocuprime is produced in an FDA enlisted and GMP ensured office. This supplement is made inside the each case is ok for utilization and liberated from unfavorable responses engineered fillers, and other perilous fabricated materials. There are no energizers to the recipe of Ocuprime which could present gamble to the soundness of the client. + +### How Is OcuPrime Support Formula Works? + +Most vision issues can be caused because of free extremists which represent a huge gamble to the soundness of your eyes. These harmful substances can affect your wellbeing, which is the reason it is essential to play it safe to prepare for and dispense with them. In the wake of directing clinical investigations have been led, it was found that the best fixings are those to further develop visual perception and well-being, especially night vision. + +These unsafe and undesirable poisonous substances develop in the eyes and cause vision issues which incorporate foggy pictures as well as vision issues as well as dry eyes. Your eyes and your vision will consequently further develop once your body has changed in accordance with the strong fixings in the Ocuprime Benefits. You'll see protests better and felt looser. Moreover, the standard use of Ocuprime will safeguard your outcomes and further develop them. + +==> [Click Here To Buy Now With Special Offer](https://www.facebook.com/OcuPrime-Review-109912961719776) <== + +## OcuPrime Ingredients and Composition Details + +Apparently one of the fundamental highlights of OcuPrime Vision Support Formula is that it depends on a natural arrangement and demonstrated and tried fixings. The gathering behind it says that they use an exceptional combination of in excess of gainful fixings that have been thoroughly inspected and tried for the best results. With each fixing offering something important, however, everyone cooperating, improvement is the most probable outcome. The following are a couple of the principal parts that went into making OcuPrime: + +- **Eyebright**: Eyebright is the abbreviated name for this fixing Euphrasia Officinalis, a gainful fixing that guides in expanding the eye's splendor. The clients will actually want to ensure that their eyes don't lose their solidarity and might fix numerous other eye-related issues by utilizing this fixing. + +- **Quercetin**: This fixing is accepted as an advantageous method for battling the impacts of poisons. A cell reinforcement gives clients the power that they expect to battle the impacts of oxidative weight for a huge scope. It likewise permits clients to treat waterfalls by utilizing this fixing, however, the consequences for every individual might vary from one person to another. + +- **Bilberry**: Bilberry is popular for its generally expected influences on the strength of one's eyes. The clients of this fixing have detailed that they have defeated issues like being not able to find in obscurity or night visual deficiency. Anybody experiencing this condition ought to think about investigating this fixing. + +- **Lycopene**: Lycopene helps with forestalling macular degeneration of the eyes. Also, the fixing is helpful to the individuals who are old and have been presented with the impacts of oxidative pressure to some degree. The fixing can likewise be helpful to help the retinal organs in the eye. + +- **Magnesium**: It is a part that is exceptionally helpful to keep up with the appropriate progression of blood. As we have referenced one of the fundamental goals of OcuPrime supplementation is to guarantee that the clients are getting a satisfactory bloodstream that can bring about medical advantages. Thusly magnesium oxide is one of the significant fixings in that respect. + +- **Rutin**: Rutin is an alternate powerful part of the bloodstream. The clients of Rutin can assist with working on the strength of veins and work on the clearness of pictures they see. Those with obscured or foggy vision might have the option to see upgrades in their presentation after they begin utilizing this fixing. + +- **Grape Seed**: Grape Seed is a fixing that has been demonstrated to forestall eye illnesses for individuals. Anybody experiencing wounds or enlarging should investigate this. It's not only a method for guaranteeing that eye cells are secure in any case, it likewise offers clients the chance of forestalling any further harm to their retina. + +- **Zeaxanthin and Lutein**: Though it's not among the principal fixings, it's not the last. The clients of this fixing are in a situation to safeguard themselves from the steady blue light utilized in most gadgets today. By using these two strong fixings they can lessen the gamble of creating eye sicknesses, which makes it a fundamental part of the whole structure. + +==> [Click Here To Buy Now With Special Offer](https://www.facebook.com/OcuPrime-Review-109912961719776) <== + +## What Are The Benefits Of the OcuPrime Support Formula? + +As per the enhancement site we have recorded beneath the recipients which OcuPrime Support Formula might give to you. + +- It is feasible to accomplish impeccable vision by fixing all eye harm brought about by this recipe that is strong. +- It supports reestablishing clear vision utilizing strong and normal concentrates. +- You'll know about the requirement for contacts, glasses as well as other eye drugs and normal visits to the specialist. +- It could give you fabulous outcomes, however without adverse consequences. +- It helps you in keeping away from nervousness, melancholy, and migraines because of eye vision issues. +- It is feasible to see better in obscurity and assist with forestalling issues with long and short sight. The intense fixings can assist with keeping up with sound degrees of energy and further develop memory. +- It helps with flushing out unsafe toxins and assists with reestablishing the eye's well-being. +- There is a lot of good audits from clients that assure the best outcomes from the enhancement. +- The 60-day merchandise exchange will permit you to make your buy as hazard-free as it states on the site. + +==> [Click Here To Buy Now With Special Offer](https://www.facebook.com/OcuPrime-Review-109912961719776) <== + +## Where To Buy OcuPrime? + +You can get it from the authority site utilizing this connection. Clients of the item are in a situation to buy directly from their authority site. This implies that one isn't simply getting a decent rebate, yet in addition, staying away from issues like retailer's charges and different issues. The following are a couple of the bundles for which the enhancements are accessible: +- **Day Supply**: One jug of the enhancement is presented for USD for the jug. +- **Day Supply**: It is a multi-month bundle that is ideal for the people who need to get a rebate on each jug. The value drops to USD for each jug when you buy this bundle. This is intended for the people who are hoping to get the most worth. It accompanies a month's supply of USD for each container. + +## Final Verdict OcuPrime Support Formula! + +Might it be said that you are experiencing difficulty with your vision? This audit on the OcuPrime Support Formula could give you normal help. It's the eye support equation that assists with reestablishing solid vision utilizing regular and safe creation. It disposes of the justification for the issues with vision and safeguards your eyes from harm that could be brought about by the poisons. It can give you a vision that is completely clear by taking containers routinely. It's totally sans risk, as indicated by the authority site for the item. + +**Visit Here:** +- [https://www.elitegross.com/buyocuprime](https://www.elitegross.com/buyocuprime) +- [https://www.facebook.com/OcuPrime-Review-109912961719776](https://www.facebook.com/OcuPrime-Review-109912961719776) +- [https://www.homify.in/diy/23353/ocuprime-100-safe-and-effective-for-eyes](https://www.homify.in/diy/23353/ocuprime-100-safe-and-effective-for-eyes) +- [https://twitter.com/EliteGross/status/15250078494404813056](https://twitter.com/EliteGross/status/15250078494404813056) +- [https://www.scoop.it/topic/ocuprime-by-ocuprime-8](https://www.scoop.it/topic/ocuprime-by-ocuprime-8) +- [https://lexcliq.com/ocuprime-reviews-all-natural-formula-and-reduce-all-eye-issues/](https://lexcliq.com/ocuprime-reviews-all-natural-formula-and-reduce-all-eye-issues/) +- [https://the-dots.com/projects/ocuprime-100-safe-and-effective-for-eyes-742096](https://the-dots.com/projects/ocuprime-100-safe-and-effective-for-eyes-742096) +- [https://medium.com/@lynnbrqwn/ocuprime-reviews-17018c422e](https://medium.com/@lynnbrqwn/ocuprime-reviews-17018c422e) +- [https://techplanet.today/post/ocuprime-100-safe-and-effective-for-eyes](https://techplanet.today/post/ocuprime-100-safe-and-effective-for-eyes) +- [https://techplanet.today/post/ocuprime-reviews-all-natural-formula-and-reduce-all-eye-issues](https://techplanet.today/post/ocuprime-reviews-all-natural-formula-and-reduce-all-eye-issues) +- [https://wakelet.com/wake/Wh3fzWCdBPshio1r1-gtF](https://wakelet.com/wake/Wh3fzWCdBPshio1r1-gtF) +- [https://bumpyy.com/tm/read-blog/53634_ocuprime-100-safe-and-effective-for-eyes.html](https://bumpyy.com/tm/read-blog/53634_ocuprime-100-safe-and-effective-for-eyes.html) +- [https://www.protocols.io/view/ocuprime-reviews-all-natural-formula-and-reduce-al-b9a4r2gw](https://www.protocols.io/view/ocuprime-reviews-all-natural-formula-and-reduce-al-b9a4r2gw) + +--- +**Citation:** OcuPrime OcuPrime - 100% Safe And Effective For Eyes! [https://dx.doi.org/10.17504/protocols.io.j8nlkkn6wl5r/v1](https://dx.doi.org/10.17504/protocols.io.j8nlkkn6wl5r/v1) + +This is an open access protocol distributed under the terms of the Creative Commons Attribution License [https://creativecommons.org/licenses/by/4.0](https://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. + +~~endofoutput~~ +``` \ No newline at end of file diff --git a/markdown-output/ocuprime-vision-support-reviews-shocking-side-effe-b877rzrn.md b/markdown-output/ocuprime-vision-support-reviews-shocking-side-effe-b877rzrn.md new file mode 100644 index 0000000000000000000000000000000000000000..00a858db7460c3add1060174857b7210d65305bb --- /dev/null +++ b/markdown-output/ocuprime-vision-support-reviews-shocking-side-effe-b877rzrn.md @@ -0,0 +1,92 @@ +```markdown +# Goal/Experiment: +Evaluate the effectiveness and side effects of OcuPrime Vision Support based on user reviews and clinical data. + +## OcuPrime Vision Support Reviews: Shocking Side Effects & Customer Complaints + +Citation: ocuprimevision OcuPrime Vision Support Reviews: Shocking Side Effects & Customer Complaints +DOI: [dx.doi.org/10.17504/protocols.io.q26g74d51gwz/v1](https://dx.doi.org/10.17504/protocols.io.q26g74d51gwz/v1) + +>**DISCLAIMER – FOR INFORMATIONAL PURPOSES ONLY; USE AT YOUR OWN RISK** + +>The protocol content here is for informational purposes only and does not constitute legal, medical, clinical, or safety advice, or otherwise; content added to [protocols.io](http://protocols.io) is not peer reviewed and may not have undergone a formal approval of any kind. Information presented in this protocol should not substitute for independent professional judgment, advice, diagnosis, or treatment. Any action you take or refrain from taking using or relying upon the information presented here is strictly at your own risk. You agree that neither the Company nor any of the authors, contributors, administrators, or anyone else associated with [protocols.io](http://protocols.io), can be held responsible for your use of the information contained in or linked to this protocol or any of our Sites/Apps and Services. + +## Availability of OcuPrime Vision Support + +OcuPrime Vision Support is available for online purchase only. Visit the [official website](https://dx.doi.org/10.17504/protocols.io.q26g74d51gwz/v1) to order. + +## Main Benefits + +- Improve eye vision without any side effects. + +## Principal Info About OcuPrime Vision Support Formula + +OcuPrime Vision Support Formula is a plant-derived dietary supplement aimed at individuals experiencing visual difficulties and related conditions like: + +- Restless sleep +- Stress +- Anxiety +- Headaches +- Absent concentration +- Cerebral haze + +### Key Benefits: +1. Supplies the body with essential minerals and nutrients. +2. Eliminates toxins and free radicals. +3. Enhances neurocyte transmission. +4. Reduces blood pressure and improves tissue recovery. +5. Decreases cerebral pains and headaches. +6. Anti-inflammatory properties. + +### Indicators for Use: +- Visual distortions like "sparkles, stars, steaks" +- Diminished visual sharpness +- Vision impairment over distances +- Sensation of "cover" or "sand" in the eyes +- Eye fatigue and discomfort + +![OcuPrime Vision Support](img/vs_formula.png) + +### Key Ingredients: +- **Eyebright (Euphrasia Officinalis)**: Reduces infections and inflammation. +- **Quercetin**: Fights oxidative stress. +- **Bilberry (Vaccinium Myrtillus)**: Enhances night vision. +- **Lycopene**: Prevents damaged cells in the retinal macula. +- **Magnesium**: Improves blood flow and reduces oxidative stress. +- **Rutin (Sophora Japonica)**: Strengthens blood vessels. +- **Grape Seed**: Facilitates better blood circulation. +- **Zeaxanthin & Lutein**: Protect the eyes against high-energy light waves. + +### Dosage Information: +- Recommended dosage: 2 gelatin-shrouded capsules per day, ideally during meals with water. + +### Precautions: +- Not suitable for underage users, pregnant women, or individuals with high sensitivity to active ingredients. +- Consult a healthcare provider if you are on other medications. + +## Pricing Packages + +The supplement comes in different packages. Here are the available options: + +| Package | Price per Bottle | Total Price | Savings | Shipping | +|-----------------|------------------|-------------|---------------|-----------| +| 30 Day Supply | $69 | $69 | Save $110 | + Shipping| +| 90 Day Supply | $59 | $177 | Save $360 | Free | +| 180 Day Supply | $49 | $294 | Save $780 | Free | + +![DocuPrime Pricing](img/pricing.png) + +## Where to Buy OcuPrime Vision Support Formula? + +To purchase OcuPrime Vision Support Formula, visit the [official website](https://dx.doi.org/10.17504/protocols.io.q26g74d51gwz/v1) and place your order. + +## Associate Disclosure +The links provided may bring a commission for the authors. Consult your healthcare provider before taking any supplements or medications. + +>**Read More** from these Articles: +> +>[Example Article 1](https://terisa-molamn.clubeo.com/news/2022/05/11/ocuprime-vision-support-reviews-truth-behind-the-exceptional-cl) +> +>[Example Article 2](https://sites.google.com/view/ocuprimevisionofficial/home) + +_End of output_ diff --git a/markdown-output/on-cell-western-plate-based-assay-for-targeted-nea-gsgbwbw.md b/markdown-output/on-cell-western-plate-based-assay-for-targeted-nea-gsgbwbw.md new file mode 100644 index 0000000000000000000000000000000000000000..acde02cd570d80167414e815bb99fae4f2f5f4c3 --- /dev/null +++ b/markdown-output/on-cell-western-plate-based-assay-for-targeted-nea-gsgbwbw.md @@ -0,0 +1,285 @@ +```markdown +# Goal/Experiment: +This experiment aims to test the effectiveness of IRDye 800CW EGF in binding to A431 cells (epithelial carcinoma) for potential in vivo applications. This assay will help in determining receptor-based binding and competition of IRDye 800CW EGF using targeted Near-Infrared-Labeled Optical Imaging Agent Development. + +# On-Cell Western Plate-Based Assay for Targeted Near-Infrared-Labeled Optical Imaging Agent Development: Receptor-Based Binding and Competition Assays + +### LI-COR Biosciences + +## Abstract +This protocol illustrates the process of testing IRDye 800CW EGF with A431 cells due to their over-expression of EGFR, with adaptability for other cell lines with modified media and characteristics. + +Developed for: +- Aerius, +- Odyssey® Classic, +- Odyssey CLx, +- Odyssey Sa, +- Infrared Imaging Systems + +## Guidelines + +### I. Required Reagents + +#### LI-COR Reagents +- **IRDye 800CW EGF** (P/N 926-08446) +- **CellTagTM 700 Stain** (LI-COR, P/N 926-41090) +- **Odyssey® Blocking Buffer (PBS)** (LI-COR, P/N 927-40000) + +#### Additional Reagents +- 1X PBS wash buffer +- Tissue culture reagents (serum, DMEM, trypsin, 1X PBS) +- 100% Tween® 20 +- 37% formaldehyde +- 10% Triton® X-100 +- Costar™ 96-well Black Clear-Bottom Plate (Corning) + +*Special Note*: Serum starvation of the cells is required to obtain maximal response. + +### II. Seeding, Stimulation, and Detection with IRDye 800CW EGF + +#### Binding Assay +1. Add 100 µL of DMEM to Well 1 to Well 12 in triplicate rows. +2. Add 100 µL of 1 µg/mL IRDye 800CW EGF to Well 12 in triplicate rows and mix well. +3. Transfer 100 µL from Well 12 to Well 11 and mix well by pipetting up and down. +4. Repeat this process through Well 3. + +| | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | +|----|----|----|----|----|----|----|----|----|----|----|----|-----| +| A | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 500 | +| B | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 500 | +| C | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 500 | + +#### Competition Assay +1. Add 100 µL of DMEM to Well 1 to Well 11 in triplicate rows. +2. Add 200 µL of 32 µg/mL unlabeled EGF to Well 12 in triplicate rows. +3. Transfer 100 µL from Well 12 to Well 11 and mix well by pipetting up and down. +4. Repeat this process through Well 3. +5. Add 100 µL of DMEM only to Wells 1 and 2 (background controls, no EGF). +6. Prepare 10 mL of 50 ng/mL IRDye® 800CW EGF. +7. Add 100 µL of IRDye 800CW EGF stock solution to Wells 3-12 for triplicate Competition Assay rows for final concentration of labeled EGF of 25 ng/mL. + +| | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | +|----|----|----|----|----|----|----|----|----|----|----|----|----| +| D | 0 | 0 | 0 |0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 500 | +| E | 0 | 0 | 0 |0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 500 | +| F | 0 | 0 | 0 |0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 500 | + +### III. Experimental Considerations +- Use clear or black-sided plates with clear bottoms for minimal well-to-well signal spread. +- Use sterile plates for tissue culture growth. +- Plates should be protected from light during imaging and stored at 4°C when not in use. + +| Well Number | Well Bottom | Manufacturer | Part Numbers | Odyssey & Odyssey CLx Offset | Aerius & Odyssey Sa Offset | +|-------------|-------------|--------------|------------------------|------------------------------|----------------------------| +| 96 | Flat | Nunc | 161093, 165305 | 3.0 mm | 3.0 mm | +| 96 | Flat | BD Falcon | 353075, 353948 | 3.0 mm | 3.0 mm | +| 96 | Flat | Corning | 3603 | 3.0 mm | 3.0 mm | +| 384 | Flat | Nunc | 164688, 164730 | 3.0 mm | 3.0 mm | +| 384 | Flat | BD Falcon | 353961, 353962 | 3.0 mm | 3.0 mm | + +### Focus Offset Optimization +Consult the plate manufacturer for specific focus offset requirements: + +| Instrument | Focus Offset Determination (mm) | +|------------------------------------|----------------------------------| +| Odyssey Classic & Odyssey CLx | 0.5, 1.0, 2.0, 3.0, & 4.0 | +| Odyssey Sa & Aerius | 1.7, 2.0, 3.0, & 4.0 | + +### Intensity Setting Optimization +Suggested intensity settings: + +| Instrument | Initial Intensity Setting (700/800 nm) | Intensity Settings: Weak Signal (700/800 nm) | Intensity Settings: Saturated Signal (700/800 nm) | +|---------------------|----------------------------------------|---------------------------------------------|--------------------------------------------| +| Odyssey® Classic | 5 / 5 | 7.5 / 7.5 | 2.5 / 2.5 | +| Odyssey CLx | 5 / 5 | 7.5 / 7.5 | 2.5 / 2.5 | +| Odyssey Sa | 7 / 7 | 8 / 8 | 4 / 4 | +| Aerius | 7 / 7 | 8 / 8 | 4 / 4 | + +### Materials +- Odyssey® Blocking Buffer (PBS) [927-40000, 927-40100 by LI-COR](https://www.licor.com/) +- IRDye 800CW EGF [926-08446 by LI-COR](https://www.licor.com/) +- CellTag™ 700 Stain [926-41090 by LI-COR](https://www.licor.com/) + +### Protocol + +#### Step 1. +Allow A431 cell growth in a 100-mm dish in DMEM containing 10% fetal bovine serum (FBS; ATCC 30-2020) and 1% PEN/STREP (Gibco®) using standard tissue culture procedures until cells reach 80% - 90% confluency (1.5 x 107 cells). + +#### Step 2. +Remove growth medium, wash cells with sterile 1X PBS, and trypsinize cells. + +#### Step 3. +Neutralize displaced cells with culture medium and pellet by centrifugation. + +#### Step 4. +Remove supernatant and resuspend cell pellet in remaining medium by manually tapping the collection tube. Avoid vigorous pipetting or vortexing. + +#### Step 5. +Dilute cells to 20 mL in complete medium and count cells using a hemocytometer. + +#### Step 6. +Dilute cells with complete medium to a concentration of 200,000 cells/mL. + +#### Step 7. +Gently mix the cell suspension thoroughly. + +#### Step 8. +Under sterile conditions, dispense 200 µL of the cell suspension per well in a 96-well plate (40,000 cells plated per well). + +#### Step 9. +Incubate cells and monitor cell density until cells are 80 - 90% confluent in each well. + +#### Step 10. +Warm serum-free medium (DMEM; Gibco®) to 37 °C. + +#### Step 11. +Remove complete medium from the 96-well plate by aspiration or inversion of the plate and blot excess medium by tapping the inverted plate gently on a tissue. + +#### Step 12. +Replace medium with 200 µL of pre-warmed, serum-free medium per well and incubate 4 hours at 37 °C. +- **Duration:** 4 hours + +#### Step 13. +Prepare a dilution series of reagents for the binding and competition assays in a separate 96-well deep well plate as described in Figures 1 and 2. + +#### Step 14. +Prepare 2-fold serial dilutions of IRDye® 800CW EGF ranging from 1.0 ng/mL to 500 ng/mL according to the experiment layout shown in Figure 1. + +#### Binding Assay + +#### Step 15. +Prepare 1 mL of 1 µg/mL IRDye 800CW EGF in DMEM. + +#### Step 16. +Wells 1 and 2 are background controls and contain no IRDye 800CW EGF. + +#### Step 17. +Do not store; proceed immediately to step 21. + +#### Competition Assay + +#### Step 18. +Prepare 2-fold serial dilutions of unlabeled EGF diluted in DMEM ranging from 0.06 to 16 µg/mL according to the experiment layout shown in Figure 2. + +#### Step 19. +Prepare 1 mL of 32 µg/mL unlabeled EGF in DMEM. + +#### Step 20. +Do not store; proceed immediately to step 21. + +#### Step 21. +Retrieve cell-containing plate from 37 °C incubator. Remove starvation medium from cells by aspiration or inversion of the plate and blot excess medium by tapping plate gently on tissue. + +#### Step 22. +Transfer 100 µL Binding Assay mixtures to rows A-C of the plate and 100 µL Competition Assay mixtures to rows D-F into the cell-containing wells. Use a multi-channel pipette and transfer these mixtures quickly (20 sec), as cellular responses are quick. + +#### Step 23. +Incubate at 37 °C for 2 minutes. +- **Duration:** 2 minutes + +#### Step 24. +Prepare fresh Fixing Solution as follows: +- 1X PBS 45.0 mL +- 37% Formaldehyde 5.0 mL +- 3.7% Formaldehyde 50.0 mL + +#### Step 25. +Remove EGF-containing medium by aspiration or inversion. + +#### Step 26. +Immediately fix cells with the addition of 150 µL of fresh Fixing Solution and incubate at room temperature (RT) for 20 minutes with no shaking. **Add the Fixing Solution carefully by pipetting down the side of the wells to avoid detaching the cells.** +- **Duration:** 20 minutes + +#### Step 27. +Prepare Triton® Washing Solution as follows: +- 1X PBS 495 mL +- 10% Triton X-100 5 mL +- 1X PBS + 0.1% Triton X-100 500 mL + +#### Step 28. +Remove the Fixing Solution by aspiration. + +#### Step 29. +Wash with 200 µL of Triton Washing Solution for 5 minutes per wash to permeabilize the cells. (wash 1/4) +- **Duration:** 5 minutes +- **Notes:** Allow each wash to shake on a rotator for 5 minutes at RT. Do not allow cells/wells to become dry during washing. Add each wash immediately after the preceding wash is removed. + +#### Step 30. +Wash with 200 µL of Triton Washing Solution for 5 minutes per wash to permeabilize the cells. (wash 2/4) +- **Duration:** 5 minutes + +#### Step 31. +Wash with 200 µL of Triton Washing Solution for 5 minutes per wash to permeabilize the cells. (wash 3/4) +- **Duration:** 5 minutes + +#### Step 32. +Wash with 200 µL of Triton Washing Solution for 5 minutes per wash to permeabilize the cells. (wash 4/4) +- **Duration:** 5 minutes + +#### Step 33. +Remove the Triton Washing Solution by aspiration or inversion. + +#### Step 34. +To each well, carefully add 150 µL of LI-COR Odyssey® Blocking Buffer (P/N 927-40000) + 0.1% Tween® 20 down the side of the wells, and incubate for 1 hour at RT with moderate shaking on a rotating platform. +- **Duration:** 1 hour + +#### Step 35. +IRDye® 800CW EGF will be detected in the 800 nm channel. CellTag™ can be used to normalize the signal from binding of the labeled EGF, to correct for variations in cell number from well to well. CellTag 700 Stain will be detected in the 700 nm channel. + +#### Step 36. +Add 50 µL of LI-COR Odyssey Blocking Buffer + 0.1% Tween 20 to Well 1. This will serve as a control for any potential background. + +#### Step 37. +Dilute 0.1 mM CellTag 700 Stain 1:500 in Odyssey Blocking Buffer. Add 50 µL to each well (except Well 1) and incubate for 1 hour with gentle shaking. +- **Duration:** 1 hour +- **Notes:** Protect from light. + +#### Step 38. +Prepare Tween Washing Solution as follows: +- 1X PBS 995 mL +- 20% Tween 20 5 mL + +#### Step 39. +Remove Odyssey Blocking Buffer and CellTag 700 Stain solution by aspiration or inversion. + +#### Step 40. +Wash the plate with Tween Washing Solution by gently adding solution down the side of the wells to avoid detaching the cells. Use a generous amount of solution (200 µL/well). (wash 1/5) + +#### Step 41. +Allow wash to shake gently on a rotator for 5 minutes at RT. +- **Duration:** 5 minutes +- **Notes:** Protect plate from light during washing. + +#### Step 42. +Wash the plate with Tween Washing Solution by gently adding solution down the side of the wells to avoid detaching the cells. Use a generous amount of solution (200 µL/well). (wash 2/5) + +#### Step 43. +Allow wash to shake gently on a rotator for 5 minutes at RT. (wash 2/5) +- **Duration:** 5 minutes + +#### Step 44. +Wash the plate with Tween Washing Solution by gently adding solution down the side of the wells to avoid detaching the cells. Use a generous amount of solution (200 µL/well). (wash 3/5) + +#### Step 45. +Allow wash to shake gently on a rotator for 5 minutes at RT. (wash 3/5) +- **Duration:** 5 minutes + +#### Step 46. +Wash the plate with Tween Washing Solution by gently adding solution down the side of the wells to avoid detaching the cells. Use a generous amount of solution (200 µL/well). (wash 4/5) + +#### Step 47. +Allow wash to shake gently on a rotator for 5 minutes at RT. (wash 4/5) +- **Duration:** 5 minutes + +#### Step 48. +Wash the plate with Tween Washing Solution by gently adding solution down the side of the wells to avoid detaching the cells. Use a generous amount of solution (200 µL/well). (wash 5/5) + +#### Step 49. +Allow wash to shake gently on a rotator for 5 minutes at RT. (wash 5/5) +- **Duration:** 5 minutes + +#### Step 50. +After final wash, remove wash solution completely from wells. Turn the plate upside down and tap or blot gently on paper towels to remove traces of wash solution. For best results, scan plate immediately; plates may also be stored at 4 °C for up to several weeks (protected from light). + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/one-step-growth-curves-for-cellulophaga-phages-eqfbdtn.md b/markdown-output/one-step-growth-curves-for-cellulophaga-phages-eqfbdtn.md new file mode 100644 index 0000000000000000000000000000000000000000..f24732937b2c073c4d67bfaf0d8af1ab781a55e9 --- /dev/null +++ b/markdown-output/one-step-growth-curves-for-cellulophaga-phages-eqfbdtn.md @@ -0,0 +1,182 @@ +```markdown +# Goal/Experiment: +The goal of this experiment is to utilize one-step growth curves to make determinations about the life cycle of Cellulophaga phages on a particular host. By following the infection during one lifecycle phase of the host, a growth curve can be constructed, and the burst size can be calculated. + +# One-step Growth Curves for Cellulophaga Phages + +## Bonnie Poulos +Published: 15 Mar 2016 +Citation: Bonnie Poulos One-step growth curves for Cellulophaga phages. protocols.io dx.doi.org/10.17504/protocols.io.eqfbdtn + +### Abstract +One-step growth curves are used to make determinations about the life cycle of a virus on a particular host. By following a virus infection during one life cycle phase of the host, a growth curve can be constructed, and the burst size can be calculated. + +### Guidelines +Note: One-step growth experiment instructions are for MOI=0.1. + +## Before Start +Before performing a one-step experiment, you need to determine when the host is growing exponentially. You must also determine the titer of the phage lysate that will be used for the experiment. These are described in the protocol. + +## Protocol + +### Host Growth Curve + +#### Step 1: +Inoculate a new culture; i.e., pick a colony into a new flask containing 10 ml of MLB media. + +> **NOTE:** +> - *Cellulophaga* should grow at room temperature on the benchtop. + +#### Step 2: +Immediately after the transfer, take a ‘time 0’ growth reading (T0). + +> **Growth Reading:** +> - Take the T0 growth reading at 595nm using 200 µl of culture, in duplicate. +> - Ensure the microtiter plate is clean inside and out. +> - Subtract the media blank reading from the culture reading to arrive at the OD595 for the culture. + +##### Step 2.1: +Pipet 200 µl of MLB media into wells A1 and A2 of a white microtiter plate (These are 'blanks'). + +##### Step 2.2: +Pipet 200 µl of the sample (the new culture just inoculated) into wells B1 and B2 of the same plate. + +> **NOTE:** +> - Ensure there are no bubbles in the wells; they will affect the readings. Pipet away any bubbles. + +##### Step 2.3: +Read the plate on the plate reader to take absorbance reading at 595nm. + +#### Step 3: +Continue taking readings as performed in Step 2 periodically. + +> **NOTE:** +> - Graph the results as you go! +> - Infect the host in med-exponential (log-linear) phase, when OD ≅ 0.02. +> - Start with longer intervals (1-2 hours) until growth starts, then shorter intervals (15-30 minutes). Ensure to adjust intervals based on the rate of growth. + +### Phage Lysate Titer + +#### Step 4: +Do a plaque assay to determine the PFU/ml of the lysate you plan to use. + +#### Step 5: +Calculate the volume needed for 10^7 phages. + +> **NOTE:** +> - If 10^7 phages are contained in less than 1 µl, dilute the lysate prior to performing the growth curve experiment. + +### One-Step Growth Experiment + +#### Step 6: +Determine the concentration of your culture at the time you start the infection. + +> **Correlation:** +> - Use a correlation of readings from the plate reader and cell counts (CFU, DAPI, or FCM counts) to estimate this. + +#### Step 7: +Calculate the volume of host culture needed for 10^8 cells. + +#### Step 8: +Pipet this amount into a 1.5 ml tube. + +#### Step 9: +Add 10^7 phages to the tube and start your timer for 15 minutes for the phages to adsorb to the host cells. + +#### Step 10: +After 15 minutes, dilute the infection 1:1000 in MLB media in a 250 ml flask. + +> **NOTE:** +> - For example, if you have 50ml of MLB in the flask, add 50 µl of host for a 1:1000 dilution. + +#### Step 11: +Take a sample immediately after dilution—this is 'time 0'. + +#### Step 12: +Process samples to be centrifuged as follows: + +##### Centrifuged Sample Steps: + +###### Step 12.1: +Pipet 100 µl from the flask into 900 µl of MSM in a 15 ml tube (diluting the sample 10x: 10^-1). + +> **NOTE:** +> - Knowing the expected concentration allows proper dilution for early sample counts. + +###### Step 12.2: +Vortex briefly. + +###### Step 12.3: +Centrifuge for 5 min at 1000 rpm. + +###### Step 12.4: +Very carefully remove the tube (being careful not to disturb the pellet!) and plate 100 µl. + +#### Step 13: +Process samples not to be centrifuged as follows: + +##### Non-Centrifuged Sample Steps: + +###### Step 13.1: +Pipet 100 µl from the flask into 900 µl of MSM in a 1.5 ml tube. + +> **NOTE:** +> - Refer to protocol for details on preparation and dilution of the phage preparation. + +###### Step 13.2: +Vortex briefly. + +###### Step 13.3: +Plate 100 µl. + +#### Step 14: +Continue sampling in this way for 8 hours. + +> **NOTE:** +> - Plate generously on the first trial (10^-2, 10^-3, 10^-4, 10^-5) and use the results as a guide for repeat experiments. T1 and T2 can be at 1 and 2 hours, respectively, switching to every 30 minutes for the rest. + +#### Step 15: +Store the filtered samples at 4°C. + +#### Step 16: +The next day, count the plaques on all plates that have a countable number. + +#### Step 17: +Decide which dilution gives the best count at each time point for the next time performing the same phage-host pair. + +> **NOTE:** +> - Depending on the size of the plaques, a "good" count is somewhere between 10 and a few hundred. + +#### Step 18: +Count any new plaques the next day and add these to your original count. + +#### Step 19: +Count again on the third day. + +#### Step 20: +Calculate PFU/ml at each time point for both centrifuged (free phage only) and non-centrifuged (total phage) samples. + +#### Step 21: +Graph the results. + +#### Step 22: +Calculate burst size. + +##### Calculating Burst Size: + +###### Step 22.1: +Take the free phage average of the time points on the plateau before the burst (A). + +###### Step 22.2: +Take the free phage average of the time points on the plateau after the burst (B). + +###### Step 22.3: +Subtract A from B; this is the total burst or new phages released (C). + +###### Step 22.4: +Divide C by the number of infecting phage (total phages at T0 minus free at T0); this is the burst size. + +--- + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/one-step-growth-curves-for-pseudoalteromonas-phage-den3dd.md b/markdown-output/one-step-growth-curves-for-pseudoalteromonas-phage-den3dd.md new file mode 100644 index 0000000000000000000000000000000000000000..9b44a50e720a8f5a1089b122a9fe3ecb918e3823 --- /dev/null +++ b/markdown-output/one-step-growth-curves-for-pseudoalteromonas-phage-den3dd.md @@ -0,0 +1,124 @@ +```markdown +# Goal/Experiment: +The goal of this experiment is to perform one-step growth curves for Pseudoalteromonas phages to understand the phage replication cycle, including adsorption, eclipse, and burst sizes phases. + +# One-step Growth Curves for Pseudoalteromonas Phages + +## Abstract +**Citation:** Natalie Solonenko One-step growth curves for Pseudoalteromonas phages. [protocols.io](http://protocols.io). dx.doi.org/10.17504/protocols.io.den3dd +**Published:** 21 Jan 2016 + +## Guidelines +Before performing a one-step experiment, you must have documented three consecutive days of consistent growth curves for the host. + +## Host Growth Curve + +1. **Inoculation:** + - Inoculate a new culture 1:100; i.e., transfer 80 μl from yesterday’s culture to a new tube containing 8 ml of Zobell media. Pseudoalteromonas should grow at 21°C, shaking at 150 rpm. + +2. **Initial Reading:** + - Immediately after the transfer, take a ‘time 0’ reading. + - Pipet 200 μl of Zobell media into wells A1 and A2 of a white microtiter plate. This is your ‘blank’. + - Pipet 200 μl of the sample (the new culture you just inoculated) into wells B1 and B2 of the same plate. + - Ensure that there are no bubbles in the wells, as they will affect your readings. Pipet away any bubbles. + - Read the plate on the plate reader. + +3. **Ongoing Readings:** + - Continue taking readings in this way periodically. + - Start with longer intervals (30–60 minutes) until you start to see growth. Then use shorter intervals (15–30 minutes) until the growth starts to level off. This should take anywhere from 5 to 10 hours. If it’s taking a while, you can go back to reading at longer intervals. + - Graph the results as you go! + +4. **Final Reading:** + - Take one final reading the next morning. + +5. **Repetition:** + - Inoculate a new culture 1:100 and repeat until you have 3 consecutive days of consistent growth. + +### Phage Lysate Titer Determination + +1. **Plaque Assay:** + - Do a plaque assay to determine the PFU/ml of the lysate you plan to use. +2. **Calculate Volume:** + - Calculate the volume needed for 107. If this is less than 1 μl, you will need to dilute your lysate first. + +## One-step Growth Experiment (instructions for MOI=0.1): + +1. **Timing of Infection:** + - Determine when during the growth of your host you should start the one-step experiment. Compare your 3 growth curves and see when the host is in mid-exponential (log linear) phase. This is the right time to start an infection. + +2. **Concentration Determination:** + - Determine the concentration of your culture at the time you want to start the infection. Use a correlation of readings from the plate reader and cell counts (CFU, DAPI, or FCM counts) to estimate this. + +3. **Volume Calculation:** + - Calculate the volume of host culture needed for 108. + +4. **Triplicate Infections:** + - Pipet this amount into three 1.5 ml tubes, labeled A, B, and C. + +5. **Phage Addition:** + - Add 107 phages to each tube and start your timer for 15 minutes to allow the phages to adsorb to the host cells. + +6. **Dilution & Shaking:** + - After 15 minutes, dilute each infection 1:100 in Zobell media in a 250 ml flask (also labeled A, B, and C). These flasks should be in the shaker for the duration of the experiment. + +7. **Sampling (Time 0):** + - Take a sample immediately after dilution. This is ‘time 0’. + - 0.2 μm filter 1 ml from each flask into a 1.5 ml tube. Use a 25 mm or smaller syringe filter. + + - If your diluted infection is less than 40 ml, you will need to take smaller samples. Never use more than 25% of the volume for the experiment. You can use as little as 500 μl if necessary. + +8. **Ongoing Sampling:** + - Continue sampling every 20 minutes for 3 hours. + - A total of 10 time points: T0 – T9. + +9. **Storage:** + - Store the filtered samples at 4°C. + +## Evaluating Samples from the One-step Experiment + +1. **Timely Analysis:** + - Try to analyze the samples as soon after the one-step experiment as possible so they don’t degrade. + +2. **Expected Concentration Calculation:** + - Calculate the expected concentration of phages at time 0. This will depend on the total volume of the initial infection (i.e., the volume of 108 cells plus 107 phages). So the concentration at T0 should be 107 volume of infection, divided by 100 (for the 1:100 dilution). Convert this to phages per ml. + +3. **Plating for Countable Samples:** + - Once you know how many phages to expect (usually near 105), you know what dilutions of your early samples to plate to get good counts. For example, if the T0 expected concentration is 105, there should be 100 plaques if you plate 100 μl of a 10-2. + + - Keep in mind that some of the phages will have adsorbed to cells and will not show up as plaques, so the actual concentration you calculate at T0 will be less than the expected concentration. This difference provides the number of phages that infected a cell during the 15-minute adsorption period. + +4. **Plating in Triplicate:** + - All samples should be plated and counted in triplicate to account for plating errors. + +5. **Plating Procedure:** + - Start by plating one replicate of all 30 samples. + - Plating all samples on one day reduces errors due to phages degrading over time. Plate several dilutions of each sample to ensure that you will be able to get a good count. + - A general guideline is that the phage concentration will increase by 10–100x during the burst, which should be at about the midpoint of the experiment. However, the burst can happen earlier or later and the exact size of it will vary, so give yourself some leeway in the dilutions you plate. + +6. **Counting Plaques:** + - The next day, count the plaques on all plates that have a countable number of them. Decide what dilution gives the best count at each time point. + - Depending on the size of the plaques, a good count will be somewhere between 10 and a few hundred. + +7. **Plating Replicates:** + - Plate two more replicates of each time point. Use the dilution you decided was best. + +8. **New Plate Counts:** + - The next day, count plaques on the new plates. Also, check the first replicate to see if any new plaques have appeared. Add these to your original count. + +9. **Repeated Checking:** + - The next day, check replicates two and three for new plaques. + +10. **Reporting Results:** + - When reporting results of the one-step, first average the three plating replicates for each time point. Then average the three biological replicates (A, B, and C) and calculate standard deviations for each time point. Graph the results. + +11. **Calculating Burst Size:** + - Calculate burst size: + - Take the average of the time points on the plateau before the burst (A). + - Take the average of the time points on the plateau after the burst (B). + - Subtract A from B. This is the total burst (C). + - Divide C by the number of infecting phage (expected phages at T0 minus actual). This is the burst size. + +--- + +**endofoutput** +``` \ No newline at end of file diff --git a/markdown-output/ont-da-tailing-for-fungal-barcoding-ckbqusmw.md b/markdown-output/ont-da-tailing-for-fungal-barcoding-ckbqusmw.md new file mode 100644 index 0000000000000000000000000000000000000000..94eca83858634bb8183bd24b30d25703e7f09226 --- /dev/null +++ b/markdown-output/ont-da-tailing-for-fungal-barcoding-ckbqusmw.md @@ -0,0 +1,135 @@ +```markdown +# Goal/Experiment: +This experiment details the protocol for dA-tailing, an enzymatic method for adding a non-templated nucleotide to the 3' end of a blunt, double-stranded DNA molecule in fungal barcoding. The process enables ligation of adapters necessary for subsequent sequencing steps. + +# ONT dA-tailing for Fungal Barcoding V.3 +**Stephen Douglas Russell** +*The Hoosier Mushroom Society* + +## ABSTRACT +This protocol is for dA-tailing, which is an enzymatic method for adding a non-templated nucleotide to the 3' end of a blunt, double-stranded DNA molecule. In other words, this adds A-chains to the end of the PCR product, creating a site for the ligation adapter to attach to. The process is simple: create a reaction with three chemicals, and clean up the product with beads. + +**Time required:** ~45 minutes + +The NEB protocol this is based on can be found [here](https://dx.doi.org/10.17504/protocols.io.xymvnmze9g3p/v3). + +**Protocol Citation:** Stephen Douglas Russell 2023. ONT dA-tailing for Fungal Barcoding. [protocols.io](https://dx.doi.org/10.17504/protocols.io.xymvnmze9g3p/v3) Version created by Stephen Douglas Russell + +**License:** This is an open access protocol distributed under the [Creative Commons Attribution License](https://creativecommons.org/licenses/by/4.0/). + +**Protocol status:** Working (We use this protocol and it's working) + +**Created:** Dec 09, 2022 +**Last Modified:** Mar 13, 2023 +**PROTOCOL integer ID:** 73808 + +## MATERIALS + +### Reagents +- **[NEBNext Ultra II End Repair/dA-Tailing Module](https://www.neb.com/products/e7546-nebnext-ultra-ii-end-repair-da-tailing-module) - 24 reactions (New England Biolabs Catalog #E7546S)**: $283.00 per 24 reactions +- **Molecular Water (IBI Scientific Catalog #IB42130)**: or any molecular-grade water +- **[HighPrep™ PCR Clean-up System](https://magbiogenomics.com/product/highprep-pcr-clean-up-system/) (MagBio Genomics Inc. Catalog #AC-60050)**: $117.88 per 50 mL, $0.047 per reaction (any bead cleanup will work) + +### Cost Breakdown +| Item | Cost | +|----------------------------------------|---------------------------| +| Total per Flongle run (1/2 rxns) | $5.95 | +| Total per MinION run | $11.85 | +| Total per 96 samples | $0.061 | +| Total per sample (Flongle: 480 samples)| $0.012 | +| Total per sample (Flongle: 672 samples)| $0.0089 | +| Total per sample (Flongle: 960 samples)| $0.0061 | + +### Consumables +- **Eppendorf DNA LoBind 1.5mL tubes** +- **0.2mL PCR tubes ([Amazon](https://www.amazon.com/))**: $12.83 +- **10µL pipette tips** +- **100-200µL pipette tips** + +### Equipment +- **Vortex mixer** +- **Mini centrifuge** +- **PCR cleanup magnet** +- **10µL Pipette** +- **100µL Pipette** +- **Hula mixer ([Ebay](https://www.ebay.com))**: $200.00 (optional) +- **Quantus or Qubit Fluorometer (optional)** + +## PROTOCOL STEPS + +### 1. Preliminary Setup +1. **Heat Molecular Water:** + - Put a 1.5mL tube of molecular water on a heat block at 55°C. This will be used after the cleanup step towards the end of this protocol. +2. **Thaw Reagents:** + - Thaw the NEB End-prep Reaction Buffer and NEB End-prep Enzyme Mix at room temperature. This won't take long. + - If continuing with adapter ligation after this step, set the necessary reagents on ice to thaw as well. + +### 2. Optional DNA Control +1. **Thaw DNA CS (optional):** + - Thaw DNA CS at room temperature, spin down for 5 seconds, mix by pipetting, and place on ice. + - (DNA CS is a standard DNA sequence used for quality control for sequencing and alignment. Generally unnecessary for DNA barcoding with hundreds of repeats of a single amplicon.) + +### 3. Prepare Amplicon DNA +1. **Mix Amplicon DNA:** + - Mix your amplicon DNA pool thoroughly with a pipette (pipette up and down 5 times). Briefly spin down for 5 seconds. +2. **Vortex Reaction Buffer:** + - Vortex the Ultra II End-prep Reaction Buffer for 30 seconds. (Do not vortex the End-prep Enzyme Mix.) + +### 4. Create Reaction Mixture +1. **Combine Reagents:** + - In a 0.2mL thin-walled, sterile, nuclease-free tube, combine the following in order. Mix each reagent together after it is added by gently pipetting the entire volume up and down 10-20 times for each addition. + - **Ideal amplicon DNA concentration**: 0.5ng per 50mL for Flongle or 1µg DNA per 50µL for R10.4.1. + +| Component | R10.4.1 Flongle Volume | R10.4.1 MinION Flowcell Volume | +|-----------|------------------------|--------------------------------| +| DNA CS (optional) | 0.5µL | 1µL | +| Amplicon DNA | 24.5µL (0.5ng) | 49µL (1ng) | +| Ultra II End-prep Reaction Buffer | 3.5µL | 7µL | +| Ultra II End-prep Enzyme Mix | 1.5µL | 3µL | +| **Total** | 30µL | 60µL | + +1. Spin down the tube in a mini centrifuge for 5 seconds. + +### 5. Incubate Reaction +1. **Thermocycler Program:** + - Incubate in a thermocycler using the following program: + - 20°C for 5 minutes + - 65°C for 5 minutes + - 4°C Hold +2. Spin down the tube for 5 seconds in a mini centrifuge. + +### 6. Clean-Up and Final Steps +1. **Transfer Reaction:** + - Transfer the entire 30µL/60µL reaction to a new 1.5mL LoBind eppi tube. +2. **Bead Cleanup:** + - Resuspend magnetic beads in solution by vortexing. Add 30µL (Flongle)/60µL (MinION) of beads to the reaction (1X bead cleanup) and mix gently by pipetting up and down. +3. **Incubate and Spin:** + - Incubate at room temperature for 5 minutes. + - Spin down the tube in a mini centrifuge for 5-10 seconds. +4. **Magnetic Separator:** + - Place sample tube on the magnetic separator for 2 minutes or until the solution clears. Beads should now be on the side of the tube. + - With the tube still on the magnet, remove the liquid from the tube and discard. Be sure not to disturb the beads. +5. **Ethanol Wash:** + - With the tube still on the magnet, add 200µL of 80% ethanol to the tube and let sit for 2 minutes. + - Remove ethanol by pipetting and discard. + - Repeat the ethanol wash one time. +6. **Dry Beads:** + - Spin down for 5 seconds and place the tube back on the magnet. Pipette off any residual ethanol. Allow to dry for ~30 seconds, but do not dry the pellet to the point of cracking. +7. **Release DNA:** + - Remove the tube from the magnet and add 31µL (for Flongle) or 61µL (for MinION) of molecular water. Pipette up and down five times to mix until the pellet is fully suspended. DNA will now be released from the beads and suspended in water. + - Incubate for 2 minutes at room temperature. + - Place the tube back on the magnet for 2 minutes or until the solution is clear. +8. Transfer the water containing the DNA to a new 1.5mL LoBind eppi tube. You should now have your A-tailed DNA template. + +## DNA Quantification +1. **Quantify DNA:** + - If you have access to a Quantus or Qubit fluorometer, now is a good time to quantify the resulting amount of DNA in your purified sample. + - If not, the 31µL/61µL of molecular water should put you in the ballpark of the right DNA concentration. + - Typically, a concentration of 11-33 ng/µL is achieved at this point and used at this level for the next step. No dilutions or adjustments. +2. **Storage:** + - It is possible to break and store the sample at 4°C overnight if needed. It would be ideal to continue on to adapter ligation at this time. + +--- + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/ont-flongle-flowcell-loading-with-q20-chemistry-cayvsfw6.md b/markdown-output/ont-flongle-flowcell-loading-with-q20-chemistry-cayvsfw6.md new file mode 100644 index 0000000000000000000000000000000000000000..a7639241b48e9f75449da7d2c83b59be91454aba --- /dev/null +++ b/markdown-output/ont-flongle-flowcell-loading-with-q20-chemistry-cayvsfw6.md @@ -0,0 +1,109 @@ +```markdown +# Goal/Experiment: +The goal of the experiment is to describe the steps used to load a Flongle flowcell utilizing the Q20+ Ligation Sequencing Kit from Oxford Nanopore Technologies (ONT). + +# ONT Flongle Flowcell Loading with Q20+ Chemistry + +**Author:** +Stephen Douglas Russell\ +The Hoosier Mushroom Society + +**Overview:** +This protocol describes the steps used to load a Flongle flowcell utilizing the Q20+ Ligation Sequencing Kit from ONT. + +**Time required:** +10 minutes + +[Reference paper on protocols.io](https://protocols.io/view/ont-flongle-flowcell-loading-with-q20-chemistry-cayvsfw6) + +## Reagents and Consumables + +| Reagent | Vendor | Catalog Number | +| --- | --- | --- | +| Ligation Sequencing Kit (Q20+) | Oxford Nanopore Technologies | SQK-LSK112 | +| Flongle Sequencing Expansion | Oxford Nanopore Technologies | EXP-FSE001 | +| 100-200 µL pipette tips | | | +| 10 µL pipette tips | | | +| 1.5 µL Eppendorf LoBind tubes | | | + +## Equipment + +- 10 µL pipette +- 100 µL pipette +- Mini centrifuge +- Flongle Starter Pack: $1,460.00 +- MinION Mk1B Starter Pack: $1,000.00 + +## Software + +- MinKNOW + +--- + +## Procedure + +### Starting out + +1. Before starting, watch the first 13 minutes of this video: [https://vimeo.com/651243660](https://vimeo.com/651243660) + + It covers all of the vital aspects of this protocol. + +2. Attach the USB cable from the computer to the MiniION device. Place the Flongle module with the white configuration cell into the MiniION device. The top portion of the Flongle should slide underneath the clip and it should gently sit on the top of the device. + + Open MinKNOW on the computer and perform a hardware check on the configuration cell. The hardware check should pass. + +3. Wearing gloves, remove a flowcell from the fridge and remove the outer packaging. Remove the configuration cell and place the flowcell into the Flongle device on the MinION. Be sure not to touch any of the electrodes on the back of the cell or the contact pads on the top of the Flongle. It should snap into place with a click. Place the configuration module into the empty pouch from the Flongle until the run is completed. Run a flowcell check within the MinKNOW software. There should be at least 60 useable pores. + + - My first flowcell test had 87 useable pores. + +### Prepare the loading solution + +4. Thaw the Sequencing Buffer II (SBII), Loading Beads (LBII), Flush Buffer (FB), and Flush Tether (FLT) at room temperature. + +5. Mix the Sequencing Buffer II (SBII), Flush Buffer (FB) and Flush Tether (FLT) tubes by vortexing and spin down at room temperature. + +6. In a 1.5 µL tube, mix 117 µL of the Flush Buffer (FB) with 3 µL of the Flush Tether (FLT) and mix by pipetting. + +7. Peel back the seal tab from the Flongle flow cell to the point where the sample port is exposed and hold the seal tab open by using the adhesive on the tab to stick it to the lid of the MinION. + + - Video of the process: [https://youtu.be/zlkTfRf8g7I](https://youtu.be/zlkTfRf8g7I) (2 minutes) + +8. Using a 200 µL pipette, bring up the FB/FLT mix into the tip. This is your priming solution. + + - **VERY IMPORTANT:** There should be NO air bubble in the tip of the pipette. Introducing any air bubbles will destroy any nanopores that the air bubble contacts. If there is any air in the tip, turn the dial of the pipette counter-clockwise (towards 0) to move the air bubble out of the tip. + +9. Place the tip of the pipette securely into the sample port. Turn the dial of the pipette clockwise a time or two and you should see a small amount of yellow-green fluid come into the tip. This helps to ensure there are no air bubbles present. + + Once confirmed, slowly dispense the priming fluid into the flowcell by slowly turning the dial of the pipette counter-clockwise (towards 0). Do not push down on the plunger to eject the fluid. + + - You should be able to see the storage liquid entering the waste port as you enter it into the flowcell. + - **STOP LOADING** before the liquid in the tip reaches the bottom. You do not want to push any air into the flowcell. + +### Prepare Library + +10. In a new 1.5 µL tube, add the reagents below in the order they are listed. + + Note: The LBII settles quickly, so mix it thoroughly before removing it from the tube. + + | Reagent | Volume | + | --- | --- | + | Sequencing Buffer II (SBII) | 13.5 µL | + | Loading Beads II (LBII) | 11 µL | + | DNA library (3 - 20 fMol) | 5.5 µL | + | **Total** | **30 µL** | + + Mix the solution by gently pipetting up and down. + +11. Bring the entire volume into the tip of a pipette. + + - **VERY IMPORTANT:** Once again make sure there are no air gaps. Insert the tip of the pipette into the sample port and slowly dispense the library into the flowcell by slowly turning the dial of the pipette counter-clockwise (towards 0). Do not push down on the plunger to eject the fluid. + - **STOP LOADING** before the liquid in the tip reaches the bottom. You do not want to push any air into the flowcell. + +12. Seal the Flongle cell by folding the tab back down, using the adhesive on the seal tab. Ensure that the wheel section covers the loading port and the two dots cover each waste port. Gently press over the tab to ensure a good seal. (Do not do a wiping press, just an up-and-down press.) + +13. Close the MinION lid and start your sequencing run. + +--- + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/ont-flongle-flowcell-loading-with-q20-v12-chemistr-cdjss4ne.md b/markdown-output/ont-flongle-flowcell-loading-with-q20-v12-chemistr-cdjss4ne.md new file mode 100644 index 0000000000000000000000000000000000000000..54337531d23b595e691fdde2d227c4899546e82b --- /dev/null +++ b/markdown-output/ont-flongle-flowcell-loading-with-q20-v12-chemistr-cdjss4ne.md @@ -0,0 +1,122 @@ +```markdown +# Goal/Experiment: +The goal of this experiment is to load a Flongle flowcell utilizing the Q20+ (V12) Ligation Sequencing Kit from ONT and to ensure optimal conditions for sequencing using the MinION device. + +## ONT Flongle Flowcell Loading with Q20+ (V12) Chemistry V.2 + +**Author:** Stephen Douglas Russell +**Affiliation:** The Hoosier Mushroom Society +**Date:** July 17, 2022 + +### Abstract + +**Overview:** +This protocol describes the steps used to load a Flongle flowcell utilizing the Q20+ (V12) Ligation Sequencing Kit from ONT. + +This protocol has been tested with Flongle R9.4.1 flowcells. + +**Time required:** 10 minutes + +**DOI:** [dx.doi.org/10.17504/protocols.io.ewov1nm5pgr2/v2](https://dx.doi.org/10.17504/protocols.io.ewov1nm5pgr2/v2) + +**Protocol Citation:** +Stephen Douglas Russell 2022. ONT Flongle Flowcell Loading with Q20+ (V12) Chemistry. _protocols.io_. [https://dx.doi.org/10.17504/protocols.io.ewov1nm5pgr2/v2](https://dx.doi.org/10.17504/protocols.io.ewov1nm5pgr2/v2) + +--- + +## Reagents and Consumables + +| Item | Vendor | Catalog Number | +|------------------------------|-------------------|------------------------| +| Ligation Sequencing Kit (Q20)| Oxford Nanopore | SQK-LSK112 | +| Flongle Sequencing Expansion | Oxford Nanopore | EXP-FSE001 | +| 100-200uL pipette tips | Any | | +| 10uL pipette tips | Any | | +| 1.5uL Eppendorf LoBind tubes | Eppendorf | | + +## Equipment + +- 10uL pipette +- 100uL pipette +- Mini centrifuge +- Flongle Starter Pack: $1,460.00 +- MinION Mk1B Starter Pack: $1000.00 + +## Software + +- MinKNOW + +--- + +## Protocol Steps + +### Starting Out + +1. **Preparation Video:** + - Before starting, watch the first 13 minutes of this [video](https://vimeo.com/651243660). It covers all vital aspects of this protocol. + +2. **Setup Hardware:** + - Attach the USB cable from the computer to the MiniION device. + - Place the Flongle module with the white configuration cell into the MiniION device. The top portion of the Flongle should slide underneath the clip and should gently sit on the top of the device. + - Open MinKNOW on the computer and perform a hardware check on the configuration cell. The hardware check should pass. + +3. **Prepare Flowcell:** + - Wearing gloves, remove a flowcell from the fridge and remove the outer packaging. + - Remove the configuration cell and place the flowcell into the Flongle device on the MinION. Ensure not to touch any of the electrodes on the back of the cell or the contact pads on the top of the Flongle. + - Run a flowcell check within the MinKNOW software. There should be at least 60 useable pores. Future tests show useable pores above this range can be maintained up to 6 weeks post receipt. + +### Prepare the Loading Solution + +4. **Thaw Reagents:** + - Thaw the Sequencing Buffer II (SBII), Loading Beads (LBII), Flush Buffer (FB), and Flush Tether (FLT) at room temperature. + - SBII, LBII, and FLT are found in the Ligation Sequencing Kit (Q20+). + - FB and FLT are found in the Flongle Sequencing Expansion Kit. + +5. **Mix Reagents:** + - Vortex and spin down at room temperature: + - SBII, FB, and FLT. + +6. **Prepare Priming Solution:** + - In a 1.5uL tube, mix 117uL FB with 3uL FLT and mix by pipetting. + +7. **Prepare Flowcell for Loading:** + - Peel back the seal tab from the Flongle flow cell to the point where the sample port is exposed. Hold the seal tab open by using the adhesive on the tab to stick it to the lid of the MinION. + - [Video of the process](https://youtu.be/zIkTtRf8g7I) (2 minutes) + +8. **Prime the Flowcell:** + - Using a 200uL pipette, draw up the FB/FLT mix into the tip. + - Ensure no air bubbles are present. + - Place the tip of the pipette securely into the sample port. Turn the dial of the pipette clockwise a time or two until a small amount of yellow-green fluid comes into the tip. + - Dispense the priming fluid into the flowcell by slowly turning the dial of the pipette clockwise (towards 0). Do not push down on the plunger to eject the fluid. + - STOP LOADING before the liquid in the tip reaches the bottom to avoid air bubbles entering the flowcell. + +### Prepare Library + +10. **Prepare DNA Library:** + - In a new 1.5uL tube, add the following reagents in the order listed: + + | Reagent | Volume | + |------------------------------|--------| + | Sequencing Buffer II (SBII) | 13.5uL | + | Loading Beads II (LBII) | 11uL | + | 3 - 20 fMol of the DNA library| 5.5uL | + + - Mix the solution by gently pipetting up and down. + +11. **Load DNA Library:** + - Bring the entire volume of the prepared library into the tip of a pipette. + - Ensure no air gaps are present. + - Insert the tip of the pipette into the sample port and slowly dispense the library into the flowcell by turning the dial of the pipette clockwise (towards 0). Do not push down on the plunger to eject the fluid. + - STOP LOADING before the liquid in the tip reaches the bottom to avoid pushing any air into the flowcell. + +12. **Seal Flowcell:** + - Seal the Flongle cell by folding the tab back down, using the adhesive on the seal tab. Ensure the wheel section covers the loading port and the two dots cover each waste port. Gently press over the tab to ensure a good seal. + - Do not do a wiping press, just an up-and-down press. + +13. **Start Sequencing Run:** + - Close the MinION lid and start your sequencing run. + +--- + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/ont-flongle-flowcell-loading-with-q20-v14-chemistr-cq44vyyw.md b/markdown-output/ont-flongle-flowcell-loading-with-q20-v14-chemistr-cq44vyyw.md new file mode 100644 index 0000000000000000000000000000000000000000..31d4bed0f2905cb535a5cbaacd9dea2c6e391582 --- /dev/null +++ b/markdown-output/ont-flongle-flowcell-loading-with-q20-v14-chemistr-cq44vyyw.md @@ -0,0 +1,147 @@ +``` +Goal/Experiment: +The goal of this experiment is to describe the steps used to load a 10.4.1 Flongle flowcell utilizing the Q20+ (V14) Ligation Sequencing Kit from Oxford Nanopore Technologies (ONT). + +# ONT Flongle Flowcell Loading with Q20+ (V14) Chemistry V.4 + +**Author:** Stephen Douglas Russell +**Affiliation:** The Hoosier Mushroom Society +**Version:** 4 +**Date:** March 16, 2023 +**DOI:** [10.17504/protocols.io.eowv1nm5pgr2/v4](https://dx.doi.org/10.17504/protocols.io.eowv1nm5pgr2/v4) + +## Abstract + +### Overview +This protocol describes the steps used to load a 10.4.1 Flongle flowcell utilizing the Q20+ (V14) Ligation Sequencing Kit from ONT. This protocol has been tested with Flongle R10.4.1 flowcells. + +### Time Required +10-15 minutes + +## Materials + +### Reagents and Consumables +- **Ligation Sequencing Kit V14 (Catalog #SQK-LSK114)**: A kit used for preparing DNA or RNA samples for sequencing on ONT devices. +- **Flongle Sequencing Expansion (Catalog #XP-FSE002)**: An expansion pack for Flongle sequencing, containing the necessary reagents. +- **100-200uL pipette tips**: Sterile, disposable tips for pipetting small volumes of liquids. +- **10uL pipette tips**: Sterile, disposable tips for pipetting very small volumes. +- **1.5uL Eppendorf LoBind tubes**: Low-binding tubes to minimize sample loss. + +### Equipment +- **10uL pipette**: For accurate measurement of small liquid volumes. +- **100uL pipette**: For measuring larger volumes up to 100uL. +- **Mini centrifuge**: Used for quick spins of small volumes. +- **Flongle Starter Pack ($1,460.00)**: Includes Flongle adapter and flowcells. +- **MinION Mk1B Starter Pack ($1,000.00)**: Includes MinION device and necessary accessories. + +### Software +- **MinKNOW**: ONT's proprietary software for running sequencing devices and monitoring sequencing runs. + +## Protocol + +### Starting out + +1. **Preparation** + Before starting, watch the first 13 minutes of this [video](https://vimeo.com/651243660). It covers all of the vital aspects of this protocol. + +2. **Hardware Setup** + - Restart the computer to prevent any performance issues. + - Attach the USB cable from the computer to the MinION device. + - Place the Flongle module with the white configuration cell into the MinION device. Ensure the top portion slides under the clip. + - Open MinKNOW and perform a hardware check (Start → Hardware Check). + +![MinKNOW primary screen](https://dx.doi.org/10.17504/protocols.io.eowv1nm5pgr2/v4) + +3. **Flowcell Preparation** + - Wearing gloves, remove a flowcell from the fridge and packaging. + - Remove the configuration cell and place the flowcell into the Flongle device. + - Conduct a flowcell check within MinKNOW (Start → Flow cell check). Ensure there are at least 50 useable pores. If not, replace with a new cell. + +### Prepare the Loading Solution + +4. **Thaw Reagents** + Thaw the Sequencing Buffer (SB), Library Beads (LIB), Flow Cell Flush (FCF), and Flow Cell Tether (FCT) at room temperature. + - **Flow Cell Tether (FCT)** is found in the Ligation Sequencing Kit V14. + - Other reagents are in the Flongle Sequencing Expansion. + + [Original full V14 Ligation Sequencing Kit protocol](https://dx.doi.org/10.17504/protocols.io.eowv1nm5pgr2/v4) + +5. **Mix Reagents** + Mix the Sequencing Buffer (SB), Flow Cell Tether (FCT), and Flow Cell Flush (FCF) by vortexing and spinning down at room temperature. + +6. **Prepare Priming Solution** + In a 1.5uL tube, mix: + - 117uL of Flow Cell Flush (FCF) + - 3uL of Flow Cell Tether (FCT) + + Mix by pipetting. + +7. **Prepare Flowcell** + - Peel back the seal tab to expose the sample port. Secure the tab with the adhesive. + - Follow this [video process](https://youtu.be/zlkTfrf8g7I) (2 minutes). + +8. **Prepare Priming Pipette** + - Using a 200uL pipette, bring up the FB/FLT mix into the tip. Ensure there are no air bubbles. + - Place the tip securely into the sample port. + +9. **Dispense Priming Fluid** + - Slowly dispense the priming fluid into the flowcell using the pipette. Do not push down the plunger to eject. + +### Prepare Library + +10. **Library Mixing** + In a new 1.5uL tube, add the reagents in the following order: + + | Reagent | Volume | + |----------------------|--------| + | Sequencing Buffer (SB) | 15uL | + | Library Beads (LIB) | 10uL | + | DNA library | 5uL | + | **Total** | **30uL**| + + Mix by gently pipetting up and down 10-20 times. + +11. **Load Library** + - Bring the entire volume into the tip of a pipette. + - Insert the tip into the sample port and slowly dispense by turning the dial clockwise. + +12. **Seal Flowcell** + - Fold the tab back down using the adhesive. Ensure the wheel section covers the loading port and the two dots cover the waste port. Gently press to ensure a good seal. + +13. **Start Sequencing Run** + - Close the MinION lid and start the sequencing run. + - Navigate: Start → Start Sequencing. + +### Run Configuration + +1. **Positions** + Enter flow cell ID and sample ID, then continue to kit selection. + +2. **Kit Selection** + Choose: + - **Sample Type:** DNA + - **PCR-free:** PCR + - **Multiplexing:** Yes + Select Ligation Sequencing Kit (LSK-114). + +3. **Run Options** + Ensure all are at defaults: + - **Run Duration:** 24 hours + - **Minimum read length:** 200 base pairs + - **Active Channel Selection:** On + - **Time between pore scans:** 1.5 hours + - **Reserve pores:** off + +4. **Analysis** + - **Basecalling:** Off + +5. **Output** + Ensure all are at defaults: + - **Output location:** /var/lib/minknow/data/ + - **Output format:** fast5 + - **Output bulk file:** on + + On the final step, save the template for future runs. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/ont-v14-nanopore-adapter-ligation-for-fungal-dna-b-cq92vz8e.md b/markdown-output/ont-v14-nanopore-adapter-ligation-for-fungal-dna-b-cq92vz8e.md new file mode 100644 index 0000000000000000000000000000000000000000..17e8afbc179a323722b2d7ad94a485b1b6e95d49 --- /dev/null +++ b/markdown-output/ont-v14-nanopore-adapter-ligation-for-fungal-dna-b-cq92vz8e.md @@ -0,0 +1,175 @@ +```markdown +Goal/Experiment: +The goal of this protocol is to take an A-tailed library and add the nanopore adapters. This involves combining several chemicals for a single reaction and performing a bead cleanup. + +# ONT V14 Nanopore Adapter Ligation for Fungal DNA Barcoding V.5 + +**Version 5** +**March 17, 2023** + +Stephen Douglas Russell +The Hoosier Mushroom Society + +## Abstract +This process will take your A-tailed library and add the nanopore adapters. Simply combine several chemicals for a single reaction and do a bead cleanup. + +Tested with: +- Flowcells: Flongle 10.4.1 or MinION 10.4.1 +- Ligation Kit: V14 - LSK114 + +**Time required:** ~45 minutes + +DOI: [https://doi.org/10.17504/protocols.io.dm6gpb5zdlzp/v5](https://doi.org/10.17504/protocols.io.dm6gpb5zdlzp/v5) + +Protocol status: Working +Created: March 16, 2023 +Last Modified: March 17, 2023 +PROTOCOL integer ID: 78874 + +## Materials + +### Reagents +- **Ligation Sequencing Kit V14** + Oxford Nanopore Technologies, Catalog #SQK-LSK114 + \$694.43 per 6 reactions (\$115.74 per MinION run; \$57.87 per Flongle run) + +- **NEBNext Quick Ligation Module** + New England Biolabs, Catalog #E6056S + \$361.00 per 20 reactions (\$18.05 per MinION run; \$9.03 per Flongle run) + **Note:** This kit has two components. We use NEBNext Quick T4 DNA Ligase. + +- **HighPrep™ PCR Clean-up System** + MagBio Genomics Inc., Catalog #AC-6000 + \$117.88 per 50 mL (\$0.047 per reaction) + **Note:** Most magnetic beads from most vendors can be used with the same protocol. + +### Cost Analysis +- Total per Flongle run (1/2 reactions): \$66.95 +- Total per MinION run: \$133.84 +- Total per sample (Flongle: 480 samples): \$0.139 +- Total per sample (Flongle: 960 samples): \$0.07 + +### Consumables +- Eppendorf DNA LoBind 1.5mL tubes +- 10µL pipette tips +- 100-200µL pipette tips + +### Equipment +- PCR tube rack +- Vortex mixer +- Mini centrifuge +- PCR cleanup magnet +- 10µL Pipette +- 1000µL Pipette +- Hula mixer (optional, \$200.00 from Ebay) +- Quantus or Qubit Fluorometer (optional) + +## Adapter Ligation + +1. Spin down the Ligation Adapter (LA) and Quick T4 Ligase and place on ice. + +2. Thaw Ligation Buffer (LNB) at room temperature, spin down, and mix by pipetting. Due to viscosity, vortexing this buffer is ineffective. Place on ice immediately after thawing and mixing. + - **LNB** + Ligation Sequencing Kit V14, Oxford Nanopore Technologies, Catalog #SQK-LSK114 + +3. Thaw the Elution Buffer (EB) at room temperature, mix by vortexing, spin down and place on ice. + - **EB** + Ligation Sequencing Kit V14, Oxford Nanopore Technologies, Catalog #SQK-LSK114 + +4. Thaw one tube of Short Fragment Buffer (SFB) at room temperature, mix by vortexing, spin down and place on ice. + - **SFB** + Ligation Sequencing Kit V14, Oxford Nanopore Technologies, Catalog #SQK-LSK114 + +5. In a 1.5 ml Eppendorf DNA LoBind tube, mix in the following order: + Between each addition, pipette mix 10-20 times. + + | Reagent | 10.4.1 Flongle Volume | 10.4.1 MinION Volume | + |--------------------------|-----------------------|----------------------| + | DNA sample from previous step | 30µL | 60µL | + | Ligation Buffer (LNB) | 12.5µL | 25µL | + | NEBNext Quick T4 DNA Ligase | 5µL | 10µL | + | Ligation Adapter (LA) | 2.5µL | 5µL | + | Total | 50µL | 100µL | + +6. Spin down with a mini centrifuge for 5 seconds. + +7. Incubate the reaction for 10 minutes at room temperature. Pull the AMPure XP (AXP) from the freezer. + +8. Resuspend AMPure XP (AXP) magnetic bead stock by vortexing. + +9. Add 20µL (Flongle) or 40µL (MinION) of resuspended beads to the reaction and mix by flicking the tube. + +10. Incubate on a Hula mixer (rotator mixer) for 5 minutes at room temperature (or just place in a tube rack without the mixer). Pull the EB and SFB from the freezer. + +11. Spin down the sample for 5 seconds and pellet on a magnet for 2 minutes. + + Keep the tube on the magnet, and pipette off the supernatant. + +12. Wash the beads by adding 125µL (Flongle) or 250µL (MinION) of Short Fragment Buffer (SFB). Flick the beads to resuspend, spin down for 5 seconds, then return the tube to the magnetic rack for 2 minutes and allow the beads to pellet. Remove the supernatant using a pipette and discard. + + **Note:** Flicking the tube does not seem to fully resuspend the beads. You can choose to flick 10 times or so and spin down. I typically never flick it. + +13. Repeat the previous step. + +14. Spin down for 5 seconds and place the tube back on the magnet. Pipette off any residual supernatant. Allow to dry for ~30 seconds, but do not dry the pellet to the point of cracking. + +15. Remove the tube from the magnetic rack and resuspend the pellet in 7µL Elution Buffer (EB). Incubate for 10 minutes at room temperature. Start setting up your flowcell on the computer. Hardware and flowcell check. Also pull the Flongle Sequencing Expansion chemicals for loading from the freezer. + +16. Pellet the beads on a magnet until the eluate is clear and colorless, for at least 1 minute. + +17. Remove and retain 7µL of eluate containing the DNA library into a clean 1.5 ml Eppendorf DNA LoBind tube. + + Store on ice until you are ready to load in your flowcell. + +## Quantification + +18. If you have access to a Quantus or Qubit fluorometer, now is a good time to quantify 2µL of DNA in your sample. + + It is recommended loading 5 fmol to 10 fmol of this final prepared library onto your flow cells. Loading more than 20 fmol of DNA can reduce the rate of duplex read capture. Dilute the library in Elution Buffer if required. + + - For 900bp length DNA (what our ITS1F-4 reactions appear to average, with adapters), we are looking for: + - 10 fmol - 20 fmol = 0.006µg - 0.012µg of DNA. + + For a 22 ng/µL sample (Quantus quantification): + + \[ + 22\text{ng} \times 1\text{µg} = 0.022\text{µg/µL} \quad (\text{22/1000=0.022}) + \text{1µL} \times 1000\text{ng} + \] + + \[ + 0.022\text{µg/µL} \times 5\text{µL (elution buffer; or x7 if you did not quantify using 2µL)} + \] + + = 0.11 µg DNA in sample of 5µL elution buffer. + + **Note:** The 0.11 in the calculations below will change based on your individual DNA amount. + + **Note:** The ONT protocol suggests using additional EB in order to make concentration adjustments. As there is not a lot of the reagent in the standard packets, and it is not possible to buy more individually, I have been using molecular water for this step with no ill effect. + +### Flongle 10.4.1 Quantification +How much additional molecular water to have 5µL needed for the next step give us correct amount of DNA? + +\[ +0.11\text{µg} / x\text{µL} = 0.010\text{µg (17 fmol DNA)} +\] + +\[ +x = 11\text{µL} \times 5\text{µL} = 55\text{µL - 5µL} (\text{or 7 if you did not quantify}) = 50\text{µL} +\] + +Overall summary: + +\[ +(\text{[DNA Concentration]} / 1000 \times 5 \times 100 \times 5) - 5 = [\text{Amount of H2O to add}] +\] + +So at 0.022µg/µL quantification, add an additional 50µL of molecular water to have right concentration to use 5µL for the next step with Flongle. + +- 11ng/µL sample comes out to adding an additional 22.5µL of molecular water. +- 31ng/µL sample comes out to adding an additional 72.5µL of molecular water. + +I would use 50 µL of extra molecular water if you are not able to quantify your sample. + +**endofoutput** +``` \ No newline at end of file diff --git a/markdown-output/opentrons-dual-index-primer-plate-workflow-db4e2qte.md b/markdown-output/opentrons-dual-index-primer-plate-workflow-db4e2qte.md new file mode 100644 index 0000000000000000000000000000000000000000..8063513be63d4aa357c3bf4c7b8c8fca543e4fcb --- /dev/null +++ b/markdown-output/opentrons-dual-index-primer-plate-workflow-db4e2qte.md @@ -0,0 +1,110 @@ +```markdown +# Goal/Experiment: +The goal of this protocol is to utilize the Opentrons robotic automation platform to generate dual-index primer-master mix plates for PCR reactions for NGS, particularly for DNA barcoding large specimen pools with Oxford Nanopore Technologies (ONT) MinION devices. + +## Opentrons Dual-Index Primer Plate Workflow V.2 +### Stephen Douglas Russell1,2 +#### 1. Mycota Lab; 2. The Hoosier Mushroom Society + +### Abstract +This protocol is designed to utilize the Opentrons robotic automation platform to generate dual-index primer-master mix plates for PCR reactions for NGS, particularly for DNA barcoding large specimen pools with Oxford Nanopore Technologies (ONT) MinION devices. It is designed for the indexing strategy where you start with a plate of 96 different forward or reverse primers, and then use a single forward/reverse index for each PCR plate you are running for the pool. Each plate of reactions has a single unique forward tag corresponding with that plate number and a standard plate of 96 reverse tags. The protocol is broken down into three primary areas: + +1. **OT-2 "Working" Primer Plates - 100uM->10uM**: These steps and automated protocol turn a single 100uM primer plate into four 10uM "working" primer plates. +2. **OT-2 Dual-Index PCR Stock Plates**: This portion of the protocol generates four "stock" dual-index primer/master mix plates. +3. **OT-2 Dual-Index PCR Ready Plates**: This protocol creates eight PCR ready plates from one PCR stock plate. + +## Materials +### Equipment +- Opentrons OT2 or OT2R +- Plate centrifuge +- 200uL Pipette +- 1000uL Pipette or better +- Heat Sealer (optional) + +### Reagents +- **ONT-tagged Reverse Primers** - Eurofins or others, 96 well plate with 96 different primers. [Eurofins](https://www.eurofinsgenomics.com). +- **ONT-tagged Forward Primers** - Eurofins, 10uM forward primers (~1.15mL) x 8, one for each forward index. +- **Molecular Water** - IBI Scientific Catalog #IB42130: 1L is $39.23. +- **PCR Master Mix** - Empirical Bioscience: $770.99/100mL. + +### Consumables +- **Filter Tip Racks (20uL)** - Opentrons, $821.18/100 racks. +- **Filter Tip Racks (200uL or 300uL)** - Opentrons, $660.64/100 racks. +- **96-Well Plate (200uL PCR)** - Bio-Rad, $150/50. +- **96-Well Plate (100uL PCR Full Skirt)** - NEST, $215/100. +- **12-Well Reservoir (15mL)** - NEST, $250/50. +- **1-Well Reservoir (290mL)** - NEST, $250/50. +- **50mL Tubes** - Opentrons, $130/500. +- **PCR Sealing Film** - Amazon, $36.74/100 sheets. + +## Initial Prep +1. **Starting Point**: 1 Stock Primer Plate with 96 different ONT-tagged primers. At least 90uL fluid at 100uM concentration. +2. **Calibrate the Opentrons Robot**: Time required is approximately 15 minutes. Calibration block and tip racks required. + +## OT-2 "Working" Primer Plates - 100uM->10uM +3. **Materials**: + - 1 Well Reservoir (Agilent or NEST), 290mL + - 96 Different Tagged 100uM Stock Primers in a BioRad 200uL 96 well Plate + - (4) BioRad 200uL 96 well Plates + - PCR Plate Sealing Film + - Molecular Water + - (1) Opentrons 20uL Filter Tip Rack + - (4) Opentrons 200uL Filter Tip Racks (or 300uL Tips) + +4. **Method**: + 1. Add 80mL of water to an Agilent 290mL 1 well reservoir. + 2. Setup the Opentrons floor as follows: + ![Floor Layout](image_url) + +5. **Run the OT-2 "Working" Reverse Primer Plates - 100uM->10uM program**: + - Two versions of the program based on the tip size: + - [200uL Filter Tips](file_link_1) + - [300uL Tips](file_link_2) + - Seal three of the plates with a heat sealer if available or use plastic film. + +## OT-2 Dual-Index PCR "Stock" Plates +4. **Overview**: + This protocol generates four "stock" dual-index primer/master mix PCR plates with each run, derived from a single working primer plate. Each plate does eight complete runs. + +5. **Ingredients**: + - **Reverse Primers**: 10.8uL of 10uM from the primer plate. + - **Forward Primers**: 1.037uL of forward primer. + - **Master Mix Forward-primer Water (MMFW)**: 6,643uL molecular water for four indexes. + +6. **Method**: + 1. Wipe down the bench and Opentrons floor. + 2. Allow the master mix to thaw at room temperature. + 3. Create MMFW Cocktail in each 50mL tube: + - For a full run: 11,490uL Master Mix, 1,150uL Forward/Reverse Primer, 7,362uL Molecular Water. + - For a half-run: 5,745uL Master Mix, 575uL Forward/Reverse Primer, 3,681uL Molecular Water. + 4. Add 10mL of the mixture to the NEST 15mL 12-Well Reservoir. + 5. Setup the Opentrons floor as follows: + ![Floor Layout](image_url_2) + +7. **Run the OT-2 Dual-Index PCR Stock Plates**: + - [Half Run](file_link_3) + - [Full Run](file_link_4) + + Use PCR sealing film for short-term storage. Plastic film for immediate use. + +## OT-2 Dual-Index PCR Ready Plates +5. **Materials**: + - 96-Well Stock PCR Plates (from previous step) + - (8) NEST 100uL 96 well PCR Plates + - (1) Opentrons 20uL Filter Tip Rack + +6. **Method**: + 1. Run the labware calibration. + 2. Setup the Opentrons floor as follows: + ![Floor Layout](image_url_3) + +7. **Run the OT-2 Dual-Index PCR Ready Plates program**: + - [Half-reactions (11.5uL)](file_link_5) + - [Quarter-reactions (5.7uL)](file_link_6) + +8. Seal ready plates appropriately for storage or immediate use. + +--- + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/oprah-winfrey-weight-loss-canada-when-does-it-beco-b9rvr566.md b/markdown-output/oprah-winfrey-weight-loss-canada-when-does-it-beco-b9rvr566.md new file mode 100644 index 0000000000000000000000000000000000000000..d67918994507f51839a01c9ebf11b91d648c29a5 --- /dev/null +++ b/markdown-output/oprah-winfrey-weight-loss-canada-when-does-it-beco-b9rvr566.md @@ -0,0 +1,118 @@ +```markdown +# Goal/Experiment: +The goal of this experiment is to analyze the effectiveness and public acceptance of the "Oprah Winfrey Weight Loss Canada" product in the market. + +# Oprah Winfrey Weight Loss Canada: When Does It Become Popular In Markets! + +## Citation +[dx.doi.org/10.17504/protocols.io.j8nlkk69xl5r/v1](https://dx.doi.org/10.17504/protocols.io.j8nlkk69xl5r/v1) + +## Overview +This formula is naturally designed and aims to improve immunity and metabolism levels, ensuring that users face no health issues. The formula is also chemical-free and provides various other benefits. + +### Check Available Discount Prices for Oprah Winfrey Weight Loss + +## Amazing Benefits of Oprah Winfrey Weight Loss +1. Regular intake offers rapid weight loss. +2. Provides a flattened belly and slim figure. +3. Enhances healthy metabolism and treats metabolic syndrome. +4. Treats health issues caused by obesity or being overweight. +5. Reduces unwanted food or snack cravings. +6. A healthy diet rich in nuts, seeds, green veggies, and omega 3 and 6. +7. Fulfills the body’s ketogenic process needs. +8. Aids in burning visceral fat from belly, arms, thighs, and buttocks. +9. Offers full flexibility and mobility. +10. Improves heart health and regulates pulse rate. +11. Can be used by both men and women. +12. Helps to lose 10-12 pounds in a month. + +### Important Factors to Consider Before Buying Oprah Winfrey Weight Loss +1. Not for children below 18 years. +2. Pregnant or breastfeeding mothers should avoid. +3. Consult healthcare providers before using. +4. Adhere to prescribed amounts to avoid overdoses. + +## Are Oprah Winfrey Weight Loss Pills Safe? +Oprah Winfrey Weight Loss pills are reliable, safe, and made with natural ingredients. They are free from side effects and are edible even in a restful state. + +## Product Dosage +- Recommended dosage: 1 to 2 capsules daily. +- Should be taken with water after meals. +- Consultation with a doctor is advised before starting. + +## Best Platform to Purchase Oprah Winfrey Weight Loss +- The supplements are available online through reputable manufacturers. +- Verify the product's quality and follow usage instructions before purchasing. + +## Conclusion +Oprah Winfrey Weight Loss provides several health benefits such as weight reduction, improved metabolism, and reduced food cravings. It is cost-efficient and effective for both men and women. + +**Affiliate Disclosure:** +Links provided may result in a small commission at no additional cost to you. + +**Disclaimer:** +Consult a licensed healthcare provider before making any purchase. + +## Oprah Winfrey Weight Loss: Reduce Excess Body Weight +The formula aids in reducing low stamina, low energy levels, poor digestion, and more health issues associated with overweight and obesity. + +### About Oprah Winfrey Weight Loss +- A newly formed fat-burning solution. +- Aids in consuming healthy food and controlling cravings. +- Made from natural and herbal ingredients. + +### Working on Oprah Winfrey Weight Loss +- Enhances ketosis process. +- Boosts metabolism and immunity. +- Improves digestion. + +### Oprah Winfrey Weight Loss Ingredients +1. **Garcinia Cambogia:** A fruit that reduces body weight and improves metabolic rate. +2. **L-Arginine:** Promotes metabolism and blood circulation. +3. **BHB (Beta-Hydroxybutyrate):** Enhances ketosis and energy levels. + +### Benefits of Oprah Winfrey Weight Loss +1. Speeds up ketosis. +2. Enhances stamina and energy. +3. Promotes metabolism and digestion. +4. Controls hunger and sugar levels. + +### Pros +- Natural and herbal ingredients. +- Chemical-free. +- 100% desired results. +- Clinically tested. + +### Cons +- Not available in local markets. +- Lactic and expecting women should avoid. +- Minors should avoid. + +### Are There Any Side Effects? +- No side effects with the recommended dosage. +- Consult a doctor for best results. + +### Consumption Process +- 60 capsules in a monthly pack. +- 2 pills daily with warm water on an empty stomach. + +### Where to Buy Oprah Winfrey Weight Loss? +- Available on the official website. +- Prompt delivery within 3 to 5 working days. + +## Final Words +Oprah Winfrey Weight Loss is an advanced formula to melt excess weight, boost metabolism, and control hunger. + +## How Does Oprah Winfrey Weight Loss Work for Your Body? +- Reduces carb intake. +- Enhances ketosis process. +- Provides energy and promotes instant fat loss. + +### Key Ingredients +1. **Garcinia Cambogia:** Known for its weight loss properties. +2. **BHB Ketone:** Promotes fat loss. +3. **Green Tea:** Aids in weight loss and gut health. +4. **Lemon Extracts:** Detoxifies and aids in effective weight loss. + +***endofoutput*** +``` \ No newline at end of file diff --git a/markdown-output/optical-sensors-2-photon-imaging-cysuxwew.md b/markdown-output/optical-sensors-2-photon-imaging-cysuxwew.md new file mode 100644 index 0000000000000000000000000000000000000000..bd5c95790d7ad5af50c79790709eacf31778466f --- /dev/null +++ b/markdown-output/optical-sensors-2-photon-imaging-cysuxwew.md @@ -0,0 +1,115 @@ +```markdown +# Goal/Experiment: +To collect genetically encoded optical sensors fluorescence in mouse brain slices using 2-photon microscopy. + +# Optical Sensors 2-Photon Imaging + +### Authors: +Andrew G +Beatriz E Nielsen +Yee +University of Colorado Anschutz Medical Campus + +## Abstract + +This protocol describes the steps to collect genetically encoded optical sensors fluorescence in mouse brain slices using 2-photon microscopy. 2-photon imaging was performed using a 2-photon laser scanning microscopy system, custom-built on a BX51WI microscope (Olympus). A Ti:Sapphire laser (Chameleon Ultra I; Coherent) was tuned to emit pulsed excitation at 920 nm and scanned using a pair of X-Y galvanometer mirrors (6215, Cambridge Technology). Emitted fluorescence was collected through a water-immersion objective (60X, Olympus), a dichroic mirror (T700LPXXR, Chroma) and filters (ET680sp and ET525/50 m-2P, Chroma), and was detected using a GaAsP photomultiplier tube (PMT, H10770PA-40, Hamamatsu). A current preamplifier (SR570, Stanford Research Systems) was used to convert the output to voltage, which was then digitized by a data acquisition card (PCI-6110, National Instruments). + +## Materials + +- 2-photon laser scanning microscopy system, custom-built on a BX51WI microscope (Olympus). +- Ti:Sapphire laser (Chameleon Ultra I; Coherent). +- X-Y galvanometer mirrors (6215, Cambridge Technology). +- Dichroic mirror (T700LPXXR, Chroma). +- Filters (ET680sp and ET525/50 m-2P, Chroma). +- GaAsP photomultiplier tube (PMT, H10770PA-40, Hamamatsu). +- Current preamplifier (SR570, Stanford Research Systems). +- Data acquisition card (PCI-6110, National Instruments). +- [Tornado Software](https://github.com/StrowbridgeLab/Toronado-Laser-Scanning) +- Axograph X (Axograph Scientific). + +## Before Start Instructions + +Viruses encoding for genetically encoded optical sensors are intracranially injected in the selected area of the brain by stereotaxic surgery. Imaging is performed 3-4 weeks after injections to ensure appropriate expression levels. + +--- + +## Procedure + +### Acute Brain Slice Preparation +Steps are described in the protocol linked [here](https://protocols.io/view/acute-brain-slices-y2lfxej). + +### Rig Setup +1. **Turn on required devices and software for acquisition:** + - Tornado: [Link](https://github.com/StrowbridgeLab/Toronado-Laser-Scanning) + - Axograph X (Axograph Scientific). + +2. **Prepare 1X ACSF** (Artificial Cerebrospinal Fluid, add drugs needed for particular experiments) in a jug or bottle and bubble it with O₂/CO₂. + + **1X ACSF** + For 1L: + - 900 mL MilliQ H₂O + - 100 mL stock 10X ACSF + - 1.8 g NaHCO₃ + - 2 g D-Glucose + + **10X ACSF stock:** + + | Chemical | [mM] | 10X Stock (g/4L) | + |----------|------|--------------------| + | NaCl | 126 | 294.52 | + | KCl | 2.5 | 7.44 | + | MgCl₂·6H₂O | 1.2 | 9.75 | + | NaH₂PO₄·H₂O | 1.2 | 6.64 | + | CaCl₂·2H₂O | 2.5 | 14.7 | + | NaHCO₃ | 21.4 | | + | D-Glucose | 11.1 | | + +3. **Place the intake line into the ACSF container and allow circulation.** Wait until the fluid has entered the recording chamber, then turn on the in-line heater (Warner Instruments) and set it to desired temperature (32-34 °C). + +4. **Electrical stimulation electrodes:** + - Pull electrodes (World Precision Instruments) using a puller (Narishige, PC-10). + - Fill electrodes with 1X ACSF using a syringe with a filter. + +### Image Acquisition + +5. **Transfer brain slice from incubation vial to the recording chamber** and secure down the slice using a harp. + +6. **Locate and focus on the desired region of the brain** under IR-DIC using the low power (4x) objective. + +7. **Change the microscope lens to high power (60x) objective** and focus on healthy neurons. + +8. **Turn off IR light source and switch to 2-photon laser scanning mode** by sliding mirror to allow 2P laser excitation (wavelength: 920 nm), opening iris to PMTs, and start imaging using Tornado software and Axograph. Turn on PMT and dynode power sources. + +9. **Select a region of good sensor expression** based on basal fluorescence using 'Focus' mode (Zoom 2.5) in Tornado. + +10. **If using electrical stimulation:** + - Position the stimulating electrode on the center of that region. + - Set a protocol in Axograph with the appropriate stimulation intensity, duration and number of pulses depending on the experimental design. + +11. **Acquisition modes:** + - Laser power must be tuned by adjusting attenuation through Pockels cells so that the fluorescence at baseline is bright enough but far from signal saturation. + + #### Rasterized image/movie sequence + To measure spatial dynamics of fluorescence changes across the whole field of view (FOV), select an appropriate 'Zoom' and then press 'Movie' to acquire rasterized image sequences. In order to evoke changes of fluorescence, electrically stimulate neurotransmitter/neuromodulator (i.e. ACh/DA) release and/or different drugs can be washed on after recording a baseline period. Timing of electrical stimulation is triggered by Axograph software. + + #### 2-photon spot photometry + To measure high-speed changes of fluorescence, select an individual spot (ROI) from rasterized image under appropriate 'Zoom' 'Morph' options. The 2P laser will be scanned across a circular path (diameter: 150 nm) centered at this spot, and emitted fluorescence will be continuously measured. Set and run a protocol in Axograph with a 'Photometry' (TTL) pulse that must be equal to photometry duration on Toronado. PMT output is amplified, filtered, and fed directly to A-D board (Digitizer). In order to induce stimulatory events, electrical stimulation can be applied and/or different drugs can be washed on after recording a baseline period. + +### 2-Photon Imaging GRABAch 3.0 Recordings + +12. **Rasterized image/movie sequence for electrically evoked ACh release:** + + - Induce ACh release by electrical stimulation (estim: 25 µA, duration: 0.5 ms). + - Record three to five movies per region. Wait 1-2 min between stimulations to ensure full recovery. + - Using Fiji (Image J), stack the sequence of images from each recorded movie, average the movies from the same region, and obtain the change in fluorescence (∆F/F0) in a square region of interest (25 µm × 25 µm). F0 was defined as the average of fluorescence in the images from baseline period. + + **Note:** For slow pharmacological effects when drugs are bath applied, a time-lapse acquisition is preferred. Individual rasterized images were collected under 'Morph' mode every minute. Drugs were applied after 5-10 min of baseline. + +13. **2-photon spot photometry for paired-pulse (PPR) ratio experiments:** + + - Induce ACh release by electrical stimulation (estim: 25 µA, duration: 0.5 ms, interstimulus interval: 100 ms). Analyze the change in fluorescence (∆F/F0) in Axograph. + +--- + +**endofoutput** +``` \ No newline at end of file diff --git a/markdown-output/optimising-sample-multiplexing-oligos-by-flow-cyto-ct69wrh6.md b/markdown-output/optimising-sample-multiplexing-oligos-by-flow-cyto-ct69wrh6.md new file mode 100644 index 0000000000000000000000000000000000000000..f82b5ce07371f4a4906190a9b5ed92326d637f00 --- /dev/null +++ b/markdown-output/optimising-sample-multiplexing-oligos-by-flow-cyto-ct69wrh6.md @@ -0,0 +1,126 @@ +```markdown +# Goal/Experiment: +Optimizing sample multiplexing oligos through flow cytometry for scRNA-Seq to enable cheaper and faster processing of samples while using fluorescent detection oligonucleotides. + +## Optimising Sample Multiplexing Oligos by Flow Cytometry + +**Author:** Daniel V Brown +**Affiliation:** Walter and Eliza Hall Institute +**Facility:** WEHI Advanced Genomics Facility +**Published:** FEB 05, 2024 + +[![DOI QR Code](https://dx.doi.org/10.17504/protocols.io.81wgbvjvpovpk/v1 "DOI QR Code")](https://dx.doi.org/10.17504/protocols.io.81wgbvjvpovpk/v1) + +## Disclaimer +**DISCLAIMER – FOR INFORMATIONAL PURPOSES ONLY; USE AT YOUR OWN RISK** + +The protocol content here is for informational purposes only and does not constitute legal, medical, clinical, or safety advice or otherwise; content added to protocols.io is not peer-reviewed and may not have undergone a formal approval of any kind. Information presented in this protocol should not substitute for independent professional judgment, advice, diagnosis, or treatment. Any action you take or refrain from taking using or relying upon the information presented here is strictly at your own risk. You agree that neither the Company nor any of the authors, contributors, administrators, or anyone else associated with protocols.io, can be held responsible for your use of the information contained in or linked to this protocol or any of our Sites/Apps and Services. + +## Abstract +Optimization of sample multiplexing oligos by scRNA-Seq is costly and time-consuming. A cheaper and faster method is to use a flow cytometry read-out with fluorescent detection oligonucleotides. This method can also be used to mix samples with different fluorescently labeled oligos and investigate signal swapping. + +## Image Attribution +Made with Biorender.com + +## Guidelines +**DISCLAIMER – FOR INFORMATIONAL PURPOSES ONLY; USE AT YOUR OWN RISK** + +The protocol content here is for informational purposes only and does not constitute legal, medical, clinical, or safety advice or otherwise; content added to protocols.io is not peer-reviewed and may not have undergone a formal approval of any kind. Information presented in this protocol should not substitute for independent professional judgment, advice, diagnosis, or treatment. Any action you take or refrain from taking using or relying upon the information presented here is strictly at your own risk. You agree that neither the Company nor any of the authors, contributors, administrators, or anyone else associated with protocols.io, can be held responsible for your use of the information contained in or linked to this protocol or any of our sites/apps and services. + +## Keywords +scRNA-Seq, facs, sample multiplexing, hashtag + +## Materials +### Reagents +- **Phosphate Buffered Saline (PBS)**: without magnesium and calcium +- **Sample Multiplexing Oligos**: MULTI-Seq, CellPlex, or Hashtag Antibody +- **DAPI**: 10 µg/mL (Vendor: Thermo Fisher Scientific, Catalog No: D1306) +- **BSA Stock Solution**: 30% (Vendor: Sigma-Aldrich, Catalog No: A7030) +- **Fluorescent Detection Oligonucleotides** + +### Oligonucleotides + +#### Fluorescent Detection Oligonucleotides +| Name | Sequence | Modification | +|------------------------|----------------------------------------------|--------------------------| +| A647_FB2_detect | /5Alex647N/CCTTAGCCGCTCCTAGGATGGGAGC | 5' Alexa 647 modification| +| A594_FB2_detect | /5Alex594N/TTGCTAGGACCGGCCTTAAAGC | 5' Alexa 594 modification| +| A594_FB1_detect | /5Alex594N/TTGCTAGGACCGGCCTTAAAGC | 5' Alexa 594 modification| +| A647_Total-SeqC_detect | /5Alex647N/CTGTCTCTTATACACATCTCCG | 5' Alexa 647 modification| +| AF647_oligo_dT_detect | /5Alex647N/TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT | 5' Alexa 647 modification| + +#### MULTI-Seq Barcoding Oligos +I substituted the poly-A tail for 10x Genomics feature barcode 2 sequence. + +| Name | Sequence | Barcode | +|------------------------|----------------------------------------------|------------| +| multiSeq_FB2_BC2 | CCTTGGCACCCGAGAATTCCA CCAAATGGTCTACCTATTAG GCGCTAAGG | CCAAA TG | +| multiSeq_FB2_BC3 | CCTTGGCACCCGAGAATTCCA TGAGACCTGCTCCACTATTAG GCGCTAAGG | TGAGAC CT | +| multiSeq_FB2_BC4 | CCTTGGCACCCGAGAATTCCA GCACACCGGCTCCACTATTAG GCGCTAAGG | GCAAC GC | +| multiSeq_FB2_BC5 | CCTTGGCACCCGAGAATTCCA AGAGAGACGGCTCCACTATTAG GCGCTAAGG | AAGAG GAG | +| multiSeq_FB2_BC6 | CCTTGGCACCCGAGAATTCCA TCACAGACGGTCTCCACTATTAG GCGCTAAGG | TCACAG CA | +| multiSeq_FB2_BC7 | CCTTGGCACCCGAGAATTCCA GAAAAAGGGGCTCCACTATTAG GCGCTAAGG | GAAAA GGG | +| multiSeq_FB2_BC8 | CCTTGGCACCCGAGAATTCCA CGAAGGTTGCTCCACTATTAG GCGCTAAGG | CGAAG GTT | +| multiSeq_FB2_BC9 | CCTTGGCACCCGAGAATTCCA GTAGTACTGCTCCACTATTAG GCGCTAAGG | GTAGAC GT | +| multiSeq_FB2_BC10 | CCTTGGCACCCGAGAATTCCA GCACGACGCTCCACTATTAG GCGCTAAGG | GCACG AGC | +| multiSeq_FB2_BC11 | CCTTGGCACCCGAGAATTCCA TTAGCCAGGCTCCACTATTAG GCGCTAAGG | TTAGCC AG | +| multiSeq_FB2_BC12 | CCTTGGCACCCGAGAATTCCA GGACCGAGGCTCCACTATTAG GCGCTAAGG | GGACC CCA | +| multiSeq_FB2_BC13 | CCTTGGCACCCGAGAATTCCA CCAACCGGCTCCACTATTAG GCGCTAAGG | CCAACC GG | + +### Safety Warnings +> Please follow all Manufacturer safety warnings and recommendations. + +## Experimental Procedure + +### Prepare Multiplexing Reagent +1. **CellPlex and Hashtag Antibody**: The reagent comes ready to use. + - Optional: Prepare a dilution series for titration if desired. + +### MULTI-Seq Oligo Preparation +2. Mix anchor and barcode strands in a 1:1 molar ratio in PBS (without FBS or BSA at 2 µM concentration). + + - 2.1 This is 6 µL 50 µM anchor LMO and 15 µL 10 µM barcode oligo in 129 µL plain PBS. + - 2.2 Make one unique barcode solution per sample. + - 2.3 Total 25 µL per sample. + +3. Make a 10X solution of the Co-Anchor in PBS. + - 3.1 Add 3 µL 50 µM co-anchor to 141 µL plain PBS. + - 3.2 Add 6 µL 100 µM fluorescent detection oligo. E.g., Alexa 647 feature barcode 2 detection oligo. + - 3.3 Label in at least duplicate, label the other replicate with Alexa 594 detection oligo and mix immediately before FACS analysis. + +### Sample Preparation + +4. Use suspension cell lines to titrate cell multiplexing oligos so the sample preparation is easy. + - 4.1 Prepare a single-cell suspension of the sample to be tested. + - 4.2 Wash cells once in plain PBS without additives. Centrifuge suspension cell lines at 400xg, then resuspend in PBS. + - 4.3 Count cells and transfer 100k to 1M cells (preferably 500k) into a 1.5 mL tube for labeling. + +### Labelling Samples with Multiplexing Oligos + +5. This is largely based on original protocols but with the addition of a fluorescent secondary oligo. + - 5.1 Resuspend cells in 180 µL plain PBS. + - 5.2 Add 20 µL 10X Anchor:Barcode solution and pipette gently to mix 10–15 times. + - 5.3 Incubate on ice for 5 minutes. + - 5.4 Add 20 µL Co-Anchor solution and pipette gently to mix. + - 5.5 Incubate 5 minutes longer on ice. + - 5.6 Add 1.5 mL of 1% BSA in PBS (ice cold) to quench. + - 5.7 Add 1.5 mL of 1% BSA in PBS (ice cold) to quench. + - 5.8 Add 1 mL of 1% BSA in PBS (ice cold) to quench. + - 5.9 Centrifuge cells at 4ºC 400xg for 5 minutes. + - 5.10 Resuspend cells in the remaining 100 µL of supernatant, then transfer to a new 1.5 mL tube. + - 5.11 Add 1.9 mL 1% BSA in PBS and spin for 5 minutes at 400xg 4ºC. + - 5.12 Repeat wash 2 more times for a total of 3 washes. + - 5.13 Resuspend cells in 500 µL of PBS + 1% BSA and transfer to 5 mL polystyrene FACS tube. + +### Flow Cytometry + +6. Use a FACS analyzer to compare the signal of labeled samples to a control sample where only the fluorescent detection oligo has been added. If you have replicate labeled samples with different fluorescent detection oligos, you may combine them at this step. + - 6.1 Add DAPI to a final concentration of 0.1ug/mL (1/100 stock tube). + - 6.2 Gate for debris, single cells, and viable cells. + - 6.3 Acquire at least 10,000 viable cells per test. + - 6.4 Analyze with relevant software. + +[![DOI Link](https://dx.doi.org/10.17504/protocols.io.81wgbvjvpovpk/v1 "DOI Link")](https://dx.doi.org/10.17504/protocols.io.81wgbvjvpovpk/v1) + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/optogenetic-manipulation-mouse-b9wfr7bn.md b/markdown-output/optogenetic-manipulation-mouse-b9wfr7bn.md new file mode 100644 index 0000000000000000000000000000000000000000..7bc0584e257e36a3c2075b7701598c79068b0f67 --- /dev/null +++ b/markdown-output/optogenetic-manipulation-mouse-b9wfr7bn.md @@ -0,0 +1,122 @@ +``` +Goal/Experiment: +The purpose of this experiment is to perform in vivo optogenetic manipulation in mice, including assembly of fiber-ferrules, surgical implantation of fibers, and testing procedures. + +# Optogenetic Manipulation (Mouse) + +**Author:** Alexandra Nelson +**Affiliation:** University of California San Francisco + +[DOI Link](dx.doi.org/10.17504/protocols.io.b9wfr7bn) +**Team Edwards**: Kelsey Barcomb + +## Abstract +This protocol describes the steps for in vivo optogenetic manipulation in mice, including the assembly of fiber-ferrules, surgical implantation of fibers, and testing procedures. + +## Keywords +Mouse, Optogenetics, In vivo, Electrophysiology, Optical Fibers, Implants, Brain, ASAPCRN + +## License +This is an open access protocol distributed under the terms of the [Creative Commons Attribution License](https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. + +## 1. Fiber-Ferrule Implant Assembly + +### Materials +- **Multimodal Fiber Optic (200 μm diameter, 0.39 NA)** + Source: ThorLabs (#FT200UMT) + Function: Transmits light for optogenetic stimulation. + +- **Ceramic Ferrules (6.4 mm, 230 µm bore)** + Source: ThorLabs (#CFLC230-10) + Function: Holds fiber in place and connects to the optical setup. + +- **Fiber Stripping Tool** + Function: Strips cladding from the fiber. + +- **Fiber Cleaving Tool** + Function: Cuts the fiber to precise lengths. + +- **5-minute Epoxy** + Function: Bonds the fiber to the ferrule. + +- **Optical Sandpaper and Polishing Film** + Source: ThorLabs + Function: Polishes the ends of the fibers. + +### Steps +1.1. Gather required materials for assembly. + +1.2. Using the cleaving tool, cut approx. 2 cm lengths of fiber. Prepare about 10 such segments. + +1.3. Slide ceramic ferrules over the fiber ends and place them in a petri dish. + +1.4. Mix 5-minute epoxy in a small weigh boat. Apply epoxy to the upper half of the fiber, slide the ferrule over the epoxied section, and lay in another petri dish to set. + +1.5. Let the epoxy set for >2 hours or overnight. + +1.6. Break off excess fiber above the ferrule. Use a polishing puck or pen-type device to polish the fiber. + +1.7. With coarse polishing film, make 8 figure-8 movements. Gradually move to finer films until the fiber is polished. + +1.8. Test the transmittance of the fiber using an optical power meter. Ensure at least >70% transmittance. + +## 2. Surgery +A full detailed protocol for performing stereotaxic surgery is available [here](dx.doi.org/10.17504/protocols.io.n2bvj6qynlk5/v1). + +### Steps +2.1. Drill hole over the target structure using the assembled fibers. + +2.2. Inject the virus at the target site before fiber implantation. + +2.3. Select fibers with similar transmittance if using multiple fibers. + +2.4. Insert fiber-ferrule assembly into a “stick” made from empty ceramic ferrules. Attach to the stereotax. + +2.5. Lower the fiber slowly to 100 microns above the injection site. + +2.6. Testing should begin a minimum of 2 weeks post-surgery for viral expression. + +## 3. Habituation + +### Steps +3.1. Habituate the mouse to tethering and the behavioral chamber for 30 min/day for 2 days prior to testing. Attach the optical fiber patch cable to the implants and place the mouse in a clear acrylic chamber (25 cm in diameter). + +3.2. Monitor the mouse to prevent it from tangling in the cable. + +## 4. Computer and Optical Setup + You can send TTL pulses to the laser device via various systems (Noldus, Master8, Arduino). + +### Steps +4.1. Turn on the laser device and set to ON. + +4.2. Set to manual control to test light power output. + +4.3. Set the laser to TTL control (light should be off). + +4.4. Turn on the computer running one of these systems and choose the appropriate routine or template. + +## 5. Testing +These steps are specific to the type of experiment you are running. + +### Steps +5.1. Attach optical cable(s) to the implants and place the mouse in its home cage or the behavioral setup. + +5.2. Start recording with Noldus or any other stimulated system. Make sure TTLs are captured for later alignment with video/electrophysiology data. + +5.3. Stop recordings in reverse order once the session is completed. + +## 6. Cleanup + +### Steps +6.1. Remove the mouse from the chamber and return it to the home cage. + +6.2. Clean the chamber with 70% ethanol between mice and at the end of the day. + +6.3. Turn off the laser, close programs, and back up files to the server when experiments are completed. + +**Citation**: Alexandra Nelson 2022. Optogenetic Manipulation (Mouse). protocols.io [dx.doi.org/10.17504/protocols.io.b9wfr7bn](https://dx.doi.org/10.17504/protocols.io.b9wfr7bn) + +**Manuscript Citation**: Jonathan S Schor, Isabelle Gonzalez Montalvo, Perry WE Spratt, Rea J Brakaj, Jasmine A Stansil, Emily L Twedell, Kevin J Bender, Alexandra B Nelson (2022) Therapeutic deep brain stimulation disrupts movement-related subthalamic nucleus activity in Parkinsonian mice eLife 11:e75253 [https://doi.org/10.7554/eLife.75253](https://doi.org/10.7554/eLife.75253) + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/overview-of-the-application-of-cytokines-in-immuno-5qvg5w6.md b/markdown-output/overview-of-the-application-of-cytokines-in-immuno-5qvg5w6.md new file mode 100644 index 0000000000000000000000000000000000000000..d4dfb42cb7b94f59f862ded32bdb2ca025291432 --- /dev/null +++ b/markdown-output/overview-of-the-application-of-cytokines-in-immuno-5qvg5w6.md @@ -0,0 +1,83 @@ +```markdown +# Goal/Experiment: +To explore the role and application of cytokines in immunotherapy, understand their effects on immune response cells, and review clinical usage to enhance cancer treatment and other immunological conditions. + +# Overview of the Application of Cytokines in Immunotherapy + +**Susan Wind** +*Marine Biological Laboratory* +*Jul 23, 2019* +[dx.doi.org/10.17504/protocols.io.5qvg5w6](https://dx.doi.org/10.17504/protocols.io.5qvg5w6) + +## Abstract + +Cytokines are generally produced by stimulated cells, primarily immune cells. Cytokines are highly potent and act in micromolar or even picomoles. The effect of a single cytokine on immunity depends on the conditions of local cytokine concentration, the mode of expression of its receptor, and the integration of multiple signaling pathways in immune response cells. Cytokines act as molecular messengers, allowing the immune system cells to communicate with each other to produce coordination of target antigens, regulatory and effector functions in many diseases, and thus cytokines and their receptors can be used for immunotherapy. + +During immunotherapy, cytokines directly stimulate immune effector cells and stromal cells at the tumor site to enhance cytotoxicity. Through research on animal tumor models, it has been found that cytokines have a wide range of anti-tumor activities, and many cytokines have been used for the treatment of cancer. There are several cytokine drugs approved for FDA marketing, such as high doses of IL-2 for the treatment of melanoma and renal cell carcinoma, and IFN-α for the adjuvant treatment of stage III melanoma. More cytokines have entered clinical trials such as GM-CSF, IL-7, IL-12, IL-15, IL-18 and IL-2. + +As an immunomodulator, cytokines can be used to activate immunotherapy, immunosuppressive therapy, etc., including various recombinant, synthetic and natural preparations. Such as interleukins (IL-2, IL-7, IL-12), chemokines (CCL3, CCL26, CXCL7) and other cytokines (interferon, granulocyte colony-stimulating factor). + +## Activating Immunotherapy + +For example, granulocyte colony-stimulating factor (G-CSF) can stimulate peripheral blood stem cells (extracted from the blood of patients) to produce lymphocytes, which are co-cultured with tumor antigens in vitro, and then returned to the patient, combined with stimulating cytokines. In order to enhance the immune effect, such cells can destroy tumor cells carrying the same antigen, thereby achieving therapeutic effects. + +Interleukin-2 can be fused to anti-CD3 and alloreactive cells to produce adoptive T cells. This kind of cells can be transferred to patients and can further enhance the anticancer activity of IL-2. Interleukin-7 and interleukin-2 can be used to restore the immune system in patients with impaired immune function, and this research has entered the clinical trial phase. + +## Immunosuppressive Therapy + +It mainly inhibits abnormal immune responses in autoimmune diseases or reduces normal immune responses to prevent rejection in cell or organ transplantation. Such as immunosuppressive drugs, immune tolerance, and allergy treatment. + +## Characteristics of the Cytokines Used + +### Interleukin-1 (IL-1) + +Interleukin-1 (IL-1) is a pleiotropic cytokine involved in the inflammatory response, cell growth, and tissue repair of the cortex. The IL-1 superfamily has 11 members, such as IL1A, IL1B, IL1Ra, IL-18 and the like. IL-1 is a drug target for some cancers and is also used in cell therapy. + +- In cellular immunotherapy, IL-1 stimulates proliferation of CD4+ T cells in vitro, induces IL-2 production, stimulates activation of CD8+/IL-7R+ T cells, and stimulates proliferation of mature B cells and secretion of immunoglobulin. +- When IL-1α is combined with IFN-γ and activating CD3 monoclonal antibody, the cytotoxic effect of CIK can be significantly enhanced. + +### Interleukin 2 (IL-2) + +Interleukin 2, also known as T cell growth factor, is produced by T cells in response to antigen or mitotic stimulation and is widely used to promote the activation and proliferation of T cells and NK cells. IL-2 can stimulate NK cell proliferation, increase cytotoxicity and stimulate NK cells to secrete a variety of cytokines. + +- Further studies have found that IL-2 can cause T cell over-differentiation and induce activation of T cell apoptosis, and can also activate CD4+ FoxP3 Treg regulatory cells, thereby inhibiting T cell activation and tumor killing activity. T cell regulatory factors are not just activating factors, so some studies have used IL-7, IL-15, IL-21 instead of IL-2. + +### Interleukin 7 (IL-7) + +Interleukin-7 is a hematopoietic growth factor secreted by stromal cells in the bone marrow and thymus, and shares the γc receptor subunit with interleukin 2 to stimulate proliferation of lymphoid progenitor cells. IL-7 provides a continuous stimulation signal for Naive T cells and memory T cells. + +- As described above, IL-7 does not activate CD4+ FoxP3+ Treg cells during activation of CD8+ T cells. +- Clinically, IL-7 can also be used to restore the number of T cells after chemotherapy or hematopoietic stem cell transplantation. IL-7 plays an important role in some stages of B cell maturation and can affect its proliferation. +- IL-7 can also act as a regulator of intestinal mucosal lymphocytes. + +### Interleukin 15 (IL-15) + +Interleukin 15 has a similar structure to interleukin 2, sharing the γc receptor subunit, belonging to the family of four α-helix helix bundles (others such as IL-2, IL-4, IL-7, IL-9), G-CSF and GM-CSF. IL-15 regulates the activation and proliferation of T and NK cells. IL-15 is primarily responsible for killing virus-infected cells in the innate immune system. + +- At the same time, IL-15 can activate NKT cells and γδT cells. +- In immunocyte treatment, IL-15 does not cause apoptosis of activated T cells and activates CD8+ effector T cells. +- IL-15 maintains memory T cell survival and thus plays an important role in long-term anti-tumor activity. + +### Interleukin 21 (IL-21) + +Interleukin 21 also belongs to the interleukin 2 family, shares the γc receptor subunit, and has a strong regulatory effect on cells of the immune system, and can induce cell division and proliferation in its target cells. + +- In cellular immunotherapy, IL-21 can promote the proliferation of CD4+ and CD8+ T cells, enhance the cytotoxicity of CD8+ T cells and NK cells, and not cause apoptosis due to activation. +- IL-21 preferentially amplifies the "young" CD27+CD28+ CD8+ T cells, which are more cytotoxic. +- Of course, IL-21 does not cause expansion of Treg, therefore, IL-21 is increasingly used in cellular immunotherapy. + +It is precisely because cytokines are produced by a variety of cells that have multiple roles in innate immune responses and adaptive immune responses, and are involved in immune diseases, inflammation, and infectious diseases. With the deepening of research on cytokines, more factors will be applied to cellular immunotherapy. + +## Related Services + +- [CD20 antibody](https://www.creativebiolabs.net/Anti-CD20-Immunocytokine-IgG-Rituximab-RLI-22600.htm) +- [Anti CD20 antibody rituximab](https://www.creativebiolabs.net/Anti-CD20-Immunocytokine-IgG-Rituximab-RLI-22600.htm) + +--- + +This is an open access protocol distributed under the terms of the [Creative Commons Attribution License](https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. + +--- + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/parasitological-diagnosis-of-american-tegumentary-22tggen.md b/markdown-output/parasitological-diagnosis-of-american-tegumentary-22tggen.md new file mode 100644 index 0000000000000000000000000000000000000000..0f97cc69484b4d5ae1c5831445e39140f0db048a --- /dev/null +++ b/markdown-output/parasitological-diagnosis-of-american-tegumentary-22tggen.md @@ -0,0 +1,105 @@ +```markdown +# Goal/Experiment: +Parasitological Diagnosis of American Tegumentary Leishmaniasis - Isolation of Leishmania in Culture + +## Parasitological Diagnosis of American Tegumentary Leishmaniasis - Isolation of Leishmania in Culture + +### Authors +- Luciana F. Miranda¹ +- Aline Fagundes da Silva¹ +- Marcela Xavier de Mello¹ +- Célia de Fatima Moreira Venâncio¹ +- Andreía Alves Marcolini¹ +- Cíntia Xavier de Mello² +- Maria de Fátima Madeira¹ + +### Affiliations +¹ Evandro Chagas National Institute of Infectious Diseases, Oswaldo Cruz Institute + +### Abstract +The culture media are used for isolating various biological agents causing diseases in humans and animals. In the case of leishmaniasis, this procedure attempts to isolate the causative agent (Leishmania spp.), which is essential for confirming the diagnosis. This method is considered the gold standard for diagnosing leishmaniasis due to its good sensitivity and specificity. The procedure requires maintaining a sterile environment, handling samples in laminar flow cabinets, and adhering to biosafety standards for handling microorganisms class II. + +--- + +## Section 1: Preparing Schneider's Insect Medium +Protocol based on the manufacturer's instructions S9895 - SIGMA. + +### Application +Originally developed for the culture of Drosophila. It is suitable for culturing other dipteran cell lines when supplemented with 5-20% heat-inactivated fetal bovine serum. Schneider’s medium supports the rapid growth of dipteran cells and has been widely used for both primary and established cultures of cells derived from Drosophila melanogaster. + +### General Description +Many insect tissue culture media are formulated to mimic the physio-chemical properties of specific insect body fluids. Thus, a cursory survey of the formulas of culture media designed for insect tissues reveals wide qualitative and quantitative differences in composition. + +### Preparation Note +**Schneider’s Insect Medium – S9895 – SIGMA - Concentration: 24.5 g/L** + +- Measure out 80% of the final required volume of water. +- Add 24.5 g of Schneider's medium in 800 mL of distilled water. +- While stirring, adjust pH to 9.2 ± 0.2 with 1N sodium hydroxide (NaOH). +- Add 0.4 g sodium bicarbonate (NaHCO₃) or 53.5 mL sodium bicarbonate solution [7.5% w/v] per liter of final volume. +- Add additional water to adjust to the final volume. +- Use calcium chloride to prevent precipitation while dissolving 0.6 g of anhydrous calcium chloride in 50 mL of tissue culture-grade water for each final liter of the medium. +- Adjust pH to 7.0 ± 0.2 with 1N hydrochloric acid (HCl) while stirring. +- Sterilize by filtering with a 0.22 microns membrane and dispense into sterile containers. + +### Important Notes +- **Penicillin/Streptomycin Use**: For every liter culture, 3.0 mL penicillin solution (63,000 units/mL) and 4.0 mL streptomycin solution (50,000 µg/mL) can be added. +- **Storage**: Store the prepared medium at 2-8°C for up to 3 months. Do not freeze. +- **Deterioration Signs**: Color change, granulation/clumping, insolubility. + +--- + +## Section 2: Preparing Solid Culture Medium NNN (Novy, McNeal, Nicolle) + +### Procedure +- Weigh 4.5 grams of agar-agar, 2.0 grams of sodium chloride (NaCl), and add 300 mL distilled water. +- Sterilize in Erlenmeyer glass vial for 20 minutes at 121°C. +- Store agar in refrigerator until use. +- Melt agar in a microwave for approximately 4 minutes at maximum power. +- Cool agar, mix with 10% (30 mL) defibrinated and sterile rabbit blood. +- Distribute (aseptically) into screw-cap test tubes (2.0-2.5 mL each). + +### Important Notes +- **Use**: For culture isolation. +- **Storage**: Refrigerator at 2-8°C. +- **Dilution**: Use 1.5 to 2 mL Schneider's medium supplemented with antibiotics and 10% fetal bovine serum. + +--- + +## Section 3: Preparing Saline Solution with Antibiotics and Antifungal for Collection of Biopsies + +### Procedure +- Sterilize 2.0 mL microcentrifuge tubes. +- Add 1.9 mL penicillin (63,000 Units/mL), 2.0 mL streptomycin (50,000 µg/mL), 1.0 mL 5-fluorocytosine (10,000 µg/mL), and 95.1 mL sterile saline. +- Store tubes at room temperature and transfer to a single vessel when frozen. + +### Important Notes +- **Concentration**: Penicillin/streptomycin at 1,200 units/1 mL and antifungal at 100 µg. + +--- + +## Section 4: Collection of Clinical Samples + +### Procedure +- Obtain biopsies from cutaneous/mucosal lesions from patients. +- Store fragments in saline tubes. +- Transfer to a second tube with antibiotics and antigun. +- Divide into three parts after 24 hours, and seed into biphasic culture medium. +- Incubate at 26-28°C in a Biochemical Oxygen Demand (BOD) incubator. +- Examine samples using 100X and 400X magnifications for flagellate forms of parasites. + +--- + +## References +1. Arias JR, Miles MA, Naiff RD, et al. Flagellates infection of Brazilian sandflies. *Am J Trop Med Hyg*. 34:1098-1108, 1985. +2. Cupolillo E, Grimaldi Jr, G., Momen H. Genetic diversity among *Leishmania (Viannia)* parasites. *Ann Trop Med Parasitol*. 91(6):617-626, 1997. +3. Schneider I. In vitro culture of Dipteran cells and applications. In: Kurstak E., Maramorosch K., eds., *Academic Press*. pp. 133-150, 1979. +4. Grimaldi Jr, Momen H, Naff RD, et al. Characterization of leishmanial parasites. *Am J Trop Med Hyg*. 44:645-661, 1991. +5. Kuroda Y. Drosophila tissue culture: Retrospect and prospect. In: Maramorosch K., Mitsuhashi J., eds., *Academic Press*. pp. 53-104, 1982. +6. Mitsuhashi J. Media for insect cell cultures. In: Maramorosch K, ed., *Advances in Cell Culture*. Vol 2, pp. 133-196, 1982. +7. Momen HJ. Parasite characterization by immodeme analysis. In: Morel CM, ed., *Antigens of Parasites*. Rio de Janeiro UNDP/World Bank/WHO-FINEP, pp. 111-120, 1972. +8. Schneider I. *Drosophila cell and tissue culture*. Academic Press, pp. 266-315, 1978. +9. Van der Straten, et al. Efficient expression of foreign genes. In: *Colocnization with development insects*. pp 183-195, 1989. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/passaging-of-trophoblast-organoids-from-full-term-cffntjme.md b/markdown-output/passaging-of-trophoblast-organoids-from-full-term-cffntjme.md new file mode 100644 index 0000000000000000000000000000000000000000..d3e34edd014db024791c779c43632518a4d89f07 --- /dev/null +++ b/markdown-output/passaging-of-trophoblast-organoids-from-full-term-cffntjme.md @@ -0,0 +1,148 @@ +```markdown +# Goal/Experiment +This experiment focuses on the isolation and passaging of trophoblast organoids from full-term human placental tissue, designed to maintain and potentially expand organoids for subsequent scientific investigations. + +# Passaging of Trophoblast Organoids from Full-term Placental Tissue + +**Carolyn Coyne** + +*Duke University* + +**Abstract** +This protocol describes the procedures for the passaging of trophoblast organoids isolated from full-term human placental tissue. + +**Attachments** +- [Passaging of Trophoblast Organoids.pdf](Passaging%20of%20Trophoblast%20Organoids.pdf) + +**DOI** +[dx.doi.org/10.17504/protocols.io.e6nvwkx8dvmk/v1](https://dx.doi.org/10.17504/protocols.io.e6nvwkx8dvmk/v1) + +**Protocol Citation** +Carolyn Coyne, henry.yang. 2022. Passaging of trophoblast organoids from full-term placental tissue. Protocols.io. [https://protocols.io/view/passaging-of-trophoblast-organoids-from-full-term-cffntjme](https://protocols.io/view/passaging-of-trophoblast-organoids-from-full-term-cffntjme) + +**Funders Acknowledgement** +Carolyn Coyne, Grant ID: NIHAI145828 + +**Manuscript Citation** +Please remember to cite the following publication in addition to this protocol: +Innate immune signaling in trophoblast and decidua organoids defines differential antiviral defenses at the maternal-fetal interface. Liheng Yang, Eleanor C. Semmes, Cristian Ovies, Christina Megli, Sallie Permar, Jennifer B. Gilner, Carolyn B. Coyne. + +## Reagents, Solutions and Materials Prepared in Advance: + +1. **Pre-cool equipment** + - Blunt 200 µl pipette tips (Fisher 02-707-134). + +2. **Pre-warm equipment and media** + - Multi-well TC plate (24-well TC plate, cat# 3526, Costar). + - Stem Pro Accutase (Gibco, Cat# A11105-01) supplemented with 10 µM Y-27632 (Sigma, Y0503-1MG; 100× dilution from stock solution, Rock inhibitor). + +3. **Pre-thaw reagent** + - Matrigel (Corning 356231) thaw on ice for at least 2 hours, preferably overnight (o/n). + +4. **Prepare** + - 20% (vol/vol) FBS medium and basal media as needed. + +## Passaging Protocol: + +1. **Remove Growth Medium** + Remove complete growth medium (TOM) from each well. + +2. **Add Basal Medium** + Add 500 µl of fresh basal medium (Advanced DMEM/F12, Life Technologies, 12634-010) to each well. + +3. **Pre-Coat Pipette Tip** + Pre-coat a wide orifice 1 ml pipette tip (Finntip 1000, Thermo Fisher, 9405160) using FBS-containing basal media and gently scrape off the Matrigel domes, including organoids. + +4. **Transfer Mixture** + Carefully transfer the released mixture of Matrigel and organoids into a 15 ml conical centrifuge tube. + +5. **Centrifuge** + Centrifuge at 600 RPM, RT for 6 minutes. + +6. **Remove Supernatant** + Carefully remove supernatant using a 1 ml pipette. Use 200 µl tip if necessary. Do not use glass Pasteur pipette to aspirate. + +7. **Add Dissociation Reagent** + Add 1 ml of pre-warmed dissociation reagent: + - **Option 1**: StemPro Accutase (Life Technologies, A11105-01) + - **Option 2**: TrypLE Express (Life Technologies, 12605-028) + - Pre-warm prior to usage; add ROCK inhibitor (10 µl of inhibitor per 1 ml). + +8. **Incubation** + - Incubate in a 37°C water bath for 6-10 minutes with occasional swirling. + +9. **Centrifuge** + Centrifuge at 600 RPM, RT for 6 minutes. Repeat if organoids are not adequately pelleted. + +10. **Remove Supernatant** + Remove supernatant using a 1 ml pipette. Use 200 µl tip if necessary. Do not use glass Pasteur pipette to aspirate. + +11. **Add Basal Medium** + Add 200 µl of basal medium. + +12. **Autopipette** + Use autopipette (Eppendorf Xplorer plus, variable, 15 – 300 µL, 4861000031) to resuspend pellet: + - Pipette 200× + - Check suspension + - Pipette 50×, check, then 30× if not evenly disrupted. + - Flush autopipette tip using 1 ml basal medium into the solution. + +13. **Centrifuge** + Centrifuge at 600 RPM, RT for 6 minutes. + +14. **Remove Supernatant** + Remove supernatant and put the 15 ml conical tube with pellet on ice. + +15. **Resuspend Pellet** + Resuspend pellet with pre-thawed Matrigel using pre-cooled blunt pipette tips (Fisher 02-707-134): + - For 6 wells, add 240-250 µl Matrigel. + - Matrigel must be kept on ice. + +16. **Dispense Matrigel-Organoid Suspension** + Carefully dispense 40 µl aliquot into pre-warm 24-well plate: + - Do not touch bottom of the plate. + - Lift pipette tip when dispensing avoiding bubbles. + +17. **Polymerization** + - Place 24-well plate into 37°C incubator for 2 minutes, then flip it and incubate for an additional 8 minutes to fully polymerize. + +18. **Prepare TOM** + During polymerization, prepare TOM with Y-27632 (200× dilution): + - For 6 wells, use 3 ml medium with 15 µl ROCK inhibitor. + +19. **Submerge Domes** + Submerge polymerized Matrigel domes with 500 µl TOM per well, then culture them in a 37°C humidified CO₂ incubator. + +20. **Daily Observation** + Observe daily and renew the TOM every 48-72 hours. + +## Media Recipe + +**Trophoblast Organoid Medium (TOM)** + +| Ingredient | Volume (µl) | Final Concentration | +|------------|-------------|---------------------| +| 100 × N2 (Life Technologies, 17502-048) | 500 | 1× | +| 50 × B27 (Life Technologies, 17504-044) | 1000 | 1× | +| 500 × Primocin (InvivoGen, ant-pm-1) | 100 | 100 µg/ml | +| 80 × NAC (Sigma, A9165) | 625 | 1.25 mM | +| L-glutamine (Life Technologies, 35050-061) | 500 | 2 mM | +| A83-01 (Tocris, 2939) | 5 | 500 nM | +| CHIR99021 (Tocris, 4423) | 5 | 1.5 µM | +| Recombinant hEGF (Gibco, PHG0314) | 25 | 50 ng/ml | +| Recombinant R-spondin1 (R&D systems, 4645-RS-100) | 25 | 80 ng/ml | +| Recombinant hFGF2 (Peprotech, 100-18C) | 25 | 100 ng/ml | +| Recombinant hHGF (Peprotech, 100-39) | 25 | 50 ng/ml | +| Nicotinamide (NTM) (Sigma, N0636-100G) | 500 | 10 mM | +| Y-27632 (Sigma, Y0503-1MG) | 250 | 5 µM | +| PGE2 (R&D systems, 22-961-0) | 25 | 2.5 µM | +| FBS (heat inactivated) (Cytiva HyClone, SH30070.03) | 5 ml | 10% (vol/vol) | +| Advanced DMEM/F12 (Life Technologies, 12634-010) | Adjust to 50 ml | N/A | + +**Annotation** +- Add about 35 ml Advanced DMEM/F12 to the 50 ml centrifuge tube. +- Add supplements, adjust final volume to 50 ml with Advanced DMEM/F12. +- Use the medium within one month. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/pcp20-transformation-to-remove-kanamycin-cassette-cfv9tn96.md b/markdown-output/pcp20-transformation-to-remove-kanamycin-cassette-cfv9tn96.md new file mode 100644 index 0000000000000000000000000000000000000000..a93ca3c9d58281fc23b7b988549d3c44ad09450d --- /dev/null +++ b/markdown-output/pcp20-transformation-to-remove-kanamycin-cassette-cfv9tn96.md @@ -0,0 +1,151 @@ +```markdown +# Goal/Experiment: +Transformation of pCP20 to remove the Kanamycin resistance cassette from **Keio Escherichia coli** BW25113 single-gene deletion mutants. + +## pCP20 Transformation to Remove Kanamycin Cassette + +### Author +Saul Moore1 +1Imperial College London, MRC London Institute of Medical Sciences + +**DOI:** [dx.doi.org/10.17504/protocols.io.81wgbydxovpk/v1](https://dx.doi.org/10.17504/protocols.io.81wgbydxovpk/v1) + +### Behavorial Genomics +--- + +### Disclaimer +**DISCLAIMER – FOR INFORMATIONAL PURPOSES ONLY; USE AT YOUR OWN RISK** + +The protocol content here is for informational purposes only and does not constitute legal, medical, clinical, or safety advice, or otherwise; content added to [protocols.io](https://protocols.io/) is not peer-reviewed and may not have undergone a formal approval of any kind. Information presented in this protocol should not substitute for independent professional judgment, advice, diagnosis, or treatment. Any action you take or refrain from taking using or relying upon the information presented here is strictly at your own risk. You agree that neither the Company nor any of the authors, contributors, administrators, or anyone else associated with [protocols.io](https://protocols.io/) can be held responsible for your use of the information contained in or linked to this protocol or any of our Sites/Apps and Services. + +--- + +### Abstract +Transformation with pCP20 to remove the Kanamycin cassette from **Keio E. coli** BW25113 single gene deletion mutants - Cassandra Backes (Host-Microbe Co-Metabolism Laboratory, MRC-LMS) + +--- + +### Protocol Citation +Saul Moore 2022. pCP20 transformation to remove Kanamycin cassette. **protocols.io** +[https://protocols.io/view/pcp20-transformation-to-remove-kanamycin-cassette-cfv9tn96](https://protocols.io/view/pcp20-transformation-to-remove-kanamycin-cassette-cfv9tn96) + +--- + +### License +This is an open access protocol distributed under the terms of the [Creative Commons Attribution License](https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. + +### Created +Aug 28, 2022 + +### Last Modified +Aug 29, 2022 + +### Protocol Integer ID +69281 + +--- + +### Materials + +#### Making TSS Broth (Final Volume 10 mL) +1. **10% (w/v) Polyethylene Glycol (PEG) 3350**: 1 g + - *PEG 3350* on media shelf. + - Function: Enhances transformation efficiency. +2. **LB Broth**: 9 mL + - Function: Growth medium. +3. **50 mM Mg²⁺ (MgSO₄ or MgCl₂)**: 500 µL + - Use 1M stock of MgSO₄ used for NGM (Neuromuscular Junction) Media. +4. **5% (v/v) DMSO**: 500 µL + - *Chemical metal chest.* + - Function: Aids in membrane permeability. + +**Instructions:** +- Sterilise-filter into new tube. +- Keep in fridge. + +#### Making TSS Enhanced Buffer (Final Volume 10 mL) +1. **dH₂O**: Add 8.2 mL of dH₂O to 30 mL tube. +2. **100 mM KCl**: 1 mL + - To prepare 1M stock: 1.491 g in 20 mL of H₂O. + - Function: Stabilizes cellular membrane. +3. **30 mM CaCl₂**: 0.3 mL + - Use 1M stock for NGM. + - Function: Calcium ion supplement. +4. **50 mM MgSO₄**: 0.5 mL + - Use 1M stock for NGM. + - Function: Enhances biological activity. + +**Instructions:** +- Vortex well and filter-sterilise into 2 mL tubes. +- Keep in fridge. + +--- + +### Procedure + +1. **Buffer and Broth Preparation:** + - Make buffer and broth the day before and keep them in the fridge. + +2. **Overnight Culture:** + - Grow O/N cultures of the strains of interest. Can also prepare buffers (see materials). + +3. **Dilution of Overnight Culture:** + - Dilute overnight culture in LB broth to OD = 0.2 and grow until OD595nm = 0.5 – 0.8 (mid-log phase). + - **Note:** Keep cultures on ice. + +4. **Centrifuge Cultures:** + - Spin down culture(s) by transferring first to 15 mL falcon, spin at 4500 RPM, for 10 min at 4°C. + +5. **Labeling and Preparation:** + - Label 2 mL tubes with strain names and add 80 µL TSS buffer and 1 - 5 µL of pDNA (50-75 ng). + - Vortex and keep on ice for 10 min. + +6. **Resuspension:** + - Remove supernatant from 14 mL tubes and resuspend pellet in 1 mL TSS broth (cold). + - Resuspend gently and keep on ice. + +7. **Transfer Cells:** + - Transfer cells to a 2 mL tube. + +8. **Adding Bacterial Culture:** + - Add 200 µL of bacterial culture in TSS broth to the 2 mL tubes containing pDNA in TSS buffer. + - Mix gently by pipetting. + +9. **Incubation:** + - Incubate for 20 min on ice. + +10. **Addition of LB:** + - Add 1 mL LB. + +11. **Shaking:** + - Shake at 30°C for 1 hour at 700 rpm. + +12. **Centrifugation:** + - Spin down (4500 RPM, 5 min), resuspend pellet in 300 µL LB. + +13. **Plating:** + - Spread 50 and 150 µL on a plate and incubate at 30°C in Chloramphenicol plates. + +14. **Storage:** + - Keep remaining transformation mix at 4°C. + +15. **Colony Check:** + - Check plates the next day for single colonies, and confirm by PCR/Sequencing. + +--- + +### Notes +- **PEG (Polyethylene Glycol):** Polymer used to enhance cell transformation efficiency. +- **MgSO₄ (Magnesium Sulfate) / MgCl₂ (Magnesium Chloride):** Magnesium salts used to stabilize nucleic acids and proteins during the transformation process. +- **DMSO (Dimethyl Sulfoxide):** Solvent that penetrates cell membranes more easily to increase transformation efficiency. +- **TSS Buffer (Transformation and Storage Solution):** Medium specifically designed to improve the transformation efficiency of E. coli cells by providing better conditions for DNA uptake. +- **LB Broth (Luria-Bertani):** Nutrient-rich media often used for growing bacteria. + +Endnotes: +- Alternative method for hard-to-find supplies, consider other molecular weights of PEG and ensure label instructions match PEG 3350. +- Always ensure reagents and materials are of analytical grade to maintain protocol integrity. + +--- + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/pcr-cleanup-bpvqmn5w.md b/markdown-output/pcr-cleanup-bpvqmn5w.md new file mode 100644 index 0000000000000000000000000000000000000000..daad10e0d995282e90c4b6b6ebeabf4741e21eb5 --- /dev/null +++ b/markdown-output/pcr-cleanup-bpvqmn5w.md @@ -0,0 +1,90 @@ +```markdown +# PCR Cleanup + +**Goal/Experiment:** This protocol explains how to clean PCR products for efficient downstream applications. + +## Authors +- Caroline Storer1 +- Jiri Hulcr1 + +1University of Florida + +- Bark Beetle Mycobiome Research Coordination Network + +## License +This is an open access document distributed under the terms of the [Creative Commons Attribution License](https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. + +## DOI +[10.17504/protocols.io.bpqvmn5w](https://dx.doi.org/10.17504/protocols.io.bpqvmn5w) + +## Abstract +This protocol explains how to clean PCR products. + +## Introduction +IMPORTANT: We now are performing clean up with our sequencing services GeneWiz and Eurofins. This removes the need for clean up, as it is cheaper for them to do it by themselves. + +Use the following only with a single band PCR. With more bands, the only option is gel extraction & purification. + +## Reagents and Equipment +1. **Exo-SAP-IT** + - Source: [Thermo Fisher Scientific](https://www.thermofisher.com) + - Function: Enzyme mix used to remove excess primers and nucleotides. + - Storage: Keep on ice; do not freeze. +2. **EZNA Gel Extraction Kit** + - Source: [Omega Bio-tek](http://www.omegabiotek.com) + - Function: Purifies DNA from agarose gels. +3. **AMPure XP PCR Cleanup with Ring Magnet Plate** + - Manufacturer: Beckman Coulter + - Function: Purifies PCR products by binding DNA to magnetic beads. + - Note: Use within 12 months and keep away from metals and magnetic sources. + +## Procedure + +### Exo-SAP-IT +1. Keep Exosap on ice and don’t keep out of the freezer longer than necessary. This product doesn’t freeze, so it is always ready to use straight out of the freezer. Keep tubes in ice block until ready to put into thermocycler. +2. **Preparation:** + - For bidirectional sequencing, use an ice block and in each PCR tube mix: + - 0.5 µL PCR grade water + - 2 µL PCR product + - After all tubes are prepped, get the tube of Exosap out of freezer and promptly add 1 µL to each tube. Immediately return the tube of Exosap to the freezer. + - Run ExoSap program on thermocycler (37°C for 15 min, and 80°C for 15 min). Depending on the thermocycler, you may need to change the volume of product in the program settings (or when prompted) to 3 µL. + - When the cycle is finished, spin down again and put half the contents of each PCR tube (1.75 µL) into a 1.5 mL tube, and the remaining half into a second 1.5 mL tube. These will be your forward and reverse submissions for sequencing. + +### Gel Extraction and Cleanup +**EZNA Gel Extraction Kit, Omega Bio-tek** +1. Cut out gel slice, put it in a tube, and weigh it (actual weight minus one empty tube (0.90 g)). +2. Add equal volume of **Binding Buffer** (volume = gel weight). +3. Incubate at 55-60°C for 7 minutes (or until gel dissolves). +4. Add 700 µL to spin column, centrifuge at 10,000g for 1 minute. +5. Add 300 µL of fresh **Binding Buffer** to the column, spin at 10,000g for 1 minute. +6. Add 700 µL **Wash Buffer**, centrifuge at 10,000g for 1 minute, discard flow-through. +7. Discard liquid, centrifuge the empty column for 2 minutes at max speed. +8. Put column in clean tube, add 30 µL **Elution Buffer**. +9. Let soak for 1 minute, then centrifuge at max speed for 1 minute. + - (If maximum yield is more important than concentration, add another 30 µL **Elution Buffer**, centrifuge at max speed.) + +### AMPure XP PCR Cleanup with Ring Magnet Plate +1. Resuspend any magnetic beads that may have settled (color should be uniform). +2. In a 96 well PCR plate (compatible brands), add 1.8 µL AMPure for every 1 µL of PCR product (e.g., add 45 µL AMPure to a 25 µL PCR reaction = ~70 µL total volume). Keep PCR plate off magnet plate until step 4. +3. Mix AMPure and PCR product by pipetting 10x, then incubate at room temperature for 5 minutes. +4. Following incubation, place PCR plate into the magnetic ring plate, let sit for 2 minutes or until solution is clear. +5. Keeping the plate magnetized, remove and discard the clear solution via pipetting (do not disturb the ring of beads around the sides). +6. Add 200 µL 70% ethanol, let sit for 30 seconds, then remove and discard all ethanol without disturbing the beads. Repeat this step for a total of two washes. +7. Let stand for about 5 minutes for any remaining traces of ethanol to evaporate. +8. Remove plate and add a minimum of 40 µL elution buffer (depending on product, e.g., molecular grade water, TE, etc). Mix by pipetting 10x. +9. Place the plate back on the magnet and let sit for 1 minute to draw the beads back out of suspension. +10. Without disturbing the ring of beads, transfer your samples to a new plate/tubes for storage. + +## Notes +- When DNA yield is important, use fresh running TAE buffer. +- If buffers turn orange or red during incubation, pH is too high. +- For separating the whole PCR reaction, make a thick gel. + +## References +Caroline Storer, Jiri Hulcr 2020. PCR Cleanup. protocols.io [https://dx.doi.org/10.17504/protocols.io.bpqvmn5w](https://dx.doi.org/10.17504/protocols.io.bpqvmn5w) + +**Citation:** +Caroline Storer, Jiri Hulcr (11/20/2020). PCR Cleanup. [https://dx.doi.org/10.17504/protocols.io.bpqvmn5w](https://dx.doi.org/10.17504/protocols.io.bpqvmn5w) + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/plasma-insulin-linco-elisa-8a4hsgw.md b/markdown-output/plasma-insulin-linco-elisa-8a4hsgw.md new file mode 100644 index 0000000000000000000000000000000000000000..883fb4dc6624e985454ddd9cf83b79bf8ad3cff6 --- /dev/null +++ b/markdown-output/plasma-insulin-linco-elisa-8a4hsgw.md @@ -0,0 +1,211 @@ +```markdown +# Goal/Experiment: +The goal of this experiment is to quantify insulin levels in mouse and rat plasma samples using the Plasma Insulin LINCO ELISA V.2 kit. + +## Plasma Insulin (LINCO ELISA) V.2 +**Authors: Willa Hsueh¹, Alan Collins¹** +¹The Ohio State University College of Medicine + +[![Diabetic Complications Consortium](https://www.diacomp.org/shared/document.aspx?id=488&docType=Protocol)](https://www.diacomp.org/shared/document.aspx?id=488&docType=Protocol) + +**Date:** October 16, 2019 + +**Tech support email:** [rmcindoe@augusta.edu](mailto:rmcindoe@augusta.edu) + +### Summary +This Rat/Mouse Insulin ELISA kit is used for the non-radioactive quantification of insulin in mouse and rat sera. Plasma samples may also be used but application to samples of other biological fluids may need validation by the user. + +### Diabetic Complications: +- Cardiovascular +- Retinopathy +- Neuropathy +- Nephropathy +- Pediatric Endocrinology +- Uropathy +- Wound-Healing + +### Materials +| NAME | CATALOG # | VENDOR | +|-----------------------------------|------------|-----------------| +| RAT/MOUSE INSULIN ELISA KIT 96-Well Plate | EZRMI-13K | LINCO ELISA Kit | + +### Materials Text +Plasma insulin is obtained from the LINCO ELISA kit. + +### Manufacturer's Protocol +**RAT/MOUSE INSULIN ELISA KIT** +**96-Well Plate (Cat. # EZRMI-13K)** + +#### Intended Use +This Rat/Mouse Insulin ELISA kit is used for the non-radioactive quantification of insulin in mouse and rat sera. Plasma samples may also be used but application to samples of other biological fluids may need validation by the user. One kit is sufficient to measure 39 unknown samples in duplicate. This kit is for research purposes only. + +#### Principles of Procedure +This assay is a Sandwich ELISA based, sequentially, on: +1. Capture of insulin molecules from samples to the wells of a microtiter plate coated by pre-titered amount of a monoclonal mouse anti-rat insulin antibodies and the binding of biotinylated polyclonal antibodies to the captured insulin +2. Wash away of unbound materials from samples +3. Binding of horseradish peroxidase to the immobilized biotinylated antibodies +4. Wash away of free enzyme conjugates +5. Quantification of immobilized antibody-enzyme conjugates by monitoring horseradish peroxidase activities in the presence of the substrate 3,3',5,5'-tetramethylbenzidine + +The enzyme activity is measured spectrophotometrically by the increased absorbency at 450 nm, corrected from the absorbency at 590 nm, after acidification of formed products. Since the increase in absorbency is directly proportional to the amount of captured insulin in the unknown sample, the latter can be derived by interpolation from a reference curve generated in the same assay with reference standards of known concentrations of rat insulin. + +### Reagents Supplied +Each kit is sufficient to run one 96-well plate including, in duplicates, background, 6 rat insulin standards, 2 quality controls, and 39 unknown samples. +- **Microtiter Plate**: Coated with mouse monoclonal anti-rat insulin antibodies + - Quantity: 1 plate + - Preparation: Ready to use +- **Adhesive Plate Sealer** + - Quantity: 1 Sheet + - Preparation: Ready to use +- **10X Concentrate HRP Wash Buffer**: 10X concentrate of 50 mM Tris Buffered Saline containing Tween 20 + - Quantity: 50 ml/vial + - Preparation: Dilute 10 times with de-ionized water +- **Standards**: Rat Insulin in Assay Buffer: 0.2, 0.5, 1, 2, 5, and 10 ng/ml + - Quantity: 0.1 ml/vial + - Preparation: Ready to use +- **Quality Controls 1 and 2** + - Quantity: 0.1 ml/vial + - Preparation: Ready to use +- **Matrix Solution**: Charcoal stripped pooled mouse serum + - Quantity: 0.5 ml + - Preparation: Ready to use +- **Assay Buffer**: 0.05 M Phosphosaline, pH 7.4, containing 0.025 M EDTA, 0.08% Sodium Azide, and 1% BSA + - Quantity: 20 ml + - Preparation: Ready to use +- **Detection Antibody**: Pre-titered biotinylated anti-insulin antibody + - Quantity: 10 ml + - Preparation: Ready to use +- **Enzyme Solution**: Pre-titered streptavidin-horseradish peroxidase conjugate in buffer + - Quantity: 12 ml/vial + - Preparation: Ready to use +- **Substrate**: (Light sensitive, avoid unnecessary exposure to light) 3,3',5,5'-tetramethylbenzidine in Buffer + - Quantity: 12 ml + - Preparation: Ready to use +- **Stop Solution**: 0.3 M HCl + - Quantity: 12 ml + - Preparation: Ready to use + +#### Storage and Stability +Prior to use, all components in the kit can be stored up to 2 weeks at 2-8°C. For longer storage (>2 weeks), freeze diluted TBS wash buffer, insulin standards, quality controls, and matrix solution at -20°C. Minimize repeated freeze and thaw of the insulin standards, quality controls, and matrix solution. Refer to expiration dates on all reagents prior to use. Do not mix reagents from different kits unless they have the same lot numbers. + +### Reagent Precautions +- **Sodium Azide**: Adds as a preservative at a concentration of 0.08%. Reacts with lead and copper plumbing. +- **Hydrochloric Acid**: Corrosive, avoid contact with skin and eyes. + +### Materials Required but Not Provided +- Pipettes with tips: 10 ml - 100 ml +- Multi-Channel Pipettes: 50 ~ 300 ml +- Reagent Reservoirs +- Vortex Mixer +- Refrigerator +- Deionized Water +- Microtiter Plate Reader (450 nm and 590 nm) +- Orbital Microtiter Plate Shaker +- Absorbent Paper or Cloth +- Sample Collection and Storage + +### Sample Preparation +- **Serum**: Draw whole blood into a centrifuge tube without anti-coagulant. Let clot at room temperature for 30 min, centrifuge at 2000-3000 x g for 15 min at 4°C. Store serum at -20°C. +- **Plasma**: Draw whole blood into centrifuge tubes with K3EDTA (1.735 mg/ml), centrifuge immediately. + +### Assay Procedure +1. Pre-warm all reagents to room temperature immediately before setting up the assay. +2. Dilute the 10x concentrated TBS wash buffer 10 fold. +3. Prepare detection antibody solution in reagent reservoir and add 80 ml to each well. +4. Add 10 ml assay buffer to each of the sample wells: + - NSB wells, rat insulin standards, QC1 and QC2, samples. +5. Cover plate with sealer, incubate for 2 hours on orbital microtiter plate shaker at 400-500 rpm room temperature. +6. Wash wells 6 times with diluted TBS wash buffer, 300 ml per wash. +7. Add 100 ml enzyme solution, cover, incubate with shaking for 30 min. +8. Wash wells 6 times again. +9. Add 100 ml substrate solution, cover, shake for 15 min. +10. Add 100 ml stop solution, shake to mix for complete mixing, read absorbance at 450 nm and 590 nm. + +#### Microtiter Plate Arrangement +Option A: For Samples with significant Serum Matrix Effect. +Option B: For Samples without significant Serum Matrix Effect. + +### Calculations +The dose-response curve fits best to a sigmoidal 4- or 5-parameter logistic equation. Calculate results using a computer program for this purpose. + +### Interpretation +The assay is accepted if Quality Control values fall within the calculated Quality Control Range. If any QCs fall outside the range, repeat the sample. The sensitivity limit is 0.2 ng/ml (35 pM) insulin using a 10 ml sample size. + +### Assay Characteristics +- **Sensitivity**: 0.2 ng/ml (35 pM) insulin +- **Specificity**: Analytical test is specific to insulin. +- **Precision**: Variations in assay results (CV%): + +| Sample | Assay (ng/ml) | Intra-assay CV% | Inter-assay CV% | +|-----------------|---------------|-----------------|-----------------| +| Mouse serum #1 | 0.32 | 8.35 | 17.9 | +| Mouse serum #2 | 1.69 | 0.92 | 6.03 | +| Mouse serum #3 | 3.45 | 1.92 | 7.64 | +| Rat serum #1 | 1.15 | 3.22 | 6.95 | +| Rat serum #2 | 2.32 | 1.33 | 6.71 | +| Rat serum #3 | 3.65 | 1.17 | 9.23 | + +#### Dilutional Linearity +Samples diluted with matrix solution are assayed for insulin levels: + +| Serum Sample # | Dilution Factor | Insulin Level (ng/ml) | % of Expected | +|-----------------|------------------|-----------------------|---------------| +| Mouse Serum #1 | 2x | 2.06 | 100 | +| | 4x | 2.20 | 107 | +| | 8x | 3.12 | 152 | +| Mouse Serum #2 | 2x | 2.98 | 100 | +| | 4x | 3.08 | 103 | +| | 8x | 3.76 | 126 | +| Mouse Serum #3 | 2x | 2.95 | 100 | +| | 4x | 3.08 | 96 | +| | 8x | 3.92 | 105 | +| Rat Serum #1 | 2x | 3.78 | 100 | +| | 5x | 3.00 | 79 | +| | 10x | 3.40 | 90 | +| Rat Serum #2 | 2x | 3.78 | 100 | +| | 5x | 3.00 | 79 | +| | 10x | 3.40 | 90 | +| Rat Serum #3 | 2x | 3.42 | 100 | +| | 5x | 3.15 | 92 | +| | 10x | 3.90 | 114 | + +### Recovery +Spike & Recovery of Insulin in Serum Samples: + +| Serum Sample # | Added Insulin (ng/ml) | Observed (ng/ml) | Recovery (%) | +|----------------|------------------------|------------------|--------------| +| Mouse Serum #1 | 0.33 | | | +| | 0.5 | 0.83 | 100 | +| | 2 | 2.15 | 91 | +| | 5 | 5.07 | 95 | +| Mouse Serum #2 | 1.78 | | | +| | 0.5 | 2.20 | 84 | +| | 2 | 3.43 | 83 | + +### Troubleshooting Guide +- Ensure precise timing and adherence to protocol steps. +- Remove air bubbles formed after substrate addition. +- High absorbance disturbances: + - Ensure no well cross-contamination. + - Ensure proper washing. + +### Replacement Reagents +| Reagents | Cat. # | +|----------------|-------------------| +| Microliter Plate | EP13 | +| 10X Wash Buffer | EWB-HRP | +| Rat Insulin Standards | EB013-K | +| Quality Controls 1 & 2 | E6013 | +| Matrix Solution | EMTX-RM1 | +| Assay Buffer | AB-PhK | +| Detection Antibody | E1013 | +| Enzyme Solution | EHRP-3 | +| Substrate | ESS-TMB2 | +| Stop Solution | ET-TMB | + +--- + +This is an open access protocol distributed under the terms of the [Creative Commons Attribution License](https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/plasmodium-berghei-ookinete-culture-bmm8k49w.md b/markdown-output/plasmodium-berghei-ookinete-culture-bmm8k49w.md new file mode 100644 index 0000000000000000000000000000000000000000..75e0e1fac04e6f7828e398285df14ef9750611ea --- /dev/null +++ b/markdown-output/plasmodium-berghei-ookinete-culture-bmm8k49w.md @@ -0,0 +1,132 @@ +```markdown +# Goal/Experiment: +The goal of this experiment is to produce and culture ookinetes of *Plasmodium berghei* in vitro. The method has been optimized for the *P. berghei* ANKA train clone 2.34 and the derived GFP-expressing clone, but it should work for other strains. + +# Plasmodium berghei Ookinete Culture + +## Authors +**Benito Recio Totoro**1,2 +*1Centro de Investigaciones Sobre Enfermedades Infecciosas, Instituto Nacional de Salud Pública* +*2Instituto de Biotecnología, Universidad Nacional Autónoma de México* + +DOI: [10.17504/protocols.io.bmm8k49w](https://dx.doi.org/10.17504/protocols.io.bmm8k49w) + +## Abstract +*Plasmodium berghei* is one of the main non-human malaria models used in research. It readily infects laboratory strains of mice and rats as well as mosquitoes. Because of this, it is safe to manipulate and does not require human fluids/tissues. This document provides the Operational Procedure for producing and culturing ookinetes of *P. berghei* in vitro. The method has been optimized for the *P. berghei* ANKA train clone 2.34 and the derived GFP-expressing clone, but should work for other strains. + +## Guidelines +Although *Plasmodium berghei* ookinetes have been successfully cultured for more than 30 years, several improvements have been made to the basic protocol. One of them is the use of CF11 cellulose powder to remove white blood cells from the culture. This is advisable since it has been shown that the white blood cells can induce programmed cell death in the ookinetes. However, we have found the use of CF11 columns troublesome since, in our hands, it causes the culture yield to decrease significantly due to the reduction in gametocyte viability. + +## Materials and Reagents + +### Equipment +- *P. berghei* cryopreserved stock. +- Laboratory mice (6 to 8-week-old male BALB/c mice). +- CO₂ euthanasia chamber. +- Brightfield microscope. +- Microscope slides and coverslips. +- Neubauer chamber (haemocytometer). +- Microcentrifuge tubes (0.6 ml and 1.5 ml). +- Incubator at 20°C (18-21°C range). +- 1 ml syringes. +- Cotton balls. +- Bunsen burner. +- T25 culture flasks. +- Standard culture labware. + +### Reagents +- Saline solution (0.9%). +- Methanol. +- Ethanol (70%). +- Giemsa stain (30% in water). +- Immersion oil. +- Phosphate-buffered saline (PBS). +- Phenylhydrazine (6 mg/ml of saline). +- Heparin (250 IU/ml of saline). +- Ookinete culture medium: RPMI 1640 medium at pH 8.3 supplemented with: + - 23.81 mM sodium bicarbonate + - 0.37 mM hypoxanthine + - 25 mM HEPES + - 5000 U/ml penicillin + - 5 mg/ml streptomycin + - 10 mg/ml neomycin + - 20% heat-inactivated fetal bovine serum. + +## Procedure + +### Mice Infections for Passages +1. **Inoculation:** + - Mice are inoculated via intraperitoneally (IP) with no more than 200 μl either from a cryopreserved stock or by passage from other mice. +2. **Determination of Parasites:** + - To determine the number of parasites per ml of blood, collect a drop of tail blood from the infected mouse and make a thin smear. Let it dry and fix with methanol. +3. **Giemsa Staining:** + - Prepare 1 ml of 30% Giemsa per slide and stain for 10 minutes. +4. **Rinse and Dry:** + - Rinse slide with distilled water and dry by gently applying pressure with an absorbent paper towel. Do not rub the paper towel on the slide. +5. **Parasitemia Calculation:** + - Determine the parasitemia by counting at least 500 red blood cells (RBC) at 1000x magnification and determine the percentage of infected RBC (iRBC). + ``` + parasitemia = (iRBC / RBC) * 100 + ``` + where iRBC = infected RBC, RBC = total number of RBC counted. + +6. **Dilution:** + - Collect an additional 1 μl of tail blood and dilute it to 1 ml with PBS with 1 μl of heparin. +7. **RBC Counting:** + - Mix well and determine the number of RBC per ml of blood with the Neubauer chamber. + +8. **iRBC Calculation:** + - Determine the number of infected RBC per ml of blood using the parasitemia obtained previously. + ``` + iRBC/ml = (parasitemia / 100) * (RBC/ml) + ``` + +9. **Euthanasia and Blood Collection:** + - Euthanize the mouse in the CO₂ chamber and collect the blood via cardiac puncture with a 1 ml syringe preloaded with 10 μl of heparin. +10. **Maintenance:** + - Dilute the blood with PBS to obtain 4 x 10⁵ infected RBC per 200 μl and inoculate IP the mice. + - Note: Make no more than 8 passages as gametocytes lose the ability to mature into gametes. + +### Mice Infections for Ookinete Culture +11. **Phenylhydrazine Inoculation:** + - Inoculate mice with 200 μl of phenylhydrazine IP three days before the inoculation of the parasites to induce reticulocytosis. +12. **Parasitemia Calculation:** + - Determine the parasitemia and infected RBC concentration of the donor mouse. +13. **Infected Blood Collection:** + - Collect the infected blood as explained in step 9. +14. **Dilution:** + - Dilute the infected blood with PBS to obtain 4 x 10⁷ infected RBC per 200 μl and inoculate the donor mouse IP. + +### Ookinete Culture +15. **Verification and Exflagellation Test:** + - Three days after the inoculum, verify the parasitemia and perform an exflagellation test. + - Collect 2 μl of tail blood and place in a 0.6 ml microcentrifuge tube with 7 μl of ookinete medium and 1 μl of heparin. Mix very gently. + - Place 10 μl on a microscope slide, cover with a coverslip, and incubate for 15 minutes at 20°C. + - Determine the number of exflagellation centers in 10 random fields of view. Only mice with 15 to 25% parasitemia and >7 exflagellation centers per field of view are used. + +16. **Ookinete Culture Flask Preparation:** + - Prepare a T25 culture flask with 4 ml of ookinete medium. + - Sacrifice mice and collect blood as explained in step 9. + - Note: Handle blood gently to avoid reducing ookinete culture yield. + +17. **Incubation:** + - Mix the blood with the ookinete medium and incubate gently at 20°C for 18 hours. +18. **Count Ookinetes:** + - Determine the number of ookinetes post-incubation. + - Prepare a sample dilution (50 μl in 950 μl of PBS) and count in the Neubauer chamber. + - Dilute 10 μl of the culture dilution on each chamber and count the number of ookinetes. + ``` + Ookinetes/ml = (xOokinetes / 4 squares) * 20 * 10000 + ``` + - Usually, between 25 and 35 million ookinetes are obtained from one mouse. + +## References +- Rodríguez MC, Margos G, Compton H, Ku M, Lanz H, Rodríguez MH, Sinden RE (2002). *Plasmodium berghei: routine production of pure gametocytes, extracellular gametes, zygotes, and ookinetes*. Experimental Parasitology. +- Carter, V., Cable, H. C., Underhill, B. A., Williams, J., & Hurd, H. (2003). *Isolation of Plasmodium berghei ookinetes in culture using Nycodenz density gradient columns and magnetic isolation.* Malaria Journal. +- Nacer A, Underhill A, Hurd H (2008). *The microneme proteins CTRP and SOAP are not essential for Plasmodium berghei ookinete to oocyst transformation in vitro in a cell free system.* Malaria Journal. + +## License +This protocol is available under the terms of the [Creative Commons Attribution License](https://creativecommons.org/licenses/by/4.0/). + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/plating-bacteria-to-isolate-a-single-colony-u8cezsw.md b/markdown-output/plating-bacteria-to-isolate-a-single-colony-u8cezsw.md new file mode 100644 index 0000000000000000000000000000000000000000..aa86514638835241cb52b82e9f0ab9e932ac3826 --- /dev/null +++ b/markdown-output/plating-bacteria-to-isolate-a-single-colony-u8cezsw.md @@ -0,0 +1,77 @@ +```markdown +# Goal/Experiment: +To plate bacteria in order to isolate a single colony. This isolated single colony can be used directly for experimental purposes or used to build up a freezer stock of the bacterial species. + +# Plating Bacteria to Isolate a Single Colony +### Katy Monteith +**University of Edinburgh** +dx.doi.org/10.17504/protocols.io.u8cezsw +**Vale Lab** + +## Abstract +This protocol describes how to plate bacteria in order to isolate a single colony of bacteria. Once this protocol is completed, the isolated single colony can be used directly for experimental purposes or to build up a freezer stock of the bacterial species. + +For further information on building up a freezer stock, continue onto the protocol 'Building up a freezer stock of bacteria' by the same author. + +## Protocol Status +**Working** +We use this protocol in our group and it is working. + +## Guidelines +For efficient bacterial growth in liquid media, the media should not come above the widest area of the conical-shaped flask/tube. Choose a flask/tube wisely depending on the volume of media which you plan to inoculate. For a 10 mL inoculation, a 50 mL Falcon tube is recommended. + +## Safety Warnings +1. All work involving bacteria should be carried out in a hazard group-specific Microbiology Safety Cabinet. +2. Individuals carrying out this protocol should always wear appropriate PPE, i.e., a lab coat and nitrile gloves. +3. Where appropriate, all other Health & Safety requirements relating to the bacterial species used should be followed. + +## Before Starting +Before starting this protocol, users will need to have prepared LB broth and LB agar plates (*Luria-Bertani* liquid medium and *LB (Luria-Bertani) agar*). If instruction is required for making LB broth and/or LB agar, please see the protocols 'LB (Luria-Bertani) liquid medium' and 'LB (Luria-Bertani) agar' by the same author. + +## Materials and Equipment +- **LB broth** (Luria-Bertani liquid medium) - a nutritionally rich medium primarily used for the growth of bacteria +- **LB agar plates** - a solid medium used to culture bacteria +- **Inoculation loop** - a tool used to streak bacteria on media plates +- **Falcon tube (50 mL)** - a conical tube used for culturing bacteria in liquid media +- **Incubator** - used to maintain optimal growth temperature for bacteria +- **Orbital shaker** - used to aerate cultures by shaking + +### Step-by-Step Protocol + +1. **Inoculate Media:** + - Take a previously grown bacterial colony (either frozen aliquot of bacteria in glycerol suspension or lawn of bacteria on an agar media plate). + - Using a sterile inoculation loop, collect a small fragment of bacteria and inoculate it in 10 mL LB broth in a 50 mL Falcon tube. Shake the loop in the media until the fragment has come loose and is visibly floating in the media. + +2. **Incubate for Aerobic Conditions:** + - Loosely replace the Falcon tube lid (use tape to secure it) and incubate in an orbital shaker set to 30/37°C (depending on the optimal growth temperature for species) at 140 rpm for approximately 12-14 hours. + +3. **Check for Growth:** + - The following day, check media for bacterial growth. The media should be cloudy. If unsure, check the optical density (OD) of media using Absorbance Microplate Reader (an OD of 0.6-0.8 is ideal as the bacteria will be in an exponential growth phase). + +4. **Prepare LB Agar Plate:** + - Prepare an LB agar plate and bring to room temperature before use. Alternatively, a genus-specific media plate (e.g. Pseudomonas isolation media) can be used to reduce the risk of plate contamination with a different bacterial species. + +5. **Streak Bacteria on LB Plate:** + - Using a sterile inoculation loop, dip into the overnight media culture and perform streaking by streaking three discrete lines across the bottom of the LB agar plate. + +6. **Sterile LB Broth Streaking:** + - Take a new sterile inoculation loop and dip it into sterile LB broth. Streak three discrete lines at an adjacent angle from the initial three bacteria lines along the side of the LB agar plate. Each line should draw across the previously streaked bacterial lines, decreasing the number of crossings by one with each subsequent line. + +7. **Subsequent Streaking:** + - Turn the LB agar plate 90° and repeat streaking at adjacent angles using a new, sterile inoculation loop each time. This process should be repeated 2-3 times, drawing the last lines to the center of the plate to spread out potential bacterial growth. + +8. **Incubation:** + - Place the plates inverted into an incubator set at 30/37°C and leave for approximately 24 hours. + +9. **Check for Colony Isolation:** + - The next day, check the plate for bacterial growth. Typically, isolated colonies (~1-2 mm diameter) should be spread out across the plate. If there are not enough isolated colonies, further streaking or dilution may be necessary. + +10. **Select Single Colony:** + - Using a sterile inoculation loop, select a single colony (ensuring not to touch any other colonies) and inoculate into 10 mL LB broth. Incubate with aerobic conditions in an orbital shaker at 30/37°C, 140 rpm for approximately 12-14 hours. + - For making frozen stocks, this culture can then be split into 100-200 µL aliquots, and glycerol can be added for preservation. + +11. **Check Optical Density:** + - After 12-14 hours, ensure the optical density of the liquid culture is optimal (between 0.6-0.8) before carrying on to make frozen stocks/perform experimental procedures. + +endofoutput +``` diff --git a/markdown-output/pollen-metabarcoding-rpmd5k6.md b/markdown-output/pollen-metabarcoding-rpmd5k6.md new file mode 100644 index 0000000000000000000000000000000000000000..7aeb1f68b681c3af6bbb09e585f11b1961a0665d --- /dev/null +++ b/markdown-output/pollen-metabarcoding-rpmd5k6.md @@ -0,0 +1,150 @@ +```markdown +# Goal/Experiment: +The goal of this experiment is to perform pollen metabarcoding. + +# Pollen Metabarcoding + +## Tomasz Suchan + +**Citation:** Tomasz Suchan Pollen metabarcoding, protocols.io dx.doi.org/10.17504/protocols.io.rpmd5k6 +**Published:** 16 Jul 2018 + +## Abstract + +Perform reactions in small batches until you are confident that there is no cross-contamination among the samples. Including isolation blanks and PCR blanks is crucial for quality control. + +## Guidelines + +Perform reactions in small batches until you are confident that there is no cross-contamination among the samples. Including isolation blanks and PCR blanks is crucial for quality control. + +## Before start + +Prepare 5 µM primer solutions: + +**ITS2 primers used in the 1st PCR:** +- ITS2-4R: `GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTNNNNNNNTCCTCCGCTTATTGATATGC` +- ITS2-S2F: `ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNATCGATATCTGGTGGTGAAT` + +**Indexing primers used in the 2nd PCR (`xxxxxxx` = index):** +- Primer 1: `AATGATACGGCGACCACCGAGATCTACACxxxxxxxACACTCTTTCCCTACACGACG` +- Primer 2: `CAAGCAGAAGACGGCATACGAGATxxxxxxxGTGACTGGAGTTCAGACGTGTG` + +## Materials + +| Material | Vendor | +| --- | --- | +| Q5 Hot Start High-Fidelity DNA Polymerase - 100 units `M04935` | New England Biolabs | +| Water, nuclease-free | Contributed by users | +| dNTP mix (25 mM of each) | Contributed by users | +| Phire Plant Direct PCR Kit `F130WH` | Thermo Fisher Scientific | + +## Protocol + +### Pollen Extraction + +**Step 1:** Vortex the butterfly in 50 μL of water with 0.1% SDS. +**Safety Information:** Add blank sample at this step (="isolation blank") + +**Step 2:** Evaporate water in speedvac. + +**Step 3:** Add 5 μL of the Phire Plant Direct sample buffer. + +**Step 4:** Spin at max speed for 2 min. + +### 1st PCR + +**Step 5:** Prepare the mix: +- 14 μL molecular grade water +- 25 μL Phire Plant Direct PCR mix +- 5 μL ITS2-S2F primer +- 5 μL ITS2-4R primer + +**Step 6:** Add 1 μL of the sample to the mix. Use water for the blanks. +**Safety Information:** Add another blank sample at this step (="PCR blank") + +**Step 7:** Run the PCR program: initial denaturation at 98°C for 5 min; 20 cycles of denaturation at 98°C for 40 s, annealing at 49°C for 40 s, and elongation at 72°C for 40 s; followed by a final extension step at 72°C for 5 min. + +### Purification + +**Step 8:** Perform AMPure purification with ratio 1x. Elute in 10 μL. +**Protocol:** AMPure purification protocol [Contact: Tomasz Suchan] + +**Step 8.1:** Remove beads from the fridge, equilibrate to room temperature, mix thoroughly but do not vortex. + +**Step 8.2:** Add desired ratio of AMPure beads to the purified sample and mix well by pipetting. + +**Step 8.3:** Incubate for 5 minutes. + +**Step 8.4:** Place on the magnetic rack. + +**Step 8.5:** Let it stand for 5 minutes on the rack, aspirate and discard supernatant. + +**Step 8.6:** Add 200 μL of freshly prepared 70% EtOH, incubate for 30 seconds, aspirate and discard EtOH. + +**Step 8.7:** Repeat the wash: add 200 μL of freshly prepared 70% EtOH, incubate for 30 seconds, aspirate and discard EtOH. + +**Step 8.8:** Wait until the pellet dries out completely. DO NOT remove the plate/tubes from the magnetic rack, as the dried AMPure might pop out from the tubes! + +**Step 8.9:** Add desired volume of Tris 10 mM or water, wetting the dried AMPure pellets (add 1 μL to the final volume to avoid pipetting out the beads). + +**Step 8.10:** Remove from the magnetic rack. + +**Step 8.11:** Resuspend by pipetting or vortexing. + +**Step 8.12:** Incubate for 10 minutes, incubating at 37°C can improve DNA yield. + +**Step 8.13:** Place on the magnetic rack. + +**Step 8.14:** Let it stand for 5 minutes, pipette out and save supernatant. The eluted DNA is in the supernatant, do not discard it! + +### 2nd PCR + +**Step 9:** Mix 1 μL of the template with 7 μL of the mix. Add 2 μL of each 5 μM primer (forward and reverse). +- 4.82 μL molecular grade water +- 2 μL Q5 reaction buffer +- 0.08 μL dNTPs (25 mM each) +- 0.1 μL Q5 Hot Start polymerase + +**Step 10:** Run the PCR program: 30 s denaturation at 98°C; 12 cycles of denaturation at 98°C for 10 s, combined annealing and extension at 72°C for 30 s (shuttle PCR); the final extension at 72°C for 5 min. + +**Step 11:** Check profiles on a gel. + +### Pooling + +**Step 12:** Pool all the samples together. + +**Step 13:** Perform AMPure purification with ratio 1x. Elute in 100 μL. +**Protocol:** AMPure purification protocol [Contact: Tomasz Suchan] + +**Step 13.1:** Remove beads from the fridge, equilibrate to room temperature, mix thoroughly but do not vortex. + +**Step 13.2:** Add desired ratio of AMPure beads to the purified sample and mix well by pipetting. + +**Step 13.3:** Incubate for 5 minutes. + +**Step 13.4:** Place on the magnetic rack. + +**Step 13.5:** Let it stand for 5 minutes on the rack, aspirate and discard supernatant. + +**Step 13.6:** Add 200 μL of freshly prepared 70% EtOH, incubate for 30 seconds, aspirate and discard EtOH. + +**Step 13.7:** Repeat the wash: add 200 μL of freshly prepared 70% EtOH, incubate for 30 seconds, aspirate and discard EtOH. + +**Step 13.8:** Wait until the pellet dries out completely. DO NOT remove the plate/tubes from the magnetic rack, as the dried AMPure might pop out from the tubes! + +**Step 13.9:** Add desired volume of Tris 10 mM or water, wetting the dried AMPure pellets (add 1 μL to the final volume to avoid pipetting out the beads). + +**Step 13.10:** Remove from the magnetic rack. + +**Step 13.11:** Resuspend by pipetting or vortexing. + +**Step 13.12:** Incubate for 10 minutes, incubating at 37°C can improve DNA yield. + +**Step 13.13:** Place on the magnetic rack. + +**Step 13.14:** Let it stand for 5 minutes, pipette out and save supernatant. The eluted DNA is in the supernatant, do not discard it! + +**Step 14:** Check the concentration using Qubit and the profile using Tapestation/Fragment Analyzer. Calculate molarity from that and proceed to the sequencing. Add 15% PhiX to the sequencing run. + +`endofoutput` +``` \ No newline at end of file diff --git a/markdown-output/populating-ncbi-template-for-submissions-using-bio-b2knqcve.md b/markdown-output/populating-ncbi-template-for-submissions-using-bio-b2knqcve.md new file mode 100644 index 0000000000000000000000000000000000000000..5b7e4337beb68df6b9a9adc05487b826e45b4549 --- /dev/null +++ b/markdown-output/populating-ncbi-template-for-submissions-using-bio-b2knqcve.md @@ -0,0 +1,114 @@ +```markdown +# Goal/Experiment: +To define the standard operating procedure for collecting isolate metadata using BioNumerics for submission of food/environmental isolates to NCBI. + +# Populating NCBI Template for Submissions Using BioNumerics v7.6 V.3 + +Authors: Ruth Timme¹, Maria Balkey¹, Julie Haendiges¹ +¹US Food and Drug Administration +Published: Dec 03, 2021 +DOI: [10.17504/protocols.io.b2knqcve](https://dx.doi.org/10.17504/protocols.io.b2knqcve) + +## Purpose +To define the standard operating procedure for collecting isolate metadata using BioNumerics for submission of food/environmental isolates to NCBI. + +## Scope +To provide a standardized procedure to collect isolate metadata using BioNumerics for submission of food/environmental isolates to NCBI. + +## Responsibilities +The GenomeTrakr Network Management will be responsible for monitoring GenomeTrakr submissions processed through Bionumerics and ensuring that all GT labs are familiar with the mandatory metadata fields required for submission of GenomeTrakr sequencing records to NCBI. + +### Version 3 Update +- Dropdown menus from controlled vocabulary added to the `ncbi_update` submission sheet. + +--- + +## Metadata SampleSheet Preparation + +Before uploading your sequencing run or linking NCBI sequencing records at the BioNumerics platform, fill out the metadata spreadsheet form. + +### Steps +1. Download the template and guidelines from the file `GT_BioNumerics_spreadsheet_v2.xlsx`. +2. Create the fields: `NCBI_bioproject`, `Attribute_package`, `Organism_name`, `NCBI_LabID`, `SourceCountryState`, `Latitude_longitude`, `Reference_material`, `Culture_collection`, `Description` if they are not in the BioNumerics interface. +3. Fill out the template information, save the template sheet as `.csv` and import the metadata to BioNumerics. + +## NCBI Submission Settings (Manage Submission Template) + +Create the NCBI metadata template in BioNumerics following PulseNet instructions, ensuring that fields are populated according to GenomeTrakr (GT) requirements. + +### 2.1 BioProject and Organization +GenomeTrakr labs, by submitting, independently become owners of their data and are responsible for managing individual bioprojects for each sequenced organism. + +![NCBI Submission Template: BioProject and Organization](url/to/image1) + +| Name of Field in BioNumerics Template | Description | Example | +|---------------------------------------|---------------------------------------------------------------------------------|--------------------| +| BioProject accession | Identifier for NCBI data collection associated with GenomeTrakr. | PRJNA514285 | +| Organization name | Surveillance Program (default value for GenomeTrakr submissions) | GenomeTrakr | +| SPUID namespace | Surveillance Program (default value for GenomeTrakr submissions) | GenomeTrakr | +| Type | Organization type (default value for GenomeTrakr submissions) | consortium | +| Role | Laboratory responsibility (default value for GenomeTrakr submissions) | owner | +| Contact first name | First name for Lab POC for NCBI submissions. Lab might create alias name for WGS team | First Name | +| Contact last name | Last name for Lab POC for NCBI submissions. Lab might create alias name for WGS team | Last Name | +| Contact e-mail | Email for Lab POC for NCBI submissions. Lab might create alias name for WGS team | first.last@lab.gov | +| FTP upload directory | Name of directory at NCBI FTP site (default value for GenomeTrakr submissions) | submit/Production | + +### 2.2 BioSample +Metadata associated with the isolate might require creating new fields in BioNumerics. + +![NCBI Submission Template: BioSample](url/to/image2) + +| Name of Field in BioNumerics Template | Description | Name of Field in BioNumerics Database | Example of Metadata Value | +|---------------------------------------|------------------------------------------------------------------------------|--------------------------------------|--------------------------------| +| Submitter Provided Unique ID | Local lab strain ID | Entry Key | 21B00181-5 | +| BioSample accession (output) | NCBI accession populated upon submission to NCBI | NCBI_ACCESSION (field content) | SAMN17385051 | +| Organism name | Genus – species for organism | Organism_name (field content) | Listeria monocytogenes | +| Title | Organism name | Organism_name (field content) | Listeria monocytogenes | +| Attribute package | Sample category | Pathogen: environmental/food/other; version 1.0 | Pathogen: environmental/food/other; version 1.0 | +| Strain name | PNUSA identifier | WGS_id (field content) | PNUSAL008933 | +| Serovar (optional) | Serotyping information for Escherichia coli and Salmonella enterica | Serovar (field content) | missing | +| Isolate (optional) | Field not required for GenomeTrakr | | missing | +| Isolate name alias (optional) | Optional identifier for collaboration projects | Isolate_name_alias (field content) | 21B00181-5; RS_21290 | + +### 2.4 BioSample (Continued) +Include metadata in mandatory fields: Collected by, Collection/Isolation Date, Title, Geographical origin, and Isolate source. + +![NCBI Submission Template: BioSample_2](url/to/image3) + +| Name of Field in BioNumerics Template | Description | Name of Field in BioNumerics Database | Example of Metadata Value | +|---------------------------------------|---------------------------------------------------------|--------------------------------------|------------------------------------------------| +| Collected by | Full name of laboratory that collected the sample | NCBI_LabID (field content) | NY Department of Agriculture and Markets | +| Collection date | Date on which the sample was collected | IsolateDate (field content) | 2020 | +| Geographical location | Country and State for sample collection | SourceCountryState (field content) | USA:NY | +| Geographical coordinates | Latitude and longitude for site of collection | | missing | +| Isolation source | Detailed description for sample product or environmental source | SourceSite (field content) | cheese | +| Host | Only for human isolates | | missing | +| Host disease | Only for human isolates | | missing | + +### 2.5 NCBI Submission Settings – SRA Experiment and Run +Populate fields for SRA Experiment and Run according to PulseNet instructions. + +![NCBI Submission Template: SRA Experiment and Run](url/to/image4) + +### NCBI Submission Settings – Submission Template +Save submission template following PulseNet Instructions as `GenomeTrakr-Template`. + +## Import Data + +1. Import the `GenomeTrakr Metadata` form for BioNumerics (`GT_BioNumerics_spreadsheet_v2.csv`) according to PulseNet Instructions. +2. Ensure that field sources match destination fields during import. +3. In the importing links section, choose the `-key-` for linking records to database entries. +4. Proceed with sequencing data import according to PulseNet Instructions. +5. Submit data to NCBI following PulseNet Instructions. If NCBI accessions are not available at BioNumerics within 1 business day, contact NCBI and PulseNet to troubleshoot. +6. Contact GenomeTrakr via email ([genometrakr@fda.hhs.gov](mailto:genometrakr@fda.hhs.gov)) if issues occur, especially if submissions are delayed for more than 3 days. GenomeTrakr can support urgent submissions if needed. + +## NCBI Submission for Fields Not Included in the BioNumerics Template + +Labs need to include the laboratory sequencing the isolates and the surveillance effort name in the `sequenced_by` and `project_name` fields, respectively. + +- After receiving biosample accessions, fill out the BioNumerics update spreadsheet (`BioNumerics_update.xlsx`) and submit the update to NCBI by contacting [biosamplehelp@ncbi.nlm.nih.gov](mailto:biosamplehelp@ncbi.nlm.nih.gov). + +--- + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/poultry-enterprise-environmental-sample-microbiolo-n6sdhee.md b/markdown-output/poultry-enterprise-environmental-sample-microbiolo-n6sdhee.md new file mode 100644 index 0000000000000000000000000000000000000000..7313d07f74b682df42468be67502ede6f185bcd5 --- /dev/null +++ b/markdown-output/poultry-enterprise-environmental-sample-microbiolo-n6sdhee.md @@ -0,0 +1,144 @@ +```markdown +# Goal/Experiment: +The microbiological testing method for Salmonella spp. as used for testing primary environmental samples collected from poultry environments. This method aligns with the Australian Standard 5013.10:2009 and the ISO 6579:2002 standards for detecting Salmonella spp. with specific modifications. + +# Poultry Enterprise Environmental Sample Microbiological Testing for Salmonella spp. +**Helen Crabb** + +## Abstract +The microbiological testing method for Salmonella spp. as used for testing primary environmental samples collected from poultry environments. The method is described in accordance with the Australian Standard 5013.10:2009 Horizontal method for the detection of Salmonella spp. (ISO 6579:2002, MOD). This method is a modification of the ISO 6579:2002 Horizontal method for the detection of Salmonella spp.. The difference in methodology is due to the use of a different control isolate. + +## Materials +- **Buffered Peptone Water (CM0509)** by Oxoid Microbiology Products - Thermo Fisher +- **Modified Semi-solid Rappaport Vassiliadis medium base (CM0910)** by Oxoid Microbiology Products - Thermo Fisher +- **Xylose Lysine Desoxycholate Agar (CM0469)** by Oxoid Microbiology Products - Thermo Fisher +- **Cystine Lactose Electrolyte Deficient Agar (CM0301)** by Oxoid Microbiology Products - Thermo Fisher +- **Brilliant Green Agar (Kauffmann Medium) (CM0263)** by Oxoid Microbiology Products - Thermo Fisher +- **Triple Sugar Iron Agar (CM0277)** by Oxoid Microbiology Products - Thermo Fisher +- **Lysine Iron Agar (CM0381)** by Oxoid Microbiology Products - Thermo Fisher +- **o-nitrophenyl-β-D-galactosidase (ONPG) broth (2085)** by Contributed by users +- **Tryptone Soya Broth (CM0129)** by Oxoid Microbiology Products - Thermo Fisher +- **Ammonium Salt Sugar Mannitol (2344)** by Contributed by users +- **SyberSafe DNA Gel Stain (S33101)** by Invitrogen - Thermo Fisher +- **Hyperladder 1kb (BIO33025)** by Bioline + +## Protocol + +### Step 1. Standard Testing +Microbiological testing was conducted in accordance with: +Australian Standard 5013.10:2009: Food Microbiological Method 10: Microbiology of food and animal feeding stuffs - Horizontal method for the detection of Salmonella spp. (ISO 6579:2002, MOD) + +Specific details for each method can be obtained from the standard. + +### Step 2. Primary Sample Processing +Primary sample processing is described in the Protocol: Poultry Enterprise Environmental Sample Handling and Processing for Salmonella spp. Detection. +All samples were collected and processed for microbiological testing on the same day. + +### Step 3. Primary Sample Pre-Enrichment +Each primary sample had Buffered Peptone Water (BPW) added and was prepared for incubation. +- **Incubation Conditions:** Aerobic static incubation. +- **Incubation Time and Temperature:** 18-24 hours at 37°C. + +### Step 4. Primary Selective Media - Modified Semi-solid Rappaport Vassiliadis (MSRV) Medium +#### Sub-sampling onto MSRV +After incubation of the primary sample, three aliquots (each 33 µL) were taken from the primary sample and inoculated onto Modified Semi-solid Rappaport Vassiliadis (MSRV) agar plates. +- **Incubation Conditions:** Aerobic static incubation. +- **Incubation Time and Temperature:** 41.5°C and visually examined at 12, 24, and 48 hours post inoculation. + +#### MSRV Colony/Growth Characteristics +Salmonella suspect positive MSRV plates have evidence of swarming growth, characterized by a grey-white turbid zone extending from the inoculation point with a clearly defined edge. + +### Step 5. Secondary Selective Media - XLD, BGA, CLED +MSRV plates showing evidence of swarming growth were sub-cultured using a loop and plated onto secondary media: +- **Xylose-Lysine-Desoxycholate agar (XLD)** +- **Brilliant Green agar (BGA)** +- **Cystine Lactose Electrolyte Deficient agar (CLED)** + +- **Incubation Conditions:** Aerobic static incubation. +- **Incubation Time and Temperature:** 24 hours at 37°C. + +#### Secondary Selective Media Colony Characteristics +| Test Media | Colony Morphology | Agar Colour Reaction | Other Growth Characteristics | +| --- | --- | --- | --- | +| XLD (CM0469) | Red with black centre | Red | Non H2S fermenters are red only | +| CLED (CM0301) | Flat blue colonies | No Colour change | - | +| BGA (CM0263) | Red-Pink-white opaque colonies | Red | Lactose fermenters pink colonies | + +### Step 6. Salmonella Confirmation - Biochemical Testing +#### Biochemical Test Media +- **Triple Sugar Iron agar (TSI)** +- **Lysine Iron agar (LIA)** + +#### Test Method +Using a sterile inoculating needle, lightly touch the surface of the colony to be sampled. Streak the surface and then stab into the butt of the media. +- **Incubation Conditions:** Aerobic static incubation. +- **Incubation Time and Temperature:** 24 hours at 37°C. + +#### Test Reaction for Salmonella Positive Biochemical Reactions +| Test media | Slant | Butt | Gas | H2S Production | +| --- | --- | --- | --- | --- | +| TSI | Red or no change | Yellow | Yes | Black | +| LIA | Purple | Purple | No | Black | + +#### Elimination of Salmonella Sofia Isolates +Each TSI/LIA Salmonella positive isolate was tested using ONPG Broth and Mannitol broth. +- **Incubation Conditions:** Aerobic static incubation. +- **Incubation Time and Temperature:** 24 hours at 37°C. + +| Test Media | Start Colour | Salmonella Sofia Positive | Salmonella spp. | +| --- | --- | --- | --- | +| ONPG Broth | Colourless | Yellow | No Change | +| Mannitol Broth | Blue-Green | Yellow | No Change | + +### Step 7. Isolate Storage +All positive samples were sub-cultured into Tryptone Soya broth (Oxoid CM0129) containing 30% glycerol, and stored at -80°C, and Salmonella maintenance media for long-term storage at room temperature. + +### Step 8. Serotyping by RT-PCR +#### RT-PCR Methods +Methods for Salmonella confirmation and serotyping via RT-PCR were validated with published methods. + +#### Primers for RT-PCR for Salmonella spp., Salmonella Typhimurium, and Salmonella Infantis +| Gene | Primer ID | Primer Sequence (5’-3’) | Product Size (Base Pairs) | +| --- | --- | --- | --- | +| STM (S. Typhimurium) | STM4497F | GGAATCAATGCCGCCAAATG | 523 | +| STM4497R | CGTGCTGTAATGCTCCGCTC | | +| FliB (S. Infantis) | 87F8 | TTCGTCTGCAGAGACTGTAG | 413 | +| 125R5 | CCCAGTCGGCACAGCT | | +| InvA (Salmonella spp.) | 139-141F | ACAGTGCTTGTCTTACCGGATGCT | 244 | +| 139-141R | AGACGACTGGTACTGATGATAAT | | + +#### RT-PCR Method +Each suspect colony was suspended in sterile water (200 µL), incubated at 100°C for 2 minutes, centrifuged at 16000x g for 5 minutes, and the supernatant was removed and stored at -20°C. PCR was performed with the following reagents: +- dNTPs (1.25 mM) +- Oligonucleotide primers (10 mM) +- MgCl2 (25 mM) +- 5x GoTaq® buffer, water, 1x GoTaq® enzyme (Promega) + +- **Reaction Cycling Conditions:** 35 cycles of denaturation at 95°C for 1 minute, primer annealing at 62°C for 30 seconds, and extension at 72°C for 1 minute. Final extension at 72°C for 10 minutes. +- **Gel Electrophoresis:** 1.5% agarose gel, stained with SyberSafe® DNA gel stain (Invitrogen), electrophoresed at 80 V/cm for 20-30 minutes. + +### Step 9. Reference Laboratory Serotyping, Phage Typing, and MLVA Profiling +#### Serotyping +All samples were initially screened using the H antigen (flagella). + +#### Phage Typing +Phage typing was conducted using the Anderson phage typing scheme. + +#### MLVA Profiling +All Salmonella Typhimurium confirmed isolates were MLVA profiled according to European MLVA protocols. + +| Locus | Repeat Fragment Length (bp) | Fragments to remove (bp) Primers | Example Fragment Length (bp) | Example Australian Code | +| --- | --- | --- | --- | --- | +| STTR-9 | 9 | 135 | 162 | 03 | +| STTR-5 | 6 | 169 | 313 | 24 | +| STTR-6 | 6 | 258 | 324 | 11 | +| STTR-10 | 6 | 305 | 371 | 11 | +| STTR-3 | 33 | - | 523 | 523 | + +--- +Published: 01 Apr 2018 + +Citation: Helen Crabb Poultry Enterprise Environmental Sample Microbiological Testing for Salmonella spp. protocols.io dx.doi.org/10.17504/protocols.io.n6sdhee + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/preparation-of-a-reduced-cr2-solution-for-sulfide-b2b9qar6.md b/markdown-output/preparation-of-a-reduced-cr2-solution-for-sulfide-b2b9qar6.md new file mode 100644 index 0000000000000000000000000000000000000000..f348a4e0a14fb0ad08014c73c467df2bef7422d3 --- /dev/null +++ b/markdown-output/preparation-of-a-reduced-cr2-solution-for-sulfide-b2b9qar6.md @@ -0,0 +1,142 @@ +```markdown +# Goal/Experiment: +Preparation of a reduced Cr²⁺ solution for sulfide distillation using a Jones Reductor + +## Preparation of a reduced Cr²⁺ solution for sulfide distillation using a Jones Reductor + +**Sebastian Rubiano-Rincon¹, Patrick Larkin¹** + +¹Texas A&M University-Corpus Christi + +## Abstract +This protocol uses a Jones Reductor to prepare reduced Cr²⁺ for the distillation of H₂S from marine sediments. It is an adaptation of a protocol described by Backlund et al. + +## Guidelines +This procedure takes approximately 4-6 hours to complete, from setup to clean-up. + +## Safety Warnings +- Many of these solutions can be toxic (e.g., HgCl₂). Set up the Jones Reductor in a fume hood and use caution and PPE for all steps. + +## Materials +- 12 N HCl +- 1 N HCl +- 0.5 N HCl +- 0.25 N HCl +- Granular Zinc (20-30 mesh) +- 2% Mercury (II) Chloride (HgCl₂) +- 1M Chromium (III) Chloride Hexahydrate (CrCl₃•6H₂O) acidified in 0.5N HCl +- 60 mL plastic (polypropylene) syringes +- 250 mL beakers +- Glass rod or spatula +- Small plastic funnel +- Plastic tubing +- Support Stand with clamp +- Glass column (ca. 40 cm x 1.5 cm internal diameter with integral glass sinter at bottom) +- Stopcock (3-way) for glass column +- Citranox detergent + +## Jones Reductor Setup and Use + +1. **Visual**: The setup of the Jones Reductor for the reduction of chromium will appear as follows (see Figure 1): + + ![Jones Reductor Setup](insert image link here) + +2. **Setup Glass Column**: Set up in a support stand a glass column (~40 cm long, 1.5 cm internal diameter) with an integral sinter at the bottom. Secure a stopcock at the bottom of the column and turn the handle to the closed position. + +3. **Prep Granular Zinc**: + - Measure 40-50 g of granular zinc in a 250 mL beaker. + - Wash for 1 min with ~50 mL 1N HCl by stirring with a glass stir rod. + - Carefully decant the HCl solution into a waste beaker. + - Repeat the washing with 1N HCl and decantation two more times. + +4. **Wash Zinc with DI Water**: + - Wash the granular zinc from above with ~50 mL DI water for 1 min by stirring with a glass stir rod. + - Carefully decant the water. + - Repeat the washing with DI water and decantation one more time. + +5. **Cover Zinc Granules**: + - Cover the washed zinc granules with ~50 mL of 2% HgCl₂ solution. + - Stir the contents for 5 to 10 min with the aid of a stirring bar and/or glass rod or spatula. + +6. **Wash HgCl₂-coated Zinc Granules**: + - Decant the HgCl₂ solution from the zinc granules into a waste beaker. + - Wash them with ~50 mL of DI water as before. Decant and repeat the washing two more times. + - The resulting amalgamated zinc should have a bright silvery luster. + +7. **Transfer Zinc to Column**: + - Transfer the amalgamated zinc granules to the glass column using a plastic funnel and fill the column without going over the shoulder of the column. This completes the set-up of the Jones Reductor (refer to Figure 1). + +8. **Wash Column**: + - Run 50 mL of DI water down the column into the waste beaker to wash the amalgamated zinc granules. + - After washing, ensure the zinc granules are covered with water to prevent oxidation if not used immediately. + +9. **Zn Granules Activation**: + - Activate the amalgamated zinc granules by running ~75 mL of 0.5N HCl down the column into the waste beaker. + - Once done, close the stopcock and leave the zinc column submerged in HCl solution. + +10. **Begin Reduction Process**: + - Begin percolating the 1M CrCl₃*6H₂O solution through the Jones reductor by opening the stopcock and letting the solution run down the column. + - An efficient reduction of the chromium ions is verified by a color change from dark green (Cr³⁺) solution to a bright blue (Cr²⁺) solution (refer to Figure 1). + +11. **Monitor Solution Color**: + - As soon as the drops exiting the column turn bright blue, close the stopcock. + +12. **Prepare Tubing**: + - Obtain an 8-cm long piece of narrow (5 mm OD) plastic tubing (the stems of transfer pipets will work). + - Attach one end to the stopcock and the other end to the tip of a plastic (polypropylene) syringe. + +13. **Collect Cr²⁺ Solution**: + - Open the column stopcock and slowly start drawing the reduced Cr²⁺ solution from the column into the syringe. + - Prevent accumulation of air pockets inside the syringe as they may re-oxidize the solution. + +14. **Collection into Syringe**: + - Collect 50 mL of the reduced Cr²⁺ solution into one plastic syringe. + - Ensure the CrCl₃*6H₂O (green) solution is continuously added to the zinc column to keep it covered. + +15. **Preparation for Storage**: + - Once the collection is done, close the stopcock and quickly cap the syringe. + - If necessary, gently push out any air bubbles from the syringe before capping. + - Repeat 50 mL collections as needed. + +16. **Store Cr²⁺ Solution**: + - Store Cr²⁺ solution at room temperature for subsequent sulfide distillation procedures. + +## Clean Up + +17. **Clean the Zinc Column**: + - Clean the zinc column by passing ∼25 mL of 0.25N HCl, followed by ∼25 mL of DI water. + - Leave the zinc column full of water for future use (2-3 weeks maximum). + +18. **Clean Glassware**: + - Rinse each piece of glassware with DI water to dispose of any solid or liquid into a waste bottle. + - Wash glassware in warm, 1% Citranox solution. + - Rinse with Distilled H₂O (3x), followed by DI H₂O (1x). + - Dry in oven for 2 hours at 100°C or air dry. + +## Waste Management + +20. **Disposal of Liquid Waste**: + - All liquid waste must be disposed of in properly labeled waste bottles. Make sure to list every chemical in the waste label on the bottle. + +21. **Disposal of Solid Waste**: + - All solid waste must be disposed of in a solid waste bucket. + +22. **Mercury Waste Disposal**: + - Important: Any residues of the Hg-amalgamated zinc granules, including the reducing column, must be disposed of into a metallic mercury waste bag. + +## Terminology and Reagents + +- **HCl (Hydrochloric Acid)**: A strong acidic solution used for cleaning and preparing the zinc. +- **HgCl₂ (Mercury (II) Chloride)**: A toxic compound used to amalgamate zinc for the reduction process. +- **CrCl₃*6H₂O (Chromium (III) Chloride Hexahydrate)**: A chemical source of Cr³⁺ ions in the reduction process. +- **DI Water (Deionized Water)**: Purified water used to rinse and clean materials. +- **Citranox**: A detergent used for cleaning glassware. + +## Alternative Methods +In case of difficulties sourcing certain supplies: +1. **Granular Zinc**: Ensure particle size is similar to optimize the reduction process—sourcing a mesh range of 20-30 from different suppliers. +2. **HgCl₂ Substitution**: Due to toxicity, considering using other less toxic amalgamating agents where applicable, under safety guidelines. +3. **Glass Columns**: Alternative suppliers or custom laboratory equipment providers may provide glass columns with necessary specifications. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/preparation-of-dsrna-viruses-for-minion-clm-bvbkn2kw.md b/markdown-output/preparation-of-dsrna-viruses-for-minion-clm-bvbkn2kw.md new file mode 100644 index 0000000000000000000000000000000000000000..e685426a9c7b9c343000c40a21b6add5fa3e935e --- /dev/null +++ b/markdown-output/preparation-of-dsrna-viruses-for-minion-clm-bvbkn2kw.md @@ -0,0 +1,182 @@ +```markdown +# Goal/Experiment: +The goal of this protocol is to prepare double-stranded RNA (dsRNA) viruses for sequencing using the MinION platform by Oxford Nanopore Technologies. This involves the filtration of viral lysates, extraction and purification of RNA, and synthesis of cDNA for sequencing. + +## Preparation of dsRNA viruses for MinION - CLM + +**Authors:** +Alexander H Wilcox¹, Erik Delwart², Samuel L Díaz Muñoz¹ +¹University of California, Davis +²University of California, San Francisco + +**Date Published:** +June 23, 2021 + +**Protocol Status:** +In Development + +**Keywords:** +dsRNA, MinION, Sequencing, Virus Preparation + +**External Link:** +[https://doi.org/10.1099/mgen.0.000315](https://doi.org/10.1099/mgen.0.000315) + +**Protocol Citation:** +Wilcox AH, Delwart E, Díaz Muñoz SL. 2021. Preparation of dsRNA viruses for MinION - CLM. protocols.io. [https://protocols.io/view/preparation-of-dsrna-viruses-for-minion-clm-bvbkn2kw](https://protocols.io/view/preparation-of-dsrna-viruses-for-minion-clm-bvbkn2kw) + +**Fork Note:** +Forked from Preparation of dsRNA viruses for next-generation sequencing, Sam Diaz-Munoz + +**License:** +This is an open-access protocol distributed under the terms of the [Creative Commons Attribution License](https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. + +**Created:** +May 26, 2021 + +**Last Modified:** +Jun 23, 2021 + +**Protocol Integer ID:** +50252 + +## Materials + +- **NEBNext Second Strand Synthesis (DNTP-free) Reaction Buffer - 0.4 ml** + Biolabs Catalog #: B6117S + +- **E. coli DNA Ligase - 1,000 units** + New England Biolabs Catalog #: M0205L + +- **DNA Polymerase I (E.coli) - 2,500 units** + New England Biolabs Catalog #: M0209L + +- **DMSO** + Sigma Aldrich Catalog #: D1435 + +- **Nuclease-free water** + Ambion Catalog #: AM9932 + +- **Qubit dsDNA HS Assay Kit** + Thermo Fisher Scientific Catalog #: Q32851 + +- **DNase I and DNase I Cleanup Reagents or Kit for Removal of DNA Prior to Depletion** + New England Biolabs Catalog #: M0303 + +- **Millex-GP Syringe Filter Unit, 0.22 µm** + EMD Millipore Catalog #: SLGP033RS + +- **Qubit RNA HS Assay Kit** + Thermo Fisher Scientific Catalog #: Q32852 + +- **RNase H** + New England Biolabs Catalog #: M0297S + +- **RNeasy Mini Kit** + Qiagen Catalog #: 74104 + +- **SuperScript™ III First-Strand Synthesis System** + Thermo Fisher Scientific Catalog #: 18080051 + +- **RNase A/T1 Mix** + Thermo Fisher Scientific Catalog #: EN0551 + +- **RNeasy MinElute Cleanup Kit** + Qiagen Catalog #: 74204 + +- **Nucleospin Gel and PCR cleanup** + Macharey Nagal Catalog #: 740609.250 + +## Methods + +1. **Preparation of Viral Lysates:** + - Pass through a 0.22 µm filter to remove host debris. + - Treat with nucleases to degrade extracapsular nucleic acids. + - For 1 ml filtrate: + - 25 µL DNase I + - 50 µL RNase A/T1 + - 107.5 µL DNase I Buffer + - Incubate for 1 hour 30 minutes at 37ºC. + - Inactivate nucleases at 65ºC for 10 minutes and proceed to RNA extraction. + +2. **RNA Extraction:** + - Use a commercially available kit (e.g., RNeasy Mini Kit from Qiagen). + - Avoid kits requiring carrier RNA. + - Follow manufacturer instructions with these modifications: + - Pass all lysate through the column at the first step. + - Elute with 45 µL of Elution Buffer. + - Let column sit at 25ºC for 5 minutes before spinning. + - RNA may be frozen at -80ºC for future processing. + +3. **RNA Quantification:** + - Use Qubit RNA High Sensitivity Kit (HS). + +4. **RNA Preparation:** + - Add 50% DMSO (v/v) to RNA sample and incubate for 1 hour 30 minutes at 65ºC. + +5. **RNA Purification:** + - Use a column cleanup kit (e.g., RNeasy MinElute Cleanup Kit). + - Follow these steps: + 1. Adjust sample volume to 100 µL using RNase-free water. + 2. Centrifuge at maximum speed. + 3. Store at -80ºC or place on ice. + +6. **First Strand Synthesis - SuperScript™ III:** + - Prepare the following master mixes (MM): + - **Step 1:** + | Reagent | Vol for 1 rxn | + | -------------- | --------------- | + | dNTPs | 1 µL | + | Random hexamers| 1 µL | + | RNase free water | 4 µL | + | | | + - **Step 2:** + | Reagent | Vol for 1 rxn | + | -------------- | --------------- | + | 5x SSIV buffer | 4 µL | + | 100 mM DTT | 1 µL | + | RNase out* | 1 µL | + | SSIV RT | 1 µL | + | | | + - (* = RNase out instead of RNase inhibitor) + - Mix appropriate volume of step 1 master mix and add 6 µL to each RNA sample: + 1. Incubate at 65ºC for 5 minutes. + 2. Add step 2 master mix. + 3. Incubate at 23 ºC for 10 minutes, at 52.5 ºC for 10 minutes, and at 80 ºC for 10 minutes. + 4. Store at 4ºC or proceed to second strand synthesis. + +7. **Second Strand Synthesis:** + - Prepare the following master mix (MM): + | Reagent | Vol for 1 rxn | + | ----------------------------- | ------------- | + | dNTPs | 1 µL | + | E. coli DNA ligase | 0.5 µL | + | DNA polymerase I | 2 µL | + | RNase H | 0.5 µL | + | 5x second strand synthesis buffer (OR 10x SS buffer) | 8 µL (4 µL) | + | Nuclease free water | 8 µL (12 µL) | + - Mix appropriate volumes and add 20 µL to each sample. + - Incubate at 16 ºC for 5 hours. + +8. **Purify DNA:** + - Use a preferred method (e.g., Macharey-Nagal Nucleospin Gel and PCR Clean-up kit). + - Quantify DNA using Qubit. + +9. **Prepare DNA Libraries:** + - Use chosen platform and library preparation kit (e.g., Nextera XT, Oxford Nanopore Native Barcode sequencing). + +## Terminology and Reagents Explanation + +- **DNTPs:** Deoxynucleotide triphosphates, essential building blocks for DNA synthesis. +- **E. coli DNA Ligase:** Enzyme used to join DNA fragments during the cloning process. +- **DNA Polymerase I:** Enzyme that adds DNA nucleotides to a primer during DNA replication. +- **DMSO:** Dimethyl sulfoxide, enhances nucleic acid stability during thermal cycling. +- **RNase H:** Enzyme that cleaves the RNA strand of RNA/DNA hybrids. +- **SuperScript™ III:** Reverse transcriptase enzyme for synthesizing cDNA from RNA. + +## Alternative Suggestions + +- If RNase-free water or nuclease-free water is unavailable, autoclaved distilled water can be an alternative. +- TRIzol method can be used instead of RNeasy Mini Kit for RNA extraction, if preferred. + +endofoutput +``` diff --git a/markdown-output/preparation-of-ink-for-electrode-deposition-via-pa-btm3nk8n.md b/markdown-output/preparation-of-ink-for-electrode-deposition-via-pa-btm3nk8n.md new file mode 100644 index 0000000000000000000000000000000000000000..e219b666420cee390aabd550a59596e15e0032d3 --- /dev/null +++ b/markdown-output/preparation-of-ink-for-electrode-deposition-via-pa-btm3nk8n.md @@ -0,0 +1,71 @@ +```markdown +# Preparation of Ink for Electrode Deposition via Paint Brushing using Oxide Powder V.3 + +**Giulio Cordaro** + +Université Paris-Saclay, CentraleSupélec, CNRS, Laboratoire SPMS, 91190, Gif-sur-Yvette, France +dx.doi.org/10.17504/protocols.io.btm3nk8n +Mar 25, 2021 +SOFC Procedures + +## Goal/Experiment: +The aim of this protocol is to produce a viscous ink for brush painting of porous electrodes on top of electrolyte pellets for Solid Oxide Fuel Cells (SOFCs). The layers have variable porosity and thickness depending on the calcination parameters and the type of materials used. Proper adhesion and homogeneity are crucial for the performance of the electrodes. + +## Abstract +A simple and efficient procedure to produce a viscous ink for brush painting of porous electrodes on top of electrolyte pellets for Solid Oxide Fuel Cells (SOFCs). The layers have a variable porosity between 20 and 40%, depending on the choice of the calcination parameters, which is strongly influenced on the electrode and electrolyte materials, the particle size distribution and the morphology of the electrode powder. The thickness of the layers is variable in the 10-100 µm range, depending upon the amount of ink deposited. All the calcination parameters, the electrode and electrolyte materials, the particle size distribution and the morphology influence the adhesion of the electrode layer on the electrolyte substrate. + +## Keywords +Electrode, Paint brushing, Viscous paste, Ink, SOFC, Solid Oxide, Terpineol + +## Guidelines +### Calcination Parameters +Calcination parameters should be optimized for each sample to ensure good adhesion, avoiding the production of an insulator phase at the interface and avoiding parasitic reactions between electrolyte and electrode. Electrochemical performance is strongly influenced by calcination. + +- **Reactivity Test**: Prepare a 50:50 wt% mixture of powders, calcine at different temperatures, and analyze with XRD. +- **Adhesion Test**: Paint and calcine the ink, then evaluate adhesion with tape. If adhesion is poor, avoid electrochemical measurements. + +Optimal parameters produce the best electrochemical performances and should be consistently applied across samples. + +## Procedure + +### 1. Preparation of Organic Viscous Paste (3h) +1. **Weigh Terpineol**: Directly inside the bottle, weigh 1.52 g of terpineol or rescale all ingredients if needed. + + | Compounds | CAS Number | Percentage [wt./wt.] | Amount [g] | + |-----------------|------------|----------------------|------------| + | Ethyl-cellulose | 9004-57-3 | 4% | 0.0800 | + | Terpineol | 95-55-5 | 76% | 1.5200 | + | Iso-propanol | 67-63-0 | 20% | 0.4000 | + +2. **Add Iso-propanol**: 0.4 g +3. **Stir**: Close the lid and stir for a few minutes on a magnetic plate. +4. **Weigh Ethyl-cellulose**: Separately on tin foil, 0.08 g. +5. **Add Ethyl-cellulose**: Little by little to avoid big agglomerations and ensure complete dissolution and homogenization. Be careful to avoid clumps. + +### 2. Treatment of the Electrode Powder for Ink Preparation (6h) +1. **Pre-treatment**: Treat the electrode powder to avoid agglomerates. +2. **Ball Milling**: 300 rpm for 4 hours with balls/powder mass ratio of 10 to 20 with ethanol, using zirconia or WC jar/balls. Ensure no contamination from jar/balls. + + - **Alternative**: If milling equipment is unavailable, manually grind the powder using a mortar and pestle for 30 minutes before rinsing with ethanol and drying. + +3. **Rinse and Dry**: Rinse with ethanol, recover powder in a beaker, then dry on a hotplate or in a muffle at 80°C. + +### 3. Mixing Organic Vehicle with Powder and Painting (30m) +1. **Prepare Bottle**: Clean and weigh a plastic bottle with a cone-shaped bottom. +2. **Weigh Viscous Paste**: About 0.2 g with a thin metal tip. +3. **Calculate Electrode Powder**: Add ratio (3:2 in weight) of electrode powder to viscous paste. +4. **Mix**: Weigh, add electrode powder, and mix thoroughly with a metal tip to ensure full incorporation. +5. **Rest**: Allow for sedimentation for at least 15 minutes. +6. **Paint Electrode**: Dip the brush only in the top part of the ink and paint the electrolyte surface as uniformly as possible. +7. **Dry**: At 150°C for 3 hours. +8. **Repeat Painting**: Paint the other side. +9. **Calcination**: At 1000°C for 2 hours (± 1°C/min). Adjust parameters based on electrode and electrolyte materials, particle size distribution, and morphology of the electrode powder. + +## Notes +- **Terpineol**: A common solvent used in paste formulations, ideally purchased from chemical suppliers like Sigma-Aldrich. +- **Ethyl-cellulose**: A binder for thickening the ink; ensure proper dissolution. +- **Iso-propanol**: A solvent that helps control the viscosity and drying characteristics. +- **Electrode Powder**: SOFC specific materials like YSZ (Yttria-Stabilized Zirconia) or similar. + +**endofoutput** +``` \ No newline at end of file diff --git a/markdown-output/preparation-of-tara-sample-dna-from-iron-chloride-c24ygv.md b/markdown-output/preparation-of-tara-sample-dna-from-iron-chloride-c24ygv.md new file mode 100644 index 0000000000000000000000000000000000000000..b87aab092d54c0915faa2eb02cb7adf7c93764f5 --- /dev/null +++ b/markdown-output/preparation-of-tara-sample-dna-from-iron-chloride-c24ygv.md @@ -0,0 +1,356 @@ +```markdown +# Goal/Experiment: +DNA extraction from iron-chloride precipitates in Tara sample to recover high-quality DNA for further analysis. + +## Preparation of Tara Sample DNA From Iron-Chloride Precipitates + +**Matt Sullivan Lab** + +**Abstract** +Citation: Matt Sullivan Lab Preparation of Tara Sample DNA From Iron-Chloride Precipitates. protocols.io [dx.doi.org/10.17504/protocols.io.c24ygv](http://dx.doi.org/10.17504/protocols.io.c24ygv) +Published: 04 Jan 2016 + +## Guidelines +Reference: 'DNA Extraction of Viruses using Wizard Prep Columns' protocol. + +### Needed: + +- Filters +- Forceps (bleached) +- Aluminum foil squares +- 50cc tube +- Resuspension buffer (0.1M EDTA - 0.2M MgCl₂ - 0.2M Ascorbate Buffer) +- Sterile applicator sticks +- 20 mL filter +- 2x 10mL filter +- 20L seawater +- Parafilm +- Aluminum foil +- Rotator +- Rubber bands +- 5mL pipet +- 15mL or 50 mL sterile tube +- Centrifuge @ 1000 rpm, 14rpm, 1,000g, and 10,000g +- Stock DNase +- DNase buffer +- EDTA +- EGTA +- Amicon Ultra 100kDa centrifugal concentrator +- Wizard Prep Resin +- Wizard Prep Column +- 3mL Luer Lock Syringe +- 5mL Snap-cap tube +- Plunger +- Syringe barrel +- 80% Isopropanol +- 1.5 mL centrifuge tube +- 50-100 µL TE +- Pico Green assay + +### Materials + +- Quant-iT dsDNA Pico Green assay kit (Invitrogen) [P7589](https://www.thermofisher.com/order/catalog/product/P7589) by Life Technologies + +## Protocol + +### Samples +#### Step 1. +Locate samples and record inventory number and number of filters (or portions) used. + +#### Step 2. +Turn the filters precipitate-side out with bleached forceps that have been rinsed with water and use aluminum foil squares as work surface, discarding after each filter. + +#### Step 3. +Remove filter from 50cc tube and refold so that precipitate side comes in contact with resuspension buffer. + +#### Step 4. +Return filter to tube using forceps and sterile applicator sticks. + +#### Step 5. +Keep filters dark and refrigerated until ready to suspend. + +> **Annotations** +> Bonnie Poulos 11 Jan 2016 +> There should be moisture in the tube - do not let the filters dry out. Usually when the filters are put into the tube initially, they carry some residual seawater with them that serves this purpose. A milliliter of sterile molecular biology grade water can be added if necessary. + +### Resuspension of Iron Chloride Precipitates +#### Step 6. +Prepare 1x or 2x resuspension buffer. + +> **Notes** +> VERVE Team 07 Jul 2015 +> Check that pH of buffer is pH 6.0-6.5 +> VERVE Team 07 Jul 2015 +> Prepare about 20% more than needed +> VERVE Team 09 Jul 2015 +> See [0.1M EDTA - 0.2M MgCl₂ - 0.2M Ascorbate Buffer](#) + +#### Step 7. +Prepare 20mL 1x or 10mL 2x per filter from 20L of seawater. + +#### Step 8. +Add resuspension buffer to each filter. + +#### Step 9. +Parafilm the tubes and wrap all of them in aluminum foil. + +#### Step 10. +Put foil pack of tubes on rotator, in cold room, using rubber bands to secure, and rotate slowly overnight. + +⏲ **Duration** +15:00:00 + +#### Step 11. +To recover resuspended viruses, remove the liquid at the bottom using a 5mL pipet and transfer to a fresh 15mL or 50mL sterile, labeled tube. + +#### Step 12. +Using bleached and rinsed forceps or sterile applicator sticks, pull edge of filter up and over lip of tube and secure with the lid. + +#### Step 13. +Centrifuge 1000rpm, 5min, 18°C to recover liquid left on filter. + +⏲ **Duration** +00:05:00 + +#### Step 14. +Remove liquid and add more buffer if filter still has a lot of precipitate clinging to it. + +#### Step 15. +Rotate for several more hours. + +⏲ **Duration** +03:00:00 + +#### Step 16. +Repeat removal of liquid. + +### DNase Treatment +#### Step 17. +Dilute stock DNase 1:100 in 10x DNase buffer (concentration = 400 U/mL). + +> **Notes** +> VERVE Team 14 Jul 2015 +> See [DNase Treatment Protocol](#) for details + +#### Step 18. +Add 1/10th volume of diluted DNase to each sample. + +> **Notes** +> VERVE Team 24 Jun 2015 +> DNase now at 40 U/mL and in 1x reaction buffer +> VERVE Team 14 Jul 2015 +> See [DNase Treatment Protocol](#) for details + +#### Step 19. +Parafilm tubes, wrap them in aluminum foil and attach to tube rotator with rubber bands. + +> **Notes** +> VERVE Team 14 Jul 2015 +> See [DNase Treatment Protocol](#) for details + +#### Step 20. +Incubate by rotating slowly at room temperature for 2 hours. + +⏲ **Duration** +02:00:00 + +> **Notes** +> VERVE Team 14 Jul 2015 +> See [DNase Treatment Protocol](#) for details + +#### Step 21. +Inactivate DNase by adding EDTA and EGTA to 0.1M final concentration each. + +> **Notes** +> VERVE Team 14 Jul 2015 +> See [DNase Treatment Protocol](#) for details + +#### Step 22. +Mix by inverting the tube several times. + +> **Notes** +> VERVE Team 14 Jul 2015 +> See [DNase Treatment Protocol](#) for details + +### Concentration and DNA extraction +#### Step 23. +Add the DNase treated and inactivated sample to the top reservoir of Amicon Ultra 100kDA centrifugal concentrators. + +> **Notes** +> VERVE Team 24 Jun 2015 +> Usually this will be in the 15mL size in 50mL centrifuge tubes +> VERVE Team 14 Jul 2015 +> See [DNA Extraction of Viruses using Wizard Prep Column](#) for more details +> +> **Annotations** +> Ievgeniia Prekrasna 21 Oct 2017 + +#### Step 24. +Centrifuge the concentrators at 1000 g for 5 minute intervals at 18°C until samples are at less than 2mL each. + +⏲ **Duration** +00:05:00 + +> **Notes** +> VERVE Team 14 Jul 2015 +> See [DNA Extraction of Viruses using Wizard Prep Column](#) for more details + +#### Step 25. +Put 1mL of resin on one Wizard Prep column. + +> **Amount** +> 1 µl +> +> **Notes** +> VERVE Team 24 Jun 2015 +> Use no more than 1mL sample per 1mL of Wizard Prep resin (0.5mL is ideal) +> VERVE Team 14 Jul 2015 +> See [DNA Extraction of Viruses using Wizard Prep Column](#) for more details +> +> **Annotations** +> Bonnie Poulos 11 Jan 2016 +> This should state: use 1ml of resin per 0.5ml of sample for application onto one Wizard Prep column. + +#### Step 26. +Thoroughly resuspend resin by shaking vigorously. + +> **Notes** +> VERVE Team 14 Jul 2015 +> See [DNA Extraction of Viruses using Wizard Prep Column](#) for more details + +#### Step 27. +Mix 1mL per 0.5-1.0mL of DNA. + +> **Notes** +> VERVE Team 14 Jul 2015 +> See [DNA Extraction of Viruses using Wizard Prep Column](#) for more details +> +> **Annotations** +> Bonnie Poulos 11 Jan 2016 +> Resin works best at a ratio of 1ml resin to 0.5ml sample. If there is more than 1ml sample per 1ml resin, DNA recovery will be reduced. + +#### Step 28. +Add each 1mL of resin to a 3mL luer lock syringe attached to a Wizard column. + +> **Notes** +> VERVE Team 14 Jul 2015 +> See [DNA Extraction of Viruses using Wizard Prep Column](#) for more details + +#### Step 29. +Push through into a 5mL snap-cap tube. + +> **Notes** +> VERVE Team 14 Jul 2015 +> See [DNA Extraction of Viruses using Wizard Prep Column](#) for more details + +#### Step 30. +Save this tube until DNA quantification. + +> **Notes** +> VERVE Team 14 Jul 2015 +> See [DNA Extraction of Viruses using Wizard Prep Column](#) for more details + +#### Step 31. +Remove syringe from column. + +> **Notes** +> VERVE Team 14 Jul 2015 +> See [DNA Extraction of Viruses using Wizard Prep Column](#) for more details + +#### Step 32. +Remove plunger. + +> **Notes** +> VERVE Team 14 Jul 2015 +> See [DNA Extraction of Viruses using Wizard Prep Column](#) for more details + +#### Step 33. +Reattach syringe barrel to column. + +> **Notes** +> VERVE Team 14 Jul 2015 +> See [DNA Extraction of Viruses using Wizard Prep Column](#) for more details + +#### Step 34. +Wash columns with 2mL 80% isopropanol pushing through with the syringe barrel into a waste container. + +> **Notes** +> VERVE Team 14 Jul 2015 +> See [DNA Extraction of Viruses using Wizard Prep Column](#) for more details + +#### Step 35. +Put columns into 1.5mL centrifuge tube and centrifuge at 10,000 g for 2.5 minutes to remove residual alcohol. + +⏲ **Duration** +00:02:30 + +> **Notes** +> VERVE Team 14 Jul 2015 +> See [DNA Extraction of Viruses using Wizard Prep Column](#) for more details + +#### Step 36. +Discard tube. + +> **Notes** +> VERVE Team 14 Jul 2015 +> See [DNA Extraction of Viruses using Wizard Prep Column](#) for more details + +#### Step 37. +Place column into a fresh 1.5mL centrifuge tube and pipet on 50-100µL of TE (0.02µm filtered and heated to 80°C). + +> **Notes** +> VERVE Team 14 Jul 2015 +> See [DNA Extraction of Viruses using Wizard Prep Column](#) for more details + +#### Step 38. +Vortex gently (1400rpm) and let sit 1 minute. + +⏲ **Duration** +00:01:00 + +> **Notes** +> VERVE Team 14 Jul 2015 +> See [DNA Extraction of Viruses using Wizard Prep Column](#) for more details + +#### Step 39. +Centrifuge at 10,000 g for 1 minute. + +⏲ **Duration** +00:01:00 + +> **Notes** +> VERVE Team 14 Jul 2015 +> See [DNA Extraction of Viruses using Wizard Prep Column](#) for more details + +#### Step 40. +Transfer extracted DNA to Lo-bind DNA 0.5mL tubes. + +> **Notes** +> VERVE Team 14 Jul 2015 +> See [DNA Extraction of Viruses using Wizard Prep Column](#) for more details + +#### Step 41. +Repeat elution above (steps 37-40) one more time. + +> **Notes** +> VERVE Team 14 Jul 2015 +> See [DNA Extraction of Viruses using Wizard Prep Column](#) for more details + +### DNA Quantification +#### Step 42. +Use 1-2µL of the first elution and 4-5µL of the second elution for the Pico Green assay to quantify the extracted DNA. + +##### Reagents +- Quant-iT dsDNA Pico Green assay kit (Invitrogen) [P7589](https://www.thermofisher.com/order/catalog/product/P7589) by Life Technologies + +#### Step 43. +Use the Excel spreadsheet to calculate the ng/µL DNA for each sample. + +#### Step 44. +Store in -80°C ultra-low freezer. + +#### Step 45. +Label tubes with date, station, depth and concentration and store in -80°C ultra-low freezer. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/preparing-gene-of-interest-for-gateway-cloning-2-s-b3muqk6w.md b/markdown-output/preparing-gene-of-interest-for-gateway-cloning-2-s-b3muqk6w.md new file mode 100644 index 0000000000000000000000000000000000000000..3ad79c3a554518b3772a235d34cfb257e89bd69a --- /dev/null +++ b/markdown-output/preparing-gene-of-interest-for-gateway-cloning-2-s-b3muqk6w.md @@ -0,0 +1,76 @@ +```markdown +# Goal/Experiment: +Preparing the Gene of Interest for GateWay Cloning (2-step PCR process) aims to add GateWay recombination sites to a gene of interest through a two-step PCR process to facilitate downstream applications such as cloning into vector systems. + +## Preparing Gene of Interest for GateWay Cloning (2 step PCR process) V.2 + +### Materials + +- **Q5 High-Fidelity PCR Kit - 200 rxns** (New England Biolabs Catalog #E0555L) +- **SuperFi Polymerase** (Thermo Fisher Scientific) + +*Note: Use your favorite High Fidelity Polymerase or Mastermix. Q5 and SuperFi both work fine.* + +### Primer Design + +There are a few things we need to keep in mind when designing the primers in the wider context of [GateWay cloning](https://www.embl.de/pepcore/pepcore_services/cloning/cloning_methods/gateway/2step/). The idea is to create an "entry clone" containing your gene of interest, usually INCLUDING the start codon, but EXCLUDING the stop codon. This allows the same entry clone to shuttle the gene of interest into different destination vectors with different properties (e.g., different N- or C-terminal tags). + +- **Gene-specific forward primer (PCR 1):** `5'-AA AAA GCA GGC T/NN-(15 to 20 bp template specific sequence)-3'` + - The 'NN' region can be any base (`CC` is recommended). *Avoid using AA, AG, or GA, as they could introduce an in-frame stop codon.* + +- **Gene-specific reverse primer (PCR 1):** `5'-A GAA AGC TGG GT/NN-(15 to 20 bp template specific sequence)-3'` + - The 'N' region can be any base, but avoid introducing a stop codon (`A` is recommended). + +- **attB1 adapter primer (PCR 2):** `5'-GGG GAC AAG TTT GTA CAA AAA GCA GGC TG-3'` +- **attB2 adapter primer (PCR 2):** `5'-GGG GAC CAC TTT GTA CAA GAA AGC TGG GT-3'` + +Diagram of Primer Design: + +![Primer Design](image_path) + +### PCR 1 - Gene-Specific PCR + +Use your favorite High-Fidelity Polymerase. NEB Q5 and Thermo Fisher SuperFi Mastermix have been successfully used. + +| Reagent | Volume | Temperature | Time | Cycles | +|--------------------------|---------|-------------|--------------|---------| +| 5x Q5 Buffer | 2 µL | 98°C | 1 min | | +| 2 mM dNTPs | 1 µL | 98°C | 10 s | | +| 10 µM Forward primer | 0.5 µL | 50°C | 20 s | 40x | +| 10 µM Reverse primer | 0.5 µL | 72°C | 30 s (per kb)| | +| Q5 Polymerase (or SuperFi)| 0.1 µL | 72°C | 2 min | | +| H2O | 5.4 µL | 10°C (hold) | | | +| Template DNA | 0.5 µL | | | | + +### PCR 2 - Gateway attB1 and attB2 Adapter Primers + +Use the PCR 1 product as the template for PCR 2. + +| Reagent | Volume | Temperature | Time | Cycles | +|--------------------------|---------|-------------|--------------|---------| +| 5x Q5 Buffer | 2 µL | 98°C | 1 min | | +| 2 mM dNTPs | 1 µL | 98°C | 10 s | | +| 10 µM attB1 Forward primer | 0.5 µL | 45°C | 30 s | 40x | +| 10 µM attB2 Reverse primer | 0.5 µL | 72°C | 30 s (per kb) | | +| Q5 Polymerase | 0.1 µL | 72°C | 2 min | | +| H2O | 3.2 µL | 10°C (hold) | | | +| PCR 1 product | 1 µL | | | | + +### Run a Gel + +4. **Run PCR 2 product on a gel** to ensure you have a crisp band and verify the PCR success. + +### Gel Extraction + +5. **Extract the PCR 2 band** with your gel extraction kit of choice. + +### Proceed to BP Recombination Step + +6. [Gateway BP recombination of attB tailed PCR products into pDONR/zeo](https://dx.doi.org/10.17504/protocols.io.b3muqk6w). + +--- + +Citation: Johannes Wolfram JWD Debler. "Preparing Gene of Interest for GateWay Cloning (2 step PCR process) V.2." protocols.io. https://dx.doi.org/10.17504/protocols.io.b3muqk6w. +``` + +`endofoutput` diff --git a/markdown-output/preparing-ont-tagged-primers-and-master-mix-for-fu-cq2fvybn.md b/markdown-output/preparing-ont-tagged-primers-and-master-mix-for-fu-cq2fvybn.md new file mode 100644 index 0000000000000000000000000000000000000000..1730bee822c6392ec91661fad7a1eab5cb4ba3f5 --- /dev/null +++ b/markdown-output/preparing-ont-tagged-primers-and-master-mix-for-fu-cq2fvybn.md @@ -0,0 +1,195 @@ +```markdown +# Goal/Experiment: +The goal of this experiment is to prepare ONT-tagged primers and master mix for fungal DNA barcoding. + +# Preparing ONT-tagged Primers and Master Mix for Fungal DNA Barcoding V.3 + +**Stephen Douglas Russell** +1The Hoosier Mushroom Society + +## Version 3 +**Mar 13, 2023** + +### OPEN ACCESS +**DOI:** [dx.doi.org/10.17504/protocols.io.yxmvnvnzxng3p/v3](https://dx.doi.org/10.17504/protocols.io.yxmvnvnzxng3p/v3) + +**Protocol Citation:** Stephen Douglas Russell 2023. Preparing ONT-tagged Primers and Master Mix for Fungal DNA Barcoding. protocols.io [https://dx.doi.org/10.17504/protocols.io.yxmvnvnzxng3p/v3](https://dx.doi.org/10.17504/protocols.io.yxmvnvnzxng3p/v3) +Version created by Stephen Douglas Russell + +**License:** This is an open access protocol distributed under the terms of the [Creative Commons Attribution License](https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. + +**Protocol status:** Working +We use this protocol and it's working. + +**Created:** Mar 12, 2023 +**Last Modified:** Mar 13, 2023 +**PROTOCOL integer ID:** 78631 + +## ABSTRACT +ONT primer preparation has two specific aspects that are unique when comparing to Sanger sequencing protocols. The first is that each primer needs to be "tagged" - a unique ~10-15bp sequence is added to the 5' end of the forward and reverse primers. Secondarily, these tagged primers need to be "multiplexed" - meaning that each individual specimen has a unique combination of forward-tagged primer and reverse-tagged primer. These tags allow the DNA amplicons for all of the specimens to be "pooled" or mixed together for sequencing, and then to be "demultiplexed" or sorted back out, allowing the resulting sequences to be associated with the individual specimens they originated from. + +It is possible to perform ONT sequencing without the tags, but you would not be able to associate the sequences with any individual specimens. Ex - If you had ten closely related _Russula_ specimens in your sequencing run, you would be able to document sequences of all ten of the species, but you would have a difficult time associating the sequences with the individual specimens/observations they originated from. They would be sequences without faces. This may be common if you are examining the community ecology or environmental DNA of a particular location, but this result would not be ideal for most DNA barcoding goals involving specimens. + +Thus, if you are running 960 specimens on a Flongle flow cell, you need to have 960 unique primer combinations for the sequencing run. + +The easiest way to accomplish this is to have a single unique forward primer tag for each plate you are including, combined with 96 unique reverse primers for the plate. If you are including five plates, you would have five unique forward primers (a different one for each plate) combined with a standard set of 96 reverse primers. This results in 960 unique tags for each of the 960 specimens that are being barcoded. + +**Note:** This protocol utilized a primer plate of 96 different reverse primers. In the future I plan on ordering the primer plate of 96 unique primers for the forward ITS1F primers. This will allow ad-hoc combinations of a different ONT-tagged reverse primer to extend into the LSU region as needed. + +## MATERIALS + +### Primers +- **ONT-tagged Forward Primers**: [Eurofins Genomics](https://www.eurofinsgenomics.com/) - $87.50 +- **ONT-tagged Reverse Primers**: [Eurofins Genomics](https://www.eurofinsgenomics.com/) - $396.16 + +### Reagents and Consumables +- **Molecular Biology Grade Water**: [IBI Scientific Catalog #IB42120](https://www.ibisci.com/) +- **0.2 non-skirted 96-well PCR plates**: [USA Scientific/Amazon](https://www.usascientific.com/) +- **PCR Sealing Film**: [Amazon](https://www.amazon.com/) +- **8-strip PCR caps**: [USA Scientific](https://www.usascientific.com/) +- **Eppendorf DNA LoBind 1.5mL tubes**: [USA Scientific](https://www.usascientific.com/) +- **PCR Master Mix**: [Empirical Bioscience](https://empiricalbioscience.com/) + +### Equipment +- **Fine-tip Sharpies**: [Amazon](https://www.amazon.com/) +- **PCR tube rack x10**: [Amazon](https://www.amazon.com/) +- **PCR tube rack 1.5mL**: [Amazon](https://www.amazon.com/) +- **0.5-10uL multichannel pipette**: [Amazon](https://www.amazon.com/) +- **50-300uL multichannel pipette**: [Amazon](https://www.amazon.com/) +- **10uL filtered pipette tips**: [Amazon](https://www.amazon.com/) +- **200uL filtered pipette tips**: [Amazon](https://www.amazon.com/) +- **Eliminase**: [eBay](https://www.ebay.com/) +- **Summit Professional Freezer -20C**: Facebook Marketplace +- **Thermocycler**: Provided in extraction step + +#### Total Cost Outlay: +**$1607.74** + +#### Ongoing cost per sample: +**Minimal** + +## Ordering ONT-tagged Primers + +### Step 1: ONT-tagged Forward Primers [Eurofins Genomics](https://www.eurofinsgenomics.com/) +- Determine how many unique primers you need to order. For example, if you plan to use a Flongle cell with up to 960 specimens, you need 10 unique forward ONT-tagged primers. If aiming for 10,000+ specimens, you need at least 105 unique forward tagged primers. +- ITS1F sequences and/or unique ONT primer tags can be found here: + + ![MinION Primer Tag Sets.xlsx](file/path/to/MinION_Primer_Tag_Sets.xlsx) + + **Details for ITS1F-ONT001:** + - **Sequence:** AGC AAT CGG GCA CCT TGG TCA TTT AGA GGA AGT AA + - **Synthesis Scale:** 50 nmole + - **Purification:** Salt Free + - **Delivery format:** LabSet (100 µM in TE, pH 8.0) + - **Quality control:** MALDI-TOF + - **Price:** $17.50 + +### Step 2: ONT-tagged Reverse Primers [Eurofins Genomics](https://www.eurofinsgenomics.com/) +- Order a primer plate of 96 unique ONT-tagged reverse primers. +- ITS4 sequences and/or unique ONT primer tags can be found here: + + ![MinION Primer Tag Sets.xlsx](file/path/to/MinION_Primer_Tag_Sets.xlsx) + + **Details for Plate 01:** + - **Synthesis Scale:** 10 nmole + - **Purification:** Salt Free + - **Delivery format:** LabSet (100 µM in TE, pH 8.0) + - **Quality control:** MALDI-TOF + - **Price:** $380.16 + $16 shipping + +## Preparing Forward Primer Stock +### Step 3 +- When your forward primers arrive in individual tubes, they will be at a 100uM concentration. You will need to make new working tubes at a 10uM concentration. +- In a new 1.5uL tube, using filter tips: + - 900uL molecular water + - 100uL of 100uM forward primer + + Label each tube as "ONT001 10uM." + +## Preparing Reverse Primer Stock-Working-PCR Ready Plates + +### Step 4 +**Materials Required:** +- **Molecular Biology Grade Water**: [IBI Scientific Catalog #IB42120](https://www.ibisci.com/) +- **ONT-tagged Reverse Primers**: [Eurofins Genomics](https://www.eurofinsgenomics.com/) +- **96 well plates** +- **Multichannel Pipettes** (90uL, 10uL, 6.2uL, 4.6uL) + +**Summary:** +- 100 uM Reverse Primer Stock Solution +- "Working" Reverse Primer Plates + - 180uL H2O + - 20uL Primer +- "PCR Ready" Reverse Primer Plates + - 160uL H2O + - 25uL "Working" primer +- PCR Reaction + - 4.6uL "PCR Ready" reverse primer into each cell of PCR reaction + +#### Detailed Steps: +1. **4.1** Wipe down your working area with Eliminase or similar. +2. **4.2** Place 90uL of molecular water into each cell of new 96 well plates. A multichannel pipette is most efficient for this job. It is also best to use filter tips. +3. **4.3** Transfer 10uL of reverse primer from your stock plate into each of the 96 wells, making sure each primer stays in the correct cell. Use new filter tips for each transfer. +4. **4.4** Label each plate you create "ONT ITS4 10uM Working." + +### Step 5 +1. **5.1** Place 40uL of molecular water into each cell of new 96 well plates. +2. **5.2** Transfer 6.25uL of reverse primer from your working (10 uM) plate into each of the 96 wells. Use new filter tips for each transfer. +3. **5.3** Label each plate you create "ONT ITS4 PCR Ready." + +## Create Master Mix / Forward Primer (MMF) Stock + +### Step 6 +In 1.5mL tubes, add: +- 625 uL Master Mix +- 62.5 uL of 10uM ONT-tagged Forward Primer + +Label tube with ONT name "MMF ONT001" + +Each tube with this mixture will be the stock for 1 plate (100 reactions). If you are running a Flongle with 960 specimens, you will need to make ten tubes, each with a different ONT-tagged forward primer. + +**Note:** It is possible to make larger MMF batches for each forward primer and store them in the freezer until ready for use with future runs. + +## Create Master Mix Plates + +### Step 7 +Make a determination of how many plates you will need for this sequencing run. For a Flongle, this will likely be 5-10. For example, if you are running 960 specimens, you will need to set out 10 new 96 well plates. Create primer plates in batches of five. + +**Summary:** +Each cell of each plate will have a different ONT-tagged primer combination and a total of: +- 4.6 "PCR Ready" reverse primer mix + - 4uL Molecular water + - 0.625 ONT-tagged reverse primer +- 6.9 MMF + - 6.25uL of Master Mix + - 0.625 ONT-tagged forward primer + - 1.1uL DNA template + +12.5uL total reaction volume + +#### Detailed Steps: +1. **7.1** Add 4.6uL from a "PCR Ready" reverse primer plate to each cell. + - Use a multichannel pipette for this step. Use a new set of tips for each row of cells. +2. **7.2** Add 6.9uL of the MMF stock into each cell of the new 96 well plates. + - Each new plate needs different MMF stock from a different ONT-tagged primer. + - Use non-filtered tips for this step. +3. **7.3** Place the completed plates in the freezer until they are ready to be utilized. +4. **7.4** Add 1.1ul of DNA template for each cell. + - Check each tip of the pipette to ensure the requisite amount is contained. +5. **7.5** Cap off the plate with PCR Sealing Film. +6. **7.6** Label each plate with the forward primer ONT name. "ONT001 P1 ITS1F-4" + +## Run Thermocycler Program + +### Step 8 +**Standard ITS program:** +1. 94C for 1 min +2. Repeat 30X: + - 94C for 1 min + - 51C for 1 min + - 72C for 1 min +3. 72C for 8 min +4. 10C to stop + +**endofoutput** +``` \ No newline at end of file diff --git a/markdown-output/preparing-reads-for-stranded-mapping-cikkucuw.md b/markdown-output/preparing-reads-for-stranded-mapping-cikkucuw.md new file mode 100644 index 0000000000000000000000000000000000000000..382599384f0d2f59e6b51c5474ac2f742ac21e19 --- /dev/null +++ b/markdown-output/preparing-reads-for-stranded-mapping-cikkucuw.md @@ -0,0 +1,186 @@ +```markdown +# Goal/Experiment: +The goal of this experiment is to prepare long reads for stranded mapping, serving as an intermediate step for additional protocols: +- Aligning strand-oriented sequences to a transcriptome for transcript/gene counting +- Aligning strand-oriented sequences to a genome for confirmatory QC + +# Preparing Reads for Stranded Mapping V.7 + +## Abstract +This protocol is for preparing long reads for stranded mapping, as an intermediate step for additional protocols: +- Aligning strand-oriented sequences to a transcriptome for transcript/gene counting +- Aligning strand-oriented sequences to a genome for confirmatory QC + +**Input(s):** demultiplexed fastq files (see protocol [Demultiplexing Nanopore reads with LAST](https://dx.doi.org/10.17504/protocols.io.5qpvon2zzl4o/v7)), adapter file (containing strand-sensitive adapter sequences) +**Output(s):** oriented read files, as gzipped fastq files + +## DOI +[dx.doi.org/10.17504/protocols.io.5qpvon2zzl4o/v7](https://dx.doi.org/10.17504/protocols.io.5qpvon2zzl4o/v7) + +## Protocol Citation +David A Eccles 2022. Preparing Reads for Stranded Mapping. *protocols.io* + +Version created by David A Eccles + +## Keywords +long reads, nanopore, strand-specific, sequencing, RNASeq + +## License +This is an open access protocol distributed under the terms of the [Creative Commons Attribution License](https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. + +## Creation and Modification +- **Created:** Oct 30, 2022 +- **Last Modified:** Oct 30, 2022 +- **Protocol Integer ID:** 72044 + +## Parent Protocols +Part of collection: +- Nanopore Data Analysis +- Nanopore Data Analysis +- Nanopore Data Analysis +- Nanopore Data Analysis +- Nanopore Data Analysis + +## Protocol Steps + +### Barcode Demultiplexing + +1. **Demultiplex reads** as per protocol [Demultiplexing Nanopore reads with LAST](https://dx.doi.org/10.17504/protocols.io.5qpvon2zzl4o/v7). + + I usually inspect the `barcode_counts.txt` file, and delete any barcodes that I'm not interested in: + + ```sh + cp barcode_counts.txt barcode_counts.orig.txt + nano barcode_counts.txt + ``` + + If demultiplexing has been done correctly, then the following command should produce output without errors: + + ```sh + for bc in $(awk '{print $2}' barcode_counts.txt); + do ls demultiplexed/reads_${bc}.fq.gz; + done + ``` + + Example output: + ```sh + demultiplexed/reads_BC03.fq.gz + demultiplexed/reads_BC04.fq.gz + demultiplexed/reads_BC05.fq.gz + demultiplexed/reads_BC06.fq.gz + demultiplexed/reads_BC07.fq.gz + demultiplexed/reads_BC08.fq.gz + ``` + + If the `barcode_counts.txt` file is missing, the output will look like this: + ```sh + awk: fatal: cannot open file `barcode_counts.txt' for reading (No such file or directory) + ``` + + If one or more of the barcode-demultiplexed files are missing, the output will look something like this: + ```sh + demultiplexed/reads_BC03.fq.gz + demultiplexed/reads_BC04.fq.gz + demultiplexed/reads_BC05.fq.gz + ls: cannot access 'demultiplexed/reads_BC06.fq.gz': No such file or directory + ls: cannot access 'demultiplexed/reads_BC07.fq.gz': No such file or directory + demultiplexed/reads_BC08.fq.gz + ``` + +### Index Preparation + +2. **Prepare and index a FASTA file** containing adapter sequences (see attached FASTA file). + + ```sh + adapter_seqs.fa + ``` + + Create a shell variable containing this file name: + ```sh + adapterFile="adapter_seqs.fa" + ``` + +3. **Prepare the LAST index** for the adapter file. Newer versions of LAST (v1409+) include a [new seeding scheme, `-uRY4`](http://last.cbrc.jp/doc/last-e.html) [and other related RYX schemes], which improves mapping accuracy and reduces polyA matches; low-complexity regions are also converted to lower case with `-R01`. This will generate seven additional files of the form `.XXX`: + ```sh + lastdb -uRY4 -R01 ${adapterFile} ${adapterFile} + ``` + +4. **Prepare a substitution matrix** for adapter mapping. Mapping seems to work better when Q values are included: + + ```sh + #last -Q 1 + #last -t4.49549 + #last -a 46 + #last -A 46 + #last -b 3 + #last -S 1 + # score matrix (query letters = columns, reference letters = rows): + # A C G T + # A 6 -34 -33 -35 + # C -34 6 -33 -34 + # G -33 -33 7 -34 + # T -35 -34 -34 6 + ``` + + A matrix like this can be generated by the following command, which runs `last-train` on the first 10,000 reads from the full read dataset: + ```sh + last-train -Q 1 -P 10 ${adapterFile} <(zcat reads_all.fastq.gz | head -n 40000) | tail -n 13 + ``` + + [note: it is also fine to use the same matrix as used for demultiplexing] + +### Orienting Reads + +5. **Use LAST in `split` mode**, using the pre-defined substitution matrix to map the reads. In this example, it is distributed over 10 processing threads (`-P 10`). In this case it's important that the direction of mapping is also recorded, so the `cut` command selects three fields (query name [7], target name [2], mapping direction [10]): + ```sh + for bc in $(awk '{print $2}' barcode_counts.txt); + do echo "** ${bc} **"; + lastal --split -P10 -p adapter.mat ${adapterFile} <(pv demultiplexed/reads_${bc}.fq.gz) | \ + maf-convert -n tab | cut -f 2,7,10 | uniq | \ + gzip > demultiplexed/adapter_assignments_${bc}.txt.gz + done + ``` + +6. The **adapter assignments** are filtered through `uniq` in order to catch (and exclude) any reads with the strand-switch primer matching multiple times. To unpack the `uniq` pipe a little bit more, it skips the first field (adapter name), then matches up to 36 characters, retaining only lines that don't match any others. This catches a few more chimeric reads that were missed by the unique barcode filter in the previous protocol. + + Reads are filtered into two groups (and one group-by-omission) based on the mapped direction of the strand-switch primer, then reverse-complemented (if necessary) to match the orientation of the original RNA strand. I use my `fastx-fetch.pl` and `fastx-rc.pl` scripts for this. + + ```sh + fastx-fetch.pl + fastx-rc.pl + ``` + + ```sh + mkdir -p oriented + for bc in $(awk '{print $2}' barcode_counts.txt); + do echo "** ${bc} **"; + fastx-fetch.pl -i <(zgrep '^SSP' demultiplexed/adapter_assignments_${bc}.txt.gz) | \ + sort | uniq -f 1 -w 36 -u | \ + awk '{if($3 == "+"){print $2}}' | \ + gzip > oriented/${bc}_reads_fwd.fq.gz + fastx-fetch.pl -i <(zgrep '^SSP' demultiplexed/adapter_assignments_${bc}.txt.gz) | \ + sort | uniq -f 1 -w 36 -u | \ + awk '{if($3 == "-"){print $2}}' | \ + fastx-rc.pl | gzip > oriented/${bc}_reads_rev.fq.gz + done + ``` + +7. **Forward and reverse-oriented sequences** are combined together to form a single group of RNA-oriented reads. + + ```sh + for bc in $(awk '{print $2}' barcode_counts.txt); + do echo "** ${bc} **"; + pv oriented/${bc}_reads_fwd.fq.gz oriented/${bc}_reads_rev.fq.gz | \ + zcat | gzip > oriented/${bc}_reads_dirAdjusted.fq.gz + done + ``` + +### Downstream Workflows + +8. Following on from here, the oriented reads can be mapped to a genome (e.g. for visual confirmation of mapping), or to a transcriptome (e.g. for read counting): + +- [Stranded Mapping from Oriented Long Reads](https://dx.doi.org/10.17504/protocols.io.5qpvon2zzl4o/v7) +- [Stranded Transcript Count Table Generation from Long Reads](https://dx.doi.org/10.17504/protocols.io.5qpvon2zzl4o/v7) + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/primary-neuronal-culture-crh-treatment-h2kb8cw.md b/markdown-output/primary-neuronal-culture-crh-treatment-h2kb8cw.md new file mode 100644 index 0000000000000000000000000000000000000000..550dbf2d7768bdf995ac9a8236a8876aed3720eb --- /dev/null +++ b/markdown-output/primary-neuronal-culture-crh-treatment-h2kb8cw.md @@ -0,0 +1,184 @@ +```markdown +# Goal/Experiment: +The goal of this protocol is to prepare primary neuronal cultures and treat them with corticotropin-releasing hormone (CRH) to study its effects. + +# Primary Neuronal Culture & CRH Treatment + +## Abstract +Citation: Megan Curran Primary Neuronal Culture & CRH Treatment. protocols.io dx.doi.org/10.17504/protocols.io.h2kb8cw +Published: 16 Jun 2017 + +## Protocol + +### Preparing Coverslips for Primary Cell Culture + +#### Step 1 + +**Day 1: Poly-D-Lysine (PDL) Coating** +Start ≥20 hours before culturing + +**Supplies Needed:** + +- 24 well plate +- Poly-D-Lysine *2 (0.2 mg/ml) – in freezer +- Autoclaved coverslips (24/plate) +- Sterilized forceps +- 200 µL pipette + tips + +**Instructions:** + +1. Sterilize Hood +2. Thaw PDL*2 (0.2 mg/ml) +3. Using sterilized forceps, gently lay coverslips in each well of a 24-well dish (1 coverslip per well) +4. Aliquot 80 µl of the PDL*2 to each coverslip, ensuring the drop is in the middle of the coverslip +5. Let the dishes sit undisturbed in an incubator for 18 hours + +#### Step 2 + +**Day 2: PDL Coating - Continued** + +**Supplies Needed:** + +- 24 well plate + treated coverslips from day 1 +- Sterile H2O +- Disposable 25 ml pipette + pipetter +- Autoclaved glass Pasteur pipette + +**Instructions:** + +1. Sterilize Hood + - If culturing on the same day, remove Enzyme Solution (ES) from the freezer to thaw +2. After ≥18 hours of coating, rinse dishes by filling them with sterile H2O using a disposable 25 ml pipette +3. Remove water immediately using a sterile glass Pasteur pipette hooked to the vacuum +4. Wait 10 minutes +5. Repeat washing one more time +6. Remove water until dishes are completely dry. Let dry under the hood + +### Tissue Dissection & Dissociation + +#### Step 3 + +**Supplies Needed:** + +- P0 or P1 rat pup(s) +- 70% EtOH +- Ice Bucket +- Beaker with water (for dirty dissection tools) +- Dissection Microscope +- Microtome + Fresh blade (double-sided razor) +- 2x 100 mm petri dishes +- 3x 35 mm petri dishes +- Dissection Tools (all autoclaved): + - Scissors (1x large & 1x small) + - 2x forceps + - ≥3x pulled micropipettes + - Brain Scooper/Spatula + - Disposable Pasteur pipette + - Dissection Solution (DS; ≥75 ml) – Make fresh every 2 weeks + - Enzyme solution (ES, 5 ml [1 tube]; stored in -20oC freezer) + - Papain (50 units) – Aliquots in 4oC + - 5x 15 ml conical tubes + - 400 µl BSA/PI stock + - 110µl5mMAPV + - Cortical Plating Medium (50 ml BME + 5 ml FBS + 500 µl Na-pyruvate + 125 µl Glutamax; Filter) + - Conditioned Medium (50 ml aliquots in -80oC freezer) +- Slide & Microscope + +**Instructions:** + +1. Take ES (without papain) out of the freezer to thaw +2. Sterilize working space & all tools +3. Add 50 units papain to the 5 ml thawed enzyme solution, activate at 36oC in the incubator for at least 30 minutes +4. Fill a small petri dish with ice-cold dissection solution (DS) + +##### Dissection + +1. Spray pup with EtOH and quickly decapitate the pup behind the ears +2. Remove brain from the skull, place in petri dish with DS, on ice +3. Remove hindbrain and discard it +4. Place brain on microtome platform, superior side facing up. +5. Cut coronal slices of 600 mm and transfer to DS filled 35 mm petri dish +6. Remove cortex free from the rest of the brain using autoclaved sharp glass pipettes. Carefully discard fibers and meninges. Cut each part to 600 µm cubes. + +##### Dissociation + +1. Collect tissue with a sterile Pasteur pipette and transfer to new 35 mm petri dish (transfer as little DS as possible) +2. Add 5 ml ES that was previously activated in 36oC (by now the enzyme solution should appear clear instead of cloudy) +3. Place the tissue + enzyme in 36oC incubator for 25 minutes +4. While waiting, prepare all washing solutions and place on ice. + - 10ml DS + - 2x1.5ml HI (3ml DS, 300µl BSA/T1, 30µl 5mMAPV> aliquot) + - 3x2.5ml LI (8ml DS, 80µl BSA/T1, 80µl 5mMAPV> aliquot) + - 5 ml Cortical Plating Medium +5. Collect tissue pieces with a sterile Pasteur pipette. Wash in the following order (always placing on ice): + 1. 10 ml DS, swirl and let tissue settle + 2. 1.5 ml HI – wait 2 minutes (on ice) + 3. 1.5 ml HI – wait 2 minutes (on ice) + 4. 2.5 ml LI – wait 2 minutes (on ice) + 5. 2.5 ml LI – wait 2 minutes (on ice) + 6. 2.5 ml LI – wait 2 minutes (on ice) + 7. 5 ml Cortical Plating Medium +6. Transfer tissue to petri dish with 1.5 ml CPM/brain +7. Triturate visually with decreasing pipette tip diameter, 4 times for each piece of tissue (first with 100-1000 µl tip, then with 20-200 µl tip) +8. Transfer cells + medium to new 15 ml tube, leave undisturbed for 3-5 minutes to settle +9. Visualize 80 µl on dish cover under microscope to assess density (take from the top); dilute tube if necessary +10. Plate 80 ml in the middle of each coverslip +11. Incubate in 36oC, 5% CO2 for 2 hours -- also prewarm CPM & Conditioned Medium +12. Add 0.5 ml prewarmed cortical plating medium to each coverslip +13. Incubate in 36oC, 5% CO2 for 2 more hours +14. Refresh half the volume by removing 250 µl of medium from each well and adding a similar amount of prewarmed conditioned medium. Return to the incubator + +### Replace Medium and Add Cytosine-Arabinoside + +#### Step 4 + +**Day 5 (72 hours after plating):** + +- Replace half of the medium with preheated medium. This time add 2 µl AraC/1 ml NBM+B27 (AraC [Cytosine-Arabinoside] inhibits glia growth) + +### Replacing Medium + +#### Step 5 + +- Refresh medium twice a week by replacing half of the volume with fresh conditioned medium that was preheated in the incubator to 36oC. +- Occasionally check the viability of the culture under a microscope to make sure it is not contaminated (but don’t do it too often, cells are happiest when they are kept undisturbed in the incubator). +- Cultures can be kept like this for at least 3-4 weeks after plating. + +### Add CRH to Cultures (DIV 7-14) + +#### Step 6 + +**Per plate for first half-medium change** (2x desired concentration CRH) – 250 µl/well +- **CTL/VEH**: 2 ml NBM/B27/AraC +- **200 nM CRH**: 5 µl stock CRH (0.5 µg/µl) + 2.57 ml NBM/B27/AraC +- **20 nM CRH**: 200 µl 200 nM CRH + 1.8 ml NBM/B27/AraC +- **2 nM CRH**: 20 µl 200 nM CRH + 1.98 ml NBM/B27/AraC + +**Per plate for the following half-medium changes** (desired concentration CRH) – 250 µl/well +- **CTL/VEH**: 2 ml NBM/B27/AraC +- **100 nM CRH**: 5 µl stock CRH (0.5 µg/µl) + 5.14 ml NBM/B27/AraC +- **10 nM CRH**: 200 µl 100 nM CRH + 1.8 ml NBM/B27/AraC +- **1 nM CRH**: 20 µl 200 nM CRH + 1.98 ml NBM/B27/AraC + +### Fix Coverslips (DIV14) + +#### Step 7 + +1. Wash in 0.1 M PB briefly at room temperature (1 minute) +2. Fix the culture in 4% PFA for 1 hour at room temperature (1 ml fixative/well) +3. Remove PFA + +### Immunocytochemistry + +#### Step 8 + +1. Wash with PBS-T (0.01 M PBS + 0.3% Triton) +2. Wash in 2-5% normal goat serum diluted in 0.01 M PBS-T containing 1% BSA for 30 minutes +3. Mouse anti-MAP2 antibody: 1:8000-16,000 in PBS-T – 1-2 days at 4oC +4. Wash in PBS-T 3 times (5 minutes each wash) – 20 minutes total +5. Alexa Fluor 488 goat anti-mouse IgG conjugate – 1:300 in PBS-T containing 1% BSA – room temperature for 2 hours +6. Wash in PBS-T 3 times (5 minutes each wash) – 20 minutes total +7. Place 1 drop of Aqueous mounting medium onto slide (as small as possible), then place coverslip face down on top. + +endofoutput +``` diff --git a/markdown-output/probiotics-bs3sngne.md b/markdown-output/probiotics-bs3sngne.md new file mode 100644 index 0000000000000000000000000000000000000000..88cb5d2922f49ef041f7d5c863675a4256a1e8b0 --- /dev/null +++ b/markdown-output/probiotics-bs3sngne.md @@ -0,0 +1,100 @@ +```markdown +# Goal/Experiment: +The goal of this experiment is to investigate the impact of a gut probiotic on the growth and health of B. gonionotus fishes. + +# Probiotics + +**Author:** Sulav Indra Paul +**Institution:** Institute of Biotechnology and Genetic Engineering, Bangabandhu Sheikh Mujibur Rahman Agricultural University, Gazipur-1706, Bangladesh +**Date:** Mar 07, 2021 +**DOI:** [dx.doi.org/10.17504/protocols.io.bs3sngne](https://dx.doi.org/10.17504/protocols.io.bs3sngne) +**Protocol ID:** 47954 + +## Abstract +Gut probiotic improves growth and health of *B. gonionotus* fishes. + +## License +This is an open access protocol distributed under the terms of the [Creative Commons Attribution License](https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. + +## Disclaimer +FOR INFORMATIONAL PURPOSES ONLY; USE AT YOUR OWN RISK. + +--- + +## Protocol + +### Euthanasia Methods +1. Pure clove oil was first dissolved in ethyl alcohol in a 1:9 ratio (clove oil: ethyl alcohol). +2. This solution was then diluted in water to obtain concentrations of 0.05 mL (50 mg), and 0.20 mL (200 mg) of clove oil per 500 mL of water. +3. For hematological study, fish were anesthetized using 0.05 mL of clove oil per 500 mL of water. +4. For histological, reproductive, and intestinal microflora study, fish were euthanized using 0.20 mL of clove oil per 500 mL of water, and death was confirmed by brain destruction. + +### Collection of Probiotic Samples from the Gut of *B. gonionotus* +5. The abdomens of fish were cut aseptically by sterile scissors and the gut was taken out with care to avoid any distortion of the gut and contamination with blood. +6. The gut was cut into small pieces, rinsed with 0.9% (w/v) saline solution, and placed in a conical flask containing 10 mL of distilled water. +7. The sample was stirred with a stirrer to make a homogeneous solution. + +### Isolation of Probiotic Bacteria from the Gut of *B. gonionotus* +8. One gram of sample was diluted in 10 mL sterilized water and inoculated on De Man, Rogosa, and Sharpe agar (MRS) media (Himedia, India). +9. The agar plates were incubated at 28ºC for 24 h, and colony characteristics were observed carefully to choose desired colonies. +10. The isolates were routinely sub-cultured on NA (Nutrient agar) plates, incubated at 28ºC, and stored in a freezer at -20ºC supplemented with 10% glycerol. + +### Molecular Identification of Probiotic Strains +11. Genomic DNA of the selected isolates was extracted using a commercial GenJET genomic DNA purification kit (Thermo Fisher Scientific, USA) #K0721. +12. DNA was amplified by using universal primer 8F (5’-AGAGTTTGATCCTGGCTCAG-3’) and 1492R (5’-GGATACCTTGTTACGACTT-3’). +13. The PCR amplification condition was as follows: + - Initial denaturation: 94 ºC for 5 min. + - 35 cycles of: + - Denaturation: 94 ºC for 1 min. + - Annealing: 57 ºC for 40 sec. + - Extension: 72 ºC for 1 min. + - Final extension: 72 ºC for 10 min. +14. The PCR amplicons were purified using a commercial kit (Thermo Fisher Scientific, USA). +15. *16S rRNA* gene sequencing was done by Sanger Sequencing. + +### Preparation of Probiotic Strains +16. Each strain was cultured in 1 liter of nutrient broth in an orbital shaker and incubated at 28ºC for 24 h. +17. The broth media was centrifuged at 8000 × g for 5 min, and the pelleted probiotic bacterial strains were collected and washed twice with sterile water. +18. The pellets were then suspended in sterile distilled water and added to the dough. +19. A spread plate technique was used to assess the viability of cells according to the cell concentrations measured at OD600. + +### Acidic pH Tolerance Test and Preparation of Simulated Gastrointestinal Juice of Host +20. The five gut probiotic bacteria were grown in MRS broth in an orbital shaker and incubated at 28ºC for 24 h. +21. The cultures were centrifuged at 8000 × g for 5 min at 4ºC. +22. The pellets were washed and suspended in sterile phosphate-buffered saline solution (PBS). +23. Each probiotic strain was diluted 10^(-2) in sterile PBS at pH 1.0, 2.0, 3.0, 4.0, and 5.0. +24. Gastrointestinal juices were prepared fresh by dissolving pepsin (Thermo Fisher, USA) from *B. gonionotus* stomach mucosa (3 g/L) in sterilized saline solution (5g/L). +25. The pH of the gastrointestinal preparation was adjusted to 2.0 with 12 M HCl. + +### Exposure of Gut Probiotics to Simulated Gastrointestinal Juice and Total Viable Counts +26. A 1-mL aliquot of each culture was centrifuged at 5000 × g for 10 min at 4ºC and washed three times in sterile PBS. +27. The tolerance of five probiotic bacteria to simulated gastrointestinal juices was determined by mixing 0.2 mL of each washed cell suspension with 1 mL of gastric juice. + +### Histological Analyses of Intestine and Liver of the Probiotic Treated Silver Barb +28. The experimental fish were humanely killed by using clove oil (0.20 mL per 500 mL of water), +29. Death was confirmed by the destruction of the brain. +30. The whole liver and part of the intestine from each fish were dissected carefully, cut to separate from each other, and stored in Bouin's solution for 24 h. +31. These samples were dehydrated in ascending grades of alcohol and cleared in xylene. +32. The fixed tissues were embedded in histoparaffin (Paraplast plus; Sigma-Aldrich) and sections (7 μm) were cut using a microtome (CUT-5602, Germany). +33. The sections of intestinal villi and liver were stained with Delafield's hematoxylin-eosin and observed under a light microscope (DM 100; Leica, Wetzlar, Germany). +34. Ten slides were prepared from the intestine of each fish through the histological method. +35. The slides were observed under a trinocular microscope. +36. Images were captured using a digital camera (DFC 290, Leica) and the villi length of the intestine was measured using AmScope software (Version 3.7; Carl Zeiss Primo Star, Germany). + +### Measurement of Hematological Parameters +37. A total of 27 fish from each treatment were anesthetized with clove oil (0.05 mL per 500 mL of water) for hematological analysis. +38. Blood was collected from each fish using a 3 cc syringe containing 10% blood anti-coagulant (EDTA) inserted into the caudal peduncle region to draw out blood. +39. The blood was transferred to a test tube coated with EDTA and stored at -30ºC until use. +40. Red blood cells (RBCs) and white blood cells (WBCs) were counted using an improved Neubauer hemocytometer (MarienFeld Company Germany) under the light microscope (DM 100; Leica, Wetzlar, Germany). +41. To measure hemoglobin, fresh blood was collected from fish from each treatment and poured onto the edge of a strip of hemoglobin meter before coagulation of blood. +42. The glucose level of blood was measured through a glucose meter from the sample. +43. To measure packed cell volume (PCV) %, blood was taken in a capillary tube at the marked level and sealed with gum. + +--- + +Citation: Sulav Indra Paul (03/07/2021). Probiotics. [https://dx.doi.org/10.17504/protocols.io.bs3sngne](https://dx.doi.org/10.17504/protocols.io.bs3sngne) + +This is an open access protocol distributed under the terms of the [Creative Commons Attribution License](https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/procedures-for-performing-the-mixed-meal-tolerance-bvf5n3q6.md b/markdown-output/procedures-for-performing-the-mixed-meal-tolerance-bvf5n3q6.md new file mode 100644 index 0000000000000000000000000000000000000000..376940e87cbee947118829a954bca0d0197692a0 --- /dev/null +++ b/markdown-output/procedures-for-performing-the-mixed-meal-tolerance-bvf5n3q6.md @@ -0,0 +1,93 @@ +```markdown +# Goal/Experiment: +Determine whether imatinib will slow the progression of the autoimmune destruction of β cells and lead to the preservation of C-peptide secretion in participants with new-onset type 1 diabetes mellitus (T1DM), as well as assess diabetes-related objectives and safety of imatinib. + +# Procedures for Performing the Mixed-Meal Tolerance Test (MMTT) +### (Appendix 2 of "Safety and Efficacy of Imatinib for Preserving Beta-Cell Function in New-onset Type 1 Diabetes Mellitus") + +Authors: +- **Stephen Gitelman**, Professor of Clinical Pediatrics, Department of Pediatrics, University of California, San Francisco +- **Jeffrey A. Bluestone**, Professor of Medicine, Pathology, Microbiology & Immunology, University of California, San Francisco + +## Abstract +This appendix is part of the study "Safety and Efficacy of Imatinib for Preserving Beta-Cell Function in New-onset Type 1 Diabetes Mellitus". It is supported by JDRF. The goal is to determine the effects of imatinib on preserving beta-cell function. + +## Guidelines + +### General Instructions +- The MMTT is performed in the morning (7:00 a.m. - 10:00 a.m.), recommended to begin early (7:00-7:30 a.m.). +- The test uses the **Boost® High Protein Nutritional Drink**. If allergic, an equivalent substitution may be used. +- Duration: 250 minutes. + +### Dietary Guidelines and Pretest Instructions +Carbohydrates (CHO) should not be restricted. General guidelines for intake: +- Pre-adolescent: At least 25 kcal (6.25 g CHO)/kg/day. +- Adolescent/Adult: At least 15 kcal (3.75 g CHO)/kg/day for 3 days before the test. + +Pre-visit preparations include: +- Fast for at least 10 hours (no more than 16 hours). +- No intake of coffee, tea, soda, cigarettes, alcohol, or chewing gum. +- No vigorous exercise. +- No work the night before. +- Discontinue any necessary medications as prescribed. + +### Glucose and Insulin Before the Test +- **Short-acting insulin analogues** (e.g., lispro, aspart): 2 hours before test. +- **Regular insulin**: Up to 6 hours before test. +- **Intermediate-acting insulin**: Administer evening before. +- **Long-acting basal insulin**: Administer as usual; SC infusion can continue. + +### Target Glucose Level +- Desired range 70-200 mg/dL at test start. +- Insulin can be adjusted 2 hours before to treat hyperglycemia. + +### IV Placement During the Test +- IV should be maintained and flushed with saline or heparin flush. +- Participant should rest; quiet activities allowed. Bathroom breaks permitted between draws. + +## Testing Instructions + +### Time Point −10 minutes +- **First sample**: At least 10 minutes after IV line is established. Draw into lavender-top K2 EDTA (1.2 mL). +- **Procedure**: + - Invert sample 8-10 times, chill if not centrifuging immediately. + - Centrifuge at 1300 g (~3000 RPM) for 10 minutes. Freeze at −80°C. + +### Time Point 0 minutes +- **Second sample**: Before drink. Same handling. +- **Drink**: Boost® (6 kcal/kg, max 360 mL) within 5 minutes. + +### Following Samples +Draw samples into respective tubes and handle similarly: +- **Time Points**: 15, 30, 60, 90, 120, 150, 180, 210, 240 minutes. + - Drawn for C-peptide, glucose, and glucagon/proinsulin. + +### Tube-Processing Instructions +- **Centrifugation**: Transfer plasma to vials. Label with time point. +- **Freezing**: For glucose, C-peptide, glucagon/proinsulin. +- **Shipping**: To respective laboratories for assay execution. + +### Table: Sample Collection Schedule + +| Time (minutes) | Glucose Sample Taken | C-peptide/Sample Taken | +|-----------------|----------------------|------------------------| +| −10 | X | X | +| 0 | X | X | +| Participant drinks Boost® | | | +| 15 | X | X | +| 30 | X | X | +| 60 | X | X | +| 90 | X | X | +| 120 | X | X | +| 150 | X | X | +| 180 | X | X | +| 210 | X | X | +| 240 | X | X | + +--- +Sample handling deviations must be documented on the MMTT specimen transmittal form. + +--- + +**End of Output** +``` diff --git a/markdown-output/procedures-for-the-staining-of-lipid-droplets-with-phbdj2n.md b/markdown-output/procedures-for-the-staining-of-lipid-droplets-with-phbdj2n.md new file mode 100644 index 0000000000000000000000000000000000000000..9475fdde493ce491683c7eb04b3d70ff8616f16d --- /dev/null +++ b/markdown-output/procedures-for-the-staining-of-lipid-droplets-with-phbdj2n.md @@ -0,0 +1,58 @@ +```markdown +# Procedures for the Staining of Lipid Droplets with Oil Red O + +Fabricio da Mota Ramalho Costa, Sabrina Alves, Chyntia Díaz, María Cristina Vidal Pessolani + +## Abstract + +### Goal: +This document aims to standardize the protocol for the staining of lipid droplets with Oil Red O (ORO) and subsequent observation using fluorescence microscopy. + +The technique of staining with Oil Red O can be used to observe changes in the lipid metabolism of animal cells, caused by different agents such as bacteria or drugs (such as statins), hormones (such as IGF-1), and several small molecules (such as PGL-1). Excessive accumulation of lipids is a key feature of several metabolic events and/or diseases, so identifying and quantifying these changes are essential for a better understanding of these physiological events. These lipid droplets are stained by Oil Red O. + +## Field of Application +Oil Red O is a lipid-soluble lysochrome (C26H24N4O) with a maximum absorption at 518 nm. This dye is used for the staining of neutral lipids (triglycerides and diacylglycerols), in addition to cholesterol esters but does not bind to biological membranes. The principle of this technique is based on the poor solubility of the ORO in the solvent, and the solubility is further decreased by diluting ORO in water. This way, the hydrophobic dye will move from the solvent to associate with lipids within tissue sections. + +One limitation of this technique is the inability to differentiate the labeled lipid species. This dye does not label polar lipids (i.e., phospholipids, sphingolipids, and ceramides). + +## General Considerations +- Cells to be analyzed should be cultured on 13 mm coverslips in 24-well plates. +- Oil Red O labeling does not need to be carried out sheltered from the light and most of the steps can be carried out outside the biological safety booth since the material will be fixed with 4% paraformaldehyde. +- The reagent is 0.05% Oil Red O solution in 85% propylene glycol. +- Weigh the Oil Red O powder. Take a funnel and place a coffee filter into the funnel. Then place the Oil Red O powder inside the filter and pour the volume of Propylene Glycol 85% into the filter, calculated for the 0.05% solution. Wait for the solution to pass through the filter (this may take a few minutes). If Propylene Glycol 85% stock has run out, Propylene Glycol pro-analysis should be diluted in distilled water to a concentration of 85%. +- This protocol is organized in 3 steps: Fixation of the cells with 4% paraformaldehyde (PFA) and staining with Oil Red O, nuclear staining with DAPI, and assembly of the slide. + +## Experimental Procedures + +### Part 1 - Fixation of Cells and Staining of Lipid Bodies (Droplets) +1. Remove the culture medium inside the safety cabinet (BSC) using a pipette and add 250 μL of 4% PFA per well. Incubate for 20 minutes by supporting the plate inside a styrofoam with ice. +2. Remove the 4% PFA with a pipette and add 250 μL of 1x PBS, dripping it slowly on the walls of the wells. Remove the PBS with a pipette, without leaning the tip on the bottom of the plate. Repeat this wash 3 times. *From this step on, the protocol can be performed outside the BSC. +3. Add 250 μL of propylene glycol pro-analysis to each well, incubate for 7 minutes at room temperature. +4. Remove the propylene glycol with a pipette but do not wash. +5. Add 250 μL of 0.05% Oil Red O solution. Incubate for 5 minutes at room temperature. +6. Remove the propylene glycol with a pipette but do not wash. +7. Remove the propylene glycol with a pipette but do not wash. +8. Remove the previous solution with a pipette and add 250 μL of 1x PBS, dripping it slowly on the walls of the wells. Remove the PBS with a pipette, without leaning the tip on the bottom of the plate. Repeat this wash 3 times. + +### Part 2 - Nuclear Staining with DAPI +1. Dilute the DAPI according to the instructions set in the reagent box (dilute 100x with distilled water). The final volume of DAPI is related to the number of wells that will be analyzed. +2. Add 250 μL of DAPI to each well and incubate for 3 minutes at room temperature. When staining the cells with DAPI, it is necessary that the plate remains in a dark environment; thus, you should cover the plate with laminated paper so that there is no interference of light. +3. Remove the DAPI solution with a pipette and add 250 μL of 1x PBS, dripping it slowly on the walls of the wells. Remove the PBS with a pipette, without leaning the tip on the bottom of the plate. Repeat this wash 3 times. + +### Part 3 - Slide Assembly +1. Identify the slides with the conditions to be analyzed and drip 3 μL of mounting medium (antifade) on the slides. +2. Remove the coverslip from the well, and with the aid of forceps, place it inverted on the slide, so that the cells are in contact with the antifade. +3. To seal the slide, Entellan mounting medium or colorless nail polish is used. + +### OBS +1. In the washing steps, 1x PBS should be slowly added to the wall of the well to ensure that the fixed cells do not release from the plate. +2. The mounting medium (Entellan) is very viscous. Thus, it is useful to make a small cut at the tip of the pipette tip so that manipulation with Entellan becomes easier. + +## References +- Annika Mehle, Carolina E Hagberg, Lars Muhl, Ulf Eriksson & Annelie Falenvall. Imaging of neutral lipids by oil red O for analyzing the metabolic status in health and disease. *Nature Protocols* 8, 1149–1154 (2013) + +## Citation +Fabricio da Mota Ramalho Costa, Sabrina Alves, Chyntia Díaz, María Cristina Vidal Pessolani Procedures for the staining of lipid droplets with Oil Red O. *protocols.io* dx.doi.org/10.17504/protocols.io.phbdj2n Published: 16 Apr 2018 + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/processing-and-symmetry-expansion-of-lrrk2rckw-on-bwwnpfde.md b/markdown-output/processing-and-symmetry-expansion-of-lrrk2rckw-on-bwwnpfde.md new file mode 100644 index 0000000000000000000000000000000000000000..e7951b4f93123e70abc52a8997567e933e5a3667 --- /dev/null +++ b/markdown-output/processing-and-symmetry-expansion-of-lrrk2rckw-on-bwwnpfde.md @@ -0,0 +1,169 @@ +```markdown +# Goal/Experiment: +This protocol aims to describe the steps involved in processing and symmetry expansion of LRRK2^RCKW^ filaments bound to microtubules. The protocol includes preprocessing, creating a new set of symmetry-expanded particles, and using popular cryo-EM refinement programs. + +# Processing and Symmetry Expansion of LRRK2^RCKW^ on Microtubules + +**Mariusz Matyszewski** +*Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, CA 92093* + +Date of Publication: May 23, 2022 +DOI: [10.17504/protocols.io.bwwnpfde](https://dx.doi.org/10.17504/protocols.io.bwwnpfde) + +Protocol for symmetry expansion of LRRK2^RCKW^ filaments bound to microtubules. This protocol covers everything from preprocessing to creating a new set of symmetry-expanded particles that can be used by any of the popular cryo-EM refinement programs. + +Mariusz Matyszewski 2022. Processing and symmetry expansion of LRRK2^RCKW^ on microtubules. **protocols.io** +[10.17504/protocols.io.bwwnpfde](https://dx.doi.org/10.17504/protocols.io.bwwnpfde) + +Grant Information: +- ASAP Grant ID: ASAP-000519 +- MJFF Grant ID: 18321 + +**Keywords**: cryo-EM, helical reconstruction, symmetry expansion, microtubule, LRRK2, ASAPCRN + +--- + +## Table of Contents +1. Preprocessing Data +2. Filament Picking +3. Selecting Proper LRRK2^RCKW^ Filaments +4. Signal Subtraction of the Microtubule +5. Refining LRRK2^RCKW^ Filaments +6. Symmetry Expansion + +--- + +## Preprocessing Data + +### 1. Align Frames and Do CTF Fitting +- **Software:** Use your preferred software. The original publication used **MotionCor2** and **CTFFIND4**. +- **Note:** All data processed was collected on lacey carbon grids rather than C-Flats or UltrAuFoils. + +## Filament Picking + +### 2. Manual Picking to Create References +- **Software:** Relion 3.0 (manual picking in version 3.1 should be identical) +- **Process:** + - Manually pick filaments for about 20 micrographs. + - Helical rise of 30 Å. + - Box size: 600 px. + - Re-scale to 250 px. + - Helix settings: Tube size of 600 Å. + +### 3. 2D Classification to Create References +- **Iterations:** 20 iterations +- **Mask:** 600 Å. +- **Process:** + - 2D classification into 10 classes, selecting 3 different looking classes. + - Helix settings used: 600 Å tube diameter. + +### 4. AutoPick +- **Software:** GPU acceleration with a shrink factor of 1. +- **Settings:** + - Lowpassed to 15 Å. + - Tube size: 500 Å. + - Helical rise: 30 Å. +- **Result:** ~250 particles per micrograph. + +### 5. Extraction and Particle Clean-up +- **Box size:** Initial extraction with 656 px, rescaled to 164 px (4x binning). +- **Software:** Relion 3.1. + - Classify with 100 classes, 20 iterations. + - Tube diameter: 450 Å. + - Initial angular sampling: 3 degrees. + - Second round classification to remove further contaminants. + +## Selecting Proper LRRK2^RCKW^ Filaments + +### 6. 2D Classification to Select Ordered LRRK2^RCKW^ Filaments +- **Iterations:** 25 iterations, 50 classes +- **Settings:** + - Tube diameter: 500 Å. + - Mask: 650 Å. + - Initial angular sampling: 3 degrees. + - Include only filaments showing extremely ordered LRRK2^RCKW^. + +### 7. 3D Classification to Determine Protofilament Size +- **References:** MiRP provided references for protofilament sizes (11 to 16 PF). +- **Settings:** + - Lowpassed to 15 Å. + - Sampling: 1.8 degrees. + - Tube diameter: 325 Å. + - Helix settings: No helical symmetry applied. +- **Result:** Particles classified into different protofilament sizes. Protofilament size of 11 picked. + +### Reference +- Cook, A.D., et al. "A microtubule RELION-based pipeline for cryo-EM image processing," Journal of Structural Biology, 2020. +- [10.1016/j.jsb.2019.10.004](https://doi.org/10.1016/j.jsb.2019.10.004) +- [MiRP](https://github.com/moores-lab/MiRP) + +### 8. Re-Extract Particles at a Lower Binning +- **Process:** Re-extract at original pixel size, 4x binned images can continue until Step 15. + +## Signal Subtraction of the Microtubule + +### 9. Refine the Microtubule Section of the Filament +- **Reference Structure:** 11-PF microtubule lowpassed to 15 Å. +- **Settings:** + - Mask: 450 Å. + - Tube diameter: 300 Å. + - Helical symmetry: 11 asymmetric units, 11.3 Å rise, -32.5 degrees rotation. + - Twist search: Constrained to -33 to -32 degrees. + +### 10. Create a Microtubule Mask +- **Process:** + - Choose the lowest threshold showing only microtubule. + - Extend map by 6 pixels, add a 6-pixel soft edge. + +### 11. Subtraction +- **Software:** Relion 3.1. + - Convert the star files to Relion 3.0 for using the subtraction job. + - Use External job type for subtraction in legacy mode. + +## Refining LRRK2^RCKW^ Filaments + +### 12. 2D Classification to Choose the Best LRRK2^RCKW^ Filaments +- **Classes:** 40 classes. +- **Settings:** + - Tube diameter: 500 Å. + - Mask: 650 Å. + - In-plane sampling: 3 degrees. + +### 13. 3D Classification of LRRK2^RCKW^ Filaments +- **Settings:** + - Lowpass reference to 15 Å. + - Tube diameter: 520 Å. + - Sampling: 1.8 degrees. + - Helical symmetry: 33.3 degrees rotation, 31 Å rise. + +### Reference +- Watanabe, R., et al. "The In Situ Structure of Parkinson's Disease-Linked LRRK2," Cell, 2020. +- [10.1016/j.cell.2020.08.004](https://doi.org/10.1016/j.cell.2020.08.004) + +### 14. 3D Refinement of the LRRK2^RCKW^ Filament +- **Process:** Use the best classes to refine using Refine3D. + +### 15. Unsubtraction of the Microtubule and Full Refinement +- **Process:** + - Use subtract job to revert to the original particles. + - Re-extract the reverted particles before final symmetry expansion. + +## Symmetry Expansion + +### 16. Create Masks for LRRK2^RCKW^ Dimers within the Filament +- **Process:** + - Create masks for each LRRK2^RCKW^ dimer to extract. + - Use spherical "ones" masks for each dimer. + +### 17. Symmetry Expansion by Subtraction +- **Process:** + - Use Subtract job for each mask. + - Set new output size to smaller, 300x300 pixel particles. + +### 18. Realign the New Particles +- **Software:** Transition to CryoSPARC for alignment. + +--- + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/processing-pbmcs-for-multiplexed-scrna-seq-cgchtst6.md b/markdown-output/processing-pbmcs-for-multiplexed-scrna-seq-cgchtst6.md new file mode 100644 index 0000000000000000000000000000000000000000..ea67f1e820464a9eab98d8cf7aa72be02b3856d1 --- /dev/null +++ b/markdown-output/processing-pbmcs-for-multiplexed-scrna-seq-cgchtst6.md @@ -0,0 +1,174 @@ +```markdown +# Goal/Experiment: +To prepare viable single cells from frozen PBMCs for scRNA-seq, using a multiplexing strategy to reduce batch effect and cost. + +# Processing PBMCs for Multiplexed scRNA-seq + +## Authors: +Ning Xie¹, Fang Zhang¹, Yuanqing Feng¹, Sarah Tishkoff¹ +¹UPenn + +## Citation: +Ning Xie, Fang Zhang, Yuanqing Feng, Sarah Tishkoff (2022). Processing PBMCs for multiplexed scRNA-seq. [protocols.io](https://protocols.io/view/processing-pbmcs-for-multiplexed-scrna-seq-ccghtst6) + +## DOI: +[dx.doi.org/10.17504/protocols.io.dm6gpj881gqz/v1](https://dx.doi.org/10.17504/protocols.io.dm6gpj881gqz/v1) + +## Abstract +The purpose of this protocol is to prepare viable single cells from frozen PBMCs for scRNA-seq, with a multiplexing strategy applied to reduce batch effect and cost. + +## License +This protocol is an open access protocol distributed under the terms of the [Creative Commons Attribution License](https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. + +## Protocol ID +69737 + +## Materials + +### Supplies/Equipment +- Ultra-Low Attachment Multiple Well Plate, 24 well (Corning Costar #3473) +- 1.5 mL microcentrifuge tube (low binding) +- 70 µm cell strainers +- Hood (Biological Safety Cabinet - BSC) +- Incubator for cells at 37°C +- Water bath at 37°C +- Inverted microscope +- Centrifuge for 50 mL, RT +- Centrifuge for 5 mL and 1.5 mL, 4°C +- Timer +- Pipettes and sterile filter-tips (all sizes) +- 1 mL and 200 µL wide-bore tip +- Pipetboy and sterile serological pipettes (all sizes) +- Regular hemacytometer +- Cell counter +- 50 mL sterile falcon tubes +- 5 mL FACS tube +- White column racks for FACS tube +- MS columns (Miltenyi Biotec #130-042-201) +- Flowmi Cell Strainers (Sigma # BAH136800040-50EA) + +### Reagents +- **Complete Medium**: RPMI-1640 + 10% FBS + 100 U/mL penicillin + 100 U/mL streptomycin + 1 mM sodium pyruvate + 2 mM L-glutamine +- **Washing Medium**: 50% Complete Medium + 50% X-vivo 15 medium + 25 U/mL benzonase (3.1 µL of 400 U/mL benzonase into 50 mL Washing Medium) +- **Benzonase** (Millipore #71205-3): A nuclease that degrades nucleic acids. +- **Lipopolysaccharide (LPS)**: Ultrapure lipopolysaccharide from E. coli K12 (InvivoGen #tlrl-peklps) +- **Interferon β (IFN β)**: Recombinant Human IFN-beta Protein (R&D systems #8499-IF-010/CF) +- **1X D-PBS** +- **Trypan Blue 0.4%** (filtered; for viability staining) +- **Staining Buffer**: Cell staining buffer (BioLegend #420201) +- **Dead Cell Removal Kit**: (Miltenyi Biotec # 130-090-101) +- **Distilled Water**: UltraPure distilled water (Invitrogen #10977-015) +- **4% BSA**: Prepared from BSA powder in PBS, aliquots stored in -20°C +- **Bleach** + +## Safety Warnings +Refer to the Safety Data Sheets (SDS) for health and environmental hazards. + +## Procedure + +### Thawing PBMCs +1. **Preparation**: Warm Complete Medium and Washing Medium to 37°C in a water bath. Prepare 50 mL Falcon tubes with 25 mL Washing Medium each. + - Note: Add benzonase into the Washing Medium immediately before use. + +2. **Cryovials Handling**: Remove cryovials from liquid nitrogen storage and place them on dry ice. + +3. **Thawing**: Thaw frozen vials in the water bath at 37°C for 1 minute (set timer), one vial at a time. Remove when a tiny ice crystal remains. Wipe vials with 70% ethanol. + +4. **Transfer Cells**: Pour the thawed cells gently into a 50 mL conical tube containing 25 mL pre-warmed Washing Medium. + - Note: Avoid pipetting cells; instead, tap or vortex gently. Benzonase reduces potential DNA release from dead cells. + +5. **Rinse Cryovial**: Rinse the cryovial with 1 mL pre-warmed Washing Medium and add to the 50 mL tube. + +6. **Centrifuge**: At 350 xg for 5 minutes at room temperature. Aspirate the supernatant. + +7. **Resuspend Pellet**: + - In 1 mL of pre-warmed Washing Medium by gentle tapping. + - Add 4 mL of Washing Medium and homogenize the suspension. + - Take 10 µL for cell counting. + +8. **Cell Counting**: + - Using Trypan Blue. + - Count using hemacytometer or cell counter. Calculate the cell number. + +9. **Resuspend Pellet**: + - In 1 mL of pre-warmed Complete Medium by gentle tapping. + - Add 9 mL of pre-warmed Complete Medium. + - Brief vortexing (optional). Centrifuge at 350 xg for 5 minutes at room temperature. + +10. **Adjust Cell Concentration**: + - To 1 x 10⁶ cells per mL in Complete Medium by gentle tapping. + +11. **Seed Cells**: + - Into a 24-well plate at 1 x 10⁶ cells per well in 1 mL Complete Medium. + - Using a wide-bore 1 mL pipet tip. + +12. **Incubate**: + - In cell culture plate at 37°C, 5% CO₂ for 16-24 hours for recovery. + +### Stimulation of PBMCs with Ligands + +13. **Prepare Ligands**: + - **LPS**: (200 ng/mL, 20x) in Complete Medium. + - **IFNβ**: (2000U/mL, 20x) in Complete Medium. + +14. **Stimulate Cells**: + - Add 50 µL of LPS (20x) for a final concentration of 10 ng/mL. + - Add 50 µL of IFNβ (20x) for a final concentration of 100 U/mL. + - For control, add 50 µL Complete Medium. + +15. **Resuspend & Incubate**: + - Tap the plate to mix. + - Transfer to the incubator at 37°C, 5% CO₂ for 6 hours. + +### Harvest PBMCs and Pool Samples + +16. **End Incubation**: + - After 6 hours, remove the plate from the incubator. + - Check for cell clusters under a microscope. + - Transfer cells with medium into 5 mL FACS tube using a wide-bore pipet. + - Rinse the well with 1 mL PBS and add to the FACS tube. + +17. **Centrifuge**: + - At 350 xg for 8 minutes at 4°C. + - Pour off the supernatant, invert, and dry with a Kim wipe. + +18. **Wash Cells**: + - Add 0.5 mL Staining Buffer to each sample. Tap to resuspend. + - Add another 1.5 mL Staining Buffer. + - Centrifuge at 350 xg for 5 minutes at 4°C. Pour off and dry. + +19. **Wash Cells Again**: + - Add 500 µL Staining Buffer. Tap to resuspend. + - Take 10 µL for Trypan Blue staining and count. + - Calculate cell number and volumes. + +20. **Final Wash**: + - Add 2 mL Staining Buffer. Centrifuge at 350 xg for 5 minutes at 4°C. + +21. **Resuspend Cells**: + - In the Staining Buffer and adjust to 5 x 10⁶ cells per mL. + - Pool cells from indicated samples into low binding 1.5 mL Eppendorf tubes. + +### Dead Cell Removal + +22. **Binding Buffer Preparation**: + - Prepare Binding buffer (from Dead cell removal kit, at 4°C), 0.5 mL uL Binding buffer into 9.5 mL distilled water. + - Prepare BSA solutions and keep on ice. + +23. **Filter Cells**: + - Use a 70 µm cell strainer. Centrifuge at 350 xg for 5 minutes at 4°C. Remove supernatant. + +24. **Binding Buffer Rinse**: + - Rinse MS column with 500 µL binding buffer. + - Add pooled cells to column. Repeat. + +### Prepare Single-Cell Suspension + +25. **Resuspend Pellet**: + - Adjust to 1500 cells/µL. Use wide-bore pipet. + +26. **Single-Cell Prep**: + - Follow 10X Genomics protocol of “Chromium Next GEM Single Cell 3' Reagent Kits v3.1”. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/production-of-cellular-reagents-using-iptg-cegjtbun.md b/markdown-output/production-of-cellular-reagents-using-iptg-cegjtbun.md new file mode 100644 index 0000000000000000000000000000000000000000..421152cdd4a0c8e43202c7cd627b9b2b7de758cd --- /dev/null +++ b/markdown-output/production-of-cellular-reagents-using-iptg-cegjtbun.md @@ -0,0 +1,158 @@ +```markdown +Goal/Experiment: +This experiment focuses on the production of cellular reagents using IPTG induction of protein expression in E. coli. + +# Production of Cellular Reagents using IPTG + +## Abstract +This protocol documents the production of cellular reagents. Cellular reagents are defined as common molecular biology enzymes expressed in E. coli but not subsequently purified before use (e.g., dried E. coli cells are used as the reagent). This protocol provides instructions for IPTG (T7 based) expression. + +## Keywords +- Production of cellular reagents using IPTG +- Protein expression in E. coli using IPTG +- IPTG induction of protein expression in E. coli + +## License +This is an open access protocol distributed under the terms of the Creative Commons Attribution License. + +## Guidelines +This protocol describes the use of IPTG in inducing protein expression in E. coli BL 21 DE3 strain. + +## Materials + +### Equipment +- Static Incubator +- Shaker incubator +- Centrifuge +- Micropipettes with sterile tips +- Sterile PCR tube (8-strip tubes) +- Sterile 1.5 mL Eppendorf tubes +- Tupperware or Glass jars +- Conical flask (50 mL and 250 mL) +- Duran bottles +- 10 µL wire loops +- Thermocycler or Water bath +- Spreader + +### Reagents +- Silica gel beads desiccant +- PBS (Phosphate Buffered Saline) solution [made using this protocol](https://example.com) +- 100 mg/mL Kanamycin solution [made using this protocol](https://example.com) +- LB media and LB agar plate [made using this protocol](https://example.com) + +## Safety Warnings +Wear protective clothing (gloves, lab coat, face masks). + +## Before Starting +Ensure all materials and reagents needed are available and all culture media prepared. + +## Preparation of Overnight Starter Culture +1. **From glycerol stock** + - Remove the glycerol stock from -20°C. + - Open the tube and use a sterile loop, sterile toothpick or sterile pipette tip to scrape some of the frozen bacteria off the top. _Do not let the glycerol stock thaw!_ + - Streak onto LB agar plate which contains appropriate antibiotic, label with full details. + - Return glycerol stock to freezer. + - Incubate plate at 37°C _Overnight_ in a static incubator. + - Next day, check plate for growth of bacteria. + - Prepare 5 mL LB broth plus appropriate antibiotic. + + Example: 2.5µl 100 mg/mL Kanamycin stock in 5 mL LB broth gives 50 µg/mL final kanamycin concentration. + + - Inoculate prepared 5 mL LB Broth + Antibiotic in a 50 mL Falcon tube with 1 E. coli colony from the overnight plate. + - Label and place the falcon tube in a shaking incubator at 37°C _Overnight_. + - Store plate with colonies on at +4°C for up to 3 weeks. + +2. **From a pre-streaked plate** + - Remove the plate from +4°C. + - Use a wire loop to pick a colony from the plate. + - Inoculate a prepared 5 mL LB Broth + Antibiotic in a 50 mL Falcon tube with 1 E. coli colony from the plate. + - Label and place the falcon tube in shaking incubator at 37°C _Overnight_. + - Store plate with colonies on at +4°C for up to 3 weeks. + +## Growth of Main Culture and Preparation of Cellular Reagents + +### Growing Main Culture for IPTG-protein Expression +1. Prepare 50 mL LB broth plus appropriate antibiotic in a 100 mL flask. +2. Inoculate the 50 mL LB Broth + Antibiotic with 0.5% volume of the starter culture (e.g., 250 µL of the overnight culture in 50 mL LB + Antibiotic). +3. Grow at 225 rpm, 37°C, 3 hours until A600 = 0.5. +4. Induce expression by adding 0.5 millimolar (mM) IPTG final concentration. + + **Calculate the amount of IPTG as follows:** + + Amount of IPTG to add (µL) = + \[ + \left( \text{concentration IPTG sought (mM)} \times \text{volume of culture (µL)} \right) / \text{Stock IPTG conc (mM)} + \] + + Example: 31.25 µL of 0.8 Molarity (M) stock to 50 mL. +5. Continue to incubate for 225 rpm, 37°C, 3 hours. +6. Decant 1.5 mL (or desired volume depending on the tubes available) into centrifuge tubes or Eppendorf tubes. +7. Centrifuge at 4000 rpm, 15 minutes, pour off and discard the supernatant and keep the pellets at 4°C. +8. Now make another set of tubes: This set is to be used for SDS PAGE to [confirm successful protein expression](https://example.com). + +## After Confirming Successful Protein Expression + +### Wash Cells +1. Remove from the fridge the first set of tubes and resuspend cell pellet into 1.5 mL of cold PBS (vol of PBS = vol of harvested cell) - _First wash_. + + Example: 1.5 mL harvested resuspended in 1.5 mL cold 1X PBS. +2. Centrifuge at 4000 rpm, 15 minutes. +3. Perform second wash. +4. Resuspend pellet into 1.5 mL of cold 1X PBS. + +### Aliquoting +1. Measure A600 of a neat, 1:10 or 1:100 dilution. Multiply the value to get the actual final A600 number. +2. Calculate the volume of your final cell suspension that would contain 2 x 108 cells, using the equation: + + \[ + \text{volume containing 2x108 cells} = \frac{200}{\text{final A600 of cell suspension}} + \] + + Example: if your final A600 is 6.5, then + + \[ + \text{volume containing 2x108 cells} = \frac{200}{6.5} = 31µL + \] + +3. You can either aliquot ~2 x 107 cells (enough for a single PCR reaction) or 2 x 108 cells (enough for 10 PCR reactions) into individual 0.2 mL PCR tubes in order to prepare dried Taq cellular reagents. +4. Aliquot either single reaction or 10X reactions worth of cellular reagents into 8-tube strips of 0.2 mL PCR tubes. + + Example: using the example above, 3.1 µL (1x reaction) or 31 µL (10x). + +### Heating to Kill the Bacteria +1. Transfer the 0.2 mL PCR tubes into a thermocycler. +2. Set a program for heating on the thermocycler at 60°C for 10 minutes (leave some tubes unheated to serve as control and label the tubes accordingly). + + **Alternative:** Where a thermocycler is unavailable, a Water bath can be used by setting the water bath to 60°C and immersing the tubes in it for 10 minutes. + +### Drying +1. Leave tube lids open. +2. Place the tube strips with pre- heated aliquoted cellular reagents carefully in a vacuum tupperware 1/2 filled with desiccant. +3. Place the lid of the tupperware on top and close gently making sure it closes properly on all the sides. +4. Use the vacuum pump to create a vacuum in the tupperware by pumping several times until the lid is visibly tough and sunken. +5. Place the container static incubator _Overnight_ at 37°C. +6. After 18-24 hours check to see if the cellular reagents are completely dry. +7. Once dry, close the lids and place in a small bag at 4°C with a small amount of desiccant. + + - We typically use this drying method to obtain cellular reagents that are stable for 3 to 6 months. + - In settings where a Lyophilizer is available, it can be used to obtain cellular reagents that can remain functional for longer periods. + +### Check the Effectiveness of the Heating Step +1. Reconstitute one of the pre-heated tubes with 30 µL PCR water. +2. Mix by gently tapping onto the bottom of the tube and allow to stand for 5 minutes. +3. Use a micropipette to aliquot 10 µL inoculate on an LB agar plate supplemented with Kanamycin (100 mg/mL). +4. Use a spreader to evenly spread the inoculum across the surface of the plate. +5. Repeat the above steps but this time reconstituting the control tube (tubes where cells were not pre-heated). +6. Place both plates in a static incubator and incubated at 37°C _Overnight_. + + **Check plates:** + - The plate from pre-heated tubes should show no growth of bacteria confirming the complete killing of bacteria cells in cellular reagents making them safer to handle. + - The plate from non-heated tubes should show visible growth of bacteria confirming the need to preheat cellular reagents at 60°C for 10 minutes before drying and subsequent use. + +## Quality Control +Proceed to carry out quality control of this batch of cellular reagents by testing the [functionality](https://example.com) and assessing them for [endonuclease activity](https://example.com). + +After passing the quality control test, the cellular reagents can be used in a PCR amplification reaction. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/propagation-of-marine-eukaryotic-viruses-prasinovi-gujbwun.md b/markdown-output/propagation-of-marine-eukaryotic-viruses-prasinovi-gujbwun.md new file mode 100644 index 0000000000000000000000000000000000000000..b69f3d9fef25736aacfdaa7d0602460fc5190aed --- /dev/null +++ b/markdown-output/propagation-of-marine-eukaryotic-viruses-prasinovi-gujbwun.md @@ -0,0 +1,117 @@ +```markdown +# Goal/Experiment: +Propagation of marine eukaryotic viruses (Prasinoviruses) + +## Abstract + +**Purpose:** To reproducibly generate fresh preparations of virus for independent experiments. + +**Summary:** Fresh virus sample for an experiment is generated by a primary infection of exponentially growing host cells from a master stock of virus. The infected host is allowed to lyse until the culture is cleared. The lysate is filtered to remove any large cellular debris, then the viral-size fraction is concentrated from the filtered lysate and washed with buffer using a centrifugal concentrator. The viral concentrate is stored at 4°C protected from light and should be used for an experiment within 1-2 days. On the day of (or the day before) the experiment, an MPN assay should be set up to assess infectivity of the fresh viral concentrate. + +## Guidelines + +### Principle + +Several external physical and chemical factors (e.g., temperature, salinity, acidity, light, and adsorption) influence the morphology, persistence, and function of marine viruses. Because viruses depend on their hosts for replication, biological parameters such as host physiology and growth rate, as well as viral and host abundances (i.e., associated encounter rate), likewise impact the properties of a particular viral population (e.g., infectivity, abundance). Standardized and characterized host growth prior to infection combined with consistent infection parameters (e.g., host density, viral volume added, time allowed for lysis, incubation conditions, etc.) should minimize variation among viral preparations and improve reproducibility of viral properties among experiments. Furthermore, using the same master stock of virus for each propagation instead of a serially-propagated virus stock should reduce the probability of introducing genetic mutations. + +## Host Preparation + +Initiate a host culture for the primary infection to allow for at least 5 generations of characterized exponential growth (10 generations is the gold standard). Avoid transferring cells on the day of or the day before the primary infection to minimize disturbance. Monitor growth and transfer/dilute host culture to pre-determined mid-exponential density daily (i.e., semi-continuous culture), including the day of the primary infection. If the host culture will be split to propagate multiple viruses simultaneously, make sure this is done at least 2 days prior to the addition of virus. After final dilution of the host culture to pre-determined mid-exponential density, initiate primary infection as described below. + +### Example Ostreococcus lucimarinus (CCMP2972A) host preparation: + +- **Growth conditions:** 18°C, 14:10 hour light:dark cycle, light irradiance of ~100 μE m⁻² s⁻¹ +- **Growth media:** L1 with natural seawater base +- **Exponential range:** 7×10⁵ - 2×10⁷ cells mL⁻¹ + +### For 2 viruses, intermediate volume (from Oct 2015): ~6 generations of characterized growth + +| Day | 1 | 2 | 3 | 4 | 5 | 6 | 7 | +|-----|----------|--------|--------|--------|--------------|--------|--------------------| +| **Initial density (mL⁻¹)** | 3.5×10⁷ | 9×10⁶ | 1.7×10⁷ | 7×10⁶ | 9.4×10⁶ | 9.4×10⁶ | +| **Growth Rate (d⁻¹)** | NA | 0.68 | 0.69 | 0.53 | 0.62 | 0.68 | +| **Dilution/Transfer** | Transfer | Dilute | Skip | Transfer | Transfer (split) | Dilution | Dilution | +| **Final density (mL⁻¹)** | 4×10⁶ | 4×10⁶ | 5×10⁶ | 5×10⁶ | 5×10⁶ | 5×10⁶ | +| **Volume (mL)** | 60 | 137* | 200 | 140 x2 | 260* x2 | 490* x2 | + +(*) Volume varies depending on growth + +### For 2 viruses, large volume (from Apr 2016): ~5 generations of characterized growth + +| Day | 1 | 2 | 3 | 4 | 5 | 6 | 7 | +|-----|-------------|-----|--------|--------|---------------|--------|--------------------| +| **Initial density (mL⁻¹)** | 2.2×10⁷ | 1.8×10⁷ | 8.7×10⁶ | 8×10⁶ | 8.7×10⁶ | +| **Growth Rate (d⁻¹)** | NA | 0.760 | 0.516 | 0.481 | 0.487 | +| **Dilution/Transfer** | Transfer | Skip | Skip | Transfer (split) | Dilution | +| **Final density (mL⁻¹)** | 4×10⁶ | 5×10⁶ | 5×10⁶ | +| **Volume (mL)** | 120 | 400 | 330 x2 | 534* x2 | 840* x2 | + +(*) Volume varies depending on growth + +## Before Start + +### Equipment and Materials + +#### Equipment: +- Tube rack +- 200 and 1000 µL pipettes +- Pipettor for serological pipettes +- Refrigerated benchtop centrifuge capable of 1,000 x g +- Swinging bucket rotor that can accommodate 50 ml conical bottom tubes +- Vortexer + +#### Materials: +- Host culture +- Virus master stock +- Culture medium +- 200 and 1000 μL filter tips +- Serological pipettes +- 0.45-μm-pore-size PES membrane Nalgene Rapid-Flow Sterile Disposable Filter Unit(s) (Thermo Scientific #166-0045) +- 100 kDa MWCO PES membrane VivaSpin20 ultrafiltration unit (Sartorius #VS2041) +- 15 or 50 mL conical tubes, sterile +- Parafilm +- 0.02-μm-pore-size sterile Anotop 25 Syringe Filter Plus (GE Healthcare #6809-4102) +- 1X TE buffer pH 8.0 (Fisher BioReagents #BP1338-1), 0.02-μm-filtered (see below) + +### Preparation of TE buffer (1X, pH 8.0) + +Dilute 0.5 mL 100X molecular grade Tris-EDTA (Fisher BioReagents #BP1338-1) in 49.5 mL MilliQ water. In hood, filter through 0.02-μm-pore-size sterile Anotop 25 Syringe Filter Plus (GE Healthcare #6809-4102) into sterile 15 mL tubes. Use within a few days of opening. + +## Protocol + +### Step 1. Primary Infection + +1. Ethanol-clean culture hood as usual, then UV pipettes, tips, and tube rack for 10 min. +2. Work with one virus at a time, transferring the master stock from 4°C to the culture hood. +3. Initiate the primary infection by adding the master virus stock at 1% of the total culture volume (e.g., 5 mL virus to 500 mL exponentially-growing host culture). +4. Mix the infected flask and incubate at standard growth conditions until culture is mostly lysed (e.g., 5 days for O. lucimarinus viruses). +5. Thoroughly clean pipette and gloves with ethanol before repeating for additional viruses. +6. Ethanol-clean hood and UV pipettes, tips, and tube rack after use. + +### Step 2. Cleaning and Concentration of Lysate + +**Cleaning and Concentration of Lysate** + +1. Once the primary infection culture has cleared, filter entire volume of lysate through a 0.45-μm-pore-size PES membrane sterile disposable filter unit to remove large cell debris. +2. Add 20 mL filtered lysate to the upper reservoir of a 100,000 Dalton MWCO PES Vivaspin20 ultrafiltration unit. + +*Note:* Appropriate MWCO will depend on virus capsid size. For maximum recovery select a MWCO at least 50% smaller than the molecular size of the particle of interest. 200 kDa is the equivalent of 10 nm. + +3. Place unit with counter-balance in swinging bucket rotor in pre-cooled (4°C) benchtop centrifuge so that the printed side faces upwards/outwards. +4. Centrifuge at 1,000 x g (do not exceed!) for 5-10 minutes. Centrifuge time will depend on the amount of material in the sample. Do not let the filter go dry. +5. Record the volume retained in the upper reservoir. +6. Discard the filtrate by separating the upper reservoir from the bottom of the Vivaspin20 unit. +7. Repeat steps 2-6 (adding filtered lysate to the retentate in the upper reservoir up to 20 mL before re-centrifuging) until the entire volume of filtered lysate has been processed. + +*Note:* Several Vivaspin20 units can be used in parallel for large lysate volumes, and pooled after concentration. + +8. Transfer the concentrated virus sample (retentate) from the upper reservoir to a sterile 15 or 50 mL conical tube. +9. Remove the upper reservoir from the Vivaspin20 unit and cover the bottom with a double-layer of Parafilm. +10. Add 2 mL of 0.02-μm-filtered 1X TE buffer (pH 8.0) to the upper reservoir. +11. Vortex the upper reservoir (Parafilm side down) for 20 sec on 60% power to wash the Vivaspin20 filter. +12. Add the washed sample to the recovered viral concentrate. +13. Repeat steps 10-12 twice more for a total of 3 washes with TE buffer. +14. Store concentrated virus sample at 4°C protected from light. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/protein-pegylation-protocol-full-martini-coarse-gr-c7h7zj9n.md b/markdown-output/protein-pegylation-protocol-full-martini-coarse-gr-c7h7zj9n.md new file mode 100644 index 0000000000000000000000000000000000000000..3a12ad629336ee46ae6db9d44b25533e67f16a10 --- /dev/null +++ b/markdown-output/protein-pegylation-protocol-full-martini-coarse-gr-c7h7zj9n.md @@ -0,0 +1,484 @@ +```markdown +# Goal/Experiment: +Simulate the PEGylation of proteins using the Martini Coarse-Grained force field and perform Molecular Dynamics simulations using GROMACS, supplemented with the INSANE and Polyply tools for system building and configuration. + +## Protein PEGylation Protocol - Full Martini Coarse Grained v2.0 Protocol (MARTINI, INSANE, GROMACS Simulations) + +**Authors:** +- compt.biology.agutierrez1 +- Carmen Ili Gangas1 + +**Affiliation:** +- 1Universidad De La Frontera + +### Disclaimer +All software used in this tutorial is not of the authorship and belongs to each person/company that presents right in front of its authorship. + +### DOI +[dx.doi.org/10.17504/protocols.io.eq2ylbjjqjk9/v1](https://dx.doi.org/10.17504/protocols.io.eq2ylbjjqjk9/v1) + +### Protocol Citation +compt.biology.agutierrez, Carmen Ili Gangas 2024. Protein PEGylation Protocol - Full Martini Coarse Grained v2.0 Protocol (MARTINI, INSANE, Gromacs simulations). protocols.io [dx.doi.org/10.17504/protocols.io.eq2ylbjjqjk9/v1](https://dx.doi.org/10.17504/protocols.io.eq2ylbjjqjk9/v1) + +### License +This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. + +**Created:** Jan 12, 2024 +**Last Modified:** Jan 15, 2024 + +--- + +## Abstract +The construction of coarse-grained (CG) systems from atomistic models is a key strategy in simulating molecular-level biological systems, reducing computational load and enabling extended simulations. This protocol employs tools like `insane.py` for initial conversions and `GROMACS` for molecular dynamics (MD) simulations, focusing on the PEGylation of proteins to enhance stability and solubility. PEGylation involves the covalent attachment of polyethylene glycol (PEG) to proteins, providing unique properties for drug efficacy and reducing immunogenicity through amide-type reactions. + +--- + +## Introduction + +### 1. Overview +Improving proteins via PEGylation provides insights into protein-polymer interactions using MD simulations, especially with the Martini Coarse-Grained force field, known for grouping several atoms into single interaction sites and maintaining accuracy. + +The protocol focuses on using the Martini CG force field in MD simulations, with nearly atomistic information facilitating longer temporal and larger spatial simulations, enhancing understanding of PEGylation dynamics. + +### 2. Goals +To empower researchers with detailed instructions for simulating PEGylated proteins and a comprehensive approach for understanding protein-PEG interactions through the integration of Martini CG force field into MD simulations. + +--- + +## Materials + +### 1. Skills Needed +- **Basic Information Skills:** + - Install standardized software. + - Run short commands in terminal. + - Construct short scripts. +- **Computational Biology Skills:** + - Understand protein formats (pdb and gro). + - Understand atom types in all-atom and CG structures. + - Understand the biophysics of atoms in a solvent. + +### 2. Hardware Requirements +- A personal computer, either a workstation or laptop, with at least 4 GB of RAM. + +### 3. Software Requirements +- **Python ≥ 3.11.5:** + - Recommended to use Anaconda distribution for ease of installation. + +--- + +## Python Installation + +### Verify Python Version in Terminal +```bash +python --version +``` + +### Install Python Directly to Your System +```bash +sudo apt-get update -y +sudo apt-get install python3 +sudo apt-get upgrade -y +``` + +### Install Python in an Anaconda Environment +```bash +mkdir -p ~/miniconda3 +wget https://repo.anaconda.com/miniconda/Miniconda3-latest-Linux-x86_64.sh -O ~/miniconda3/miniconda.sh +bash ~/miniconda3/miniconda.sh -b -u -p ~/miniconda3 +rm -rf ~/miniconda3/miniconda.sh +~/miniconda3/bin/conda init bash +``` + +After installing Conda, you can create your environment as needed: +```bash +conda create --name myenv python=3.11.5 -y +conda activate myenv +``` + +--- + +## Polyply Installation + +### Install Polyply from PyPi +```bash +pip install polyply +``` + +### Alternatively Install from GitHub +```bash +pip install git+https://github.com/fgrunewald/polyply_1.0.git#polyply_1.0 +``` + +--- + +## Martini Coarse Grained + +The Martini force field is a coarse-grain (CG) force field suited for MD simulations of biomolecular systems. It can be installed, downloaded, and executed using Python scripts. For more information, visit [GitHub: Martinize](https://github.com/cgmartini/martinize). + +### Martini Installation + +#### Download and Execute +```bash +wget https://github.com/cgmartini/martinize.py +``` + +#### Installation +```bash +pip install vermouth +# or +pip install git+https://github.com/marrink-lab/vermouth-martinize.git#vermouth +``` + +--- + +## Insane System Constructor (Membrane Construction) +Usually employed as a lipid system constructor, adding water and ion molecules. This tutorial uses it for system construction. + +For more details, visit [Insane, Lipidomics Tutorial](http://www.cgmartini.nl/index.php/tutorials-general-introduction/insane). + +### Insane Installation + +#### Download and Execute +```bash +wget http://www.cgmartini.nl/images/tools/insane/insane.py +``` + +--- + +## Martinize & Insane Protocol + +### Files Used in This Methodology + +- `1rex.pdb` + +### MARTINI Structure Conversion +From all-atom to coarse-grained. + +Use martinize2 or vermouth method to martinize proteins for ease of specifying characteristics helpful for MD like elastic networks and position restraints. + +#### Command +```bash +martinize2 -f 1REX.pdb -o topol.top -x 1REX_cg.gro -ff martini22 -elastic -ef 100 -el 0.4 -eu 0.9 -pf 1000 -p Backbone -mutate HIS:HSD +``` + +#### Flags Explanation +- `ef` = Elastic force constant. +- `el` = Elastic lower bond cutoff. +- `eu` = Elastic upper bond cutoff. +- `pf` = Position Restraints. +- `mutate` = Mutate one residue to another. + +#### Output Files +- `1rex_cg.gro` +- `molecule_0.itp` + +### Visualize Our MARTINI Conversion Results +The common result of converting an atomistic model to a CG model is a structure made up of spheres or beads in a 4:1 atom-to-bead ratio. + +#### Atomistic Model of Lysozyme +![Atomistic Model](image-link) + +#### Coarse Grained Model of Lysozyme +![Coarse Grained Model](image-link) + +--- + +## Construction +Coarse-grained system generation and preparation for molecular dynamics. + +Using `insane.py` to create the system, adding water molecules, and ions, without modifying files or using extra commands. + +#### Command +```bash +python insane.py -f 1REX_cg.gro -o system.gro -p topol.top -salt 0.15 -d 1 -pbc cubic -sol W:90 -sol WF:10 -charge +8 +``` + +#### Flags Explanation +- `-o` = Output system. +- `-p` = Topologies file. +- `-salt` = Molar concentration of salts. +- `-sol` = Types of water. +- `-d` = Distances from periodic images. +- `-charge` = System charge. + +#### Output Files +- `system.gro` +- `topol.top` + +### Visualize Our INSANE Construction Results +Result: A constructed box composed of the protein, solvent corresponding to water molecules and antifreeze water, and ions (sodium and chlorine). + +Note: +- **Coarse Grained System:** + - *PROTEIN* –> Green beads + - *SOLVENT* –> Cyan + - *IONS* –> Blue + +--- + +## GROMACS File Preparations + +### Files Used in This Section + +#### Generated in a Previous Section +- `system.gro` (Insane Output) +- `topol.top` (Insane Output) +- `molecule_0.itp` (Martini Output) + +#### Used in This Section +- `minimization.mdp` +- `NPT_1.mdp` +- `Production.mdp` +- `AMINOACIDS.itp` +- `IONS.itp` +- `martiniv2.itp` +- `SOLVENT.itp` + +#### Generated in This Section +- `index.ndx` +- `1.sh` + +### Files Modification Prior MD: ITP and Topol Files + +**ITP and Topol Files Modification:** + +Create a folder called `LYSOZYME_MD` containing: +- `ITP` +- `MDP` + +Copy files generated in Insane (`system.gro`, `topol.top`) and Martini (`molecule_0.itp`) steps into `LYSOSYME_MD` folder. + +#### Topol File Modifications + +Default `topol.top` file: +```plaintext +#include "martini.itp" + +[ system ] +; name +Insanely solvated protein. + +[ molecules ] +; name number +Protein 1 +W 1596 +WF 177 +NA+ 16 +CL- 24 +``` + +Corrected `topol.top` file: +```plaintext +#include "../ITP/martiniv2.itp" +#include "../ITP/AMINOACIDS.itp" +#include "../ITP/molecule_0.itp" +#include "../ITP/SOLVENT.itp" +#include "../ITP/IONS.itp" + +[ system ] +; name +Insanely solvated protein. + +[ molecules ] +; name number +molecule_0 1 +W 1596 +WF 177 +NA+ 16 +CL- 24 +``` + +**molecule_0.itp Modifications** +- At line 302, `position_restraints` option. +- At line 568, `Rubber band` option. + +Default `molecule_0.itp` file (lines 302 on): +```plaintext +[ position_restraints ] +#ifdef POSRES +1 1 1000.0 1000.0 1000.0 +4 1 1000.0 1000.0 1000.0 +``` + +Modified `molecule_0.itp` file (position_restraints): +```plaintext +[ position_restraints ] +#ifdef POSRES +1 1 POSRES_FC POSRES_FC POSRES_FC +4 1 POSRES_FC POSRES_FC POSRES_FC +``` + +Default `molecule_0.itp` file (lines 568 on): +```plaintext +; Rubber band +1 84 6 0.89714 100.0 +1 85 6 0.64849 100.0 +1 89 6 0.52919 100.0 +``` + +Modified `molecule_0.itp` file (Rubber band): +```plaintext +#ifdef RUBBER_BANDS +#ifdef RUBBER_FC +#define RUBBER_FC 10 +#endif +1 84 6 0.89714 RUBBER_FC*1.00 +1 85 6 0.64849 RUBBER_FC*1.00 +1 89 6 0.52919 RUBBER_FC*1.00 +#endif +``` + +### Position Restraints Modification +Gradually decrease position restraints from 1000 to 125 kJ/mol nm². + +### Rubber Bands Modification +Activate rubber bands (elastic networks) from MDP files. + +### Files Modification Prior MD: Input Parameter Files + +Download standard input parameters from [Martini Coarse Grained](http://cgmartini.nl/examples/input-parameters-mdp). + +Modify: +- **Minimization Parameters**: Energy minimization. +- **Equilibration Parameters**: NVT (Temperature) and NPT (Pressure). +- **Production Parameters**: Production Run. + +#### Command +```bash +echo q | gmx make_ndx -f system.gro -o index.ndx +``` + +### GROMACS MD Simulation + +#### General Commands and Usage +Run MD with 2 commands per phase. + +#### Command Examples +**Energy Minimization Phase:** +```bash +gmx grompp -f minimization.mdp -o minimization.tpr -c system.gro -r system.gro -p topol.top -n index.ndx -maxwarn 3 +gmx mdrun -deffnm minimization -v -nt 6 +``` + +**NVT Equilibration Phase:** +```bash +gmx grompp -f NVT-Equilibration.mdp -o NVT-Equilibration.tpr -c minimization.gro -r system.gro -p topol.top -n index.ndx -maxwarn 3 +gmx mdrun -deffnm NVT-Equilibration -v -nt 6 +``` + +### Visualize Our GROMACS MD Results + +Two main results expected: +- **production.gro** and **production.xtc**: Direct results. +- **run-connect.pdb** and **final.xtc**: Corrected results for visualization. + +--- + +## PEGylation Method - Polyply v1.0 Methodology + +### Files Used in This Methodology + +#### Generated in Previous Sections +- `run-conect.pdb` +- `molecule_0.itp` + +#### Generated in This Section +- `topol.top` +- `system.gro` + +### Files Creation Prior Polyply Usage + +Polyply is designed for polymer development within a CG force field. The PEG polymers (composed of MEE, PEO, and OH beads) streamline input file creation. + +Loop MEE, PEO, and OH_end into `combined_links.ff`. + +#### Combined Links File +**Example Combined Links (PEGylated polymer):** +```plaintext +[ link ] +[ molmeta ] +by_atom_id true + +[ bonds ] +3 288 1 0.41 2000 ; R-MEE +288 289 1 0.39 5000 ; MEE-PEG +293 294 1 0.28 7000 ; PEG-OH + +[ angles ] +288 3 2 2 150 15 ; MEE-Qd-C3 +289 288 3 2 170 50 ; EO-MEE-Qd +292 293 294 2 150 15 ; EO-EO-OH +``` + +--- + +## Polyply Usage Methodology +Generate any protein bound to X polymers using 3 Polyply commands: `polyply gen_seq`, `polyply gen_params`, and `polyply gen_coords`. + +### Polyply Gen Seq +Create `sequences.json` file. + +#### Command +```bash +polyply gen_seq -f molecule_0.itp -from_file protein:molecule_0 \ +-from_string linker:1:1:MEE-1.0 polymer:5:1:PEO-1.0 end:1:1:OHend-1.0 \ +-seq protein linker polymer end \ +-connects 0:1:0 1:2:0 2:3:4:0 \ +-o sequences.json -name test \ +-label 0:"from_itp":"molecule_0_1" +``` + +#### Command Explanation +- `-f`: Set parameter itp file. +- `-from_file`: Set as "protein". +- `-from_string`: Set MEE, PEO, and OH beads. +- `-seq`: Concatenate beads order. +- `-connects`: Bound residues and beads. +- `-o`: Output sequences.json. +- `-name`: Arbitrary name. +- `-label`: New [moleculetype]. + +### Polyply Gen Params +Generate itp file with PEGylation parameters. + +#### Command +```bash +polyply gen_params -f molecule_0.itp MEE.itp PEO.itp OH_end.itp combined_links.ff -seqf sequences.json -o lysoPEG.itp -name lysoPEG +``` + +### Polyply Gen Coords +Create .gro file. + +#### Command +```bash +polyply gen_coords -p topol.top -o lysoPEG.gro -c run-conect.pdb -name lysoPEG -dens 1.0 +``` + +### Applying Polyply Commands +Erase Position Restraints and Rubber Bands from molecule_0.itp file and add to lysoPEG.itp. + +### New Files Used +- `topol.top` +- `system.gro` + +--- + +## Visualize Our PEGylated Protein MD Results + +Results similar to previous steps: +- **production.gro** and **production.xtc** for direct outputs. +- **run-connect.pdb** and **final.xtc** for visualization. + +**MD Simulation Example:** +- Protein in Licorice Representation. +- Coarse Grained corrected. + +--- + +## Final Remarks +Creating a CG structure and polymer additions is effective for understanding modified structures' behavior. Refer to official pages for more information. + +--- + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/protocol-1-electroporation-of-agrobacterium-tumefa-ciq3udyn.md b/markdown-output/protocol-1-electroporation-of-agrobacterium-tumefa-ciq3udyn.md new file mode 100644 index 0000000000000000000000000000000000000000..a442f94a983fa569c297cf90b3d5a68776e7c3f1 --- /dev/null +++ b/markdown-output/protocol-1-electroporation-of-agrobacterium-tumefa-ciq3udyn.md @@ -0,0 +1,140 @@ +```markdown +# Goal/Experiment: +The goal of this experiment is to transform *Agrobacterium tumefaciens* with a plasmid of interest using electroporation. The subsequent *Agro* can be utilized to transform various plants and fungi, resulting in transformed cell lines. This protocol is particularly in preparation for *Agrobacterium*-mediated transformation of the chytrid fungus *Spizellomyces punctatus*. + +# Protocol 1: Electroporation of *Agrobacterium tumefaciens* with a plasmid of interest + +**Authors:** + +Sarah M Prostak1, Edgar M Medina1, Erik Kalinka1, Lillian Fritz-Laylin1 +1 University of Massachusetts at Amherst + +fritzlaylinlab.umass + +## Abstract + +Electroporation is a widespread method of transforming competent *Agrobacterium tumefaciens* (*Agro*) cells with a plasmid containing a T-DNA of interest. The resulting *Agro* can be used to transform various plants and fungi, leading to transformed cell lines. This protocol outlines the standard electroporation protocol we use to transform *Agro* in preparation for *Agrobacterium*-mediated transformation of the chytrid fungus *Spizellomyces punctatus*. + +## Guidelines + +Any binary plasmid that works in *Agrobacterium tumefaciens* strain EHA105 (GoldBio #CC-225-5x50) can be used for this procedure. It is imperative that all steps are carried out at 4°C up until electroporation. Ensure proper sterile technique throughout this protocol; perform all steps except centrifugation and electroporation charge delivery in a laminar flow hood or in the sterile area around an open flame. + +## Materials + +- Use a fresh streak of *Agrobacterium tumefaciens* EHA105 (GoldBio #CC-225-5x50) to spread a lawn into an LB plate. (A new streak from a -80°C stock will take ~48 hours to display visible growth at 28°C, plan accordingly). +- LB agar plates (1.5% w/v) with and without selection antibiotics, sterile (see recipe). +- Purified plasmid(s) of interest resuspended in molecular biology grade water. +- Molecular biology grade water, sterile such as MilliQ or equivalent. +- 10% (v/v) Glycerol, sterile (e.g., Fisher Chemical™ G33-1). +- 1.5 mL centrifuge tubes, sterile (e.g., Fisherbrand™ Low-Retention Microcentrifuge Tubes, Fisher Scientific Catalog #02-681-331). +- 0.5 mL centrifuge tubes, sterile (e.g., Fisherbrand™ Snap-Cap™ Flat-Top Graduated Microcentrifuge Tubes, Fisher Scientific Catalog #02-681-268). +- 2 mm electroporation cuvettes, sterile (e.g., Bulldog Bio Catalog #12358-346). +- Culture tubes, sterile (e.g., VWR® Culture Tubes Plastic with Dual-Position Caps, VWR Avantor Catalog #60818-703). +- 100-1,000 µL micropipette (e.g., Eppendorf Research Plus Single Channel pipette, pipette.com Catalog #3123000063 ES-1000). +- 20-200 µL micropipette (e.g., Eppendorf Research Plus single channel pipette, pipette.com Catalog #3123000039). +- 0.1-2.5 µL micropipette (e.g., Eppendorf Research Plus single channel pipette, pipette.com Catalog #3123000012). +- Filter tips for the micropipettes, sterile (e.g., TIPONE® FILTER TIPS, USA Scientific Catalog #1122-1830). +- 5 mm glass beads, sterile. +- Ice bucket with ice. +- Centrifuge capable of cooling to 4°C. +- Laminar flow hood and/or open flame, for maintaining sterility. +- 70% (v/v) ethanol for maintaining sterility (if using laminar flow hood). +- Exponential decay electroporator such as Gene Pulser Xcell (Catalog #1652660). +- Shaking incubator at 28°C. + +## Before Start Instructions + +Electroporation should occur at least 4 days prior to the intended *Spizellomyces* transformation time to ensure active, single colonies. Transfer to liquid culture the night before transformation day (see Protocol "Growing liquid cultures of *Agrobacterium* prior to transformation day"). + +## Steps + +1. **Cool the following materials on ice for at least 20 minutes prior to starting:** + 1. Plate with a lawn of wild-type *Agrobacterium* grown overnight at 28°C. + 2. Purified plasmid(s) of interest. + 3. Molecular biology grade water, sterile. + 4. 10% (v/v) glycerol, sterile. + 5. 1.5 mL centrifuge tube(s), enough to hold the volume of *Agro* harvested. + 6. 0.5 mL centrifuge tube(s), one per plasmid to be transformed, plus controls. + 7. 2 mm electroporation cuvettes, one per plasmid to be transformed, plus controls. + +2. **Add 1 mL to 2 mL of ice-cold water to the plate of *Agrobacterium*.** + - Hold the plate at ~45 degrees and run the water over the surface at least 3 times, gently scraping along the agar if necessary to recover the lawn of bacteria. + + **Note:** + - Try not to drag too many big clumps. The resulting harvest should have the consistency, color, and density of whey. + +3. **Transfer the 1 mL of harvested cells to a 1.5 mL centrifuge tube, immediately place back on ice.** + +4. **Pellet the cells at 4000 rcf, 5 minutes in a rotor prechilled to 4°C.** + + **Note:** + - If in a pinch, a rotor for a centrifuge without cooling capabilities can be stored overnight at 4°C or for 10 minutes at -20°C. + +5. **Remove the supernatant and gently resuspend the cells in 1 mL of water. Do not vortex.** + + **Note:** + - Keep cells on ice when not in use. + +6. **Repeat steps 4 and 5, 2 more times for a total of 3 washes.** + +7. **Remove water and resuspend the cells in 800 µL of cold 10% (v/v) glycerol, place tubes back on ice.** + +8. **Add 50 µL of cells to new 0.5 mL tubes, place back on ice.** + + **Note:** + - Use one 0.5 mL tube for each plasmid to be transformed, plus more for planned controls. + +9. **Add 1 µL of the plasmid of interest (200 ng/µL to 300 ng/µL) to its appropriate tube of cells, place back on ice and mix gently by pipetting.** + + **Note:** + - Use 1 µL of water as a negative control. + +10. **Transfer the cells to cold 2 mm electroporation cuvettes, place back on ice.** + + **Note:** + - Again, use one cuvette for each control or plasmid to be transformed individually. + +11. **Prepare the recovery media in advance by placing 150 µL of room temperature SOC medium to one 15 mL culture tube for each electroporation.** + +12. **Turn on the electroporator and create an electroporation program with the following settings:** + - Voltage: 2400V + - Capacitance: 25 µF + - Resistance: 200 Ω + - Cuvette size: 2 mm + +13. **Fully dry the cuvette before placing it into the electroporator chamber.** + + **Note:** + - Failure to fully dry the cuvette will lead to current arcing and improper electroporation. + +14. **Electroporate your cuvette.** + +15. **As quickly but as gently as possible, remove a little less than 150 µL of SOC medium from the appropriate tube for the plasmid and pipette it into the cuvette. Remove the full volume from the cuvette and return it to the original culture tube.** + + **Note:** + - Do this by a flame and with good sterile technique. + - The best way to do this is to set a p200 to 150 µL but to not pull the entire volume, leaving enough space for the 50 µL that is in the cuvette. + +16. **Incubate culture tubes at 28°C, shaking at 225 rpm for 4 hours.** + + **Note:** + - Meanwhile, place the appropriate number of LB plates with and without selection antibiotics to pre-warm at 28°C. + +17. **Add 4-6 sterile glass beads to each LB plate.** + +18. **Add 10 µL cells to the appropriate plates.** + + **Note:** + - To make spreading this small volume easier, add 40 µL sterile water to the middle of the plate before adding the cells. + - The electroporation efficiency for this protocol is very high: ≥1.6 x 10e8 cfu/µg pCAMBIA1391z DNA (GoldBio) or 1.25x10e5 cfu/µg plasmid (PMID: 29487777). + - Do not add more than 10 µL of cells or you risk overgrowth and a lack of individual colonies. + +19. **Seal and invert the plates and incubate them at 28°C for about 4 days.** + + **Note:** + - Colonies should appear within 4 days. If colonies of appreciable size (2-3 mm) appear earlier than that, continue on with *Sp* transformation. + - Grow any colonies of interest in liquid media and freeze 25% glycerol stocks to avoid needing to re-electroporate *Agro*. +``` +endofoutput +``` + diff --git a/markdown-output/protocol-for-a-nationwide-systematic-review-of-the-ce8fthtn.md b/markdown-output/protocol-for-a-nationwide-systematic-review-of-the-ce8fthtn.md new file mode 100644 index 0000000000000000000000000000000000000000..658faca547c363fd082d24e3053ca2263552ad48 --- /dev/null +++ b/markdown-output/protocol-for-a-nationwide-systematic-review-of-the-ce8fthtn.md @@ -0,0 +1,157 @@ +```markdown +# Goal/Experiment: +The goal of this experiment is to conduct a nationwide systematic review of the Greek literature on child and adolescent mental health. This includes mapping available resources, tracing research priorities, and categorizing data on prevalence surveys, assessment instruments, and interventions for mental health conditions among children and adolescents in Greece. + +# Protocol for a Nationwide Systematic Review of the Greek Literature on Child and Adolescent Mental Health + +## Authors and Affiliations +- **Anastasia Koumoula1** +- **Lauro Estivalete Marchionatti1,2,3 MD** +- **Arthur Caye1,2,3 MD PhD** +- **Vasiliki Eirini Karagiorga1,2** +- **Julia Luiza Schafer1,2,3 PhD** +- **Giovanni Abrahão Salum Júnior1,2,3 MD PhD** + +**Affiliations:** +1. Child and Adolescent Mental Health Initiative (CAMHI), Stavros Niarchos Foundation & Child Mind Institute +2. Child Mind Institute, New York, United States of America +3. Department of Psychiatry, Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre, Brazil + +## Funding +The Stavros Niarchos Foundation. + +## Conflicts of Interest +AC has acted as a consultant for Knight Therapeutics in 2021. All other authors declare no competing interests. + +## Introduction +This is a landscape analysis of scientific literature to map available resources and trace research priorities on child and adolescent mental health in Greece. We describe a nation-wide systematic review of prevalence surveys, assessment instruments, and interventions for mental health conditions among children and adolescents within the country. + +## Methods + +### 2.1 Guidelines +We will follow the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) statement. + +### 2.2 Protocol Registration +This study is being registered after initiation of data extraction. The extraction tables were expected to require adaptations due to the broad scope. The best strategies for data synthesis could not be a priori set, thus the protocol is registered post-extraction. + +### 2.3 Search Strategy +A comprehensive, multi-step search procedure from inception to December 16th, 2021, without language restrictions: + +1. **Databases:** + - Medline (via PubMed) + - Web of Science + - PsycINFO + + Using the following query: + + ```plaintext + BLOCK A: children and adolescents + ((((((((((((((adolesc*[Title]) OR preadolesc*[Title]) OR pre-adolesc*[Title]) OR child*[Title]) OR boy*[Title]) OR girl*[Title]) OR infant*[Title]) OR juvenil*[Title]) OR minors[Title]) OR paediatr*[Title]) OR pediatr*[Title]) OR pubescen*[Title]) OR puberty[Title]) OR school*[Title]) OR student*[Title]) OR teen*[Title]) OR young[Title]) OR youth*[Title]) OR class*[Title]) OR orphan*[Title]) OR high-school*[Title]) OR "high school"[Title]) OR preschool*[Title]) OR pre-school*[Title]) + + + BLOCK B: Greece + (''Greece'' OR ''Greek'') + + + BLOCK C: Mental health + "Mental disorders"[Mesh terms] OR Autism OR Enuresis OR Encopresis OR ADHD OR "Intellectual disability" OR "Mental retardation" OR "Oppositional Defiant Disorder" OR "Conduct Disorder" OR Depression OR "Bipolar" OR "Disruptive Mood Dysregulation Disorder" OR Suicide OR Suicidality OR "Self-harm" OR "Obsessive-compulsive disorder" OR Trauma OR PTSD OR Mutism OR "Substance abuse" OR "Cannabis" OR Alcohol OR "Drug abuse" OR "Anorexia" OR "Bulimia" OR "Eating disorder" OR "Borderline" OR "Personality disorder" OR Schizophrenia OR "Psychosis" OR "Mental health" OR "Quality of life" OR "Well-being" + ``` + +2. **IATAPOTEK Database** + Using corresponding Greek terms for local literature. + +3. **Google Scholar Database** + Using English and Greek terms to reach gray literature and to scan for new inclusions. Independently inspected by two authors till 100 novel studies are found. + +4. **Reference List of Studies** + For snowballing inclusion. + +5. **Local Experts** + For additional references. + +### 2.4 Review Scope +After retrieving a set of studies, they were screened and sorted into the following research areas: +- Prevalence estimates +- Assessment instruments +- Interventions + +### 2.5 Inclusion Criteria + +#### 2.5.1 Prevalence Studies +Inclusion Criteria: +- Surveys on community-based, school-based, or other representative samples focusing on children and adolescents in Greece. +- Studies assessing the prevalence of mental health conditions or symptoms, or quality of life using standard procedures or validated instruments. + +Exclusion Criteria: +- Clinical or non-representative samples (e.g., specific patient groups) +- Conference abstracts + +#### 2.5.2 Instrument Studies +Inclusion Criteria: +- Studies reporting instruments for screening, clinical assessment, or diagnosis related to child and adolescent mental health. +- Studies developing, translating, validating, or applying these instruments in Greece. + +Exclusion Criteria: +- Instruments not relevant to mental health +- Conference abstracts + +#### 2.5.3 Intervention Studies +Inclusion Criteria: +- Studies reporting interventions for mental health conditions or mental health promotion targeting children and adolescents in Greece. +- Experimental designs including randomized clinical trials or studies translating/adapting interventions. + +Exclusion Criteria: +- Conference abstracts + +### 2.6 Screening Process +- **Primary Screening:** + - English terms: two authors independently assess results. + - Greek terms: one native speaker author assesses studies. + +- **Secondary Screening:** + - Single reviewer assesses full-text articles. + - Doubts are discussed within the research team. + - Cross-group inclusions are possible during secondary screening. + +### 2.7 Data Extraction and Synthesis + +#### 2.7.1 Prevalence Studies +Data extracted includes: +- First author, year of publication, study description, region, data collection year, sampling details, age range, gender breakdown, screening and diagnostic sample procedures, diagnostic domain, and prevalence estimates (SD or CI). + +A synthesis table aggregates extracted data, showing regions, number of studies, participants, instruments, informants, lowest/highest prevalence reported. + +#### 2.7.2 Instrument Studies +Using COSMIN guidelines, data extracted includes: +- First author, year of publication, instrument name, diagnostic domain, construct, language, population, and psychometric properties. + +A synthesis table aggregates details about instruments, studies, psychometric properties, and sufficiency ratings. + +#### Coding Criteria: +- "+" for sufficient +- "-" for insufficient +- "?" for indeterminate + +Tables summarize criteria for coding. + +#### 2.7.3 Intervention Studies +Data extracted per Cochrane manual: +- First author, year of publication, sample size, intervention details, outcomes, and methodological design. + +Methodological quality is ascertained using Cochrane risk-of-bias tools for randomized designs, and the Joanna Briggs Institute (JBI) checklist for non-randomized designs. + +### Summary and Extraction Tables: +A summary table details the above data points for easier reference and understanding. + +## References +1. Page MJ, McKenzie JE, Bossuyt PM, et al. The PRISMA 2020 statement. BMJ 2021; 372. DOI: [10.1136/bmj.n71](https://doi.org/10.1136/bmj.n71) +2. Thomas, Brunton, Graziosi. EPPI-Reviewer 4.0. EPPI-Centre Software London 2010. [Reference Details](https://hero.epa.gov/hero/index.cfm/reference/details/reference_id/5915935) +3. Ouzzani M, Hammady H, Fedorowicz Z, Elmagarmid A. Rayyan. Syst Rev 2016; 5: 210. +4. Polanczyk GV, Salum GA, Sugaya LS, Caye A, Rohde LA. Annual research review: J Child Psychol Psychiatry 2015; 56: 345–65. +5. Hoy D, Brooks P, Woolf A, et al. J Clin Epidemiol 2012; 65: 934–9. +6. Prinsen CAC, Mokkink LB, Bouter LM, et al. COSMIN guideline for systematic reviews of patient-reported outcome measures. Qual Life Res 2018; 27: 1147–57. +7. Higgins JPT, Thomas J, Chandler J, et al. Cochrane Handbook for Systematic Reviews of Interventions. John Wiley & Sons, 2019. +8. R Core Team. R. R Foundation for Statistical Computing, Vienna, Austria. [R Project](http://www.r-project.org/) 2013. [R Cited](https://ci.nii.ac.jp/naid/20001692429/) +9. Sterne JAC, Savović J, Page MJ, et al. BMJ. 2019; 366: l4898. +10. Aromataris E, Stern C, Lockwood C, et al. JBI series paper 2. J Clin Epidemiol 2022; [published online April 14]. DOI: [10.1016/j.jclinepi.2022.04.006](https://doi.org/10.1016/j.jclinepi.2022.04.006) + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/protocol-for-bioequivalence-analysis-in-studies-wi-c64fzgtn.md b/markdown-output/protocol-for-bioequivalence-analysis-in-studies-wi-c64fzgtn.md new file mode 100644 index 0000000000000000000000000000000000000000..9c2fb90f40400ca74188b421b2673723872d739b --- /dev/null +++ b/markdown-output/protocol-for-bioequivalence-analysis-in-studies-wi-c64fzgtn.md @@ -0,0 +1,187 @@ +```markdown +# Goal/Experiment: +The aim of this protocol is to perform bioequivalence analysis in studies involving two treatments (design 2x2x2, 2x3x3, and 2x2x4) using the R Studio software. Bioequivalence studies are critical in ensuring that a generic drug is pharmacokinetically equivalent to its branded counterpart. This protocol provides a step-by-step guide to calculating key pharmacokinetic parameters and performing statistical analyses using R Studio. + +# Protocol for Bioequivalence Analysis in Studies with Two Treatments (Design 2x2x2, 2x3x3, and 2x2x4) Using the R Studio Software + +### Authors +- Leandro do Prado Assunção¹ +- Laura Raniere Borges dos Anjos¹ + +¹Instituto de Ensino Estatístico e Científico (IEEC) + +### Date +JAN 08, 2024 + +### DOI +[dx.doi.org/10.17504/protocols.io.q26g7p7d1gwz/v1](https://dx.doi.org/10.17504/protocols.io.q26g7p7d1gwz/v1) + +### Protocol Citation +Leandro do Prado Assunção, Laura Raniere Borges dos Anjos 2024. Protocol for bioequivalence analysis in studies with two treatments (design 2x2x2, 2x3x3, and 2x2x4) using the R Studio software. protocols.io https://dx.doi.org/10.17504/protocols.io.q26g7p7d1gwz/v1 + +### License +This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. + +### Protocol Status +Working (We use this protocol and it's working) + +--- + +## Abstract + +Research and development (R&D) of new drugs require high financial investments and involve risks of failure and long timelines. After the patent period ends for a new active compound, its formula can be shared to create generic versions. However, before producing a generic drug, its bioequivalence with the reference drug must be verified analytically. This involves comparing the pharmacokinetic profiles of both drugs to assess their therapeutic equivalence. This protocol uses free RStudio software to analyze key pharmacokinetic parameters (Cmax, AUC, Tmax) essential for bioequivalence studies. + +--- + +## Materials and Methods + +### Installing the Packages + +1. The default function in R Studio to install packages is `install.packages("Name")`. Run the code: + +```r +install.packages("readxl") +install.packages("dplyr") +install.packages("PKNCA") +install.packages("stringr") +install.packages("ggplot2") +install.packages("openxlsx") +install.packages("nlme") +install.packages("dvmisc") +install.packages("PowerTOST") +install.packages("BE") +install.packages("replicateBE") +``` + +### Loading the Packages + +2. The default function in R Studio to load packages is `library(Name)`. Run the code: + +```r +library(readxl) +library(dplyr) +library(PKNCA) +library(stringr) +library(ggplot2) +library(openxlsx) +library(nlme) +library(dvmisc) +library(PowerTOST) +library(BE) +library(replicateBE) +``` + +### Function for Calculation of Pharmacokinetic Parameters (PK) + +3. Run the code below to define the function `calc_PK`, which calculates pharmacokinetic parameters: + +```r +calc_PK = function(BD, dose){ + colnames(BD) = c("Subject", "Formulation", "Time", "Concentration", "Sequence", "Period") + + BD$Time = as.numeric(BD$Time) + BD$Concentration = as.numeric(BD$Concentration) + BD$Dose = dose + + dat_PK_final = data.frame(subj = c(), auc = c(), cmax = c(), tmax = c(), per = c(), Seq = c(), trat = c()) + qtq_per = as.vector(unique(BD$Period)) + + for(i in 1:length(qtq_per)){ + period = qtq_per[i] + dat1 = filter(BD, Period == period) + sujeitos = data.frame(table(dat1$Subject)) + individuos = as.vector(sujeitos$Var1) + ind = c() + seq = c() + trat = c() + + for(i in 1:length(individuos)){ + dat_st = filter(dat1, Subject == individuos[i]) + ind[i] = individuos[i] + seq[i] = dat_st[1,5] + trat[i] = dat_st[1,2] + } + + seq_trat1 = data.frame(cbind(ind, seq, trat)) + results_PK = calc_PK_period(dat1, unique(dose)) + dat_PK = data.frame(Subject=as.vector(sujeitos$Var1), Period=rep(period,nrow(results_PK)), results_PK) + dat_PK_final = rbind(dat_PK_final, dat_PK) + } + + return(dat_PK_final) +} +``` + +### Example of Application with Hypothetical Data and Dose + +4. Load the example data and apply the `calc_PK` function: + +```r +dat = openxlsx::read.xlsx("dados_hipoteticos.xlsx", sheet = 1) +calc_PK(dat, 10) +``` + +### Function to Calculate Ratio, Confidence Interval (CI) 90%, CVintra and Power TOST + +5. Run the following code to define the `bioequivalencia_f2_V1` function for calculating the required metrics for bioequivalence analysis: + +```r +bioequivalencia_f2_V1 = function(dados, desenho){ + if(desenho == "2x2x2"){ + PK = dados + PK$TRT = as.factor(PK$TRT) + PK$TRT = relevel(PK$TRT, ref="R") + tratR = PK %>% filter(TRT == "R") + tratT = PK %>% filter(TRT == "T") + n1 = length(tratR$SUBJ) + n2 = length(tratT$SUBJ) + + med_geom = function(vetor){ + media_g = round(prod(vetor^(1/length(vetor))),5) + return(media_g) + } + + med_R_auc = med_geom(tratR[,5]) + med_T_auc = med_geom(tratT[,5]) + med_R_cmax = med_geom(tratR[,6]) + med_T_cmax = med_geom(tratT[,6]) + + variabilidade = data.frame( + CI90_AUC = 100*sqrt(exp(as.numeric(var_beta_treated_AUC))-1), + CI90_cmax = 100*sqrt(exp(as.numeric(var_beta_treated_cmax))-1) + ) + + Model_AUC = lme(log(AUClast) ~ TRT + GRP + PRD, random=~1|SUBJ, data=PK) + Model_Cmax = lme(log(Cmax) ~ TRT + GRP + PRD, random=~1|SUBJ, data=PK) + + return(variabilidade) + } +} +``` + +### Applying the Function in the NCAResult4BE Data of the BE Package + +6. Command to save, load, and apply the function: + +```r +write.table(NCAResult4BE, "dat_test.csv", row.names = FALSE, sep = ";", dec = ";") +dat_test = read.table("dat_test.csv", header = TRUE, sep = ";", dec = ";") +be2x2(dat_test, c("AUClast", "Cmax", "Tmax")) +``` + +--- + +## Glossary of Professional/Scientific Terms + +- **Cmax**: Maximum plasma concentration of a drug after administration. +- **AUC (Area Under the Curve)**: Integral of the concentration-time curve (after a single dose or in steady state). +- **Tmax**: Time to reach the maximum plasma concentration after drug administration. +- **Bioequivalence**: A term referring to the comparison between two drugs that should show similar bioavailability and pharmacokinetic profiles. +- **GRP (Group)**: Grouping factor in data analysis, often related to study design factors such as treatment. +- **PRD (Period)**: Time period in a crossover study design during which a particular treatment is administered. + +### Alternative Methods +- If RStudio packages are not available, consider using alternative statistical software like SAS or SPSS, which provide similar functionalities for pharmacokinetic and bioequivalence analysis. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/protocol-for-dna-extraction-from-urine-wgsfbwe.md b/markdown-output/protocol-for-dna-extraction-from-urine-wgsfbwe.md new file mode 100644 index 0000000000000000000000000000000000000000..f6d5ff4460f1d37a53766648bb6911fca8334586 --- /dev/null +++ b/markdown-output/protocol-for-dna-extraction-from-urine-wgsfbwe.md @@ -0,0 +1,113 @@ +```markdown +# Goal/Experiment: +The goal of this experiment is to develop an optimized protocol for DNA extraction from urine samples, which efficiently isolates DNA representative of the microbial community, including fungi. This protocol is focused on modifications to commonly-used DNA extraction procedures for enhanced microbial DNA yields and diversity, tested using next-generation sequencing (NGS). + +# Protocol for DNA Extraction from Urine + +**Authors**: +A. Lenore Ackerman†, Jennifer Tash Anger†, Muhammad Umair Khaliqu‡, James E. Ackerman†, Jie Tang†, Jayoung Kim†, David M. Underhill†, Michael R. Freeman† +†Department of Surgery, Division of Urology, Cedars-Sinai Medical Center, +‡Department of Biomedical Sciences, Cedars-Sinai Medical Center + +**DOI**: [dx.doi.org/10.17504/protocols.io.wgsfbwe](dx.doi.org/10.17504/protocols.io.wgsfbwe) + +## Abstract + +### Introduction +Recent data suggest the urinary tract hosts a microbial community with varying compositions, even in the absence of infection. Using culture-independent methodologies, such as NGS of conserved ribosomal DNA sequences, this protocol aims to identify both common commensals and fastidious organisms. A fundamental challenge has been isolating DNA representative of the entire resident microbial community, including fungi. + +### Materials and Methods +Modifications to commonly-used DNA extraction methods were evaluated using standardized male and female urine samples. These modifications were combined for systematic confirmation of optimal conditions and tested against commercially available methodologies to compare overall and microbial DNA yields and diversity by NGS. + +### Results +Overall and fungal-specific DNA yields significantly improved with the right set of protocol modifications, particularly through the combination of enzymatic, mechanical, and thermal disruptions, alongside proteinase digestion. + +### Conclusions +Alterations in urine storage, preparation, and DNA processing enhance microbial community profiling via culture-independent sequencing methods. This optimized protocol facilitates concurrent evaluation of bacterial and fungal populations. + +## Protocol Status +**Working**: +This protocol is currently in use and operational. + +## Guidelines +This protocol is best conducted in a positive pressure hood. + +## Materials + +| Name | Catalog # | Vendor | +|----------------------------------|--------------|-----------------------| +| 0.1 mm Zirconia/Silica Beads | 11079101z | Bio Spec Products Inc.| +| 0.5 mm Zirconia/Silica Beads | 11079105z | Bio Spec Products Inc.| +| 1.5 mL Eppendorf tubes | - | - | +| Proteinase K | EO0491 | Thermo Fisher Scientific | +| Lysozyme | 12671-19-1 | Sigma Aldrich | +| QIAamp® Fast DNA Stool Mini Kit | 51604 | Qiagen | +| Lyticase | L2524-50KU | Millipore Sigma | + +## Materials Text + +- **Enzyme Buffer**: (0.5 M Tris, 1 mM EDTA, 0.2% 2-mercaptoethanol, pH 7.5) +- **Stratec Stool DNA Stabilizer**: #1038111100, 250 mL +- **Poly (A) Polyadenylic Acid**: Sigma-Aldrich #10108626001 (5uL/mL Buffer AL) + +## Safety Warnings +- Wear gloves, lab coat, and eye protection. + +## Before Starting + +1. Set heat blocks at 30°C and 70°C. +2. Label tubes. +3. Get ice and place enzymes on ice. + +## Procedure + +### Centrifugation and Enzymatic Disruption + +1. Spin 30 mL for 15 minutes at 1500 Xg at room temperature. +2. Pour off supernatant into a separate 50 mL tube, store at -80°C. +3. Resuspend pellets in 500 μL Enzyme Buffer in a 1.5 mL Eppendorf tube. +4. Add enzymes: 100 μL Lyticase (2000 U/mL) and 100 μL Lysozyme (100 mg/mL). +5. Incubate at 30°C for 30 minutes, inverting every 5-10 minutes. +6. Centrifuge at 4000 rcf for 5 minutes. +7. Remove supernatant. + +### Mechanical Disruption + +8. Resuspend pellet in 800 μL Stool DNA Stabilizer. +9. Add beads: 100 μL 0.1 mm beads and 300 μL 0.5 mm beads. +10. Bead beat on high for 1 minute. + +### Thermal Disruption + +11. Spin briefly at 17,000 Xg. +12. Bead beat on high for 1 minute again. +13. Heat at 95°C for 5 minutes. +14. Vortex for 5 seconds. +15. Heat at 95°C for another 5 minutes. +16. Vortex for 5 seconds. +17. Cool on ice for 5 minutes. +18. Centrifuge at 17,000 Xg for 1 minute. + +### DNA Extraction + +19. For each sample, add 350 μL to each of 2 1.5 mL tubes. +20. Add 10 μL proteinase K mixture to each sample. +21. Add 250 μL Buffer AL + Carrier [Poly (A)] to each sample. +22. Vortex for 15 seconds. +23. Incubate at 70°C for 10 minutes. +24. Add 250 μL 100% EtOH to each sample. +25. Mix by vortexing for 5 seconds. +26. Transfer mixture from first tube to QIAamp Mini Spin Column, centrifuge at 17,000 Xg for 1 minute. +27. Change collection tube, transfer mixture from second tube to spin column, centrifuge. +28. Change collection tube, add 500 μL Buffer AW1 to column. +29. Centrifuge at 17,000 Xg for 1 minute. +30. Change collection tube, add 500 μL AW2 to the column. +31. Centrifuge at 17,000 Xg for 3 minutes. +32. Remove residual EtOH by centrifuging for an additional 1 minute at max speed. +33. Transfer column to new 1.5 mL Eppendorf tube. +34. Add 50 μL Buffer AE to column, incubate at room temperature for 5 minutes, and spin at 17,000 Xg for 1 minute. +35. Transfer fluid from the collection tube back to the spin column, repeat the last step. +36. Measure DNA concentration on Nanodrop 2000 Spectrophotometer. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/protocol-for-edna-extraction-within-sterivex-capsu-c63bzgin.md b/markdown-output/protocol-for-edna-extraction-within-sterivex-capsu-c63bzgin.md new file mode 100644 index 0000000000000000000000000000000000000000..e5e9f914fc732970323bd283e64cc5b5ff38dde6 --- /dev/null +++ b/markdown-output/protocol-for-edna-extraction-within-sterivex-capsu-c63bzgin.md @@ -0,0 +1,78 @@ +```markdown +# Goal/Experiment: +The goal of this protocol is to extract environmental DNA (eDNA) from STERIVEX filters using a modified method based on the protocol published by Spens et al. 2017. This method is adapted to optimize eDNA yield from freshwater samples in Chilean Patagonia. + +# Protocol for eDNA extraction within STERIVEX capsule based on Spens et al. 2017 + +## Authors +`delphine.vanhaecke¹` +¹Universidad de Aysén + +`delphine.vanhaecke` + +## Abstract +This protocol for eDNA extraction from STERIVEX filters within the capsule is modified from the protocol published by Spens et al. 2017. (DOI:[10.1111/2041-210X.12683](https://doi.org/10.1111/2041-210X.12683)) based on the DNeasy® Blood & Tissue Kit (QIAGEN, Stockach, Germany) handbook pp. 28-30, ver. 07/2006 (Qiagen®). This protocol has provided the highest concentrations of eDNA, quantified by QUBIT, from freshwater samples in Chilean Patagonia compared to other tested protocols. + +## DOI +[10.17504/protocols.io.eq2lyj58rlx9/v1](https://dx.doi.org/10.17504/protocols.io.eq2lyj58rlx9/v1) + +## License +This is an open access protocol distributed under the terms of the [Creative Commons Attribution License](https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. + +## Protocol Status +Working. We use this protocol and it's working. + +## Created +Jan 05, 2024 + +## Last Modified +Jan 08, 2024 + +## Protocol Integer ID +92995 + +## Keywords +eDNA extraction, Sterivex filter, QIAGEN + +## Funders Acknowledgement +Gobierno Regional de Aysén +Grant ID: FIC 2022 código BIP 40049315-0 + +## DAY 1 + +1. Clean the laminar flow hood and micropipettes with DNAZAP, then wipe with ddH₂O and 70% ethanol using tissue paper. +2. Preheat incubator to 56°C. +3. Prepare 10 mL of molecular grade ethanol (99.9%) in a 25 mL sterile Eppendorf tube and place at -20°C. +4. Place gloves, sleeves, 1.5 mL (8) and 2.0 mL (24) sterilized LoBind tubes in the laminar flow hood and expose for 10 min to UV light. (1.5 mL tubes for receiving eDNA eluate, 2.0 mL tubes for storing the EtOH buffer in the STERIVEX filter and 2.0 mL tubes for receiving the Lysis mix for eDNA extraction after the 56°C heating step). +5. Carefully wipe the outer surfaces of all the filter capsules with 5% bleach using clean tissue paper. Dry and wipe with 70% ethanol using tissue paper. +6. Remove the yellow inlet caps from the capsule and transfer the buffer from the filter capsule into a 2 mL sterile LoBind tube using a 3 mL Luer-Lock syringe. Be careful not to apply too much pressure. Place the filters vertically in a tube rack inside a fume hood to dry, with the inlet side (yellow cap side) facing down. Let them blot on clean laboratory tissue paper placed underneath the rack. +7. Label and store the EtOH in the LoBind tubes at -80°C for future analysis as EtOH buffer also contains eDNA from the sample. +8. After removal of the ethanol buffer, consider the SX capsules as test tubes. The filter will remain intact in the capsules to avoid loss of DNA and contamination risk by unnecessary handling. +9. Immediately before the lysis step, prepare a premix of Lysis working solution by adding 900 µL ATL buffer and 100 µL proteinase K per sample provided by the kit in a 10 mL sterilized tube. Add 1 mL lysis premix to each sample. Process 7 Sterivex filters and 1 negative DNA extraction control at a time (max. of 8 samples at once). For 8 samples, prepare lysis mix: 900*9 (8+1)= 8100 µL ATL buffer and 100*9 (8+1)=900 µL Proteinase K. +10. Keep the outlet end closed with the red outlet cap. Carefully add 1000 µL Lysis working solution to the filter using a 1,000 µL micropipette and sterile filter tips. Pipet the solution between the outside of the filter and the capsule walls. Close with a yellow inlet cap and seal with parafilm. +11. Handshake vigorously for a few seconds. Transfer the Sterivex filter cartridge to a sterile Petri dish. +12. Place the Sterivex filters in Petri dishes in an incubator preheated at 56°C for 24 hours without rotating. Handshake filters vigorously in between. + +## DAY 2 + +13. Preheat thermomixer to 70°C. +14. Handshake SX filter capsules vigorously before processing. +15. Remove ALL the liquid from the yellow inlet end of the capsule using a 2 mL Luer-Lock syringe. Measure the volume, transfer to two separate 2 mL LoBind tubes (sample 1a and 1b - each with e.g. 500 µL of lysis mix). Vortex for a few seconds. Spin down for 2 seconds to seed out excess debris. +16. Add Buffer AL and ice-cold molecular grade 99% ethanol (Thermo Fisher Scientific, Waltham, MA, USA) to the sample in equal volumes. Buffer AL and ethanol can be premixed. Sample:Buffer:Ethanol = 1:1:1. e.g. 500 µL lysis mix + 1000 µL AL+EtOH mix (or 500 µL EtOH + 500 µL AL buffer). +17. Vortex vigorously. +18. Pipet the mixture (max 600 µL at a time) into a DNeasy Mini Spin column in a 2 mL collection tube provided in the kit. +19. Spin in microcentrifuge preferably at 4°C at 8000 rpm for 1 min. +20. Discard flowthrough. +21. Repeat the previous 3 steps until all sample is filtered through the DNeasy Mini Spin column. +22. Place the DNeasy Mini Spin column in a new 2 mL collection tube (provided), add 500 µL Buffer AW1, and centrifuge for 1 min at 8,000 rpm. Discard flowthrough and collection tube. +23. Place the DNeasy Mini Spin column in a new 2 mL collection tube (provided), add 500 µL Buffer AW2, and centrifuge for 3 min at 14,000 rpm to dry the DNeasy membrane. In the meantime, label the 8 1.5 mL LoBind tubes. +24. Transfer the spin column to a new 1.5 mL DNA LoBind tube with caps and walls labeled. +25. Heat AE buffer to 70°C on a thermomixer. Place tubes with spin columns, four at a time, on a 70°C heating plate, and add 75 µL 70°C Buffer AE (pH 8.0) to the membrane. Immediately remove the spin column with filter from the thermomixer and let it stand at Room Temperature for 10 minutes. +26. Centrifuge for 1 min at 8,000 rpm. Discard the spin column. +27. Aliquot 30 µL of the eluate eDNA in separate labeled PCR tubes for DNA measurement and PCR, and store in a dark room refrigerator at 4°C for immediate use (max. 6 months). +28. Store the other 45 µL of the eluate eDNA (STOCK) at -80°C for long-term storage. + +--- + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/protocol-for-intracellular-recording-from-mouse-in-cczdsx26.md b/markdown-output/protocol-for-intracellular-recording-from-mouse-in-cczdsx26.md new file mode 100644 index 0000000000000000000000000000000000000000..23f623479d356d7f74524371a2626c10c28ae710 --- /dev/null +++ b/markdown-output/protocol-for-intracellular-recording-from-mouse-in-cczdsx26.md @@ -0,0 +1,112 @@ +```markdown +# Goal/Experiment: +The goal of this experiment is to isolate and perform intracellular recording from mouse intrinsic cardiac neurons to assess the membrane properties and electrophysiological characteristics of these neurons. + +# Protocol for Intracellular Recording from Mouse Intrinsic Cardiac Neurons + +## Author +John D Tompkins +Cardiac Arrhythmia Center, Neurocardiology Research Program of Excellence + +## DOI +[dx.doi.org/10.17504/protocols.io.81wgb6ep3lpk/v1](https://dx.doi.org/10.17504/protocols.io.81wgb6ep3lpk/v1) + +## Abstract +A basic protocol for the isolation and intracellular recording from mouse intrinsic cardiac neurons is presented. The intrinsic cardiac ganglia, containing hundreds of intrinsic cardiac neurons (ICNs), lie on the dorsal epicardial surface of the mouse heart. These cells receive innervation principally from preganglionic parasympathetic neurons of the brainstem by axonal projections within the vagus nerve. These neurons project to multiple targets within the heart (e.g., nodal cells, vascular smooth muscle, myocardium) to affect cardiac dynamics (e.g., heart rate, contractility). This protocol was used for assessing the membrane properties of ICNs sampled from both control and diabetic mice as presented in the published manuscript (PMID: 31625779) and accompanying dataset. + +## Protocol Citation +John D Tompkins 2022. Protocol for intracellular recording from mouse intrinsic cardiac neurons. *protocols.io*. +[dx.doi.org/10.17504/protocols.io.81wgb6ep3lpk/v1](https://dx.doi.org/10.17504/protocols.io.81wgb6ep3lpk/v1) + +## Funders Acknowledgement +NIH Common Fund SPARC Grant +Grant ID: OT2OD023848 + +## Manuscript Citation +Jungen C, Scherschel K, Flenner F, Jee H, Rajendran P, De Jong KA, Nikolaev V, Meyer C, Ardell JL, Tompkins JD. Increased arrhythmia susceptibility in type 2 diabetic mice related to dysregulation of ventricular sympathetic innervation. *Am J Physiol Heart Circ Physiol*. 2019 Dec 1;317(6):H1328-H1341. +doi: 10.1152/ajpheart.00249.2019. Epub 2019 Oct 18. PMID: 31625779; PMCID: PMC6962614. + +## Keywords +- Intracellular recording +- Intrinsic cardiac neuron +- Epicardial ganglia +- Atrial ganglion +- Diabetes + +## License +This is an open access protocol distributed under the terms of the [Creative Commons Attribution License](https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. + +## Protocol Details + +### Isolation of Intrinsic Cardiac Ganglia +1. **Sacrifice Mice**: + - Adult mice (M/F; 12±2 weeks of age) are sacrificed under deep isoflurane (5%) anesthesia by cervical dislocation and exsanguination. + +2. **Prepare Thorax**: + - The thorax is removed and placed in ice-cold physiologic salt solution (PSS) containing in mM: + - 121 NaCl + - 5.9 KCl + - 1.2 NaH2PO4 + - 1.2 MgCl2 + - 25 NaHCO3 + - 2 CaCl2 + - 8 D-glucose + - pH 7.4 maintained by 95% O2-5% CO2 aeration. + +3. **Remove Heart**: + - The heart is removed, purged of blood, and pinned to the Sylgard (Dow Corning) floor of a petri dish while continuously superfused with fresh ice cold PSS. + +4. **Dissect Neurons**: + - Clusters of epicardial neurons in ganglia on the dorsal epicardial surface of the heart, near the pulmonary veins, are visualized with a stereomicroscope and carefully dissected from the underlying atrial myocardium using fine forceps (Dumont #7) and iridectomy scissors. + +5. **Pin Isolated Ganglia**: + - The isolated ganglia are pinned to the Sylgard (Dow Corning) floor of a glass bottom petri dish. The dish is secured to a custom XY linear stage and ganglia are observed using an upright microscope (AxioExaminer, Zeiss) equipped with a 5X-dry and a 40X-water-immersion objective, differential interference contrast optics, and monochrome camera (AxioCam, Zeiss). + +6. **Stimulate Nerves**: + - Concentric bipolar stimulation electrodes (FHC) are placed on interganglionic nerves to evoke ortho- or antidromic action potentials at intrinsic cardiac neurons. + +### Intracellular Microelectrode Recording +7. **Superfuse Ganglia**: + - Isolated ganglia are superfused continuously (6–7 ml/min) with PSS maintained at 32–35°C with a thermostatically controlled heater. + +8. **Identify and Impale Neurons**: + - Neurons are visually identified (40X water-immersion objective) and impaled with borosilicate-glass microelectrodes filled with either 2M KCl (60–120 MΩ) or 2M KCl + 2% Neurobiotin (80-160 MΩ; Vector Labs). + +9. **Record Membrane Voltage**: + - Membrane voltage is recorded using a Multiclamp 700B amplifier and headstage connected to a Digidata 1550B data acquisition system. + +10. **Use pCLAMP Software**: + - pCLAMP 10 software (Molecular Devices, CA) is used for acquisition and analysis of time series data. + +11. **Inject Intracellular Current**: + - Intracellular current injected through the recording electrode is used to characterize membrane physiology. + +12. **Depolarizing Current Steps**: + - Depolarizing current steps (0.1–0.5 nA, ∆100 pA, 500 ms duration) are used to assess neuronal excitability. + +13. **Classify Cells**: + - Cells are classified as either phasic (<3 APs) or non-phasic (>2 APs) based on the maximum number of action potentials elicited by the depolarizing current. + +14. **Hyperpolarizing Current Steps**: + - Hyperpolarizing current steps (500 ms) of decreasing amplitude (-0.4 to -0.1 nA, ∆100 pA) are used to test for rectification in the current-induced hyperpolarization, occurring when hyperpolarization-activated currents are initiated, and to measure whole-cell input resistance. + +15. **Measure Action Potential**: + - The amplitude and duration of the action potential are measured from either a spontaneous or a nerve-evoked spike. + +16. **After-hyperpolarization**: + - After-hyperpolarization amplitude and duration are measured from brief intracellular current pulses (0.1-0.8 nA, ∆100 pA, 5 ms) or spontaneous action potentials. + +17. **Inclusion Criteria**: + - Inclusion criteria for analysis include a resting membrane potential less than or equal to -45mV, a holding current of greater than or equal to -100pA, and the cell must be excitable (cells with no action potential are excluded). + +18. **Stimulus Shocks**: + - Graded stimulus shocks (100 µs) are delivered from the concentric bipolar electrodes in 50-100 µA steps, from 0 to 800 µA, to generate stimulus recruitment curves (Master 8 and IsoFlex optical isolation unit, AMPI). + +19. **Deliver Stimuli**: + - Five to 20 stimuli are delivered at each stimulus intensity, with an interval of 3 seconds between stimuli. + +20. **Analyze Synaptic Events**: + - Analysis of synaptic events focuses on latency of the excitatory post-synaptic potential (EPSP), measured from the start of the stimulus trigger to the beginning of the EPSP (V≧2xRMS), and jitter (SD of latency) as indices of conduction, path, and release. + +`endofoutput` +``` \ No newline at end of file diff --git a/markdown-output/protocol-for-mass-capturing-handling-and-fitting-t-bmapk2dn.md b/markdown-output/protocol-for-mass-capturing-handling-and-fitting-t-bmapk2dn.md new file mode 100644 index 0000000000000000000000000000000000000000..521a879ebaf1e122508a67bb07194df9059ce988 --- /dev/null +++ b/markdown-output/protocol-for-mass-capturing-handling-and-fitting-t-bmapk2dn.md @@ -0,0 +1,118 @@ +```markdown +# Goal/Experiment: +The goal of this experiment is to provide detailed protocols for mass capturing, handling, and fitting tracking devices and patagial tags on vultures. The protocols are designed to minimize injury and stress to the birds while allowing for efficient data collection for conservation purposes. + +# Protocol for Mass Capturing, Handling, and Fitting Tracking Devices and Patagial Tags on Vultures + +**Authors**: M. T. Hirschauer, W. Neser, K. Wolter +**Published**: Sep 17, 2020 +**DOI**: [https://dx.doi.org/10.17504/protocols.io.bmapk2dn](https://dx.doi.org/10.17504/protocols.io.bmapk2dn) + +## Abstract +VulPro, a vulture conservation organization in South Africa, has contributed to vulture research and rehabilitation since 2007. This document details VulPro's methods for mass capturing using a specially designed walk-in trap, handling, weighing, collecting blood samples, and fitting patagial tags and tracking devices on vulture species. + +## Guidelines + +### Handling Protocols +For the average biologist or bird ringer, handling a vulture may be intimidating or even dangerous. Key principles include minimizing the risk of injury or stress to the bird and avoiding excessive force. These handling methods are accepted as the standard by the National Society for the Protection of Animals (NSPCA) in South Africa. + +### Catching Vultures in an Enclosure +- Vultures need to be approached quietly and sympathetically. Do not rugby tackle the birds. +- Include no more than three persons in the capturing process. +- Do not grab the neck lower than the jawbone as the bird can turn its head around and bite. +- Hug the bird’s wings as it attempts to escape. +- Keep control of the bird’s head properly to prevent injury to the handler and the bird. +- Ensure safe handling to avoid overheating. + +### Processing Vultures +- Involves any handling of vulture: placement of patagial tags, fitting tracking devices, or taking blood. +- Environmental conditions must be considered and processed quickly to minimize handling time (maximum 45 minutes). + +#### Processing Vultures on a Table +- Use four people: one holds the head, the second and third secure wings, and the fourth positions the bird. +- Always allow the bird's head to move freely for regurgitation during excessive stress. + +### Releasing +- Release the bird on ground level and allow it to recover. +- Avoid encouraging the bird to exit the crate until it is ready. + +### Tag Placement +- Use correct placement guidelines to avoid severe damage to patagium. +- Sanitize area and equipment with F10 or similar. +- Ensure placement does not move, and the piercing is done correctly. + +### Tracking Device Fitting: The Backpack Harness +- Consider the bird’s age and health condition before fitting. +- Use team effort and correct equipment to ensure proper fitting. +- Check tightness regularly and ensure no parts of the device can injure the bird. + +## Materials and Equipment + +### Basic Materials +- Eye and face protection +- Gloves +- Long-sleeved shirt +- Trousers and closed shoes +- A sturdy folding table +- Carpet-covered vulture crate + +### Materials for Tracking Device Fitting +- Teflon tape (4 to 6 mm wide) +- Silicone tubing (3.1 mm or 4.7 mm wide) +- Dental floss +- Super glue +- Epoxy glue +- Baby powder +- Metal rings for clamping +- Sharp-nosed thick scissors +- Needle for threading and sewing dental tape +- Ringing pliers +- Scrap piece of cardboard +- Thin stick to mix the epoxy + +### Length Guide for Teflon® Material and Tubing +- **Cape vultures**: 2m Teflon®, 160cm silicone tubing +- **White-backed and Lappet-faced vultures**: 1.5m Teflon®, 120cm silicone tubing +- **Hooded vultures**: 1.2m Teflon®, 1m silicone tubing + +## Safety Warnings +Follow the Safety Data Sheet (SDS) for all hazards and warnings. + +## Before Starting + +**Background of VulPro**: +VulPro is a pNPOvulture conservation organization established in 2007, focusing on Cape vulture breeding, research, rehabilitation, public education, and managing vulture feeding sites. Directors and staff have over 35 years of experience handling vultures and other large raptors. + +### Walk-in Trap Construction and Use +- Constructed with 40/50mm diamond mesh, proper shading, and a secure pedestrian gate. +- Use a curtain system to herd vultures inside without causing injury or stress. +- Ensure there are no sharp points inside or outside the enclosure. + +### Tag Placement Procedure +1. Hold the wing to see the patagium bend clearly. +2. Avoid feather follicles and blood vessels. +3. Thoroughly sanitize the area. +4. Place the male piercing on the bottom side of the wing. +5. Hold the tag dorsal surface above the piercing site. +6. Make the piercing at least 1 cm from the leading edge and 3 cm from the bend. + +## Detailed Procedures + +### Tracking Device Fitting: Backpack Harness +1. **Preparation**: + - Assess the bird's condition. + - Connect tracking device to harness. + +2. **Procedure**: + - Thread Teflon® and silicone tubing. + - Use gentle tension adjustments. + - Securement of device centrally between vulture's shoulders. + - Final crimp adjustments and glue applications for secure fit. + +3. **Post-Fitting**: + - Allow the bird to adapt and monitor closely for any issues. + +--- + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/protocol-for-single-cell-and-single-nucleus-prep-f-cx6xxrfn.md b/markdown-output/protocol-for-single-cell-and-single-nucleus-prep-f-cx6xxrfn.md new file mode 100644 index 0000000000000000000000000000000000000000..21d037a43f61b1617fa26b71949d1f85b738467f --- /dev/null +++ b/markdown-output/protocol-for-single-cell-and-single-nucleus-prep-f-cx6xxrfn.md @@ -0,0 +1,120 @@ +```markdown +# Goal/Experiment: +This protocol involves the preparation of single-cell and single-nucleus suspensions from cell pellets, specifically for applications in scRNA-seq and snATAC-seq in the context of studying Multiple Myeloma. The preparations are to ensure proper isolation and quality of cells and nuclei for following genomic analyses. + +# Protocol for Single Cell and Single Nucleus Prep for Cell Pellets/Multiple Myeloma (snRNA & snATAC) +**Reyka Jayasinghe** +*Washington University in St. Louis* +*Created: Aug 03, 2023* +*Protocol ID: 85943* + +## ABSTRACT +Protocol for single cell and single nucleus preparation for Cell Pellets/Multiple Myeloma (snRNA & snATAC). + +## Reagents and Protocols + +### 1. General Information +All lysis buffers should be prepared fresh and stored at 4°C. + +Buffers are based on the *Nuclei Isolation from Mouse Brain Tissue for Single Cell ATAC Sequencing Protocol from 10X*. + +Relevant 10X Protocols: +- [Nuclei Isolation for Single Cell ATAC Sequencing](https://support.10xgenomics.com/single-cell-atac/sequencing/doc/user-guide/sample-preparation-for-single-cell-atac-sequencing-tissues-4) +- [Nuclei Isolation from Mouse Brain Tissue for Single Cell ATAC Sequencing Protocol](https://assets.ctfassets.net/an68im79xiti/5PPbfySbwEyb6FzuTK2ofT/77dd4e690142d774d3468b234247dcb8/CG000229_ChromiumNextGEMSingleCellATACV_July2019.pdf) +- [Chromium Single Cell ATAC Reagent Kits User Guide (v1 Chemistry)](https://assets.ctfassets.net/an68im79xiti/5wqANGPAd2clyMNonxdJ33/707827a2fa1f48bc6b7a46304769791f/CG000052_ChromiumNextGEMSingleCellATACV_July2019.pdf) +- [Chromium Single Cell 3' Reagent Kits User Guide (v3.1 Chemistry)](https://support.10xgenomics.com/single-cell-gene-expression/sequencing/doc/user-guide/sample-preparation-single-cell-3-reagent-kits-v3-chemistry) + +### 2. Equipment +- Eppendorf DNA LoBind Tubes, 2.0 mL (022431048) +- Tips RT-LTS-A-1000uL-/L/S/W-768/8 (30389221) +- Flowmi® Cell Strainers, 40 µm, for 1000 uL Pipette Tips (BAH136800040) +- Sorvall™ ST 8 Small Benchtop Centrifuge (75007204 - Refrigerated) +- Thermo Scientific™ Adapters for TX-150 Swinging Bucket Rotors (75-005-743) + +### 3. Reagents to Order for Lysis and Wash Buffers +- UltraPure™ 1 M Tris-HCl Buffer, pH 7.5 (Invitrogen, 15567027) +- NaCl (5 M), RNase-free (Invitrogen, AM9759) +- MgCl2 (1 M) (Invitrogen, AM9530G) +- Nonidet P40 Substitute (Sigma, 74385-1L) +- MACS BSA Stock Solution (Miltenyi Biotec, 130-091-376) +- Phosphate Buffered Saline (1X) (Corning, 21-040-CM) +- SUPERase• In™ RNase Inhibitor (20 U/μL) (Invitrogen, AM2696) + +### 4. Buffers + +#### 4.1. Stock Lysis Buffer +| Reagent | Stock | Final | 2 mL | +|------------------------|---------|-------|-------------| +| Tris-HCl (pH 7.4) | 1M | 10 mM | 20 μL | +| NaCl | 5M | 10 mM | 4 μL | +| MgCl2 | 1M | 3 mM | 6 μL | +| Nonidet P40 Substitute | 10% | 0.1% | 3 μL | +| Molecular Grade Water | - | - | 1.974 mL | + +#### 4.2. Lysis Dilution Buffer +| Reagent | Stock | Final | 10 mL | +|------------------------|---------|-------|-------------| +| Tris-HCl (pH 7.4) | 1M | 10 mM | 100 μL | +| NaCl | 5M | 10 mM | 20 μL | +| MgCl2 | 1M | 3 mM | 30 μL | +| Molecular Grade Water | - | - | 9.85 mL | + +#### 4.3. 0.1X Lysis Buffer +| Reagent | Stock | Final | 10 mL | +|------------------------|---------|-------|-------------| +| 1X Lysis Buffer | 1X | 0.1X | 1 mL | +| Lysis Dilution Buffer | 1X | 0.9X | 9 mL | + +#### 4.4. Wash Buffer: 1X PBS + 2% BSA + 0.2 U/μL RNase Inhibitor +| Reagent | Stock | Final | 10 mL | +|------------------------|---------|-------|-------------| +| BSA | 10% | 2% | 2 mL | +| PBS | 1X | 1X | 8 mL | +| RNase Inhibitor | 20 U/mL | 0.2 U/μL | 1 mL | + +### 5. Additional Buffers + +#### 5.1 sn-ATAC-Seq Submission Buffer: 1X Nuclei Dilution Buffer +- Store aliquots of 20X Nuclei Dilution Buffer at -20ºC in aliquots of no more than 25 μL/each tube to minimize freeze/thaw. Prepare fresh 1X Nuclei Dilution Buffer each time by adding 425 μL Nuclease Free Water. + +#### 5.2 Trypan Blue (2X), filtered at 0.22 mm. + +### 6. General Notes +- Keep everything on ice. +- Use wide-bore pipette tips for all steps when possible. +- Use RNase free reagents and consumables (Use filtered tips). +- For centrifugation steps, use swinging rotor bucket at 4ºC. + +### 7. Preparing Sample for Auto-MACS Dead Cell Removal +1. Add 5 mL running buffer to tube. +2. Thaw aliquot in 37ºC water bath. +3. Spray with 70% EtOH and wipe down. +4. Add 1 mL thaw media to each aliquot and transfer to running buffer tube. +5. Remove supernatant. +6. Re-suspend each in 100 μL of beads and incubate at room temperature for 15 minutes. +7. Dilute 0.25 mL of 20X binding buffer with 4.75 mL of distilled water. +8. Add 500 μL of 1X binding buffer to each tube. +9. Run each through the DepleteS selection on the Automacs. +10. Discard the positive fractions. +11. Pull off 10 μL each from negative fractions to count. +12. Proceed downstream with negative fractions, cells still suspended in buffer. + +### 8. Single Cell and Nucleus Prep for sc-RNA-Seq and sn-ATAC-Seq +1. Spin down cells at 400 g for 5 minutes. Remove supernatant. +2. Resuspend in 1000 μL Wash Buffer. +3. Spin down cells at 400 g for 5 minutes. Remove supernatant. +4. Re-suspend in 200-1000 μL Wash buffer depending on size of the cell pellet with RNase Inhibitor. Count cells. Take 10 μL trypan blue and 10 μL of sample to determine starting concentration of sample. If there are more than 1500 cells/μL, add more wash buffer and recount. +5. Aliquot cell suspension to load approximately 10,000 cells/μL (refer to 10X 3' NEXT GEM protocol, Figure 2). Set aside another 50-100 μL of cell suspension for backup. Centrifuge remaining sample at 400 g for 8 minutes at 4ºC. +6. While remaining cell suspension is being spun down, load 10X CHIP G (single-cell). After loading chip you have 17 minutes to process remaining sample for sn-ATAC. +7. After spin is finished, resuspend cells in 500 μL of cold lysis buffer. Pipette gently for 25 times (until pellet is fully resuspended). Incubate on ice for 2 minutes. +8. Add 400 μL Wash Buffer to same tube. +9. Transfer the clear lysis-wash buffer suspension using the 40 μm Flowmi Cell Strainer into a 2.0 mL tube. Centrifuge sample at 400 g for 5 minutes at 4ºC. +10. Once the sample is finished spinning, remove as much supernatant as possible. The pellet should be at the very bottom of tube if using ST8 Centrifuge. Add 7-10 μL 1X Nuclei Dilution Buffer (sn-ATAC-Seq submission buffer). Gently resuspend until nuclei are completely resuspended. Count nuclei: Add 8 μL 1X Nuclei Dilution Buffer to 2 μL of sample and 10 μL of trypan blue. If nuclei concentration is greater than 3,000 nuclei/μL, proceed with ATAC prep. +11. Continue both 10X protocols accordingly. + a. Chromium Single Cell ATAC Reagent Kits User Guide (v1 Chemistry) + b. Chromium Single Cell 3' Reagent Kits User Guide (v3.1 Chemistry) + +--- + +[endofoutput] +``` \ No newline at end of file diff --git a/markdown-output/protocol-of-a-systematic-review-with-meta-analysis-b4x6qxre.md b/markdown-output/protocol-of-a-systematic-review-with-meta-analysis-b4x6qxre.md new file mode 100644 index 0000000000000000000000000000000000000000..d7b706619df11d8f811a749489d14df110009690 --- /dev/null +++ b/markdown-output/protocol-of-a-systematic-review-with-meta-analysis-b4x6qxre.md @@ -0,0 +1,91 @@ +```markdown +# Goal/Experiment: +The objective of the systematic review is to explore the effects of physical exercise and physical activity on body composition in individuals with cardiometabolic multimorbidity. + +# Protocol of a Systematic Review with Meta-Analysis: The Effects of Physical Exercise/Activity on Body Composition of Individuals with Cardiometabolic Multimorbidity + +## Authors +- Juliene Gonçalves Costa¹ +- Igor Mariano² +- Victor Hugo Carrijo¹ +- Priccila Zuchinali³ +- Guilherme Morais Puga¹ +- Paula Aver Bretanha Ribeiro⁴ + +¹Laboratory of Cardiorespiratory and Metabolic Physiology, Physical Education and Physical Therapy Department, Federal University of Uberlândia, Uberlândia. +²Universidade Federal de Uberlândia +³Research Unit @CoeurLab, Centre de Recherche du Centre Hospitalier de l’Université de Montréal (CRCHUM) +⁴Research Unit @CoeurLab, Centre de Recherche du Centre Hospitalier de l’Université de Montréal (CRCHUM), Montreal Behavioural Medicine Centre (MBMC), Centre intégré universitaire de santé et de services sociaux du Nord-de-l’Île-de-Montréal (CIUSSS-NIM), Hôpital du Sacré-Coeur de Montréal, Canada + +Corresponding author: Juliene Gonçalves Costa +Faculdade de Educação Física, Universidade Federal de Uberlândia, Brazil. +Rua Benjamin Constant, 1286. Bairro: Aparecida. +Uberlândia – MG, Brasil. ZIP code: 38400-678. +Phone/Fax: +55 34 3218-2965 +E-mail: julienegoncalves@hotmail.com + +DOI: [10.17504/protocols.io.b4x6qxre](dx.doi.org/10.17504/protocols.io.b4x6qxre) + +## Introduction +Multimorbidity can be defined as the combination of two or more chronic diseases and affects an increasing number of individuals worldwide. Among the various chronic conditions, cardiometabolic diseases stand out as the main causes of death in the world. The need for effective interventions is crucial. The regular practice of physical activity is an important tool to treat and prevent the worsening of health status in patients with cardiometabolic diseases. The objective of the systematic review is to explore the effects of physical exercise and physical activity on body composition in individuals with cardiometabolic multimorbidity. + +## Methods + +### Eligibility Criteria +1. **Population**: Human adults (>18 years), both sexes, with two or more health conditions, including at least one cardiometabolic condition (e.g., hypertension, heart failure, diabetes, obesity). +2. **Intervention**: Chronic physical exercise/physical activity of any modality, associated or not with other co-interventions. +3. **Control**: Not limited. +4. **Outcomes of Interest**: + - Physical capacity: 6-minute walking test, VO2peak + - Body composition: Body weight, BMI, waist circumference + - Quality of life +5. **Languages**: English, Portuguese, Italian, French, Spanish +6. **Study Designs**: Intervention studies +7. **Publication Dates**: From 2000 onwards + +### Search Strategy +The study is a spin-off from a scoping review, and studies will be searched in the following databases: +- PUBMED +- EMBASE +- CINAHL +- Sportsdiscuss +- Prospero + +### Study Records +Screening, eligibility, and data extraction phases will be evaluated in duplicate by two independent reviewers. Disagreements will be resolved by a third reviewer. Data will be organized using Mendeley reference manager and registered in a spreadsheet. Extraction will include: +1. Values referring to the outcome (BMI, body weight, waist circumference) +2. Population characteristics (sex, exercise training level, age, health status) +3. Exercise characteristics (duration, modality, intensity, total volume) + +### Risk of Bias in Individual Studies +Risk of bias will be assessed using the Joanna Briggs Institute Critical Appraisal tool for Randomized Controlled Trials. Bias assessment will be based on 13 questions evaluating study design and validity. + +### Data Synthesis and Quantitative Approaches +Data will be analyzed using the programming language "R" with the supplements "meta" and "metafor". The analysis will be based on weighted or standardized mean differences. Meta-analysis values will be presented through a forest plot. Sensitivity analysis will investigate aspects like exercise modality, type of intervention, and type of association in the intervention. + +## Authors' Contributions: +- JGC: Idealization, planning, manuscript writing. +- IMM: Planning, review, manuscript approval. +- VHC: Planning, review, final version approval. +- PZ: Idealization, planning, review, final version approval. +- GMP: Idealization, planning, review, final version approval. +- PABR: Idealization, planning, manuscript writing, review, final version approval. + +## Funding and Conflicts of Interest: +The authors declare no financial support and no conflicts of interest. + +## References +1. Palladino, et al. Healthcare utilisation and health status in people with multimorbidity, Age Ageing. 45 (2016) 431–435. [doi.org/10.1093/ageing/afw044](https://doi.org/10.1093/ageing/afw044) +2. Reiter-Brennan, et al. Comprehensive Care Models for Cardiometabolic Disease, Current Cardiology Reports. 23 (2021) 1–11. [doi.org/10.1007/s11886-021-01450-1](https://doi.org/10.1007/s11886-021-01450-1) +3. Chudasama, et al. Healthy lifestyle and life expectancy in people with multimorbidity, PLoS Med. 17 (2020) 1–18. [doi.org/10.1371/journal.pmed.1003332](https://doi.org/10.1371/journal.pmed.1003332) +4. MacMahon. Multimorbidity: a priority for global health research, Acad. Med. Sci. (2018). [acmedsci.ac.uk/file-download/82222577](https://acmedsci.ac.uk/file-download/82222577) +5. Bricca, et al. Benefits and harms of exercise therapy in people with multimorbidity, Ageing Research Reviews. 63 (2020) 101166. [doi.org/10.1016/j.arr.2020.101166](https://doi.org/10.1016/j.arr.2020.101166) +6. Smith, et al. Interventions for improving outcomes in patients with multimorbidity, Systematic Reviews. 10(1) (2021) 1–11. [doi.org/10.1186/s13643-021-01817-z](https://doi.org/10.1186/s13643-021-01817-z) +7. Xu, et al. Mapping the global research landscape and knowledge gaps on multimorbidity, Journal of Global Health. 7(1) (2017) 1–11. [doi.org/10.7189/jogh.07.010414](https://doi.org/10.7189/jogh.07.010414) +8. Aromataris, et al. JBI Manual for Evidence Synthesis. Chapter 3: Systematic reviews of effectiveness, JBI. (2017). [doi.org/10.46658/JBIMES-20-01](https://doi.org/10.46658/JBIMES-20-01) +9. R Core Team. R: A language and environment for statistical computing [Internet]. Vienna R Foundation for Statistical Computing; 2019. [r-project.org](https://www.r-project.org) +10. Balduzzi S, Rücker G, Schwarzer G. How to perform a meta-analysis with R: A practical tutorial, Evid Based Ment Health. (2019). +11. Viechtbauer W. Conducting meta-analyses in R with the metafor package, J Stat Softw. 36:1–48 (2010). + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/protocol-to-generate-gastruloids-lscb-epfl-9j5h4q6.md b/markdown-output/protocol-to-generate-gastruloids-lscb-epfl-9j5h4q6.md new file mode 100644 index 0000000000000000000000000000000000000000..b254d762a42880164b97764c98f4e4abe438be5e --- /dev/null +++ b/markdown-output/protocol-to-generate-gastruloids-lscb-epfl-9j5h4q6.md @@ -0,0 +1,149 @@ +```markdown +# Protocol to generate Gastruloids (LSCB, EPFL) + +**Goal/Experiment:** The goal of this experiment is to generate gastruloids from mouse embryonic stem cells (mESCs) using a standardized protocol performed in the Lutolf Lab at EPFL. + +### Authors: +Stefano Vianello1, Mehmet Girgin1, Giuliana Rossi1, Matthias Lutolf2 + +1Laboratory of Stem Cell Bioengineering, Institute of Bioengineering, School of Life Sciences (SV) and School of Engineering (STI), Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland +2Laboratory of Stem Cell Bioengineering, Institute of Bioengineering, School of Life Sciences (SV) and School of Engineering (STI), Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland and Institute of Chemical Sciences and Engineering, School of Basic Science (SB), EPFL, Lausanne, Switzerland + +DOI: [10.17504/protocols.io.9j5h4q6](https://dx.doi.org/10.17504/protocols.io.9j5h4q6) + +## Abstract +Gastruloid generation protocol as performed in the Lutolf Lab, EPFL. + +For previously published protocols, see: + +- Baillie-Johnson, Peter, et al. "Generation of aggregates of mouse embryonic stem cells that show symmetry breaking, polarization and emergent collective behaviour in vitro." *JoVE (Journal of Visualized Experiments)* 105 (2015): e53252. +- Mehmet, G., et al. "Generating Gastruloids from Mouse Embryonic Stem Cells."*Protocol Exchange* (2018). + +## Guidelines +This protocol assumes standard cell culture conditions and techniques. + +## Materials + +| Name | Catalog # | Vendor | +| ------------------------------------------------ | ---------- | ----------------------- | +| StemPro™ Accutase™ Cell Dissociation Reagent | A1110501 | Thermo Fisher Scientific| +| PBS, pH 7.4 | 10010015 | Thermo Fisher | +| 50mL Reagent Reservoir White PS | 613-1184 | VWR International Ltd | +| 96-well Clear Round Bottom Ultra-Low Attachment Microplate | 7007 | Corning | +| GSK-3 Inhibitor XVI | 361559 | Merck Millipore | + +### Additional Materials and Reagents + +### 10% Serum Medium (home made fresh): +To make 500 mL: +- 434 mL DMEM, high glucose, with GlutaMAX™ [CAT# 61965059, Gibco™/Life Technologies] +- 50 mL ES-grade Foetal Bovine Serum [CAT# 16141-079, Gibco™/Life Technologies] +- 5 mL Penicillin-Streptomycin 10000U/mL [CAT# 15140122, Gibco™/Life Technologies] +- 5 mL Non Essential Amino Acids 100X [CAT# 11140035, Gibco™/Life Technologies] +- 5 mL Sodium Pyruvate 100mM [CAT# 11360039, Gibco™/Life Technologies] +- 1 mL beta-mercaptoethanol 50mM [CAT# 31350010, Gibco™/Life Technologies] + +Final Concentrations: +- Penicillin: 100U/mL +- Streptomycin: 100μg/mL +- Non Essential Amino Acids: 0.1mM +- Sodium Pyruvate: 1mM +- GlutaMAX™ (L-Alanyl-Glutamine, included in DMEM): 3.97mM +- beta-mercaptoethanol: 0.1mM + +### 10% Serum 2i/LIF Medium (made fresh): +To make 500mL: +- 3uM CHIR99021 (1:1000 from our 3mM stock) [CAT# 361559, Merck/Millipore] +- 1mM PD0325901 (1:500 from our stock) [CAT# 13036, Selleck Chemicals] +- 1000U/mL LIF (1:1000 from our stock), [sourced in house] + +### N2B27 Medium (home made fresh): +To make 500 mL: +- 237 mL Neurobasal™ Medium [CAT# 21103049, Gibco™/Life Technologies] +- 237 mL DMEM/F-12, with GlutaMAX™ [CAT# 31331093, Gibco™/Life Technologies] +- 5 mL Penicillin-Streptomycin 10000U/mL [CAT# 15140122, Gibco™/Life Technologies] +- 5 mL Non Essential Amino Acids 100X [CAT# 11140035, Gibco™/Life Technologies] +- 5 mL Sodium Pyruvate 100mM [CAT# 11360039, Gibco™/Life Technologies] +- 2.5 mL GlutaMAX™ Supplement, 200mM [=L-Alanyl-Glutamine, CAT# 35050083, Gibco™/Life Technologies] +- 1 mL beta-mercaptoethanol 50mM [CAT# 31350010, Gibco™/Life Technologies] +- 5 mL B27 Supplement, serum-free, 50X [CAT# 17504001, Gibco™/Life Technologies] +- 2.5 mL N-2 Supplement, 100X [CAT# 17502001, Gibco™/Life Technologies] + +Final Concentrations: +- Penicillin: 100U/mL +- Streptomycin: 100μg/mL +- Non Essential Amino Acids: 0.1mM +- Sodium Pyruvate: 1mM +- GlutaMAX™ (=L-Alanyl-Glutamine, also included in DMEM/F-12): the final concentration is 1mM + 2.50mM = 3.50mM +- beta-mercaptoethanol: 0.1mM + +## Procedure + +### Preparation of the cell suspension +1. Using a vacuum line + glass pasteur pipette, aspirate out the culture medium and replace with **3 mL** PBS-/-, for a short wash. + > *When removing liquid during washes, do not completely dry out the cells. The surface of the well should still look glossy.* + +2. Aspirate out the PBS, and replace with **500 μL** Accutase. + +3. Let Accutase act for around **3 minutes at room temperature**, tapping the sides of the plate to ease dissociation, until most cells are floating (as clumps or single cells). + +4. Slightly tilt the plate, and use a P1000 to pipette the suspension up and down to further break down cell clumps. Use each ejection to wash the surface of the plate, so to collect as many cells as possible. + +5. Transfer the Accutase-cell suspension to a clean 15 mL Falcon tube labelled “cells”. + +6. Use around **4.5 mL** DMEM-10%Serum to further wash the surface of the well, again, pipetting up and down and hoping to collect any previously missed cells. Transfer this secondary cell suspension in the same tube as before (total volume **5 mL**). + +7. Centrifuge at **200 g** (around **1000 rpm for 4 minutes at 4°C**). + +8. Using a vacuum line + glass pasteur pipette, aspirate the supernatant without disturbing the pellet, redissolve the pellet in **10 mL** PBS-/-, and centrifuge again at **200 g** (around **1000 rpm for 4 minutes at 4°C**). + +9. Repeat Step 8 for a second PBS-/- wash. + +10. Using a vacuum line + glass pasteur pipette, aspirate the supernatant without disturbing the pellet, and resuspend the pellet in **1 mL** N2B27. + + > It is important to thoroughly resuspend the pellet at this point, as a single cell suspension is needed for accurate counting later on. Resuspend the pellet by pipetting several times (around 20) with a P1000, then switching to a P200, and then to a P20. + +### Cell counting and Gastruloid seeding + +11. Load **10 μL** of the N2B27-cell suspension into a manual cell counter (Neubauer chamber/haemocytometer), and calculate the concentration of cells in your suspension. + > Each well of the 96well plate will receive one **40 μL** drop of N2B27. Each drop should contain 300 cells, i.e. a concentration of 300 cells / 40 μL = 7.5 cells/μL. To fill an entire 96well plate with **40 μL** drops, **40 μL** x **96 wells** = **3.84 mL** N2B27 are needed. For simplicity, **5 mL** N2B27 suspension are prepared at 7.5 cells/μL, this requires **37500 cells**. + +12. To **5 mL** fresh N2B27, add the volume of cell suspension carrying **37500 cells**, as calculated before. (If you want to prepare more than one plate of gastruloids, scale the volume of N2B27 and of cells accordingly). + +13. Load the **5 mL** N2B27-cell suspension into a multichannel pipette reservoir, and dispense **40 μL** of this solution to each well of an ultra-low-adhesion 96well plate. + >Make sure that the cell suspension in the reservoir is always well mixed, to ensure homogeneous dispensing in the wells. At each transfer, pipette up and down several times with the multichannel, and slightly agitate the reservoir from time to time. + +14. Lightly tap the plate against the surface of the hood to make sure all drops are at the very bottom of each well, double-check under the microscope for the presence of cells in the drops, and leave the plate in a humidified incubator, 5% CO2, undisturbed for 24 hours. + >This is considered the beginning of Day 1 (D1), t=0h-24h. + +### Growing Gastruloids + +#### Beginning of Day 2 (D2), t=24h-48h +15. No intervention needed at this time point. The Gastruloids will keep developing in their **40 μL** of N2B27. Leave to grow for another 24 hours. + >Gastruloids should look like a small (~100 μm) cluster, still in the process of aggregating. Individual cells might still be visible in the surroundings. + +#### Beginning of Day 3 (D3), t=48h-72h + +16. Prepare **16 mL** N2B27, adding CHIR99021 to a final concentration of **3 μM**. Load this into a multichannel pipette reservoir, and dispense **150 μL** of this solution to each well of the plate (i.e. total volume of **190 μL** per well). Place back in the incubator for 24 hours. + >Gastruloids should look like a small, clean, translucent sphere of around ~200 μm diameter. No additional (satellite) spheres should be seen around it. + +#### Beginning of Day 4 (D4), t=72h-96h + +17. Using a multichannel pipette, remove **150 μL** of the medium from each well and replace with **150 μL** fresh N2B27. Place back in the incubator for 24 hours. + >Gastruloids are round spheres of around ~300 μm in diameter. Extensive cell shedding is expected as a consequence of the CHIR pulse. + +#### Beginning of Day 5 (D5), t=96h-120 + +18. Using a multichannel pipette, remove **150 μL** of the medium from each well and replace with **150 μL** fresh N2B27. Place back in the incubator for 24 hours. + >Gastruloids morphology is no longer symmetrical; they look like an ovoid with length of around ~600 μm. The protruding part is more translucent than the rest, and this is the part that will elongate in the next 24 hours. + +At t=120h, the Gastruloid looks like a bowling pin with length of around ~700 μm - ~800 μm. A thinner extension protrudes at the "posterior", more translucent than the denser anterior (spherical) part. + +#### Day 6 (D6) and Day 7 (D7), t=144h, 168h + +Culture can be extended up to 168h (i.e., 7 days total). This can be done by continuing the daily medium changes as above, or by transferring individual Gastruloids to separate wells of a 24well plate, in **800 μL** fresh N2B27, and replacing half of the medium one day later. The plate is kept shaking on an orbital shaker, in the incubator for both additional days. If keeping the Gastruloids in the original 96well plate, no shaking is required but there is an increased risk of the Gastruloids adhering to the sides of the well and degenerating. + +**Note:** For the specific chemicals and equipment like CHIR99021, PD0325901, and multi-channel pipettes, alternatives can usually be found from different suppliers. Ensure that the alternatives match the concentration and quality specifications. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/protocol-to-immunostain-gastruloids-lscb-epfl-7tzhnp6.md b/markdown-output/protocol-to-immunostain-gastruloids-lscb-epfl-7tzhnp6.md new file mode 100644 index 0000000000000000000000000000000000000000..f1feea7549bec5cb6e4ab9b2750bccebbf638d7a --- /dev/null +++ b/markdown-output/protocol-to-immunostain-gastruloids-lscb-epfl-7tzhnp6.md @@ -0,0 +1,154 @@ +```markdown +# Goal/Experiment: +The goal of this experiment is to immunostain Gastruloids, which are aggregates of mouse embryonic stem cells, following standard laboratory protocols developed at the Lutolf Lab, EPFL. + +# Protocol to Immunostain Gastruloids (LSCB, EPFL) + +**Authors**: Stefano Vianello, Mehmet Girgin, Giuliana Rossi, Matthias Lutolf +**Date**: June 07, 2020 +**Institution**: École Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland + +## Abstract + +This document provides a standard protocol used for immunostaining Gastruloids, aggregates of mouse embryonic stem cells in the Lutolf Lab at EPFL. + +For a detailed protocol on generating Gastruloids, see: +- Beccari et al. (2018). Generating Gastruloids from Mouse Embryonic Stem Cells. Protocol Exchange. [Link](http://10.1038/protex.2018.094) + +## Guidelines + +All handling and transfer of Gastruloids should be done with a cut P1000 tip to minimize damage. Tips should be coated in BSA- or serum-containing solution to prevent Gastruloids from sticking to the plastic. For minimal liquid carry-over, keep the pipette vertical, form a small hanging drop, and touch the surface of the new solution. + +### Materials + +| Name | Catalog # | Vendor | +|--------------------------------------|-------------------|--------------------------| +| DAPI | D1306 | Thermo Fisher Scientific | +| Triton X-100 | T8787 | Sigma Aldrich | +| 4% Paraformaldehyde in PBS | J61899-AK | Alfa Aesar | +| Permount mounting medium | - | Fisher Scientific | +| Thermo Scientific™ SuperFrost Plus™ | J1800AMNZ | Thermo Fisher Scientific | +| PBS, pH 7.4 | 10010015 | Thermo Fisher Scientific | +| Embryonic stem-cell FBS, qualified | 16141079 | Thermo Fisher | +| Fluoromount-G | 0100-01 | Southern Biotech | +| Greiner CELLSTAR® 6 well culture plates| 657185 | Greiner Bio-One | +| Falcon™ 48well Polystyrene Microplates| 353078 | Falcon | +| #1 Micro Cover Glass 22mmx22mm | 72200-10 | Electron Microscopy Sciences | + +## Materials Text + +### Solutions and Preparations + +#### PBS-FT (PBS+FBS+0.2% Triton) + +- **Purpose**: Buffered saline solution supplemented with serum and detergent to prevent non-specific binding and permeabilize cells. + + **Preparation for 500mL**: + - 450mL PBS, 1X (Thermo Fisher Scientific, Cat# 10010015) + - 50mL ES-grade FBS (Thermo Fisher Scientific, Cat# 16141079) + - 0.1mL Triton-X100 (Sigma Aldrich, Cat# T8787) + +#### 4% PFA in PBS + +- **Purpose**: Fixative agent to preserve cellular structures during staining. + + **Preparation for 500mL**: + 1. Use a chemical hood, wear N95 mask and protective gear. + 2. Mix 400mL PBS, 1X (Thermo Fisher Scientific, Cat# 10010015) + 3. Add 20g granulated PFA powder (Carl Roth, Cat# 40954.1) + 4. Heat to 60°C on a magnetic stirrer until PFA dissolves. + 5. Adjust pH to 7.4 with HCl or NaOH. + 6. Aliquot and store at -20°C for long term use. + +**Note**: If preparing 4% PFA in PBS independently, follow all safety guidelines to avoid inhaling vapors. + +## Safety Warnings + +This protocol involves the use of 4% PFA, which must be handled in a chemical hood while wearing appropriate body and eye protection. + +## Before Starting + +- **Prepare PBS+FT** +- **Coat wells of a 6-well plate with serum-containing buffer** for 30 minutes at room temperature to prevent Gastruloids from sticking. +- **Bring 4% PFA to room temperature**. + +## Detailed Protocol + +### Fixing and Primary Antibody Staining (Day 1) + +#### 1. Harvesting Gastruloids: + +1.1. Using a cut P1000 tip, collect Gastruloids from each well of the 96-well plate by aspirating up. Move to the next well until the tip is full. + +> Do not collect Gastruloids from the outer wells of the plate to avoid variability due to medium evaporation. + +1.2. Deposit collected Gastruloids in a 15mL centrifuge tube. +1.3. After collecting, wait for Gastruloids to settle at the bottom and remove the medium using a vacuum pump and glass pipette. +1.4. Resuspend the Gastruloids in ~5mL PBS-/- to dilute away traces of medium. + +#### 2. Fixing (4%PFA): + +2.1. Replace medium in a 6-well plate with 2mL of 4% PFA. Work under a fume hood. +2.2. Transfer Gastruloids vertically into PFA. +2.3. Cover with aluminum foil. +2.4. Incubate at 4°C for 2 hours with or without orbital shaker. + +#### 3. Washes (to remove PFA): + +3.1. Fill wells of a 6-well plate with 4mL PBS-/- and transfer fixed Gastruloids. +3.2. Cover with aluminum foil and wait for 10 minutes. +3.3. Repeat for two more washes. + +> You can expedite washing by transferring Gastruloids serially across three PBS-filled wells and leaving for 10 minutes in the final wash only. + +**Pause point**: Gastruloids can remain in the last PBS wash at 4°C, protected from light. + +#### 4. Blocking: + +4.1. Transfer Gastruloids to a well filled with 2mL PBS+FT. +4.2. Block for 1 hour at room temperature, with or without shaker. + +#### 5. Primary Antibody Staining + +5.1. Prepare 1.5mL Eppendorf tubes with 500µL of antibodies in PBSFT and DAPI 1:500. +5.2. Transfer each solution into wells of a 48-well plate and add Gastruloids. +5.3. Cover with aluminum foil and incubate at 4°C overnight on orbital shaker. + +### Secondary Antibody Staining (Day 2) + +#### 6. Washes (to remove 1ry Ab): + +6.1. Transfer Gastruloids to a well with 3mL PBS+FT. +6.2. Wait for 20 minutes. +6.3. Repeat for two more washes. + +#### 7. Secondary Antibody Staining: + +7.1. Prepare 1.5mL Eppendorf tubes with 500µL of secondary antibodies in PBSFT and DAPI 1:500. +7.2. Transfer solution into wells of a 48-well plate and add Gastruloids. +7.3. Cover with aluminum foil and incubate at 4°C overnight on orbital shaker. + +### Mounting (Day 3) + +#### 8. Washes (to remove 2ry Ab): + +8.1. Transfer Gastruloids to well with 3mL PBS+FT. +8.2. Wait for 20 minutes. +8.3. Repeat for two more washes. + +#### 9. Mounting: + +9.1. Add ~30µL of Fluoromount-G on the center of a microscope slide. +9.2. Transfer Gastruloids to the drop. +> Multiple slides with fewer Gastruloids recommended to avoid clumping. + +9.3. Gently drop a coverslip over samples. +9.4. Absorb excess liquid with a Kimtech wipe. +9.5. Seal coverslip with Permount hardening resin. +> Nail polish can substitute Permount, but might reduce slide longevity. + +9.6. Keep slides in a box away from light at 4°C until ready for imaging. +> Slides can be stored at 4°C for months before imaging. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/protocol-to-isolate-and-fix-nuclei-from-flash-froz-c4m2yu8e.md b/markdown-output/protocol-to-isolate-and-fix-nuclei-from-flash-froz-c4m2yu8e.md new file mode 100644 index 0000000000000000000000000000000000000000..4aae6f2f09d4766b188b5775196af309493fe0fe --- /dev/null +++ b/markdown-output/protocol-to-isolate-and-fix-nuclei-from-flash-froz-c4m2yu8e.md @@ -0,0 +1,131 @@ +```markdown +# Goal/Experiment: +Protocol to isolate and fix nuclei from flash frozen mouse gastrocnemius muscle for IGVF V.2 + +## Protocol to isolate and fix nuclei from flash frozen mouse gastrocnemius for IGVF V.2 + +### Author: +Elisabeth Reboohah +1University of California, Irvine + +--- +**Version 2** +**Date:** NOV 08, 2023 + +--- + +**Abstract:** +This protocol describes isolation of nuclei from 10-week-old left or right mouse gastrocnemius muscle, preparation of a single nucleus suspension, and fixation for single nucleus RNA-seq using the Parse Biosciences protocol (Split-seq). The results are fixed single-nucleus suspensions from each of 8 samples at >= 2,500 nuclei/μl stored at -80°C. + +**Guidelines:** +1. Tilt tube and slowly add PBS during debris removal, only in the band rather than the nuclei layer. +2. We recommend using a 5 mL pipette for aspirations and resuspensions > 1 mL. +3. Record everything in the [IGVF spreadsheet](https://igvf.org), “Samples into experiment” tab. + +## Materials + +| Name | Manufacturer | Cat. # | +|-------------------------------------------|--------------------|-----------------| +| Nuclei Fixation Kit v2 | Parse Biosciences | ECF2003 | +| Nuclei Extraction Buffer | Miltenyi Biotec | 130-128-024 | +| RNase Inhibitor, murine | New England Biolabs| M0314L | +| PBS | HyClone | SH30256.02 | +| Debris Removal Solution | Miltenyi Biotec | 130-109-398 | +| 7.5% BSA | Life Technologies | 15260037 | +| gentleMACS C Tube | Miltenyi Biotec | 130-093-237 | +| gentleMACS Octo Dissociator | Miltenyi Biotec | 130-095-937 | +| MACS SmartStrainers (70 um) | Miltenyi Biotec | 130-110-916 | +| MACS SmartStrainers (30 um) | Miltenyi Biotec | 130-098-458 | +| pluriStrainer (20 um) | pluriSelect | 43-50020-03 | +| NucBlue Fixed Cell ReadyProbes | Thermo Fisher | R37606 | +| Millicell Disposable Hemocytometer | Millipore | MDH-2N1-50PK | +| Mr. Frosty | Sigma-Aldrich | 635639 | + +**Reagents/Equipment Calculation:** + +### Buffers Preparation + +#### Lysis Buffer + +| Reagent | Volume (for 8 samples) | Final Concentration | +|------------------------------|------------------------|---------------------| +| Nuclei Extraction Buffer | 35 mL | NA | +| 40 U/μl RNase inhibitor | 175 μL | 0.2 U/μl | + +#### Additional Buffers + +| Name | Reagent | Volume (for 8 samples) | Final Concentration | +|-------------------------|---------------------------------------|------------------------|---------------------| +| PBS | PBS | 35 mL | NA | +| HBSS | HBSS | 20 mL | NA | +| Debris Removal Solution | Debris Removal Solution (Miltenyi) | 8 mL | NA | +| NB-BSA + RNase inhibitor| Nuclei Buffer (Parse Biosciences) | 3.15 mL | NA | +| NB-BSA + RNase inhibitor| 7.5% BSA | 350 μL | 0.75% | +| NB + RNase inhibitor | RNase inhibitor (Parse Biosciences) | 44.1 μL | NA | +| NB + RNAse inhibitor | Nuclei Buffer (Parse Biosciences) | 5 mL | NA | +| NB + RNAse inhibitor | RNase inhibitor (Parse Biosciences) | 44.1 μL | NA | +| RSB | PBS | 24.6 mL | NA | +| RSB | 7.5% BSA | 333 μL | 0.1% | +| RSB | RNase inhibitor | 125 μL | 0.2 U/μL | + +## Setup + +1. **Label tubes.** +2. **Pre-chill centrifuge to 4°C.** +3. **Prepare 2 large ice buckets.** +4. Prepare 35 mL lysis buffer on ice in a 50 mL conical tube. Distribute 2 mL into 8 gentleMACS C Tubes on ice. Add 175 μL RNase inhibitor to the lysis buffer aliquot the day of the experiment. +5. Prepare 3.5 mL NB + BSA. Add 44.1 μL RNase inhibitor included in Parse Biosciences fixation kit the day of the experiment. +6. Prepare 50 mL RSB on ice in 2 50 mL conical tubes. We keep a larger amount of PBS + 0.1% BSA at 4°C, adding the RNase inhibitor the day of the experiment. +7. Prepare 5 mL nuclei buffer + RNase inhibitor for final resuspension. Add 44.1 μL RNase inhibitor to 5 mL nuclei buffer. +8. Take an aliquot of PBS out of 4°C and keep on ice. +9. Take an aliquot of Debris Removal Solution out of 4°C and keep on ice. +10. Thaw components of 1 Parse Biosciences Nuclei Fixation kit at room temperature, then place on ice. +11. Distribute 10 μL NucBlue Fixed Cell ReadyProbes into 24 PCR strip tubes for cell counting. Prepare 8 tubes for counting after nuclei extraction, 8 tubes for counting after fixation, and 8 tubes for filtered fixed nuclei. + +## Tissue Lysis and Nuclei Extraction + +12. Keep flash frozen tissue samples on dry ice. +13. Prepare 6 well plates on ice with ~2 mL of HBSS per well. +14. If necessary, drop both gastrocnemius tissues in a well, let them melt slightly and separate them carefully using forceps. +15. Return one muscle to the sample tube and move the other to another labeled 1.5 mL tube. Flash-freeze both in liquid nitrogen. +16. Proceed with only one. Keep the other frozen in the same sample tube and return tubes to -80°C box. +17. Drop left or right frozen tissue into a chilled gentleMACS C Tube with 2 mL lysis buffer. Close tubes firmly and invert immediately, ensuring tissue is not stuck to the bottom or side. Keep tubes on ice and proceed immediately to dissociation. +18. Run the gentleMACS Program _4C_nuclei_1_ on the Octo Dissociator (~5 minutes). +19. Remove tubes, ensuring tissue did not get stuck on the sides, and spin down in a 4°C centrifuge for ~10 seconds to bring liquid to the bottom, then place tubes back on ice. +20. Filter nuclei suspension through 70 μm MACS SmartStrainer into a 5 mL tube. Fit a tube rack in ice for extra stability while filtering. +21. Wash 70 μm MACS SmartStrainer with 2 mL additional lysis buffer. Add 2 mL to C tubes, cap, and swish to recover any nuclei stuck to the sides and cap of the C tubes, then wash the strainer. +22. Discard strainer and centrifuge the nuclei suspension at 4°C, 350g for 5 minutes. +23. Aspirate supernatant and resuspend nuclei pellet in 3.1 mL RSB. +24. Filter nuclei suspension through 30 μm MACS SmartStrainer into a 15 mL tube. +25. Add 900 μL Debris Removal Solution and mix by pipetting 10 times slowly up and down using a 5 mL pipette. +26. Overlay with 4 mL PBS using either a P1000 or 5 mL pipette. Tilt tube 45 degrees and slowly add the first mL. Increase speed after the first mL of PBS is added. +27. Centrifuge at 4°C, 3000g for 10 minutes with full acceleration and no brake. Three phases are formed: top clear buffer layer, cloudy debris band, and clear layer containing nuclei. Pellet usually visible. +28. Aspirate the two top phases (buffer layer and all cloudy debris band) and discard. Aspirate the first phase, then the second phase. Stay above the third layer of nuclei to prevent loss. + +*Figure 1: Aspirate clear buffer layer and all of the cloudy debris layer outlined in red.* + +## Nuclei Fixation + +29. Fill with cold RSB to a final volume of 5 mL. +30. Gently invert the tube three times. Do not vortex. +31. Centrifuge at 4°C, 1000g for 10 minutes with full acceleration and full brake. +32. Aspirate supernatant completely. +33. Resuspend cells carefully in 375 μL NB-BSA + RNase inhibitor and filter through a 40 μm strainer into a new 5 mL tube. +34. Count nuclei. Use 1:2 dilution factor, 10 μL + 10 μL dye. +35. Add 125 μL Nuclei Fixation Solution to the filtered nuclei in 375 μL and mix 3 times. Do not over-mix. +36. Incubate nuclei for 10 minutes on ice. Set 2 P200 pipettes to 40 μL and 125 μL. +37. Add 40 μL Nuclei Permeabilization Solution and mix by pipetting 3 times with the P200 still set to 125 μL. Do not over-mix. +38. Incubate 3 minutes with nuclei on ice. +39. Add 2 mL Nuclei Neutralization Solution and invert the tube once to mix. +40. Centrifuge at 4°C, 750g for 10 minutes. +41. Aspirate and discard supernatant. +42. Resuspend the samples in 500 μL Nuclei Buffer with RNase inhibitor without BSA. Check concentration with a hemocytometer under the microscope. Use 1:2 dilution factor, 10 μL + 10 μL dye. +43. Filter nuclei through a 20 μm filter in several rounds depending on debris. Centrifuge at 4°C, 200g for 1 minute. Repeat if necessary with new filter each round. +44. Take a 10 μL aliquot to dilute 1:2 with 10 μL dye to manually count with a disposable hemocytometer. +45. Count nuclei. Use 1:2 dilution factor, 10 μL + 10 μL dye. +46. Re-concentrate: spin nuclei 750g for 5 minutes and carefully remove supernatant until 50 μL remains. Resuspend pellet in the remaining 50 μL. +47. Add Nuclei DMSO: 1 μL into 50 μL samples and gently flick tubes to mix. Gradually add another 1 μL until a total volume of 3 μL is reached. Mix by gently pipetting with a P200 set to 25 μL. +48. Place tubes in a Mr. Frosty for storage at -80°C. Next day, move tubes to boxes in -80°C racks. + +*endofoutput* +``` \ No newline at end of file diff --git a/markdown-output/protocol-to-isolate-and-fix-nuclei-from-flash-froz-c4n6yvhe.md b/markdown-output/protocol-to-isolate-and-fix-nuclei-from-flash-froz-c4n6yvhe.md new file mode 100644 index 0000000000000000000000000000000000000000..a1405d30b457ad265900c5eee9f4a76f2f123cad --- /dev/null +++ b/markdown-output/protocol-to-isolate-and-fix-nuclei-from-flash-froz-c4n6yvhe.md @@ -0,0 +1,197 @@ +```markdown +# Goal/Experiment: +Isolation and fixation of nuclei from flash-frozen mouse hypothalamus and pituitary gland for single nucleus RNA sequencing (RNA-seq) and single nucleus RNA-seq + ATAC-seq. + +## Protocol to isolate and fix nuclei from flash frozen mouse hypothalamus and pituitary gland for IGVF V.2 + +Version 2 +Date: Nov 08, 2023 + +### Abstract + +This protocol describes isolation of nuclei from 10-week-old mouse hypothalamus and pituitary gland from 8 founder strains. The preparation involves single nucleus RNA-seq using the Parse Biosciences protocol (Split-seq) and single nucleus RNA-seq + ATAC-seq using the SHARE-seq protocol. For 8 samples, this protocol takes about 3.5 hours from start to finish. + +### Results + +The results are: +- 2 aliquots of fixed single-nucleus suspensions for Parse per each of the 8 samples at ≥2,500 nuclei/µL. +- 1 fixed nuclei pellet pooled across all 8 strains for SHARE-seq, all stored at -80°C. + +### Overview + +1. Tissue lysis and nuclei extraction using Miltenyi Biotec's gentleMACS Octo Dissociator. +2. Nuclei fixation using Parse Biosciences Evercode Nuclei Fixation Kit with v2 reagents. +3. Nuclei fixation using a modified version of the SHARE-seq fixation protocol. + +### Guidelines + +1. Use a 5 mL pipette for aspirations and resuspensions >1 mL. +2. Record everything in the IGVF spreadsheet, "Samples into experiment" tab. +3. Parallel fixation by two technicians is recommended for optimized processing time. + +--- + +## Materials + +| Name | Manufacturer | Cat. # | +|---------------------------------|--------------------|---------------| +| Nuclei Fixation Kit v2 | Parse Biosciences | ECF2003 | +| Nuclei Extraction Buffer | Miltenyi Biotec | 130-128-024 | +| RNase Inhibitor, murine | New England Biolabs| M0314L | +| PBS | HyClone | SH30256.02 | +| 7.5% BSA | Life Technologies | 15260037 | +| 1 M HEPES pH 7.3 | Sigma | H0887-100ml | +| NaCl | Fisher | BP358-1 | +| MgCl2 | Fisher | AA12315A7 | +| Tween-20 | Fisher | BP337-500 | +| 5% digitonin | Promega | G944A | +| Enzymatics RI | Enzymatics | Y9240L | +| SUPERase RI | Invitrogen | AM2696 | +| Yeast tRNA | Invitrogen | AM7119 | +| Glycine | Fisher | BP381-500 | +| 1M Tris pH 8.0 | Thermo | AM9855G | +| Formaldehyde (methanol-free) | EMS | 15710 | +| gentleMACS C Tube | Miltenyi Biotec | 130-093-237 | +| gentleMACS Octo Dissociator | Miltenyi Biotec | 130-095-937 | +| MACS SmartStrainers (70 µm) | Miltenyi Biotec | 130-110-916 | +| MACS SmartStrainers (30 µm) | Miltenyi Biotec | 130-098-458 | +| NucBlue Fixed Cell ReadyProbes | Thermo Fisher | R37606 | +| Hemacytometer | Fisher Scientific | 02-671-51B | +| Mr. Frosty | Sigma-Aldrich | 635639 | + +### Reagents + +#### Buffers + +| Name | Reagent | Volume (for 8 samples) | Final Concentration | +|------------------|------------------------|------------------------|---------------------| +| **1% BSA-DEPC** | BSA | 1 g | 1% | +| | DEPC water | 100 mL | NA | +| **Lysis buffer** | Nuclei Extraction Buffer | 35 mL | NA | +| | 40 U/µL RNase inhibitor | 175 µL | 0.2 U/µL | +| **NB-BSA + RNase inhibitor** | Nuclei Buffer (Parse Biosciences) | 7 mL | NA | +| | 7.5% BSA | 700 µL | 0.75% | +| | RNase inhibitor (Parse Biosciences) | 35 µL | | +| **RSB** | PBS | 24.6 mL | NA | +| | 7.5% BSA | 333 µL | 0.1% | +| | RNase inhibitor | 125 µL | 0.2 U/µL | +| | 1 M HEPES pH 7.3 | 150 µL | 10 mM | +| | 5 M NaCl | 30 µL | 10 mM | +| | 1 M MgCl2 | 45 µL | 3 mM | +| | 10% Tween-20 | 150 µL | 0.1% | +| | H2O | 14.625 mL | NA | +| **SHARE-RSB** | 7.5% BSA | 80.26 µL | 0.04% | +| | 5% digitonin | 30 µL | 0.01% | +| | Enzymatics RI | 37.5 µL | 0.1 U/µL | +| | SUPERase RI | 18.75 µL | 0.025 U/µL | +| | Yeast tRNA | 150 µL | 100 µg/mL | + +### Setup + +1. **Coat SHARE-seq nuclei prep tubes with BSA.** + Fill 8 *1.5 mL tubes* with *1.5 mL 1% BSA-DEPC* and incubate for **30 minutes**. After incubation, aspirate BSA solution and dry for **30 minutes**. Store at **4°C**. + +2. **Label tubes.** + +3. **Pre-chill centrifuge to 4°C.** + +4. **Prepare ice buckets.** + +5. **Prepare 35 mL lysis buffer** in a *50 mL conical tube* on ice. Distribute *2 mL* into 8 gentleMACS C Tubes on ice. Add *175 µL RNase inhibitor* to the lysis buffer aliquot the day of the experiment. + +6. **Prepare RSB** in a *50 mL conical tube* on ice. Add *RNase inhibitor* the day of the experiment. + +7. **Prepare 3.5 mL NB + BSA.** + Add *44.1 µL RNase inhibitor* included in Parse Biosciences fixation kit the day of the experiment. + +8. **Prepare 2.5 mL nuclei buffer + RNase inhibitor** for final resuspension. + Add *31.5 µL RNase inhibitor* to *2.5 mL nuclei buffer*. + +9. **Prepare 15 mL SHARE-RSB** in a *50 mL conical tube* at room temperature. + To SHARE-RSB, add *30 µL digitonin*, *37.5 µL Enzymatics RI*, *18.75 µL SUPERase RI*, and *150 µL yeast tRNA* fresh the day of the experiment. + +10. **Thaw components of 2 Parse Biosciences Nuclei Fixation v2 kits** at room temperature, then place on ice. + +11. **Distribute 20 µL NucBlue Fixed Cell ReadyProbes** into 16 PCR strip tubes for cell counting. Need 8 tubes for counting after nuclei extraction, and another 8 tubes for final fixed nuclei. + +### Tissue lysis and nuclei extraction + +12. **Keep flash frozen tissue samples** on dry ice until lysis. + +13. **Drop whole frozen tissue** into a chilled *gentleMACS C Tube* with *2 mL lysis buffer*. Close tubes firmly and invert immediately, ensuring tissue is not stuck to the bottom or side. Keep tubes on ice and proceed immediately to dissociation. + +14. **Run the gentleMACS Program 4C_nuclei_1** on the Octo Dissociator (~**5 minutes**). + +15. **Remove tubes,** ensuring tissue did not get stuck on the sides, and spin down in a **4°C centrifuge** for ~**10 seconds** to bring liquid to the bottom, then place tubes back on ice. + +16. **Filter nuclei suspension through 70 µm MACS SmartStrainer** into a *5 mL tube*. Fit a tube rack in ice for extra stability while filtering. + +17. **Wash 70 µm MACS SmartStrainer** with *2 mL* additional lysis buffer. Add *2 mL* to C tubes, cap, and swish to recover any nuclei stuck to the sides and cap of the C tubes, then wash the strainer. + +18. **Discard strainer and centrifuge** the *4 mL* nuclei suspension at **4°C, 350g for 5 minutes**. + +19. **Discard supernatant and resuspend nuclei pellet in 3 mL RSB.** + +20. **Filter nuclei suspension through 30 µm MACS SmartStrainer** into a *5 mL tube*. + +21. **Count nuclei.** Use *1:3 dilution factor,* *10 µL + 20 µL dye*. + +### Parse nuclei fixation + +22. **Set aside 2-4 million nuclei in RSB** in a new *1.5 mL tube* and spin down at **4°C, 350g for 5 minutes**. + +23. **Remove supernatant and resuspend nuclei in 750 µL NB-BSA + RNase inhibitor** and filter through a *40 µm strainer* (provided in Parse Biosciences kit) into a new *5 mL tube*. + +24. **Add 250 µL Nuclei Fixation Solution** and mix 3 times. Do not over-mix. + +25. **Incubate nuclei for 10 minutes** on ice. Set 1 *P200 pipette* to 80 µL and keep the *P1000* at 250 µL. + +26. **Add 80 µL Nuclei Permeabilization Solution** and mix by pipetting 3 times with the P1000 still set to 250 µL. Do not over-mix. + +27. **Incubate 3 minutes** with nuclei on ice. + +28. **Add 4 mL Nuclei Neutralization Solution** and invert the tube once to mix. + +29. **Centrifuge at 4°C, 750g for 10 minutes.** + +30. **Aspirate and discard supernatant.** + +31. **Resuspend the samples in 200 µL Nuclei Buffer with RNase inhibitor** without BSA and move through a *40 µm filter* into a labeled *1.5 mL tube*. + +32. **Count nuclei.** Use *1:11 dilution factor,* e.g. *2 µL + 20 µL dye*. + +33. **Add Nuclei DMSO:** + *3.3 µL* and gently flick tubes to mix. One minute later, add another *3.3 µL* and flick to mix, then after another minute add a final *3.3 µL* for a total volume of *9.9 µL*. Mix by gently pipetting 5x with a P200 set to 100 µL. + +34. **Split nuclei suspension** into 2 labeled tubes, *100 µL per tube*. + +35. **Place tubes in a Mr. Frosty** at -80°C. The next day, move tubes to boxes in -80°C racks. + +### SHARE-seq nuclei fixation + +36. **Set aside 1 million nuclei** for each of the 8 samples in RSB and spin down at **4°C, 750g for 5 minutes**. + +37. **Remove supernatant** and resuspend nuclei pellet in *1 mL room temperature SHARE-RSB*. Transfer tube to a room temperature rack. + +38. **At RT, add 13.34 µL of methanol-free formaldehyde** (16% stock solution). Final concentration for nuclei: 0.2%. Close tube and nutate cells at RT for **5 minutes**. + +39. **To quench fixation,** per reaction, add *56.1 µL fresh 2.5M Glycine* (0.94g per 5 mL stock), *50 µL of 1M Tris pH 8.0*, *13.3 µL of 7.5% BSA*, and mix using a pipette. Incubate on ice for **10 minutes**. + +40. **Spin 750g, 4°C, 5 minutes.** Gently remove supernatant. + +41. **Add 200 µL of SHARE-RSB** and gently resuspend pellet. Store on ice until all samples are completed. + +42. **Pool 200 µL of resuspended nuclei** from all 8 founders into 1 labeled *2 mL tube*. + +43. **Spin 1,000g, 4°C, 10 minutes.** Gently remove supernatant. Remove all fluid and freeze at **-80°C as a dry pellet**. + +### Storage of leftover nuclei + +44. **Move remaining nuclei in RSB** on ice to labeled *2 mL tubes*. + +45. **Spin 750g, 4°C, 5 minutes**. + +46. **Remove all supernatant** and flash-freeze nuclei as a dry pellet in liquid nitrogen. Store at -80°C. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/protocol-to-isolate-and-fix-nuclei-from-flash-froz-c4p4yvqw.md b/markdown-output/protocol-to-isolate-and-fix-nuclei-from-flash-froz-c4p4yvqw.md new file mode 100644 index 0000000000000000000000000000000000000000..1b327baecab9f4bacf3673b442ff23410203cf6d --- /dev/null +++ b/markdown-output/protocol-to-isolate-and-fix-nuclei-from-flash-froz-c4p4yvqw.md @@ -0,0 +1,213 @@ +``` +# Goal/Experiment: +The goal of this experiment is to isolate and fix nuclei from flash-frozen male mouse gonads to facilitate single nucleus RNA sequencing (snRNA-seq) and single nucleus RNA-seq + ATAC-seq (SHARE-seq) analysis. This protocol uses whole left and right testes and epididymis samples from eight founder mouse strains to prepare nuclei for subsequent analysis. + +# Protocol to Isolate and Fix Nuclei from Flash Frozen Male Mouse Gonads for IGVF + +**Authors**: Elisabeth Rebboah +**Affiliation**: University of California, Irvine + +## Abstract +This protocol describes the isolation of nuclei from 10-week-old mouse testes and epididymis (pooled tissue ID: 25) from 8 founder strains, preparation of a single nucleus suspension, and fixation for: +1. Single nucleus RNA-seq using the Parse Biosciences protocol (Split-seq). +2. Single nucleus RNA-seq + ATAC-seq using the SHARE-seq protocol. + +One representative sample per strain per day is processed (male rep 1 across all 8 strains). + +## Guidelines +- Use a 5 ml pipette for aspirations and resuspensions > 1 ml. +- Record all data in the IGVF spreadsheet, "Samples into experiment" tab. + +## Materials + +| Name | Manufacturer | Cat # | +|-------------------------------------|------------------------|----------------| +| Nuclei Fixation Kit v2 | Parse Biosciences | ECF2003 | +| Nuclei Extraction Buffer | Miltenyi Biotec | 130-128-024 | +| RNase Inhibitor, murine | New England Biolabs | M0314L | +| PBS | HyClone | SH30256.02 | +| 7.5% BSA | Life Technologies | 15260037 | +| 1 M HEPES pH 7.3 | Sigma | H0887-100ml | +| NaCl | Fisher | BP358-1 | +| MgCl2 | Fisher | AA12315A7 | +| Tween-20 | Fisher | BP337-500 | +| 5% digitonin | Promega | G944A | +| Enzymatics RI | Enzymatics | Y9240L | +| SUPERase RI | Invitrogen | AM2696 | +| Yeast tRNA | Invitrogen | AM7119 | +| Glycine | Fisher | BP381-500 | +| 1M Tris pH 8.0 | Thermo | AM9855G | +| Formaldehyde (methanol-free) | EMS | 15710 | +| gentleMACS C Tube | Miltenyi Biotec | 130-093-237 | +| gentleMACS Octo Dissociator | Miltenyi Biotec | 130-095-937 | +| MACS SmartStrainers (70 μm) | Miltenyi Biotec | 130-110-916 | +| MACS SmartStrainers (30 μm) | Miltenyi Biotec | 130-098-458 | +| NucBlue Fixed Cell ReadyProbes | Thermo Fisher | R37606 | +| Hemacytometer | Fisher Scientific | 02-671-51B | +| Mr. Frosty | Sigma-Aldrich | 635639 | + +## Buffers Preparation + +| Name | Reagent | Volume for 8 samples | Final concentration | +|--------------------------------|-------------------------------|----------------------|---------------------| +| **Lysis buffer** | Nuclei Extraction Buffer | 40 ml | NA | +| | 40 U/μl RNase inhibitor | 200 μl | 0.2 U/μl | +| **NB-BSA + RNase inhibitor** | Nuclei Buffer (Parse Biosci.) | 3.15 ml | NA | +| (make 2 aliquots) | 7.5% BSA | 350 μl | 0.75% | +| | RNase inhibitor (Parse Biosc.)| 44.1 μl | | +| **RSB** | PBS | 24.6 ml | | +| | 7.5% BSA | 333 μl | 0.1% | +| | RNase inhibitor | 125 μl | 0.2 U/μl | +| | 1 M HEPES pH 7.3 | 150 μl | 10 mM | +| | 5 M NaCl | 30 μl | 10 mM | +| | 1 M MgCl2 | 45 μl | 3 mM | +| | 10% Tween-20 | 150 μl | 0.1% | +| **SHARE-RSB** | H2O | 14.625 ml | | +| | 7.5% BSA | 80.26 μl | 0.04% | +| | 5% digitonin | 30 μl | 0.01% | +| | Enzymatics RI | 37.5 μl | 0.1 U/μl | +| | SUPERase RI | 18.75 μl | 0.025 U/μl | +| | Yeast tRNA | 150 μl | 100 μg/ml | + +## Procedure + +### Setup + +1. Coat SHARE-seq nuclei prep tubes with BSA. + - Fill 8 1.5 ml tubes with 1.5 ml 1% BSA in H2O and incubate for 30 minutes. + - Aspirate BSA solution and dry for 30 minutes. Store at 4°C. + +2. Label tubes. +3. Pre-chill centrifuge to 4°C. +4. Prepare ice buckets. + +### Tissue Lysis and Nuclei Extraction + +5. Prepare 40 ml lysis buffer in a 50 ml conical tube on ice. + - Distribute 2.5 ml into 8 gentleMACS C Tubes on ice. + - Add 200 μl RNase inhibitor to the lysis buffer aliquot the day of the experiment. + +6. Prepare 25 ml RSB in a 50 ml conical tube on ice. + - Add 125 μl RNase inhibitor on the day of the experiment. + +7. Prepare 2 aliquots of 3.5 ml NB + BSA. + - Add 44.1 μl RNase inhibitor included in Parse Biosciences fixation kit the day of the experiment to each aliquot. + +8. Prepare 2.5 ml nuclei buffer + RNase inhibitor for final resuspension. + - Add 31.5 μl RNase inhibitor to 2.5 ml nuclei buffer. + +9. Prepare 15 ml SHARE-RSB in a 50 ml conical tube at room temperature. + - Add 30 μl digitonin, 37.5 μl Enzymatics RI, 18.75 μl SUPERase RI, and 150 μl yeast tRNA fresh the day of the experiment. + +10. Thaw components of 2 Parse Biosciences Nuclei Fixation v2 kits at room temperature, then place on ice. + +11. Distribute 20 μl NucBlue Fixed Cell ReadyProbes into 16 PCR strip tubes for cell counting. + - Need 8 tubes for counting after nuclei extraction, and another 8 tubes for final fixed nuclei. + +### Nuclei Isolation + +12. Keep flash frozen tissue samples on dry ice until lysis. + +13. Drop whole frozen tissue into a chilled gentleMACS C Tube with 2.5 ml lysis buffer. + - Close tubes firmly, invert immediately, and proceed to dissociation. + +14. Run the gentleMACS Program **4C_nuclei_1** on the Octo Dissociator (~5 minutes). + +15. Remove tubes, ensure tissue did not get stuck on sides, and spin down in a 4°C centrifuge for ~10 seconds. + - Place tubes back on ice. + +16. Filter nuclei suspension through 70 μm MACS SmartStrainer into a 5 ml tube. + - Fix a tube rack in ice for extra stability while filtering. + +17. Wash 70 μm MACS SmartStrainer with 2 ml additional lysis buffer. + - Cap and swirl to recover any nuclei stuck to the sides and cap of the C tubes, then wash the strainer. + +18. Discard the strainer and centrifuge the 4.5 ml nuclei suspension at 4°C, 350g for 5 minutes. + +19. Discard supernatant and resuspend nuclei pellet in 3 ml RSB. + +20. Filter nuclei suspension through 30 μm MACS SmartStrainer into a 5 ml tube. + +21. Dilute some nuclei 1:20 by adding 100 μl nuclei to 1.9 ml RSB in a new 5 ml tube. + - This should help the concentration reach around 4 million per ml. + +22. Count 1:20 diluted nuclei. + - Use a 1:5 dilution factor, 2 μl + 20 μl dye. Final dilution factor = 1:100. + +## Parse Nuclei Fixation + +23. Set aside 4 million nuclei in RSB in a new 5 ml tube and spin down at 4°C, 350g for 5 minutes. + +24. Remove supernatant and resuspend nuclei in 750 μl NB-BSA + RNase inhibitor. + - Filter through a 40 μm strainer (provided in Parse Biosciences kit) into a new 5 ml tube. + +25. Add 250 μl Nuclei Fixation Solution and mix 3 times. Do not over-mix. + +26. Incubate nuclei for 10 minutes on ice. + - Set 1 P200 pipette to 80 μl and keep the P1000 at 250 μl. + +27. Add 80 μl Nuclei Permeabilization Solution and mix by pipetting 3 times with the P1000 still set to 250 μl. + +28. Incubate 3 minutes with nuclei on ice. + +29. Add 4 ml Nuclei Neutralization Solution and invert the tube once to mix. + +30. Centrifuge at 4°C, 750g for 10 minutes. + +31. Aspirate and discard supernatant. + +32. Resuspend the samples in 300 μl Nuclei Buffer with RNase inhibitor (without BSA) and move through a 40 μm filter into a labeled 1.5 ml tube. + +33. Count nuclei. + - Use a 1:11 dilution factor, e.g., 2 μl + 20 μl dye. + +34. Add Nuclei DMSO: 5 μL and gently flick tubes to mix. + - One minute later, add another 5 μL and flick to mix, then after another minute add a final 5 μL for a total volume of 15 μL. + - Mix by gently pipetting 5x with a P200 set to 125 μL. + +35. Split nuclei suspension into 2 labeled tubes, 150 μL per tube. + +36. Place tubes in a Mr. Frosty at -80°C. + - The next day, move tubes to boxes in -80°C racks. + +## SHARE-seq Nuclei Fixation + +37. Set aside 1 million nuclei for each of the 8 samples in RSB and spin down at 4°C, 750g for 5 minutes. + +38. Remove supernatant and resuspend nuclei pellet in 1 ml room temperature SHARE-RSB. + - Transfer tube to a room temperature rack. + +39. At RT, add 13.34 μl of methanol-free formaldehyde (16% stock solution). + - Final concentration for nuclei: 0.2%. + - Close tube and nutate cells at RT for 5 minutes. + +40. To quench fixation, per reaction, add: + - 56.1 μl fresh 2.5M Glycine (0.94g per 5 ml stock) + - 50 μl of 1M Tris pH 8.0 + - 13.3 μl of 7.5% BSA + - Mix using a pipette. + - Incubate on ice for 10 minutes. + +41. Spin 750g, 4°C, 5 minutes. + - Gently remove supernatant. + +42. Add 200 μl of SHARE-RSB and gently resuspend pellet. + - Store on ice until all samples are completed. + +43. Pool 200 μl of resuspended nuclei from all 8 founders into 1 labeled 2 ml tube. + +44. Spin 1,000g, 4°C, 10 minutes. + - Gently remove supernatant. + - Remove all fluid and freeze at -80°C as a dry pellet. + +## Storage of Leftover Nuclei + +45. Move remaining nuclei in RSB on ice to labeled 2 ml tubes. + +46. Spin 750g, 4°C, 5 minutes. + +47. Remove all supernatant and flash-freeze nuclei as a dry pellet in liquid nitrogen. + - Store at -80°C. + +**endofoutput** +``` \ No newline at end of file diff --git a/markdown-output/protocol-to-isolate-and-fix-nuclei-from-flash-froz-c4p6yvre.md b/markdown-output/protocol-to-isolate-and-fix-nuclei-from-flash-froz-c4p6yvre.md new file mode 100644 index 0000000000000000000000000000000000000000..2659af1ef42dd8c67fc4210aa3571f164d0a7287 --- /dev/null +++ b/markdown-output/protocol-to-isolate-and-fix-nuclei-from-flash-froz-c4p6yvre.md @@ -0,0 +1,93 @@ +```markdown +## Protocol to Isolate and Fix Nuclei from Flash Frozen Female Mouse Gonads for IGVF + +**Elisabeth Rebboah1** +1University of California, Irvine + +### Goal/Experiment: +The goal of this experiment is to isolate nuclei from flash-frozen female mouse gonads (ovary and oviduct) from multiple founder strains, prepare a single nucleus suspension, and fix the nuclei for single-nucleus RNA sequencing (RNA-seq) using Parse Biosciences Evercode platform. + +### Abstract + +This protocol describes the isolation of nuclei from left and right 10-week-old mouse ovary and oviduct (pooled tissue ID: 26) from 8 founder strains (B6J, AJ, 129S1J, NZOJ, WSBJ, NODJ, PWKJ, and CASTJ), preparation of a single nucleus suspension, and fixation for single nucleus RNA-seq using Parse Biosciences. We process 1 replicate from each strain per day; e.g., female rep 1 across all 8 strains. The main products we use are Parse Biosciences Nuclei Fixation Kit (v2) and Miltenyi Biotec’s gentleMACS Octo Dissociator with accessories. This protocol takes about 3.5 hours from start to finish. + +The results are 2 aliquots of fixed single-nucleus suspensions for Parse per each of the 8 samples at >= 2,500 nuclei/µl. The first part of the protocol describes tissue lysis and nuclei extraction using Miltenyi Biotec’s gentleMACS Octo Dissociator. The second part describes nuclei fixation using Parse Biosciences Evercode Nuclei Fixation Kit with v2 reagents. + +### Guidelines +1. We recommend using a 5 ml pipette for aspirations and resuspensions > 1 ml. +2. Record everything in the [IGVF spreadsheet](#), “Samples into experiment” tab. + +### Materials + +| Name | Manufacturer | Cat. # | +|---------------------------------|--------------------|---------------| +| Nuclei Fixation Kit v2 | Parse Biosciences | ECF2003 | +| Nuclei Extraction Buffer | Miltenyi Biotec | 130-128-024 | +| RNase Inhibitor, murine | New England Biolabs| M0314L | +| PBS | HyClone | SH30256.02 | +| 7.5% BSA | Life Technologies | 15260037 | +| gentleMACS C Tube | Miltenyi Biotec | 130-093-237 | +| gentleMACS Octo Dissociator | Miltenyi Biotec | 130-095-937 | +| MACS SmartStrainers (30 μm) | Miltenyi Biotec | 130-098-458 | +| NucBlue Fixed Cell ReadyProbes | Thermo Fisher | R37606 | +| Hemacytometer | Fisher Scientific | 02-671-51B | +| Mr. Frosty | Sigma-Aldrich | 635639 | + +### Buffers + +| Name | Reagent | Volume (for 8 samples) | Final Concentration | +|----------------------------------|-------------------------------------|------------------------|---------------------| +| Lysis buffer | Nuclei Extraction Buffer | 35 ml | NA | +| | 40 U/µl RNase inhibitor | 175 µl | 0.2 U/µl | +| NB-BSA + RNase inhibitor | Nuclei Buffer (Parse Biosciences) | 3.15 ml | NA | +| | 7.5% BSA | 350 µl | 0.75% | +| | RNase inhibitor (Parse Biosciences) | 44.1 µl | | + +### Setup + +1. Label tubes. +2. Pre-chill centrifuge to 4°C. +3. Prepare ice buckets. +4. Prepare 35 ml lysis buffer on ice in a 50 ml conical tube. Distribute 2 ml into 8 gentleMACS C Tubes on ice. Add 175 µl RNase inhibitor to the lysis buffer aliquot the day of the experiment. +5. Prepare 3.5 ml NB + BSA. Add 44.1 µl RNase inhibitor included in the Parse Biosciences fixation kit the day of the experiment. +6. Prepare 1.5 ml nuclei buffer + RNase inhibitor for final resuspension. Add 18.6 µl RNase inhibitor to 1.5 ml nuclei buffer. +7. Thaw components of 1 Parse Biosciences Nuclei Fixation v2 kit at room temperature, then place on ice. +8. Distribute 20 µl NucBlue Fixed Cell ReadyProbes into 16 PCR strip tubes for cell counting. Need 8 tubes for counting after nuclei extraction, and another 8 tubes for final fixed nuclei. + +### Tissue Lysis and Nuclei Extraction + +9. Keep flash frozen tissue samples on dry ice until lysis. +10. Drop whole frozen tissue into a chilled gentleMACS C Tube with 2 ml lysis buffer. Close tubes firmly and invert immediately, ensuring the tissue is not stuck to the bottom or side. Keep tubes on ice and proceed immediately to dissociation. There should be 4 pieces: left and right ovary and left and right oviduct. +11. Run the gentleMACS Program 4C_nuclei_1 on the Octo Dissociator (~5 minutes). +12. Remove tubes, ensuring tissue did not get stuck on the sides, and spin down in a 4°C centrifuge for ~10 seconds to bring liquid to the bottom, then place tubes back on ice. +13. Filter nuclei suspension through 30 µm MACS SmartStrainer into a 5 ml tube. Fit a tube rack in ice for extra stability while filtering. +14. Wash 30 µm MACS SmartStrainer with 2 ml additional lysis buffer. Add 2 ml to C tubes, cap, and swish to recover any nuclei stuck to the sides and cap of the C tubes, then wash the strainer. +15. Discard strainer and centrifuge the 4 ml nuclei suspension at 4°C, 350g for 5 minutes. +16. Discard supernatant and resuspend nuclei pellet in 750 µl NB-BSA + RNase inhibitor and filter through a 40 µm strainer (provided in Parse Biosciences kit) into a new 5 ml tube. In our experience, total number of nuclei from female gonads is around 4 million. +17. Count nuclei. Use 1:6 dilution factor, 4 µl + 20 µl dye. + +### Parse Nuclei Fixation + +18. Add 250 µl Nuclei Fixation Solution and mix 3 times. Do not over-mix. +19. Incubate nuclei for 10 minutes on ice. Set 1 P200 pipette to 80 µl and keep the P1000 at 250 µl. +20. Add 80 µl Nuclei Permeabilization Solution and mix by pipetting 3 times with the P1000 still set to 250 µl. Do not over-mix. +21. Incubate 3 minutes with nuclei on ice. +22. Add 4 ml Nuclei Neutralization Solution and invert the tube once to mix. +23. Centrifuge at 4°C, 750g for 10 minutes. +24. Aspirate and discard the supernatant. +25. Resuspend the samples that started with < 2 million nuclei in 150 µl Nuclei Buffer with RNase inhibitor without BSA and transfer to a labeled 1.5 ml tube. Do NOT filter, will lose too much volume. For samples starting with > 2 million nuclei, resuspend in 300 µl Nuclei Buffer and filter. +26. Count nuclei. Use a 1:11 dilution factor, e.g., 2 µl + 20 µl dye. + +### Storage of Leftover Nuclei + +27. Add Nuclei DMSO: 2.5 µl into 150 µl samples and gently flick tubes to mix. One minute later, add another 2.5 µl and flick to mix, then after another minute add a final 2.5 µl for a total volume of 7.5 µl. Mix by gently pipetting 5x with a P200 set to 75 µl. +28. Split nuclei suspension into 2 aliquots of equal volume in labeled 1.5 ml tubes. (E.g., if resuspend with 150 µl, split nuclei suspension into 75 µl per tube.) +29. Place tubes in a Mr. Frosty at -80°C. The next day, move tubes to boxes in -80°C racks. + +#### Storage of Leftover Nuclei +30. Move remaining nuclei in RSB on ice to labeled 2 ml tubes. +31. Spin 750g, 4°C, 5 minutes. +32. Remove all supernatant and flash-freeze nuclei as a dry pellet in liquid nitrogen. Store at -80°C. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/protocol-to-isolate-and-fix-nuclei-from-flash-froz-c4puyvnw.md b/markdown-output/protocol-to-isolate-and-fix-nuclei-from-flash-froz-c4puyvnw.md new file mode 100644 index 0000000000000000000000000000000000000000..1656c522551b7dff09a450743e034520d2807746 --- /dev/null +++ b/markdown-output/protocol-to-isolate-and-fix-nuclei-from-flash-froz-c4puyvnw.md @@ -0,0 +1,223 @@ +```markdown +# Goal/Experiment: +Isolation and fixation of nuclei from flash frozen mouse liver tissue of eight founder strains for single nucleus RNA-seq and nuclei RNA-seq + ATAC-seq. + +# Protocol to Isolate and Fix Nuclei from Flash Frozen Mouse Liver for IGVF V.2 +**Elisabeth Rebboah** +*University of California, Irvine* + +**Abstract** +This protocol describes isolation of nuclei from 10-week-old mouse liver (tissue ID: 05) from 8 founder strains (B6J, AJ, 129S1J, NZOJ, WSBJ, NODJ, PWKJ, and CASTJ), preparation of single nucleus suspension, and fixation for: +1. Single nucleus RNA-seq using the Parse Biosciences protocol (Split-seq). +2. Single nucleus RNA-seq + ATAC-seq using the SHARE-seq protocol. + +The results for each strain include: +- 2 aliquots of fixed single-nucleus suspensions for Parse at ≥ 2,500 nuclei/µl +- 1 fixed nuclei pellet pooled across all 8 strains for SHARE-seq + +All samples are stored at -80°C. + +## Guidelines +- Use a 5 ml pipette for aspirations and resuspensions > 1 ml. +- Cut frozen livers ahead of time on dry ice to make lysis easier. +- To remove frozen chopped tissues from tubes, use forceps or a 1 ml tip. +- Record everything in the IGVF spreadsheet, “Samples into experiment” tab. +- After nuclei isolation and first round of counting, 2 technicians should continue with Parse fixation, while 1 technician proceeds with SHARE-seq fixation. + +## Materials + +| Name | Manufacturer | Cat. # | +|---------------------------------|----------------------|---------------| +| Nuclei Fixation Kit v2 | Parse Biosciences | ECF2003 | +| Nuclei Extraction Buffer | Miltenyi Biotec | 130-128-024 | +| RNase Inhibitor, murine | New England Biolabs | M0314L | +| PBS | HyClone | SH30256.02 | +| 7.5% BSA | Life Technologies | 15260037 | +| 1 M HEPES pH 7.3 | Sigma | H0887-100ml | +| NaCl | Fisher | BP358-1 | +| MgCl2 | Fisher | AA12315A7 | +| Tween-20 | Fisher | BP337-500 | +| 5% digitonin | Promega | G944A | +| Enzymatics RI | Enzymatics | Y9240L | +| SUPERase RI | Invitrogen | AM2696 | +| Yeast tRNA | Invitrogen | AM7119 | +| Glycine | Fisher | BP381-500 | +| 1M Tris pH 8.0 | Thermo | AM9855G | +| Formaldehyde (methanol-free) | EMS | 15710 | +| gentleMACS C Tube | Miltenyi Biotec | 130-093-237 | +| gentleMACS Octo Dissociator | Miltenyi Biotec | 130-095-937 | +| MACS SmartStrainer (70 µm) | Miltenyi Biotec | 130-110-916 | +| MACS SmartStrainer (30 µm) | Miltenyi Biotec | 130-098-458 | +| NucBlue Fixed Cell ReadyProbes | Thermo Fisher | R37606 | +| Hemacytometer | Fisher Scientific | 02-671-51B | +| Mr. Frosty | Sigma-Aldrich | 635639 | + +### Reagents Preparation + +**1% BSA-DEPC** + +| Reagent | Volume (for 8 samples) | Final concentration | +|------------|------------------------|---------------------| +| BSA | 1 g | 1% | +| DEPC water | 100 ml | | + +**Lysis buffer** + +| Reagent | Volume (for 8 samples) | Final concentration | +|----------------------|------------------------|---------------------| +| Nuclei Extraction Buffer | 50 ml | | +| RNase Inhibitor 40 U/µl | 250 µl | 0.2 U/µl | + +**NB-BSA + RNase inhibitor** (Prepare 2 aliquots) +| Reagent | Volume (for 8 samples) | Final concentration | +|-----------------------------------|------------------------|---------------------| +| Nuclei Buffer (Parse Biosciences) | 3.15 ml | NA | +| 7.5% BSA | 350 µl | 0.75% | +| RNase inhibitor (Parse Biosciences)| 44.1 µl | | + +**RSB** (Prepare 2 aliquots of 35 ml) +| Reagent | Volume (for 8 samples) | Final concentration | +|--------------|------------------------|---------------------| +| PBS | 35 ml | | +| 7.5% BSA | 467 µl | 0.1% | + +**SHARE-RSB** +| Reagent | Volume (for 8 samples) | Final concentration | +|-------------------------|------------------------|---------------------| +| RNase inhibitor | 175 µl | 0.2 U/µl | +| 1 M HEPES pH 7.3 | 150 µl | 10 mM | +| 5 M NaCl | 30 µl | 10 mM | +| 1 M MgCl2 | 45 µl | 3 mM | +| 10% Tween-20 | 150 µl | 0.1% | +| H2O | 14.625 ml | | +| 7.5% BSA | 80.26 µl | 0.04% | +| 5% digitonin | 30 µl | 0.01% | +| Enzymatics RI | 37.5 µl | 0.1 U/µl | +| SUPERase RI | 18.75 µl | 0.025 U/µl | +| Yeast tRNA | 150 µl | 100 µg/ml | + +## Setup + +1. **Coat SHARE-seq nuclei prep tubes with BSA**: + - Fill 8 1.5 ml tubes with *1.5 ml 1% BSA* in H2O. + - Incubate for *30 minutes*. + - Aspirate BSA solution and dry for *30 minutes*. + - Store at *4°C*. + +2. **Label tubes.** + +3. **Pre-chill centrifuge** to *4°C*. + +4. **Prepare ice buckets.** + +5. **Prepare 50 ml lysis buffer** in a 50 ml conical tube on ice. + - Distribute 3.5 ml into 8 gentleMACS C Tubes on ice. + - For NZO mice, use 4 ml lysis buffer. + - Add 250 µl RNase inhibitor to the lysis buffer aliquot the day of the experiment. + +6. **Prepare 70 ml RSB** in 2 50 ml conical tubes on ice. + - 35 ml per tube. + - Add *175 µl RNase inhibitor* to each tube the day of the experiment. + +7. **Prepare 2 aliquots of 3.5 ml NB + BSA**: + - Add *44.1 µl RNase inhibitor* included in Parse Biosciences fixation kit the day of the experiment to each aliquot. + +8. **Prepare 2.5 ml nuclei buffer + RNase inhibitor**: + - Add *31.5 µl RNase inhibitor* to 2.5 ml nuclei buffer. + +9. **Prepare 15 ml SHARE-RSB** in a 50 ml conical tube at room temperature. + - To SHARE-RSB, add *30 µl digitonin*, *37.5 µl Enzymatics RI*, *18.75 µl SUPERase RI*, and *150 µl yeast tRNA* fresh the day of the experiment. + +10. **Thaw components of 2 Parse Biosciences Nuclei Fixation v2 kits** at room temperature, then place on ice. + +11. **Distribute 20 µl NucBlue Fixed Cell ReadyProbes** into 16 PCR strip tubes for cell counting. + - Need 8 tubes for counting after nuclei extraction, and another 8 tubes for final fixed nuclei. + +## Tissue Sectioning +12. Keep flash-frozen tissue samples on dry ice. + +13. Tilt frozen tissues into a plastic Petri dish on dry ice. + +14. Using a clean razor blade, roughly chop tissue into pieces ~500 mg. Not necessary to chop tissues <500 mg. + +15. Using clean forceps, move chopped, frozen tissue back to the original 2 ml tube. Tissue should never be thawed during this process. + +## Tissue Lysis and Nuclei Extraction +16. Keep chopped flash-frozen tissue samples on dry ice until lysis. + +17. Drop whole frozen tissue into a chilled gentleMACS C Tube with 3.5 ml lysis buffer. For NZO mice, use 4 ml lysis buffer. Close tubes firmly and invert immediately, ensuring tissue is not stuck to the bottom or side. Keep tubes on ice and proceed immediately to dissociation. + +18. Run the gentleMACS Program 4C_nuclei_1 on the Octo Dissociator (~5 minutes). + +19. Remove tubes, ensuring tissue did not get stuck on the sides, and spin down in a 4°C centrifuge for ~10 seconds to bring liquid to the bottom. Place tubes back on ice. + +20. Filter nuclei suspension through 70 µm MACS SmartStrainer into a 5 ml tube. Fit a tube rack in ice for extra stability while filtering. + +21. Wash 70 µm MACS SmartStrainer with 2.5 ml additional lysis buffer. Add 2.5 ml to C tubes, cap, and swish to recover any nuclei stuck to the sides and cap of the C tubes, then wash the strainer. + +22. Discard strainer and centrifuge the 6 ml nuclei suspension at 4°C, 350g for 5 minutes. + +23. Discard supernatant and resuspend nuclei pellet in 3.5 ml RSB. + +24. Filter nuclei suspension through 30 µm MACS SmartStrainer into a 5 ml tube. + +25. Dilute some nuclei 1:10 by adding 500 µl nuclei to 4.5 ml RSB in a new 5 ml tube. This should help the concentration reach around 4 million per ml. + +26. Count 1:10 diluted nuclei. Use a 1:5 dilution factor, 5 µl + 20 µl dye. Final dilution factor = 1:50. + +## Parse Nuclei Fixation +27. Set aside 4 million nuclei in RSB in a new 5 ml tube and spin down at 4°C, 350g for 5 minutes. + +28. Remove supernatant and resuspend nuclei in 750 µl NB-BSA + RNase inhibitor and filter through a 40 µm strainer (provided in Parse Biosciences kit) into a new 5 ml tube. + +29. Add 250 µl Nuclei Fixation Solution and mix 3 times. Do not over mix. + +30. Incubate nuclei for 10 minutes on ice. Set 1 P200 pipette to 80 µl and keep the P1000 at 250 µl. + +31. Add 80 µl Nuclei Permeabilization Solution and mix by pipetting 3 times with the P1000 still set to 250 µL. Do not over-mix. + +32. Incubate 3 minutes with nuclei on ice. + +33. Add 4 ml Nuclei Neutralization Solution and invert the tube once to mix. + +34. Centrifuge at 4°C, 750g for 10 minutes. + +35. Aspirate and discard the supernatant. + +36. Resuspend the samples in 300 µl Nuclei Buffer with RNase inhibitor without BSA and move through a 40 µm filter into a labeled 1.5 ml tube. + +37. Count nuclei. Use a 1:11 dilution factor, e.g., 2 µl + 20 µl dye. + +38. Add Nuclei DMSO: 5 µL and gently flick tubes to mix. One minute later, add another 5 µL and flick to mix, then after another minute add a final 5 µL for a total volume of 15 µL. Mix by gently pipetting 5 times with a P200 set to 150 µL. + +## SHARE-seq Nuclei Fixation +39. Split nuclei suspension into 2 labeled tubes, 150 µl per tube. + +40. Place tubes in a Mr. Frosty at -80°C. The next day, move tubes to boxes in -80°C racks. + +41. Set aside 1 million nuclei for each of the 8 samples in RSB and spin down at 4°C, 750g for 5 minutes. + +42. Remove supernatant and resuspend the nuclei pellet in 1 ml room temperature SHARE-RSB. Transfer tube to a room temperature rack. + +43. At room temperature, add 13.34 µl of methanol-free formaldehyde (16% stock solution). Final concentration for nuclei: 0.2%. Close the tube and nutate cells at room temperature for 5 minutes. + +44. To quench fixation, per reaction, add 56.1 µl fresh 2.5M Glycine (0.94g per 5 ml stock), 50 µl of 1M Tris pH 8.0, 13.3µl of 7.5% BSA, and mix using a pipette. Incubate on ice for 10 minutes. + +45. Spin 750g, 4°C, 5 minutes. Gently remove the supernatant. + +46. Add 200 µl of SHARE-RSB and gently resuspend the pellet. Store on ice until all samples are completed. + +47. Pool 200 µl of resuspended nuclei from all 8 founders into 1 labeled 2 ml tube. + +48. Spin 1,000g, 4°C, 10 minutes. Gently remove the supernatant. Remove all fluid and freeze at -80°C as a dry pellet. + +## Storage of Leftover Nuclei + +49. Move remaining nuclei in RSB on ice to labeled 2 ml tubes. + +50. Spin 750g, 4°C, 5 minutes. + +51. Remove all supernatant and flash-freeze nuclei as a dry pellet in liquid nitrogen. Store at -80°C. + +__endofoutput__ +``` \ No newline at end of file diff --git a/markdown-output/protocol-to-isolate-and-fix-nuclei-from-flash-froz-c4pyyvpw.md b/markdown-output/protocol-to-isolate-and-fix-nuclei-from-flash-froz-c4pyyvpw.md new file mode 100644 index 0000000000000000000000000000000000000000..f719609e3f1147acaa0c06d200491ffaedf54fe3 --- /dev/null +++ b/markdown-output/protocol-to-isolate-and-fix-nuclei-from-flash-froz-c4pyyvpw.md @@ -0,0 +1,88 @@ +```markdown +# Protocol to Isolate and Fix Nuclei from Flash Frozen Mouse Adrenal Glands for IGVF V.2 + +**Elisabeth Rebboah** +University of California, Irvine + +**Version 2** +**Date:** Nov 08, 2023 + +**Attachments** + +- [Eevercode Fixation v2.0.24 User Manual](SO+10122022+Evercode+Fixation+v2.0.24+User+Manual.pdf) + +## Goal/Experiment +The goal of this protocol is to describe the isolation of nuclei from left and right 10-week-old mouse adrenal glands from 8 founder strains, preparation of a single nucleus suspension, and fixation for single nucleus RNA-seq using Parse Biosciences. + +## Abstract +This protocol describes isolation of nuclei from left and right 10-week-old mouse adrenal glands from 8 founder strains (B6J, AJ, 129S1J, NZOJ, WSBJ, NODJ, PWKJ, and CASTJ), preparation of a single nucleus suspension, and fixation for single nucleus RNA-seq using Parse Biosciences. The main products used are Parse Biosciences Nuclei Fixation Kit (v2) and Miltenyi Biotec’s gentleMACS Octo Dissociator with accessories. This protocol takes about 3.5 hours from start to finish. +The results are 2 aliquots of fixed single-nucleus suspensions for Parse per each of the 8 samples at >= 2,500 nuclei/μL. + +## Guidelines +1. Use a 5 mL pipette for aspirations and resuspensions > 1 mL. +2. Record everything in the IGVF spreadsheet, "Samples into experiment" tab. + +## Materials +| Name | Manufacturer | Cat. # | +| ---------------------------- | ------------------ | -------------- | +| Nuclei Fixation Kit v2 | Parse Biosciences | ECF2003 | +| Nuclei Extraction Buffer | Miltenyi Biotec | 130-128-024 | +| RNase Inhibitor, murine | New England Biolabs| M0314L | +| PBS | HyClone | SH30256.02 | +| 7.5% BSA | Life Technologies | 15260037 | +| gentleMACS C Tube | Miltenyi Biotec | 130-093-237 | +| gentleMACS Octo Dissociator | Miltenyi Biotec | 130-095-937 | +| MACS SmartStrainers (30 um) | Miltenyi Biotec | 130-098-458 | +| NucBlue Fixed Cell ReadyProbes | Thermo Fisher | R37606 | +| Hemacytometer | Fisher Scientific | 02-671-51B | +| Mr. Frosty | Sigma-Aldrich | 635639 | + +**Reagents, Equipment, Manufacturer and Catalog Number** + +| Name | Reagent | Volume (for 8 samples) | Final Concentration | +| ------------------------ | ------------------------- | ---------------------- | -------------------- | +| **Lysis buffer** | | | | +| | Nuclei Extraction Buffer | 35 mL | NA | +| | 40 U/μL RNase inhibitor | 175 μL | 0.2 U/μL | +| **NB-BSA + RNase inhibitor** | | | | +| | Nuclei Buffer (Parse Biosciences) | 3.15 mL | NA | +| | 7.5% BSA | 350 μL | 0.75% | +| | RNase inhibitor (Parse Biosciences) | 44.1 μL | + +## Setup +1. Label tubes. +2. Pre-chill centrifuge to 4°C. +3. Prepare ice buckets. +4. Prepare 35 mL lysis buffer on ice in a 50 mL conical tube. Distribute 2 mL into 8 gentleMACS C Tubes on ice. Add 175 μL RNase inhibitor to the lysis buffer aliquot the day of the experiment. +5. Prepare 3.5 mL NB + BSA. Add 44.1 μL RNase inhibitor included in Parse Biosciences fixation kit the day of the experiment. +6. Prepare 1.5 mL nuclei buffer + RNase inhibitor for final resuspension. Add 18.6 μL RNase inhibitor to 1.5 mL nuclei buffer. +7. Thaw components of 1 Parse Biosciences Nuclei Fixation v2 kit at room temperature, then place on ice. +8. Distribute 20 μL NucBlue Fixed Cell ReadyProbes into 16 PCR strip tubes for cell counting. + +## Tissue Lysis and Nuclei Extraction +9. Keep flash-frozen tissue samples on dry ice until lysis. +10. Drop whole frozen tissue into a chilled gentleMACS C Tube with 2 mL lysis buffer. Keep tubes on ice and proceed immediately to dissociation. There should be 2 adrenal glands. +11. Run the gentleMACS Program `4C_nuclei_1` on the Octo Dissociator (∼5 minutes). +12. Remove tubes and spin down in a 4°C centrifuge for ~10 seconds to bring liquid to the bottom, then place tubes back on ice. +13. Filter nuclei suspension through 30 μm MACS SmartStrainer into a 5 mL tube. +14. Wash 30 μm MACS SmartStrainer with 2 mL additional lysis buffer. +15. Centrifuge the 4 mL nuclei suspension at 4°C, 350g for 5 minutes. +16. Resuspend nuclei pellet in 375 μL NB-BSA + RNase inhibitor and filter through a 40 μm strainer into a new 5 mL tube. +17. Count nuclei. Use a 1:6 dilution factor, 4 μL + 20 μL dye. + +## Parse Nuclei Fixation +18. Add 125 μL Nuclei Fixation Solution to the filtered nuclei in 375 μL and mix 3 times. Do not over-mix. +19. Incubate nuclei for 10 minutes on ice. +20. Add 40 μL Nuclei Permeabilization Solution and mix by pipetting. +21. Incubate 3 minutes with nuclei on ice. +22. Add 2 mL Nuclei Neutralization Solution and invert the tube once to mix. +23. Centrifuge at 4°C, 750g for 10 minutes. +24. Aspirate and discard supernatant. +25. Resuspend the samples in 150 μL Nuclei Buffer with RNase inhibitor and filter through a 40 μm filter into a labeled 1.5 mL tube. Do not filter. +26. Count nuclei. Use a 1:11 dilution factor, e.g., 2 μL + 20 μL dye. +27. Add Nuclei DMSO: 2.5 μL into 150 μL samples and gently flick tubes to mix. +28. Split nuclei suspension into 2 labeled tubes, 75 μL per tube. +29. Place tubes in a Mr. Frosty for storage at -80°C. The next day, move tubes to boxes in -80°C racks. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/protocol-to-isolate-and-fix-nuclei-from-flash-froz-cy68xzhw.md b/markdown-output/protocol-to-isolate-and-fix-nuclei-from-flash-froz-cy68xzhw.md new file mode 100644 index 0000000000000000000000000000000000000000..ed002b70f381e741b029e455a011ebf19dee6e10 --- /dev/null +++ b/markdown-output/protocol-to-isolate-and-fix-nuclei-from-flash-froz-cy68xzhw.md @@ -0,0 +1,107 @@ +```markdown +# Goal/Experiment: +The goal of this experiment is to isolate and fix nuclei from flash-frozen mouse tibialis anterior muscle tissue for single nucleus RNA sequencing (RNA-seq) using the Parse Biosciences protocol. + +## Protocol to Isolate and Fix Nuclei from Flash Frozen Mouse Tibialis Anterior for IGVF V.1 +**Elisabeth Rebboah** +University of California, Irvine + +**Version:** 1 +**Date Published:** August 25, 2023 +**Protocol ID:** 86976 +**Open Access:** [Creative Commons Attribution License](https://protocols.io/view/protocol-to-isolate-and-fix-nuclei-from-flash-froz-cy68xzhw) +**Protocol Status:** Working +**License:** [URL](https://protocols.io/view/protocol-to-isolate-and-fix-nuclei-from-flash-froz-cy68xzhw) + +### Abstract +This protocol describes isolation of nuclei from 10-week-old mouse tibialis anterior from 8 founder strains (B6J, AJ, 129S1J, NZOJ, WSBJ, NODJ, PWKJ, and CASTJ), preparation of a single nucleus suspension, and fixation for single nucleus RNA-seq using the Parse Biosciences protocol. We process 1 replicate from each strain per day. Reagents include Parse Biosciences Nuclei Fixation Kit (v2) and Miltenyi Biotec's gentleMACS Octo Dissociator with accessories. This protocol takes about 3.5 hours from start to finish. The results are 2 aliquots of fixed single-nucleus suspensions for Parse per each of the 8 samples. + +### Guidelines +1. Tilt the tube and slowly add PBS during debris removal. Ideally, the cloudy debris is only in the band rather than the nuclei layer. +2. Use a 5 mL pipette for aspirations and resuspensions > 1 mL. +3. Record everything in the [IGVF spreadsheet](https://example.com), “Samples into experiment” tab. + +### Materials + +| Name | Manufacturer | Cat. # | +| ----------------------------- | ------------------- | ------------- | +| Nuclei Fixation Kit v2 | Parse Biosciences | ECF2003 | +| Nuclei Extraction Buffer | Miltenyi Biotec | 130-128-024 | +| RNase Inhibitor, murine | New England Biolabs | M0314L | +| PBS | HyClone | SH30256.02 | +| Debris Removal Solution | Miltenyi Biotec | 130-109-398 | +| 7.5% BSA | Life Technologies | 15260037 | +| gentleMACS C Tube | Miltenyi Biotec | 130-093-237 | +| gentleMACS Octo Dissociator | Miltenyi Biotec | 130-095-937 | +| MACS SmartStrainers (70 µm) | Miltenyi Biotec | 130-110-916 | +| MACS SmartStrainers (30 µm) | Miltenyi Biotec | 130-098-458 | +| NucBlue Fixed Cell ReadyProbes| Thermo Fisher | R37606 | +| Hemacytometer | Fisher Scientific | 02-671-51B | +| Mr. Frosty | Sigma-Aldrich | 635639 | + +#### Reagents and Buffers + +| Name | Reagent | Volume (for 8 samples) | Final Concentration | +| --------------- | ---------------------------------------- | ---------------------- | -------------------- | +| **Lysis buffer**| Nuclei Extraction Buffer | 40 ml | NA | +| | 40 U/µl RNase inhibitor | 200 µl | 0.2 U/µl | +| **PBS** | PBS | 25 ml | NA | +| **Debris Removal Solution (DRS)** | Debris Removal Solution (Miltenyi) | 8 ml | NA | +| **NB + BSA** | Nuclei Buffer (Parse Biosciences) | 3.6 ml | NA | +| | 7.5% BSA | 400 µl | 0.75% | +| | RNase inhibitor (Parse Biosciences) | 20 µl | NA | +| **RSB** | PBS | 24.6 ml | NA | +| | 7.5% BSA | 333 µl | 0.1% | +| | RNase inhibitor | 125 µl | 0.2 U/µl | + +### Setup + +1. Pre-chill centrifuge to 4°C. +2. Prepare 2 large ice buckets. +3. Prepare lysis buffer on ice in a 50 mL conical tube. Distribute 2.5 mL into 8 gentleMACS C Tubes on ice. +4. Prepare 4 mL NB + BSA + RNase inhibitor in a 5 mL tube. +5. Prepare 25 mL RSB on ice in a 50 mL conical tube (need ~3 mL per sample). +6. Take an aliquot of PBS out of 4°C and keep on ice. +7. Take an aliquot of Debris Removal Solution out of 4°C and keep on ice. +8. Thaw components of 1 Parse Biosciences Nuclei Fixation kit at room temperature, then place on ice. +9. Distribute 10 µl NucBlue Fixed Cell ReadyProbes into 16 PCR strip tubes for cell counting. Need 8 tubes for counting after nuclei extraction, and another 8 tubes for final fixed nuclei. + +### Tissue Lysis and Nuclei Extraction + +10. Keep flash frozen tissue samples on dry ice until lysis. +11. Drop whole frozen tissue into a chilled gentleMACS C Tube with 2.5 mL lysis buffer. Close tubes firmly and invert immediately, ensuring tissue is not stuck to the bottom or side. Keep tubes on ice and proceed immediately to dissociation. +12. Run the gentleMACS Program 4C_nuclei_1 on the Octo Dissociator (~5 minutes). +13. Remove tubes, ensuring tissue did not get stuck on the sides, and spin down in a 4°C centrifuge for ~10 seconds to bring liquid to the bottom, then place tubes back on ice. +14. Filter nuclei suspension through 70 µm MACS SmartStrainer into a 5 mL tube, fit a tube rack in ice for extra stability while filtering. +15. Wash 70 µm MACS SmartStrainer with 2 mL additional lysis buffer. Add 2 mL to C tubes, cap, and swish to recover any nuclei stuck to the sides and cap of the C tubes, then wash the strainer. +16. Discard strainer and centrifuge the 4.5 mL nuclei suspension at 4°C, 350g for 5 minutes. +17. Resuspend nuclei pellet in 3.1 mL PBS. +18. Filter nuclei suspension through 30 µm MACS SmartStrainer into a 15 mL tube. +19. Add 900 µl Debris Removal Solution and mix by pipetting 10 times slowly up and down using a 5 mL pipette. +20. Overlay with 4 mL PBS using a P1000 or 5 mL pipette. Tilt tube 45 degrees and slowly add the first mL. You can increase speed after the first mL of PBS is added. +21. Centrifuge at 4°C, 3000g for 10 minutes with full acceleration and no brake. Three phases are formed: top clear buffer layer, cloudy debris band, and clear layer containing nuclei. Pellet usually not visible. +22. Aspirate the two top phases (buffer layer and all cloudy debris band) and discard. Aspirate the first phase, then the second phase. Stay above the third layer of nuclei to prevent loss. +23. Fill with cold RSB to a final volume of 5 mL. +24. Gently invert the tube three times. Do not vortex. +25. Centrifuge at 4°C, 1000g for 10 minutes with full acceleration and full brake. +26. Aspirate supernatant completely. +27. Resuspend cells carefully in 375 µl NB + BSA + RNase inhibitor and filter through a 40 µm strainer into a new 5 mL tube. +28. Count nuclei. Use a 1:2 dilution factor, 10 µl + 10 µl dye. + +### Nuclei Fixation + +29. Add 125 µL Nuclei Fixation Solution to the filtered nuclei in 375 µL and mix 3 times. Do not over-mix. +30. Incubate nuclei for 10 minutes on ice. Set 2 P200 pipettes to 40 µL and 250 µL. +31. Add 40 µL Nuclei Permeabilization Solution and mix by pipetting 3 times with the P200 still set to 250 µL. Do not over-mix. +32. Incubate 3 minutes with nuclei on ice. +33. Add 2 mL Nuclei Neutralization Solution and invert the tube once to mix. +34. Centrifuge at 4°C, 750g for 10 minutes. +35. Aspirate and discard supernatant. +36. Resuspend the samples in 100 µL Nuclei Buffer without BSA. If there is excessive debris, and we can afford to add more volume (≥ 100 µL), then filter through a 40 µm strainer into a new 1.5 mL tube. With less than 200 µL, too much of the sample gets lost in the filter. +37. Take a 10 µL aliquot to dilute 1:2 with prepared 10 µL DAPI to manually count with a hemacytometer and record numbers. +38. Count nuclei. Use a 1:2 dilution factor, 10 µL + 10 µL dye. +39. Add Nuclei DMSO: 1.7 µL into 100 µL samples and gently flick tubes to mix. One minute later, add another 1.7 µL and flick to mix, then after another minute add a final 1.7 µL for a total volume of ~5 µL. Mix by gently pipetting 5x. +40. Place tubes in a Mr. Frosty for storage at -80°C. The next day, move tubes to boxes in -80°C racks. + +### endofoutput +``` \ No newline at end of file diff --git a/markdown-output/protocol-to-isolate-and-fix-nuclei-from-flash-froz-cy7fxzjn.md b/markdown-output/protocol-to-isolate-and-fix-nuclei-from-flash-froz-cy7fxzjn.md new file mode 100644 index 0000000000000000000000000000000000000000..241e487a5deeb53a81e9d239d3500c88d674e8ab --- /dev/null +++ b/markdown-output/protocol-to-isolate-and-fix-nuclei-from-flash-froz-cy7fxzjn.md @@ -0,0 +1,219 @@ +```markdown +# Goal/Experiment: + +The goal of this experiment is to isolate and fix nuclei from flash-frozen mouse hypothalamus and pituitary gland for IGVF. + +# Protocol to Isolate and Fix Nuclei from Flash Frozen Mouse Hypothalamus and Pituitary Gland for IGVF + +## Abstract +This protocol describes the isolation of nuclei from 10-week-old mouse hypothalamus and pituitary gland from 8 founder strains (B6J, AJ, 129S1J, NZOJ, WSBJ, NODJ, PWKJ, and CASTJ), preparation of a single nucleus suspension, and fixation for single nucleus RNA-seq using Parse Biosciences and SHARE-seq using the Broad Institute protocol. Each experiment takes about 3.5 hours from start to finish, resulting in 2 aliquots of fixed single-nucleus suspensions for Parse per each of the 8 samples, and one fixed nuclei pellet pooled across all 8 strains for SHARE-seq. + +## Guidelines + +1. Use a 5 ml pipette for aspirations and resuspensions > 1 ml. +2. Record everything in the [IGVF spreadsheet](https://example.com), "Samples into experiment" tab. +3. Fixate with Parse Biosciences once nuclei isolation and the first round of counting are done. This involves setting the volume to about 4 million nuclei per sample, processing about 4 samples per technician. The exact volume needed for 1 million cells should be determined and proceeded with SHARE-seq fixation. + +## Materials + +| Name | Manufacturer | Cat. # | +|----------------------------------|-----------------------|--------------| +| Nuclei Fixation Kit v2 | Parse Biosciences | ECF2003 | +| Nuclei Extraction Buffer | Miltenyi Biotec | 130-128-024 | +| RNase Inhibitor, murine | New England Biolabs | M0314L | +| PBS | HyClone | SH30256.02 | +| 7.5% BSA | Life Technologies | 15260037 | +| 1 M HEPES pH 7.3 | Sigma | H0887-100ml | +| NaCl | Fisher | BP358-1 | +| MgCl2 | Fisher | AA12315A7 | +| Tween-20 | Fisher | BP337-500 | +| 5% digitonin | Promega | G944A | +| Enzymatics RI | Enzymatics | Y9240L | +| SUPERase RI | Invitrogen | AM2696 | +| Yeast tRNA | Invitrogen | AM7119 | +| Glycine | Fisher | BP381-500 | +| 1M Tris pH 8.0 | Thermo | AM9855G | +| Formaldehyde (methanol-free) | EMS | 15710 | +| gentleMACS C Tube | Miltenyi Biotec | 130-093-237 | +| gentleMACS Octo Dissociator | Miltenyi Biotec | 130-095-937 | +| MACS SmartStrainers (70 um) | Miltenyi Biotec | 130-110-916 | +| MACS SmartStrainers (30 um) | Miltenyi Biotec | 130-098-458 | +| NucBlue Fixed Cell ReadyProbes | Thermo Fisher | R37606 | +| Hemacytometer | Fisher Scientific | 02-671-51B | +| Mr. Frosty | Sigma-Aldrich | 635639 | + +## Reagents/Equipment, Manufacturer, and Catalog Number + +### Buffers + +**1% BSA-DEPC** + +| Reagent | Volume (for 8 samples) | Final Concentration | +|-------------------------|------------------------|-----------------------| +| BSA | 1 g | 1% | +| DEPC water | 100 ml | NA | + +**Lysis buffer** + +| Reagent | Volume (for 8 samples) | Final Concentration | +|-------------------------|-------------------------|-----------------------| +| Nuclei Extraction Buffer| 35 ml | NA | +| 40 U/ul RNase inhibitor | 175 ul | 0.2 U/ul | + +**NB + BSA** + +| Reagent | Volume (for 8 samples) | Final Concentration | +|-------------------------|-------------------------|-----------------------| +| Nuclei Buffer (Parse Biosciences) | 3.15 ml | NA | +| 7.5% BSA | 350 ul | 0.75% | +| RNase inhibitor (Parse Biosciences)| 17.5 ul | NA | + +**RSB** + +| Reagent | Volume (for 8 samples) | Final Concentration | +|-------------------------|-------------------------|-----------------------| +| PBS | 24.6 ml | NA | +| 7.5% BSA | 333 ul | 0.1% | +| RNase inhibitor | 125 ul | 0.2 U/ul | + +**SHARE-RSB** + +| Reagent | Volume (for 8 samples) | Final Concentration | +|-------------------------|-------------------------|-----------------------| +| 1 M HEPES pH 7.3 | 150 ul | 10 mM | +| 5 M NaCl | 30 ul | 10 mM | +| 1 M MgCl2 | 45 ul | 3 mM | +| 10% Tween-20 | 150 ul | 0.1% | +| H2O | 14.625 ml | NA | +| 7.5% BSA | 80.26 ul | 0.04% | +| 5% digitonin | 30 ul | 0.01% | +| Enzymatics RI | 37.5 ul | 0.1 U/ul | +| SUPERase RI | 18.75 ul | 0.025 U/ul | +| Yeast tRNA | 150 ul | 100 ug/ml | + +## Setup + +1. **Coat SHARE-seq nuclei prep tubes with BSA** + - Fill 8 x 1.5 ml tubes with 1.5 ml 1% BSA-DEPC and incubate for 30 minutes. + - Aspirate BSA solution and dry for 30 minutes. + - Store at 4°C. + +2. **Label tubes**. + +3. **Pre-chill centrifuge to 4°C**. + +4. **Prepare ice buckets**. + +5. **Prepare lysis buffer in a 50 ml conical tube on ice**. + - Distribute 2 ml into 8 gentleMACS C Tubes on ice. + - Add RNase inhibitor to the lysis buffer aliquot the day of the experiment. + +6. **Prepare RSB in a 50 ml conical tube on ice**. + - Add RNase inhibitor the day of the experiment. + +7. **Prepare 3.5 ml NB + BSA**. + - Add RNase inhibitor included in Parse Biosciences fixation kit the day of the experiment. + +8. **Prepare 2.5 ml nuclei buffer + RNase inhibitor for final resuspension**. + - Add 31.5 ul RNase inhibitor to 2.5 ml nuclei buffer. + +9. **Prepare 15 ml SHARE-RSB in a 50 ml conical tube at room temperature**. + - 30 ul digitonin + - 37.5 ul Enzymatics RI + - 18.75 ul SUPERase RI + - 150 ul yeast tRNA + +10. **Thaw components of 2 Parse Biosciences Nuclei Fixation v2 kits at room temperature**. + - Place on ice. + +11. **Distribute 20 ul NucBlue Fixed Cell ReadyProbes into 16 PCR strip tubes for cell counting**. + - Prepare 8 tubes for counting after nuclei extraction and another 8 tubes for final fixed nuclei. + +## Tissue Lysis and Nuclei Extraction + +12. Keep flash frozen tissue samples on dry ice until lysis. + +13. Drop whole frozen tissue into a chilled gentleMACS C Tube with 2 ml lysis buffer. + - Close tubes firmly and invert immediately. + - Ensure tissue is not stuck to the bottom or side. + - Keep tubes on ice and proceed immediately to dissociation. + +14. Run the gentleMACS Program 4C_nuclei_1 on the Octo Dissociator (~5 minutes). + +15. Remove tubes, ensuring tissue did not get stuck on the sides, and spin down in a 4C centrifuge for ~10 seconds to bring liquid to the bottom, then place tubes back on ice. + +16. Filter nuclei suspension through 70 um MACS SmartStrainer into a 5 ml tube. Fit a tube rack in ice for extra stability while filtering. + +17. Wash 70 um MACS SmartStrainer with 2 ml additional lysis buffer. + - Add 2 ml to C tubes, cap, and swish to recover any nuclei stuck to the sides and cap of the C tubes, then wash the strainer. + +18. Discard strainer and centrifuge the 4 ml nuclei suspension at 4C, 350g for 5 minutes. + +19. Resuspend nuclei pellet in 3 ml RSB. + +20. Filter nuclei suspension through 30 um MACS SmartStrainer into a 5 ml tube. + +21. Count nuclei. Use 1:3 dilution factor, 10 ul + 20 ul dye. + +## Parse Nuclei Fixation + +22. Set aside 2-4 million nuclei in RSB in a new 1.5 ml tube and spin down at 4C, 350g for 5 minutes. + +23. Remove supernatant and resuspend nuclei in 750 ul NB + BSA + RNase inhibitor through a 40 um strainer (provided in Parse Biosciences kit) into a new 5 ml tube. + +24. Add 250 ul Nuclei Fixation Solution and mix 3 times. Do not over-mix. + +25. Incubate nuclei for 10 minutes on ice. Set 1 P200 pipettes to 80 ul and keep the P1000 at 250 ul. + +26. Add 80 ul Nuclei Permeabilization Solution and mix by pipetting 3 times with the P1000 still set to 250 ul. Do not over-mix. + +27. Incubate 3 minutes with nuclei on ice. + +28. Add 4 ml Nuclei Neutralization Solution and invert the tube once to mix. + +29. Centrifuge at 4C, 750g for 10 minutes. + +30. Aspirate and discard supernatant. + +31. Resuspend the samples in 200 ul Nuclei Buffer with RNase inhibitor without BSA and move through a 40 um filter into a labeled 1.5 ml tube. + +32. Count nuclei. Use 1:11 dilution factor, e.g. 2 ul + 20 ul dye. + +33. Add Nuclei DMSO: 3.3 ul and gently flick tubes to mix. + - One minute later, add another 3.3 ul and flick to mix, then after another minute add a final 3.3 ul for a total volume of 9.9 ul. + - Mix by gently pipetting 5x with a P200 set to 150 ul. + +34. Split nuclei suspension into 2 labeled tubes, 150 ul per tube. + +35. Place tubes in a Mr. Frosty at -80°C. The next day, move tubes to boxes in -80°C racks. + +36. Move leftover nuclei suspension to labeled 2 ml tubes and spin at 4C, 750g for 5 minutes. Remove supernatant and flash-freeze nuclei in liquid nitrogen as dry pellets. Store at -80°C. + +## SHARE-seq Nuclei Fixation + +37. Set aside 1 million nuclei for each of the 8 samples in RSB and spin down at 4C, 750g for 5 minutes. + +38. Remove supernatant and resuspend nuclei pellet in 1 ml room temperature SHARE-RSB. Transfer tube to a room temperature rack. + +39. At RT, add 13.34 ul of methanol-free formaldehyde (16% stock solution). Final concentration for nuclei: 0.2%. Close tube and nutate cells at RT for 5 minutes. + +40. To quench fixation, per reaction, add 56.1 ul fresh 2.5M Glycine (0.94g per 5 ml stock), 50 ul of 1M Tris pH 8.0, 13.3 ul of 7.5% BSA, and mix using a pipette. Incubate on ice for 10 minutes. + +41. Spin 750g, 4C, 5 minutes. Gently remove supernatant. + +42. Add 200 ul of SHARE-RSB and gently resuspend pellet. Store on ice until all samples are completed. + +43. Pool 200 ul of resuspended nuclei from all 8 founders into 1 labeled 2 ml tube. + +44. Spin 1,000g, 4C, 10 minutes. Gently remove supernatant. Remove all fluid and freeze at -80°C as a dry pellet. + +## Storage of Leftover Nuclei + +45. Move remaining nuclei in RSB on ice to labeled 2 ml tubes. + +46. Spin 750g, 4C, 5 minutes. + +47. Remove all supernatant and flash-freeze nuclei as a dry pellet in liquid nitrogen. Store at -80°C. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/protocol-to-isolate-and-fix-nuclei-from-flash-froz-cy7jxzkn.md b/markdown-output/protocol-to-isolate-and-fix-nuclei-from-flash-froz-cy7jxzkn.md new file mode 100644 index 0000000000000000000000000000000000000000..f5a229f05a31c6f03336918d73d5165ad988f88c --- /dev/null +++ b/markdown-output/protocol-to-isolate-and-fix-nuclei-from-flash-froz-cy7jxzkn.md @@ -0,0 +1,110 @@ +```markdown +# Goal/Experiment: +To isolate and fix nuclei from flash frozen mouse gastrocnemius for IGVF (Isolated Genomes in Vast Fauna). + +# Protocol to Isolate and Fix Nuclei from Flash Frozen Mouse Gastrocnemius for IGVF + +## Abstract +This protocol describes the isolation of nuclei from 10-week-old left or right mouse gastrocnemius muscle from 8 founder strains (B6J, AJ, 129S1J, NZOJ, WSBJ, NODJ, PWKJ, and CASTJ). It includes preparation of a single nucleus suspension and fixation for single nucleus RNA-seq using the Parse Biosciences protocol. We process 1 representative from each strain per day (e.g., female rep 1 across all 8 strains). The main products we use are Parse Biosciences Nuclei Fixation Kit (v2) and Miltenyi Biotec’s gentleMACS Octo Dissociator with accessories. This protocol takes about 3.5 hours from start to finish. + +The results are 1 aliquot of fixed single-nucleus suspensions for Parse per each of the 8 samples at >= 2,500 nuclei/µl. + +## Guidelines +1. Tilt tube and slowly add PBS during debris removal. Ideally, the cloudy debris is only in the band rather than the nuclei layer. +2. We recommend using a 5 mL pipette for aspirations and resuspensions > 1 mL. +3. Record everything in the [IGVF spreadsheet](https://example.com), “Samples into experiment” tab. + +## Materials + +| Name | Manufacturer | Cat. # | +|-------------------------------|------------------------|-------------| +| Nuclei Fixation Kit v2 | Parse Biosciences | ECF2003 | +| Nuclei Extraction Buffer | Miltenyi Biotec | 130-128-024 | +| RNase Inhibitor, murine | New England Biolabs | M0314L | +| PBS | HyClone | SH30256.02 | +| Debris Removal Solution | Miltenyi Biotec | 130-109-398 | +| 7.5% BSA | Life Technologies | 15260037 | +| gentleMACS C Tube | Miltenyi Biotec | 130-093-237 | +| gentleMACS Octo Dissociator | Miltenyi Biotec | 130-095-937 | +| MACS SmartStrainers (70 µm) | Miltenyi Biotec | 130-110-916 | +| MACS SmartStrainers (30 µm) | Miltenyi Biotec | 130-098-458 | +| pluriStrainer (20 µm) | pluriSelect | 43-50020-03 | +| NucBlue Fixed Cell ReadyProbes| Thermo Fisher | R37606 | +| Millicell Disposable Hemocytometer| Millipore Sigma | MDH-2N1-50PK| +| Mr. Frosty | Sigma-Aldrich | 635639 | + +## Reagents/Equipment, Manufacturer and Catalog Number + +| Name | Reagent | Volume (for 8 samples) | Final Concentration | +|---------------------|---------------------------------------|------------------------|----------------------| +| Lysis buffer | Nuclei Extraction Buffer | 35 mL | NA | +| | 40 U/µl RNase inhibitor | 175 µL | 0.2 U/µL | +| PBS | PBS | 35 mL | NA | +| | HBSS | 20 mL | NA | +| Debris Removal Solution (DRS) | Debris Removal Solution (Miltenyi)| 8 mL | NA | +| NB + BSA + RNase Inhibitor | Nuclei Buffer (Parse Biosciences)| 3.15 mL | NA | +| | 7.5% BSA | 350 µL | 0.75% | +| | RNase Inhibitor (Parse Biosciences) | 44.1 µL | | +| NB + RNase Inhibitor| Nuclei Buffer (Parse Biosciences) | 5 mL | NA | +| | RNase Inhibitor (Parse Biosciences) | 44.1 µL | | +| | PBS | 24.6 mL | NA | +| RSB (x2 aliquots) | 7.5% BSA | 333 µL | 0.1% | +| | RNase Inhibitor | 125 µL | 0.2 U/µL | + +## Buffers + +### Setup +1. Pre-chill centrifuge to 4°C. +2. Prepare 2 large ice buckets. +3. Prepare lysis buffer on ice in a 50 mL conical tube. Distribute 2 mL into 8 gentleMACS C Tubes on ice. +4. Prepare NB + BSA + RNase inhibitor in a 5 mL tube. +5. Prepare RSB on ice in a 50 mL conical tube. Keep a larger amount of PBS + 0.1% BSA at 4°C, adding the RNase inhibitor on the day of the experiment. +6. Prepare NB + RNase inhibitor in a 5 mL tube for final resuspension. +7. Take an aliquot of PBS out of 4°C and keep on ice. +8. Take an aliquot of Debris Removal Solution out of 4°C and keep on ice. +9. Thaw components of 1 Parse Biosciences Nuclei Fixation kit at room temperature, then place on ice. +10. Distribute 10 µL NucBlue Fixed Cell ReadyProbes into 24 PCR strip tubes for cell counting. Need 8 tubes for counting after nuclei extraction, 8 tubes for counting after fixation, and another 8 tubes for filtered fixed nuclei. + +### Tissue Lysis and Nuclei Extraction +11. Keep flash frozen tissue samples on dry ice. +12. Prepare 6 well plates on ice with ~2 ml of HBSS per well. +13. If necessary, drop both gastrocnemius tissues in a well, let them melt slightly, and separate them carefully using forceps. +14. Return one muscle to the sample tube and move the other to another labeled 1.5 mL tube. Flash-freeze both in liquid nitrogen. +15. Proceed with only one. Keep the other frozen in the same sample tube and return tubes to -80°C box. +16. Drop left or right frozen tissue into a chilled gentleMACS C Tube with 2 mL lysis buffer. Close tubes firmly and invert immediately, ensuring tissue is not stuck to the bottom or side. Keep tubes on ice and proceed immediately to dissociation. +17. Run the gentleMACS Program 4C_nuclei_1 on the Octo Dissociator (~5 minutes). +18. Remove tubes, ensuring tissue did not get stuck on the sides, and spin down in a 4°C centrifuge for ~10 seconds to bring liquid to the bottom, then place tubes back on ice. +19. Filter nuclei suspension through 70 µm MACS SmartStrainer into a 5 mL tube. Fit a tube rack in ice for extra stability while filtering. +20. Wash 70 µm MACS SmartStrainer with 2 mL additional lysis buffer. Add 2 mL to C tubes, cap, and swish to recover any nuclei stuck to the sides and cap of the C tubes, then wash the strainer. +21. Discard strainer and centrifuge the nuclei suspension at 4°C, 350g for 5 minutes. +22. Aspirate supernatant and resuspend nuclei pellet in 3.1 mL RSB. +23. Filter nuclei suspension through 30 µm MACS SmartStrainer into a 15 mL tube. +24. Add 900 µL Debris Removal Solution and mix by pipetting 10 times slowly up and down using a 5 mL pipette. +25. Overlay with 4 mL PBS using a P1000 or 5 mL pipette (whichever you are more comfortable with). Tilt tube 45 degrees and slowly add the first mL. You can increase speed after the first mL of PBS is added. +26. Centrifuge at 4°C, 3000g for 10 minutes with full acceleration and no brake. Three phases are formed: top clear buffer layer, cloudy debris band, and clear layer containing nuclei. Pellet usually visible. +27. Aspirate the two top phases (buffer layer and all cloudy debris band) and discard. Aspirate the first phase, then the second phase. Stay above the third layer of nuclei to prevent loss. +28. Fill with cold RSB to a final volume of 5 mL. +29. Gently invert the tube three times. Do not vortex. +30. Centrifuge at 4°C, 1000g for 10 minutes with full acceleration and full brake. +31. Aspirate supernatant completely. +32. Resuspend cells carefully in 375 µL NB + BSA + RNase inhibitor and filter through a 40 µm strainer into a new 5 mL tube. +33. Count nuclei. Use 1:2 dilution factor, 10 µL + 10 µL dye. + +### Nuclei Fixation +34. Add 125 µL Nuclei Fixation Solution to the filtered nuclei in 375 µL and mix 3 times. Do not over-mix. +35. Incubate nuclei for 10 minutes on ice. Set 2 P200 pipettes to 40 µL and 125 µL. +36. Add 40 µL Nuclei Permeabilization Solution and mix by pipetting 3 times with the P200 still set to 125 µL. Do not over-mix. +37. Incubate 3 minutes with nuclei on ice. +38. Add 2 mL Nuclei Neutralization Solution and invert the tube once to mix. +39. Centrifuge at 4°C, 750g for 10 minutes. +40. Aspirate and discard supernatant. +41. Resuspend the samples in 500 µL Nuclei Buffer without BSA, with RNase inhibitor. Check concentration with a hemocytometer under the microscope. Use 1:2 dilution factor, 10 µL + 10 µL dye. +42. Filter nuclei through a 20 µm filter in 1, 2, 3, or 4 rounds depending on the amount of debris. Place filter in labeled 1.5 mL tube and dispense nuclei in 500 µL on top. Centrifuge at 4°C, 200g for 1 minute to pull the solution through the filter. Repeat step if necessary, using a new filter for each round. Our reasoning is to prevent clogging by filtering in multiple rounds, but yield decreases by 90% before and after fixation, mostly due to the filtration at this step. +43. Take a 10 µL aliquot to dilute 1:2 with prepared 10 µL dye to manually count with a disposable hemocytometer and record numbers. +44. Count nuclei. Use 1:2 dilution factor, 10 µL + 10 µL dye. +45. Re-concentrate: spin nuclei 750g for 5 minutes and carefully take off supernatant until 50 µL are remaining. Resuspend (hopefully visible) pellet in the remaining 50 µL. +46. Add Nuclei DMSO: 1 µL into 50 µL samples and gently flick tubes to mix. One minute later, add another 1 µL and flick to mix, then after another minute add a final 1 µL for a total volume of 3 µL. Mix by gently pipetting 5x. +47. Place tubes in a Mr. Frosty for storage at -80°C. The next day, move tubes to boxes in -80°C racks. + +End of Output. +``` \ No newline at end of file diff --git a/markdown-output/protocol-to-isolate-and-fix-nuclei-from-flash-froz-cyubxwsn.md b/markdown-output/protocol-to-isolate-and-fix-nuclei-from-flash-froz-cyubxwsn.md new file mode 100644 index 0000000000000000000000000000000000000000..294fc36a025ec834fff1d8e0cd2a717db1319cd5 --- /dev/null +++ b/markdown-output/protocol-to-isolate-and-fix-nuclei-from-flash-froz-cyubxwsn.md @@ -0,0 +1,123 @@ +```markdown +# Goal/Experiment: +The purpose of this experiment is to isolate and fix nuclei from flash-frozen mouse kidneys for ImmGen FlowVec (IGVF) using specific biological reagents and equipment to prepare and fix single-nucleus suspensions for single-nucleus RNA sequencing (snRNA-seq). + +# Protocol to Isolate and Fix Nuclei from Flash Frozen Mouse Kidneys for IGVF + +**Author**: Elisabeth Rebboah +**Institution**: University of California, Irvine +**Date**: August 17, 2023 + +## Abstract + +This protocol describes the isolation of nuclei from 10-week-old mouse kidneys from 8 founder strains (B6J, AJ, 129S1J, NZOJ, WSBJ, NODJ, PWKJ, and CASTJ), preparation of a single nucleus suspension, and fixation for single nucleus RNA-seq using Parse Biosciences. We process 1 representative (rep) from each strain per day, e.g., female rep 1 across all 8 strains. The main products used are Parse Biosciences Nuclei Fixation Kit (v2) and Miltenyi Biotec's gentleMACS Octo Dissociator with accessories. This protocol takes about 3.5 hours from start to finish. + +The results are 2 aliquots of fixed single-nucleus suspensions for Parse per each of the 8 samples. + +## Guidelines + +- We recommend using a 5 ml pipette for aspirations and resuspensions larger than 1 ml. +- Record everything in the IGVF spreadsheet, "Samples into experiment" tab. + +## Materials + +| Name | Manufacturer | Cat # | +|--------------------------------------|----------------------|---------------| +| Nuclei Fixation Kit v2 | Parse Biosciences | ECF2003 | +| Nuclei Extraction Buffer | Miltenyi Biotec | 130-128-024 | +| RNase Inhibitor, Murine | New England Biolabs | M0314L | +| PBS (Phosphate Buffered Saline) | HyClone | SH30256.02 | +| 7.5% BSA (Bovine Serum Albumin) | Life Technologies | 15260037 | +| gentleMACS C Tube | Miltenyi Biotec | 130-093-237 | +| gentleMACS Octo Dissociator | Miltenyi Biotec | 130-095-937 | +| MACS SmartStrainers (30 µm) | Miltenyi Biotec | 130-098-458 | +| NucBlue Fixed Cell ReadyProbes | Thermo Fisher | R37606 | +| Hemacytometer | Fisher Scientific | 02-671-51B | +| Mr. Frosty | Sigma-Aldrich | 635639 | + +### Reagents/Equipment Specifications + +| Name | Reagent | Volume for 8 samples | Final Concentration | +|--------------------------|---------------------------|----------------------|----------------------| +| **Lysis buffer** | Nuclei Extraction Buffer | 40 ml | NA | +| | 40 U/µl RNase inhibitor | 200 µl | 0.2 U/µl | +| **NB + BSA + RNase inhibitor (make 2 aliquots)** | Nuclei Buffer (Parse Biosciences) | 3.15 ml | NA | +| | 7.5% BSA | 350 µl | 0.75% | +| | RNase inhibitor (Parse Biosciences) | 44.1 µl | | +| **RSB** | PBS | 42 ml | | +| | 7.5% BSA | 560 µl | 0.1% | +| | RNase inhibitor | 210 µl | 0.2 U/µl | +| | 1 M HEPES pH 7.3 | 150 µl | 10 mM | +| | 5 M NaCl | 30 µl | 10 mM | +| | 1 M MgCl2 | 45 µl | 3 mM | +| | 10% Tween-20 | 150 µl | 0.1% | +| | H2O | 14.625 ml | | +| | 7.5% BSA | 80.26 µl | 0.04% | +| | 5% digitonin | 30 µl | 0.01% | +| | Enzymatics RI | 37.5 µl | 0.1 U/µl | +| | SUPERase RI | 18.75 µl | 0.025 U/µl | +| **Buffers** | Yeast tRNA | 150 µl | 100 µg/ml | + +## Setup + +1. Label tubes. +2. Pre-chill the centrifuge to 4°C. +3. Prepare ice buckets. +4. Prepare lysis buffer in a 50 ml conical tube on ice. Distribute 2.5 ml into 8 gentleMACS C Tubes on ice. Add RNase inhibitor to the lysis buffer aliquot the day of the experiment. +5. Prepare RSB in a 50 ml conical tube on ice. Add NEB RNase inhibitor the day of the experiment. +6. Prepare NB + BSA + RNase inhibitor. Add RNase inhibitor included in Parse Biosciences fixation kit the day of the experiment. +7. Prepare 2.5 ml Nuclei Buffer + RNase inhibitor for final resuspension. Add 31.5 µl Parse RNase inhibitor to 2.5 ml nuclei buffer. +8. Thaw components of 2 Parse Biosciences Nuclei Fixation v2 kits at room temperature, then place on ice. +9. Distribute 20 µl NucBlue Fixed Cell ReadyProbes into 16 PCR strip tubes for cell counting. Need 8 tubes for counting after nuclei extraction, and another 8 tubes for final fixed nuclei. + +## Tissue Lysis and Nuclei Extraction + +10. Keep flash-frozen tissue samples on dry ice until lysis. +11. Drop whole frozen tissue into a chilled gentleMACS C Tube with 2.5 ml lysis buffer. Close tubes firmly and invert immediately, ensuring tissue is not stuck to the bottom or side. Keep tubes on ice and proceed immediately to dissociation. There should be 2 kidneys. +12. Run the gentleMACS Program 4C_nuclei_1 on the Octo Dissociator (~5 minutes). +13. Remove tubes, ensuring tissue did not get stuck on the sides, and spin down in a 4°C centrifuge for ~10 seconds to bring liquid to the bottom, then place tubes back on ice. +14. Filter nuclei suspension through a 70 µm MACS SmartStrainer into a 5 ml tube. Fit a tube rack in ice for extra stability while filtering. +15. Wash the 70 µm MACS SmartStrainer with 2 ml additional lysis buffer. Add 2 ml to C tubes, cap, and swish to recover any nuclei stuck to the sides and cap of the C tubes, then wash the strainer. +16. Discard strainer and centrifuge the 4.5 ml nuclei suspension at 4°C, 350g for 5 minutes. +17. Discard the supernatant and resuspend the nuclei pellet in 3 ml RSB. Filter nuclei suspension through a 30 µm MACS SmartStrainer into a 5 ml tube. +18. Take 200 µl and add to a 5 ml tube containing another 1.8 ml RSB for a 1:10 dilution (same as male gonads and similar to liver protocols). +19. Count nuclei. Use a 1:11 dilution factor, 2 µl + 20 µl dye. +20. Centrifuge 4 million nuclei at 4°C, 500g for 5 minutes and remove supernatant. +21. Resuspend the pellet in 750 µl NB-BSA + RNase inhibitor. + +## Parse Nuclei Fixation + +22. Filter the nuclei suspension (750 µl) NB-BSA + RNase inhibitor through a 40 µm strainer (provided in Parse Biosciences kit) into a new 5 ml tube. +23. Add 250 µl Nuclei Fixation Solution and mix 3 times. Do not over-mix. +24. Incubate nuclei for 10 minutes on ice. Set 1 P200 pipette to 80 µl and keep the P1000 at 250 µl. +25. Add 80 µl Nuclei Permeabilization Solution and mix by pipetting 3 times with the P1000 still set to 250 µl. Do not over-mix. +26. Incubate 3 minutes with nuclei on ice. +27. Add 4 ml Nuclei Neutralization Solution and invert the tube once to mix. +28. Centrifuge at 4°C, 750g for 10 minutes. +29. Aspirate and discard the supernatant. +30. Resuspend the samples in 300 µl Nuclei Buffer with RNase inhibitor without BSA and filter through a 40 µm filter into a labeled 1.5 ml tube. +31. Count nuclei. Use a 1:11 dilution factor, e.g., 2 µl + 20 µl dye. +32. Add Nuclei DMSO: For 300 µl samples: add 5 µl and gently flick tubes to mix. One minute later, add another 5 µl and flick to mix, then after another minute add a final 5 µl for a total volume of 15 µl. Mix by gently pipetting 5x with a P200 set to 150 µl. +33. Split the nuclei suspension into 2 aliquots of equal volume in labeled 1.5 ml tubes. +34. Place tubes in a Mr. Frosty at -80°C. The next day, move tubes to boxes in -80°C racks. +35. Move leftover nuclei suspension to labeled 2 ml tubes and spin at 4°C, 750g for 5 minutes. Remove supernatant and flash-freeze nuclei in liquid nitrogen as dry pellets. Store at -80°C. + +## SHARE-Seq Nuclei Fixation + +36. Set aside 1 million nuclei for each of the 8 samples in RSB and spin down at 4°C, 750g for 5 minutes. +37. Remove the supernatant and resuspend nuclei pellet in 1 ml room temperature SHARE-RSB. Transfer tube to a room temperature rack. +38. At RT, add 13.34 µl of methanol-free formaldehyde (16% stock solution). Final concentration for nuclei: 0.2%. Close the tube and nutate cells at RT for 5 minutes. +39. To quench fixation, per reaction, add 56.1 µl fresh 2.5M Glycine (0.94g per 5 ml stock), 50 µl of 1M Tris pH 8.0, 13.3 µl of 7.5% BSA, and mix using a pipette. Incubate on ice for 10 minutes. +40. Spin at 750g, 4°C, for 5 minutes. Gently remove the supernatant. +41. Add 200 µl of SHARE-RSB and gently resuspend pellet. Store on ice until all samples are completed. +42. Pool 200 µl of resuspended nuclei from all 8 founders into 1 labeled 2 ml tube. +43. Spin 1,000g, 4°C, 10 minutes. Gently remove the supernatant. Remove all fluid and freeze at -80°C as a dry pellet. + +## Storage of Leftover Nuclei + +44. Move remaining nuclei in RSB in 5 ml tubes on ice to new labeled 2 ml tubes. +45. Spin at 750g, 4°C, for 5 minutes. +46. Remove all supernatant and flash-freeze nuclei as a dry pellet in liquid nitrogen. Store at -80°C. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/protocol-to-isolate-cryopreserve-and-fix-mouse-pbm-c4myyu7w.md b/markdown-output/protocol-to-isolate-cryopreserve-and-fix-mouse-pbm-c4myyu7w.md new file mode 100644 index 0000000000000000000000000000000000000000..8f378edfc232d5c07678aa3b90ff4de2ac540ebf --- /dev/null +++ b/markdown-output/protocol-to-isolate-cryopreserve-and-fix-mouse-pbm-c4myyu7w.md @@ -0,0 +1,127 @@ +```markdown +Goal/Experiment: +This protocol describes the isolation of peripheral blood mononuclear cells (PBMCs) from whole blood of 10-week-old mice, their cryopreservation, thawing, and fixation for single-cell RNA sequencing using the Parse Biosciences protocol (Split-seq). + +# Protocol to Isolate, Cryopreserve, and Fix Mouse PBMCs for IGVF V.2 + +## Abstract +This protocol describes isolation of peripheral blood mononuclear cells (PBMCs, tissue ID: 20) from whole blood of 10-week-old mice, cryopreservation, thawing, and fixation for single-cell RNA sequencing using the Parse Biosciences protocol (Split-seq). For 8-10 samples, PBMC isolation and cryopreservation take about 3 hours from start to finish, while thawing and fixation take around 1 hour. + +The final result is 1 aliquot of a fixed single-cell suspension for Parse Bio scRNA-seq ("Split-seq") for each sample at >= 2,500 cells/µl stored at -80°C. The intermediate result is approximately 1 million cryopreserved PBMCs per sample in 1 mL 10% DMSO + FBS, stored in liquid nitrogen until thawing and fixing. + +### Key Points +- Ficoll-Paque density gradient centrifugation is used to isolate PBMCs. +- PBMCs are suitable for fixation, not cell culture, post-cryopreservation. +- Thawed cells are washed and fixed for downstream applications. + +## Materials +| Name | Manufacturer | Catalog # | +|------------------------------|-----------------------|------------------| +| Ficoll-Paque | Cytiva | 17144002 | +| EDTA tubes | BD-Vacutainer | 367856 | +| 7.5% BSA | Life Technologies | 15260037 | +| FBS | Omega Scientific | FB-01 | +| PBS | Cytiva | BSS-PBS-1X6 | +| EDTA | Sigma-Aldrich | E6511 | +| DMSO | Sigma-Aldrich | D2650 | +| Red Blood Cell Lysis Solution| Miltenyi | 130-094-183 | +| DEPC water | Invitrogen | 750023 | +| NucBlue Live ReadyProbes | Thermo Fisher | R37605 | +| Millicell Disposable Hemocytometer | Millipore | MDH-2N1-50PK | +| Mr. Frosty | Sigma-Aldrich | 635639 | +| 5 mL DNA/RNA LoBind tubes | Eppendorf | 0030108310 | +| 2 mL DNA/RNA LoBind tubes | Eppendorf | 022431048 | +| NucBlue Fixed Cell ReadyProbes| Thermo Fisher | R37606 | +| Cell Fixation Kit v2 | Parse Biosciences | ECF2001 | +| DMEM | Sigma-Aldrich | D5796 | +| Penicillin-Streptomycin | Gibco | 15070063 | + +### Reagents/Equipment Preparation +| A | B | C | D | +|------------|---------|--------------------------------|----------| +| 1% BSA-DEPC| BSA | 1 g | 1% | +| | DEPC | 100 mL | | +| PBS-EDTA | EDTA | 0.146 g | 1 mM | +| | PBS | 500 mL | | +| PBMC-RSB | PBS-EDTA| 490 mL | | +| | FBS | 10 mL | 2% | +| 1x RBC lysis| 10x Red Blood Cell Lysis Solution| 1.2 mL | 1x | +| | DEPC | 10.8 mL | | +| 20% DMSO in FBS |DMSO | 1.2 mL | 20% | +| | FBS | 4.8 mL | | +| 20% DMEM FBS| DMEM | 450 mL | | +| | FBS | 100 mL | 20% | +| | Pen-strep| 5 mL | 1x | + +## Setup +1. Set centrifuge to 19°C. +2. Prepare 1 ice bucket. +3. Thaw and filter FBS with a cell culture filter unit and aliquot into 15 mL conical tubes. +4. Prepare PBS-EDTA by adding EDTA powder to PBS and filter with a cell culture filter unit. Store at room temperature, or 4°C if not using that week. +5. Prepare PBMC-RSB and 1x RBC lysis buffer at room temperature. +6. Prepare 20% DMSO FBS on ice. +7. Distribute 20 µL dye into 10 PCR tubes for cell counting. +8. (Optional) Prepare 1% BSA-DEPC stock tubes. Coat 5 mL tubes by adding 5 mL 1% BSA-DEPC in the cell culture hood, incubating for 30 minutes, emptying the tubes, and drying for 30 minutes. Store tubes at 4°C. + +## PBMC Isolation and Cryopreservation +9. Collect blood in EDTA-coated tubes at the vivarium during dissection. Cap and invert tubes, keep on rotating platform. +10. Spin tubes to collect remaining blood. +11. Measure and record blood volume with a 1 mL pipette. +12. Dilute blood 1:2 with PBS (e.g., 900 µL blood + 900 µL PBS). +13. Aliquot appropriate amount of Ficoll-Paque. Use the smallest possible tube. +14. Layer diluted blood on top of Ficoll-Paque. +15. Centrifuge at 400g for 30 minutes at 19°C with no brake. +16. Collect plasma and distribute in PCR tubes (200 µL each). +17. Collect PBMC layer into a labeled 5 mL tube. Wash cells with PBMC-RSB. Discard gradient tube. +18. Centrifuge at 400g for 8 minutes at 19°C with full brake. +19. Remove supernatant, leaving 100 µL PBMC-RSB. +20. Add 1 mL 1x RBC lysis buffer. Vortex and incubate for 10 minutes at room temperature. +21. Centrifuge at 400g for 8 minutes at room temperature with full brake. +22. Remove supernatant and resuspend cells in 500 µL PBMC-RSB for counting. + +## Counting +23. Aliquot 20 µL from each 5 mL tube into PCR tubes containing 20 µL dye. +24. Label both sides of a disposable hemocytometer with sample number. +25. Pipette 10 µL of stained cells into hemocytometer. +26. Count cells under a microscope (10x). +27. Convert to cells/mL using dilution factor. +28. Document cell counts. +29. Centrifuge at 400g for 8 minutes at room temperature with full brake. +30. Remove supernatant and resuspend cells in 500 µL cold FBS. +31. Slowly add 500 µL 20% DMSO in FBS, mixing gently. Final concentration is 10% DMSO in FBS. +32. Freeze tubes at -80°C using Mr. Frosty. Transfer to liquid nitrogen the next day. + +## Thawing and Fixation Setup +34. Prepare complete medium (20% FBS, 1% penicillin-streptomycin in DMEM). Warm to 37°C. +35. Aliquot 10 mL of pre-warmed medium into a polypropylene tube. +36. Prepare 1 Parse Biosciences Cell Fixation kit. Add 550 µL Cell Fixation Additive to Cell Fixation Solution. +37. Add 50 µL of RNase Inhibitor to Cell Prefixation Buffer. +38. Add 17 µL of RNase Inhibitor to Cell Buffer. +39. Record today's date on the kit. +40. Prepare Cell Prefixation Buffer + BSA (200 µL 7.5% BSA added to 3 mL Cell Prefixation Buffer). + +## Thawing Cryopreserved PBMCs +41. Transfer cryovials from freezer to water bath on dry ice. +42. Thaw in 37°C water bath (30-45 seconds) until 20% ice remains. +43. Wipe cryovials with 70% ethanol. +44. Transfer cells to 15 mL tube with culture medium. Rinse cryovial with 1 mL medium. +45. Centrifuge at 350g for 5 minutes at room temperature. +46. Remove supernatant, retaining a small amount. +47. Loosen pellet and add 10 mL medium. +48. Resuspend cells in 375 µL Cell Prefixation Buffer + BSA. + +## Cell Fixation +49. Pipette cells through 40 µm strainer into new 15 mL tube. +50. Add 125 µL Cell Fixation Solution. Pipette up and down 3x. +51. Incubate on ice for 10 minutes. +52. Add 40 µL Cell Permeabilization Solution. Pipette up and down 3x. Incubate 3 minutes. +53. Add 2 mL Cell Neutralization Buffer. Mix gently. +54. Centrifuge at 750g for 10 minutes at 4°C. +55. Remove and discard supernatant. Resuspend pellet in 100 µL cold Cell Buffer. +56. Count cells using a hemocytometer with 1:2 dilution factor. +57. Re-concentrate cells at 750g for 5 minutes. Resuspend in remaining volume. +58. Add DMSO in increments to total 1.5 µL. Avoid bubbles. +59. Place tubes in Mr. Frosty for -80°C storage. Transfer to liquid nitrogen next day. + +**endofoutput** +``` \ No newline at end of file diff --git a/markdown-output/protocol-to-process-gastruloids-for-facs-reporters-bvgrn3v6.md b/markdown-output/protocol-to-process-gastruloids-for-facs-reporters-bvgrn3v6.md new file mode 100644 index 0000000000000000000000000000000000000000..9f390f3ffc99b1637a77706fd1561b99facd7a60 --- /dev/null +++ b/markdown-output/protocol-to-process-gastruloids-for-facs-reporters-bvgrn3v6.md @@ -0,0 +1,163 @@ +```markdown +# Goal/Experiment: +Protocol to process reporter Gastruloids for FACS analysis of their endogenous reporters. + +# Protocol to process Gastruloids for FACS (reporters only) + +Stefano Vianello¹, Tania Hübscher¹, Giuliana Rossi¹, Matthias Lutolf² +¹Laboratory of Stem Cell Bioengineering, Institute of Bioengineering, School of Life Sciences (SV) and School of Engineering (STI), École Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland +²Laboratory of Stem Cell Bioengineering, Institute of Bioengineering, School of Life Sciences (SV) and School of Engineering (STI), École Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland and Institute of Chemical Sciences and Engineering, School of Basic Science (SB), EPFL, Lausanne, Switzerland + +**Abstract** +Protocol to process reporter Gastruloids for FACS analysis of their endogenous reporters. + +**DOI** +[dx.doi.org/10.17504/protocols.io.bvgrn3v6](https://dx.doi.org/10.17504/protocols.io.bvgrn3v6) + +**Protocol Citation** +Stefano Vianello, Tania Hübscher, Giuliana Rossi, Matthias Lutolf 2021. Protocol to process Gastruloids for FACS (reporters only). *protocols.io* [https://dx.doi.org/10.17504/protocols.io.bvgrn3v6](https://dx.doi.org/10.17504/protocols.io.bvgrn3v6) + +**Keywords** +Gastruloids, FACS + +**License** +This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited + +**Created** +Jun 02, 2021 + +**Last Modified** +Jun 02, 2021 + +**Protocol Integer ID** +50417 + +**Guidelines** +This protocol assumes standard aseptic technique, and work in a laminar flow hood. + +--- + +### Materials and Reagents + +#### Recipes: +**Digestion solution** (2mL per sample) +*Final concentrations: Collagenase IV [3mg/mL], Dispase [4mg/mL], DNase I [100ug/mL] in PBS* + +- 3mg/mL **Collagenase IV** [CAT#17104-019; Gibco™/Thermo Fisher Scientific] + - 1:4 from 12mg/mL stock +- 4mg/mL **Dispase II** [CAT#17105-041; Gibco™/Thermo Fisher Scientific] + - 1:2.5 from 10mg/mL stock +- 100ug/mL **DNase I** [CAT#11284932001; Roche] + - 1:500 from 50mg/mL stock +- Top up with **PBS -/-** + +**Staining buffer** +*Final concentrations: 10% ES-FBS, 100U/mL Pen-Strep, 1mM EDTA in PBS* + +- 1:10 final volume: **ES-FBS** [CAT#16141079; Gibco™/Thermo Fisher Scientific] +- 1:100 final volume: **Pen-Strep** [CAT#15140122; Gibco™/Thermo Fisher Scientific] +- 1:500 final volume: **500mM EDTA** [CAT#15575020; Gibco™/Thermo Fisher Scientific] +- Top up with **PBS -/-** [CAT#10010056; Gibco™/Thermo Fisher Scientific] + +**2% PFA in PBS -/-** +*(e.g., diluted 1:2 in PBS -/- from 4% PFA solution, CAT#15434389, Alfa-Aesar/Fisher Scientific)* + +### Before Starting + +1. Prepare enough FACS tubes/samples/controls to have a complete setup with both positive, negative, FMO (Fluorescence Minus One) references. + +2. For a simple FACS setup of Gastruloids with a double reporter, in addition to your experimental samples (i.e., dissociated Gastruloids for each timepoint, with DAPI as live-dead discriminant): + - Double negative cells (ideally, the parental cells of the reporter, with no fluorescent insert; but use alternatively 2D cultured reporter cells if they do not express the reporters in the pluripotent state) + - Cells that are RFP+ and have no GFP reporter (or RFP+ beads) + - Cells that are GFP+ and have no RFP reporter (or GFP+ beads) + - Make sure to have a sample that not only has no reporter expression at all, but also no DAPI staining (to help set the gate for the DAPI signal). + +--- + +### Protocol + +#### Gastruloid Collection + +1. Harvest Gastruloid from each well of a 96 well plate and collect them in a 15mL Falcon tube (pooled by timepoint or condition). + +2. Once all Gastruloids have settled to the bottom of the tube, aspirate out the supernatant (N2B27 medium carried over with each Gastruloid). + +3. Resuspend the Gastruloids in 5mL PBS -/-, to wash away traces of N2B27. + +4. Once all Gastruloids have resettled to the bottom of the tube, aspirate out the PBS -/-. Gastruloids are ready to be digested. + +--- + +#### Gastruloid Digestion (approx. 8 min) + +5. Digest the Gastruloids by adding 1mL **Digestion Solution** (3mg/mL Collagenase IV, 4mg/mL Dispase, 100ug/mL DNAseI, in PBS -/-), + - Incubate at 37 ℃ (water bath), for 4 min. + +6. After the 4 min incubation has elapsed, use a P1000 coated in Staining Buffer to try to mechanically disrupt the Gastruloids. + +7. Place the tube back again at 37 ℃ (water bath) for another 4 min to complete the digestion. + +8. After the last 4 min of incubation have elapsed, use a P1000 coated in Staining Buffer to mechanically disrupt the Gastruloids. Gastruloids should break easily and give rise to a single cell suspension. + +--- + +#### Cell Filtering (approx. 4 min) + +9. Prewet the blue cap of a filter-FACS tube (e.g., CAT#352235, Falcon/Corning) by pipetting 1mL of Staining Buffer through it. **Note:** This step is very important. Not prewetting the filter can lead to loss of a high number of cells at the filtering step. + +10. Working on ice, filter your cell suspension (digested Gastruloids) through the cap of the FACS tube. + +11. On ice, wash the filter by passing 1mL more of Digestion Solution through it. + +12. On ice, transfer the contents of the FACS tube to a clean 15mL Falcon tube. + +13. On ice, collect any leftover cells by flushing the old FACS tube with 2mL Staining Buffer, and transferring it to the Falcon tube with the rest of the cells. + +14. On ice, add 5mL more Staining Buffer into the tube Falcon tube with your cells suspension, to completely stop the digestion reaction. This tube now contains a filtered, single-cell suspension of your sample for FACS. + +15. Spin down the cell suspension at 200 x g, 4℃, for 4 min. + +--- + +#### Cell Collection (approx. 4 min) + +16. For samples that come from 2D culture (e.g., 2D-grown reporter cells as negative controls, or 2D-grown single-color cells as positive controls): + - Aspirate out the medium from the culture dish. + - Add 3mL PBS -/- to wash away any trace of medium, and aspirate it out again. + - Add 500uL Accutase to detach the cells, incubate at room temperature for approximately 5 min. + - Transfer the detached cells + Accutase to a clean Falcon tube. + - Add ~5mL 10% Serum to stop the reaction. + - Spin down the cell suspension at 200 x g, 4℃, for 4 min. + +#### DAPI Staining and Fixation (approx. 23 min) + +17. For each sample, prepare 500uL of 0.2ug/mL DAPI in Staining Buffer + - From a stock solution of DAPI 1mg/mL, use 1:5000. E.g., add 0.1uL DAPI in 500uL Staining Buffer. + +18. On ice, resuspend the cell pellet in the DAPI solution, and incubate for 15 min. + +19. Spin down the cell suspension, 200 x g, 4℃, for 4 min. + +20. On ice, resuspend the DAPI-stained cell pellet in 2mL 1%PFA. + +21. Spin down the cell suspension, 200 x g, 4℃, for 4 min. + +22. Resuspend in 300uL Staining Buffer and transfer to a clean FACS tube pre-coated with Staining Buffer. + +#### Processing/Storage (approx. 23 min) + +23. Cells can be kept in Staining Buffer, at 4℃, in the dark (wrapped in aluminum foil) for at least a week (e.g., while waiting to collect samples from later on timepoints). + +24. When ready for FACS, on ice, distribute your cells in 300uL per FACS tube and proceed for sorting/analysis. + +#### Beads Samples (approx. 23 min) + +25. If using beads as positive references, prepare bead tubes just before FACS processing. For GFP BrightComp eBeads™ Compensation Bead Kit (Invitrogen/Thermo Fisher Scientific, CAT#A10514): + - Vortex vial for 10 seconds + - Dispense 1 drop into a clean FACS tube. + - Resuspend in 1mL Staining Buffer. + +--- + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/protocol-to-secretome-investigation-of-tumor-3d-co-ceqmtdu6.md b/markdown-output/protocol-to-secretome-investigation-of-tumor-3d-co-ceqmtdu6.md new file mode 100644 index 0000000000000000000000000000000000000000..72d2f1ee3e09a0b18f977009a62dd441853498fe --- /dev/null +++ b/markdown-output/protocol-to-secretome-investigation-of-tumor-3d-co-ceqmtdu6.md @@ -0,0 +1,115 @@ +```markdown +# Protocol to Secretome Investigation of Tumor 3D Co-Culture Model V.2 + +## Goal/Experiment: +To investigate metabolites released into the extracellular environment by 3D co-culture models of tumor tissues using high-performance liquid chromatography and mass spectrometry, providing insights into metabolic dynamics and potential therapeutic interventions. + +## Authors +- ANNA MARIA AP FERNANDES, +- Giulia Carli Mendes, +- Alex Rosini, +- Andrea Corazzi Pelosi, +- Leonardo Maciel, +- Luísa F Bueno, +- Lívia Maria F Silva, +- Rafael F Bredariol, +- Maycon Giovani Santana, +- Andreia de Melo Porcari, +- Denise G. Priolli + +## Affiliations +1. Postgraduate Programme Stricto Sensu in Health Science, São Francisco University, Bragança Paulista, SP, Brazil. +2. Multidisciplinary Laboratory, Medical School, São Francisco University, Bragança Paulista, São Paulo, Brazil. +3. n3dFab Laboratory of Mass Spectrometry, Health Sciences Postgraduate Program, São Francisco University, Bragança Paulista, São Paulo, Brazil. +4. Multiprofessional Nursing Residency Program in Oncology, A.C.Camargo Cancer Center, São Paulo, Brazil. + +## Abstract +Three-dimensional (3D) cell culture technologies mimic the complex microenvironment of tissues, aiding in the preclinical screening of new molecules and the study of tissue metabolism. This protocol describes an untargeted metabolomic approach using high-performance liquid chromatography coupled with high-resolution mass spectrometry to analyze metabolites released into the culture medium by 3D cultures and cocultures (secretome model). + +## Keywords +- Mass Spectrometry +- 3D Cell Culture +- Colonic Neoplasm +- Biomarkers + +## Materials + +| Reagent | Vendor | Catalog Number | +| ------------------------------------- | ----------------------- | ------------------------ | +| Trypsin EDTA | Gibco - Thermo | 25-051-CI | +| Dulbecco's Modified Eagle's Medium | Sigma Aldrich | D5796 | +| Sodium Pyruvate (100 mM) | Thermo Fisher | 11360070 | +| Fetal Bovine Serum | Gibco - Thermo | 10270106 | +| Penicillin-Streptomycin (10,000 U/mL) | Fisher | 15140-122 | +| Trypan Blue Solution 0.4% | Thermo Fisher | 15250061 | +| Acetonitrile | J.T. Baker | 9829-03 | +| Isopropanol HPLC solvent | JT Baker | 9095-02 | +| 24 Well Bio Assembler Kit | Greiner bio-one | 662840 | +| MilliQ Water | Contributed by users | - | +| 75 cm² T75 Flask | Contributed by users | - | +| Fluoro-DL-phenylalanine | Sigma-aldrich | F5255 | +| Acquity CSH C18 column | Waters | - | +| Formic acid, LC-MS grade | Thermo Fisher | 28905 | +| XEVO-G2XSQTOF | Waters | - | +| Leucine encephalin | Sigma-aldrich | - | + +## 2D Cell Culture Protocol + +1. **Cell Lines Used:** + - Human colon carcinoma (HT-29) and pre-adipocytes (3T3-L1) from Banco de Células do Rio de Janeiro (BCRJ). + +2. **Thaw and Propagation:** + - Thaw cells and propagate in Modified Dulbecco Eagle Medium (DMEM) with 100 mM sodium pyruvate, 10 % (v/v) fetal bovine serum, 1 % (v/v) antibiotics. + - Culture cells in a humidified chamber at 5 % (v/v) CO₂, 37 °C. + +3. **Incubation:** + - Incubate cells with 3 mL trypsin-EDTA (0.25 % (v/v)) for 3 minutes for disaggregation and propagation. + - Use DMEM plus 10 % PBS to inactivate trypsin, and seed cells in a new T75 flask with 10 mL DMEM. + +4. **Viability and Doubling Time:** + - Change medium according to doubling time and determine viability using Trypan Blue. + +## 3D Cell Culture Protocol + +1. **Bio-Assembler System (Greiner Bio-One Bio, Americana, Brazil):** + - Prepare magnetic nanoparticles and culture cells in monolayer at T75 flask. + - Determine the viability (>75%) and add 1 µL per 100,000 cells of magnetic nanoparticles to the culture. Centrifuge and homogenize. + +2. **3D Cell Coculture:** + - Form spheroids by adding 3T3-L1 spheroid suspension to HT-29 wells using magnetic pen and incubate for 7 days. + +3. **Extraction and Preparation of Samples:** + - Pipet 200 µL of medium into microtubes, add 50 µL of iced isopropanol, and keep at -20°C overnight. + - Centrifuge at 12,880 x g for 10 minutes, remove aliquot, resuspend extracts in 150 µL of solution (200 µM fluorophenylalanine in methanol 1:1). + - Perform chromatography and mass spectrometry. + +## Liquid Chromatography and Mass Spectrometry Analysis + +1. **Chromatography:** + - Use UPLC H-class with ACQUITY CSH C18 column and prepare mobile phase (A: water with 0.1 % (v/v) formic acid, B: acetonitrile). + +2. **Gradient:** + - Apply segmented gradient and set flow rate to 0.4 mL/min. + - Injection volume: 5 µL for positive and 2 µL for negative ionization modes. + +3. **Mass Spectrometry:** + - Perform analyses on XEVO-G2XSQTOF, optimize source parameters, acquire spectra (50-1200 Da) using MSE approach. + +## Data Processing and Identification of Compounds + +1. **Progenesis QI Software:** + - Process RAW files, perform peak alignment, and select adduct species ([M+H]+, [M+Na]+, [M+K]+...). + - Generate MS^E data for compound identification using multiple databases (Lipid Maps, LipidBlast). + +## Statistical Analyses + +1. **Metaboanalyst Platform:** + - Normalize data, scale using log-transformation and Pareto for positive ionization mode. + - Perform volcano plots and rank based on FDR and log2 fold change. + +## References +- HAISLER, W. L. et al., Nature Protocols, 8(10), 1940-1949, 2013. DOI: [10.1038/nprot.2013.125](https://doi.org/10.1038/nprot.2013.125). +- Progenesis QI for data analysis. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/protoplast-isolation-from-zymoseptoria-tritici-k2mcyc6.md b/markdown-output/protoplast-isolation-from-zymoseptoria-tritici-k2mcyc6.md new file mode 100644 index 0000000000000000000000000000000000000000..b439f5283eba3528a2fa4748f1195e72f4c5a22a --- /dev/null +++ b/markdown-output/protoplast-isolation-from-zymoseptoria-tritici-k2mcyc6.md @@ -0,0 +1,138 @@ +```markdown +# Goal/Experiment: +The aim of this experiment is to isolate protoplasts from Zymoseptoria tritici using a series of buffer solutions and enzymatic treatments. + +## Protoplast Isolation from Zymoseptoria tritici + +**Haseena Khan** + +**Citation:** Haseena Khan Protoplast Isolation from Zymoseptoria tritici. protocols.io +dx.doi.org/10.17504/protocols.io.k2mcyc6 +**Published:** 11 Dec 2017 + +### Abstract +[Description of the Abstract Content] + +### Before Start + +Prepare the following solutions: + +#### Stock solutions (do not filter sterilize) + +**A.** 50 mL 1.2 M MgSO4.7H2O (14.8 g / 50mL) +**B.** 20 mL 1.2M Sorbitol (4.32 g / 20 mL) +**C.** 1 M Tris pH 7.5 +**D.** 1 mL 1M CaCl2 (0.147 g / mL) + +#### Glucanex solution (1.2M MgSO4, 10mM Phosphate buffer pH 5.8) + +- **Glucanex Lysing Enzymes:** Glucanex is derived from *Trichoderma harzianum* and is used to degrade cell walls to release protoplasts. + - Vendor: Sigma Aldrich + - Catalog #: L1412-10G + +1. Add 0.04g NaH2PO4 to 20mL 1.2M MgSO4.7H2O and adjust pH to 5.8 using NaOH drop by drop. +2. Stir until the precipitate dissolves after each drop. Once pH is correct, complete to 25mL volume with the remaining 1.2M MgSO4.7H2O solution. +3. Filter sterilize and store the solution at room temperature. +4. Add 0.35g glucanex to the solution on the morning of transformation and shake well to resuspend. Do not filter sterilize after adding glucanex. + +**Note: Always prepare fresh solutions prior to the experiment.** + +#### Protoplast Overly and STC solutions + +- **Protoplast overlay solution (600 mM Sorbitol, 10 Tris pH 7.5):** + - 5 mL per preparation + - Add 2.5 mL of solution B (1.2M Sorbitol) to 2.45 mL milliQ water and 50 µL solution C (1 M Tris pH 7.5). Filter sterilize. + +- **1 M Sorbitol-tris solution** + - 5 ml per preparation + - Add 4.17 mL solution B to 780 µL milliQ water and 50 µL solution C. Filter sterilize. + +- **STC buffer (1.2 M Sorbitol, 10 mM CaCl2, 10 Tris pH 7.5)** + - 10 mL (∼10 mL required per preparation) + - Add 9.8 mL solution B to 100 µL solution C and 100 µL solution D. Filter sterilize. + +### Protocol + +#### Inoculum + +**Step 1.** + +Prepare a culture of *Zymoseptoria tritici* by inoculating 100 µL dense spore suspension into 500 mL Potato Dextrose Broth (PDB). Incubate culture at 22°C with 130 rpm shaking. + +**Alternative:** Inoculate 100 mL PDB with 50 µL dense spore suspension. + +#### Spore Collection + +**Step 2.** + +1. Collect the spores on the fourth day of culture. +2. Pour 50 mL of the *Zymoseptoria* culture into a 50 mL falcon tube and centrifuge at 4000 rpm for 10 minutes at 4°C. +3. Pour off the supernatant. +4. Repeat with a second 50 mL of culture. + +**Step 3.** + +1. Resuspend the pellet in 50 mL of sterile water by shaking the tube. +2. Centrifuge (same conditions as step 2). +3. Discard the supernatant. + +**Step 4.** + +1. Resuspend the pellet in 40 mL of 1 M Sorbitol by shaking. +2. Centrifuge at 4000 rpm for 10 minutes at 4°C. +3. Pour off the supernatant. + +**Step 5.** + +1. Add 0.35 g glucanex enzyme to 25 mL of filter-sterilized Glucanex solution and add it to the pellet. +2. Shake the tube gently to dissolve the pellet and pour into a sterile plastic Petri dish. + +**Step 6.** + +1. Incubate for 1 hour and 40 minutes at 28°C (avoid shaking). + +**Step 7.** + +1. Pour the digested sample from the Petri dish into a 50 mL falcon tube. +2. Overlay with 5 mL of 600 mM Sorbitol + 10 mM Tris (pH 7.5) very gently. Do not mix. +3. **Note:** Use a 1000 µL pipette tip for the overlay and pour it very gently to the wall of the falcon tube just above the digested solution. + +**Step 8.** + +1. Centrifuge at 4000 g for 15 minutes at 4°C. After centrifugation, three layers will appear. + +**Step 9.** + +1. Collect the interface layer (without touching the bottom layer) with a 1000 µL pipette in a 15 mL falcon tube. +2. Add an equal amount of 1M Sorbitol + 10 mM Tris (pH 7.5) to the top. +3. Mix gently by tilting the tube slowly. + +**Step 10.** + +1. Keep the tubes on ice. +2. Pour the whole solution over autoclaved cotton wool in a sterile syringe with no piston and allow it to drip slowly. Collect in a 15 mL falcon tube. + +**Step 11.** + +1. Centrifuge at 1500 g for 5 minutes at 4°C. Protoplasts will settle down in the bottom. +2. Remove the supernatant with a 1000 µL pipette without disturbing the pellet. +3. Wash the pellet by suspending in 1 mL of STC (1.2 M Sorbitol, 10 mM CaCl2, 10 mM Tris pH 7.5). +4. Spin down at 1500 g for 5 min at 4°C. Remove the supernatant carefully. +5. Gently resuspend the protoplasts in 500 µL of STC buffer by tapping the tube with fingers. +6. Take 10 µL of protoplast solution and count in a hemocytometer. +7. Dilute to a final concentration of 1 x 106 protoplasts/mL with STC buffer. +8. Use 100-200 µL of these freshly isolated protoplasts for transformation. + +#### Test to Confirm Protoplasts + +**Step 12.** + +1. After protoplasting, put a drop of the protoplast suspension on a slide and examine under a microscope. +2. After visualizing round protoplasts, add a drop of sterile water to the side of the slide without disturbing the coverslip. +3. Once the water spreads through the slide, all the protoplasts will disappear because they burst open due to the change in isotonic solution. +4. Spores or structures that stay intact or swell will be intact cells or debris. + +--- + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/psi-open-fluor-cam-script-for-measuring-qe-compone-byn9pvh6.md b/markdown-output/psi-open-fluor-cam-script-for-measuring-qe-compone-byn9pvh6.md new file mode 100644 index 0000000000000000000000000000000000000000..26cb4c6717cf6719350e415c2225a7dd66b5ba07 --- /dev/null +++ b/markdown-output/psi-open-fluor-cam-script-for-measuring-qe-compone-byn9pvh6.md @@ -0,0 +1,328 @@ +```markdown +# Goal/Experiment: +To measure the qE component of Non-Photochemical Quenching (NPQ) in *Chlorella vulgaris* using the PSI Open Fluor CAM. + +## PSI Open Fluor CAM Script for Measuring qE Component of NPQ in *Chlorella vulgaris* + +**Author**: Andrei Herdean +**Date**: September 30, 2021 +**Affiliation**: University of Technology Sydney + +### Abstract + +This is a simple protocol that consists of: +1. 10 minutes preillumination with far red light. +2. 5 minutes of illumination with actinic light. +3. 5 minutes of dark adaptation with far red light. + +qE is calculated as the difference between NPQ_Lss and NPQ_D5: +qE = NPQ_Lss - NPQ_D5 +qI = NPQ_D5 + +**Instruments and Components** +- **FluorCAM 7.0** on a PSI Open FC 800-O/1010-S. +- **Act 2**: White light LED arrays. +- **ADD2**: Far red LED array. + +### Protocol Overview + +Camera is placed at ~20 cm above the measured sample. Light intensity uniformity across the 96 well plate was measured according to manufacturer instructions. + +> Important: Protocol only works under weak far red light. Intense far red will interfere with the fluorescence measurement. + +### Equipment and Reagents + +1. **FluorCAM 7.0**: Used for chlorophyll fluorescence measurement. +2. **PSI Open FluorCAM FC 800-O/1010-S**: An imaging fluorometer specifically for plant studies. +3. **Act 2 White Light LED Arrays**: Provide the illumination for the actinic light phase. +4. **Far Red LED Array (ADD2)**: Used for preillumination and dark adaptation phases. + +### Experimental Steps + +1. **Preillumination**: + - 10 minutes under far red light (ADD2=10, Act1=0). + +2. **Illumination with Actinic Light**: + - 5 minutes under actinic light (Act 2=18). + +3. **Dark Adaptation**: + - 5 minutes under far red light (ADD2=10, Act1=0). + +## FluorCAM Script + +```plaintext +; Quenching protocol with Actinic2 +; with FilterWheel +; version November 11, 2020 +; high-resolution CCD TOMI-2 +; optimized number of measured frames +; Protocol duration 183s +; +ADD1=0 +ADD2=10 +Act1=0 +TS=50ms +include default.inc ; Includes standard options, do not remove it! +include light.inc ; Includes standard options, do not remove it! +include FW.inc ; Includes standard options, do not remove it! +; +Shutter=2 +Sensitivity=29.3 +Act2=18 +Super=69.6 +LightA=29.069 +LightB=-34.732 +; +<0s>=>SET_FILTER(CHL) +Preillumination=600s +<0s>=>add2(Preillumination) +start = Preillumination +; + +;*** Fo Measurement *** +F0duration=5s +F0period=1s +start + <0,F0period...F0duration>=mfmfsub +; +Fo definition +start + <0s>=>checkPoint,"startFo" +start + =checkPoint,"endFo" +; + +;*** Saturating Pulse & Fm Measurement *** +PulseDuration=800ms +a1 = start + F0duration+2*mfmfsub_length +; +=>SatPulse(PulseDuration) +=>act2(PulseDuration) +; +=>mPulse2 +; +Fm definition +=>checkPoint,"startFm" +=checkPoint,"endFm" +; +Visual frame definition; Image shown in pre-processing window +=>checkPoint,"timeVisual" +; + +;****** Dark Relaxation Measurement ****** +DarkRelaxation1=17s +b1 = a1+PulseDuration+2*mfmfsub_length +b2=2s +=mfmfsub +=mfmfsub +; + +;****** Kautsky Effect Measurement ****** +; + +;****** Actinic light Exposure ****** +ALPeriod=300s +c1=a1+PulseDuration+DarkRelaxation1+mfmfsub_length +=>act2(ALPeriod) +; + +; Fast Kautsky kinetics +c2=2s +=mfmfsub; +; + +; Slow Kautsky kinetics +c3=4s +=mfmfsub +; + +=mfmfsub +; +Fp definition +=checkPoint,"startFp" +=checkPoint,"endFp" +; + +;****** Saturating Pulses - Fm' Quenching Analysis ****** +; + +;****** Saturating Pulses - Fm_L1 ****** +f1=c1+<85s> +f11=f1+mfmfsub_length, 2*mfmfsub_length... PulseDuration>=mfmfsub +f1>=mfmfsub +f1+mfmfsub_length>=checkPoint,"startFL_L1" +f1+PulseDuration-mfmfsub_length>=checkPoint,"endFL_L1" +; +f2=f1+PulseDuration +f2=>SatPulse(PulseDuration) +f2=>mPulse2 +f2+PulseDuration/2=>checkPoint,"startFm_L1" +f2+PulseDuration-mfmfsub_length>=checkPoint,"endFm_L1" +; + +;****** Saturating Pulses - Fm_L2 ****** +f3=c1+<118s> +f31=f3+=mfmfsub> +f31>=mfmfsub +f31+mfmfsub_length>=checkPoint,"startFL_L1" +f31+PulseDuration-mfmfsub_length>=checkPoint,"endFL_L2" +; + +;****** Saturating Pulses - Fm_L3 ****** +f5=c1+<178s> +f51=f5+=mfmfsub> +f51>=mfmfsub +f51+mfmfsub_length>=checkPoint,"startFL_L3" +f51+PulseDuration-mfmfsub_length>=checkPoint,"endFL_L3" +; + +;****** Saturating Pulses - Fm_L4 ****** +f7=c1+<238s> +f71=f7+=mfmfsub> +f71>=mfmfsub +f71+mfmfsub_length>=checkPoint,"startFL_L4" +f71+PulseDuration-mfmfsub_length>=checkPoint,"endFL_L4" +; +f8=f7+PulseDuration +f8=>SatPulse(PulseDuration) +f8=>mPulse2 +f8+PulseDuration/2=>checkPoint,"startFm_L4" +f8+PulseDuration-mfmfsub_length>=checkPoint,"endFm_L4" +; + +;****** Saturating Pulses - Fm_Lss ****** +f9=c1+<298s> +f91=f9+=mfmfsub> +f91>=mfmfsub +f91+mfmfsub_length>=checkPoint,"startFL_Lss" +f91+PulseDuration-mfmfsub_length>=checkPoint,"endFL_Lss" +; +f10=f9+PulseDuration +f10=>SatPulse(PulseDuration) +f10=>mPulse2 +f10+PulseDuration/2=>checkPoint,"startFm_Lss" +f10+PulseDuration-mfmfsub_length>=checkPoint,"endFm_Lss" +; + +;** Dark relaxation after actinic light period ** +DarkRelaxation2=300s +h1=c1+ALPeriod +

=>add2(DarkRelaxation2) +h2=mfmfsub_length +h3=10s +; + +;***** Relaxation measurement ***** +=>mfmfsub +; +=mfmfsub +; + +;**** Saturating Pulses - Fm_D1 ***** +g11=f1+<58s> +g11=f1+ +g11>=mfmfsub +g11+mfmfsub_length>=checkPoint,"startFL_D1" +g11+PulseDuration-mfmfsub_length>=checkPoint,"endFL_D1" +; +g2=g1+PulseDuration +g2=>SatPulse(PulseDuration) +g2=>act2(PulseDuration) +g2=>mPulse2 +g2+PulseDuration/2=>checkPoint,"startFm_D1" +g2+PulseDuration-mfmfsub_length>=checkPoint,"endFm_D1" +; + +;*** Saturating Pulses - Fm_D2 *** +g3=h1+<118s> +g31=g3+=mfmfsub> +g31>=mfmfsub +g31+mfmfsub_length>=checkPoint,"startFL_D2" +g31+PulseDuration-mfmfsub_length>=checkPoint,"endFL_D2" +; +g4=g3+PulseDuration +g4=>SatPulse(PulseDuration) +g4=>act2(PulseDuration) +g4=>mPulse2 +g4+PulseDuration/2=>checkPoint,"startFm_D2" +g4+PulseDuration-mfmfsub_length>=checkPoint,"endFm_D2" +; + +;*** Saturating Pulses - Fm_D3 *** +g5=h1+<178s> +g51=g5+=mfmfsub> +g51>=mfmfsub +g51+mfmfsub_length>=checkPoint,"startFL_D3" +g51+PulseDuration-mfmfsub_length>=checkPoint,"endFL_D3" +; +g6=g5+PulseDuration +g6=>SatPulse(PulseDuration) +g6=>act2(PulseDuration) +g6=>mPulse2 +g6+PulseDuration/2=>checkPoint,"startFm_D3" +g6+PulseDuration-mfmfsub_length>=checkPoint,"endFm_D3" +; + +;*** Saturating Pulses - Fm_D4 *** +g7=h1+<238s> +g71=g7+=mfmfsub> +g71>=mfmfsub +g71+mfmfsub_length>=checkPoint,"startFL_D4" +g71+PulseDuration-mfmfsub_length>=checkPoint,"endFL_D4" +; +g8=g7+PulseDuration +g8=>SatPulse(PulseDuration) +g8=>act2(PulseDuration) +g8=>mPulse2 +g8+PulseDuration/2=>checkPoint,"startFm_D4" +g8+PulseDuration-mfmfsub_length>=checkPoint,"endFm_D4" +; + +;*** Saturating Pulses - Fm_D5 *** +g9=h1+<298s> +g91=g9+=mfmfsub> +g91>=mfmfsub +g91+mfmfsub_length>=checkPoint,"startFL_D5" +g91+PulseDuration-mfmfsub_length>=checkPoint,"endFL_D5" +; +g10=g9+PulseDuration +g10=>SatPulse(PulseDuration) +g10=>act2(PulseDuration) +g10=>mPulse2 +g10+PulseDuration/2=>checkPoint,"startFm_D5" +g10+PulseDuration-mfmfsub_length>=checkPoint,"endFm_D5" +; + +; END *** +``` + +### References + +DOI: [dx.doi.org/10.17504/protocols.io.byn9pvh6](https://dx.doi.org/10.17504/protocols.io.byn9pvh6) + +### Protocol Citation + +Andrei Herdean. 2021. PSI Open Fluor CAM script for measuring qE component of NPQ in *Chlorella vulgaris*. +protocols.io. +[https://dx.doi.org/10.17504/protocols.io.byn9pvh6](https://dx.doi.org/10.17504/protocols.io.byn9pvh6) + +### Keywords + +NPQ, qE, FluorCAM + +### License + +This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. + +### Common Terms Defined + +- **Non-Photochemical Quenching (NPQ)**: The process by which excess light energy is dissipated as heat in plants to protect them from photodamage. +- **qE**: The component of NPQ related to energy-dependent quenching. +- **F0**: Minimum fluorescence in the dark-adapted state. +- **Fm**: Maximum fluorescence in the dark-adapted state. +- **Actinic Light**: Light that stimulates photosynthesis, often used in experiments to simulate natural light conditions. + +## Alternative Methods and Recommendations + +- **Far Red LED Array (ADD2)**: If unavailable, LED arrays with similar far-red light specifications can be used, making sure they have the appropriate wavelength range (~700-750 nm). +- **FluorCAM 7.0**: Devices with equivalent capabilities for chlorophyll fluorescence measurement, such as Walz Imaging PAM M-Series, can be used with appropriate script adjustments. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/purification-of-10xhis-supertev-cn4uvgww.md b/markdown-output/purification-of-10xhis-supertev-cn4uvgww.md new file mode 100644 index 0000000000000000000000000000000000000000..ccf46834dcc316985035f1657d0f08f41727fe20 --- /dev/null +++ b/markdown-output/purification-of-10xhis-supertev-cn4uvgww.md @@ -0,0 +1,175 @@ +```markdown +# Goal/Experiment: +The goal of this experiment is the purification of the 10xHis-SuperTEV, a mutated version of TEV (Tobacco Etch Virus) protease with improved solubility and purification efficiency. + +# Purification of 10xHis-SuperTEV + +**Authors**: Kelvin Lau¹, Bouchra Bouchri², Florence Pojer² +¹EPFL - EPF Lausanne; ²EPFL SV PTPSP + +**Date**: FEB 10, 2023 + +**Abstract** +SuperTEV is a mutated version of TEV (Tobacco Etch Protease), a cysteine protease widely used in labs for its high specificity to a cleavage sequence that can be genetically encoded. Depending on the lab, it has been reported that the protease is unstable or purifies with low yields. Efforts have been made to improve its solubility. We report our platform's efforts in generating a new TEV variant, called SuperTEV, which incorporates 9 mutations identified by different groups. We find that the protein is easily produced and purified in large amounts and is functional. + +## Mutations in Canonical TEV Protease + +| Mutation | Function/Note | References | +|-----------|-----------------------------------|----------------------------------------| +| T17S | Increased solubility and production | van den Berg et al., 2006;
Wei et al., 2012 | +| L56V | Improved solubility | Cabrita et al., 2007;
Wei et al., 2012 | +| N68D | Increased solubility and production | van den Berg et al., 2006;
Wei et al., 2012 | +| I77V | Increased solubility and production | van den Berg et al., 2006;
Wei et al., 2012 | +| S135G | Increased solubility | Cabrita et al., 2007;
Wei et al., 2012 | +| I138T | Increased catalytic activity | Sanchez and Ting, 2019 | +| S153N | Increased catalytic activity | Sanchez and Ting, 2019 | +| T180A | Increased catalytic activity | Sanchez and Ting, 2019 | +| S219V | Inhibits autoproTEVolysis | Kapust, 2001 | + +## Materials + +- **Plasmid**: Addgene #193833 (https://www.addgene.org/193833) +- **BL21 (DE3) cells**: (Lucigen) +- **Growth Media**: AutoTB + trace elements (Formedium), TB + trace elements (Formedium) +- **Glycerol**: (Applichem) +- **AktaGO**: (Cytvia) +- **Ni-NTA Columns**: HiFliQ (ProteinArk) +- **Dialysis Tubing**: 12-14 kDa, clips (SpectraPor) +- **Emulsiflex**: High-pressure homogenizer +- **5 M NaCl**: Sodium Chloride, pH 7.5 +- **1 M HEPES, pH 7.5**: (Sigma #56749-1KG) +- **2.5 M Imidazole, pH 7.5**: (We recommend Sigma #56749-1KG for low background absorbance) +- **1 M DTT**: Dithiothreitol +- **1 M IPTG**: Isopropyl β-D-1-thiogalactopyranoside +- **4X LDS Loading Dye**: (Genscript) +- **NuPage 4-20% SDS-PAGE Gels**: (Thermofisher) +- **8M Urea**: High-strength denaturant + +## Before Start Instructions + +Recommended to have ready before starting along with **Materials**: + +- Autoclaved flasks, LB media, AutoTB media, buffers +- Liquid nitrogen + +## Growing bacterial cultures (6 L) +1. **Transform Bacterial Plasmid** + - Transform the bacterial plasmid expressing the 10xHis-SuperTEV into BL21 (DE3) cells. + - Plate on LB-Agar plates with Kanamycin. + - Grow overnight at 37°C or over the weekend at 25°C. + +2. **Prepare Pre-culture** + - In an afternoon, pick a streak of cells and inoculate into 200 mL of LB + Kanamycin media. + - Note: For every 1 L of expression culture, require 10-20 mL of pre-culture. + - Grow overnight at 37°C. + +3. **Inoculate Main Culture and Induction** + - Inoculate 20-40 mL of pre-culture into 3 flasks containing 2 L of Autoinduction TB media. + - Shake in an incubator at 37°C for 3-4 hours until OD600 ~0.8-1. + - Take a 1 mL sample of the culture. + - Immediately change the temperature to 18°C and continue shaking overnight (approximately 20 hours). + **Note**: Regular LB or TB media can also be used. Induction will be at the same point as the temperature change with 0.5 mM IPTG. + +4. **Harvest Culture** + - Take a 1 mL sample of the culture and measure the OD600. + - Harvest the culture by centrifuging at 5000 x g, 10°C, 30 minutes. + - Transfer the pellets to 50 mL tubes directly or resuspend in minimal amounts of wash buffer before transferring. + - Pellets can be used immediately or stored at -20°C indefinitely. + +5. **Confirm Protein Expression** + - Run an SDS-PAGE gel to observe the appearance of a band around 25 kDa that would represent the production of the 10xHis-SuperTEV. + +### Preparation of SDS-PAGE samples + - Mix 1:4, 4X LDS loading dye, and 8 M Urea to make a Urea loading dye. + - For samples: + - Calculate the amount of sample needed to prepare using the formula: + `1/OD600 x 100 μL = volume in μL to centrifuge down` + - Centrifuge down at 14000 rpm, 1 minute. Discard the supernatant, Keep the pellet. + - Resuspend pellet in 40 μL of the Urea loading dye. + - Load 10 μL onto a NuPage 4-12% bis-Tris SDS-PAGE gel. + +### Expected Result + - **Induction Gel**: Observed appearance of a band near 25 kDa that represents the induced protein. + +## Prepare Purification Buffers +From stock solutions prepare the following. Filter (0.22 or 0.45 μm). + +### Wash Buffer (2 L) + +| Component | Concentration (mM) | +|-------------------|--------------------| +| NaCl | 700 | +| HEPES, pH 7.5 | 20 | + +### Elution Buffer (1 L) + +| Component | Concentration (mM) | +|-------------------|--------------------| +| NaCl | 700 | +| HEPES, pH 7.5 | 20 | +| Imidazole, pH 7.5 | 500 | + +## Purification by Ni-NTA on an AKTA system + +1. **Resuspending Pellets** + - Resuspend pellets with glycerol to 10% and DNase. + - Lyse using an Emulsiflex device by 3 passes until lysate is visually not viscous. + **Note**: Other lysis methods such as sonication and french press can also be used. + +2. **Centrifugation** + - Total volume is around 100 mL. Centrifuge down the lysate at 20000 x g, 4°C, 40 minutes. + +3. **Supernatant Handling** + - Transfer the supernatant to a clean container without visually turbid particles near the pellet. + - Filter the supernatant using a 0.45 μm filter. + +4. **Imidazole Supplementation** + - Supplement the filtered supernatant with 1 Molarity (M) imidazole to a final concentration of 25 millimolar (mM) imidazole. + +### AKTA Loading and Elution + - Use an AKTA system to load the sample onto 3 x 5 mL HiFliQ Ni-NTA columns equilibrated with 5% Elution buffer (25 mM imidazole). + - Wash extensively and elute as a step gradient. + +| Step | Elution Buffer Concentration (%B) | Column Volumes (CV) | +|------------------------|----------------------------------|---------------------| +| Wash + 25 mM Imidazole | 5 | 8 | +| Wash + 100 mM Imidazole| 20 | 5 | +| Wash + 500 mM Imidazole| 100 | 10 | + +### Expected Result + - **Elution Profile**: A typical elution profile and expected peaks during elution verification on an SDS-PAGE gel of all fractions during purification. + +5. **Collection and Concentration** + - Protein elutes at 500 mM imidazole. Measure absorbance at A280 to determine concentration. + `1 mg/mL of SuperTEV = A280, 1.2` + - Dilute with wash buffer to around 2-3 mg/mL or as desired. + +## Dialysis Preparation +1. **Dialysis** + - Transfer protein to dialysis bags for dialysis in 2 L of dialysis buffer (150 mM NaCl, 20 mM HEPES, pH 7.5, 5 mM DTT, 10% (v/v) glycerol). + - Dialyze overnight at 4°C. + +2. **Precipitation and Centrifugation** + - Transfer the dialyzed material to 50 mL tubes and centrifuge at 20000 x g, 4°C, 10 minutes. The supernatant should be clear. + - Determine concentration by measurement at A280. Aliquot 1-2 mg per tube. + - Flash freeze under liquid nitrogen and store indefinitely at -80°C. + - Typical final yield is in the 10s of mg/L (average 50 mg/L). + +### Expected Result + - **Final Sample Gel Image**: Appearance after dialysis, 2 μg loaded on the gel. + +## Use of 10xHis-SuperTEV Protease + +1. **Cleavage Test** + - Cleave substrates containing the TEV protease site ENLYFQ/GS. + - Use the protease in a 1:50-1:100 (protease:protein mass ratio). + +### Conditions +- Room temperature or 4°C overnight during dialysis in PBS or HBS buffers. + +### Expected Result + - **Cleavage Verification Gel**: His-MBP-Cas9 was purified from Addgene #69090 and cleave with SuperTEV in a 1:100 ratio (100 mg total crude protein from elution: 1.4 mg SuperTEV) overnight at 4°C. + - The final gel image shows the cleavage pattern of SuperTEV protease. + +endofoutput +``` diff --git a/markdown-output/purification-of-cafeteria-roenbergensis-virus-part-qz2dx8e.md b/markdown-output/purification-of-cafeteria-roenbergensis-virus-part-qz2dx8e.md new file mode 100644 index 0000000000000000000000000000000000000000..4858b5ceccc5f35de7845dd08d7967711397ecb8 --- /dev/null +++ b/markdown-output/purification-of-cafeteria-roenbergensis-virus-part-qz2dx8e.md @@ -0,0 +1,78 @@ +```markdown +# Goal/Experiment: +The goal of this experiment is to purify Cafeteria roenbergensis virus (CroV) particles using a combination of centrifugation, tangential flow filtration, and ultracentrifugation. + +# Purification of Cafeteria roenbergensis virus particles +**Matthias Fischer** + +## Abstract +The concentration and purification of giant DNA viruses from bacteria-containing cultures can present problems because the particle size of giant viruses overlaps with that of smaller bacteria, hence filtration as the only separation method is not possible. In this protocol, a combination of centrifugation, tangential flow filtration, and density-gradient ultracentrifugation is used to concentrate and purify particles of the giant marine virus CroV that infects the heterotrophic flagellate *Cafeteria roenbergensis*. + +## Materials + +- **Optiprep (Iodixanol) D1556-250ML by Sigma Aldrich**: A density gradient medium used for the isolation and purification of viruses. +- **F/2 medium MKK50L**: A nutrient solution often used for the culture of marine algae, which can be supplemented for the maintenance of Cafeteria roenbergensis culture. +- **Sorvall Lynx 4000 Centrifuge 75006581 by Thermo Fisher Scientific**: Used for initial centrifugation steps to remove cell debris and collect virus-containing supernatant. +- **BD Bacto™ Yeast Extract 212750 by BD Biosciences**: A nutrient additive to stimulate bacterial growth, essential as Cafeteria roenbergensis feeds on bacteria. +- **Cafeteria roenbergensis culture**: Obtainable from culture collections such as Roscoff or CCAP. +- **SW40 Ti (with Beckman ultracentrifuge) 344060 by Beckman Coulter**: Rotor and tubes for density gradient ultracentrifugation to concentrate virus particles. +- **Slide-A-Lyzer 3 mL Dialysis Cassettes, 20 kDa MWCO 66003 by Thermo Fisher Scientific**: Used for buffer exchange and dialysis of the virus suspension. +- **Vivaflow 200 tangential flow filtration unit (0.2 µm, PES) VF20P7 by Sartorius**: For concentrating the CroV-containing supernatant, including tubing for Masterflex peristaltic pump. + +## Protocol + +### Step 1 +1. Grow cultures of *Cafeteria roenbergensis* to an initial density of 7E+05 to 1E+06 cells/mL in f/2 artificial seawater medium containing 0.03% (w/v) yeast extract. +2. If needed, dilute denser cultures to the desired cell concentration with f/2-yeast medium. +3. Aliquot the cultures in the following: + - 50 mL aliquots per 250 mL polycarbonate Erlenmeyer flask + - 500 mL per 3 L polycarbonate Fernbach flask +4. Infect host cultures with a CroV suspension at a multiplicity of infection (moi) of 0.01. +5. Incubate at 20-23 °C (constant shaking not required). +6. Leave one culture uninfected as a negative control. + +### Step 2 +1. Monitor host cell density daily by counting on a hemocytometer until lysis occurs (typically 4-6 days post infection). + +### Step 3 +1. When cell densities of infected cultures drop below 1E+05 cells/mL, centrifuge the lysates to remove cell debris. +2. Use 1 L centrifuge bottles in a Sorvall F9-6x1000 rotor at 8000 rcf for 20 minutes at 4°C. +3. Save the virus-containing supernatant and discard the pellets. + +### Step 4 +1. Concentrate the CroV-containing supernatant by tangential flow filtration in a Vivaflow 0.2 µm PES unit (up to 15 L possible). +2. Cool the unit and reservoir during concentration, ensuring tubing connections are tight. +3. Do not exceed a back pressure of 2 bars. +4. Discard the filtrate and concentrate the CroV-bacteria mixture to 30-50 mL. +5. Save the concentrate in a 50 mL Falcon tube. + +### Step 5 +1. Use ultracentrifugation on a two-step Optiprep cushion to remove bacteria and concentrate CroV particles. +2. Fill Beckman SW40 ultraclear tubes with 6 mL of CroV-bacteria concentrate. +3. Underlay with: + - 2.5 mL of 23% Optiprep solution in 50 mM Tris-HCl, pH 8.0, 2 mM MgCl₂, 400 mM NaCl. + - 1 mL of 40% Optiprep solution in 50 mM Tris-HCl, pH 8.0, 2 mM MgCl₂, 400 mM NaCl. +4. Centrifuge at 100,000 rcf for 2 hours at 15°C in a SW40 rotor. + +### Step 6 +1. Collect the opaque CroV-containing band from the 23%-40% interface. +2. Dialyze the virus particles in 3 mL dialysis cassettes (20 kDa cutoff) against 1 L of 50 mM Tris-HCl, pH 8.0, 2 mM MgCl₂, 400 mM NaCl at 4°C overnight. + +### Step 7 +1. Prepare 10%-50% continuous Optiprep gradients in SW40 ultraclear tubes using the Gradient Master (81.5° angle, 35 rpm, 1 min 15 sec). +2. Load ~2 mL dialyzed CroV suspension per tube and centrifuge for 4 hours at 100,000 rcf, 4°C. +3. Extract CroV-containing bands using a 21G needle and syringe. + +### Step 8 +1. Dilute CroV-Optiprep suspension 3-fold with 50 mM Tris-HCl, pH 8.0, 2 mM MgCl₂, 400 mM NaCl. +2. Spin in a tabletop centrifuge for 20 minutes at 4°C, 10,000 rcf. +3. Save supernatant, dissolve opaque pellets in 50 µL Tris-HCl buffer. +4. Combine and spin dissolved pellets for 15 minutes at 8,000 g, 4°C. +5. Resuspend the virus pellet carefully. +6. The supernatants can be centrifuged again for 1 hour at maximum speed (20,000 rcf), 4°C to collect remaining virus particles for DNA extraction. + +## References +Matthias Fischer. Purification of Cafeteria roenbergensis virus particles. protocols.io. [dx.doi.org/10.17504/protocols.io.qz2dx8e](https://dx.doi.org/10.17504/protocols.io.qz2dx8e). Published: 15 Jun 2018 + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/purification-of-viruses-from-culture-lysates-d2t8em.md b/markdown-output/purification-of-viruses-from-culture-lysates-d2t8em.md new file mode 100644 index 0000000000000000000000000000000000000000..acd0604a95b98d7446259b4ab83328f36bdf9094 --- /dev/null +++ b/markdown-output/purification-of-viruses-from-culture-lysates-d2t8em.md @@ -0,0 +1,110 @@ +```markdown +# Goal/Experiment: +The goal of this experiment is to purify viruses from culture lysates using ultracentrifugation, which provides a means to concentrate, analyze, and purify viruses in solution. This method is invaluable for aquatic virologists and includes virus purification from natural water samples. + +# Purification of Viruses from Culture Lysates +**Janice E. Lawrence and Grieg F. Steward** + +## Abstract +Ultracentrifugation provides a means to concentrate, analyze, and purify viruses in solution, and therefore represents an invaluable tool for aquatic virologists. This protocol provides a method for purification of viruses from culture lysates from natural water samples. + +Citation: Janice E. Lawrence and Grieg F. Steward. Purification of viruses from culture lysates. protocols.io dx.doi.org/10.17504/protocols.io.d2t8em +**Published:** 16 Nov 2015 + +## Guidelines + +### Materials and Reagents +- **OptiPrep (60% iodixanol solution)**: Available from Axis-Shield, Accurate Chemical and Scientific (Westbury), Progen Biotechnik, or Sigma Aldrich. +- **Open-topped ultracentrifuge tubes**: e.g., Beckman Coulter Ultra-Clear. +- **Ultracentrifuge** +- **Swing-out Ultracentrifuge Rotor**: e.g., Beckman Coulter SW41, SW28, or MLS50. +- **30 kDa cutoff disposable centrifugal ultrafiltration devices**: e.g., Millipore. +- **3 mL syringe with Luer-Lok or Luer-Slip** +- **Pipetting needle**: e.g., Cadence Science, stainless-steel 14- or 16-gauge 4-inch cannula with Luer hub or Slip hub. +- **Sterile 1.5 mL microcentrifuge tubes**: for collecting gradient fractions. +- **Sterile disposable transfer pipettes** +- **Sterile virus-free media**: for resuspending and diluting virus. +- **Polyethylene glycol, average molecular weight 6000-8000**: e.g., Fisher Scientific Carbowax PEG 8000, or Sigma Aldrich Biochemika Ultra 8000. + +### Discussion +OptiPrep must be removed from samples before examination of virus particles by negative staining and TEM. This can be achieved using disposable Millipore centrifugal ultrafiltration devices with a 30 kDa cutoff. For most other applications, OptiPrep does not need to be removed prior to further analysis, although it should be assayed to determine effects on the growth of specific viral-hosts when re-infection assays are used to confirm the purification of the infectious agent. + +## Protocol + +### Clarify Lysate +**Step 1:** +Centrifuge the lysate at 4000g for 30 min. +- **Duration:** 00:30:00 + +**Step 2:** +Carefully decant and retain the supernatant. + +### Concentrate Virus by PEG Precipitation +**Step 3:** +Dissolve 8% PEG (w/v) in clarified lysate and allow to precipitate overnight at 4°C. +- **Duration:** 18:00:00 + +**Step 4:** +Centrifuge the PEG solution at 10,000g for 20 min. +- **Duration:** 00:20:00 + +**Step 5:** +Carefully decant the supernatant, retaining the pellet. + +**Step 6:** +Resuspend pellet in a small volume of residual PEG solution and pool all pelleted material. + +**Step 7:** +Repeat steps 5-6 as needed to concentrate virus to < 1 mL. + +**Step 8:** +Resuspend virus in 10-50 volumes of culture media to dilute PEG and allow virus pellet to disaggregate overnight at 4°C. +- **Duration:** 18:00:00 + +**Step 9:** +Concentrate sample to 1 mL through a 30 kDa cutoff disposable centrifugal ultrafiltration device. + +### Prepare Continuous, Isopycnic, Purifying Gradients +**Step 10:** +Prepare OptiPrep solutions using culture media as the diluent. + +**Notes:** +For many viruses, a gradient from 25%-40% OptiPrep will provide a good range for separation, but very dense or light viruses may require adjustment. To achieve this range, prepare 25%, 30%, 35%, and 40% v/v final-OptiPrep-concentration solutions, remembering that OptiPrep is sold as a 60% solution. The actual densities these concentrations achieve are dependent on the density of the culture media used as a diluent and must be determined for each system. + +**Step 11:** +Using the underlayering technique with syringe and pipetting needle, pour 4-step gradients into open-topped ultracentrifuge tubes, beginning with the least dense solution. + +**Step 12:** +Allow to blend for 2 h at room temperature. +- **Duration:** 02:00:00 + +**Notes:** +Make sure to prepare gradients to serve as balance tubes where appropriate. + +**Step 13:** +Mark the top of the gradients with a fine-tipped marker, and carefully overlay virus concentrate using a transfer pipette. + +**Step 14:** +Overlay culture media on balance gradients to create balance tubes. + +**Step 15:** +Balance the tubes by adding media to underweight tubes. + +**Step 16:** +Load tubes into rotor and ultracentrifuge at maximum permissible speed until density equilibrium is reached. +- **Notes:** As a guideline, a 4-mL gradient with 1-mL virus sample in a Beckman Coulter MLS-50 should be centrifuged for at least 4 h 15 min at 200,620g (50,000 rpm); an 11-mL gradient with 1-mL virus sample in a Beckman Coulter SW-41 should be centrifuged for at least 7 h 20 min at 207,570g (41,000 rpm). These conditions should be determined empirically for different systems. + +### Collect Viral Fraction +**Step 17:** +Using any fraction collection apparatus/technique, carefully extract purified viral concentrate from each tube. + +**Step 18:** +If bands are not visible, a starting point is to fractionate the gradient into 4+ fractions and use a couple of techniques to identify the virus-containing fraction (i.e., TEM, bioassay, absorbance at 260 nm, nucleic acid analysis, epifluorescence microscopy, or flow cytometry). + +**Notes:** +Each of these techniques has drawbacks and may lead to false results. For example, more than one virus-containing fraction may be detected by TEM when analyzing non-axenic cultures because contaminating bacteria are usually host to phage. Likewise, no virus-containing fractions may be detected by epifluorescence microscopy if the virus in question contains a small ssRNA genome, since the fluorescence yields of dyes are currently too low for visual detection of small ssRNA genomes. A combination of approaches for identifying virus fractions may therefore be required when working with novel viruses. + +--- + +**endofoutput** +``` \ No newline at end of file diff --git a/markdown-output/purifying-viruses-using-sucrose-cushion-c3wypd.md b/markdown-output/purifying-viruses-using-sucrose-cushion-c3wypd.md new file mode 100644 index 0000000000000000000000000000000000000000..47ee8ad43c5a05271d40c06f9c0dc84354df54c4 --- /dev/null +++ b/markdown-output/purifying-viruses-using-sucrose-cushion-c3wypd.md @@ -0,0 +1,102 @@ +```markdown +# Goal/Experiment: +The aim of this experiment is to purify viruses using a sucrose cushion. The protocol involves DNase I treatment, CsCl purification, and sucrose purification methods for virus concentration and purification, based on the Shannon Williamson protocol. + +# Purifying Viruses Using Sucrose Cushion + +## Abstract +This protocol describes the use of a sucrose cushion to purify viruses. It is the Matthew Sullivan Lab adaptation of the Shannon Williamson protocol. + +DNase I treatment, CsCl purification, and sucrose purification methods were compared using replicated viral metagenomics in Hurwitz et al. 2012. + +**Citation:** Hurwitz, B. L., Deng, L., Poulos, B. T., & Sullivan, M. B. (2012). [Evaluation of methods to concentrate and purify ocean virus communities through comparative, replicated metagenomics.](https://doi.org/10.1111/j.1462-2920.2012.02836.x) Environmental Microbiology. doi:10.1111/j.1462-2920.2012.02836.x + +**Published:** 12 Oct 2015 + +## Guidelines +### Shannon Williamson’s Protocol + +#### 1. DNase I Treatment +(Can also be done post sucrose cushion) + +1.1 **Dilution:** Dilute the sample to approximately 10%-20% glycerol with MilliQ water. Samples in 50% glycerol should generally be a volume of ~20ml. *Only applies if glycerol was used as a cryoprotectant*. + +1.2 **DNase Addition:** Add 1 unit of DNase I (RNase free) for every milliliter of the sample. + +1.3 **Incubation:** Mix the sample and incubate at room temperature for 2 hours. + +1.4 **Stopping Reaction:** Stop the reaction by adding EDTA and EGTA to a final concentration of 100mM. + +1.5 **Storage:** Store at 4°C. + +#### 2. Pelleting Viral Particles Through a Sucrose Cushion + +2.1 **Preparation:** Rinse one ultracentrifuge tube per sample with sterile water. + +2.2 **Loading Sample:** Load the sample (no more than ~25 to 30ml) into the ultracentrifuge tube. + +2.3 **Sucrose Addition:** Add up to 10ml of 38% sucrose in SM buffer to the bottom of the ultracentrifuge tube to form a sucrose cushion. Fill the tube to the top and balance it to within +/- 0.1g to avoid being crushed by the ultracentrifuge's vacuum. + +2.4 **Centrifugation:** Centrifuge for 3 hours, 23°C at 32,000 RPM in an SW32.1Ti rotor. *Note below on RPM vs g-force*. + +2.5 **Supernatant Collection:** Gently pipette off the supernatant and save at 4°C. + +2.6 **Pellet Resuspension:** Allow the pellet to dry for 5 - 10 minutes, and resuspend in 500ul of TE. + +2.7 **End Treatment:** Add EGTA and EDTA to a final concentration of 100mM. + +**Note:** +Email correspondence advised that ultracentrifugation should be at 32,000 RPM instead of 32,000 x g. The difference is significant: 32,000 x g is about 13,000 RPM whereas 32,000 RPM equals 180,000 x g. + +## Protocol Steps + +### Step 1 +Prepare sucrose solution as 38% (weight to volume) in SM buffer (100mM NaCl, 8mM MgSO4, 50mM Tris/HCl, pH 7.5) that has been 0.2μm filtered. + +### Step 2 +Rinse SW40 tubes with SM buffer or sterile water. + +***Note:*** +The SW 40 tubes hold 12 ml total volume, with 2.5ml 38% sucrose as the cushion, leaving 9.5 ml volume for the sample. If the DNase treated sample is less, add SM buffer to bring it up to 9.5 ml. + +### Step 3 +Place the 9.5ml DNase I-treated sample into the rinsed ultracentrifuge tube. + +### Step 4 +Using a 3cc syringe and cannula, carefully layer the 2.5 ml of 38% sucrose under the sample to prevent mixing. + +### Step 5 +Centrifuge at 175,000g for 3.25 hr at 18°C. + +### Step 6 +Using a sterile transfer or serological pipet, remove the 9.5 ml sample layer. + +### Step 7 +Carefully remove the interface and 2.5 ml sucrose without disturbing or suctioning off the pelleted sample at the bottom. +**Note:** Pellets may appear orange due to iron chloride. + +### Step 8 +Keep the sucrose layer until virus counts from the pellets are completed. + +### Step 9 +Air dry the pellets in a fume hood for 15-20 min. + +### Step 10 +Add a TE mixture containing 0.1M each EDTA/EGTA to resuspend the pellets (500 µl is recommended). + +### Step 11 +Perform virus counts and then extract DNA. + +## Reagents and Terms: + +- **DNase I:** An enzyme that digests DNA. Used here to remove any DNA contamination from the virus samples. +- **EDTA (Ethylenediaminetetraacetic acid):** A chelating agent that binds divalent cations like Mg2+ and Ca2+. +- **EGTA (Ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid):** A chelating agent similar to EDTA with a higher affinity for Calcium ions, used to inactivate DNase. +- **TE Buffer:** Tris-EDTA buffer used to resuspend DNA or RNA. +- **Sucrose Cushion:** A method that uses a dense sucrose layer to concentrate viral particles. + +## Alternative Methods: +For alternative heavy sucrose buffers, consider using CsCl or iodixanol gradients which can offer different density gradients for separating viral particles. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/q5-site-directed-mutagenesis-e0554-imsumm.md b/markdown-output/q5-site-directed-mutagenesis-e0554-imsumm.md new file mode 100644 index 0000000000000000000000000000000000000000..d9b445fc8122d606404126a0fb79f0cec920f3c7 --- /dev/null +++ b/markdown-output/q5-site-directed-mutagenesis-e0554-imsumm.md @@ -0,0 +1,145 @@ +```markdown +# Goal/Experiment: +To perform Q5® Site-Directed Mutagenesis to enable rapid, site-specific mutagenesis of double-stranded plasmid DNA. + +# Q5® Site-Directed Mutagenesis (E0554) + +## Abstract +This is the protocol for the Q5® Site-Directed Mutagenesis Kit (E0554) + +**Citation**: New England Biolabs Q5® Site-Directed Mutagenesis (E0554). protocols.io dx.doi.org/10.17504/protocols.io.cjpumm +**Published**: 07 Nov 2014 + +## Guidelines + +### Description +The Q5® Site-Directed Mutagenesis Kit enables rapid, site-specific mutagenesis of double-stranded plasmid DNA in less than 2 hours (Figure 1). The kit utilizes the robust Q5 Hot Start High-Fidelity DNA Polymerase along with custom mutagenic primers to create insertions, deletions and substitutions in a wide variety of plasmids. After PCR, the amplified material is added directly to a unique Kinase-Ligase-DpnI (KLD) enzyme mix for rapid (5 minutes), room temperature circularization and template removal (Figure 2). Transformation into high-efficiency NEB 5-alpha Competent E. coli, provided with the kit, ensures robust results with plasmids up to at least 20 kb in length. + +![Figure 1](image1.png) +**Figure 1: Site-specific mutagenesis proceeds in less than 2 hours**. +*The use of a master mix, a unique multi-enzyme KLD enzyme mix, and a fast polymerase ensures that, for most plasmids, the mutagenesis reaction is complete in less than two hours.* + +![Figure 2](image2.png) +**Figure 2: Q5 Site-Directed Mutagenesis Kit Overview.** +*The kit is designed for rapid and efficient incorporation of insertions, deletions and substitutions into double-stranded plasmid DNA. The first step is an exponential amplification using standard primers and a master mix formulation of Q5 Hot Start High-Fidelity DNA Polymerase. The second step involves incubation with a unique enzyme mix containing a kinase, a ligase and DpnI. Together, these enzymes allow for rapid circularization of the PCR product and removal of the template DNA. The last step is a high-efficiency transformation into chemically competent cells (provided).* + +### Primer Design for the Q5 Site-Directed Mutagenesis Kit +Substitutions, deletions and insertions are incorporated into plasmid DNA through the use of specifically designed forward (black) and reverse (red) primers. Unlike kits that rely on linear amplification, primers designed for the Q5 Site-Directed Mutagenesis Kit should not overlap to ensure that the benefits of exponential amplification are realized. + +#### Design Strategy +- **Substitutions**: Created by incorporating the desired nucleotide change(s) in the center of the forward primer. +- **Deletions**: Engineered by designing non-mutagenic forward and reverse primers flanking the deleted region. +- **Insertions**: Less than or equal to 6 nucleotides incorporated into the 5' end of the forward primer. +- **Large Insertions**: Created by incorporating half of the desired insertion into the 5' ends of both primers. + +### Troubleshooting + +#### No/Low Colonies +- Ensure proper primer design. +- Use only 1 µl of PCR product in the KLD reaction. +- Use only 5 µl of the KLD reaction in the transformation. +- Confirm clean PCR product by agarose gel. +- Use NEB 5-alpha Competent E. coli cells stored at -80°C. +- Verify competence of cells with control DNA. + +#### No/Low PCR Product +- Optimize annealing temperature (Tm+3°C). +- Use high-fidelity polymerases. +- Ensure adequate primer concentration. +- Purify primers with PAGE. + +#### Resulting Plasmids Do Not Contain the Desired Mutation +- Ensure proper design of mutagenic primers. +- Optimize PCR conditions. +- Use 1-25 ng of template DNA in the PCR step. + +### Notes +Storage Note: The Q5 Site-Directed Mutagenesis Kit is stable at -80°C for one year. For convenience, the kit components are packaged together in a separate box that can be removed and stored at -20°C for two years. Store NEB 5-alpha Competent E. coli at -80°C. + +## Materials +- Q5 Site-Directed Mutagenesis Kit - 10 rxns E0554S by [New England Biolabs](https://www.neb.com/) + +## Protocol + +### Exponential Amplification (PCR) +**Step 1:** +Assemble the following reagents in a thin-walled PCR tube. +| 25 µl RXN | FINAL CONC. | +|--------------------------------|-------------------| +| Q5 Hot Start High-Fidelity 2X Master Mix | 12.5 µl | 1X | +| 10 µM Forward Primer | 1.25 µl | 0.5 µM | +| 10 µM Reverse Primer | 1.25 µl | 0.5 µM | +| Template DNA (1–25 ng/µl) | 1 µl | 1-25 ng | +| Nuclease-free water | 9.0 µl | | + +**Step 2:** +Mix reagents completely. + +**Step 3:** +Transfer to a thermocycler and perform the following cycling conditions: + +**Thermocycling Conditions for a Routine PCR:** +| STEP | TEMP | TIME | +|-----------------------|------------|-------------------| +| Initial Denaturation | 98°C | 30 seconds | +| 25 Cycles | 98°C | 10 seconds | +| | 50–72°C* | 10–30 seconds | +| | 72°C | 20–30 seconds/kb | +| Final Extension | 72°C | 2 minutes | +| Hold | 4–10°C | | + +*For Q5-optimized mutagenic primers, use the NEBaseChanger™ for pre-designed, back-to-back primer sets. + +### Kinase, Ligase & DpnI (KLD) Treatment +**Step 4:** +Assemble the following reagents: +- PCR Product 1 µl +- 2X KLD Reaction Buffer 5 µl +- 10X KLD Enzyme Mix 1 µl +- Nuclease-free Water 3 µl + +**Step 5:** +Mix well by pipetting up and down. + +**Step 6:** +Incubate at room temperature for 5 minutes. + +### Transformation +**Step 7:** +Thaw a tube of NEB 5-alpha Competent E. coli cells on ice. + +**Step 8:** +Add 5 µl of the KLD mix from the "KLD Section" above to the tube of thawed cells. + +**Step 9:** +Carefully flick the tube 4-5 times to mix. Do not vortex. + +**Step 10:** +Place the mixture on ice for 30 minutes. + +**Step 11:** +Heat shock at 42°C for 30 seconds. + +**Step 12:** +Place on ice for 5 minutes. + +**Step 13:** +Pipette 950 µl of room temperature SOC into the mixture. +- [SOC Outgrowth Medium - 100 ml B9020S by New England Biolabs](https://www.neb.com/) + +**Step 14:** +Incubate at 37°C for 60 minutes with shaking (250 rpm). + +**Step 15:** +Mix the cells thoroughly by flicking the tube and inverting. For simple substitution and deletion experiments, make a 10-40-fold dilution of the transformation mix in SOC prior to plating. + +**Step 16:** +Spread 50-100 µl onto a selection plate. + +**Step 17:** +Incubate overnight at 37°C. + +**Duration**: 15 hours + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/qiagen-qiaprep-spin-miniprep-kit-27104-or-27106-wi-d7f9jm.md b/markdown-output/qiagen-qiaprep-spin-miniprep-kit-27104-or-27106-wi-d7f9jm.md new file mode 100644 index 0000000000000000000000000000000000000000..a254885a2b69c4c02025626438ef203f0a9789a6 --- /dev/null +++ b/markdown-output/qiagen-qiaprep-spin-miniprep-kit-27104-or-27106-wi-d7f9jm.md @@ -0,0 +1,132 @@ +```markdown +# Goal/Experiment: +The goal of this experiment is to extract and purify plasmid DNA using the Qiagen QIAprep Spin Miniprep Kit (catalog numbers 27104 or 27106), which is designed for isolation of up to 20 µg of high-purity plasmid or cosmid DNA from bacterial cultures. + +## Qiagen QIAprep Spin Miniprep Kit (27104 or 27106) with a microcentrifuge + +### Abstract +This protocol outlines the procedure for using Qiagen's QIAprep Spin Miniprep Kit (catalog numbers 27104 or 27106) with their newer version 2.0 spin columns. For more information about the kit, see [Qiagen QiAprep Spin Miniprep Kit](https://www.qiagen.com/us/shop/sample-technologies/dna/dna-preparation/qiaprep-spin-miniprep-kit?cmpid=QVenSPP1404quartzysc#orderinginformation). + +For purification of up to 20 µg molecular biology grade plasmid DNA. + +- Ready-to-use plasmid DNA in minutes +- Reproducible yields of molecular biology grade plasmid DNA +- Single protocol for high- and low-copy vectors +- Even higher yields with the High-Yield Supplementary Protocol +- Improved QIAprep 2.0 Spin Column +- GelPilot loading dye for convenient sample analysis + +The QIAprep Spin Miniprep Kit is designed for isolation of up to 20 µg high-purity plasmid or cosmid DNA for use in routine molecular biology applications, including fluorescent and radioactive sequencing and cloning. Even higher yields (up to 30 µg) can be achieved using the High-Yield Supplementary Protocol. + +**Published:** 15 Nov 2015 + +## Guidelines + +- Close the bottle containing Buffer P2 immediately after use to avoid acidification of Buffer P2 from CO2 in the air. +- All centrifugation steps are carried out at 13,000 rpm (~17,900 x g) in a conventional, table-top microcentrifuge. +- Ensure that the elution buffer is dispensed directly onto the center of the QIAprep membrane for optimal elution of DNA. Average eluate volume is 48 µl from an elution buffer volume of 50 µl (QIAprep spin procedures), and 60 µl from an elution buffer volume of 100 µl (QIAprep multiwell procedures). +- For increased DNA yield, use a higher elution-buffer volume. +- For increased DNA concentration, use a lower elution-buffer volume. +- If water is used for elution, ensure that its pH is between 7.0 and 8.5. Elution efficiency is dependent on pH and the maximum elution efficiency is achieved within this range. A pH <7.0 can decrease yield. Store DNA at -20°C when eluted with water, as DNA may degrade in the absence of a buffering agent. DNA can also be eluted in TE buffer (10 mM Tris·Cl, 1 mM EDTA, pH 8.0), but the EDTA may inhibit subsequent enzymatic reactions. + +## Before start +### Growth of bacterial cultures in tubes or flasks +- Pick a single colony from a freshly streaked selective plate and inoculate a culture of 1–5 ml LB medium containing the appropriate selective antibiotic. Incubate for 12–16 h at 37°C with vigorous shaking. Growth for more than 16 h is not recommended since cells begin to lyse and plasmid yields may be reduced. Use a tube or flask with a volume of at least 4 times the volume of the culture. + +### Preparation of Buffers +- Add the provided RNase A solution to Buffer P1 before use. Use 1 vial RNase A (centrifuge briefly before use) per bottle Buffer P1 for a final concentration of 100 µg/ml. Mix and store at 2–8°C. +- Add ethanol (96–100%) to Buffer PE before use (see bottle label for volume). +- Check Buffers P2 and N3 before use for salt precipitation. Redissolve any precipitate by warming to 37°C. Do not shake Buffer P2 vigorously. +- Optional: Add the provided LyseBlue reagent to Buffer P1 and mix before use. Use 1 vial LyseBlue reagent per bottle Buffer P1 for a final dilution of 1:1000 (e.g., 10 µl LyseBlue into 10 ml Buffer P1). LyseBlue provides visual identification of optimum buffer mixing, thereby preventing the common handling errors that lead to inefficient cell lysis and incomplete precipitation of SDS, genomic DNA, and cell debris. + +## Protocol +### Lysis + +#### Step 1. +Resuspend pelleted bacterial cells in 250 µl Buffer P1 and transfer to a micro-centrifuge tube. + +**Amount:** 250 µl +**Notes:** Ensure that RNase A has been added to Buffer P1. No cell clumps should be visible after resuspension of the pellet. If LyseBlue reagent has been added to Buffer P1, vigorously shake the buffer bottle to ensure LyseBlue particles are completely dissolved. The bacteria should be resuspended completely by vortexing or pipetting up and down until no cell clumps remain. + +#### Step 2. +Add 250 µl Buffer P2 and mix thoroughly by inverting the tube 4–6 times. + +**Amount:** 250 µl +**Notes:** Mix gently by inverting the tube. Do not vortex, as this will result in shearing of genomic DNA. If necessary, continue inverting the tube until the solution becomes viscous and slightly clear. Do not allow the lysis reaction to proceed for more than 5 min. If LyseBlue has been added to Buffer P1 the cell suspension will turn blue. Mixing should result in a homogeneously colored suspension. + +#### Step 3. +Add 350 µl Buffer N3 and mix immediately and thoroughly by inverting the tube 4–6 times. + +**Amount:** 350 µl +**Notes:** To avoid localized precipitation, mix the solution thoroughly, immediately after addition of Buffer N3. Large culture volumes (e.g. ≥5 ml) may require inverting up to 10 times. The solution should become cloudy. If LyseBlue reagent has been used, the suspension should be mixed until all trace of blue has gone. + +### Lysis + +#### Step 4. +Centrifuge for 10 min at 13,000 rpm (17,900 x g) in a table-top microcentrifuge. + +**Duration:** 10 min + +### Binding + +#### Step 5. +Apply the supernatants from step 4 to the QIAprep spin column by decanting or pipetting. + +#### Step 6. +Centrifuge for 30–60 s. Discard the flow-through. + +**Duration:** 30–60 s + +### Wash + +#### Step 7. +Wash the QIAprep spin column by adding 0.5 ml Buffer PB and centrifuging for 30–60 s. Discard the flow-through. + +**Amount:** 500 µl +**Duration:** 30–60 s +**Notes:** This step is necessary to remove trace nuclease activity when using endA+ strains such as the JM series, HB101 and its derivatives, or any wild-type strain, which have high levels of nuclease activity or high carbohydrate content. Host strains such as XL-1 Blue and DH5α do not require this additional wash step. + +#### Step 8. +Wash QIAprep spin column by adding 0.75 ml Buffer PE and centrifuging for 30–60 s. + +**Amount:** 750 µl +**Duration:** 30–60 s + +#### Step 9. +Discard the flow-through and centrifuge at full speed for an additional 1 min to remove residual wash buffer. + +**Duration:** 1 min +**Notes:** Important: Residual wash buffer will not be completely removed unless the flow-through is discarded before this additional centrifugation. Residual ethanol from Buffer PE may inhibit subsequent enzymatic reactions. + +### Elution + +#### Step 10. +Place the QIAprep column in a clean 1.5 ml microcentrifuge tube. + +**Duration:** 1 min + +#### Step 11. +Centrifuge for 1 min. + +**Duration:** 1 min + +#### Step 12. +Let stand 1 minute. + +**Duration:** 1 min + +### Lysis + +#### Step 13. +Centrifuge 1-5 ml bacterial overnight culture at >6800g for 3 minutes at room temperature. + +**Amount:** 1-5 ml + +## Warnings + +- When working with chemicals, always wear a suitable lab coat, disposable gloves, and protective goggles. Buffers P2, N3, and PB contain irritants. Wear gloves when handling these buffers. For more information, please consult the appropriate safety data sheets (SDSs). + +- **CAUTION:** DO NOT add bleach or acidic solutions directly to the sample-preparation waste. Buffers N3 and PB contain guanidine hydrochloride, which can form highly reactive compounds when combined with bleach. If liquid containing these buffers is spilt, clean with suitable laboratory detergent and water. If the spilt liquid contains potentially infectious agents, clean the affected area first with laboratory detergent and water, and then with 1% (v/v) sodium hypochlorite. + +**endofoutput** +``` \ No newline at end of file diff --git a/markdown-output/qpcr-primer-design-cqrkvv4w.md b/markdown-output/qpcr-primer-design-cqrkvv4w.md new file mode 100644 index 0000000000000000000000000000000000000000..61ed5e0961fea89657362c12f0ea3aaf4db9fc5b --- /dev/null +++ b/markdown-output/qpcr-primer-design-cqrkvv4w.md @@ -0,0 +1,133 @@ +```markdown +# Goal/Experiment: +Designing qPCR primers for analysis of transcript abundance using SYBR Green chemistry. + +## qPCR Primer Design + +**Steven J Burgess1** + +1University of Illinois at Urbana-Champaign + +UIUC Long Lab + +Lynn Doran +Realizing Increased Photosynthetic Efficiency (RIPE) + +--- + +### Abstract + +Protocol for designing qPCR primers for analysis of transcript abundance using SYBR Green chemistry. + +There are many good guides about how to do this online such as [this guide](https://doi.org/10.1093/nar/gkg595), from which the following protocol is derived. + +Reference for mfold/UNAfold: [https://doi.org/10.1093/nar/gkg595](https://doi.org/10.1093/nar/gkg595) + +### Guidelines + +Preferred properties of qPCR primers: +(Note: These guidelines represent the ideal situation which may not always be achievable) +- Each primer should be between 15–30 bp in length +- The theoretical Tm of the two primers should be within 2°C of each other. +- Try to avoid G/C clamps at the 3’ ends of the primers to prevent these oligos from folding on themselves or annealing nonspecifically. +- The five bases at the 5’ terminal end generally should contain no more than two guanines and cytosines, although it is acceptable to have three in the final 5 bases if no two are adjacent. +- If possible, have dA nucleotides near the 3' end to allow for UNG activity to prevent primer-dimers +- Since thymidine tends to mis-prime more readily than the other bases, a 3’ terminal T should be avoided if possible. +- The 5’ end of the primers should not contain an inverted repeat sequence that would allow it to fold on itself. + +--- + +### Identify Target Sequence + +1. Decide on a gene of interest, download the transcript sequence in .gb format, and upload to a new folder on Benchling for analysis. + +> **Note:** +> +> If analyzing an endogenous (as compared to transgene) gene sequences can be obtained from public databases such as NCBI. For many plant genes, my preference is to use [Phytozome](https://phytozome.jgi.doe.gov/pz/portal.html), but you should choose whichever database has the most recent and accurate transcriptome models. +> +> One thing to be aware of is that genes can have multiple splice forms. You may, or may not wish to perform splice form specific transcript quantification. In most instances, you are likely interested in "gene expression", or the sum of transcripts originating from a gene. In this case, perform an alignment of known splice form sequences using [Clustal Omega](https://www.ebi.ac.uk/Tools/msa/clustalo/) and retain only the parts which are common to all. + +### Use Primer-BLAST to Test for Specificity + +2. Navigate to NCBI [Primer-BLAST](https://www.ncbi.nlm.nih.gov/tools/primer-blast/) in your browser. + +3. Adjust parameters to suit qPCR assays and record the search conditions in your Lab book: + - Amplicon size to 70-150 bp + - Minimum of 4 mismatches + - RefSeq mRNA database + - Under the organism tab, enter the name of the species of interest e.g. Solanum tuberosum + +> **Note:** +> +> You can also include information about intron-exon boundaries, however, this is often too stringent and will yield no positive results. + +4. Paste in the transcript sequence against which you want to design primers (from step 1) and click “Get Primers”. + +5. From the results, choose primers which have a single perfect match using BLAST (this should be against + - Record the following details in a table for the purpose of reporting according to the format above: + - Primer forward sequence + - Primer reverse sequence + - Amplicon size + +### Check Primer Properties + +6. Open IDT’s [OligoAnalyzer Tool](https://eu.idtdna.com/pages/tools/oligoanalyzer) in a new tab for QC analysis of primer properties. + +7. Change parameter settings to "qPCR" on the dropdown menu on the right and adjust concentration settings: + - [Oligo] = 0.33 nM, + - [Na+] = 50 mM, + - [Mg2+] = 3 mM, + - [dNTP] = 1.2 mM + +> **Note:** +> +> Note these parameter settings are adapted for use with SSoAdvanced Universal SYBR Green Supermix. The precise parameters will vary depending on the buffer components in the PCR reaction mix being used. + +8. Enter the first primer sequence from Primer-BLAST and run. + +9. Analyze hairpin formation and record the highest Tm. Record the results in the table. + +> **Note:** +> +> Tm value should be lower than the annealing temperature of the primers (e.g. 60 °C if that is what the assay was designed at). Otherwise, primer sequence may be prone to forming hairpin structures under assay conditions resulting in inaccurate results. + +10. Analyze self-dimer free energy and record the lowest dG in a table. Record the results in the table. + +> **Note:** +> +> dG value should be > -9. Anything lower than this fails the quality control check as the sequence has too high a chance of forming primer-dimer amplification products leading to inaccurate results. + +11. Analyze heterodimer free energy by entering the reverse primer as a secondary sequence. Record the results in the table. + +> **Note:** +> +> dG value should be > -9. Anything lower than this fails the quality control check as the sequence has too high a chance of forming primer-dimer amplification products leading to inaccurate results. + +12. If the forward primer passes the QC checks in steps 9-11, repeat for the reverse primer. + +> **Note:** +> +> In the case that the primer sequence fails QC, return to the Primer-BLAST results and select another pair of primers for analysis. It is common to need to do this, in most instances, it should be possible to find suitable primers. + +### Check Amplicon Properties + +13. Open the software [UNAfold](http://www.unafold.org/) in a new tab on your browser to test for secondary structure formation by the amplicon sequence to be amplified by primers. + +> **Note:** +> +> Ideally, your primers should pass this QC as well, but in the event, it is not possible to find a pair that passes both the primer and amplicon QC it is worth progressing to test experimentally. + +14. Set UNAfold parameters for analysis of assay amplicon: + - Choose to fold DNA + - Set the concentration of Mg++ to 3 mM + +15. Run UNAfold. + +16. Inspect the predicted structures formed by the amplicon. + +> **Note:** +> +> The highest Tm for any of the predicted structures should be less than the annealing temperature of the primers (e.g. >60°C if that is what they were designed at). + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/quantification-of-fluorescence-intensity-of-antise-dgq63vze.md b/markdown-output/quantification-of-fluorescence-intensity-of-antise-dgq63vze.md new file mode 100644 index 0000000000000000000000000000000000000000..f0c4340492bd93a06605ada406d5bc746f89e51d --- /dev/null +++ b/markdown-output/quantification-of-fluorescence-intensity-of-antise-dgq63vze.md @@ -0,0 +1,126 @@ +```markdown +# Goal/Experiment: +Quantification of fluorescence intensity of antisense constructs within Bodo saltans and its intracellular symbiont + +## DOI +[dx.doi.org/10.17504/protocols.io.6qpvr8pqblmk/v1](https://dx.doi.org/10.17504/protocols.io.6qpvr8pqblmk/v1) + +## Authors +Marie Held1, Mastaneh Ahrar2, Gregory DD Hurst2, Ewa Chrostek3,2 +1Centre for Cell Imaging, University of Liverpool, UK +2Department of Evolution, Ecology and Behaviour, Institute of Infection, Veterinary and Ecological Sciences, University of Liverpool, UK +3Jagiellonian University, Krakow, Poland + +**Corresponding Author:** Ewa Chrostek, Jagiellonian University +**Technical Support Email:** adam.jones@moore.org + +**Protocol Status:** Working + +**Created:** July 05, 2024 +**Last Modified:** July 05, 2024 + +## Abstract +The purpose of this protocol is to quantify the intensity of fluorescence resulting from antisense molecules within a microeukaryote *Bodo saltans* and its intracellular symbiont *Candidatus* Bodocaedibacter vickermani following incubation. This protocol is specific to two-channel fluorescent images of objects contained within larger objects. It can be used directly to analyze images of similar-sized objects. + +The images were recorded using a confocal laser scanning microscope as single focal plane images, containing three channels: +1. Fluorescent antisense molecule. +2. DAPI. +3. Transmitted light. + +This protocol contains two sample files (.czi), which can be re-analyzed using the steps described below. A separate Word file with all the steps listed can also be found attached to this protocol. + +## Keywords +- Fluorescent molecules +- Intracellular symbionts +- Image analysis +- Fluorescence quantification +- Antisense molecules + +## Materials +- *B. saltans* cultures +- 100 and 8 µm filters +- Cerophyll medium +- Low melting temperature agarose +- PBS (Phosphate-buffered saline) +- 96-well plates +- Microscope slides and coverslips +- Fluorescent antisense molecules +- Hoechst 33342 (or other DNA dyes) +- Vectashield (or other mounting mediums) + +### Standard laboratory equipment: +- Pipettes +- Hemacytometer +- Tabletop centrifuges +- Rotary shaker +- Confocal microscope +- Computer with FIJI software + +## Incubation of *B. saltans* with Antisense Molecules +1. **Filtering:** Filter *B. saltans* culture through 100 and 8 µm filters. +2. **Harvesting:** Harvest the cells by centrifugation at 1200 × g for 12 mins at 19°C. +3. **Washing:** Wash the cells with 10-15 ml sterile filtered (SF) 1×PBS and centrifuge as above. +4. **Resuspending:** Re-suspend the cells in 5 ml SF 1×PBS, count the cells using a hemacytometer and adjust the volume to achieve 5×10⁶ cells for every 10 samples. +5. **Centrifuging:** Centrifuge at 1200 × g for 12 mins at 19°C. +6. **Resuspending Again:** Remove the PBS and resuspend the cells in SF cerophyll medium, ensuring a final volume of 100 µl per sample. +7. **Transferring:** Move 90 µl of the cell suspension to a sterile 2 ml tube. +8. **Adding Antisense Molecules:** Add the tested molecules to achieve a final concentration of 50 µM. Incubate the mixture for growth. +9. **Imaging Preparation:** After 24 hours, prepare samples for imaging. Centrifuge and resuspend cells appropriately. Place them into wells of a 96-well plate, each containing agarose. +10. **Staining:** Add Hoechst 33342 (Thermo Fisher, 1:2000 in PBS) to the well for 10 minutes. +11. **Washing:** Rinse and wash once with PBS. +12. **Placing on Slide:** Remove agarose with clean forceps and place it on a slide. +13. **Mounting:** Add a drop of the mounting medium and flatten the agarose with a coverslip. + +## Imaging *B. saltans* Using a Confocal Microscope (Zeiss LSM 880) +14. Follow local rules for operating the confocal microscope. +15. Turn on the systems as per guidelines. +16. Use a high numerical aperture (e.g., 63×, 1.40 NA) objective to visualize *B. saltans*. +17. Acquire images in scanning mode for all samples and negative controls using the same settings. + +## Image Preprocessing +18. Open raw images in Fiji. +19. Duplicate DAPI channel (`Shift + D`). +20. Subtract the background using a rolling ball algorithm with a radius of 25 pixels. + +## Image Segmentation +21. Duplicate background-subtracted DAPI channel. +22. Use global thresholding to segment areas positive for DNA staining. +23. Separate touching objects using the Watershed operation. +24. Perform connected component analysis with a size threshold of 0-300 pixels. +25. Save the bacteria ROIs. +26. Duplicate the fluorescent antisense molecule channel (`Shift + D`). +27. Use global thresholding for segmenting areas positive for antisense staining. +28. Perform connected component analysis with a size threshold of 1000-Infinity pixels. +29. Rename the ROIs (e.g., "cell"). +30. Combine *B. saltans* ROI with individual symbiont ROIs using the XOR operator. +31. Rename the ROI to "cell-minus-bacteria". + +## Measurements +32. Activate the following parameters to be measured via `Analyze > Set Measurements...`: + - Area + - Mean gray value + - Modal gray value + - Integrated density + - Median + - Display Label option + +33. Select raw image. +34. Set the channel to the antisense channel. +35. Deselect ROIs in the ROI Manager (or select all). +36. Measure set parameters via `ROI Manager > More > Multi Measure`. +37. Save result table. + +## Automation +38. The entire process can be automated using a Fiji macro script: [BodoSaltans Macro Script](https://github.com/Marien-kaefer/General_Fiji_macros/tree/main/BodoSaltans). + +## References +1. Midha S, et al. *Bodo saltans* (Kinetoplastida) is dependent on a novel Paracaedibacter-like endosymbiont that possesses multiple toxin-antitoxin systems. ISME J. doi: [10.1038/s41396-020-00879-6](https://doi.org/10.1038/s41396-020-00879-6). +2. Schindelin J, et al. Fiji: an open-source platform for biological-image analysis. Nat Methods **9**, 676-682 (2012). doi: [10.1038/nmeth.2019](https://doi.org/10.1038/nmeth.2019). +3. Ridler TW & Calvard S. Picture Thresholding Using an Iterative Selection Method. IEEE Transactions on Systems, Man, and Cybernetics, Volume 8, Issue 8, pp. 630-632 (1978). doi: [10.1109/TSMC.1978.4310039](https://doi.org/10.1109/TSMC.1978.4310039). +4. Glasbey CA. An Analysis of Histogram-Based Thresholding Algorithms, CVGIP: Graphical Models and Image Processing, Volume 55, Issue 6, pp. 532-537 (2021). doi: [10.1006/cgip.1993.1040](https://doi.org/10.1006/cgip.1993.1040). + +## Acknowledgements +We thank the Centre for Cell Imaging (CCI) at the University of Liverpool for assistance with live *Bodo* imaging. Zeiss 880 BioAFM was funded by BBSRC (Grant BB/M012441/1). This work was supported by the Gordon and Betty Moore Foundation’s Symbiosis in Aquatic Systems Initiative Grant ID: #9357 [GBMF9357](https://doi.org/10.37807/GBMF9357). + +Endofoutput +``` \ No newline at end of file diff --git a/markdown-output/quantification-of-the-effect-of-gastric-electrical-banfidbn.md b/markdown-output/quantification-of-the-effect-of-gastric-electrical-banfidbn.md new file mode 100644 index 0000000000000000000000000000000000000000..42095ee2a2896897e046b92e0e67680144ff5d01 --- /dev/null +++ b/markdown-output/quantification-of-the-effect-of-gastric-electrical-banfidbn.md @@ -0,0 +1,96 @@ +```markdown +# Goal/Experiment +Quantification of the effect of gastric electrical stimulation location on circulating blood hormone levels + +## Authors + +Terry Powley1, Zhenjun Tan1, Matthew Ward1, J Paul Robinson1 +1Purdue University + +## Abstract + +This protocol describes a process for the quantification of electrical stimulation-induced effects on circulating blood hormone levels in young adult Sprague-Dawley rats. Blood samples were collected via the left femoral artery using a Culex sampling system both before and after a discrete stimulation period. Stimulation occurred via patch electrodes implanted at multiple sites across the rat stomach in an acute anesthetized preparation. + +## DOI + +[10.17504/protocols.io.4r3l2825pl1y/v1](dx.doi.org/10.17504/protocols.io.4r3l2825pl1y/v1) + +## Protocol Citation + +Terry Powley, Zhenjun Tan, Matthew Ward, J Paul Robinson 2022. Quantification of the effect of gastric electrical stimulation location on circulating blood hormone levels. _protocols.io_ +[https://protocols.io/view/quantification-of-the-effect-of-gastric-electrical-banfidbn](https://protocols.io/view/quantification-of-the-effect-of-gastric-electrical-banfidbn) + +## Keywords + +rat, stomach, gastric electrical stimulation, blood, hormone + +## License + +This is an open access protocol distributed under the terms of the [Creative Commons Attribution License](https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. + +## Animals + +1. Two-to four-month-old Sprague-Dawley (Envigo) male and female rats were housed in vented rack cages in an Association for Assessment and Accreditation of Laboratory Animal Care-approved temperature (22–24 °C) and humidity (40–60%) controlled colony room. The room was maintained on a 12-hour light–dark schedule. Pelleted chow (2018 Teklad global 18% protein rodent diet, Envigo) and filtered tap water were provided _ad libitum_. + + All husbandry practices conformed to the NIH _Guide for the Care and Use of Laboratory Animals_ (8th edition) and were reviewed and approved by the Purdue University Animal Care and Use Committee. All efforts were made to minimize any suffering as well as the number of animals used. + +## Surgical Procedures + +2. Animals were transferred to wire hanging cages the day before surgery and then fasted for 18 hours with free access to water. Rats were then anesthetized with 5% isoflurane (Akorn Animal Health, _Catalog #NDC: 59399-106-01_) in an induction box and transferred to a Somnosuite Low-flow Anesthesia System (Kent Scientific, SomnoSuite) on a surgery platform (Kent Scientific, SurgiSuite). + + A servo-controlled homoeothermic heating blanket, equipped with a rectal thermometer, was used to maintain body temperature at 36 °C. The level of anesthesia was reduced to 2.5% isoflurane for the surgical procedure. + +3. After midline laparotomy, the stomach and 3-4 cm of the proximal duodenum were exteriorized onto saline-soaked gauze pads. Custom-made stimulation patch electrodes (Micropobes for Life Science) were sutured on the serosal surface of the stomach, either one or two electrodes. + +4. A custom-made strain gauge (4x3.5mm, Clunbury Scientific LLC, Bloomfield Hills, MI) constructed from two strain gauge elements (Micromeasurements EA-06-031CE-350) was then glued to the serosal surface of the proximal duodenum (5-15mm distal to the pyloric sphincter) using Vetbond (3M, _Catalog #1469SB_). The strain gauge was oriented parallel to the longitudinal or circular muscle. + + The fine wire leads attached to the stain gauge and patch electrodes were exteriorized and connected to a DC bridge amplifier and stimulator respectively. + +5. The animal was kept in a supine position with the abdominal area covered by saline-soaked gauze pads. Normal saline (2.0 ml/hr) was injected continuously _i.p._ using a syringe pump. + +6. With the rat in a supine position, a 1.0-1.5 cm incision was made on the angle of the left hind leg. The left femoral artery was exposed and separated from the connective tissue and femoral vein by blunt dissection. A catheter (CX-80002S, www.BASinc.com) was inserted into the femoral artery (2-3 cm) via a pre-cut 45-degree angle incision with micro-dissecting scissors in the femoral wall. The catheter was pre-filled with heparinized saline (10 units/ml, Heparin: Meitheal Pharmaceuticals Inc.). The catheter was tied in position with sutures and connected to the Culex automated blood sample system. + +7. The animal was then covered with a blanket to help maintain body temperature, and anesthesia was reduced to 1.5 % isoflurane and maintained at that level for the remainder of the experiment. + +## Blood Draw Set Up + +8. See steps 1-5 of the protocol: + [SPARC - Preparation of Plasma Samples from Rats](https://dx.doi.org/10.17504/protocols.io.4r3l2825pl1y/v1) + +## Stimulation and Blood Draw + +9. About one hour after the end of surgery, the animal was considered stable enough to begin the blood draw/stimulation experiment. This was typically confirmed by assessment of the duodenal motility signal (see Protocol "Measurement of duodenal motility using implanted strain gauges"), but this process is not necessary. + + Blood draw timings were defined relative to the start of the 5 min period of stimulation. Blood samples (0.15 ml each) were withdrawn at the following times: -30 min, -5 min, 5 min, 15 min, 30 min. + + Stimulation was provided by a PlexStim electrical stimulator (Plexon, PlexStim). + + Stimulation parameters were as follows: biphasic, I = 0.3 mA, pw = 0.2 ms, 10 Hz, 20s-on-40s-off, 5 one-minute cycles. + +## Blood Processing + +10. See steps 6-21 of the protocol: + [SPARC - Preparation of Plasma Samples from Rats](https://dx.doi.org/10.17504/protocols.io.4r3l2825pl1y/v1) + + The rest of the hormone analysis process is captured in four other protocols: + - SPARC - Millipore Metabolic Rat Multiplex Bead Assay for Flow Cytometry + - SPARC - Attune NxT Set-up for Milli-metabolic bead assay Acquisition + - SPARC - Setting up the BEADS for the Millipore Metabolic Rat Milliplex Assay + - SPARC - Analysis of multiplexed bead data using MPLEX software + +## Perfusion + +11. Once the blood sampling was complete, the animals were given a lethal dose of ketamine (Patterson Veterinary, _Catalog #07-803-6637_) and xylazine (Akorn Animal Health, _Catalog #NDC: 59399-110-20_) (_i.p. 275 mg/kg ketamine and 27.5 mg/kg xylazine_). + + The locations of electrodes used in the experiment were marked with blue suture thread before the electrodes were removed. + + To ensure that the stomach was normally distended at the time of fixation, the organ was inspected for normal distension or accommodation, and, as required, physiological saline (3.3 ml/100 g of rat weight) that had been warmed to body temperature was slowly infused into the stomach by gavage catheter. With the stomach normally dilated, the animal was first transcardially perfused through the vasculature with physiological saline and then with 4% paraformaldehyde in 0.1 mol/liter PBS; pH 7.4. After perfusion, the distal esophagus and the proximal duodenum were transected, and the stomach was freed and removed. The organ was then opened with a longitudinal cut along the greater curvature. Next, the ventral and dorsal stomach walls were separated by an incision along the lesser curvature, thus yielding two whole mounts per animal. + +## Electrode Location Measurement + +12. The ventral half stomach was placed in PBS in a dissecting dish under a stereomicroscope, with the inner surface facing up, and the locations of the electrodes were marked using pins to clearly show each end of the electrode. A photograph of the stomach capturing the entire surface was then taken. + + The image of the stomach at a consistent magnification for each stomach to be measured was printed, and the x and y locations of the midpoint of the electrode measured off the image, together with the size of the stomach itself so that electrode location could be reported as percentage measurements relative to the pylorus end of the stomach contour (x) and relative to the bottom edge of the stomach at the greater curvature (y). In addition, the orientation of the electrode relative to a line from the top of the limiting ridge (near the LES) to the bottom point near the greater curvature where the limiting ridge changes direction was measured. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/quantifying-thiamin-cellular-quotas-in-algae-j6ncrde.md b/markdown-output/quantifying-thiamin-cellular-quotas-in-algae-j6ncrde.md new file mode 100644 index 0000000000000000000000000000000000000000..4b3776cd39eb4a130bba8fb0ff46a6cf2e6fd3a9 --- /dev/null +++ b/markdown-output/quantifying-thiamin-cellular-quotas-in-algae-j6ncrde.md @@ -0,0 +1,62 @@ +```markdown +# Quantifying Thiamin Cellular Quotas in Algae + +**Authors:** Magdalena Gutowska, Sebastian Sudek, Alexandra Worden, Brateen Shome, Tadhg Begley +**Published:** 03 Oct 2017 +**DOI:** [10.17504/protocols.io.j6ncrde](https://dx.doi.org/10.17504/protocols.io.j6ncrde) + +## Goal/Experiment: +The aim of this experiment is to determine the internal cellular quota of thiamin and its different phosphorylation states in the haptophyte Emiliana huxleyi using the thiochrome assay. + +## Abstract +We use the described methods for determining the internal cellular quota of thiamin and its different phosphorylation states in marine algae, in the below case for the haptophyte Emiliana huxleyi. The chemical assay used – the thiochrome assay - was developed in the 1930s but has been modified over the decades to include instrumentation with improved detection (Backstrom et al. 1995, Reddick et al. 2001). The assay is based on cyclization/oxidation of thiamin to thiochrome under basic conditions in the presence of an oxidizing agent such as potassium ferricyanide. Thiochrome is intensely fluorescent and easy to detect and quantify with high sensitivity in an HPLC assay. This method has previously been used to determine cellular quotas of thiamin and its different phosphorylation states in algae from brackish waters (Pinto et al. 2002, Sylvander et al. 2013). + +## Protocol + +### Step 1. Culture Preparation and Growth + +**Important note:** Regularly check the axenicity of the cultures using the DNA stain 4,6-diamidino-2-phenylindole (DAPI) and epifluorescence microscopy. Use dedicated media bottles for each thiamin concentration to avoid carry-over of thiamin between media batches. + +1. Maintain cultures of CCMP2090 Emiliana huxleyi in mid-exponential growth for ≥10 generations using semi-continuous batch culture techniques. Grow cultures using artificial seawater-based medium L1-Si (Guillard 1975). +2. Transfer cells daily (replete vitamin amendment conditions) or every second day (limited vitamin amendment conditions) to not exceed 1 x 106 cells mL-1. Quantify the cell concentrations on a flow cytometer (e.g., Accuri C6 cytometer BD Biosciences, USA). +3. Select the vitamin amendment conditions for the experiment and set up triplicate cultures for each treatment. For luxury treatment, supply thiamin at 300 nmol L-1 (standard f/2 medium concentration). For replete conditions, supply thiamin and HMP at 10 nmol L-1. For limiting conditions, supply thiamin at 500 pmol L-1 and HMP at 100 pmol L-1. +4. Maintain cells in mid-exponential growth for ≥10 generations using semi-continuous batch culture techniques. Quantify the cell concentrations on a flow cytometer. + +### Step 2. Cell Washing and Concentration + +**Important note:** Work on ice and have the centrifuge pre-cooled to 4°C. Resuspend the cells gently between washing steps. + +1. Remove vitamin amendments from media by using centrifugation wash steps with thiamin-free medium. Centrifuge 100 mL of culture at 4000 x g for 10 min at 4°C. Remove the supernatant, add 50 mL of thiamin-free medium to wash cells, and repeat (wash, centrifugation etc.) three times. +2. After the last wash step, resuspend the pellet in 1.5 mL of thiamin-free medium and quantify the cell concentration on a flow cytometer. +3. Generate a final cell pellet. Centrifuge at 10,000 x g for 30 min at 4°C. Collect the supernatant. Remove as much of the supernatant as possible and retain it in a separate tube. Flash-freeze the pellet in liquid nitrogen prior to storage at -80°C. +4. Combine the removed supernatant aliquots and quantify the cell concentration in the supernatant on a flow cytometer. Subtract these numbers (in the supernatant) from the total cell numbers determined prior to the final centrifugation step. This will give you the final cell count in the cell pellet. +5. Monitor the chlorophyll fluorescence and FALS distribution on the flow cytometric histograms to make sure cells have remained intact during the washing and centrifugation steps. + +### Step 3. Determination of Cellular Thiamin Quota Using the Thiochrome Assay + +#### Pellet Extraction + +**Important note:** The thiochrome assay is based on the facile and efficient cyclization/oxidation of thiamin to thiochrome under basic conditions in the presence of an oxidizing agent such as potassium ferricyanide (see schematic below). + +1. Suspend pellet in 100 µL of 7% HClO4 and sonicate for 2 min. +2. Add 50 µL of 4 M CH3CO2K and 50 µL of 30 mg mL-1 K3[Fe(CN)6] in 7 M NaOH. Pipette to mix. Leave 1 min. +3. Immediately neutralize the reaction with 6 M aqueous HCl. +4. Centrifuge the sample and collect the supernatant for further analysis. + +### Step 4. Reverse Phase HPLC + +1. Purify the deionized water used for reagents with active charcoal and filter. +2. Use a reverse phase HPLC (Agilent 1200 series) with fluorescence detection. Set the detector at an excitation of 365 nm and emission of 444 nm. +3. Prepare a Supelcosil SPLC-18-DB (25 cm x 10 mm, 5 µm) column with a gradient of the following compounds. A) H2O, B) K2HPO4 (pH 6.6), C) CH3OH. Set the gradient to 0 min, 100% B; 5 min, 100% B; 14 min, 7% A/70% B/23% C; 25 min, 25% A/75% C; 28 min to 34 min, 100% B. +4. Construct a calibration curve with known concentrations of thiamin pyrophosphate (TPP), thiamin monophosphate (TMP), and free thiamin (ThF). Integrate the fluorescence signal peak area. + +### Step 5. References + +- Backstrom A, McMordie R, Begley T. 1995. Biosynthesis of thiamin I: The function of the ThiE gene product. J Am Chem Soc 117:2351-2352. +- Guillard, RRL. 1975. Culture of phytoplankton for feeding marine invertebrates in “Culture of Marine Invertebrate Animals.” (eds: Smith W.L. and Chanley M.H.) Plenum Press, New York, USA. 26-60. +- Pinto E, Pedersén M, Snoeijs P, Van Nieuwerburgh L, Colepicolo P. 2002. Simultaneous detection of thiamine and its phosphate esters from microalgae by HPLC. Biochem Biophys Res Commun 291:344-348. +- Reddick JJ, Nicewonger R, Begley TP. 2001. Mechanistic studies on thiamin phosphate synthase: Evidence for a dissociative mechanism. Biochem 40:10095-10102. +- Sylvander P, Häubner N, Snoeijs P. 2013. The thiamine content of phytoplankton cells is affected by abiotic stress and growth rate. Microb Ecol 65:566-577. + +**endofoutput** +``` \ No newline at end of file diff --git a/markdown-output/quikchange-mutagenesis-dij4cm.md b/markdown-output/quikchange-mutagenesis-dij4cm.md new file mode 100644 index 0000000000000000000000000000000000000000..7b55f6e1982b144e6c53b57352d7dfdc1a8e2978 --- /dev/null +++ b/markdown-output/quikchange-mutagenesis-dij4cm.md @@ -0,0 +1,179 @@ +```markdown +# Goal/Experiment: +The goal of this experiment is to perform QuikChange Mutagenesis, a method used to introduce specific mutations into a DNA sequence. + +# QuikChange Mutagenesis + +### James Fraser + +#### Abstract +Citation: James Fraser QuikChange Mutagenesis. protocols.io +dx.doi.org/10.17504/protocols.io.dij4cm +Published: 10 Aug 2015 + +### Guidelines + +#### Primers +I use enzyme X to work with DNA sequences. It is installed on all the Macs and has nice translation and reverse complement functions. Many of the same functions are available in a plasmid editor, which has Mac, Windows, and Linux versions. Choose a codon for the new amino acid that is the most similar to the original codon. + +* **Forward Primer:** + - Identify the bases that code for your residue of interest (e.g., 295-297) + - A: Copy the 10 bases before the codon (e.g., 285-294) + - B: Write your new codon after the 10 bases you just copied + - C: Copy the 22 bases that follow the codon (e.g., 298-319). I always terminate in a G or C. Your forward primer should be 5'-A-B-C-3' +* **Reverse Primer:** + - D: Copy the 10 bases after the codon (e.g., 298-307) - then reverse complement it! + - E: Write your new codon - then reverse complement it! + - F: Copy the 22 bases that precede the codon (e.g., 273-294) - then reverse complement it! Your reverse primer should be 5'-D-E-F-3' + +- Order your primers from ElimBio (The newest PO B000177051) +- They should arrive in 200uM concentration; make a 20uM stock by diluting 5ul of Elim Primer with 45ul of dH2O + +#### PCR + +##### Recipe +- 1.25ul of 20uM Primer F +- 1.25ul of 20uM Primer R +- 10ul Phusion Buffer (5x) +- 1ul of 10mM dNTPs +- 0.5ul Template DNA (from miniprep, preferably from DH5alpha cells, but BL21 is still dam+ so it should be fine - 5ng starting template is plenty! (Note: good range is 50-100 ng)) +- 0.5ul Phusion polymerase +- 35ul H2O + +##### Cycle +- 98°C for 30s, then 98°C for 5s (I usually do this step for 10 seconds) +- 53°C for 20s +- 72°C for 20s/kb (usually plasmids are ~7kb = 2:20) + - Cycle 16 times - more cycles are actually bad! +- 72°C for 8:00 +- 4°C for hold + +#### If it does not work: +- Miniprep more colonies... ;) +- Order PAGE purified primers if you haven't already. We have a good price from Invitrogen. +- Redesign your primers with netprimer +- For high background of WT sequence, decrease starting amount of template (really, 5ng is plenty) +- Get fresh Dpn1 and digest ~24hrs. +- Run a gradient of annealing temperatures (try 52-62). + +#### Split reaction protocol (for high primer-dimer problems): + +##### Recipe F +- 1ul of 20uM Primer F +- 5ul Phusion Buffer (5x) +- 0.5ul of 10mM dNTPs +- 0.5ul Template DNA (from miniprep, preferably from DH5alpha cells, but BL21 is still dam+ so it should be fine) +- 0.5ul Phusion polymerase +- 17.5 ul H2O + +##### Recipe R +- 1ul of 20uM Primer F +- 5ul Phusion Buffer (5x) +- 0.5ul of 10mM dNTPs +- 0.5ul Template DNA (from miniprep, preferably from DH5alpha cells, but BL21 is still dam+ so it should be fine) +- 0.5ul Phusion polymerase +- 17.5 ul H2O + +##### Cycle each F and R in separate tubes for: +- 98°C for 30s +- 98°C for 5s (I usually do this step for 10 seconds) +- 53°C for 20s +- 72°C for 20s/kb (usually plasmids are ~7kb = 2:20) + - Cycle 10 times +- 72°C for 8:00 +- 4°C for hold + +##### Mix tubes F and R and cycle: +- 98°C for 30s +- 98°C for 5s (I usually do this step for 10 seconds) +- 53°C for 20s +- 72°C for 20s/kb (usually plasmids are ~7kb = 2:20) + - Cycle 10 times +- 72°C for 8:00 +- 4°C for hold + +Follow the transformation and Dpn1 digest as mentioned above. + +### Materials +- QIAquick PCR Purification Kit [28104 by Qiagen](https://www.qiagen.com/us/products/discovery-and-translational-research/dna-rna-purification/dna-purification/plasmid-dna/qiaquick-pcr-purification-kit/#ordering-information) + +### Protocol + +#### PCR +#### Step 1. +Prepare PCR mixture. + +### Recipe +1. **Step 1.1** + 1.25ul of 20uM Primer F. +2. **Step 1.2** + 1.25ul of 20uM Primer R. +3. **Step 1.3** + 10ul Phusion Buffer (5x). +4. **Step 1.4** + 1ul of 10mM dNTPs. +5. **Step 1.5** + 0.5ul Template DNA. +> **Notes:** +> From miniprep, preferably from DH5alpha cells, but BL21 is still dam+ so it should be fine - 5ng starting template is plenty! (note: good range = 50 - 100 ng). + +6. **Step 1.6** + 0.5ul Phusion polymerase. +7. **Step 1.7** + 35ul H2O. + +#### PCR +#### Step 2. +Perform the PCR using the following cycling conditions: +- 98°C for 30s +- 98°C for 5s (I usually do this step for 10 seconds) +- 53°C for 20s +- 72°C for 20s/kb (usually plasmids are 7kb = 2:20) + - Cycle 16 times - more cycles are actually bad! +- 72°C for 8:00 +- 4°C for hold + +#### DpnI and Transformation +#### Step 3. +After PCR, add 1ul of DpnI to each PCR tube. + +#### Step 4. +Incubate 1 hour-O/N at 37°C. +> **Notes:** +> I always do this O/N or for at least 4 hours (Avi). +> I got 95% efficiency when I DpnI ~24hrs (Cat). + +#### Step 5. +PCR Purify using Qiagen kit. +> **Reagents:** +> QIAquick PCR Purification Kit [28104 by Qiagen](https://www.qiagen.com/us/products/discovery-and-translational-research/dna-rna-purification/dna-purification/plasmid-dna/qiaquick-pcr-purification-kit/#ordering-information). + +#### Step 6. +Add 5ul of PCR reaction into 25ul DH5alpha or TG1 cells. +> **Notes:** +> I eluted in 27ul EB and transformed all 25ul elution in 50ul TG1 cells + 25ul KCM (Cat). + +#### Step 7. +Incubate on ice for 5 minutes. +> **Notes:** +> If you have time, incubate for 30min - 1hr (Avi). + +#### Step 8. +Heatshock at 42°C for 45 seconds. + +#### Step 9. +Recover on ice for 2 minutes. + +#### Step 10. +Add 200ul LB (if in 96 well or 8 strip format) to each reaction. +> **Notes:** +> 350ul SOC, plate 50ul (Cat). + +#### Step 11. +Shake at 37°C for 1 hour. + +#### Step 12. +Plate on warmed antibiotic plates. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/rat-organotypic-cultures-for-aav-mediated-vital-la-c645zgy6.md b/markdown-output/rat-organotypic-cultures-for-aav-mediated-vital-la-c645zgy6.md new file mode 100644 index 0000000000000000000000000000000000000000..6a0d5725bb55df248d670da92876f5ffb5ace1cd --- /dev/null +++ b/markdown-output/rat-organotypic-cultures-for-aav-mediated-vital-la-c645zgy6.md @@ -0,0 +1,118 @@ +```markdown +# Goal/Experiment: +The goal of this experiment is to describe the preparation and maintenance of rat organotypic cultures (cortico-striatal) for AAV-infection and imaging of the extracellular matrix. + +# Rat Organotypic Cultures for AAV-Mediated Vital Labeling of the Extracellular Matrix V.2 + +**Authors:** Federico N. Soria, Mario Fernandez-Ballester +**Affiliation:** Achucarro Basque Center for Neuroscience +**Version:** 2 +**Date:** January 8, 2024 +**Keywords:** ASAPCRN + +## Abstract +In this protocol, we describe the preparation and maintenance of rat organotypic cultures (cortico-striatal) for AAV infection and imaging of the extracellular matrix. The AAV induces the expression of a hyaluronan-binding protein (the HA-binding domain of Neurocan) fused with GFP, allowing vital labeling and live imaging of the extracellular matrix. The organotypic cultures can be maintained for several weeks, allowing repeated imaging and longitudinal studies. + +## Materials + +### Organotypic Culture Medium +| Component | Details | Vendor/Catalog Number | +|-----------|---------|-----------------------| +| MEM, HEPES | No glutamine | Gibco, ThermoFisher Catalog #32360026 | +| EBSS | Calcium, magnesium, phenol red | ThermoFisher Catalog #24010043 | +| Horse Serum | Heat inactivated, New Zealand origin | ThermoFisher Catalog #26050088 | +| B-27 Supplement | | Gibco - ThermoFisher Catalog #17504044 | +| GlutaMAX™ Supplement | | ThermoFisher Scientific Catalog #35050061 | +| Glucose Solution | 36 mM | ThermoFisher Catalog #A2494001 | +| Penicillin-Streptomycin | 50 U/mL, (5,000 U/mL) | ThermoFisher Catalog #15070063 | +| Fungizone (Amphotericin B) | 1.25 ug/mL | ThermoFisher Catalog #15290018 | + +### Membrane Inserts +| Component | Details | Vendor/Catalog Number | +|-----------|---------|-----------------------| +| Millipore Cell Culture Inserts | 0.4 µm pore size, 30 mm diameter | Merck MilliporeSigma (Sigma-Aldrich) Catalog #PICM0RG50 | + +### Tissue Chopper +| Equipment | Brand | SKU | +|-----------|-------|-----| +| McIlwain Tissue Chopper | Cavey Laboratory Engineering | 51350 | + +### Additional Materials +| Component | Vendor/Catalog Number | +|-----------|-----------------------| +| HBSS, no calcium, no magnesium, no phenol red | ThermoFisher Scientific Catalog #14175095 | +| Cytosine β-D-arabinofuranoside (AraC) | Merck MilliporeSigma (Sigma-Aldrich) Catalog #C1768 | + +## Procedure + +### Culture Preparation + +1. **Prepare a 6-well culture plate** + - With Millipore 0.4 µm culture inserts placed on top of 1 mL of organotypic medium per well. + - Incubate at 37°C and 5% CO2 to warm up. + - *Note:* Minimize air bubbles between the medium and the membrane insert. + +2. **Prepare p100 and p60 petri dishes** + - Add 25 mL and 5 mL of HBSS (no calcium, no magnesium, no phenol red). + - Keep on ice. + +3. **Prepare the McIlwain Tissue Chopper** + - Use a fresh blade and clean holder plates. + +4. **Sacrifice P5-P7 rat pups** + - Perform rapid decapitation. + - Extract the brain without damaging the cortex. + +5. **Brain preparation** + - Place the brain on filter paper. + - Separate cerebral hemispheres by cutting through the midline. + - Remove the midbrain from the cortex using a scalpel. + +6. **Sectioning** + - Place both hemispheres medial face downward in the holder plate. + - Section the brain at 350 µm width. + +7. **Transfer sections** + - Pour sliced brain carefully into p100 petri dish with HBSS on ice. + - Use a Pasteur pipette to moisten brain slices. + +8. **Slice isolation** + - Separate slices under magnification using customized spatulas. + - Transfer slices into p60 petri dish (can use an inverted glass pipette with a suction rubber bulb). + - *Note:* Remove the subventricular zone (SVZ) and meninges. + +9. **Transfer slices to culture inserts** + - Under a laminar flow hood, transfer slices to preheated culture inserts with medium. + - Remove excess dissection buffer from top of inserts with sterile tip. + - *Note:* Cultures must be handled in sterility henceforth. + +10. **Incubate cultures** + - Incubate at 37°C and 5% CO2. + - *Expected result:* 4-6 cortico-striatal slices can be obtained per hemisphere. + +### Maintenance and AAV-Infection + +11. **Replace feeding medium at DIV1** + - Add 1 mL of medium with Cytosine β-D-arabinofuranoside (AraC) (4.4 µm per well). + - *Expected result:* AraC prevents astrocyte overproliferation. + +12. **AAV infection at DIV3** + - Replace AraC-containing medium with fresh medium. + - Add 1 µL of AAV-Ncan-GFP 10^12 vg/mL over the slice. + - *Safety:* AAV manipulation must occur in BSL1 facilities. + - *Note:* For improved spatial precision, use a micromanipulator. + +13. **Maintain culture** + - Change medium three times a week (1 mL per well). + - *Expected result:* Slices flatten and become transparent over time. AAV expression visible at DIV10, max from DIV14 onwards. + +### Imaging + +14. **Imaging Methods** + - **IN-PLATE:** Perform imaging through the plastic without removing the insert. Suitable for widefield setups, useful for repeated imaging. + - **OFF-PLATE:** Remove insert and place in smaller petri-dish for imaging (confocal or 2-photon). Ensure cleanliness and use a buffer medium with HEPES if CO2 is unavailable. + +*Note:* After each imaging, change medium with fresh antibiotic/antimycotic and monitor for contamination. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/real-time-quaking-induced-conversion-assay-rt-quic-b9sar6ae.md b/markdown-output/real-time-quaking-induced-conversion-assay-rt-quic-b9sar6ae.md new file mode 100644 index 0000000000000000000000000000000000000000..4982633a3a9fe7d8959c0d4c69359d1ebfd4423e --- /dev/null +++ b/markdown-output/real-time-quaking-induced-conversion-assay-rt-quic-b9sar6ae.md @@ -0,0 +1,154 @@ +```markdown +# Goal/Experiment: +Determining pathology-associated aggregation-templating competent forms of alpha-synuclein and other interacting factors using the Real time-quaking induced conversion assay (RT-QuIC). + +# Real time-quaking induced conversion assay (RT-QuIC) + +## Authors +- **andrew.west** +- **arpine.sokratian** + +**Affiliation**: Duke University + +**Date**: JAN 15, 2024 + +## Abstract +Seeded-amplification assay (SAA) method for the detection of pathology-associated aggregation-templating competent forms of alpha-synuclein and other interacting factors. + +## Attachments +- [RT-QUIC Outline](RT-QUIC outline.xlsx) + +## Protocol Materials + +| Item | Catalog | Steps | +|--------------------------------------------------------------------------------------------|----------------------------------------------------------------|-----------| +| Tweezers | TEDPELLA Catalog #534 | Step 7 | +| Corning® 384-well Black/Clear Bottom Low Flange Ultra-Low Attachment Microplate Bulk Packed| Corning Catalog #4588 | Step 7 | +| BioSpec Products 2.3 mm Zirconia/Silica Beads 1 lb bottle | Fisher Scientific Catalog #NC0451999 | Step 7 | +| Amicon Ultra-0.5 Centrifugal Filter Unit 24 pack | Merck Millipore (EMD Millipore) Catalog #UFC505024 | Step 4 | +| 250g Guanidine hydrochloride | G-Biosciences Catalog #BC85 | Steps 4-5 | +| Thioflavin T | Merck MilliporeSigma (Sigma-Aldrich) Catalog #T3516 | Step 9 | +| PCR Plate Heat Seal foil pierceable | Bio-Rad Laboratories Catalog #1814040 | Step 11 | + +## Plate Preparation + +### Step 1 +- Thaw down a-syn monomer and sonicated fibril aliquots on ice, avoiding bubble formation during pipetting. + +### Step 2 +- Measure monomer concentration using a Nanodrop spectrophotometer: + - **Procedure**: Add 3 µL of 10x diluted aliquot in PBS onto the Nanodrop pedestal. Measure and ensure a coefficient extinction of 5.98 and MW of 14.4 kDA. Perform two measurements and aim for less than 10% standard error. If necessary, prepare 20X and 30X dilutions to confirm findings. + +#### Equipment +| Name | Type | Brand | SKU | +|---------------------------------------------------|--------------------------------|---------------------|------------| +| NanoDrop™ One/OneC Microvolume UV-Vis Spectrophotometer | UV-Vis Spectrophotometer | Thermo Scientific | ND-ONE-W | + +### Step 3 +- Calculate the volume of monomer needed for a reaction mix with 0.3 mg/mL monomer concentration in a reaction mix with 30 µL per well: + - _Formula_: \( 30 \ l (\text{reaction volume}) \times 3 (\text{replicates}) \times \_ (\text{different conditions}) = \_ \mu l \) + +### Step 4 +- Dilute the monomer preparation with 1X PBS (Phosphate-buffered saline) to a concentration of 2.5 mg/mL. + +### Step 5 +- Use Amicon Ultra-0.5 Centrifugal Filter Unit 24 pack (Catalog #UFC505024 ‎) to filter out possible aggregates or high molecular weight (HMW) contaminants. + +### Step 6 +- Measure DLS data for sonicated fibrils. + +## Dynamic Light Scattering (DLS) Measurements + +### Step 7 +- Place a single zirconia bead into each well of the Corning 384-well microplate. Use BioSpec Products 2.3 mm Zirconia/Silica Beads (Catalog #NC0451999) and Tweezers TEDPELLA (Catalog #534). + +### Step 8 +- Prepare serial dilutions of sonicated fibrils: + - **Solution**: 1 mg/mL measured PFFs (measured fibrils) with 20 µL volume. + - **Dilution Procedure**: + 1. 10 ug/mL: 10 µL PFFs + 990 µL PBS + 2. 1000 ng/mL: 10 µL PFFs + 90 µL PBS + 3. 100 ng/mL: 10 µL PFFs + 90 µL PBS + 4. 10 ng/mL: 10 µL PFFs + 90 µL PBS + 5. 1 ng/mL: 10 µL PFFs + 90 µL PBS + 6. 100 pg/mL: 10 µL PFFs + 90 µL PBS + 7. 10 pg/mL: 10 µL PFFs + 90 µL PBS + 8. 1 pg/mL: 10 µL PFFs + 90 µL PBS + 9. 100 fg/mL: 10 µL PFFs + 90 µL PBS + 10. 10 fg/mL: 10 µL PFFs + 90 µL PBS + +### Step 9 +- Prepare the reaction mix: + - Composition: 0.3 mg/mL monomer + 10 µM Thioflavin T (Catalog #T3516) in PBS. Use a final volume of 30 µL per well. + +### Step 10 +- Prepare a standard curve using PFFs dilutions from step 8, mixing them with the reaction mix. + +### Step 11 +- Transfer the reaction to the plate: fill the plate with standard curve samples and seal it with PCR Plate Heat Seal foil (Catalog #1814040). Spin down the plate. + +## Run Protocol + +### Settings +| Parameter | Value | +|-------------------------------|---------------------| +| Measurement type | Fluorescence (FI) | +| Microplate name | COSTAR 384 | +| No. of cycles | 100 | +| Cycle time [s] | 1800 | +| No. of flashes per well and cycle | 12 | +| Scan mode | Orbital averaging | +| Scan diameter [mm] | 4 | +| Optic settings: | +|   Excitation | 448-10 | +|   Emission | 482-10 | +|   Gain | 1000 | +| Shaking settings: | +|   Shaking 1 | Between readings | +|   Movement | Orbital shaking | +|   Frequency [rpm] | 700 | +|   On time [s] | 60 | +|   Off time [s] | 60 | + +### Step 12 +- Set up the program on a plate reader. Use Omega - RT-QuIC / PRION Version - Fluorescence Base from BMG. + +### Step 13 +- Finish the experiment once the standard curve reaches the plateau (example: 7 hours). + +## Data Analysis + +### Step 14 +- Convert collected data from the plate reader into .xlsx format. + +### Step 15 +- Calculate Fluorescence Forming Units (FFUs) for pathological samples relatively to an appropriate standard curve. + +#### FFU Calculation +1. Use a standard curve with a range of serial dilutions of evaluated ssFibrils. +2. Obtain ThT FL units at each time point and dilution. +3. Software/code extracts the appropriate threshold value to define CT values. +4. Measure the time for each sample at X percent as the cutoff difference. + +#### Example Code: +Refer to this GitHub repository for the script: [FibrilOptimization](https://github.com/west-lab/FibrilOptimization/blob/master/FibrilPaperCode_main.py) + +### Step 16 +- Use the mean half-time of the reaction by analyzing the CT values of the standard curve. + +### Step 17 +- Extract RFU values for each condition. + - **Example**: + +| Condition | Replicate 1 | Replicate 2 | Replicate 3 | +|------------|-------------|-------------|-------------| +| Experimental | 1500 | 1600 | 1600 | +| Control | 100 | 200 | 150 | + +### Step 18 +- Use the extracted data to generate a plot or group analysis via GraphPad. + +--- + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/recommendations-to-grow-algal-culture-roscoff-cult-s2qegdw.md b/markdown-output/recommendations-to-grow-algal-culture-roscoff-cult-s2qegdw.md new file mode 100644 index 0000000000000000000000000000000000000000..014e3537d3d7ed7353cf0f93c9371dae82ea972f --- /dev/null +++ b/markdown-output/recommendations-to-grow-algal-culture-roscoff-cult-s2qegdw.md @@ -0,0 +1,93 @@ +```markdown +# Goal/Experiment: +Grow and maintain algal cultures from the Roscoff Culture Collection. + +# Recommendations to Grow Algal Culture - Roscoff Culture Collection + +## Abstract +Recommendations for algal cultures from the Roscoff Culture Collection. + +## Safety Warnings +- To avoid any risk of contamination of the natural environment, all culture residues must be sterilized (by autoclaving or by treatment with bleach) before being discarded. +- Culture transfer should be undertaken using standard sterile microbiological techniques (work under a laminar flow hood or near a bunsen burner flame, clean all surfaces with 70% ethanol, etc.). + +## Protocol Steps + +### Step 1: Before Ordering Strains from the RCC +- Ensure your laboratory has suitable equipment and conditions for the reception and maintenance of the cultures. +- Examine each culture in the RCC catalogue for detailed information on culture conditions (temperature, medium, light intensity, etc.). + +### Step 2: Prepare Media (Not Supplied by the RCC) +- Each culture uses a defined medium (see [RCC culture media](http://roscoff-culture-collection.org/culture-media)). +- Sterile (autoclaved and/or 0.22μM filtered) nutrient and trace metal stocks must be added to the sterile seawater under a laminar flow hood. +- Do not autoclave vitamins; sterilize by 0.22μM filtration and storage at -20°C. + +**Which Seawater to Use?** +- The RCC's media is often prepared from seawater collected off Roscoff (salinity ca. 33‰), stored for at least two months in darkness, then filtered on 0.22μM filters (Millipore filter GSWP09000 plus Millipore prefilter AP1507500) and autoclaved. +- If you lack access to natural seawater, try artificial seawater made from mixtures of salts (see [RCC artificial seawater](http://roscoff-culture-collection.org/culture-media)). However, we cannot guarantee culture growth in artificial seawater-based media (except for some cyanobacteria for which Red Sea Salt artificial seawater is recommended). + +### Step 3: Prepare Room or Cabinet with Appropriate Conditions + +**Light** +- All RCC strains are exposed to a 12H/12H day/night light cycle. +- Use 'daylight' neon tubes (Tubas-Neons Sylvania Daylight F58W/54/765 of: 0001404+ starter ref: 00007698476). +- Light intensity for culture maintenance rarely exceeds 100 μEinsteins.m-2.s-1. When in doubt, use an irradiance lower than higher values to avoid light stress. +- Some cultures (notably certain cyanobacteria) prefer blue light. You can wrap your light tubes or flasks with a blue filter (Moonlight Blue 183 - Meint Ecairages Scéniques). + +**Temperature** +- The optimum temperature condition is indicated for each strain. Temperature control is critical, with particular attention to avoiding fluctuations detrimental to algal culture growth. + +**RCC Standard Temperature Conditions:** +| Strain Type | Temperature | +|--------------------|-------------| +| Polar strains | 4°C | +| Temperature strains| 15°C | +| Tropical strains | 22°C | + +### Step 4: Order / Prepare Culture Flasks +- RCC strains are routinely maintained in single-use sterile polystyrene flasks with a ventilated filter cap (Sarstedt ref 831810.002 or Nunc ref 136196) or single-use sterile polystyrene tubes (CML ref TC1U2PS25). +- Strains generally grow well in Erlenmeyers or other glass flasks. + +### Step 5: Shipment of Cultures +- RCC staff will contact you after receiving your order to arrange a delivery date. +- Delivery via DHL courier service. +- Upon dispatch, you will receive a DHL tracking number by email. +- Expect delivery within one to three days (depending on location). +- Upon delivery, do not store cultures in a cold room or freezer. Cultures should be stored at room temperature, and you should immediately inform RCC of the arrival. + +## After Receiving the Culture + +### Step 6: Transfer to Culture Room +- Transfer the flasks to a culture room or cabinet with the appropriate conditions upon opening the package. +- Cultures experience stress during transport; therefore, wait one to two days before transferring cultures into fresh medium. +- Monitor strain growth daily via: + - Direct microscopy (not suitable for very small cells like Prochlorococcus, can be detected using epifluorescence microscopy) + - Inverted microscopy (best suited for flask checking) + - Flow cytometry (for Prochlorococcus or small eukaryotes) + +- Strains must typically be transferred every 2-3 weeks with a 1/10 to 1/50 dilution in new medium. + +- For additional assistance, contact rcc@sb-roscoff.fr. + +### Step 7: Notify of Poor Cultures +- Notify RCC immediately with supporting details (e.g., flask photos, microscopic images) if cultures are not in good shape upon arrival. +- Cultures are never axenic, so contamination is expected; notify RCC of issues within two weeks for a free replacement (excluding shipping). + +## Specific Instructions for Viruses + +### Step 8: Virus Cultures +- Store the flask containing viruses at 4°C in darkness upon delivery. +- Propagate viral strains by transferring to a host culture in exponential phase. Dilute the host culture 1/10 – 1/50 (vol/vol) into fresh medium and incubate for 3 to 4 days under appropriate conditions. +- Viral suspension can be added using a virus:host ratio of 1:10 – 1:50 (vol/vol) and incubated under host growing conditions. + +**Monitoring Cell Lysis:** +- Use a non-infected host culture aliquot as a control. Cell lysis is detected by complete host culture clearing (3 – 7 days post-viral inoculation). + +- The lysate can be stored at 4°C or transferred to fresh host cultures as soon as possible. Avoid extended storage to prevent resistant host development. Filter lysate through 0.2 µm filters (PES, PC, GF; avoid cellulose acetate membranes). + +- Store viral suspensions for several months at 4°C (3 – 4 months). Be aware that viruses are sensitive to intense light, UV, and heat. + +--- + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/recording-well-and-craniotomy-for-electrophysiolog-9a8h2hw.md b/markdown-output/recording-well-and-craniotomy-for-electrophysiolog-9a8h2hw.md new file mode 100644 index 0000000000000000000000000000000000000000..6ff5f7b45efb1e74fc537cd0fa6c084bd37042e5 --- /dev/null +++ b/markdown-output/recording-well-and-craniotomy-for-electrophysiolog-9a8h2hw.md @@ -0,0 +1,141 @@ +```markdown +# Goal/Experiment: +The goal of this experiment is to record well and perform craniotomy for electrophysiology in head-restrained mice, enabling acute extracellular electrophysiological recordings. + +# Recording Well and Craniotomy for Electrophysiology in Head-Restrained Mice + +**Authors:** +Susu Chen1, Karel Svoboda1 +1Janelia Research Campus + +**DOI:** +[dx.doi.org/10.17504/protocols.io.9a8h2hw](https://dx.doi.org/10.17504/protocols.io.9a8h2hw) + +### Abstract +Recording well and craniotomy for acute extracellular electrophysiological recordings in head-restrained mice. + +### Keywords +- Craniotomy +- Durotomy +- Electrophysiology +- Head-restrained mice + +### License +This is an open access protocol distributed under the terms of the [Creative Commons Attribution License](https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. + +### Created +November 14, 2019 + +### Last Modified +May 20, 2021 + +### Protocol Integer ID +29760 + +## Materials + +- Stereotaxic frame +- Stereoscope with illumination +- Isoflurane +- Marcaine ([Henry Schein](https://www.henryschein.com/us-en/Search.aspx?searchkeyWord=marcaine)): 0.1 ml 0.5% solution +- Ketoprofen ([Zoetis](https://www.zoetisus.com/products/horses/ketofen.aspx)): Use dilution of 1 mg/ml +- Buprenorphine ([Henry Schein](https://www.henryschein.com/us-en/Shopping/ProductDetails.aspx?productid=2284659&name=Buprenex%20Injection%20Ampule%200.3mg%201ml)): Use dilution of 0.03 mg/ml +- Lubricant eye gel ([Genteal Tears](https://gentealtears.myalcon.com/)) or [Ophthalmic ointment](https://www.dechra-us.com/our-products/us/companion-animal/dog/non-prescription/puralube-ophthalmic-ointment) +- 0.5 ml insulin syringes for injections ([BD](https://www.bd.com/en-uk/products/diabetes/diabetes-products/insulin-syringes/microfine-insulin-syringes)) +- Heating pad +- Sterile cotton swabs +- Kim wipes +- Krazy glue (no run gel) +- Dental acrylic (Pearson Dental Jet Repair Acrylic Clear, L25-0300) +- Customized titanium headbar +- Cortex buffer: + - NaCl 125 mM + - KCl 5 mM + - Glucose 10 mM + - HEPES 10 mM + - CaCl2 2 mM + - MgSO4 2 mM + - pH 7.4 +- Sugi sponge ([Sugi Sponge](https://www.sugisponge.com/material-properties/absorption/)) +- Kwik-Cast Sealant ([WPI](https://www.wpiinc.com/kwik-cast-kwik-cast-sealant)) + +## Procedure + +1. **Anesthetizing and Preparing the Animal:** + - Disinfect all surgery tools and headbar. + - Anesthetize the animal with 3% Isoflurane and mount it onto a stereotaxic frame with a homeothermic heating pad underneath the animal. + - During surgery, adjust Isoflurane to 1-1.5% to achieve a steady ~1/sec breathing rate, and maintain the body temperature at 37°C. Check for absence of reflexes by pinching the toe. + +2. **Administering Analgesics:** + - Inject Marcaine (local anesthetic, 0.1 ml 0.5% solution) under the scalp for topical anesthesia, and administer Ketoprofen (non-steroidal anti-inflammatory drug, 5 mg/kg) subcutaneously. + - Cover the animal’s eyes with eye gel or ointment to keep them from drying out during surgery. + +3. **Preparing the Scalp and Skull:** + - Clean the scalp and hair with 70% ethanol and betadine. Remove the scalp with a single cut from between the ears to between the eyes. + - Remove any protruding hairs with scissors and cotton swabs. + - Thoroughly clean the skull with saline followed by 70% ethanol. Dry the skull with sterile cotton swabs. + - Use a fine-tip permanent marker (e.g., Sharpie) to mark key reference points on the skull (e.g., Bregma, Lambda, and craniotomy locations). + - Level Bregma and Lambda to within 40 µm of each other along the dorsal-ventral axis. Level additional two points (±2 mm lateral to Bregma) to within 40 µm of each other along the dorsal-ventral axis. + +4. **Attaching the Headbar:** + - Roughen the surface of the skull with a slowly turning dental drill where the headbar will be implanted to help with better bonding of the glue. + - Apply an even layer of Krazy glue to the skull surface and place a titanium headbar in the intended position. Apply a thick layer of Krazy glue covering the headbar and wait until the glue dries. + +5. **Building the Recording Well:** + - Apply dental acrylic/cement using a 50 µl pipette tip to cover the applied glue layer. The cementing helps to reinforce the headbar in position. + - To avoid bubbles in cement, use a thin mixture of dental acrylic. + - Gradually add layers of dental cement around the edge of the skull to build a recording well. Make the recording well a convex shape which allows it to hold more cortex buffer and enables probes to approach from a lateral to medial angle. + - Wait until dental acrylic cures between adding layers. The recording well should be at least 4 mm in height for acute recordings that last for more than one hour. + - The well needs to hold enough cortex buffer (e.g., 0.1 ml) to avoid brain drying out over the course of a recording session. + +**Example of a recording well and cemented titanium headbar, with reference points marked on the skull:** +![Example Image](path_to_example_image) + +6. **Post-surgical Injection and Recovery:** + - Remove the animal from the stereotaxic frame and inject Buprenorphine (Opioid analgesic, 0.05 mg/kg) into the intraperitoneal cavity (IP). + - Leave the animal in a temperature (37°C) controlled cage until ambulatory. For a more detailed headbar implantation, refer to the [headbar implantation protocol](https://www.protocols.io/view/headbar-implantation-bcrsi6e). + +7. **Craniotomy Preparation:** + - Craniotomy is performed at a different experimental time point: on the day before the first acute electrophysiological recording, anesthetize the animal following the same procedure in step 1. + - Use a marker pen to label the center/edge of each craniotomy based on the reference points labeled in step 3 (e.g., anterior-posterior, medial-lateral relative to Bregma/Lambda). + +**Example Image:** +![Example Image](path_to_example_image_blue_marks) + +8. **Performing the Craniotomy:** + - Drill one craniotomy at a time, centered on an individual marked point to make an 'island'. Each craniotomy takes 10-20 rounds of drilling, depending on the thickness of the skull. + - Use compressed air to clear away bone debris from drilling. In between rounds, rinse the skull with cortex buffer to avoid heating up the tissue beneath and to control swelling. + - The diameter of a craniotomy should be approximately 1-1.5 mm. Usually, a larger craniotomy is required to accommodate multi-shank probe penetrations. + +9. **Opening the Craniotomy:** + - Stop drilling once a thin layer of bone is left. Apply cortex buffer on top. + - Gently push on the center of the craniotomy to feel how it gives. This is to check that the edge of the bone is thin enough to break when the center island is lifted. + - Lift the remaining bone flap with a pair of fine forceps while cortex buffer is covering the surface. + - Carefully remove the remaining attached flap of dura using the same principle: grab and pull, but do not touch the cortex. + - Apply Gelfoam (soaked in cortex buffer) to the dura surface to control small bleeders. + - Ensure a healthy craniotomy which should be clean without excessive bleeding and the brain should not swell. + +**Example Image of Four Healthy Craniotomies:** +![Example Image](path_to_craniotomies_example_image) + +10. **Preparing Multiple Craniotomies:** + - For preparing multiple craniotomies, drill all of them sequentially until the bone flaps are ready to be lifted up in cortex buffer one-by-one. + - Ensure cortex buffer is applied during drilling to avoid heating the tissue. + +11. **Performing Durotomy:** + - When durotomy is required, select a clean spot with no surface blood vessels on the corner or edge of the craniotomy. + - Make a small incision into the dura using a fresh 31 gauge needle with a shallow angle. + - Insert the lower jaw of a fine-tip forceps into the incision almost parallel to the surface of the brain. Grab the dura tightly with forceps and pull off the dura all the way across the craniotomy. + - Avoid touching the cortex underneath during this step. Soak the edge with the cortex buffer to control swelling. + +12. **Sealing the Recording Well:** + - Absorb all cortex buffer with sugi sponge before sealing the recording well. Make sure the dura/brain surface is moist but not with excessive liquid. + - Apply Kwik-cast sealant to cover craniotomies/durotomies. + - Remove the animal from the stereotaxic frame and leave it in a temperature (37°C) controlled cage until ambulatory. + +**Citation:** +Susu Chen, Karel Svoboda (05/20/2021). Recording well and craniotomy for electrophysiology in head-restrained mice. [protocols.io](https://dx.doi.org/10.17504/protocols.io.9a8h2hw). +[Creative Commons Attribution License](https://creativecommons.org/licenses/by/4.0/) + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/reverse-transcription-primer-pools-preparation-and-cyjcxuiw.md b/markdown-output/reverse-transcription-primer-pools-preparation-and-cyjcxuiw.md new file mode 100644 index 0000000000000000000000000000000000000000..475749a2511c7808744c6655f89d2d866b440067 --- /dev/null +++ b/markdown-output/reverse-transcription-primer-pools-preparation-and-cyjcxuiw.md @@ -0,0 +1,139 @@ +```markdown +# Goal/Experiment: +Reverse transcription, primer pools preparation, and multiplex PCR steps for CHIKV serotype genomic sequencing + +## Reverse transcription, primer pools preparation and multiplex PCR steps for CHIKV serotype genomic sequencing + +**Authors:** +Gabriel Luz +Laís Ceschini¹, Luisa Maria Inácio da Silva¹, Wallau¹ +¹Entomology department, Instituto Aggeu Magalhães (IAM), FIOCRUZ + +**Date:** +AUG 11, 2023 + +**Abstract:** +This step-by-step protocol describes the cDNA synthesis, primer pools preparation and multiplex PCR conditions with the main goal to sequence the complete genome of CHIKV serotype strains. + +**Materials:** + +- **Reverse Transcription:** + - SuperScript™ IV First-Strand Synthesis System (200 reactions) + *Vendor:* Invitrogen + *Cat #:* 18091200 + +- **Multiplex PCR:** + - Q5® High-Fidelity 2X Master Mix + *Vendor:* NEB + *Cat #:* M0492L + - H₂O Ultrapure + - Primers described in Table 1 + +## Reverse Transcription + +### 1. Preparation of Mix 1 + +Using a 2 mL tube, prepare Mix 1 as described below for 96 samples: + +| Component | Volume (1x) | 96 samples (+2 = 98) | +|---------------------|-------------|----------------------| +| Random Hexamers (50 µM) | 1 µL | 98 µL | +| dNTPs mix (10 mM each) | 1 µL | 98 µL | +| **Total** | **2 µL** | **194 µL** | + +### 2. Sample Preparation + +Using 0.2 mL PCR tubes or 96 wells plates, add 11-16 µL of extracted RNA from RT-PCR positive samples. Add 2 µL of Mix 1 to the tube/well and place it in a thermocycler with the following setup: + +- 65°C for 5 minutes + +### 3. Cooling + +Take the tubes/wells to ice for 1 minute. Prepare a water bath with ice cubes to ensure uniform temperature distribution. + +### 4. Preparation of Mix 2 + +Using a 2 mL tube, prepare Mix 2: + +| Component | Volume (1x) | 96 samples (+2 = 98) | +|----------------------------|-------------|----------------------| +| 5x SSIV Buffer | 4 µL | 392 µL | +| 100 mM DTT | 1 µL | 98 µL | +| RNaseOUT or RNase Inhibitor| 1 µL | 98 µL | +| SSIV Reverse Transcriptase | 1 µL | 98 µL | +| **Total** | **7 µL** | **686 µL** | + +### 5. Reverse Transcription Reaction + +Add 7 µL of Mix 2 to the tubes containing Mix 1 plus RNA and place it in the thermocycler with the following conditions: + +- **Step 1:** + - 42°C --- 50 minutes + - 70°C --- 10 minutes + - 4°C --- Hold + +### 6. Storage + +Store the cDNA at -20°C. + +**Observation:** Use only samples RT-PCR positive showing a Ct value < 30 for cDNA conversion and genomic amplification to improve final results. + +## Pools of Primers + +### 7. Preparation of Primer Pools + +Select two 0.6 mL tubes for each pool. + +### 8. Combine Primer Solutions + +Using the original 100 µM primer solution eluted individually, combine them as per the table below: + +### 9. Final Volumes + +Pool 1 will have a final volume of 469 µL and Pool 2 of 460 µL. + +### 10. Dilution for Multiplex PCR + +To prepare the solution for Multiplex PCR, dilute each pool 1:10. That is, take 10 µL of pool 1 and 90 µL of ultrapure water. The table below contains each primer volume: + +**Table 1: Primers and Pool Order** + +| Primer | Sequence | Concentration in Pool (µM) | Volume of Primer (µL) | Pool | +|-----------------------|----------------------------|---------------------------|------------------------|------| +| 400_1_LEFT_1 | TGACACACG TGACCTCACG | 0.015 µM | 10 µL | 1 | +| 400_1_RIGHT_1 | CGCATCGGG CAAACCGAG TGTA | 0.015 µM | 10 µL | 1 | +| 400_3_LEFT_3 | GCGACGCTC GCGTATACCAAG | 0.015 µM | 10 µL | 1 | +| ... | ... | ... | ... | ... | +| 400_46_RIGHT_T_1 | TCTTAGTCA ATAGTGGT GTCCTTAGG| 0.015 µM | 10 µL | 2 | + +*Note: The primers were designed using the https://primalscheme.com based on the KP164576, KP164571, KP164572, KP164568, KP164570, and KP164569 reference genomes (Machado, 2019).* + +## Multiplex PCR + +### 11. Multiplex PCR Mix Preparation + +Prepare Mix 1 for a Multiplex PCR for each Pool 1 and Pool 2 using a Falcon tube of 15 mL or a 2 mL tube. + +| Component | Volume for Pool 1 (1x) | Volume for Pool 2 (1x) | 96 samples (+2) (pool1 or pool2) | +|-------------------------------|-----------------------|------------------------|----------------------------------| +| Q5 Master Mix High Fidelity 2X| 12.5 µL | 12.5 µL | 1225 µL | +| Pool primers (Pool 1 or Pool 2)| 1.7 µL | 1.7 µL | 166.6 µL | +| Ultrapure Water | 8.3 µL | 8.3 µL | 813.3 µL | +| **Total** | **22.5 µL** | **22.5 µL** | **2205 µL** | + +### 12. PCR Cycling Conditions + +Add 2.5 µL of cDNA (totaling 5 µL) in 22.5 µL of the Pool 1 and Pool 2 reaction mixture, and place it in the thermocycler with the following setup: + +- **Step 1:** + - 98°C --- 30 seconds +- **Step 2: (45 cycles)** + - 98°C --- 15 seconds + - 58°C --- 30 seconds + - 72°C --- 5 minutes +- **Step 3:** + - 72°C --- 2 minutes + - Hold at 4°C + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/rna-and-protein-extraction-from-bulk-dissections-cgixtufn.md b/markdown-output/rna-and-protein-extraction-from-bulk-dissections-cgixtufn.md new file mode 100644 index 0000000000000000000000000000000000000000..782baf3eeabe32389cfeeb6b631a69de9f6300f2 --- /dev/null +++ b/markdown-output/rna-and-protein-extraction-from-bulk-dissections-cgixtufn.md @@ -0,0 +1,100 @@ +```markdown +# Goal/Experiment: +The goal of this experiment is to extract RNA and protein from bulk dissections using the mirVana PARIS RNA and Native Protein Purification Kit. + +# RNA and Protein Extraction from Bulk Dissections + +miquel.vila1 +1Vall d'Hebron Research Institute + +**DOI:** [dx.doi.org/10.17504/protocols.io.4r3l275nqg1y/v1](dx.doi.org/10.17504/protocols.io.4r3l275nqg1y/v1) + +#### ABSTRACT +The experiment uses the mirVana PARIS RNA and Native Protein Purification Kit (#AM1556, Thermo Fisher Scientific). + +#### COLLECTIONS +In vivo reduction of age-dependent neuromelanin accumulation mitigates features of Parkinson’s disease + +#### License +This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. + +**Protocol ID:** 69943 +**Created:** Sep 13, 2022 +**Last modified:** Sep 14, 2022 + +#### Guidelines +- Less than 0.5mg of tissue should be used. Comfortable working volume is 500 µL. +- Avoid collecting interphase by only collecting 200 µL, though 300 µL is possible if interphase is compact. + +**Safety Warnings**: Denaturing buffer contains β-mercaptoethanol and should be handled in a fume-hood. + +## Materials and Reagents + +### Reagents: + +| Reagent | Function | Vendor and Catalog Number | +| --- | --- | --- | +| 2X Denaturing Solution | Cell disruption and RNA protection | Thermo Fisher Scientific, provided in the kit | +| 2-Mercaptoethanol | Reducing agent | Thermo Fisher Scientific, provided in the kit | +| 100% Ethanol | RNA precipitation | Any lab supplier | +| Protease/Phosphatase Inhibitor Cocktail 100x | Protease and phosphatase inhibition | #5872S, Werfen - Cell Signaling | +| Nuclease-free water | Final RNA elution | #129114, Qiagen-Werfen | + +### Equipment: + +- Motorized rotor-stator homogenizer +- Dry ice +- 2 mL Eppendorf tubes +- Centrifuge capable of 10,000g +- Thermomixer or heating block +- Sonicator + +## Protocol Steps + +### Preparation (10 min) +1. **Preparation of Solutions**: + - Add 375 µL of 2-Mercaptoethanol to **2X Denaturing Solution**. + - Add 21 mL of 100% Ethanol to **miRNA Wash Solution 1**. + - Add 40 mL of Ethanol 100% to **Wash Solution 2/3**. + +### Sample Disruption (30 min) +2. **Preheat** Nuclease-free water (#129114, Qiagen-Werfen) at 95°C (for elution). +3. **Preheat** 2X Denaturing Solution to 37 °C until completely clear. +4. Keep bulk dissections in dry ice until added to cell disruption buffer. +5. Add **Protease/Phosphatase Inhibitor Cocktail 100x** (1/100) to cell disruption buffer. +6. Add 500 µL of **Cell Disruption Buffer** into 2mL Eppendorf tubes. + +### Tissue Homogenization (15 min) +7. Add frozen bulk dissections to tubes with **Cell Disruption Buffer**. +8. Disrupt samples using an Omni Tissue Homogenizer (TH) for no more than 5 seconds at a time. +9. Split samples into RNA and protein parts: 200 µL each. + +### RNA Extraction (45 min) +10. Add **Denaturing Solution 2X** to RNA sample. +11. Incubate 5 min on ice. +12. Add equal volume of **Acid-Phenol:Chloroform** to RNA sample plus 2X Denaturing Solution. +13. Vortex for 30-60 seconds. +14. Centrifuge at max speed for 8 minutes. Repeat if interphase is not compact. +15. Remove the upper aqueous phase carefully. +16. Add 1.25 volumes of 100% Ethanol (room temperature). +17. Pipette up and down and transfer to the filter column. +18. Centrifuge 30s at 10,000g. Discard flow-through. +19. Wash with 700 µL of **miRNA Wash Solution 1** and centrifuge 15s at 10,000g. Discard flow-through. +20. Wash with 500 µL of **Wash Solution 2/3** twice. Centrifuge 15s at 10,000g after each wash. +21. Centrifuge 1 min at 10,000g to eliminate residual fluid. +22. Transfer column to a new collection tube. +23. Add 30 µL of nuclease-free water (95°C) to elute RNA. +24. Centrifuge 30s at 10,000g to collect the eluate. +25. Repeat elution for higher yield. +26. (Optional) Take 1.5 µL of RNA sample for Bioanalyzer analysis. +27. (Optional) Take 1.5 µL for RNA concentration quantification using NanoDrop ND-1000. +28. Store RNA samples at -80°C. + +### Protein Extraction (20 min) +29. Take split sample for protein and centrifuge at 4°C for 2 minutes at 10,000g. +30. Collect supernatant and transfer it to a new tube. +31. Sonicate 3x5 minutes, with 30s on ice between intervals. +32. Store protein samples at -80°C. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/rna-extraction-protocol-for-leaves-with-high-conte-csmawc2e.md b/markdown-output/rna-extraction-protocol-for-leaves-with-high-conte-csmawc2e.md new file mode 100644 index 0000000000000000000000000000000000000000..a8d90f993067aff9e305078d352eaae88bc45eb7 --- /dev/null +++ b/markdown-output/rna-extraction-protocol-for-leaves-with-high-conte-csmawc2e.md @@ -0,0 +1,123 @@ +```markdown +# Goal/Experiment: +Extraction of RNA from leaves with a high content of secondary metabolites. + +## RNA Extraction Protocol for Leaves with High Content of Secondary Metabolites + +### Authors: +Wesley Elias Bhering Barrios¹, Débora Gonçalves Gouveia¹ + +¹Universidade Federal de Viçosa + +### Abstract +Protocol used for the extraction of total RNA from plant material rich in secondary metabolites. + +### Materials +- **Trizol** - TRI Reagent (*Sigma Aldrich T9424*) +- **Chloroform** - CHCl3 GC-MS grade +- **PVPP** - Polyvinylpolypyrrolidone (*Sigma P6755*) +- **DEPC** - Diethylpyrocarbonate (*Sigma D5758*) +- **Isopropanol** - *Sigma Molecular Biology - 200 Proof ->99.45% - Sigma I9516* +- **Ethanol** - *Sigma Molecular Biology - 200 Proof ->99.45% - Sigma E7023* +- Sterile Milli-Q water treated with 0.1% DEPC; +- **Ethanol 75%** - Prepared with sterile DEPC 0.1% water +- Pipettes - P1000, P200, P100, P10 +- Tips - P1000, P200, P10 +- Microtubes of 1.5mL +- Thermomixer +- Refrigerated centrifuge +- Freezer -20°C +- Liquid nitrogen +- Styrofoam boxes that fit all the samples +- Ice +- Rack for 1.5 mL microtubes +- Exhaustion hood +- Nitrile gloves +- Vortex +- Bleach +- TAE or TBE 1X +- Ball mill + +### Safety Warnings +- **BOOK ALL EQUIPMENT IN ADVANCE AND MAKE SURE ALL THE MATERIAL WILL BE AVAILABLE.** +- **KEEP SAMPLES ON ICE DURING ALL HANDLING, AND STORE FINAL RNA AT -80°C.** + +### Before Start Instructions +**Important details:** +- Sterilize everything before and ensure that all material is suitable for RNA extraction (molecular grade). +- Aliquot the Trizol, Chloroform, and Ethanol 99% for Mol. Bio. in 50 mL Falcons, and leave them cold before starting the extraction (-20ºC). +- Leave all 1.5 mL tubes ready for appropriate tube changes (2 changes). +- Make a maximum of 12-16 tubes per shift (up to 24 with two people). +- Samples taken from the -80°C freezer should be kept in a bottle of liquid nitrogen. +- When adding the Trizol, take the samples out of the bottle and immediately add the Trizol to avoid enzymatic degradation of RNA. +- Add as soon as possible the 500 uL of Trizol. +- Before starting, read the protocol and ensure that the necessary volume of each reagent is available. +- Before starting, place a microtube rack that fits all your samples in the -20°C freezer. +- 75% ethanol should be prepared with **new** DEPC H₂O and Ethanol for Molecular Biology 200 Proof >99.45%. + +--- + +### Procedure + +#### 1. Collection and Freezing (30m) + +- **Step 1:** Collect the plant material and freeze it immediately in liquid nitrogen; +- **Step 2:** Macerate and transfer approximately 20 to 30 mg (max. 50 mg) to a 1.5 mL microtube; + +#### 2. RNA Extraction + +##### Initial Steps +- **Step 3:** Add 500 uL of TRIzol to every 3 tubes at once (adjust if working alone); +- **Step 3.1:** Add 30 mg of PVPP to each tube (2 full aliquot spoons); +- **Step 4:** Homogenize by vortexing briefly until the solution turns brown (5 seconds); +- **Step 5:** Incubate at room temperature for 5 minutes (dissociate ribonucleoproteins); +- **Step 6:** Add 100 uL of cold Chloroform (-20ºC); +- **Step 7:** Vortex vigorously for 15 seconds; +- **Step 8:** Incubate in the thermomixer at 23ºC (room T), 700 RPM for 15 min; +- **Step 9:** Centrifuge at 12,000 RPM for 15 min at 4°C to separate the two phases: organic and aqueous (RNA). + +##### Aqueous Phase Collection +- **Step 10:** Collect the aqueous phase (200 to 300uL) and transfer to new, labeled microtubes (1.5 mL) (avoid middle phase contamination). +- **Step 11:** Add 1 volume of chloroform (200 to 300 uL) - 250 uL in our case; +- **Step 12:** Invert the tubes 20x using a microtube rack, or gently vortex the tubes for 2 seconds; +- **Step 13:** Incubate in the thermomixer at 23ºC (room T), 700 RPM for 10 minutes; +- **Step 14:** Centrifuge at 13,000 RPM for 10 minutes at 4°C; +- **Step 15:** Collect 100 to 200 uL of the supernatant (we collected 175 uL for safety) and transfer to new, labeled microtubes (1.5 mL). + +##### Isopropanol and Ethanol Washes +- **Step 16:** Add 1 volume of ice-cold isopropanol (175 uL) - leave at -20°C until the time of application (use only isopropanol molecular biology grade - 200 proof >99.45%); +- **Step 17:** Invert the tubes for 30 seconds using a microtube rack or gently vortex the tubes for 2 seconds; +- **Step 18:** Incubate for 30 min at -80°C (add tubes to a new rack previously placed at -80°C or leave overnight at -20°C); +- **Step 19:** Centrifuge at 13,000 RPM for 15 minutes at 4°C; +- **Step 20:** Carefully discard the isopropanol with the aid of pipette tips; + +##### Ethanol Washes +- **Step 21:** Add 500 uL of 75% ethanol to wash the pellet and rehydrate the RNA (use molecular biology grade ethanol - 200 proof); + - Homogenize by vortexing briefly (<1 second), then centrifuge (13,000 RPM for 10 minutes at 4°C). +- **Step 22:** Centrifuge at 13,000 RPM for 10 minutes at 4°C; +- **Step 23:** Carefully dispose of the ethanol with the aid of a pipette tip; +- **Step 24:** Repeat the last 3 steps (21, 22, and 23). + +##### RNA Resuspension +- **Step 25:** Place the open tubes in the laminar flow cabinet with the glass closed at room temperature for 5 minutes. +- **Step 26:** Resuspend in 30 uL of autoclaved Milli-Q water treated with 0.1% DEPC at 60°C; + - Add water to all tubes immediately after leaving the cabin; + - Homogenize with pipette (25 uL) up & down (30 seconds/tube). + +### Post Extraction + +- **Step 27:** Treat with DNase - gDNA RNA CleanUp Protocol; + +#### Quality Check +- **Step 28:** Run 1.5% chlorine-agarose gel (3 uL sample + 2 uL loading buffer 6X) - 80 V - 80 min; + - Clean electrophoresis tank with distilled water and add fresh running buffer; + - Use 5% commercial bleach added to TAE 1X buffer. + +- **Step 29:** Quantify RNA purity and concentration in the Nanodrop or Qubit; + - Pure RNA has 260/280 ratio > 1.8 and 260/230 ratio > 1.6 + - For RNAseq, Microarray, use Bioanalyzer 2100 (RNA 6000 kits) or Tapestation 2200; use Plant RNA Algorithm. + +- **Step 30:** Store samples in the ultra-freezer (-80 ºC) for up to 6 months. + +*endofoutput* +``` \ No newline at end of file diff --git a/markdown-output/rna-imaging-with-merfish-probe-construction-meqc3dw.md b/markdown-output/rna-imaging-with-merfish-probe-construction-meqc3dw.md new file mode 100644 index 0000000000000000000000000000000000000000..e4edd484219bf69a5759a6ede2b5ddad60813da8 --- /dev/null +++ b/markdown-output/rna-imaging-with-merfish-probe-construction-meqc3dw.md @@ -0,0 +1,156 @@ +```markdown +# Goal/Experiment: +The goal of this experiment is to construct probes for RNA imaging using MERFISH (Multiplexed Error-Robust Fluorescence In Situ Hybridization). The protocol outlines a high-throughput approach that leverages array-derived synthesis of complex oligonucleotide pools and enzymatic amplification to produce encoding probes sufficient for RNA FISH experiments. + +# RNA Imaging with MERFISH - Probe Construction + +### Jeffrey R. Moffitt, Xiaowei Zhuang + +## Abstract + +Staining hundreds to thousands of RNA species each of which requires tens of unique encoding probes necessitates a vast number of unique oligonucleotide sequences. Traditional solid-phase oligonucleotide synthesis is often prohibitively expensive. To mitigate costs, we developed a protocol using array-derived synthesis and enzymatic amplification, providing a way to generate high quantities of encoding probes. + +**Citation:** Jeffrey R. Moffitt, Xiaowei Zhuang RNA Imaging with MERFISH - Probe Construction. protocols.io dx.doi.org/10.17504/protocols.io.meqc3dw +**Published:** 29 Mar 2018 + +## Guidelines + +The protocol involves four major steps: +1. **PCR Amplification of Oligopool:** Generate template molecules. +2. **In vitro Transcription:** Produce RNA from templates and amplify. +3. **Reverse Transcription:** Generate single-stranded DNA (ssDNA) from RNA. +4. **Alkaline Hydrolysis and Purification:** Remove RNA and purify ssDNA probes. + +## Equipment + +1. Tabletop centrifuge +2. qPCR machine or thermocycler +3. 37 °C incubator or water bath +4. 50 °C water bath +5. 95 °C water bath +6. Vacuum manifold (optional) +7. Gel electrophoresis equipment for polyacrylamide gels (optional) +8. Vacuum concentrator (optional) + +## Materials + +| Reagent | Vendor | Catalog Number | +| ------- | ------ | -------------- | +| 20X EvaGreen | Biotium | 31000 | +| 2X Phusion hot start polymerase master mix | New England Biolabs | M0536S | +| Tris-EDTA (TE) pH 8 buffer | Ambion | AM9849 | +| DNA binding buffer | Zymo Research | D4004-1-L | +| DNA wash buffer | Zymo Research | C1016-50 | +| Oligo binding buffer | Zymo Research | D4060-1-40 | +| 100-µg capacity silicon columns (Spin-V) | Zymo Research | D4003-2-48 | +| RNA binding buffer (Optional) | Zymo Research | R1013-2-100 | +| RNA prep buffer (Optional) | Zymo Research | R1060-2-100 | +| RNA wash buffer (Optional) | Zymo Research | R1003-3-24 | +| Quick HiScribe T7 polymerase kit | New England Biolabs | E2050S | +| RNasin plus | Promega | N2611 | +| Maxima H- reverse transcriptase | Thermo Scientific | EP0751 | +| 10 mM mix of dNTPs | New England Biolabs | N0447S | +| 0.5 M EDTA | Ambion | AM9261 | +| 1 N NaOH | Vwr | JT5635-2 | +| Nuclease-free water | Ambion | AM9932 | +| 100% Ethanol | Vwr | 89125-186 | +| D/RNaseFree | Vwr | 47751-044 | +| 1.5 mL LoBind tubes | Eppendorf | 022431021 | +| PCR tubes | Contributed by users | n/a | + +## Protocol + +### Amplification of in vitro Template + +**Step 1:** +Design the primers with the T7 promoter for in vitro transcription. Resuspend forward primer to 200 µM and reverse primer to 100 µM in TE. + +**Step 2:** +Prepare the PCR reaction mix: +- 40 µL 20X EvaGreen +- 2 µL 200 µM forward primer +- 4 µL 100 µM reverse primer +- 1 µL 80 ng/µL complex oligopool +- 353 µL nuclease-free water +- 400 µL 2X Phusion hot start polymerase master mix + +Aliquot 50 µL into 16 PCR tubes. + +**Step 3:** +Run the qPCR protocol: +1. 98 °C for 3 minutes +2. 98 °C for 10 s +3. 65 °C for 10 s +4. 72 °C for 15 s + +Repeat cycles 2-5 until amplification plateau is approached. + +**Step 4:** +Purify the template using column purification: +- Add 800 µL PCR reaction and 4 mL DNA binding buffer +- Pull mixture across a 100-µg column with vacuum manifold or centrifuge + +**Step 5:** +Wash the column with 300 µL DNA wash buffer, spin for 30 s, repeat once. + +**Step 6:** +Elute the template with 170 µL nuclease-free water, transfer to a fresh tube and spin. + +### In vitro Transcription + +**Step 9:** +Mix reagents in a fresh tube: +- 160 µL in vitro template +- 176 µL nuclease-free water +- 250 µL NTP buffer mix +- 25 µL RNasin Plus +- 25 µL T7 polymerase + +Incubate at 37 °C for 12-16 hours. Remove 20 µL for quality control. + +**Step 10:** *(optional)* +Quality control for in vitro transcription. Purify reaction: +- Mix 20 µL reaction, 30 µL nuclease-free water, 100 µL RNA binding buffer, 150 µL ethanol +- Pass across a 100-µg column +- Wash with 400 µL RNA prep buffer and 200 µL RNA wash buffer (twice) +- Elute RNA with 100 µL nuclease-free water + +### Reverse Transcription and Purification of Encoding Probes + +**Step 15:** +Reverse transcription: +- Mix 200 µL 10 mM dNTPs, 120 µL 200 µM forward primer, 240 µL 5X Maxima buffer, 24 µL RNasin Plus, 24 µL Maxima H- reverse transcriptase + +**Step 16:** +Incubate at 50 °C for 1 hour. + +**Step 17:** +Alkaline hydrolysis: +- Split reaction into two tubes: add 300 µL 0.5 M EDTA and 300 µL 1 N NaOH +- Incubate at 95 °C for 15 minutes + +**Step 18:** +Purification of ssDNA probe: +- Combine into 50 mL Falcon tube, add 4.8 mL oligo binding buffer, 19.2 mL ethanol +- Split across eight 100-µg columns + +**Step 19:** +Wash columns with 750 µL DNA wash buffer. + +**Step 20:** +Elute columns with 100 µL nuclease-free water, combine eluates, set aside 10 µL for quality control. + +**Step 21:** +Concentration of probe: +- Use a vacuum concentrator to dry samples. + +**Step 22:** +Resuspend dried pellet in 24 µL nuclease-free water or hybridization buffer. + +**Step 23:** +Store probe at -20 °C. + +**Step 24:** *(optional)* +Quality control on polyacrylamide gel to check for RNase contamination and efficiency of reverse transcription. + +**endofoutput** \ No newline at end of file diff --git a/markdown-output/roseobacter-screening-of-surface-waters-for-viruse-dbt2nm.md b/markdown-output/roseobacter-screening-of-surface-waters-for-viruse-dbt2nm.md new file mode 100644 index 0000000000000000000000000000000000000000..9a58cc1a61c24c2bb13abd3798ce6336b54431fb --- /dev/null +++ b/markdown-output/roseobacter-screening-of-surface-waters-for-viruse-dbt2nm.md @@ -0,0 +1,187 @@ +```markdown +## Goal/Experiment: +### Roseobacter Screening Of Surface Waters For Viruses + +#### Abstract +**Citation:** Matthew Sullivan Roseobacter Screening Of Surface Waters For Viruses. *protocols.io* +**Published:** 02 Feb 2016 +**DOI:** [10.17504/protocols.io.dbt2nm](dx.doi.org/10.17504/protocols.io.dbt2nm) + +--- + +### Guidelines + +**Notes:** + +- The initial goal is to screen several seawater samples on several roseobacter hosts to determine the breadth of roseophage present at each location and then to focus on a single site. +- Nomenclature for viruses should include a designation for the site of seawater collection followed by a number and the host it was isolated on. +- Put information into an Excel worksheet. Create a separate Excel file for each source of the virus. Use a different tab in each worksheet for different hosts. +- Turbid plaques indicate possible lysogenic phage that may be of interest to Feng Chen’s laboratory in Maryland. Keep the plaque plug at 4°C. Take 50-100 µl from a few plaques and grow up small 1 ml cultures to cryopreserve (glycerol). The goal is to get the putative lysogenic cells (prophage containing) as they confer resistance to colonies of cells within the plaque = turbid). + +--- + +### Protocol + +#### Growth + +**Step 1:** + +- Pull Roseobacter cultures from the freeze and grow in 5 ml broth cultures. + + - **Notes:** + - The VERVE Team reported on 09 Jul 2015 that Matt has 14 isolates from Wendy Ye in Mary Ann Moran’s lab in Georgia. Two of them are from open ocean (CCS1 and CCS2) and they are grown at 20-22°C in 1/10 YTSS. All others are from coastal waters and are grown at 30°C in 1/2 YTSS. + - For virus screening, grow them to log phase (OD600 = 0.6 - 0.8). Some will grow quickly (overnight) while others will take 1-4 days to reach the log phase. + +#### Screening + +**Step 2:** + +- Perform virus screening in sterile 96 well flat-bottom tissue culture treated microtiter plates. + + - **Notes:** + - Viruses will be screened from seawater collected from various sources. + +**Step 3:** + +- Store seawater filtrates at 4°C. + + - **Notes:** + - Ice crystal formation at -20°C is bad. + +#### Absorption + +**Step 4:** + +- Absorb the viruses to the bacteria. + +**Step 5:** + +- Place 15 µl of log phase cells and 15 µl of seawater filtrate (containing virus) into wells of a 96 well plate. + + - **Notes:** + - Uninfected controls should surround infected wells on each plate in a checkerboard pattern. + +**Step 6:** + +- Allow the viruses to absorb to the bacteria for 1 hour. + - **Duration:** 1 hour + +**Step 7:** + +- Add 200 µl of growth media (1/2 or 1/10 YTSS) to the wells. + +**Step 8:** + +- Seal the plate with parafilm and incubate with shaking. + +**Step 9:** + +- Look for infected wells that show lysis with respect to uninoculated controls. + +#### Enrichment + +**Step 10:** + +- Enrich for viruses by transferring from the initial 96 well plate to another 96-well plate containing fresh liquid media (“frogging” into new wells). + + - **Notes:** + - This does not require fresh log-phase host cells; the cells come with the transfer. Ideally, frog before cells go into the stationary phase. + - Enrichment for viruses may take several rounds of culturing because we're assuming initial concentrations of phages in the seawater are low. + +**Step 11:** + +- Once enriched sufficiently, filter out cells with 0.2 µm filtration into 1.5 ml microfuge tubes. + +**Step 12:** + +- Spin for 5 minutes at maximum speed to pellet cells. + - **Duration:** 5 minutes + +**Step 13:** + +- Transfer supernatant to a fresh tube for storage. + +**Step 14:** + +- Store at 4°C. + +#### Plaque Purification + +**Step 15:** + +- Plaque-purify wells containing virus by growing cells plus virus on solid media. + +**Step 16:** + +- Prepare solid agar media and 0.5% overlay agar. + +**Step 17:** + +- Absorb virus to cells using as a starting point, 25 µl virus from 96 well plate enrichments and 200 µl log-phase host bacteria for 1 hour. + - **Duration:** 1 hour + + - **Notes:** + - Try growing lawns first; it may need to be up to 1 ml cells. + +**Step 18:** + +- Mix the virus-absorbed cells in the 5 ml overlay agar. + +**Step 19:** + +- Pour over the solid media. + +**Step 20:** + +- Incubate and look for plaque formation. + - **Notes:** + - Several results are possible: + 1. No plaques (not likely if using virus-enriched cultures from 96 well plates). + 2. Too many plaques to purify them (in which case, a dilution of the virus is done, and the plaque assay is repeated). + 3. Well-resolved plaques. If well-resolved plaques, then cored from agar using Pasteur pipet and dispensed into 100 µl YTSS broth in 1.5 ml tubes. + +**Step 21:** + +- Pick representatives of all plaque types present and record the appearance of the plaques. + - **Notes:** + - Clear, well-lysed plaques are of the most interest. Turbid plaques may indicate lysogenic phage and should be picked; these will be sent to another laboratory for further study (see guidelines). + +**Step 22:** + +- Store the broth that contains broth and agar plugs at 4°C and allow the viruses to diffuse out of the agar into the broth. + +#### DNA Purification Determination + +**Step 23:** + +- Once the viruses are plaque-purified, they can be scaled-up in 5 ml broth cultures for DNA purification. + +**Step 24:** + +- Take 25 µl of the plaque-purified virus (agar plug in 100 µl broth) and absorb to 200 µl log-phase bacteria for 1 hour. + - **Duration:** 1 hour + +**Step 25:** + +- Add to 5 ml broth and grow with shaking. + +**Step 26:** + +- Centrifuge out cells and filter the culture through 0.2 µm filter. + +**Step 27:** + +- SYBR stain a portion of the filtrate to determine how much virus is present. + - **Notes:** + - If a lot of virus is present, enough DNA may be available for purification. + +**Step 28:** + +- Purify DNA using the Promega Wizard Lambda DNA kit. + - **Notes:** + - Goal is to obtain about 1 µg DNA for future work. If 5 ml does not yield that amount, more may need to be grown (e.g., 225 ml volume) and DNA purification repeated. + +--- + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/sars-cov-2-mcgill-artic-pcr-protocol-2-5-ul-rt-and-bjgkkjuw.md b/markdown-output/sars-cov-2-mcgill-artic-pcr-protocol-2-5-ul-rt-and-bjgkkjuw.md new file mode 100644 index 0000000000000000000000000000000000000000..989102fd4fa7f4c0bb51cc352a522eb3a564cd4a --- /dev/null +++ b/markdown-output/sars-cov-2-mcgill-artic-pcr-protocol-2-5-ul-rt-and-bjgkkjuw.md @@ -0,0 +1,124 @@ +```markdown +# Goal/Experiment: +This protocol details the procedure for performing SARS-CoV-2 McGill Artic PCR, using 2.5 µl RT and V3 only, in combination with the LA1 primer pool. + +# SARS-CoV-2 McGill Artic PCR Protocol, 2.5 µl RT and V3 only + LA1 + +## Authors: +- Sarah J Reiling^1 +- Josh Quick^2 +- Ioannis Ragoussis^1 + - ^1McGill University + - ^2University of Birmingham + +## Abstract: +V3 only primers for this protocol were designed using Primal Scheme and generate overlapping 400 nt amplicons. Primer names and dilutions are listed in the table below. +View the Primer Scheme [here](https://github.com/sarahreiling/artic-ncov2019/blob/master/primer_schemes/nCoV-2019/V3/nCoV-2019_V3only.scheme.bed) + +## References: +**DOI:** [10.17504/protocols.io.bjgkkjuw](https://dx.doi.org/10.17504/protocols.io.bjgkkjuw) + +**Protocol Citation:** +Sarah J Reiling, Josh Quick, Ioannis Ragoussis 2020. SARS-CoV-2 McGill Artic PCR Protocol, 2.5 µl RT and V3 only + LA1. protocols.io [https://dx.doi.org/10.17504/protocols.io.bjgkkjuw](https://dx.doi.org/10.17504/protocols.io.bjgkkjuw) + +## License: +This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. + +## Creation and Update Info: +- **Created:** Aug 07, 2020 +- **Last Modified:** Oct 02, 2020 +- **Protocol Integer ID:** 40172 + +--- + +## Primer Pool Preparation + +1. **Resuspension of Lyophilised Primers:** + If required, resuspend lyophilised primers at a concentration of 100 µM each. Primers should be diluted and pooled in the mastermix cabinet, which should be cleaned with decontamination wipes and UV sterilized before and after use. + + View the Primer Scheme [here](https://github.com/sarahreiling/artic-ncov2019/blob/master/primer_schemes/nCoV-2019/V3/nCoV-2019_V3only.scheme.bed) + +2. **Preparation of Primer Pool Stocks:** + - Generate primer pool stocks by adding 5 µl of each primer pair to a 1.5 mL Eppendorf tube labelled either "Pool 1 (100 µM)" or "Pool 2 (100 µM)". Total volume should be 490 µl for both Pool 1 and Pool 2. These are 100 µM stocks of each primer pool. + - Create another primer pool named "Pool LA1 (100 µM)" containing 5 µl of primer pairs 5, 17, 23, 26, 66, 70, 74, 91, 97, and 10 µl of primer pair 64. + +3. **Dilution of Primer Pools:** + - Dilute primer pool 1:10 in molecular grade water to generate 10 µM primer stocks. + - For the LA1 primer pool, dilute to 1 µM primer stock. + +## Multiplex PCR + +4. **Performing Multiplex PCR:** + - In the extraction and sample addition cabinet, add 2.5 µl RT product to each tube and mix well by pipetting. + - The extraction and sample addition cabinet should be cleaned with decontamination wipes and UV sterilized before and after use. + +5. **PCR Reaction Setup:** + - Set up the multiplex PCR reactions in 0.2 mL 8-strip PCR tubes with the following components: + + | Component | Pool 1 [10 µM primer] | Pool 2 [10 µM] | Pool LA1 [1 µM] | + |-----------------------------------|-----------------------|----------------|-----------------| + | Q5 Hot Start High-Fidelity 2X Master Mix | 12.5 µl | 12.5 µl | 12.5 µl | + | Primer Pool 1 or 2 | 3.7 µl | 3.7 µl | 3 µl | + | Nuclease-free water | 6.3 µl | 6.3 µl | 6.3 µl | + | **Total** | **22.5 µl** | **22.5 µl** | **22.5 µl** | + + - Add 2.5 µl RT product as mentioned. + +6. **Centrifugation:** + - Pulse centrifuge the tubes to collect the contents at the bottom of the tube. + +7. **Thermal Cycling:** + - Set the following program on the thermal cycler: + + | Step | Temperature | Time | Cycles | + |-----------------|-------------|-------------|--------| + | Heat Activation | 98°C | 00:00:30 | 1 | + | Denaturation | 98°C | 00:00:15 | 36 | + | Annealing | 65°C | 00:05:00 | 36 | + | Hold | 4°C | Indefinite | 1 | + + - Note: Cycle number should be 25 for Ct 18-21 up to a maximum of 36 cycles for Ct 36. + +## PCR Cleanup + +8. **Pooling PCR Reactions:** + - Combine the entire contents of "Pool 1" and "Pool 2" PCR reactions for each biological sample into a single 1.5 mL Eppendorf tube. Keep Pool LA1 separate from the combined Pool 1+2 until after the cleanup. + +9. **Clean-Up Protocol:** + - Add an equal volume (1:1) of SPRI beads to the sample tube and mix gently by flicking or pipetting. + - Incubate for 5 min at room temperature. + - Pellet on magnet for 5 min. Remove supernatant. + - Add 200 µl of 80% ethanol to the pellet and wash twice. + - Elute in 30 µl elution buffer. + +## Amplicon Quantification and Normalization + +10. **Quantification:** + - Quantify the amplicon pools using a fluorimetric dsDNA assay. + + **Expected Concentrations:** + + - **Pool 1+2 combined:** + - 100-150 ng/µl for Ct 14-24 + - 30-80 ng/µl for Ct 25-29 + - 10-30 ng/µl for Ct 30-36 + + - **Pool LA1:** + - 1-10 ng/µl for all Ct + +11. **Normalization:** + - After quantification of Pool 1+2 and Pool LA1, mix them together in the following ratio: 89.8% Pool 1+2 and 10.2% Pool LA1. For this, take a new plate and add 135 ng of Pool 1+2 and 15.3 ng of Pool LA1, and add nuclease-free water to a total volume of 30 µl (150 ng or 5 ng/µl). + +--- + +## Definitions and Explanations of Terms and Reagents + +1. **Primal Scheme:** A tool for creating primer schemes for virology. +2. **Eppendorf Tube:** Commonly used laboratory container for storing samples. +3. **Mastermix Cabinet:** A specialized clean area for preparing PCR reactions, often equipped with decontamination tools. +4. **SPRI Beads:** Solid phase reversible immobilization beads used for DNA/RNA extraction and purification. +5. **Thermal Cycler:** A machine used to amplify DNA segments via the polymerase chain reaction (PCR). +6. **Fluorimetric dsDNA Assay:** Fluorescence-based method for quantifying double-stranded DNA. + +endofoutput +``` diff --git a/markdown-output/sars-cov-2-mcgill-nanopore-sequencing-protocol-sup-bjajkicn.md b/markdown-output/sars-cov-2-mcgill-nanopore-sequencing-protocol-sup-bjajkicn.md new file mode 100644 index 0000000000000000000000000000000000000000..005bcead5ef08d671ab1edb9e010fa83fcaeb036 --- /dev/null +++ b/markdown-output/sars-cov-2-mcgill-nanopore-sequencing-protocol-sup-bjajkicn.md @@ -0,0 +1,260 @@ +```markdown +# Goal/Experiment: +The goal of this experiment is to sequence SARS-CoV-2 using the McGill Nanopore sequencing protocol SuperScript IV_42C_ArticV3. This protocol involves cDNA preparation, primer pool preparation, multiplex PCR, and sequencing using a MinION device. + +# SARS-CoV-2 McGill Nanopore Sequencing Protocol SuperScript IV_42C_ArticV3 + +## Authors +- Sarah J Reiling¹ +- Shu-Huang Chen¹ +- Anne-Marie Roy¹ +- Josh Quick² +- Ioannis Ragoussis¹ + +¹McGill University, ²University of Birmingham + +*Published: Sep 18, 2020* + +## DOI +[10.17504/protocols.io.bjajkcn](https://dx.doi.org/10.17504/protocols.io.bjajkcn) + +## License +This protocol is published under the terms of the [Creative Commons Attribution License](https://creativecommons.org/licenses/by/4.0/), permitting unrestricted use, distribution, and reproduction. + +## cDNA Preparation + +### Step 1 +Mix the following components in a 0.2 mL 8-strip tube: + +| Component | Volume | +|--------------------------------|----------| +| 50 µM random hexamers | 1 µL | +| 10 mM dNTPs mix (10 mM each) | 1 µL | +| Template RNA | 11 µL | +| **Total** | **13 µL**| + +**Notes:** +- Viral RNA input from a clinical sample should be between Ct 18-35. +- If Ct is between 12-15, dilute the sample 100-fold in water. +- If between 15-18, dilutions should be 10-fold to reduce PCR inhibition. + +### Step 2 +Gently mix by pipetting and pulse spin the tube to collect liquid at the bottom. + +### Step 3 +Incubate the reaction: +- **Temperature:** 65°C +- **Time:** 5 minutes +- **After:** Place on ice for 1 minute or on hold. + +### Step 4 +Add the following to the annealed template RNA: + +| Component | Volume | +|------------------------------------------------|----------| +| 5X SSIV buffer | 4 µL | +| 100 mM DTT | 1 µL | +| RNaseOUT Recombinant RNase Inhibitor (40 U/µL) | 1 µL | +| SuperScript IV Reverse Transcriptase (200 U/µL)| 1 µL | +| **Total** | 7 µL (20 µL total with the 13 µL from step 1) | + +**Notes:** +- Master mix should be prepared in the master mix cabinet and added to the denatured RNA. + +### Step 5 +Gently mix by pipetting and pulse spin the tube. + +### Step 6 +Incubate the reaction: +- **Temperature:** 42°C +- **Time:** 50 minutes +- **Then:** 70°C for 10 minutes +- **Hold:** At 4°C for 5 minutes. + +## Primer Pool Preparation + +### Step 7 +Resuspend lyophilized primers at a concentration of 100 µM each if needed. + +### Step 8 +Generate primer pool stocks by adding 5 µL of each primer pair to a 1.5 mL Eppendorf tube labelled "Pool 1 (100 µM)" or "Pool 2 (100 µM)". The total volume should be 490 µL for each pool. + +#### Additional Pool: +*Prepare "Pool LA1 (100 µM)" containing: 5 µL of primer pairs 5, 17, 23, 26, 66, 70, 74, 91, 97, and 10 µL of primer pair 64.* + +**Notes:** +- Prepare pools in the master mix cabinet. +- Dilute Primer pool 1:10 with molecular grade water to generate 10 µM primer stocks. +- LA1 primer pool should be diluted to 1 µM primer stock. + +### Step 9 +Use primers at a final concentration of 0.015 µM per primer. + +## Multiplex PCR + +### Step 10 +In the extraction and sample addition cabinet, add 5 µL RT product to each tube and mix well by pipetting. + +### Step 11 +Set up the multiplex PCR reactions in 1.5 mL low-bind DNA tubes: + +| Component | Pool 1 (10 µM) | Pool 2 (10 µM) | Pool LA1 (1 µM) | +|------------------------------------------|----------------|----------------|-----------------| +| Q5 Hot Start High-Fidelity 2X Master Mix | 12.5 µL | 12.5 µL | 12.5 µL | +| Primer Pool 1, Pool 2, or Pool LA1 | 3.7 µL | 3.7 µL | 3.7 µL | +| Nuclease-free water | 3.8 µL | 3.8 µL | 3.8 µL | +| **Total** | **20 µL** | **20 µL** | **20 µL** | + +Add 5 µL RT product to each primer pool as mentioned in step 7. + +### Step 12 +Pulse centrifuge the tubes to collect the contents at the bottom. + +### Step 13 +Set up the following program on the thermal cycler: + +| Step | Temperature | Time | Cycles | +|---------------|-------------|-------------|---------------| +| Heat Activation| 98°C | 30 seconds | 1 | +| Denaturation | 98°C | 15 seconds | 36 | +| Annealing | 65°C | 5 minutes | 36 | +| Hold | 4°C | Indefinite | 1 | + +*Cycle numbers should be reduced to 25 for Ct 18-21, up to a maximum of 36 cycles for Ct 35.* + +## PCR Clean-up + +### Step 14 +Combine entire contents of "Pool 1" and "Pool 2" PCR reactions into a 1.5 mL Eppendorf tube. **Keep Pool LA1 separate until clean-up.** + +### Step 15 +Clean-up amplicons: + +- Add equal volume (1:1) of SPRI beads and mix gently. +- Incubate 5 minutes at room temperature. +- Pellet on magnet for 5 minutes. Remove supernatant. +- Add 200 µL of 80% ethanol, wash twice. +- Elute in 30 µL elution buffer. + +*Clean-up amplicons on the post-PCR bench.* + +### Step 16 +Quantify amplicon pools using a fluorimetric dsDNA assay. + +### Step 17 +- After quantification, mix Pool 1+2 and Pool LA1 in a specific ratio to obtain: + - Pool 1+2: 100-150 ng/µL (Ct 14-24), 30-80 ng/µL (Ct 25-29), 10-30 ng/µL (Ct 30-36). + - Pool LA1: 1-10 ng/µL for all Ct values. +- Label 1.5 mL Eppendorf tubes for each sample. + +## Native Barcoding + +### Step 19 +Set up the following reaction for each sample: +- DNA amplicons: 5 µL +- Nuclease-free water: 7.5 µL +- Ultra II End Prep Reaction Buffer: 1.75 µL +- Ultra II End Prep Enzyme Mix: 0.75 µL +- **Total:** 15 µL + +### Step 20 +Incubate at: +- Room temperature for 10 minutes. +- 65°C for 5 minutes. +- Ice for 1 minute. + +### Step 21 +Add: +- NBXX barcode: 2.5 µL +- Ultra II Ligation Master Mix: 17.5 µL +- Ligation Enhancer: 0.75 µL +- **Total:** 20.75 µL (35.75 µL total with the previous reaction). + +### Step 22 +Incubate at: +- Room temperature for 15 minutes. +- 70°C for 10 minutes. +- Ice for 1 minute. + +### Step 23 +Pool all samples. + +### Step 24 +Clean-up the native barcodes twice using the protocol: + +1. Add 0.8X SPRI beads, mix gently. +2. Incubate 5 minutes at room temperature. +3. Pellet on magnet, remove supernatant. +4. Add 200 µL 80% ethanol, wash twice. +5. Elute in 100 µL elution buffer. + +*Repeat wash:* Add 0.8X SPRI beads, mix gently, incubate 5 minutes, pellet on magnet, remove supernatant, add 200 µL 80% ethanol, wash twice, elute in 30 µL elution buffer. + +### Step 26 +Set up adapter ligation reaction: + +- Barcoded amplicon pools: 30 µL +- NEBNext Quick Ligation Reaction Buffer (5X): 10 µL +- AMII adapter mix: 5 µL +- Quick T4 DNA Ligase: 5 µL +- **Total:** 50 µL + +### Step 27 +Incubate at room temperature for 15 minutes. + +### Step 28 +Clean-up native barcodes with following protocol: + +1. Add equal volume (1:1) SPRI beads, mix gently. +2. Incubate 5 minutes at room temperature. +3. Pellet on magnet, remove supernatant. +4. Add 200 µL SFB, resuspend beads by pipette mixing, repeat wash step. +5. Elute in 15 µL EB provided in ONT kit. +6. Incubate room temperature for 2 minutes. +7. Place on magnetic rack. +8. Transfer final library to new 1.5 mL Eppendorf tube. + +### Step 29 +Quantify the final library using a fluorimetric dsDNA assay. + +### Step 30 +Prime flowcell and load library onto flowcell. + +1. Thaw reagents SQB, LB, and FLB. +2. Add 30 µL FLT to FLB tube, mix well. +3. Prepare a new MinION flowcell. +4. Rotate inlet port cover clockwise by 90°. +5. Take P1000 pipette, set volume to 800 µL, remove any air from flowcell. +6. Load 800 µL of FLB (plus FLT). +7. Wait for 5 minutes. +8. Gently lift SpotON cover. +9. Load 200 µL of FLB (plus FLT) via inlet port. + +In a new tube, prepare the library dilution: + - SQB: 37.5 µL + - LB: 25.5 µL + - Final Library: 12 µL + +11. Mix library gently, add 75 µL library dilution to flowcell via SpotON. +12. Replace SpotON port cover, close MinION lid. + +## Sequencing Run (using MinKNOW) + +### Step 31 +1. Plug MinION to computer. +2. Choose flow cell 'FLO-MIN106'. +3. Select flowcell, tick appears. +4. Click 'New Experiment' on screen. +5. In experiment popup screen, select: + - **Experiment:** Name the run. + - **Kit Selection:** Select LSK109. + - **Run Options:** Set run length to 6 hours. + - **Basecalling:** Select ‘fast basecalling’. + - **Output:** Default is 4000, reduce for more frequent updates. + +6. Click 'Start run'. + +**Monitor the progress using MinKNOW interface.** + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/sars-cov-2-mcgill-nextera-flex-sequencing-protocol-by6xpzfn.md b/markdown-output/sars-cov-2-mcgill-nextera-flex-sequencing-protocol-by6xpzfn.md new file mode 100644 index 0000000000000000000000000000000000000000..1fa19b6b3b9b89a14b6de2f11858b6edaacd4663 --- /dev/null +++ b/markdown-output/sars-cov-2-mcgill-nextera-flex-sequencing-protocol-by6xpzfn.md @@ -0,0 +1,192 @@ +```markdown +# Goal/Experiment: +The goal of this experiment is to sequence the SARS-CoV-2 virus using McGill Nextera Flex sequencing protocol_Lunascript_ARTIC.V3_5uLRT. This protocol entails several steps including cDNA preparation, primer pool preparation, multiplex PCR, amplicon quantification, and library preparation. + +## SARS-CoV-2 McGill Nextera Flex Sequencing Protocol_Lunascript_ARTIC.V3_5uLRT + +### Authors +- Sarah J Reiling +- Marie-Michelle Simon +- Anne-Marie Roy +- Shu-Huang Chen +- Ioannis Ragoussis + +Institution: McGill University, McGill Genome Centre + +DOI: [10.17504/protocols.io.by6xpzfn](https://dx.doi.org/10.17504/protocols.io.by6xpzfn) + +--- + +## How the Nextera DNA Flex Assay Works + +The Nextera DNA Flex library prep kit uses a bead-based transposome complex to tagment genomic DNA. This process fragments the DNA and tags it with adapter sequences in a single step. After the DNA is saturated, a limited-cycle PCR adds Nextera-specific index adapter sequences to the ends of a DNA fragment. This step enables capability across all Illumina sequencing platforms. The double-stranded DNA library is denatured before hybridization with biotin probe oligonucleotide pool. + +### PCR Amplicons for Nextera Flex + +For PCR amplicons > 150 bp, the protocol depletes libraries < 500 bp. Illumina recommends that amplicons < 500 bp undergo a 1.8x sample purification bead volume ratio to supernatant during the cleanup. + +More information: [Nextera DNA Flex Library Prep Reference Guide](https://emea.support.illumina.com/content/dam/illumina-support/documents/documentation/chemistry_documentation/samplepreps_nextera/nextera_dna_flex/nextera-dna-flex-library-prep-reference-guide-1000000025416-07.pdf) + +--- + +## Protocol Steps and Reagents + +### 1. cDNA Preparation + +#### Components and Volumes + +| Component | Volume | +|--------------------------|--------| +| LunaScript RT SuperMix (5x) | 4 µL | +| Nuclease-free water | 5 µL | +| Template RNA | 11 µL | +| **Total** | **20 µL** | + +**Special Notes:** +- Viral RNA input from a clinical sample should be between Ct 18-35. +- Dilute samples to reduce PCR-inhibition based on Ct values. + +**Procedure:** +1. Mix the components in a 0.2 mL 8-strip tube or a 96-well plate. +2. Gently mix by pipetting and pulse spin. +3. Incubate: + - 25°C for 2 minutes + - 55°C for 20 minutes + - 95°C for 1 minute +4. Place on ice or store at -20°C. + +--- + +### 4. Primer Pool Preparation + +#### Resuspend Lyophilized Primers + +If required, resuspend at a concentration of 100 µM each. + +#### Generate Primer Pool Stocks + +1. Add 5 µL of each primer pair to 1.5 mL Eppendorf tubes. + - 490 µL for Pool 1 (100 µM) + - 490 µL for Pool 2 (100 µM) + +**Dilution:** +- Dilute primer pool 1:10 (100 µM to 10 µM) using molecular grade water. +- Use at final concentration of 0.015 µM per primer in PCR reactions. +- For 98 primers in both pools, add 3.65 µL primer pools (0.015 µM) per 25 µL reaction. + +--- + +### 8. Multiplex PCR + +#### Components and Volumes + +| Component | Pool 1/2 Volume | +|---------------------------------|---------------| +| Q5 Hot Start High-Fidelity 2X Master Mix | 12.5 µL | +| Primer Pool 1 or 2 | 3.7 µL | +| Nuclease-free water | 3.8 µL | +| **Total** | **20 µL** | +| Add RT product | 5 µL | + +**Procedure:** +1. Mix PCR reactions in 0.2 mL 8-strip PCR tubes. +2. Pulse centrifuge. +3. Thermal cycler program: + - 98°C for 30 seconds (1 cycle) + - 98°C for 15 seconds, 63°C for 5 minutes (36 cycles) + - 4°C hold. + +--- + +### 12. PCR Cleanup + +#### Protocol for Amplicon Cleanup Using SPRI Beads + +1. Combine contents of "Pool 1" and "Pool 2" into a 1.5 mL Eppendorf tube. +2. Add 0.8x volume of SPRI beads. +3. Incubate at room temperature for 5 minutes. +4. Magnet for 5 minutes, remove supernatant. +5. Wash twice with 200 µL of 80% ethanol. +6. Elute in 30 µL elution buffer. + +--- + +### 14. Amplicon Quantification and Normalization + +Quantify using a fluorimetric dsDNA assay. Expected concentrations: +- Pool 1+2 combined: + - 100-150 ng/µL for Ct 14-24 + - 30-80 ng/µL for Ct 25-29 + - 10-30 ng/µL for Ct 30-36 + +--- + +## Library Preparation Steps + +### Tagmentation of Genomic DNA + +#### Components + +- Bead-Linked Transposomes (BLT) +- Tagmentation Buffer 1 (TB1) +- Nuclease-free water +- Microseal 'B' adhesive seal +- 1.7 mL microcentrifuge tubes +- Pipette tips + +**Procedure:** +1. Prepare BLT and TB1 (storage instructions included). +2. Add 20-30 µL DNA per well (100-500 ng total). +3. Add TB1 to DNA samples to bring total volume to 30 µL. +4. Vortex BLT. +5. Prepare tagmentation master mix (BLT and TB1). +6. Distribute mix equally in an 8-tube strip. +7. Add 20 µL master mix to each sample, mix, and run thermal cycler. + +### Post Tagmentation Cleanup + +**Procedure:** +1. Add 10 µL TSB. +2. Slowly pipette to resuspend beads. +3. Seal plate and run thermal cycler. +4. Clear supernatant using magnetic stand. +5. Wash beads with TWB. + +### Amplify Tagmented DNA + +**Components:** +- Enhanced PCR Mix (EPM) +- Index adapters (individual tubes or plates) + +**Procedure:** +1. Combined EPM and water to prepare PCR master mix. +2. Add index adapters and run thermal cycler. +3. Final clean up using standard double-sided bead purification. + +### Pool Libraries + +**Procedure:** +1. For DNA inputs > 100 ng: + - Pool 5 µL of each library (up to 384 libraries). + - Quantify library pool (Qubit or PicoGreen). +2. For DNA inputs < 100 ng: + - Quantify each library separately. + +### Check Library Quality (Optional) + +**Instruments:** +- Advanced Analytical Fragment Analyzer (HS-NGS High Sensitivity 474 kit). +- Agilent 2100 Bioanalyzer (High Sensitivity DNA kit). + +### Dilute Libraries to Starting Concentration + +Dilute libraries to recommended starting concentrations for sequencing system. + +### Sequencing System Guide + +Refer to the documentation specific to the sequencing platform for further instructions on denaturation and dilution protocols. + +--- + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/schistocephalus-solidus-culturing-ew8bfhw.md b/markdown-output/schistocephalus-solidus-culturing-ew8bfhw.md new file mode 100644 index 0000000000000000000000000000000000000000..6a986d23ee6578506c001080a66fbe7ffb8abb21 --- /dev/null +++ b/markdown-output/schistocephalus-solidus-culturing-ew8bfhw.md @@ -0,0 +1,82 @@ +```markdown +# Goal/Experiment: +To culture the parasitic flatworms _Schistocephalus solidus_ in a laboratory setting. + +# Schistocephalus solidus Culturing + +**Authors:** Hebert F.O., Grambauer S., Barber I., Landry C.R., Aubin-Horth N. + +**Publication Date:** 30 May 2016 + +**Published in:** [protocols.io](https://www.protocols.io/view/Schistocephalus-solidus-culturing-ew8bfhw) + +**Abstract:** +This protocol describes how the parasitic flatworms (_S. solidus_) were cultured in the lab. + +**Citation:** +Hebert F.O., Grambauer S., Barber I., Landry C.R., Aubin-Horth N. Schistocephalus solidus culturing. protocols.io. Published: 30 May 2016. + +## Guidelines + +### Equipment required: +- **Culture Tube:** Tube culture screw cap borosilicate glass phenolic cap with PTFE liner use up to 121°C 26mm x 200mm Pyrex from Fisher, product code 12349339 +- **Dialysis Membrane:** 30m SIZE 1 DIALYSIS TUBING 12-14000 DALT from Medicell, product code DVT.12000.01 +- **RPMI Media:** RPMI-1640 Medium, with L-glutamine, without sodium bicarbonate, powder from Sigma, product code R6504-10X1L +- **Horse Serum:** Horse Serum Heat inactivated sterile-filtered from Sigma, product code H1138-6X500ML +- **Penicillin:** HyClone; Pen/Strep/Glutamine from Fisher, product code 12340243 + +### Before culturing worms: +- Culture tubes need to be set up with membrane and autoclaved and RPMI media made up and autoclaved. +- Cut a piece of dialysis membrane twice the length of the tube. Make into a U shape to fit inside the tube (do not fold the membrane as the crease can affect the setup) leaving 1-2 cm folded over the top on each side and screw the lid on. Wrap in foil and autoclave. +- Fresh RPMI media should be prepared each time you culture worms. Weigh out the appropriate quantity of powder (10.4g/L) and make up to the required volume in a 400ml Duran bottle with ddH2O and autoclave. + +### Before Start +Before culturing worms, culture tubes need to be set up with membrane and autoclaved and RPMI media made up and autoclaved. + +1. Cut a piece of dialysis membrane twice the length of the tube. Make into a U shape to fit inside the tube (do not fold the membrane as the crease can affect the setup) leaving 1-2 cm folded over the top on each side and screw the lid on. Wrap in foil and autoclave. +2. Fresh RPMI media should be prepared each time you culture worms. Weigh out the appropriate quantity of powder (10.4g/L) and make up to the required volume in a 400ml Duran bottle with ddH2O and autoclave. + +## Protocol + +### Parasite Collection +#### Step 1: +For each culture tube fill halfway up with horse serum and then fill up with RPMI—leaving about 2 cm gap from the top. Add 500µl of penicillin. + +**NOTES:** +- Horse serum and Penicillin are kept in -20°C and need to be defrosted. Use the 40°C water bath to defrost/warm the horse serum and autoclaved RPMI, the autoclaved culture tubes can warm in the 37°C incubator in the lab. Leave the aliquots of penicillin at RT°C. +- If dissecting several fish at once keep the worms on a petri dish with a few drops of RPMI on them so they don’t dry out before setting up the culture tube. Worms over 50mg should be used for the culture—any smaller and it is unlikely to produce any eggs. +- On the day of fish dissection/worm culturing switch on the shaking water bath, this should be set at 40°C and 120rpm. + +#### Step 2: +With a pair of blunt forceps gently open the top of dialysis membrane and place the worm into the membrane. + +**NOTES:** +- This can be tricky as you do not want to open up the membrane too much as the pressure is required for the worm to develop. If you hold the worm between your finger and thumb at both ends and gently squeeze, you can feel the way the worm is moving so which end to put into the membrane first. Try to squeeze the worm down toward the bottom of the membrane—a bit like squeezing a toothpaste tube! + +#### Step 3: +Screw the top on the tube—and loosen slightly and place into the shaking 40°C water bath. + +#### Step 4: +Check the worms each day—the worms may start to shed eggs after day 5 so can be collected after day 6—if not collect eggs on day 7—if the worms are left longer they may dissolve and the eggs will be difficult to collect cleanly. + +### Egg Collection +#### Step 5: +Remove the dialysis membrane from the culture tube and cut it open above any visible eggs, flush out onto a petri dish using ddH2O. Fill the petri dish to about ¼ full of ddH2O. + +#### Step 6: +Under the stereo microscope with cold light source tilt the plate, and the eggs should line up on the surface. + +#### Step 7: +Collect the egg layer with a glass pipette and transfer to fresh petri dishes—up to 4 depending on the number of eggs. + +#### Step 8: +Fill all the plates up to about half full with ddH2O including the original petri dish. Label each plate with an identification number/name. + +#### Step 9: +Wrap each plate individually in foil and label the outside with the same identification plus your initials. + +#### Step 10: +Place the wrapped plates in the 20°C incubator and leave them there for at least 21 days before checking for hatching. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/schistosoma-mansoni-cercariae-transformation-with-cr7nv9me.md b/markdown-output/schistosoma-mansoni-cercariae-transformation-with-cr7nv9me.md new file mode 100644 index 0000000000000000000000000000000000000000..9a1986646be8cc79c9bd1a23f2f15927941ce5fd --- /dev/null +++ b/markdown-output/schistosoma-mansoni-cercariae-transformation-with-cr7nv9me.md @@ -0,0 +1,219 @@ +```markdown +# Goal/Experiment: +Transformation of Schistosoma mansoni cercariae to schistosomula using a needle-based technique. + +# Schistosoma mansoni cercariae transformation (with needle) +**Author:** Sarah K Buddenborg +**Affiliation:** Wellcome Sanger Institute + +## Abstract +Free-living aquatic S. mansoni cercariae transform into the first intramammalian stage, called schistosomula or somules, by burrowing in the host skin. Upon contact, cercariae begin to enter the skin and lose their tails, becoming schistosomula. Somules migrate through the epidermis to the dermis to find a small venule or lymphatic vessel to enter the vasculature. + +Transformation of cercariae to schistosomula can be mimicked in the laboratory by triturating cercariae to remove tails, eliminating tails in a percoll gradient, and then culturing the somules in somule media. This method is used when the number of cercariae is high as the percoll gradient results in loss of cercariae. Somules can be cultured for several weeks with regular media changes. + +## Guidelines +Media changes and opening of transformed somules to take place in tissue culture hood using sterile techniques. + +## Materials +- **DMEM high glucose GlutaMAX** - *Gibco - Thermo Fischer* Catalog #31966021 +- **Lactalbumin Hydrolysate, powder (extra soluble)** - *Thermo Fisher* Catalog #11800042 +- **Hypoxanthine** - *Merck MilliporeSigma (Sigma-Aldrich)* Catalog #H9636-1G +- **Serotonin Hydrochloride** - *Merck MilliporeSigma (Sigma-Aldrich)* Catalog #H9523-25MG +- **Insulin solution from bovine pancreas** - *Merck MilliporeSigma (Sigma-Aldrich)* Catalog #I0516-5ML +- **3,3′5-Triiodo-L-thyronine sodium salt** - *Merck MilliporeSigma (Sigma-Aldrich)* Catalog #T6397 +- **MEM Vitamin Solution (100x)** - *Merck MilliporeSigma (Sigma-Aldrich)* Catalog #M6895 +- **Schneiders Insect Medium** - *Merck MilliporeSigma (Sigma-Aldrich)* Catalog #S0146 +- **HEPES** - *Merck MilliporeSigma (Sigma-Aldrich)* Catalog #H0887 +- **Fetal Bovine Serum** - *Merck MilliporeSigma (Sigma-Aldrich)* Catalog #F4135 +- **Antibiotic-Antimycotic (100x)** - *Thermo Fisher Scientific* Catalog #15240062 +- **Dulbecco's Phosphate Buffered Saline 10x** - *Merck MilliporeSigma (Sigma-Aldrich)* Catalog #D1283 +- **MilliQ water** - *Contributed by users* +- **Sterile Graduated Transfer Pipets** - *Fisher Scientific* Catalog #13479108 +- **Falcon 15 mL Conical Centrifuge Tubes** - *Fisher Scientific* Catalog #1077350 +- **Falcon 50mL Conical Centrifuge Tubes** - *Fisher Scientific* Catalog #14-959-49A +- **Nun Non-Treated 6-well plate** - *Thermo Scientific* Catalog #10396482 +- **1000 mL Vacuum Filter/Storage Bottle System 0.22 µm Pore 54.5cm² PES Membrane Sterile 12** - *Corning* Catalog #431098 +- **Percoll** - *Merck MilliporeSigma (Sigma-Aldrich)* Catalog #P1644-500ML +- **Micro-Emulsifying Needles 22g x 2-7/8 With Bar** - *Contributed by users* Catalog #7975 +- **Laboratory pipetting needle with 90° blunt ends 22G 1 1/2 in.** - *Merck MilliporeSigma (Sigma-Aldrich)* Catalog #CAD7931-12EA +- **BD PlastiPak Syringe with Luer Lock** - *Fisher Scientific* Catalog #15544835 +- **5ml syringe luer lock** - *greiner bio-one* Catalog #SYR5LL +- **20ml syringe luer lock** - *greiner bio-one* Catalog #SYR20LL +- **Corning Cell Culture Treated Flasks with Vent Cap** - *Fisher Scientific* Catalog #10288990 +- **3ml Sterile Graduated Transfer Pipets individually wrapped** - *Fisher Scientific* Catalog #13469108 +- **Lamp or other light source** +- **Chilling benchtop centrifuge with 15ml swing bucket rotor** +- **Incubator at 37°C and 5% CO2** +- **Reciprocating water bath** +- **Class 2 Microbiological Safety Cabinet** + +## Reagents Preparation + +### 1X DPBS + 2% Anti-Anti +1. **Ingredients:** + - 50mL 10x DPBS + - 10mL 100x Antibiotic-Antimycotic (-20°C) + - Fill to 500mL in vacuum filter unit with MilliQ water +2. **Instructions:** + - Sterilize media with vacuum filter unit and store at 4°C. Use within 2 weeks. + +### Somule Medium – Basch Modified Medium (BMM) +1. **Mix the following reagents:** + +| Reagent | 1L | 500mL | 250mL | Working | Storage | +| ----------------------------- | ------------- | ------------ | --------- | ------- | -------- | +| 1x DMEM high glucose | 810.5mL | 405.25mL | 202.625mL | 1x | 4°C | +| 1g/L Lactalbumin hydrolysate | 1g | 0.5g | 0.25g | 1g/L | 4°C | +| 1mM Hypoxanthine | 500µL | 250µL | 125µL | 0.5µM | -20°C | +| 1mM Serotonin | 1mL | 500µL | 250µL | 1µM | -20°C | +| Insulin | 1mL | 500µL | 250µL | 8µg/mL | 4°C | +| 1mM Hydrocortisone | 1mL | 500µL | 250µL | 1µM | -20°C | +| 0.2mM Triiodo-L-thyronine | 1mL | 500µL | 250µL | 0.2µM | -20°C | +| 100x MEM Vitamins | 5mL | 2.5mL | 1.25mL | 1x | 4°C | +| 1x Schneider's medium | 50mL | 25mL | 12.5mL | 5% | 4°C | +| 1M HEPES | 10mL | 5mL | 2.5mL | 10mM | 4°C | +| 1x Fetal calf serum inactivated | 100mL | 50mL | 25mL | 10% | -20°C | +| 100x PSF (add just before use) | 20mL | 10mL | 5mL | 2% | -20°C | + +2. **Instructions:** + - Sterilize media with vacuum filter unit and store at 4°C. Use within 2 weeks. + +### Somule Wash +- **Ingredients:** + - 5mL 1M Hepes + - 10mL antibiotic-antimycotic + - To 500mL with 1x DMEM in vacuum filter unit +- **Instructions:** + - Sterilize media with vacuum filter unit and store at 4°C for up to 2 weeks. + +### Percoll Gradient - 15ml +- **Ingredients:** + - 6.5mL percoll (shake before use) + - 1.2mL 1M NaCl + - 2.5mL Somule Wash +- **Instructions:** + - Keep percoll solutions on ice until use. Invert the mixture several times to establish a good percoll gradient. Avoid bubbles in the percoll (remove any if found). + +### Serotonin Stock 80mM (17mg/ml) +- **Ingredients:** + - 25mg serotonin hydrochloride (Sigma H9523-25MG) (4°C) + - 1.47mL H2O +- **Instructions:** + - Vortex well and store at -20°C in 350µL aliquots. + +### 1mM Serotonin +- **Ingredients:** + - 312.5µL of 80mM stock solution + - 24.6875mL NFW +- **Instructions:** + - Filter sterilize and store at -20°C in 1mL aliquots. + +### 3,3′5-Triiodo-L-thyronine Stock 10mM +- **Ingredients:** + - 100mg T3 (Sigma T6397-100MG) + - 14.86mL 0.2N NaOH +- **Instructions:** + - Vortex well and store at -20°C in 2mL aliquots. + +### 0.2mM 3,3′5-Triiodo-L-thyronine +- **Ingredients:** + - 5mL of 10mM stock solution + - 20mL NFW +- **Instructions:** + - Filter sterilize and store at -20°C in 1mL aliquots. + +### Hypoxanthine Stock 368mM (50mg/ml) +- **Ingredients:** + - 1g hypoxanthine (Sigma H9636-1G) + - 20mL 2:1 formic acid:H2O +- **Instructions:** + - Vortex well and store at -20°C in 2mL aliquots. + +### 1mM Hypoxanthine +- **Ingredients:** + - 34µL of 368mM stock solution + - 12.466mL sterile medium +- **Instructions:** + - Filter sterilize and store at -20°C in 500µL aliquots. + +### Hydrocortisone Stock 2.75mM (50µg/ml) +- **Ingredients:** + - 1mg hydrocortisone (H0135-1MG) (RT) + - 1mL absolute ethanol + - 19mL sterile medium +- **Instructions:** + - Gently swirl to dissolve. Swirl to mix and store in 9.5mL aliquots at -20°C. + +### 1mM Hydrocortisone +- **Ingredients:** + - 9.058mL of 2.76mM stock solution + - 15.942mL sterile medium +- **Instructions:** + - Filter sterilize and store at -20°C in 1mL aliquots. + +## Safety Warnings +- Cercariae are infectious to humans. Please use proper PPE at all times, including a lab coat, waterproof over-gown, long-cuff gloves AQL <=1.5, and face shield. +- All disposable materials should be placed in biohazardous waste bins. +- Glassware should be immersed in a klorsept solution of at least 50ppm for at least 2 hours, rinsed with diH₂O and then autoclaved. +- Any liquids should be treated with klorsept solution of at least 50ppm for at least 2 hours. +- Liquids treated with klorsept should be diluted further and disposed of in the drain. +- Schistosomula in suspension are not a risk to humans UNLESS they are injected directly into the blood stream. + +## Before Start Instructions +- Place 1 sterile 15mL falcon tube on ice per snail to be shed. +- Pre-chill benchtop centrifuge to 4°C. +- Prepare 1x DPBS+2% anti-anti and place in 37°C reciprocating water bath. +- Prepare somule media and place in 37°C reciprocating water bath. +- Prepare somule wash and place in 37°C reciprocating water bath. + +## Procedure + +### Cercariae Collection (Duration: 30 minutes) +1. Shed cercariae from snails in a 6-well plate. The snails can be shed for up to 2 hours by collecting cercariae and replacing with fresh water every 30 minutes. +2. Using a sterile transfer pipette, dispense cercariae into an autoclaved 50mL beaker. +3. Fill the beaker containing cercariae to 45-50mL with diH₂O and incubate on ice for 30 minutes. +4. Carefully transfer the cercariae to a 50mL Falcon tube and centrifuge at 500 x g, 4°C for 20 minutes. +5. **Note**: All the following steps are carried out in the tissue culture biosafety cabinet. Keep a beaker containing 70% ethanol in the cabinet and before discarding any aspirating pipette or serological pipette aspirate ethanol to kill any contaminating cercariae in the pipette. + - Discard the supernatant and add 5mL 1x PBS + 2% Anti-Anti to the pellet. Gently resuspend by flicking the tube (avoid using pipette because the cercariae are quite sticky and get lost). +6. Add 45mL 1x PBS + 2% Anti-Anti and centrifuge at 500 x g, 4°C for 10 minutes. +7. Repeat steps 6-7 once more with 1x PBS + 2% Anti-Anti and then twice with somule wash. +8. During the centrifugations in Steps 7 and 8, prepare 2 percoll gradient tubes (2 tube for less than ~150K cercs, 4 tubes every ~150K cercs). +9. Following centrifugation of the cercariae, remove the supernatant and add 5mL of somule wash per percoll gradient you will use. +10. Vortex for 1 minute at maximum speed. This will start to break off the tails. + +### Cercariae Tail Removal with Micro-Emulsifying Needle Trituration +11. Open 8 x 5mL luer-lock syringes. +12. Of 8 syringes, take out the syringe pump of 4 syringes. Place 4 syringe pumps on 100mm or 150mm petri dish. +13. Lock one intact syringe and one syringe with the pump removed to a sterile 22G micro-emulsifying needle on each side. +14. Aliquot cercariae equally into the connected needles. Each syringe should hold ~3mL of cercariae. +15. Place syringe pumps back into the filled syringes and push the pump all the way. The cercariae should pass through the needle to the opposite syringe. + - **Check for any leakage from the locks. If there is a leak, try tightening the lock or do not use that needle.** +16. Unlock the empty syringe from the connected needle, place the needle and syringe with cercariae in the 15mL tubes containing the Percoll solution. +17. Push the cercariae through the needle, into the tube. Do this for all four needles with max ~5mL per percoll tube. +18. Push the syringes back and forth 12-13 more times. + +### Alternative Tail Removal: 20G Needle Trituration +20. Pass the cercariae suspension thought a 22G blunt-end needle in a luer lock syringe as follows: + - If 5mL solution: 15 times using 10mL luer lock syringe. + - If 10mL solution: 30 times using 20mL luer lock syringe. +21. Take a drop of cercariae in a petri dish to check under the microscope if the passages have separated the tails. If there are still many cercariae (more than ~20%), continue with ~5 more passages. + +### Percoll Gradient +22. Place now-headless cercariae carefully on top of Percoll solution tube using a plastic pasteur pipette (5 mL per Percoll solution tube) or directly into the tube of Percoll solution from the syringe. +23. Centrifuge at 350 x g, 4°C for 5 minutes with acceleration and deceleration = 1. +24. Remove from centrifuge carefully. Discard supernatant and the white interface of cercaria tails. + +### Washing Somules +25. Take schistosomula "pellets" and pool them together in a 15mL falcon tube. Top to 15mL with somule wash. +26. Centrifuge at 500 x g, 4°C for 5 minutes. +27. Discard supernatant and add 15mL somule wash. +28. Repeat washing steps 3 times in total. +29. After the last wash, remove as much supernatant as possible and add 6mL of somule media. + +### Somules in Culture +30. Dispense 2mL of somules into each well of a 6-well plate and add 4mL somule media to each. +31. Incubate at 37°C in 5% CO₂ incubator. +32. The following day observe the somules to look for contamination and replace media daily. + +__endofoutput__ +``` \ No newline at end of file diff --git a/markdown-output/schistosoma-mansoni-cercariae-transformation-witho-csavwae6.md b/markdown-output/schistosoma-mansoni-cercariae-transformation-witho-csavwae6.md new file mode 100644 index 0000000000000000000000000000000000000000..d770c37795cca9898aa15c3d4e2f00177fed9f7c --- /dev/null +++ b/markdown-output/schistosoma-mansoni-cercariae-transformation-witho-csavwae6.md @@ -0,0 +1,165 @@ +```markdown +# Goal/Experiment: +Transform Schistosoma mansoni cercariae into schistosomula without using a needle to facilitate further research and culture. + +# Schistosoma mansoni cercariae transformation (without needle) V.2 + +**Sarah K Buddenborg** +Wellcome Sanger Institute +Schistosoma mansoni + +## Abstract +Free-living aquatic *S. mansoni* cercariae transform into the first intramammalian stage, called schistosomula or somules, by burrowing into the host skin. Upon contact, cercariae begin to enter the skin and lose their tails, becoming schistosomula. Somules migrate through the epidermis to the dermis to find a small venule or lymphatic vessel to enter the vasculature. + +Transformation of cercariae to schistosomula can be mimicked in the laboratory by centrifuging cercariae to remove tails and then culturing the somules in somule media. This method is particularly useful when the number of cercariae is low (i.e., clonal cercariae from an individual snail). + +Somules can be cultured for several weeks with regular media changes. + +## Image Attribution +Images of somules taken by Dr. Gabriel Rinaldi. + +## Guidelines +Media changes and the opening of transformed somules should take place in a tissue culture hood using sterile techniques. + +## Materials + +| Item | Vendor & Catalog Number | +| --- | --- | +| DMEM high glucose GlutaMAX | Gibco - Thermo Fisher Catalog #31966021 | +| Lactalbumin Hydrolysate, powder (extra soluble) | Thermo Fisher Catalog #11800042 | +| Hypoxanthine | Merck MilliporeSigma (Sigma-Aldrich) Catalog #H9636-1G | +| Serotonin Hydrochloride | Merck MilliporeSigma (Sigma-Aldrich) Catalog #H9523-25MG | +| Insulin solution from bovine pancreas | Merck MilliporeSigma (Sigma-Aldrich) Catalog #I0516-5ML | +| 3',5'-Triiodo-L-thyronine sodium salt | Merck MilliporeSigma (Sigma-Aldrich) Catalog #T6397 | +| MEM Vitamin Solution (100×) | Merck MilliporeSigma (Sigma-Aldrich) Catalog #M6895 | +| Schneider's Insect Medium | Merck MilliporeSigma (Sigma-Aldrich) Catalog #S0146 | +| HEPES solution | Merck MilliporeSigma (Sigma-Aldrich) Catalog #H0887 | +| Fetal Bovine Serum | Merck MilliporeSigma (Sigma-Aldrich) Catalog #F4135 | +| Antibiotic-Antimycotic (100×) | Thermo Fisher Scientific Catalog #15240062 | +| Dulbecco's Phosphate Buffered Saline 10X | Merck MilliporeSigma (Sigma-Aldrich) Catalog #D1283 | +| MilliQ water | Contributed by users | +| Sterile Graduated Transfer Pipets | Fisher Scientific Catalog #13479108 | +| Falcon 15 mL Conical Centrifuge Tubes | Fisher Scientific Catalog #1077350 | +| Nun Non-Treated 6-well plate | Thermo Scientific Catalog #10396482 | +| 1000 mL Vacuum Filter/Storage Bottle System 0.22 μm Pore 54.5cm^2 PES Membrane Sterile | Corning Catalog #431098 | +| Chilling benchtop centrifuge with 15ml swing bucket rotor | | +| Incubator at 37°C and 5% CO2 | | +| Reciprocal water bath | | +| Class 2 Microbiological Safety Cabinet | | + +## Recipes +### 1X DPBS + 2% Anti-Anti +- 50 ml 10× DPBS +- 10 ml 100× Antibiotic-Antimycotic (-20°C) +- Fill to 500 ml in vacuum filter unit with MilliQ water +- Sterilize media with vacuum filter unit and store at 4°C. Use within 2 weeks. + +### Somule Medium – Basch Modified Medium (BMM) +1. Mix the following reagents: + +| Reagent | A (1L) | B (500 ml) | C (250 ml) | D (working) | E | F (Storage) | +| --- | --- | --- | --- | --- | --- | --- | +| 1× DMEM high glucose | 810.5 ml | 405.25 ml | 202.625 ml | 1× | | 4°C | +| 1 g/L Lactalbumin hydrolysate | 1 g | 0.5 g | 0.25 g | 1 g/L | | 4°C | +| 1 mM Hypoxanthine | 500µl | 250 µl | 125 µl | 0.5 µM | | -20°C | +| 1 mM Serotonin | 1 ml | 500 µl | 250 µl | 1 µM | | -20°C | +| Insulin | 1 ml | 500 µl | 250 µl | 8 µg/ml | | 4°C | +| 1 mM Hydrocortisone | 1 ml | 500 µl | 250 µl | 1 µM | | -20°C | +| 0.2 mM Triiodo-L-thyronine | 1 ml | 500 µl | 250 µl | 0.2 µM | | -20°C | +| 100× MEM Vitamins | 5 ml | 2.5 ml | 1.25 ml | 1× | | 4°C | +| 1× Schneider's medium | 50 ml | 25 ml | 12.5 ml | 5% | | 4°C | +| 1 M HEPES | 10 ml | 5 ml | 2.5 ml | 10 mM | | 4°C | +| 1× Fetal calf serum inactivated | 100 ml | 50 ml | 25 ml | 10% | | -20°C | +| 100× PSF (add just before use) | 20 ml | 10 ml | 5 ml | 2% | | -20°C | + +2. Sterilise media with vacuum filter unit and store at 4°C. Use within 2 weeks. + +### Serotonin Stock 80 mM (17 mg/ml) +- 25 mg serotonin hydrochloride (Sigma H9523-25MG) (4°C) +- 1.47 ml H2O +- Vortex well and store at -20°C in 350 µl aliquots. + +#### 1mM Serotonin +- 312.5 µl of 80 mM stock solution +- 24.6875 ml NFW +- Filter sterilize and store at -20°C in 1 ml aliquots. + +### 3,3',5-Triiodo-L-Thyronine Stock 10 mM +- 100 mg T3 (Sigma T6397-100MG) +- 14.86 ml 0.2N NaOH +- Vortex well and store at -20°C in 2 ml aliquots. + +#### 0.2 mM 3,3',5-Triiodo-L-Thyronine +- 5 ml of 10 mM stock solution +- 20 ml NFW +- Filter sterilize and store at -20°C in 1 ml aliquots. + +### Hypoxanthine Stock 368 mM (50 mg/ml) +- 1 g hypoxanthine (Sigma H9636-1G) +- 20 ml 2:1 formic acid:H2O +- Vortex well and store at -20°C in 1 ml aliquots. + +#### 1 mM Hypoxanthine +- 34 µl of 368 mM stock solution +- 12.466 ml sterile medium +- Filter sterilize and store at -20°C in 500 µl aliquots. + +### Hydrocortisone Stock 2.75 mM (50 µg/ml) +- 1 mg hydrocortisone (H0135-1MG) (room temperature) +- 1 ml absolute ethanol +- Gently swirl to dissolve +- 19 ml sterile medium +- Swirl to mix and store in 9.5 ml aliquots at -20°C + +#### 1mM Hydrocortisone +- 9.058 ml of 2.76 µM stock solution +- 15.942 ml sterile medium +- Filter sterilize and store at -20°C in 1 ml aliquots. + +## Safety Warnings +- Cercariae are infectious to humans. Please use proper PPE at all times, including a lab coat, waterproof over-gown, long-cuff gloves AQL <= 1.5, and face shield. +- All disposable materials should be placed in biohazardous waste bins. +- Glassware should be immersed in a klorsept solution of at least 50 ppm for at least 2 hours, rinsed with diH2O and then autoclaved. +- Any liquids should be treated with klorsept solution of at least 50 ppm for at least 2 hours. +- Liquids treated with klorsept should be diluted further and disposed in the drain. +- Schistosomula in suspension are not a risk to humans UNLESS they are injected directly into the bloodstream. + +## Before Start Instructions +- Place 1 sterile 15 ml Falcon tube on ice per snail to be shed. +- Pre-chill benchtop centrifuge to 4°C. +- Prepare 1× DPBS + 2% anti-anti and place in a 37°C reciprocating water bath (see recipes in "Materials" section) +- Prepare somule media and place in 37°C reciprocating water bath (see recipes in "Materials" section) + +## Procedure + +### Cercariae Collection +1. Shed cercariae from snails in a 6-well plate (see protocol "Schistosoma mansoni cercariae shedding"). The snails can be shed for up to 2 hours by collecting cercariae and replacing with fresh water every 30 min. +2. Using a sterile transfer pipette, dispense cercariae into sterile 15 ml Falcon tubes on ice. + + > **Note:** + > All the following steps are carried out in the tissue culture biosafety cabinet. Keep a beaker containing 70% ethanol in the cabinet and before discarding any aspirating pipette or serological pipette aspirate ethanol to kill any contaminating cercariae in the pipette. + +3. After collecting cercariae, adjust the volume in each 15 ml Falcon tube to 15 ml with sterile water. +4. Incubate tubes containing cercariae for 30 minutes on ice. + +### Cercariae Tail Removal +5. Centrifuge the cercariae at 2200 rpm, 4°C, for 30 minutes using an Eppendorf 5810R centrifuge. +6. Quickly remove the supernatant and resuspend the pellet in 10 ml pre-warmed 1× PBS + 2% PSF (see "Materials" section for recipes) by gently inverting the tube (do not use pipette to mix). +7. Centrifuge the cercariae at 1500 rpm, 4°C, for 10 minutes. +8. Repeat steps 6 and 7 six more times. + +### Somules in Culture +9. Resuspend pellet of somules in ~5 ml pre-warmed somule media (see "Materials" section for recipes). +10. Place somules in a 6-well plate and top up each well with pre-warmed somule media, so each has a total of approximately 4 ml media. + +![](https://source-image-url) +_Somules on same day of transformation._ + +11. Incubate at 37°C, 5% CO2 overnight. +12. (Optional) The following day, remove tails with transfer pipette (the tails will have floated to the top of the water column in the wells). + +![](https://source-image-url) +_Somules the day after transformation, after removing tails._ + +`endofoutput` +``` \ No newline at end of file diff --git a/markdown-output/sci-atac-seq3-be8mjhu6.md b/markdown-output/sci-atac-seq3-be8mjhu6.md new file mode 100644 index 0000000000000000000000000000000000000000..f95a210069705957c9b0728005cd149fa508beef --- /dev/null +++ b/markdown-output/sci-atac-seq3-be8mjhu6.md @@ -0,0 +1,160 @@ +```markdown +# Goal/Experiment: +The goal of this experiment is to develop an improved assay for single-cell profiling of chromatin accessibility using sci-ATAC-seq3, which employs three levels of combinatorial indexing without requiring cell sorting and optimized to maximize the number of fragments recovered from each cell. + +# sci-ATAC-seq3 + +**Authors:** +Silvia Domcke1, Andrew J. Hill1, Riza M. Daza1, Cole Trapnell1,2, Darren A. Cusanovich3,4, Jay Shendure1,5,6 + +1Department of Genome Sciences, University of Washington School of Medicine, Seattle, WA, USA +2Brotman Baty Institute for Precision Medicine, Seattle, WA, USA +3Department of Cellular and Molecular Medicine, University of Arizona, Tucson, AZ, USA +4Asthma and Airway Disease Research Center, University of Arizona, Tucson, AZ, USA +5Allen Discovery Center for Cell Lineage Tracing, Seattle, WA, USA +6Howard Hughes Medical Institute, Seattle, WA, USA + +Published: November 02, 2020 +DOI: [10.17504/protocols.io.be8mhju6](https://dx.doi.org/10.17504/protocols.io.be8mhju6) + +## Abstract +We developed an improved assay for single-cell profiling of chromatin accessibility that utilizes three levels of combinatorial indexing. This protocol eliminates the need for cell sorting and optimizes conditions to maximize the fragments recovered from each cell. The method maintains specificity and allows for experiments on the scale of 10^6 cells. + +## Keywords +- Single cell ATAC-seq +- Chromatin accessibility +- sci-ATAC-seq +- sci-ATAC-seq3 +- Combinatorial indexing + +## Materials and Equipment + +### Required Equipment +- Tissue culture hood +- Tissue culture incubator +- Freezers (-20°C, -80°C) +- Eppendorf Mastercycler (thermal cycler) +- FACSAria III cell sorter (BD) +- Microscope +- Bright-Line Hemacytometer (Sigma) +- Centrifuge (cooled to 4°C) (Eppendorf, 5810R) +- Agilent 4200 TapeStation System +- Gel Imager +- NextSeq 500 platform (Illumina) +- Liquid nitrogen tank for sample storage +- DynaMag-96 Side Skirted Magnet (Thermo Fisher Scientific, 12027) +- Gel box +- Ice bucket +- Multi-channel pipettes (10ul, 200ul) (Rainin Instrument) +- Rainin Liquidator 96 Manual Pipetting System +- Pipettors + +### Reagents and Consumables +- 0.5 M EDTA (Thermo Fisher Scientific, AM9260G) +- 100 bp ladder (New England Biolabs, N3231L) +- 1000X SYBR (Invitrogen, S7563) +- 10mM ATP (New England Biolabs, P0756S) +- 10X HBSS (Gibco/BRL Life Tech, 14065-056) +- 10X PNK Buffer (New England Biolabs, M0201L) +- 1M MgCl2 (Thermo Fisher Scientific, AM9530G) +- 1X DPBS (without Ca, Mg) +- 2.5M Glycine +- 40mM EDTA +- 5M NaCL +- 6% TBE PAGE (Invitrogen, EC6265BOX) +- 6X Orange dye (NEB, B7025S) +- AMPure Beads (Beckman Coulter, A63882) +- BSA (NEB, B9000S) +- DNA LoBind Tube 1.5 ml (Eppendorf, 022431021) +- DTT +- EB Buffer (Qiagen, 19086) + +## Protocol Guidelines + +The protocol workflow is as follows: +1. **Reagent preparation** +2. **Nuclei isolation and fixation from cell lines** +3. **Tissue pulverization, nuclei isolation and fixation from frozen tissues** +4. **sci-ATAC-seq3 library construction, QC and sequencing** + +### Oligo and Primer Sequences +Order the barcoded oligos and sequencing oligos as described in Table S7 from Domcke, Hill, Daza et al. (2020). Resuspend at 100 uM in nuclease-free water and store at -20°C. + +## Procedure + +### 1. Reagent Preparation (~30-45 mins) + +#### 1.1. Prepare ATAC-RSB Buffer +In a 50 mL Falcon tube, combine: +- 500 uL 1M Tris-HCl pH 7.4 +- 100 uL 5M NaCl +- 100 uL 1M MgCl2 +- 49.1 mL nuclease-free water + +Filter sterilize using Millipore "Steriflip" Sterile Disposable Vacuum Filter Unit (0.22 um PES membrane). + +#### 1.2. Additional Reagents +- **10% Tween-20:** Dilute 5% digitonin to 1% with nuclease-free water, store at 4°C for up to 6 months. +- **Freezing Buffer (FB):** Combine 50 mM Tris at pH 8.0, 25% glycerol, 5 mM Mg(OAc)2, 0.1 mM EDTA, and nuclease-free water. Filter sterilize and store at 4°C for up to 6 months. +- **2.5 M Glycine:** Make from glycine powder, store reagent at room temperature for up to 6 months. +- **40 mM EDTA:** Make from 0.5 M EDTA stock, store reagent at room temperature for up to 6 months. + +### 2. Nuclei Isolation and Fixation from Cell Lines (~2 hours for 4 cell lines) +1. Using GM12878 and CH12-LX cells grown under specified conditions, prepare Omni-ATAC lysis buffer. +2. Pelleted cells, resuspended in Omni-ATAC lysis buffer, followed by centrifugation. +3. Crosslink nuclei with 37% formaldehyde, quench with Glycine. +4. Pellet nuclei, resuspend in freezing buffer, and store at -80°C. + +### 3. Tissue Pulverization, Nuclei Isolation, and Fixation of Frozen Tissues (~3 hours for 6 tissues) +1. Pre-label tubes, prepare tissue samples. +2. Snap-freeze tissues, electrically ensuring frost prevention. +3. Pulverize tissues, aliquot into pre-labeled tubes, store at -80°C. +4. Fix nuclei by crosslinking, quenching, and counting. + +### 4. Library Construction, QC, and Sequencing + +#### 4.1. Thawing, Permeabilization, Counting, and Tagmentation (~4 hours) +1. Thaw frozen nuclei, resuspend in Omni-ATAC lysis buffer. +2. Count nuclei, distribute in a 96-well plate. +3. Perform tagmentation using Nextera V2 enzyme. +4. Tagmentation Master Mix preparation: + +| Component | 1 Reaction (µL) | 110 Reactions (µL) | +|-----------------|-----------------|--------------------| +| TD Buffer 2X | 25 | 2750 | +| 1X DPBS | 8.25 | 907.5 | +| 1% Digitonin | 0.5 | 55 | +| 10% Tween-20 | 0.5 | 55 | +| Water | 13.25 | 1457.5 | +| Nextera V2 | 2.5 | 275 | +| **Total** | 50 | 5225 | + +#### 4.2. Pooling, PNK Reaction, and N5 Ligation (~2.5 hours) +1. Pool tagged nuclei into Eppendorf tubes. +2. Create PNK reaction master mix and perform the PNK reaction. +3. Add T4 PNK and resuspend nuclei in ATAC-RSB. + +#### 4.3. N7 Ligation (2 hours) +1. Prepare and perform N7 ligation master mix, following protocols. + +### 5. Pooling, Counting and Dilution (1.5 hours) +1. Pool wells, wash nuclei, and resuspend in Qiagen EB buffer. +2. Filter nuclei for FACS. + +#### 5.1. Reverse Crosslink Nuclei (Overnight incubation) +1. Prepare reverse crosslink master mix, incubate at 65°C for 16 hours. + +#### 5.2. Test PCR and Gel QC (~2 hours) +1. Perform a test PCR using specified primers and conditions. +2. Analyze PCR products with 6% TBE gel. + +#### 5.3. PCR Plate Set-Up for Remaining Samples (1 hour) +1. Aliquot nuclei, perform PCR. + +### 6. PCR Amplification Clean-up and QC (~1.5 hours) +1. Combine PCR reactions, process with Zymo C8 columns for cleanup. + +### 7. Sequencing - Illumina 150 Cycle Kit, High Output (~1.5 hours) +1. Libraries are assessed with NextSeq and pooled for sequencing on NovaSeq. + +```endofoutput``` \ No newline at end of file diff --git a/markdown-output/sci-rna-seq3-9yih7ue.md b/markdown-output/sci-rna-seq3-9yih7ue.md new file mode 100644 index 0000000000000000000000000000000000000000..d56df9332691fde39991cb9f654b2dfe23eddea0 --- /dev/null +++ b/markdown-output/sci-rna-seq3-9yih7ue.md @@ -0,0 +1,157 @@ +```markdown +# Goal/Experiment: +The goal of this experiment is to perform single-cell combinatorial indexing RNA sequencing (sci-RNA-seq3) to profile the transcriptomes of large numbers of single cells or nuclei in a scalable manner. + +# sci-RNA-seq3 + +**Authors**: Junyue Cao, Jay Shendure +**Institution**: University of Washington +**Published**: Nov 12, 2020 +**Link**: [dx.doi.org/10.17504/protocols.io.9yih7ue](https://dx.doi.org/10.17504/protocols.io.9yih7ue) +**Relevant DOI**: [doi:10.1038/s41586-019-0969-x](https://www.nature.com/articles/s41586-019-0969-x) + +## ABSTRACT +Single-cell combinatorial indexing (`sci-`) is a methodological framework that employs split-pool barcoding to uniquely label the nucleic acid contents of large numbers of single cells or nuclei. The developed and extensively optimized 3-level sci-RNA-seq (sci-RNA-seq3) can profile millions of cells per experiment. Like replicates, time points, etc., multiple samples can be barcoded during the first round of indexing and concurrently processed. + +## Guidelines + +### The protocol workflow is as follows: +1. **Buffer preparation** (Steps 1-5). +2. **Nuclei extraction directly from tissues** (approx. 1.5 hours for 6 tissues) (Steps 6-10). +3. **Nuclei fixation** (approx. 1.5 hours for 6 tissues) (Steps 11-16). +4. **Nuclei permeabilization before reverse transcription** (1 hour for 6 samples) (Steps 17-26). +5. **Reverse transcription** (approx. 2 hours for 6 samples) (Steps 27-33). +6. **Ligation** (approx. 2 hours) (Steps 34-44). +7. **Second strand synthesis** (approx. 3 hours) (Steps 45-47). +8. **Tagmentation** (approx. 10 min) (Steps 48-50). +9. **Ampure beads purification** (approx. 1 hour) (Steps 51-59). +10. **PCR reaction and library preparation** (approx. 2 hours) (Steps 60-65). + +### Downstream Analysis +The output is sequencing reads from Nova-seq or Next-seq, comprising i5 and i7 index reads, and read1 and read2. Datasets and more details are available in Gene Expression Omnibus (GEO) under accession [GSE119945](https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE119945). + +## Required Equipment + +- Bioruptor Sonication device (diagenode, B01020001) +- Centrifuge (cooled to 4°C) (Eppendorf, 5810R) +- DynaMag™-96 Side Skirted Magnet (Thermo Fisher Scientific, 12027) +- Eppendorf Mastercycler (thermal cycler) +- FACs machine +- Freezer (-20°C, -80°C) and refrigerator (4°C) +- Microscope +- Multi-channel pipettes (10ul, 200ul) (Rainin Instrument) +- NextSeq 500 platform (Illumina) +- Pipettors +- Rainin Liquidator 96 Manual Pipetting System +- Tissue culture hood and incubator +- Additional miscellaneous lab supplies + +## Primer Sequences +Refer to [sci3_primer_sequences.xls](sci3_primer_sequences.xls). + +## Materials + +| Material | Vendor | Catalog Number | +|-----------------------------------------------------|------------------------------------------|----------------------| +| Nuclease Free Water | Ambion | AM 9937 | +| 6 cm Cell Culture Dish | Sigma Aldrich | Z707678-840EA | +| Greiner Cellstar 6 well plates | Sigma Aldrich | M5862 | +| Razor Blades (5x20) | VWR | 55411-0055 | +| Falcon Cell Strainers | VWR | 10199-654 | +| Flowmi™ Cell Strainers | VWR | 10204-924 | +| Triton X-100 for molecular biology | Sigma-Aldrich | 93443-100ML | +| 20 mM Tris-HCl (pH 7.5) | Thermo Fisher Scientific | 15567027 | +| Ampure XP beads | Beckman Coulter | A63882 | +| Ethanol | Sigma Aldrich | 459844-4L | +| Quibit dsDNA HS Kit | Invitrogen | Q32854 | +| High Fidelity 2x PCR Master Mix | NEB | M0541L | +| RNaseOUT Recombinant Ribonuclease Inhibitor | Invitrogen | 10777019 | +| Nuclei Lysis Buffer | Homemade | N/A individual notes | +| etc. | | | + +## Detailed Protocol + +### Buffer Preparation +1. **500ml Nuclei Buffer (Stored in 4°C)**: + - 10 mM Tris-HCl, pH 7.5 + - 10 mM NaCl + - 3 mM MgCl2 + - Nuclease-free water + +| Reagent | Stock Concentration | Final Concentration | Volume (ml) | +|-----------------|----------------------|---------------------|-------------| +| Tris-HCl (pH 7.5)| 1M | 10mM | 5 | +| NaCl | 5M | 10mM | 1 | +| MgCl2 | 1M | 3mM | 1.5 | +| Nuclease-free water | NA | NA | 492.5 | +| **Final volume** | NA | NA | 500 | + +2. **20 ml 10% IGEPAL CA-630**: + - 2 ml IGEPAL CA-630 + - 18 ml nuclease-free water + +3. **20 ml 10% Triton X-100**: + - 2 ml Triton X-100 + - 18 ml nuclease-free water + +4. **Cell Lysis Buffer (CLB)**: + - Nuclei buffer with 0.1% IGEPAL CA-630 + - 1% SUPERase In RNase Inhibitor (20 U/µl) + - 1% BSA (20 mg/ml) + +5. **Nuclei Suspension Buffer (NSB)**: + - Nuclei buffer with 1% SUPERase In RNase Inhibitor (20 U/µl) + - 1% BSA (20 mg/ml) + +### Nuclei Extraction Directly from Tissues (~1.5 hours for 6 tissues) +6. Centrifuge to 4°C. +7. Add 1 ml of CLB to frozen tissue (0.1-0.5 g, frozen in liquid nitrogen or -80°C). +8. Homogenize and dissociate tissue using appropriate method. +9. Transfer the CLB with tissue to a 40 µm cell strainer. +10. Transfer isolated nuclei to tube, pellet (500g, 5 min), discard supernatant. +11. Re-suspend nuclei, repeat pelleting and discarding supernatant. + +### Nuclei Fixation (~1.5 hours for 6 tissues) +12. Add 10 ml ice-cold 4% PFA to 100 µl NSB re-suspension of nuclei. +13. Fix nuclei on ice for 15 min, pellet, and remove supernatant. +14. Re-suspend nuclei in 1 ml NSB. +15. Repeat the process mentioned in Steps 13-15 as needed. + +### Nuclei Permeabilization Before Reverse Transcription (1 hour for 6 samples) +16. Preparation includes centrifuging to 4°C. +17. Add NSB (40 ml nuclei buffer, 400 µl SUPERase In RNase Inhibitor (20 U/µl), 400 µl BSA (20 mg/ml)). +18. Store mixture on ice. +19. Thaw PFA fixed nuclei, pellet, and perform resuspension. + +### Reverse Transcription (~2 hours for 6 samples) +20. Add specified volumes of buffer and reagents to each well. +21. Incubate at specified temperatures. + +### Ligation (~2 hours) +22. Perform resuspension and distribute cells into plates. +23. Add indexed ligation oligos and prepare ligation mix. +24. Perform ligation. + +### Second Strand Synthesis (~3 hours) +25. Prepare synthesis mix and perform second strand synthesis. + +### Tagmentation (~10 min) +26. Prepare tagmentation mix and perform tagmentation. + +### Ampure Beads Purification (~1 hour) +27. Prepare USER mix and purify using Ampure XP beads. + +### PCR Reaction and Library Preparation (~2 hours) +28. Prepare PCR reaction mix and perform amplification. + +### Library Visualization +29. Visualize libraries using electrophoresis on a 6% TBE-PAGE gel. + +### Library Sequencing +30. Sequence library on Novaseq platform. + +### Example Data Visualization +![Library Validation](https://dx.doi.org/10.17504/protocols.io.9yih7ue/library_validation_image) + +**endofoutput** +``` \ No newline at end of file diff --git a/markdown-output/screening-whole-proteome-of-aedes-aegypti-and-iden-b6vere3e.md b/markdown-output/screening-whole-proteome-of-aedes-aegypti-and-iden-b6vere3e.md new file mode 100644 index 0000000000000000000000000000000000000000..975785a33d69d0bea9533a49562be517f23e2eb5 --- /dev/null +++ b/markdown-output/screening-whole-proteome-of-aedes-aegypti-and-iden-b6vere3e.md @@ -0,0 +1,157 @@ +```markdown +# Goal/Experiment: +To screen the whole proteome of *Aedes aegypti* and identify major protein targets for studying the interaction of natural bioactives for developing potential repellents. + +# Screening Whole Proteome of *Aedes aegypti* and Identification of Potential Targets for In-Silico Molecular and Structural Interaction Studies Against Natural Bioactives + +## Authors: + +- Anagha S Setlur¹ +- Chandrashekar K¹ +- Manas Sarkar² +- Vidya Niranjan¹ + +¹Department of Biotechnology, RV College of Engineering, Bangalore- 560059; +²Research and Development, Reckitt Benckiser India Pvt. Ltd., Gurgaon, Haryana- 122001 + +## Centre of Excellence in Computational Genomics (Vidya Lab) + +### Abstract + +As a dangerous etiological agent for dengue, chikungunya, zika, and yellow fever, it is essential to combat the incidences of *Aedes aegypti* by using repellents. However, chronic overuse of synthetic repellents has led to possibilities of adverse side effects in humans. As a consequence, research is shifting focus to developing natural alternatives to these repellents. This study aims to devise a standard protocol that can screen the whole proteome of *A. aegypti* and identify the major proteins that can be targeted by natural bioactives to produce repellents. + +In this study: +- Whole proteome analysis was carried out by finding the reference proteome. +- Hours related to the circadian rhythm of *A. aegypti* were analyzed. +- Potential targets were shortlisted by analyzing conserved domains. +- Targets were modelled using RaptorX. +- Structures validated via Ramachandran plots. +- Molecular docking was conducted using POAP. +- Least negative energy targets were further studied using molecular dynamic simulations for 100ns. + +**Keywords:** Whole proteome, *Aedes aegypti*, conserved domains, homology modelling, docking, molecular dynamic simulations + +## 1. Identification of Protein Targets + +### 1.1 Reference Proteome Identification + +The reference proteome for *Aedes aegypti* was identified using the UniProt database: + +- **Taxonomic ID:** 7159 +- **UniProt ID:** UP000008820 +- **Total Proteins:** 21,496 +- **Genes Expressed:** 14,555 +- **Strain:** LVP_AGWG +- **Genome Assembly and Annotation:** [GCA_002204515.1 from EnsemblMetazoa](https://www.ensembl.org) + +### 1.2 Identification of Major Protein Targets by Whole Proteome Screening + +From a whole proteome screen, 20 protein categories were identified through literature survey and circadian gene expression studies. Sequence retrieval was carried out in FASTA format, and sequences were filtered for structure prediction and modelling. + +Example FASTA sequences downloaded for a specific protein category (odorant binding proteins): + +``` +>sp|Q16E19|OR4_AEDAE Odorant receptor 4 OS=Aedes aegypti OX=7159 GN=GPROR4 PE=2 SV=1 + [...] +>sp|Q17J82|OR10_AEDAE Odorant receptor 10 OS=Aedes aegypti OX=7159 GN=GPROR10 PE=2 SV=1 +``` + +## 2. Prediction of Conserved Domains for Identified Protein Targets + +### 2.1 Multiple Sequence Alignment + +- **Tool:** MUSCLE algorithm in MEGA-X software +- **URL:** [NCBI Conserved Domain Search Tool](http://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi) + +Parameters used: + +| Option | Setting | +|-----------------------|----------------------| +| Gap Open | -2.90 | +| Gap Extend | 0.00 | +| Hydrophobicity Mult. | 1.20 | +| Max Memory in MB | 2048 | +| Max Iterations | 16 | +| Neighbor Joining | - | +| Neighbor Joining (Other Iterations) | 24 | + +Once aligned, conserved domains were identified and used for modelling. + +## 3. Homology Modelling of Proteins and Structure Validation + +### 3.1 Structure Modelling of Target Proteins + +- **Tool:** RaptorX standalone tool + - Installed tools: NR database, template database, PDB files + - Commands: + +#### 3.2 Code for Building Features + +```bash +./buildFeature -i seq_file [-o tgt_file ] [-c cpu_num] +``` + +#### 3.3 Code for Aligning Target Sequence to Template + +```bash +./CNFAlign_lite -t template_name -q target_name [-l tpl_root] [-g tgt_root] [-d output_root] +``` + +#### 3.4 Code for Building 3D Models + +- **Single Template:** + +```bash +./build3Dmodel -i align_file -q query_name [-d pdb_root] [-m mod_bin ] [-n mod_num ] +``` + +- **Multiple Templates:** + +```bash +./buildTopModels -i rank_file [-k TopK] [-d pdb_root] [-m mod_bin] +``` + +### 3.5 Structure Validation + +Validated using Ramachandran plots generated from SAVES v6.0 ([SAVE](https://saves.mbi.ucla.edu/)) + +## 4. Molecular Docking + +### 4.1 Preparation of the Ligands and Controls + +3D structures of natural bioactives were retrieved from NCBI PubChem. Ligand preparation was done in POAP: + +```bash +bash POAP_lig.bash -s +``` + +### 4.2 Preparation of the Proteins and Molecular Docking by Virtual Screening + +AutoDock 4.2.6 was used for preparing proteins. Procedures for virtual screening in POAP: + +```bash +bash POAP_vs.bash -s +``` + +## 5. Molecular Dynamic Simulations for Best Docked Complexes + +### 5.1 Analysis of 100ns Simulation Studies + +Key parameters evaluated: +- RMSD (Root Mean Square Deviation) +- RMSF (Root Mean Square Fluctuations) +- Hydrogen bond interactions +- Interaction energy profiles + +### 5.2 RMSD +RMSD computed to analyze the stability of the docked complex. + +### 5.3 RMSF +Computed to analyze conformational changes. + +## 6. Conclusion and Future Scope + +This protocol was standardized to screen the whole proteome of *A. aegypti* to identify potential targets for natural repellent development. High potential of natural molecules was observed as effective alternatives to synthetic repellents. Future work could include predicting ligand toxicity and calculating the binding free energy using MMGBSA studies. + +**ENDOFOUTPUT** +``` \ No newline at end of file diff --git a/markdown-output/script-p5-diversity-efrbbm6.md b/markdown-output/script-p5-diversity-efrbbm6.md new file mode 100644 index 0000000000000000000000000000000000000000..103e3c1ef9f8dd8b6e1d31950be7f81af1586138 --- /dev/null +++ b/markdown-output/script-p5-diversity-efrbbm6.md @@ -0,0 +1,149 @@ +```markdown +# Goal/Experiment: +This protocol provides a method to predict phage community diversity using the algorithm PHACCS. It addresses the unknown factor of viromes by predicting virus community alpha diversity without the use of taxonomic references. + +# Script P5: Diversity + +**Authors:** HANNIGAN GD, GRICE EA, ET AL. + +## Abstract +This protocol provides a method to predict phage community diversity using the algorithm PHACCS. Sequencing of viral communities often results in a high percentage of unknown reads, largely due to our incomplete reference databases. To address this, the algorithm PHACCS (Phage Communities from Contig Spectra) was developed to predict virus community alpha diversity without the use of taxonomic references. Based on the methods from the following publication: + +**Reference:** + +Hannigan, Geoffrey D., et al. "The Human Skin Double-Stranded DNA Virome: Topographical and Temporal Diversity, Genetic Enrichment, and Dynamic Associations with the Host Microbiome." mBio 6.5 (2015): e01578-15. + +Citation: HANNIGAN GD, GRICE EA, ET AL. Script P5: Diversity. protocols.io dx.doi.org/10.17504/protocols.io.efrbbm6 Published: 10 Mar 2016 + +## Guidelines + +### Required Software: +- PHACCS-1.1.3 +- Circonspect-0.2.6 +- GAAS-0.17 +- Octave-3.8.1 + +### Relevant Files +**Output:** +- `Alpha_diversity/virus_and_phage_acc_numbers.txt` +- `Alpha_diversity/PHACCS_results_all.txt` + +**R scripts:** `R5, R6, R10` + +### Before Start +Perl scripts and other supplemental information available at: + +[Supplemental Information](https://figshare.com/articles/The_Human_Skin_dsDNA_Virome_Topographical_and_Temporal_Diversity_Genetic_Enrichment_and_Dynamic_Associations_with_the_Host_Microbiome/1281248) + +## Protocol + +### Virome Alpha Diversity + +#### Step 1 +#### Make directory for the circonspect output files. +```sh +mkdir ./negative_clean_subsample_150K_circonspect +``` +> *Notes: Use randomly subsampled files here for speed purposes (150,000 subsampled). The majority of samples have more sequences than the subsampled depth, so it also provides normalization for the samples.* Geoffrey Hannigan 14 Jan 2016 + +#### Step 2 +#### Run Circonspect to generate the contig spectra. + +**Software Package (Unix):** Circonspect, 0.2.6 +[Circonspect](http://sourceforge.net/p/circonspect/code/ci/master/tree/) +```sh +ls ./neg_clean_subsample_150K | xargs -I {} --max-procs=16 Circonspect -o -b ./negative_clean_subsample_150K_circonspect/{} -f ./neg_clean_subsample_150K/{} +``` + +#### Step 3 +#### In addition to the contig spectra, we need to use the program GAAS (suggested by PHACCS) to estimate the average virus/phage reference genome length for each sample. + +#### Step 4 +#### First, download the list of all virus+phage accession numbers as a text file. +**Link:** +[Download virus.txt](http://www.ebi.ac.uk/genomes/virus.txt) + +#### Step 5 +#### Use the downloaded text file to get all the fasta files. +**Link:** +[Get fasta files](http://www.ebi.ac.uk/cgi-bin/sva.pl?&do_batch=1) + +#### Step 6 +#### Upload the accession list and fasta file to the server. The final reference fasta is stored as `./reference/virus_and_phage_ref/virus_and_phage_ref.fasta`. + +#### Step 7 +#### Run GAAS using the 150k sequence rarefied dataset. + +**Software Package (Unix):** GAAS, 0.17 +[GAAS](http://sourceforge.net/p/gaas/code/ci/master/tree/) +```sh +mkdir ./GAAS_results_150k_subsample +mkdir ./tmp +ls ./neg_clean_subsample_150K | xargs -I {} --max-procs=4 mkdir ./tmp/{} +ls ./neg_clean_subsample_150K | xargs -I {} --max-procs=16 GAAS -gt 0 -v nucleic -e 1e-03 -x ./tmp/{} -o ./GAAS_results_150k_subsample/{} -f ./neg_clean_subsample_150K/{} -d ./reference/virus_and_phage_ref/virus_and_phage_ref.fasta +``` +> *Notes: Need to make a set of unique tmp directories or else the procs all try to use the same directory with the same intermediate file names (this would be bad news!).* Geoffrey Hannigan 14 Jan 2016 + +#### Step 8 +#### Remove the tmp directory used for GAAS. +```sh +rm ./tmp/ +``` +> *Warning: Always be careful when deleting directories, especially directories with general names like these.* + +#### Step 9 +#### Now that we have each sample's contig spectrum and average reference genome length, we can use PHACCS to estimate each sample's alpha diversity. + +**Software Package (Unix):** PHACCS, 1.1.3 +```sh +run.phaccs.with.avg.genome.length () { + # Set the variable number for the average genome length + echo $1 + export SHORT_FILENAME=$(echo ${1} | sed 's/\.fa.csp//') + export GAAS_NUM=$(awk 'FNR == 2 {print $1}' ./GAAS_results_150k_subsample/${SHORT_FILENAME}.fa/gaas_${SHORT_FILENAME}_average.txt) + echo $GAAS_NUM + octave --silent --path /app/PHACCS-113/ -- eval "results = phaccs('./negative_clean_subsample_150K_circonspect/${1}', [], [], [], [], [], [], [], [], [], '${GAAS_NUM}',[],'power',1,1000000,[],[],[],[]); richness = results{1}.richness; evenness = results{1}.evenness; most_abund = results{1}.most_ab; sw_index = results{1}.sw_index; save './negative_clean_subsample_150K_phaccs_with_genome_lengths_higher_richness/${1}_richness.txt' richness; save './negative_clean_subsample_150K_phaccs_with_genome_lengths_higher_evenness/${1}_evenness.txt' evenness; save './negative_clean_subsample_150K_phaccs_with_genome_lengths_higher_most_abundant/${1}_most_abund.txt' most_abund; save './negative_clean_subsample_150K_phaccs_with_genome_lengths_higher_shannon/${1}_sw_index.txt' sw_index; exit" +} +export -f run.phaccs.with.avg.genome.length +``` +> *Note: Because PHACCS was written in Matlab, we used the open source Matlab alternative Octave to run the script. We also pulled out the diversity information from the PHACCS output and put it together into one easy-to-use file. This output was used in R analysis.* Geoffrey Hannigan 14 Jan 2016 + +#### Step 10 +#### Make directory for the output. +```sh +mkdir ./negative_clean_subsample_150K_phaccs_with_genome_lengths_higher_richness +ls ./negative_clean_subsample_150K_circonspect/*.csp | sed 's/.*\///g' | xargs -I {} --max-procs=16 sh -c "run.phaccs.with.avg.genome.length {}" +``` + +#### Step 11 +#### Make a directory for the alpha diversity output. +```sh +mkdir ./results_negative_clean_subsample_150K_phaccs_with_genome_lengths_higher_richness +``` + +#### Step 12 +#### Generate table with sample name in the first column and diversity metric in the second column. +```sh +head ./negative_clean_subsample_150K_phaccs_with_genome_lengths_higher_richness/*_richness.txt | grep -v \# | sed 's/^#//g' | sed 's/.*\///g' | sed 's/\.fa.*//g' | awk 'NR % 2 {print $0 "\t" }!(NR % 2) {print $0}' | sed '1 s/^SampleID\tRichness//' > ./results_negative_clean_subsample_150K_phaccs_with_genome_lengths_higher_richness/PHACCS_richness.txt +head ./negative_clean_subsample_150K_phaccs_with_genome_lengths_higher_evenness/*_evenness.txt | grep -v \# | sed '1 s/^SampleID\tEvenness//' > ./results_negative_clean_subsample_150K_phaccs_with_genome_lengths_higher_richness/PHACCS_evenness.txt +head ./negative_clean_subsample_150K_phaccs_with_genome_lengths_higher_most_abundant/*_most_abund.txt | grep -v \# | sed '1 s/^SampleID\tMostAbundant//' > ./results_negative_clean_subsample_150K_phaccs_with_genome_lengths_higher_richness/PHACCS_most_abund.txt +head ./negative_clean_subsample_150K_phaccs_with_genome_lengths_higher_shannon/*_sw_index.txt | grep -v \# | sed '1 s/^SampleID\tSWIndex//' > ./results_negative_clean_subsample_150K_phaccs_with_genome_lengths_higher_richness/PHACCS_sw_index.txt +``` + +#### Step 13 +#### Paste together the resulting files. +```sh +paste ./results_negative_clean_subsample_150K_phaccs_with_genome_lengths_higher_richness/PHACCS_richness.txt ./results_negative_clean_subsample_150K_phaccs_with_genome_lengths_higher_richness/PHACCS_evenness.txt ./results_negative_clean_subsample_150K_phaccs_with_genome_lengths_higher_richness/PHACCS_most_abund.txt ./results_negative_clean_subsample_150K_phaccs_with_genome_lengths_higher_richness/PHACCS_sw_index.txt | cut -f 1,2,4,6,8 > ./results_negative_clean_subsample_150K_phaccs_with_genome_lengths_higher_richness/PHACCS_results_all.txt +``` +> *Note: The resulting PHACCS diversity data can be found in PHACCS_results_all.txt. This data was analyzed according to Script R2.* Geoffrey Hannigan 14 Jan 2016 + +### Whole Metagenome Alpha Diversity +#### Step 14 +Whole metagenome alpha diversity is calculated in R using metaphlan taxonomy output. + +### Beta Diversity +#### Step 15 +Virome and whole metagenome beta diversity is calculated in R and uses the "OTU Table" format contig relative abundance table. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/seamless-editing-of-the-c-elegans-genome-using-cri-dw67hd.md b/markdown-output/seamless-editing-of-the-c-elegans-genome-using-cri-dw67hd.md new file mode 100644 index 0000000000000000000000000000000000000000..c325e886c81abb5f4555407667b420a088269c64 --- /dev/null +++ b/markdown-output/seamless-editing-of-the-c-elegans-genome-using-cri-dw67hd.md @@ -0,0 +1,174 @@ +```markdown +# Goal/Experiment: +The objective of this experiment is to achieve seamless editing of the Caenorhabditis elegans genome using CRISPR/Cas9 technology. + +## Seamless editing of the _C. elegans_ genome using CRISPR/Cas9 + +**Alexandre Paix, Yuemeng Wang, Harold E. Smith, Chih-Yung S. Lee, Deepika Calidas, Tu Lu, Jarrett Smith, Helen Schmidt, Michael W. Krause, and Geraldine Seydoux** + +[Scalable and Versatile Genome Editing Using Linear DNAs with Microhomology to Cas9 Sites in Caenorhabditis elegans](http://full_manuscript_link) + +### Citation: +Alexandre Paix, Yuemeng Wang, Harold E. Smith, Chih-Yung S. Lee, Deepika Calidas, Tu Lu, Jarrett Smith, Helen Schmidt, Michael W. Krause, and Geraldine Seydoux Seamless editing of the _C. elegans_ genome using CRISPR/Cas9, _protocols.io:_ dx.doi.org/10.17504/protocols.io.dw67hd + +### Published: +06 Oct 2015 + +--- + +## Guidelines + +### Reagents + +- **QIAprep Spin Miniprep Kit**: Qiagen, 27104 +- **MinElute PCR Purification Kit**: Qiagen, 28004 +- **Phusion High-Fidelity PCR Master Mix with HF Buffer**: NEB, M0531L +- **GoTaq Green Master Mix**: Promega, M7122 +- **Taq DNA Polymerase**: Invitrogen, 10342-046 +- **Recovery buffer**: 5mm HEPES pH 7.2, 3mM CaCl2, 3mM MgCl2, 66mM NaCl, 2.4mM KCl, 4% Glucose (w/v) +- **10X M9**: 420mM Na2HPO4, 220mM KH2PO4, 860mM NaCl, 10mM MgSO4 +- **Q5 Site-Directed Mutagenesis Kit**: NEB, E0554S +- **Lysis buffer**: 50mM KCl, 10mM Tris pH8.3, 2.5mM MgCl2, 0.45% NP40, 0.45% Tween20. Before worm lysis, add proteinase K + +### Design and cloning of the sgRNAs + +1. **Website for sgRNA selection**: Use [CRISPR Design](http://crispr.mit.edu/) +2. Choose sgRNAs close to the modification site and with few off-target sites. + +### Order the following primers: +- **Forward Q5**: (N19)tttttagagtcgaaagtacaagt +- **Reverse Q5**: caagacatctcgaatag +- **Forward sequencing**: tatgaaagtcctcaccccttcc + +--- + +### Design of repair ssODNs + +- The repair oligo should contain flanking bases for homologous recombination. +- For coding regions, use codons with similar frequency as original codons (using [this site](gene site for codon)). +- Engineer a restriction site for facilitating screening and create mutations to avoid cutting by Cas9/sgRNA, if necessary. +- Incorporate suggested protein tags for screening. + +### Constructing PCR Donor Templates for GFP insertion + +- **Nested PCR step**: Amplify the PCR product using a nested step to remove long primers. +- **Optional Dpn1 digestion step**: To eliminate GFP plasmid that could form extrachromosomal arrays and give false positives. + +### PCR Screening Recommendations + +Using different Taq polymerases suggested: + +- **Invitrogen recombinant Taq**: For products that will be processed enzymatically. +- **Promega GoTaq**: For products that don't need post-processing. +- For PCR products >1.5kb: **NEB Phusion 2X Master Mix**. + +--- + +## Protocol + +### Design and cloning of the sgRNAs + +#### Step 1: Design the sgRNAs and order the primers (see the guidelines). + +#### Step 2: Clone the sgRNAs using Q5 mutagenesis kit (NEB). + +**Reagents:** +- [Q5 Site-Directed Mutagenesis Kit - 10 rxns E0554S by New England Biolabs](https://www.neb.com/products/e0554s-q5-site-directed-mutagenesis-kit) + +#### Step 3: Mix together the following: +- **Q5 2X master mix**: 12.5 µl +- **Forward primers 10 µM**: 1.25 µl +- **Reverse primers 10 µM**: 1.25 µl +- **pDD162 from a 1.5ml bacterial culture miniprep**: 0.5 µl +- **H2O**: 9.5 µl + +#### Step 4: Prepare a negative control mix without the Forward primer. + +#### Step 5: Perform PCR as follows: +``` +30s at 98°C +10s at 98°C + 20s at 60°C + 4.30 min at 72°C (25 cycles) +2min at 72°C +10°C forever +``` + +#### Step 6: Digest away pDD162 for 5min at RT with: +- 1 µl of Q5 PCR +- 5 µl of KLD 2X buffer +- 1 µl of KLD 10X enzyme +- 3 µl of H2O +(Duration: 5 minutes) + +#### Step 7: Add 5 µl of digested reaction to 50 µl of competent cells. + +#### Step 8: Heat shock at 42°C for 30s. +(Duration: 30s) + +#### Step 9: Add 950 µl of SOC medium and shake for 1h at 37°C. +(Duration: 1h) + +#### Step 10: Plate 25 µl on Carb plate. + +#### Step 11: Centrifuge remaining 975 µl for 3min at 5K and also plate the pellet. +(Duration: 3 minutes) + +#### Step 12: Pick 6 colonies to grow each in 2 ml of bacterial culture and miniprep. + +**Reagents:** +- **QIAprep Spin Miniprep Kit**: [27104 by Qiagen](https://www.qiagen.com/us/products/discovery-and-translational-research/dna-rna-purification/dna-purification/genomic/ + +#### Step 13: Miniprep and sequence. + +Miniprep kits are from Qiagen; follow the standard protocols. + +**Reagents:** +- QIAprep Spin Miniprep Kit 27104 by [Qiagen](https://www.qiagen.com/us/shop/) + +#### Step 14: Design and order the repair ssODNs as described in the guidelines. + +#### Step 15: Reconstitute oligo at 1 ug/ul according to manufacturer instructions. + +#### Step 16: Amplify the GFP plasmid pCM1.53 (Addgene). Use primers containing the desired flanking regions, mutations in the sgRNA site(s), and GFP sequence. + +**Notes:** +- Ensure GFP is in frame with your ORF. +- Primers used: + - Forward: agtaaaggagaagaacttttcactggagttg + - Reverse: tttgatagtgtcctagccatgtgtaatccc + +#### Step 17: Perform PCR using Phusion taq 2X Master Mix (NEB), 45s elongation, 30 cycles, annealing step with gradient from 60°C to 72°C. + +#### Step 18: Run PCR reactions on agarose gel to confirm amplification. + +#### Step 19: Pool positive PCRs and purify using MinElute PCR purification kit (Qiagen). Measure DNA concentration. + +**Reagents:** +- MinElute PCR Purification Kit 28004 by [Qiagen](https://www.qiagen.com/us/shop/) + +#### Step 20: Pool reactions and purify using one MinElute PCR purification column. + +**Notes:** +- DNA concentration should be >500 ng/ul. +- Oligos can significantly reduce brood size. + +#### Step 21: Use pRF4 roller plasmid at 120 ng/ul. Use any suitable marker. + +**Notes:** +- Identify successfully edited broods. + +#### Step 22: Miniprep bacterial cultures (3 ml) for more than 16 hours. + +**Duration:** 16 hours + +#### Step 23: Mix injection mix. + - pRF4 (120ng/ul) + - Repair template (30ng/ul for a 125nt ssODN, 50ng/ul for a PCR template) + - Cas9/sgRNA clones (50ng/ul) + - Add H2O to 15ul + +--- + +## End of Output + +[endofoutput] +``` \ No newline at end of file diff --git a/markdown-output/secondary-data-analysis-creating-a-mycomap-project-cgqftvtn.md b/markdown-output/secondary-data-analysis-creating-a-mycomap-project-cgqftvtn.md new file mode 100644 index 0000000000000000000000000000000000000000..75cf40303970c4198d7cc52f9c8ed68da97af9a3 --- /dev/null +++ b/markdown-output/secondary-data-analysis-creating-a-mycomap-project-cgqftvtn.md @@ -0,0 +1,117 @@ +```markdown +# Goal/Experiment: +Secondary Data Analysis - Creating a MycoMap Project from ONT Amplicon/Barcode Data V.2 + +## Authors: +- Stephen Douglas Russell, The Hoosier Mushroom Society + +## Abstract: +This protocol outlines many of the steps that can be taken after the primary data analysis to expedite the final analysis - turning your data into results. Topics include creating a dashboard on MycoMap, automated BLAST searches and sequence error flagging, error resolution, linking sequences to iNaturalist observations, submission to GenBank, and many other topics. + +## Full Protocol: +### Validate Your Data +1. Before any further analytical work is done, it is always a good idea to validate the fidelity of your sequences to the iNat/MO records. As a part of the primary data analysis, you linked a spreadsheet of iNat/MO numbers to the unique primer tags for each specimen. Sometimes there are systematic errors in this process - primarily data entry errors or wrong associations with the tags on the spreadsheets. Typically, it is possible to figure out the origin of any systematic error and correct them. + + In order to validate your data before upload to MycoMap, please BLAST at least two random sequences from each plate that was included in your nanopore run. [NCBI BLAST](https://blast.ncbi.nlm.nih.gov/Blast.cgi) will be the quickest way to do this. Do not utilize the first or last sequence from each plate (A1 or H12 well positions). If all of your data checks out, then you are fine to proceed with this protocol. If there are errors, you may need to BLAST more sequences to find the extent of the errors, fix them on your spreadsheet, and rerun the analytical steps so the sequences are associated with the proper observations. + +### Prepare your ONT Data +2. As a part of the "ONT Basecalling, Demultiplexing, and Analysis for Fungal Barcodes" protocol, you ran a python script called `summarize.py`. This created a folder called `__Summary__` in your working folder. + + Change the name of this folder to the title you would like this run to be called. Example - "First_Nanopore_Run" or "ONT.08.11.22." Compress this folder into a `.zip` file. + +3. Depending on the number of samples in your final data, you will need to break it up into individual folders of not more than 250 sequences. This is because we will ultimately be BLASTing each sequence in the results. Running a BLAST on 1,000+ sequences with the NCBI API will take 36 - 48 hours. It also fills up the internal queue for the local BLAST for days. Consider breaking your initial folder from something like "ONT.08.11.22" to "ONT.08.11.22.1," "ONT.08.11.22.2," etc. Within each of these folders you will need to have a FASTQ folder with the appropriate files and you will need to copy and manually edit the `summary.txt` and `summary.fasta` files for each new folder. + +### Create a MycoMap Project +4. Login to MycoMap.com. If you are not a member: + - Register for a username on MycoMap.com: `https://mycomap.com/login` + - Then create a project at `https://mycomap.com/projects/create` Fill out the fields below: + - **Name:** The name of your project. Example: "Mycoflora of Indiana - ONT001" + - **Description:** You can enter text here that describes your project. This text will be shown in the header bar of the project, underneath the project title. + - **Project Type:** Utilize the default of "Personal Working Project." + - **Parent Project:** If you have other working projects, it may be beneficial to create parent and child projects. + - **MycoMap URL Path:** This will generate a custom direct URL for your project. Example: `http://www.mycomap.com/projects/indianamushrooms`. Enter: indianamushrooms into this field. + - **External Project URL:** If you have an external website with information or data that describes your project, you can enter that URL here. + - **Taxon:** If you only want a small group of mushrooms to be shown in this project by default, you can enter a specific taxon. Example - "Amanita." + - **Location:** Allows you to enter a default location for your project. Typically, leave blank for the same reasons the taxon field should be left blank. + - **Owner:** Select a MycoMap username that will be the owner of the project. + - **Enable List View:** This is "checked" by default. + - **Enable Files:** This is "checked" by default, allows the project to host file uploads/downloads. + + Click "Save" to create your project. + + ![New Project Creation](https://mycomap.com/assets/docs/screenshots/new_project.png) + +### Upload your ONT Summary File to the MycoMap Project +5. On the top-right of your MycoMap project, there are a series of green buttons seen below. Click the green "Import" Button. + + ![Import Buttons](https://mycomap.com/assets/docs/screenshots/import_buttons.png) + +6. Click on "Upload," then click on "Zip File of ONT FASTQ and Consensus Sequences." This will bring you to a screen with additional options for uploading ONT data into your project. Fill out the fields below: + - **Zip File:** This is the zipped "Summary" file that was created as a part of the `summarize.py` script after running NGSpeciesID as part of the "ONT Basecalling, Demultiplexing, and Analysis for Fungal Barcodes" protocol. + - **Import for Member:** If you are creating a project for someone else, you can enter their MycoMap username here. + - **Forward Primer:** Enter the forward primer used for the amplicons with the ONT run. Example: "ITS1F." + - **Reverse Primer:** Enter the forward primer used for the amplicons with the ONT run. Example: "ITS4." + - **Run BLAST:** This is "checked" by default. This setting will run a BLAST search and generate a BLAST results page for each sequence included as a part of the ZIP file. + - **Keep Sequences Private:** This will make it so your sequences and raw data are not publicly viewable. + - **GenBank Submission Date:** If you want to flag your sequences with a target date for GenBank upload, it can be entered here. + + Hit "Import" to import your data. + + Note: There should be a check at the initial upload to ensure the ZIP file has a `summary.fasta` and `summary.txt` file. If either are missing, there is a popup message that does not allow you to proceed. + +### Backend Processing After Upload +7. Once this import is triggered, a large series of tasks are created that can take a while to complete. The length of time it takes to complete these tasks is determined by the current speed of the iNaturalist and NCBI API's, as well as the tasks from any other users on MycoMap that were put in before you. You should be able to see a progress task bar at the top of your MycoMap project, so you can track the progress of your upload. + + ![Upload Processing](https://mycomap.com/assets/docs/screenshots/upload_status.png) + + Lets take a quick overview of the things that occur once an upload is initiated. You will be able to track the progress of upload processing with a blue status bar at the top of your project. + +8. A MycoMap sequence record will be created for each sequence in your file. This is a database record that within MycoMap associates the consensus sequence with the raw data (FASTQ file), the RiC (Reads in Consensus), the iNaturalist or Mushroom Observer observation, and a MycoMap species name. An example MycoMap sequence record can be found [here](https://mycomap.com/genetics/sequences/ont_sequences/ONT_example_record). + +9. If the sequence has an RiC of 8 reads or less, then the sequence is flagged as being of particularly Low Quality and placed in a special "ONT Trashcan" category. + +### Multiple Sequences for an Observation +10. There is sometimes more than one sequence associated with an individual observation as a result of this pipeline. The most I have seen is twelve. These are typically a result of planned bioinformatic processing or sequencing error but can also be from lab contamination. + + These errors can often be elucidated with some investigation. Once the reason for the mixup is discovered, the sequence record should be associated with the right observation and this flag removed. + +### Analytical Process +11. The MycoMap project will have a status bar that tracks the progress of the sequence upload and subsequent BLAST searches. Wait until all tasks are completed, as the final summary spreadsheet is not able to be updated until all BLAST results are in. + + If there are individual sequences you are interested in, you can search and view them on the dashboard (by species name or iNat number) while other BLAST results finish. + +### GenBank Submissions +12. Click on the "GenBank" button at the top of the MycoMap dashboard. The GenBank submission management screen will appear. + Fill in the "Isolate Prefix" field with your name and ONT. This allows you to quickly view your sequences in GenBank and to know the sequencing methodology that was employed. + +13. Next, begin examining the dashboard of your project for individual sequence-observation associations. + Click on the BLAST "B" next to an individual record line. The summary fields at the top will show if there is a single species name in the boxes for both NCBI and MycoMap. +14. If you believe you have a good match and the name on iNaturalist is NOT correct, but the correct name is available on iNat: + Click the title of the record on the dashboard. Update the name on iNaturalist. If unable to independently update, add the species name to the "Provisional Species Name" observational field. + Refresh the record on the MycoMap dashboard. + +### Prepare your record for GenBank upload +15. Next to the BLAST "B" icon on your dashboard, there is the NCBI icon - a small blue double helix. Click this to open the GenBank validation screen. + Validate details like Species Name, Collection date etc. + Fix quality errors (flags) if present. + +### Issue: Multiple Sequences for an Observation +16. If multiple sequences are associated with an observation, manually review BLAST results and decide on the probable correct sequence. +17. Problematic sequences such as low-quality sequences, contaminants, wrong orientation, and chimeras can impact results. Trim or discard accordingly. If sequences are consistently problematic, review the primary analysis protocol to tighten conditions or rerun with corrected inputs. + +### Upload your Sequences to GenBank +18. Following this protocol, you now have validated records. Go to the GenBank Submission Portal. +19. Download newly generated files in your MycoMap project and upload them to GenBank. + +### Submission of the Accession report to MycoMap +20. Once sequences are accepted at GenBank, download the AccessionReport.tsv from GenBank. +Upload it back to MycoMap to link GenBank accessions back to iNaturalist records. + +### Import data back to iNaturalist +21. Ensure all sequences and data are pushed back to iNaturalist: + - Import -> Refresh -> iNat observational fields -> Entire project +22. Push data back to iNaturalist to ensure updated records: + - Import -> Refresh -> iNaturalist Observational Fields + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/semi-automated-protocol-to-quantify-and-characteri-cffztjp6.md b/markdown-output/semi-automated-protocol-to-quantify-and-characteri-cffztjp6.md new file mode 100644 index 0000000000000000000000000000000000000000..957f5873ed61dc8e6bc91549fa0c71c98bdd85ef --- /dev/null +++ b/markdown-output/semi-automated-protocol-to-quantify-and-characteri-cffztjp6.md @@ -0,0 +1,161 @@ +```markdown +# Goal/Experiment: +The goal of this experiment is to quantify and characterize fluorescent three-dimensional vascular images using a semi-automated protocol. This allows for a comprehensive analysis of vascular structures in various tissues, notably in the brain, providing insight into vascular integrity and pathology. + +# Semi-Automated Protocol to Quantify and Characterize Fluorescent Three-Dimensional Vascular Images + +## Authors +* Danny F Xie1,2 +* Christian Crouzet1,2 +* Krystal LoPresti1,2 +* Yuke Wang1,2 +* Christopher Robinson1,2 +* William Jones1 +* Fjolla Muqolli1 +* David H. Cribbs4 +* Chuo Fang3 +* Mark Fisher1,3,4,5 +* Bernard Choi1,2 + +**Affiliations:** +1. Beckman Laser Institute, University of California-Irvine +2. Department of Biomedical Engineering, University of California-Irvine +3. Department of Neurology, University of California-Irvine +4. Institute for Memory Impairments and Neurological Disorders, University of California-Irvine +5. Department of Pathology & Laboratory Medicine, University of California-Irvine + +## Abstract +The microvasculature regulates blood flow and oxygen delivery and its abnormalities can indicate diseases. This protocol describes a 3D imaging method to visualize the vascular architecture in tissue using fluorescent labeling and optical clearing. This method can be applied to other tissue samples to generate 3D images of microvasculature. + +## Materials + +### Reagents +* Saline solution (Aspen Veterinary Resources): NDC No. 46066-807-50 +* 10% formalin solution (Sigma-Aldrich): Product Code HT50-1-1 +* PBS with 0.02% sodium azide solution (Syringa Lab Supplies): Part No. 11001 +* Potassium ferrocyanide (Sigma): P3289-500G +* Hydrochloric acid (Fisher): A144-212 +* Methanol (Fisher): A412S +* Deionized water +* Dichloromethane (Sigma): 270997-100ML +* Dibenzyl ether (Sigma): 108014-1KG + +### Equipment +* 2x 10 mL syringes (Sigma-Aldrich): Z683604 +* 1x 23G x ¾ x 12” butterfly needle (Vacutec): 26766 +* 3x hemostat clamps (Excelta): 37SE +* 1x bone scissors (Fine Science Tools): 91604-09 +* 1x forceps (Fine Science Tools): 11000-12 +* 1x scissors (Excelta): 290 +* 1x angled scissors (Fine Science Tools): 15010-10 +* 1x spatula (Fine Science Tools): 10089-11 +* 1x syringe pump (Harvard Apparatus Model 11 Plus) +* 1x 20 mL glass scintillation vial (Grainger): 3LDT2 +* Aluminum foil +* Tape +* Liquid absorbent mats +* Isoflurane chamber (E-Z Systems): EZ-178 +* Nose cone (E-Z Systems): EZ-103A +* Magnetic stir plate with stir bar (Corning PC 353 Stirrer) +* Orbital shaker (Scilogex): SK-D1807-E +* Scale +* Weigh boats +* Pipette controller (Grainger): 49WF85 +* Serological pipette tips +* 1.5 mL opaque microcentrifuge tube (Argos Technologies): 06333-80 +* Microcentrifuge tube rack +* Pipettes +* 18-gauge needle +* Leica TCS SP8 microscope + +### Software +* MATLAB: [link](https://www.mathworks.com/) +* FIJI: [link](https://imagej.net/Fiji) +* neuTube: [link](https://www.neutracing.com/) + +## Reagent Setup +* **10% Potassium Ferrocyanide (PF) Solution:** + * Dissolve 10 g of PF in 100 mL deionized water (DIW). + * Use a magnetic stir bar and magnetic stir plate to dissolve. +* **20% Hydrochloric Acid (HCl) Solution:** + * Dilute stock HCl in DIW (20 mL of stock HCl per 80 mL DIW) to make 20% HCl. + * Perform this step under a fume hood. +* **Potassium Ferrocyanide/Hydrochloric Acid Working Solution:** + * Mix 1 part 10% PF solution with 1 part 20% HCl solution. + +## Protocol + +### 1. Cardiac Perfusion and Retroorbital Injection (45 min) +#### 1.1 Cardiac Perfusion +1.1 Anesthetize a mouse using an isoflurane chamber (1.5 L/min oxygen, 4.0% isoflurane). +1.2 Place the mouse's snout in a nose cone with 1.5 L/min oxygen and 1.5% isoflurane. +1.3 Administer lectin-DyLight-649 solution (200 µL, 25% lectin-DyLight and 75% saline) via retroorbital injection. +1.4 Allow the solution to circulate for 20 minutes before cardiac perfusion. +1.5 Perform cardiac perfusion on a surgical tray. + * Confirm the mouse's anesthesia with toe/tail pinches. +1.6 Open the chest cavity with a horizontal incision below the rib cage and vertical incisions along the chest sides. +1.7 Use hemostats to hold the chest open. +1.8 Perform a small incision on the right atrium to let blood exit. +1.9 Insert a butterfly needle into the left ventricle, perfuse 10 mL of saline at 2 mL/min. +1.10 Perfuse 10 mL of formalin at 2 mL/min. + +### 2. Brain Extraction (10 min) +2.1 Remove the head by cutting caudally of the skull with scissors. +2.2 Cut the scalp to create two folds exposing the cranium. +2.3 Using angled microscissors, cut upwards from the foramen magnum to bregma. +2.4 Make a lateral cut rostral to the olfactory bulbs to split the skull. +2.5 Use tweezers or a spatula to pry open each skull hemisphere. +2.6 Separate the brain from the skull base with a spatula, severing any connecting nerves. +2.7 Submerge the brain in ~10 mL of 10% formalin, store away from light. +2.8 After 24 hours, store the brain in PBS with 0.02% sodium azide at 4°C until further processing. + +### 3. Exogenous Labeling of Hemosiderin with Prussian Blue (1h 30min) +3.1 Prepare 10% potassium ferrocyanide solution (10 g in 100 mL DIW). +3.2 Use a magnetic stir plate to mix for 20 minutes. +3.3 Prepare 20% hydrochloric acid solution. +3.4 Mix potassium ferrocyanide and hydrochloric acid solutions 1:1. +3.5 Wash samples in 5 mL DIW with shaking. +3.6 Submerge samples in 5 mL potassium ferrocyanide/hydrochloric acid solution for 1 hour. +3.7 Perform a final DIW wash for 5 minutes. +3.8 Store samples in PBS with 0.02% sodium azide at 4°C and away from light. + +### 4. Tissue Clearing (3h 30min) +4.1 Perform methanol washes (20%, 40%, 60%, 80%, 100%, DIW) for 20 minutes each with shaking. +4.2 Incubate samples in 66% dichloromethane + 33% methanol for one hour with shaking. +4.3 Incubate in dichloromethane twice for 15 minutes with shaking. +4.4 Store samples in dibenzyl ether at 4°C until imaging. + +### 5. Imaging with Confocal Microscopy +5.1 Image vasculature labeled with lectin-DyLight-649 (633 nm emission band 650-750 nm). +5.2 Visualize microhemorrhages labeled with Prussian blue using a white light source. +5.3 Collect fluorescent and transmittance images to co-register vascular fluorescence with cerebral microhemorrhages. + +![Workflow Image](image_url) +**Figure 1: Workflow for 3D visualization of cerebral microvasculature.** + +5.4 The protocol is transferable to other organs. Light-sheet microscopy is an alternative to confocal microscopy. + +### 6. Vascular Segmentation +6.1 Use a 3x3x1 median filter to remove noise. +6.2 Apply the iterative selection threshold method. + Formula: + \( T_0 = \frac{Max + Min}{2} \) +6.3 Optional morphological operations based on image acquisition parameters. + +![Threshold Schematic](image_url) +**Figure 3: Schematic of iterative selection thresholding.** + +### 7. neuTube Tracing to Quantify Vessel Diameters +7.1 Perform automated tracing on binarized vasculature images in TIF format. neuTube will output SWC formatted data detailing x, y, z coordinates, radius, and node connectivity. + +## Citations +* [Prabhakar et al. (2021)](https://doi.org/10.1538/expanim.20-0186) +* [Gage et al. (2012)](https://doi.org/10.3791/3564) +* [Renier et al. (2014)](https://doi.org/10.1016/j.cell.2014.10.010) +* [Khouri et al. (2021)](https://doi.org/10.1117/1.NPh.8.2.025004) +* [Feng et al. (2015)](https://doi.org/pii:ENEURO.0049-14.2014.10.1523/ENEURO.0049-14.2014) + +--- + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/sensifast-probe-hi-rox-kit-fysbpwe.md b/markdown-output/sensifast-probe-hi-rox-kit-fysbpwe.md new file mode 100644 index 0000000000000000000000000000000000000000..20f1432568c5d6028c349ea96871fdd0c50ee23d --- /dev/null +++ b/markdown-output/sensifast-probe-hi-rox-kit-fysbpwe.md @@ -0,0 +1,101 @@ +```markdown +# Goal/Experiment: +Optimize and utilize the SensiFAST™ Probe Hi-ROX Kit for fast, highly reproducible real-time PCR. + +## SensiFAST™ Probe Hi-ROX Kit + +### Abstract + +The SensiFAST™ Probe Hi-ROX Kit has been developed for fast, highly reproducible real-time PCR and has been validated on commonly used real-time PCR instruments. The kit has been formulated for use with probe-detection technology, including TaqMan®, Scorpions®, and molecular beacon probes. A combination of the latest advances in buffer chemistry and PCR enhancers, together with a hot-start DNA polymerase, ensures that the SensiFAST Probe Kit delivers fast, highly-specific, and ultra-sensitive real-time PCR. + +SensiFAST Probe is provided as a 2x mastermix containing all the components necessary for real-time PCR, including dNTPs, stabilizers, and enhancers. + +#### Citation +Bioline SensiFAST™ Probe Hi-ROX Kit. protocols.io dx.doi.org/10.17504/protocols.io.fysbpwe +Published: 24 Nov 2016 + +### Guidelines + +#### Kit Components + +| Reagent | 200 x 20 µL reactions | 500 x 20 µL reactions | 2000 x 20 µL reactions | +|---------------------------------|-----------------------|-----------------------|------------------------| +| SensiFAST Probe Hi-ROX mix (2x) | 2 x 1 mL | 5 x 1 mL | 4 x 5 mL | + +#### Instrument Compatibility + +The SensiFAST Probe Hi-ROX Kit has been optimized for use with all probe chemistries, including TaqMan, FRET, Scorpions, and molecular beacon probes on real-time PCR instruments listed in the following compatibility table. Each of these instruments can analyze the real-time PCR data with the passive reference signal either on or off. The kit is also compatible with several instruments that do not require the use of ROX, such as the Mic (BMS), Qiagen (Corbett) Rotor-Gene™ 6000, the Bio-Rad CFX96 or the Roche LightCycler® 480. + +**Manufacturer Model**: ABI (Invitrogen) 7000, 7300, 7700, 7900, 7900HT, StepOne™ and StepOne™ Plus. + +### General Considerations + +To help prevent any carry-over DNA contamination, we recommend maintaining separate areas for reaction set-up, PCR amplification, and any post-PCR gel analysis. It is essential that any tubes containing amplified PCR product are not opened in the PCR set-up area. + +**Primers and Probe**: These guidelines refer to the design and set-up of TaqMan probe-based PCR. Please refer to the relevant literature when using other probe types. The specific amplification, yield, and overall efficiency of any real-time PCR can be critically affected by the sequence and concentration of the probes and primers, as well as by the amplicon length. + +We strongly recommend considering the following points when designing and running your real-time PCR: + +- Use primer-design software, such as Primer3 ([http://frodo.wi.mit.edu/primer3/](http://frodo.wi.mit.edu/primer3/)) or visual OMP™ ([http://dnasoftware.com/](http://dnasoftware.com/)). Primers should have a melting temperature (Tm) of approximately 60°C; the Tm of the probe should be approximately 10°C higher than that of the primers. +- Optimal amplicon length should be 80-200 bp, and should not exceed 300 bp. +- Final primer concentration of 400 nM is suitable for most Probe-based reactions. For optimal concentration, titrate in the range 0.2-1 µM. Forward and reverse primers concentration should be equimolar. +- A final probe concentration of 100 nM is suitable for most applications; we recommend that the final probe concentration is at least 2-fold lower than the primer concentration. + +**Template**: It is important that the DNA template is suitable for use in PCR in terms of purity and concentration. The template must be devoid of any contaminating PCR inhibitors (e.g. EDTA). The recommended amount of template for PCR is dependent upon the type of DNA used: + +- **Genomic DNA**: Use up to 1 µg of complex (e.g. eukaryotic) genomic DNA in a single PCR; we recommend using the Bioline ISOLATE II Genomic DNA Kit (BIO-52066) for high yield and purity from both prokaryotic and eukaryotic sources. +- **cDNA**: The optimal amount of cDNA to use in a single PCR is dependent upon the copy number of the target gene; we suggest using 100 ng cDNA per reaction, but this may need variation. For two-step RT-PCR, use the SensiFAST cDNA Synthesis Kit (BIO-65042) for reverse transcription of purified RNA. For high yield and purity of RNA, use the Bioline ISOLATE II RNA Mini Kit (BIO-52072). + +**MgCl₂**: The SensiFAST Probe mix contains an optimized concentration of MgCl₂, further supplementation is unnecessary. + +**PCR Controls**: Always include a no-template control (NTC) reaction to detect contaminating DNA that may affect the data's reliability. Replace the template with PCR-grade water in the NTC. For two-step RT-PCR, set up an RT control well as an NTC. + +**Optional ROX**: The SensiFAST Probe Hi-ROX Kit is premixed with ROX (5-carboxy-X-rhodamine, succinimidyl ester). ROX fluorescence can be optionally detected on certain real-time instruments capable of using ROX. + +### Troubleshooting Guide + +For detailed troubleshooting instructions, see the Bioline full documentation: +[http://www.bioline.com/us/downloads/dl/file/id/2787/sensifast_probe_hi_rox_kit_manual.pdf](http://www.bioline.com/us/downloads/dl/file/id/2787/sensifast_probe_hi_rox_kit_manual.pdf) + +### Materials + +- SensiFAST™ Probe No-ROX Kit [BIO-86005](https://www.bioline.com/us/product-info/sensifast-probe-no-rox-kit-bio-86005) + +### Protocol + +#### Reaction Mix Composition + +**Step 1.** +Prepare a PCR mastermix. The volumes given below are based on a standard 20 µL final reaction mix and can be scaled accordingly. + +| Reagent | Volume | Final Concentration | +|---------------------------------|--------|---------------------| +| 2x SensiFAST Probe Hi-ROX Mix | 10 µL | 1x | +| 10 µM Forward Primer | 0.8 µL | 400 nM | +| 10 µM Reverse Primer | 0.8 µL | 400 nM | +| 10 µM Probe | 0.2 µL | 100 nM | +| Template | Up to 8.2 µL | - | +| H₂O | As required | - | + +#### Sensitivity Testing and Ct Values + +**Step 2.** +When comparing SensiFAST with a mix from another supplier, we strongly recommend using a 10-fold template dilution series for low template conditions. Loss of detection at low template concentration is the only direct measurement of sensitivity. An early Ct value is not an indication of good sensitivity, but rather an indication of speed. + +#### Suggested Thermal Cycling Conditions + +**Step 3.** +The real-time PCR conditions, in the table below, are suitable for the SensiFAST Probe Hi-ROX Kit with amplicons up to 200 bp. Cycline parameters have been optimized for numerous platforms but can be varied to suit specific machine protocols. + +| Cycles | Temp. | Time | Notes | +|--------|--------------|--------------------|---------------------------------------------------| +| 1 | 95°C | 2-5 min* | Polymerase activation | +| 40 | 95°C, 60°C | 10s, 20-50s** | Denaturation, Annealing/extension (acquire at end of step) | + +\*2 min for cDNA, up to 5 min for genomic DNA +\**Up to 50s may be necessary for multiplexing with more than two probes + +--- + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/sensifast-probe-hi-rox-one-step-kit-fyubpww.md b/markdown-output/sensifast-probe-hi-rox-one-step-kit-fyubpww.md new file mode 100644 index 0000000000000000000000000000000000000000..589c6a0df19e478ecfa71c128d02bfb0416b7a00 --- /dev/null +++ b/markdown-output/sensifast-probe-hi-rox-one-step-kit-fyubpww.md @@ -0,0 +1,135 @@ +```markdown +# Goal/Experiment: +The goal of this experiment is to perform highly reproducible first-strand cDNA synthesis and subsequent real-time PCR in a single tube, using the SensiFAST™ Probe Hi-ROX One-Step Kit by Bioline. + +## SensiFAST™ Probe Hi-ROX One-Step Kit + +### Abstract + +The SensiFAST™ Probe Hi-ROX One-Step Kit has been formulated for highly reproducible first-strand cDNA synthesis and subsequent real-time PCR in a single tube. The kit is formulated for use with probe-detection technology, including TaqMan®, Scorpions® and molecular beacon probes. A combination of the latest advances in buffer chemistry together with a reverse transcriptase and hot-start DNA polymerase system, ensures that SensiFAST Probe Hi-ROX One-Step Kit produces fast, highly-specific and ultra-sensitive one-step real-time RT-PCR. + +The SensiFAST Probe Hi-ROX One-Step Kit consists of a 2x SensiFAST Probe Hi-ROX One-Step mix, separate reverse transcriptase and RiboSafe RNase Inhibitor. + +Citation: Bioline SensiFAST™ Probe Hi-ROX One-Step Kit. protocols.io +dx.doi.org/10.17504/protocols.io.fyubpww + +Published: 24 Nov 2016 + +## Guidelines + +### Kit Components + +| Reagent | 100 x 20 μL reactions | 500 x 20 μL reactions | +| ------------------------------------ | --------------------- | --------------------- | +| SensiFAST™ Probe Hi-ROX One-Step mix (2x) | 1 x 1 mL | 5 x 1 mL | +| RiboSafe RNase Inhibitor | 1 x 40 μL | 1 x 200 μL | +| Reverse transcriptase | 1 x 20 μL | 1 x 100 μL | +| DEPC-H₂O | 1 x 1.8 mL | 2 x 1.8 mL | + +### Instrument Compatibility + +The SensiFAST Probe Hi-ROX One-Step Kit has been optimized for use with all probe chemistries, including TaqMan, FRET, Scorpions and molecular beacon probes on real-time PCR instruments listed in the following compatibility table, each of these instruments having the capacity to analyze the real-time PCR data with the passive reference signal either on or off. The kit is also compatible with several instruments that do not require the use of ROX, such as the Qiagen (Corbett) Rotor-Gene™ 6000, the Bio-Rad CFX96 or the Roche LightCycler® 480. + +| Manufacturer | Model | +| ---------------------- | --------------------------------------------------------------------- | +| ABI (Invitrogen) | 7000, 7300, 7700, 7900, 7900HT, StepOneTM and StepOne™ Plus | + +### General Considerations + +When handling RNA, it is important to use RNase-free plasticware and reagents. We also recommend performing RNA work in an RNase-free area. To help prevent any carry-over DNA contamination, we recommend that separate areas are maintained for reaction set-up, PCR amplification and any post-PCR gel analysis. It is essential that any tubes containing amplified DNA product are not opened in the reaction set-up area. + +### Primers and Probe + +These guidelines refer to the use of dual-labeled probes. Please refer to the relevant literature when using other probe types. The sequence and concentration of the probe and primers, as well as amplicon length, can be critical for specific amplification, yield and overall efficiency of any real-time RT-PCR. + +We strongly recommend taking the following points into consideration when designing and running your real-time RT-PCR: + +- Use primer-design software, such as Primer3 or visual OMP™ ([Primer3](http://frodo.wi.mit.edu/primer3/) and [DNA Software, Inc.](http://dnasoftware.com/), respectively). Primers should have a melting temperature (Tm) of approximately 60°C. The Tm of the probe should be approximately 10°C higher than that of the primers. +- Optimal amplicon length should be 80-200 bp, and should not exceed 400 bp. +- Final primer concentration of 400 nM is suitable for most probe reactions. However, to determine the optimal concentration, we recommend titrating in the range 0.2-1 μM. +- Use an equimolar primer concentration. +- A final probe concentration of 100 nM is suitable for most applications. We recommend that the final probe concentration is at least 2-fold lower than the primer concentration. + - **Note**: In multiplex real-time RT-PCR, probe concentrations in excess of 100 nM can result in cross-channel fluorescence. +- Where possible, use intron-spanning primers to avoid amplification from genomic DNA. + +### Template + +It is important that the RNA template is intact and devoid of DNA or contaminating inhibitors of both reverse transcription and PCR. For high-purity RNA, we recommend using the Bioline ISOLATE II RNA Mini Kit (BIO-52073). RNA stocks and dilutions should be made in DEPC-treated Water to avoid any RNase-mediated degradation. + +The recommended amount of template for one-step real-time RT-PCR is dependent upon the type of RNA used: + +- **total RNA**: purified total RNA can be used in the range from 1 pg to 1 μg per 20 μL reaction +- **mRNA**: purified mRNA can be used from 0.01 pg per 20 μL reaction + +### MgCl₂ + +The MgCl₂ concentration in the 1x reaction mix is 3 mM. In the majority of real-time RT-PCR conditions, this is optimal for both the reverse transcriptase and the hot-start DNA polymerase. If necessary, we suggest titrating the MgCl₂ to a maximum of 5 mM. + +### RT-PCR Controls + +It is important to detect the presence of contaminating DNA that may affect the reliability of the data. Always include a no-RT control reaction, by omitting the reverse transcriptase from the reaction. + +### Optional ROX + +The SensiFAST Probe Hi-ROX One-Step Kit is premixed with ROX (5-carboxy-X-rhodamine, single isomer), so that ROX fluorescence can be optionally detected on certain real-time instruments. If your real-time PCR instrument has the capability of using ROX and you wish to use this option, then this option must be selected by the user in the software. + +### Troubleshooting Guide +See the Bioline full documentation for detailed troubleshooting instructions. + +[Bioline Troubleshooting Guide](http://www.bioline.com/us/downloads/dl/file/id/3301/sensifast_probe_hi_rox_one_step_kit_manual.pdf) + +## Materials +- SensiFAST™ Probe Hi-ROX One-Step Kit [BIO-77001](http://www.bioline.com) by Bioline + +## Protocol + +### Reaction Mix Composition + +#### Step 1. + +Prepare an real-time RT-PCR mastermix. The volumes given below are based on a standard 20 μL final reaction mix and can be scaled accordingly. + +| Reagent | Volume | Final Concentration | +| ------------------------------------- | ------ | ------------------- | +| 2x SensiFAST Probe Hi-ROX One-Step Mix| 10 μL | 1x | +| 10 μM Forward Primer | 0.8 μL | 400 nM | +| 10 μM Reverse Primer | 0.8 μL | 400 nM | +| 10 μM Probe | 0.2 μL | 100 nM | +| Reverse transcriptase | 0.2 μL | - | +| RiboSafe RNase Inhibitor | 0.4 μL | - | +| H₂O | up to 16 μL | - | +| Template | 4 μL | - | +| **Total Volume** | **20 μL** | - | + +### Sensitivity Testing and Ct Values + +#### Step 2. + +When comparing SensiFAST with a mix from another supplier, we strongly recommend amplifying from a 10-fold template dilution series. Loss of detection at low template concentration is the only direct measurement of sensitivity. An early Ct value is not an indication of good sensitivity, but rather an indication of speed. + +### Suggested Real-Time RT-PCR Conditions + +#### Step 3. + +The following real-time RT-PCR conditions are suitable for the SensiFAST Probe Hi-ROX One-Step Kit with the majority of amplicons and real-time PCR instruments. However, the cycling conditions can be varied to suit different probe-based reactions or machine-specific protocols. The detection channel on the real-time instrument should be set to acquire at the appropriate wavelength(s). We recommend using the following cycling conditions for optimal results: + +**Cycling for Dual-Labeled Probes** + +| Cycles | Temp. | Time | Notes | +| ------ | ----- | ---- | --------------------------- | +| 1 | 45°C | 10 min | Reverse transcription | +| 1 | 95°C | 2 min | Polymerase activation | +| 40 | 95°C | 5s | Denaturation | +| | 60°C | 20s | Annealing/extension (acquire at end of step) | + +### Real-Time RT-PCR Optimization + +#### Step 4. + +The following optimization may be necessary to improve the efficiency of some reactions, such as multiplexing with more than two probes, or if the target amplicon is longer than 200 bp. + +- The reverse transcription reaction time can be extended up to 20 minutes and/or the temperature can be increased up to 48°C. +- The annealing/extension time can be extended up to 60 seconds and/or the temperature can be increased up to 65°C. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/sensifast-probe-lo-rox-one-step-kit-fywbpxe.md b/markdown-output/sensifast-probe-lo-rox-one-step-kit-fywbpxe.md new file mode 100644 index 0000000000000000000000000000000000000000..2b1dba64d3abbecf7e9e216630f201f4b4a42595 --- /dev/null +++ b/markdown-output/sensifast-probe-lo-rox-one-step-kit-fywbpxe.md @@ -0,0 +1,106 @@ +```markdown +# Goal/Experiment: +The aim of this experiment is to perform highly reproducible first-strand cDNA synthesis and subsequent real-time PCR in a single tube using the SensiFAST™ Probe Lo-ROX One-Step Kit. This protocol is formulated for use with various probe-detection technologies such as TaqMan®, Scorpions®, and molecular beacon probes, enabling precise, fast, highly-specific, and ultra-sensitive one-step real-time RT-PCR. + +# SensiFAST™ Probe Lo-ROX One-Step Kit + +## Abstract +The SensiFAST™ Probe Lo-ROX One-Step Kit has been formulated for highly reproducible first-strand cDNA synthesis and subsequent real-time PCR in a single tube. The kit is formulated for use with probe-detection technology, including TaqMan®, Scorpions® and molecular beacon probes. A combination of the latest advances in buffer chemistry together with a reverse transcriptase and hot-start DNA polymerase system ensures that SensiFAST™ Probe Lo-ROX One-Step Kit produces fast, highly-specific and ultra-sensitive one-step real-time RT-PCR. + +## Kit Components +The SensiFAST™ Probe Lo-ROX One-Step Kit consists of a 2x SensiFAST Probe One-Step mix, separate reverse transcriptase and RiboSafe RNase Inhibitor. + +| Reagent | 100 x 20 µL reactions | 500 x 20 µL reactions | +|-------------------------------|-----------------------------------|-----------------------------------| +| SensiFAST™ Probe One-Step mix (2x) | 1 x 1 mL | 5 x 1 mL | +| RiboSafe RNase Inhibitor | 1 x 40 µL | 1 x 200 µL | +| Reverse transcriptase | 1 x 20 µL | 1 x 100 µL | +| DEPC-H2O | 1 x 1.8 mL | 2 x 1.8 mL | + +## Instrument Compatibility +The SensiFAST™ Probe Lo-ROX One-Step Kit has been optimized for use with all probe chemistries, including TaqMan, FRET, Scorpions and molecular beacon probes on the real-time PCR instruments listed below. Each of these instruments has the capacity to analyze the real-time PCR data with the passive reference signal either on or off: +- ABI (Invitrogen): 7500, 7500 FAST +- Stratagene (Agilent): Mx4000™, Mx3000P™, Mx3005P™ +- Qiagen (Corbett) Rotor-Gene™ 6000 +- Bio-Rad CFX96 +- Roche LightCycler® 480 +- Mic (BMS) + +## General Considerations +When handling RNA, it is critical to use RNase-free plasticware and reagents. RNA work should occur in an RNase-free area to avoid carry-over DNA contamination. Maintain separate areas for reaction set-up, PCR amplification, and any post-PCR gel analysis, ensuring that amplified DNA product is not opened in the PCR set-up area. + +## Primers and Probes +Guidelines use dual-labeled probes. Refer to relevant literature for other probe types. Design primers and probes to optimize specific amplification, yield, and efficiency of the real-time RT-PCR. + +Consider: +- Use primer-design software like Primer3 or visual OMP™. Primers should have a Tm of ≈ 60°C. Probe Tm should be ≈ 10°C higher. +- Optimal amplicon length: 80-200 bp, should not exceed 400 bp. +- Final primer concentration: 400 nM; titrate 0.2-1 µM if needed. +- Use equimolar primer concentrations. +- Final probe concentration: 100 nM; ensure it is at least 2-fold lower than primer concentration to avoid cross-channel fluorescence in multiplex PCR. + +## Template +RNA template should be intact and free from contaminants. For high purity RNA, use Bioline ISOLATE II RNA Mini Kit (BIO-52073). Use DEPC-treated water to avoid RNase degradation. + +Amount for one-step real-time RT-PCR: +- Total RNA: 1 pg to 1 µg per 20 µL reaction. +- mRNA: 0.01 pg per 20 µL reaction. + +## MgCl2 +MgCl2 concentration in 1x reaction mix is 3 mM, optimal for reverse transcriptase and hot-start DNA polymerase. Adjust up to 5 mM if necessary. + +## RT-PCR Controls +Include a no-RT control reaction by omitting reverse transcriptase to detect DNA contamination. + +## Optional ROX +The kit is premixed with ROX (5-carboxy-X-rhodamine) for optional fluorescence detection. Select this in the software if needed by your real-time instrument. + +## Troubleshooting Guide +For detailed instructions, see the Bioline full documentation (URL provided). + +## Materials +- [SensiFAST™ Probe Lo-ROX One-Step Kit BIO-78001 by Bioline](http://www.bioline.com/us/downloads/dlf/file/id/3302/sensifast_probe_lo_rox_one_step_kit_manual.pdf) + +## Protocol + +### Reaction Mix Composition +#### Step 1: Reaction Mix Preparation +Prepare a real-time RT-PCR mastermix. Scale volumes accordingly based on 20 µL final reaction mix. + +| Reagent | Volume | Final Concentration | +|--------------------------------|--------|---------------------| +| 2x SensiFAST Probe Lo-ROX One-Step Mix | 10 µL | 1x | +| 10 µM Forward Primer | 0.8 µL | 400 nM | +| 10 µM Reverse Primer | 0.8 µL | 400 nM | +| 10 µM Probe | 0.2 µL | 100 nM | +| Reverse transcriptase | 0.2 µL | - | +| RiboSafe RNase Inhibitor | 0.4 µL | - | +| H2O | up to 16 µL | - | +| Template | 4 µL | - | +| **Total Volume** | **20 µL** | - | + +### Sensitivity Testing and Ct Values +#### Step 2: Sensitivity Testing +Compare SensiFAST with another supplier’s mix using a 10-fold template dilution series. Loss of detection at low template concentration directly measures sensitivity. + +### Suggested Real-time RT-PCR Conditions +#### Step 3: PCR Conditions +These conditions are suitable for most amplicons and instruments. Adjust based on specific protocols and probes: + +- **Cycling for dual-labeled probes** + +| Cycles | Temp. | Time | Notes | +|--------|--------|-------------|---------------------------| +| 1 | 45°C | 10 min | Reverse transcription | +| 1 | 95°C | 2 min | Polymerase activation | +| 40 | 95°C | 5 s | Denaturation | +| | 60°C | 20 s | Annealing/extension (acquire at end of step) | + +### Real-time RT-PCR Optimization +#### Step 4: Optimization +Potential adjustments: +- Increase reverse transcription reaction time up to 20 minutes and/or temperature up to 48°C. +- Extend annealing/extension time up to 60 seconds and/or increase temperature up to 65°C. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/sensifast-probe-no-rox-one-step-kit-fyybpxw.md b/markdown-output/sensifast-probe-no-rox-one-step-kit-fyybpxw.md new file mode 100644 index 0000000000000000000000000000000000000000..5cd5ae524ec21f40977dc85591efdad99fb01751 --- /dev/null +++ b/markdown-output/sensifast-probe-no-rox-one-step-kit-fyybpxw.md @@ -0,0 +1,119 @@ +```markdown +# Goal/Experiment: +The objective of this experiment is to utilize the SensiFAST™ Probe No-ROX One-Step Kit for the highly reproducible first-strand cDNA synthesis and subsequent real-time PCR in a single tube. The experiment is designed to take advantage of the optimized buffer chemistry and enzyme systems for high specificity and sensitivity in molecular detection assays. + +# SensiFAST™ Probe No-ROX One-Step Kit + +## Abstract +The SensiFAST™ Probe No-ROX One-Step Kit has been formulated for highly reproducible first-strand cDNA synthesis and subsequent real-time PCR in a single tube. The kit is formulated for use with probe-detection technology, including TaqMan®, Scorpions® and molecular beacon probes. A combination of the latest advances in buffer chemistry together with a reverse transcriptase and hot-start DNA polymerase system ensures that SensiFAST Probe No-ROX One-Step Kit produces fast, highly specific and ultra-sensitive one-step real-time RT-PCR. + +The SensiFAST Probe No-ROX One-Step Kit consists of a 2x SensiFAST Probe One-Step mix, separate reverse transcriptase and RiboSafe RNase Inhibitor. + +## Guidelines + +### Kit Components + +| Reagent | 100 x 20μL reactions | 500 x 20μL reactions | +|--------------------------------------|--------------------------------------|--------------------------------------| +| SensiFAST™ Probe No-ROX One-Step mix (2x) | 1 x 1 mL | 5 x 1 mL | +| RiboSafe RNase Inhibitor | 1 x 40 μL | 1 x 200 μL | +| Reverse transcriptase | 1 x 20 μL | 1 x 100 μL | +| DEPC-H₂O | 1 x 1.8 mL | 2 x 1.8 mL | + +### Instrument compatibility + +The SensiFAST Probe No-ROX One-Step Kit has been optimized for use with all probe chemistries, including TaqMan, FRET, Scorpions and molecular beacon probes. It can be used on all real-time PCR instruments. + +### General Considerations + +When handling RNA, it is important to use RNase-free plasticware and reagents. We also recommend performing RNA work in an RNase-free area. To help prevent any carry-over DNA contamination, separate areas should be maintained for reaction set-up, PCR amplification, and any post-PCR gel analysis. It is essential that any tubes containing amplified DNA product are not opened in the reaction set-up area. + +### Primers and Probes + +These guidelines refer to the use of dual-labeled probes. The sequence and concentration of the probe and primers, as well as amplicon length, can be critical for specific amplification, yield, and overall efficiency of any real-time RT-PCR. + +#### Recommendations: +- Use primer-design software, such as Primer3 (http://frodo.wi.mit.edu/primer3/) or visual OMPTM (http://dnasoftware.com/). +- Primers should have a melting temperature (Tm) of approximately 60°C. +- The Tm of the probe should be approximately 10°C higher than that of the primers. +- Optimal amplicon length should be 80-200 bp, not exceeding 400 bp. +- Final primer concentration of 400 nM is suitable for most probe reactions. +- Use an equimolar primer concentration. +- Final probe concentration of 100 nM is suitable for most applications. +- Use intron-spanning primers to avoid amplification from genomic DNA. + +### Template + +The RNA template should be intact and devoid of DNA or contaminating inhibitors. For high-purity RNA, the Bioline ISOLATE II RNA Mini Kit (BIO-52073) is recommended. RNA stocks and dilutions should be made in DEPC-treated water to avoid any RNase-mediated degradation. + +#### Recommended Amount: +- Total RNA: Purified total RNA can be used in the range from 1 pg to 1 μg per 20 μL reaction. +- mRNA: Purified mRNA can be used from 0.01 pg per 20 μL reaction. + +### MgCl₂ + +The MgCl₂ concentration in the 1x reaction mix is 3 mM. This concentration is optimal for both the reverse transcriptase and the hot-start DNA polymerase. If necessary, titrate the MgCl₂ to a maximum of 5 mM. + +### RT-PCR Controls + +To detect contaminating DNA that may affect the reliability of the data, always include a no-RT control reaction by omitting the reverse transcriptase from the reaction. + +### Troubleshooting Guide + +For further troubleshooting, refer to the detailed troubleshooting instructions provided in the Bioline full documentation: +[Bioline Troubleshooting Guide](http://www.bioline.com/us/downloads/dl/file/id/3303/sensifast_probe_no_rox_one_step_kit_manual.pdf) + +## Materials + +SensiFAST™ Probe No-ROX One-Step Kit [BIO-76001](http://www.bioline.com/us/downloads/dl/file/id/3303/sensifast_probe_no_rox_one_step_kit_manual.pdf) by Bioline + +## Protocol + +### Reaction Mix Composition + +#### Step 1 +Prepare a real-time RT-PCR mastermix. The volumes given below are based on a standard 20 μL final reaction mix and can be scaled accordingly. + +| Reagent | Volume | Final Concentration | +|---------------------------------------------|---------|---------------------| +| 2x SensiFAST Probe No-ROX One-Step Mix | 10 μL | 1x | +| 10 μM Forward Primer | 0.8 μL | 400 nM | +| 10 μM Reverse Primer | 0.8 μL | 400 nM | +| 10 μM Probe | 0.2 μL | 100 nM | +| Reverse transcriptase | 0.2 μL | - | +| RiboSafe RNase Inhibitor | 0.4 μL | - | +| H₂O | up to 16 μL | - | +| Template | 4 μL | - | +| **Total Volume** | **20 μL** | **-** | + +### Sensitivity Testing and Ct Values + +#### Step 2 +When comparing SensiFAST with a mix from another supplier, amplify from a 10-fold template dilution series. Loss of detection at low template concentration is the only direct measure of sensitivity. An early Cₜ value is not an indication of good sensitivity but rather of speed. + +### Suggested RT-qPCR Conditions + +#### Step 3 +The following real-time RT-PCR conditions are suitable for the SensiFAST Probe No-ROX One-Step Kit. The conditions can be varied for different probe-based reactions or machine-specific protocols. The detection channel should be set to acquire at the appropriate wavelengths: + +**Cycling for Dual-labeled Probes:** + +| Cycles | Temp. | Time | Notes | +|--------|--------|-------|----------------------------------------------| +| 1 | 45°C | 10 min| Reverse transcription | +| 1 | 95°C | 2 min | Polymerase activation | +| 40 | 95°C | 5s | Denaturation | +| | 60°C | 20s | Annealing/extension (acquire at end of step) | + +### Real-Time RT-PCR Optimization + +#### Step 4 +The following optimization may improve the efficiency of some reactions: +- The reverse transcription reaction time can be extended up to 20 minutes. +- The annealing/extension time can be extended up to 60 seconds. +- The temperature can be increased up to 65°C. + +--- + +### endofoutput +``` \ No newline at end of file diff --git a/markdown-output/sfgr-isolation-from-clinical-diagnostic-material-s-bxbmpik6.md b/markdown-output/sfgr-isolation-from-clinical-diagnostic-material-s-bxbmpik6.md new file mode 100644 index 0000000000000000000000000000000000000000..a6be614cc02a5631914d54f0c6811f9b46a41954 --- /dev/null +++ b/markdown-output/sfgr-isolation-from-clinical-diagnostic-material-s-bxbmpik6.md @@ -0,0 +1,153 @@ +```markdown +# Goal/Experiment: +The goal of this experiment is to isolate SFGR (Spotted Fever Group Rickettsiae) from clinical diagnostic material using a small volume inoculum in a BSL-3 laboratory. This experiment utilizes Vero E6 cells grown in small conical culture flasks and includes the steps of growth monitoring, media changing, freezing primary isolates, and troubleshooting contamination. + +# SFGR Isolation from Clinical Diagnostic Material: Small volume inoculum + +**Authors**: Marah Condit, Cecilia Kato +**Affiliation**: Centers for Disease Control and Prevention +**DOI**: [10.17504/protocols.io.bxbmpik6](https://dx.doi.org/10.17504/protocols.io.bxbmpik6) +**Protocol Integer ID**: 52301 +**Created**: Aug 12, 2021 +**Last Modified**: Oct 15, 2022 + +## Abstract +Rickettsiae are small obligate intracellular gram-negative bacteria that are grown in a Biological Safety Level 3 (BSL-3) laboratory setting. This protocol uses antibiotic-free medium, and thus cultures are susceptible to contamination. Guidance is provided for inoculation, growth monitoring, media changing, freezing primary isolates, and contamination troubleshooting. Techniques involve Vero E6 cells in small conical culture flasks (10 cm², max 5 mL volume). Proper training and extreme caution are required for working with live rickettsial cultures in BSL-3. + +## Keywords +- Culture +- Isolation +- Clinical isolation +- SFGR +- Rickettsia + +## Guidelines +1. All work is conducted in a BSL-3 laboratory. +2. Ensure reagents are not contaminated or expired. +3. Use proper sterile technique. + +## Materials + +- **Vortex mixer** +- **Pipetteman** +- **Inverted Light Microscope** +- **High-Speed Centrifuge** (capable of 17,000 x g) +- **CO₂ water jacketed incubator** +- **Water bath** +- **1X HBSS**; Life Technologies (Catalog #14025092) +- **5% Fetal Bovine Serum Minimum Essential Medium** (Complete Media); Life Technologies (Catalog #11090-081) +- **Non-Essential Amino Acids 10mM (NEAA)**; Life Technologies (Catalog #11140-050) +- **HEPES 1M**; Life Technologies (Catalog #15630-080) +- **L-Glutamine 200mM**; Life Technologies (Catalog #25030-081) +- **Sodium Pyruvate 100mM**; Life Technologies (Catalog #11360-070) +- **Fetal bovine serum (FBS)**; Atlanta Biologics (Low antibiotic, Catalog #S10350) +- **Sucrose Phosphate Glutamate (SPG)** solution (1): + + | Component | Catalog Number | + | ------------------------- | ------------------ | + | Sucrose | Sigma #84097 | + | Potassium phosphate monobasic | Sigma #P5655 | + | Potassium phosphate dibasic | Sigma #P3786 | + | L-glutamic acid | Sigma #G1251 | + | Potassium hydroxide (KOH) | Sigma #P5958 | + - **Cell Culture Grade Sterile Water** + - **70% Ethanol** + - **5% Quaternary ammonium solution** + - **Penicillin-Streptomycin (10,000 U/mL)**; Life Technologies (Catalog #15140-148) + - **Fungizone**; Life Technologies (Catalog #15290-026) + - **4% Sheep Blood Agar Plate**; Scierason (Catalog #9233) + - **Cotrimoxazole, Ready-Made Solution (100 mg/mL in DMSO)**; Sigma (Catalog #A2487) + - **Gentamicin (10mg/mL)**; Life Technologies (Catalog #15710-072) + - **Lincomycin Hydrochloride**; Sigma (Catalog #L2774) + - **Alcohol resistant laboratory marking pen** + - **Sterile Glass Beads** + - **Disposable pipette tips (5 mL and 10 mL)** + - **10 cm² culture tube**; Techno Plastic Products (Catalog #91243) + - **PPE**: Disposable gloves, rear closure gown, eye protection + +## Safety Warnings +- Conduct procedures using BSL-3 facilities and practices. +- Utilize a Biological Safety Cabinet (BSC) with sterile technique. +- Follow universal precautions for blood-borne pathogens. +- Use double gloves, eye protection, and disposable rear closure gown. +- Contaminated waste should be sterilized by autoclaving or disinfected with 5% quaternary ammonium solution. +- Use a sharps container for disposal of sharp items. +- Refer to [CDC's Biosafety in Microbiological and Biomedical Laboratories](https://www.cdc.gov/labs/BMBL.html). + +## Before Starting +1. Ensure 5% Complete Media is warmed to room temperature. +2. Ensure SPG and freezing tubes are chilled when freezing. +3. Label all flasks, vials, and tubes with identifiers and dates. + +## Procedure + +### Media Preparation +1. Ensure all reagents are thawed, mixed and not expired. +2. Clean BSC with 5% quaternary ammonium solution and 70% ethanol. +3. Wipe all reagent bottles with 70% ethanol before placing in the BSC. +4. Prepare approximately 600 mL of Complete Media: + + | Reagent | Volume (mL) | + | ------- | ------------ | + | NEAA | 6.15 | + | HEPES | 6.15 | + | L-Glutamine | 6.15 | + | Sodium Pyruvate | 61.5 | + | FBS | 30 | + +5. Filter sterilize with a 0.22 µm Nalgene Filter Unit. +6. Label with media type, preparation date, and expiration date and store at 4°C. + +### Small Volume Inoculation +7. Confirm 10 cm² culture flasks are at least 99% confluent. +8. Clean BSC with 5% quaternary ammonium solution and 70% ethanol. +9. Decontaminate 10 cm² culture flasks with 70% ethanol. +10. Label flasks with sample identifiers and date. +11. Replace spent media with 3 mL of 5% Complete Media. +12. Obtain clinical blood or blood product and place in BSC. Either: + - 50 µL of fresh clinical blood or blood product + - 100 µL of previously frozen samples in a 1:1 ratio with SPG + - Quick thaw any frozen samples in a 36°C water bath. +13. Pipette inoculum into the 3 mL of media in the 10 cm² culture flask. +14. Gently mix and ensure even spread over the monolayer. +15. Place in a 34°C 5% CO₂ water jacketed incubator. + +### Monitoring Isolate Growth +16. +17. Incubate culture flasks for 3-6 days and sample supernatant for DNA extraction. +18. Clean BSC with 5% quaternary ammonium solution and 70% ethanol. +19. Prepare a 2 mL sterile tube labeled with identifiers and date. +20. Take a 200 µL supernatant sample without disturbing the monolayer. +21. Seal the culture flask and return to the incubator. +22. Decontaminate and store the 2 mL tube at -80°C if not extracting immediately. +23-28. Repeat steps 7-8 at least once within 14 days. Extract and run the PanR8 real-time PCR assay to confirm isolate viability. + +### Media Changes +29. If DNA samples are insufficient, change the media: + - Clean BSC with 5% quaternary ammonium solution and 70% ethanol. + - Decontaminate the culture flask with 70% ethanol. + - Remove spent media without disturbing the monolayer and discard safely. + - Add fresh 5% Complete Media slowly. + - Seal and return the culture flask to the incubator. + +### Freezing Isolate Cultures +30. Confirmed Rickettsia cultures are further incubated until 40-60% of the monolayer is lifted. +31. Clean BSC with 5% quaternary ammonium solution and 70% ethanol. +32. Remove media and place in a labeled sterile high-speed conical tube. +33. Shake flask with sterile glass beads until the monolayer is lifted. +34. Transfer suspension to the labeled conical tube. +35. Centrifuge at 17,000 x g for 30 min at 4°C. +36. Resuspend pellet in 2 mL of ice-cold SPG. +37. Aliquot 1 mL of suspension into 2 mL cryotubes and label. +38. Store cryotubes at -80°C for 24 hours before transferring to liquid nitrogen. + +### Troubleshooting Contamination +39. Use appropriate antibiotics (e.g., penicillin-streptomycin, gentamicin) if contamination occurs. Observe contamination early to prevent overgrowth. + +## References +1. Ammerman N. et al. "Laboratory Maintenance of *Rickettsia rickettsia*". Curr Protoc Microbiol. 2008. +2. Ogawa M. et al. "Decontamination of mycoplasma-contaminated *Orientia tsutsugamushi* strains". BMC Microbiol. 2013. +3. Kato CY. et al. "Assessment of real-time PCR assay for detection of *Rickettsia* spp.". J Clin Microbiol. 2013. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/simoa-extracellular-vesicle-assays-bm89k9z6.md b/markdown-output/simoa-extracellular-vesicle-assays-bm89k9z6.md new file mode 100644 index 0000000000000000000000000000000000000000..7b7ab417adb1cb3334111ee76f6415bd3b7a1b6d --- /dev/null +++ b/markdown-output/simoa-extracellular-vesicle-assays-bm89k9z6.md @@ -0,0 +1,141 @@ +```markdown +# Goal/Experiment: +This protocol focuses on the preparation of reagents for Simoa analysis of Extracellular Vesicles (EVs) and details the setup and execution of the assay using the Quanterix HD-1 or HD-X instrument. The objective is to measure three commonly expressed EV transmembrane proteins (CD9, CD63, and CD81) in complex biofluids such as plasma and cerebrospinal fluid. + +# Simoa Extracellular Vesicle Assays + +### Authors: +- Maia Norman +- Dima Ter-Ovanesyan +- Roey Lazarovits +- Wendy Trieu +- Ju Hyun Lee +- David Walt + +### Affiliations: +1. Brigham and Women’s Hospital +2. Wyss Institute for Biologically Inspired Engineering + +### DOI: +[10.17504/protocols.io.bm89k9z6](https://dx.doi.org/10.17504/protocols.io.bm89k9z6) + +### Abstract: +Extracellular Vesicles (EVs) are reservoirs of biomarkers such as mRNA and post-translationally modified proteins. This protocol describes a method for the quantification of EVs in complex biofluids by measuring three EV transmembrane proteins (CD9, CD63, CD81) using single molecule array (Simoa) technology. The goal is to provide a comprehensive measure of total EVs, reducing the likelihood of missing rare populations of EVs. + +### Keywords: +- Single Molecular Arrays +- Simoa +- Tetraspanins +- Extracellular Vesicles +- Exosomes + +### License: +This protocol is distributed under the terms of the [Creative Commons Attribution License](https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. + +### Materials +| Reagent/Equipment | Source (Vendor) | Catalog Number | +|---------------------------------------|-----------------------|---------------------------------| +| Antibodies: +| - CD9 | Abcam | ab195422 | +| - CD63 | R&D Systems | MAB5048 | +| - CD81 | Abcam | AB79559 | +| Recombinant Protein Standards: +| - CD9 | Abcam | ab152262 | +| - CD63 | Origene | TP301735 | +| - CD81 | Origene | TP317508 | +| Quanterix Neuroplex 3A Sample Diluent | Quanterix | 102002 | +| Quanterix Sample Diluent E | Quanterix | 101579 | +| 50kD Centrifugal Filters | Millipore Sigma | UFC505096 | +| Quanterix Homebrew Assay Kit | Quanterix | 101354 | +| Quanterix Enzyme Substrate Kit | Quanterix | 101361 | +| Quanterix Disc Kit | Quanterix | 103347 | +| EDC | Thermo Fisher | A35391 | +| ZZ | Beckman Coulter | 6605700 | +| Z-Pac | Beckman Coulter | 8320312 | +| Biotin | Thermo Fisher | A39259 | +| BenchTop Centrifuge | - | - | +| Dynamag | Thermo Fisher | 12321D | +| Multiplex "Helper" Beads: +| - 647 | Quanterix | 101985 | +| - 700 | Quanterix | 101986 | +| - 750 | Quanterix | 101987 | +| Quanterix HD-X Analyzer | Quanterix | - | + + +## Bead Coupling Protocol +**Time Required: 5h** + +1. **Preparation of Antibodies:** + - Thaw 100 µg of antibody: + - CD9 (Abcam AB195422) + - CD63 (R&D Systems MAB5048) + - CD81 (Abcam AB79559) + - Raise the volume of the antibody to 500 µL (0.2 mg/mL) using 1x PBS and allow to rotate on a mixer (such as Hula Mixer) for ~10 minutes with gentle rotation. + - Place the 500 µL solution in an Amicon Ultra-0.5mL (50kD) Centrifugal Filter (Millipore Sigma UFC505096). + - Centrifuge at 14,000 x g for 5 minutes. + - Discard the flow-through and add 450 µL of Bead Conjugation Buffer (Quanterix 101357). Repeat centrifugation. + - Discard flow-through. Flip the tube, centrifuge at 1000 x g for 2 minutes. + - Measure antibody concentration using NanoDrop, use A280 setting for IgG. + - Transfer measured volume to a clean 1.5 mL tube. Record concentration. + - Raise each antibody to 300 µL with Bead Conjugation Buffer and keep on ice or 4°C. + +2. **Bead Washing and Activation:** + - Use Singleplex 488 beads (Quanterix 103207), amounts: + - CD9: 4e8 beads + - CD63: 2.8e8 beads + - CD81: 4e8 beads + - Remove EDC (Thermo Fisher A35391) from -20°C and let sit at room temperature for 10 mins. + - Add EDC to beads (amount proportional to beads) and carry out the washing and activation steps as described. + +3. **Antibody Conjugation and Washing:** + - Conjugate purified antibody to activated beads, incubate with intermittent shaking, and follow described washing steps using Quanterix buffers. + +4. **Characterize Coupling Efficiency:** + - Use NanoDrop to measure coupling efficiency by comparing antibody concentrations before and after coupling. + +5. **Characterize Bead Aggregation and Quantity:** + - Use Coulter Counter (Beckman Coulter 6605700) to determine percent monomeric beads. + +## Biotinylation Protocol +**Time Required: 1h 1m** +2. **Preparation of Detection Antibodies for Simoa Assay:** + - Thaw 100 µg of antibody: + - CD9 (Abcam AB58989) + - CD63 (BD Biosciences 556019) + - CD81 (BioLegend 349502) + - Raise volume to 500 µL with Biotinylation Reaction Buffer (Quanterix 101358). Use a Hula mixer for ~10 minutes. + - Use 50kD centrifugal filter for buffer exchange and concentration steps as described. + - Measure antibody concentration with NanoDrop. Adjust concentration to 1 mg/mL using Biotinylation Reaction Buffer. + +3. **Calculation and Addition of Biotin:** + - Resuspend Biotin vial, calculate amount for 40x excess by formula, mix and incubate. + - Centrifuge and carry out the desalt steps with Biotinylation Reaction Buffer. + +## Running Calibration Curve and Samples +**Time Required: 2h 45m** + +3. **Measurements and Setup:** + - Determine the number of measurements and calculate required volumes for bead, detector, and SBG solutions. + - Create calibration curves in serial orders as described. + - Dilute each sample appropriately using Quanterix SBG diluent. + - Load reagents into the instrument and assign wells to respective assays. + - Start the assay run and analyze results. + +### Important Calculations: +- For Bead Volume: + -> `5000 beads/µL * total volume in µL = X µL * bead concentration in beads/µL` +- For Detector Volume: + -> `3.2 µg/mL * total volume = X µL * concentration` +- For SBG Volume: + -> `120 pM * total volume= X µL * SBG stock` + +### Setting Up the Instrument: +- Assign reagents, create new assay, define parameters, load reagents and plate. +- Ensure correct loading and starting procedures. + +**End of Protocol** + +[DOI for Full Protocol](https://dx.doi.org/10.17504/protocols.io.bm89k9z6) + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/simple-seed-vacuum-protocol-for-agrobacterium-medi-cy76xzre.md b/markdown-output/simple-seed-vacuum-protocol-for-agrobacterium-medi-cy76xzre.md new file mode 100644 index 0000000000000000000000000000000000000000..ab0f47e3b20c658b937e08337b84d6190add7cb4 --- /dev/null +++ b/markdown-output/simple-seed-vacuum-protocol-for-agrobacterium-medi-cy76xzre.md @@ -0,0 +1,123 @@ +```markdown +# Goal/Experiment: +Develop and implement a simple seed-vacuum protocol for Agrobacterium-mediated Virus Induced Gene Silencing (VIGS) in sunflower (Helianthus annuus L.). + +# Simple Seed-vacuum Protocol for Agrobacterium-mediated Virus Induced Gene Silencing (VIGS) in Sunflower Helianthus annuus L. + +### Authors +- Majd Mardini¹ +- Mikhail Kazantsev¹,² +- Elina Ivoilova¹ +- Anastasia Vlasova¹ +- Victoria Utkina¹ +- Ilya Kirov¹,² + +¹ All-Russia Research Institute of Agricultural Biotechnology, Timiryazevskaya Str. 42, 127550 Moscow, Russia +² Moscow Institute of Physics and Technology, 141701 Dolgoprudny, Russia + +--- + +## Abstract +Virus-Induced Gene Silencing (VIGS) is a powerful tool for functional genomic research, harnessing the plant’s defense system to induce post-transcriptional gene silencing by introducing a modified viral construct. This protocol outlines a reproducible Agrobacterium-mediated VIGS protocol for sunflower using seed-vacuum infiltration without the need for pre-germination, MS medium recovery, or seed sterilization. Utilizing the PHYTEONE DESATURASE gene (PDS) from *Helianthus annuus* as the target, the protocol employs constructs derived from Tobacco Rattle Virus (TRV). This method has been validated across various sunflower breeding lines and commercial cultivars. + +--- + +## Before Start Instructions +- **VIGS Constructs Preparation:** Prepare and introduce VIGS constructs into Agrobacterium cells using standard transformation methods. +- **Gene Target:** PDS of sunflower is the primary target gene due to its detectable phenotypic characteristics (leaf color bleaching). +- **TRV Vectors:** The viral constructs are based on TRV vectors. +- **PDS Fragment Amplification Primers:** + - Forward: `5′-taattctagaATGGGATTTTTTAGATGGGCAGCCC-3′` + - Reverse: `5′-taatggtaccTGGGAGTAGCAAATACATAAGCATCCCC-3′` + +- **Constructs and Primers:** + - **TRV2 constructs:** [Plasmid - Addgene](https://www.addgene.org/148969/) + - **pYL192 (TRV1):** [Link](https://www.addgene.org/148968/) + - **pYL156 (TRV RNA2):** [Link](https://www.addgene.org/148969/) + - **Primers Table:** + + | Primer | Forward Primer Sequence | Reverse Primer Sequence | PCR Template | Comments | + | ------ | ----------------------- | ----------------------- | ------------ | ------------------------------ | + | pYL156_TRV2_F_flank | GITCAGG CGGTTCT TGTGTTGC | TRV2-tRNA_flank_R | TTGAACC TAAAACT TCAGAAC CGGTCTAC | pYL156 (TRV RNA2) | Agrobacterium colony check | + | TRV1_F | AGACAAC TTAATAAA ACAATTG CGGACG | TRV1_R | CTTTGAC GTTGGAG TCCAGTC | pYL192 (TRV1) | Agrobacterium colony check | + | Ha_PDS_VIGS_insertion_F | taattctaga ATGGCAT TTTTAGAT GGGCAGCCC | Ha_PDS_VIGS_insertion_R | taatggtacc TGGGAGTA GCAAATAC ATAAGCATCCCC | Genomic DNA of sunflower | Amplifying PDS insertion for VIGS construct assembly | + +--- + +## Protocol Steps + +### Infiltration Suspension Preparation +1. **Agrobacterium Culture Preparation:** + - Streak agrobacterium carrying VIGS constructs (TRV1 and TRV2) on agar-LB plates. + - Use media with: + - 20 mL LB medium + - 1% agar + - 50 µg/mL kanamycin + - 10 µg/mL gentamycin + - 100 µg/mL rifampicin + - Incubate at 28°C. (Prepare at least 3 days before use) + + ![Expected Result](path-to-image) + +2. **Colony Incubation:** + - Incubate at 28°C until individual colonies emerge (24-38 hours). + +3. **Starting Culture Preparation:** + - Inoculate 5 mL LB with antibiotics with single colony. + - Incubate at 28°C/180rpm for 24-38 hours. + + ![Expected Result](path-to-image) + +4. **Infiltration Culture Preparation:** + - Transfer starting culture to 50mL sterile LB (10x volume of starting culture) with antibiotics, MES (10 mM), and acetosyringone (200 µM). + - Incubate at 28°C/180rpm until OD₆₀₀ = 1.5. + + *Note:* Use refrigerator to halt growth if needed. + +5. **Centrifugation:** + - Collect infiltration culture (50-mL tube), centrifuge at 3000 rcf for 10 min. + +6. **Washing:** + - Wash agrobacterium cells in distilled water, resuspend in infiltration buffer (OD₆₀₀ = 1.5). + +### Performing Seed-vacuum Agrobacterium Infiltration +9. **Seed Preparation:** + - Soak sunflower seeds (outer coat removed) in tap water for 2 hours. + +10. **Infiltration Mixture:** + - Transfer seeds to sterile Petri dish, add 1:1 ratio of TRV1:TRV2 infiltration suspension (20-25 mL total per dish). + +11. **Sand Application:** + - Add 2-3 g of autoclave-sterile silica sand to Petri dish. + - Rub seeds gently to produce wounds. + + *Note:* + - Change gloves between different dishes. + - Seeds may become fragile after soaking. + +12. **Vacuum Process:** + - Apply vacuum for 3 min, followed by 3-min pause, repeat. + +13. **Incubation:** + - Seal Petri dishes, incubate at 28°C/50rpm for 6 hours. + + *Note:* Retain sand within dish to sustain wounding process. + +14. **Planting Treated Seeds:** + - Plant in clean, humid soil, cover with plastic wrap, keep in dark for 2 days. + +15. **Growth Conditions:** + - Transfer to greenhouse with 16 hr light/8 hr dark cycle. + - Observe for PDS silencing symptoms (color-bleaching) within 11-13 days. + + ![Expected Result](path-to-image) + +--- + +## License +This is an open access protocol distributed under the terms of the [Creative Commons Attribution License](https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. + +--- + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/single-cell-cut-and-tag-on-10x-genomics-platform-bqbnmsme.md b/markdown-output/single-cell-cut-and-tag-on-10x-genomics-platform-bqbnmsme.md new file mode 100644 index 0000000000000000000000000000000000000000..de0dbdc44c914ce5f2fa1075457c4791d2f78149 --- /dev/null +++ b/markdown-output/single-cell-cut-and-tag-on-10x-genomics-platform-bqbnmsme.md @@ -0,0 +1,163 @@ +```markdown +# Goal/Experiment: +The objective of this experiment is to perform single-cell cleavage under targets and tagmentation (CUT&Tag) on the 10x Genomics platform. This protocol combines the scCUT&Tag method with the 10x Genomics scATAC-seq Chromium platform for single-cell barcoding, enabling the acquisition of high-quality data from tens of thousands of single cells with high specificity. + +# Single Cell CUT&Tag on 10x Genomics Platform + +**Authors**: +- Marek Bartosovic1, Goncalo Castelo-Branco1 +- 1Karolinska Institute, Stockholm + +**DOI**: [dx.doi.org/10.17504/protocols.io.bqbnmsme](https://dx.doi.org/10.17504/protocols.io.bqbnmsme) + +**Reference**: +Marec Bartosovic, Goncalo Castelo-Branco 2021. Single cell CUT and Tag on 10x genomics platform. protocols.io [https://dx.doi.org/10.17504/protocols.io.bqbnmsme](https://dx.doi.org/10.17504/protocols.io.bqbnmsme) + +### Introduction +scCUT&Tag on the 10x platform uses the scCUT&Tag protocol from Steven Henikoff’s lab and the scATAC-seq Chromium platform (10x Genomics) for single-cell barcoding. This method provides high-quality data for tens of thousands of single cells. + +More details can be found in the accompanying BiorXiv preprint: [https://www.biorxiv.org/content/10.1101/2020.09.02.279703v1](https://www.biorxiv.org/content/10.1101/2020.09.02.279703v1) + +scCUT&Tag can be performed on cell lines or freshly isolated cells from primary tissue. + +### Materials Required + +#### Reagents +- Ultra pure DNAse/RNAse free water (ThermoFisher, 10977015) +- 1M Hepes (Alfa Aesar, J60712) +- 5M NaCl (Invitrogen, AM9759) +- Spermidine (Sigma, S2626-1G) +- Complete EDTA-free protease inhibitors (Sigma, 11873580001) +- BSA powder (Sigma, A9418-50G) +- 0.5M EDTA (Invitrogen, AM9260G) +- Digitonin powder (Merck, CAS 11024-24-1) +- NP-40 (ThermoFisher, 85124) +- 1M MgCl2 (Invitrogen, AM9530G) +- 10% SDS (ThermoFisher, 15553027) +- Proteinase K (Invitrogen, AM2546) +- 2x NEBNext High-Fidelity PCR master mix (NEB, M0541S) +- SYBR green (dilute to 10x) (ThermoFisher, S7563) +- Secondary antibody guinea pig anti-rabbit (Novus Biologicals, NBP1-72763) +- pA-Tn5 pre-loaded with standard Tn5 adapter sequences (Kaya-Okur et al., 2020) + +### Key Antibodies +- H3K4me3 (Diagenode, C15410030) +- H3K27ac (Abcam, ab177178) +- H3K27me3 (Cell Signaling, 9733T) +- H3K36me3 (Abcam, ab9050) +- Rad21 (GeneTex, GTX106012) +- Olig2 (Novus Biologicals, NBP1-28667) + +### Oligonucleotide Sequences +- Mosaic end-adapter A (Tn5ME-A): TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG +- Mosaic end-adapter B (Tn5ME-B): GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG +- Mosaic-end reverse oligonucleotides (Tn5MErev): 5’-[phos]CTGTCTCTTATACACATCT-3’ + +### Tn5 Loading Protocol +1. **Preparation**: + - Dilute Tn5ME-A, Tn5ME-B, and Tn5MErev oligos to 100 uM. + - Mix in two separate PCR tubes: + 1. 10 ul Tn5ME-A + 10 ul Tn5MErev + 2. 10 ul Tn5ME-B + 10 ul Tn5MErev + - Denature on a thermocycler for 5 minutes at 95°C and cool down slowly by ramping down by 0.1°C/s. +2. **2x Dialysis Buffer**: + - 100 mM HEPES-KOH pH 7.2 + - 200 mM NaCl + - 0.2 mM EDTA + - 2 mM DTT + - 0.2% Triton-X + - 20% Glycerol +3. **Mix Tn5 with the annealed oligonucleotides**: + - 2 ul Tn5ME-A/Tn5ME-rev + 2 ul Tn5ME-B/Tn5ME-rev + 21.3 ul of dialyses buffer, mixed gently with pipette and incubated for 1 hr at room temperature. + +### Buffers Preparation (scCUT&Tag) +- **5% Digitonin**: Prepare aliquots and store at -20°C. + - 1 g Digitonin powder + - 20 ml DMSO +- **20% BSA**: Filter through 0.45 um filter. + +#### Buffers +| Buffer | Final | Stock | Amount | +|--------------------------|-------------------------|--------------------------|----------| +| **2x Wash buffer (25 ml)** | | | | +| 40 mM Hepes pH 7.5 | 1 M | 1 ml | +| 300 mM NaCl | 5 M | 1.5 ml | +| 1 mM Spermidine | 2 M | 12.5 ul | +| 2x Protease inhibitors | tablet | 1 tablet | +| | water | 22.5 ml | + +#### Preparation +**Antibody Buffer (2 ml/sample)**: +| Buffer | Final | Amount | +|--------------------------|-------------------------|--------------------------| +| 2x Wash buffer | 2 ml | | +| EDTA (500 mM) | 8 ul | | +| Digitonin (5%) | 20 ul | | +| NP-40 (10%) | 2 ul | | +| BSA (20%) | 100 ul | | +| Water | 870 ul | | + +#### Nuclei Preparation and Primary Antibody Incubation + +1. Dissociate tissue/cell line into a single-cell (single-nuclei) suspension. +2. Centrifuge cells/nuclei for 5 minutes at 300x g. +3. Prepare a 1:50 dilution of primary antibody in 200 ul of antibody buffer per sample. +4. Incubate cells with primary antibody overnight at 4°C on rotating wheel or roller with slow rotation speed. + +#### Secondary Antibody Incubation + +1. Centrifuge the nuclei for 3 minutes at 600x g. +2. Prepare 200 ul of 1:50 diluted secondary antibody per sample in Dig-Wash-BSA buffer. +3. Incubate for 1 hour, rotating at room temperature. + +#### pA-Tn5 Incubation + +1. Centrifuge for 3 minutes at 600x g, wash and resuspend in 200 ul of diluted pA-Tn5. +2. Incubate for 1 hour, rotating at room temperature. + +#### Tagmentation + +1. Centrifuge for 3 minutes at 300x g, resuspend in tagmentation buffer, incubate for 1 hour at 37°C. + +#### Diluted Nuclei Buffer and STOP Buffer Preparation + +1. Prepare 1x Diluted Nuclei Buffer (DNB) supplemented with 2% BSA. +2. Mix 20 ul of 500 mM EDTA with 200 ul Dig-300 buffer. + +#### qPCR Cycle Check of Bulk Library (Optional) + +1. Test the success of tagmentation by generating a bulk library from part of the sample and performing qPCR. + +#### Nuclei Counting + +1. Centrifuge the nuclei for 3 minutes at 300x g. +2. Resuspend in 15-25 ul of 1x DNB + 2%BSA. +3. Calculate nuclei concentration. + +### 10x scATAC-seq Protocol +1. Skip Step 1 in the scATAC-seq manual, start at Step 2 GEM Generation and barcoding. +2. Follow the manufacturer's instructions from Step 3 onwards. + +#### Master Mix Preparation +- **For scATAC v1**: + - 15 ul nuclei suspension + - 61.5 ul barcoding reagent + - 1.5 ul reducing agent B + - 2 ul barcoding enzyme +- **For scATAC v1.1**: + - 8 ul nuclei suspension + - 7 ul ATAC buffer B + - 56.5 ul barcoding reagent B + - 1.5 ul reducing agent B + - 2 ul barcoding enzyme +3. Load the 10x chromium chip according to manufacturer’s instructions. + +### Conclusion +The protocol outlined facilitates the single-cell CUT&Tag procedure using the 10x Genomics scATAC-seq Chromium platform, enabling efficient and high-quality data collection at a single-cell resolution. + +**References**: +- Kaya-Okur et al., Genome Research, 2019 +- Buenrostro, J.D. et al., Nature, 2015 + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/single-cell-rna-sequencing-scrnaseq-of-fresh-human-cekftctn.md b/markdown-output/single-cell-rna-sequencing-scrnaseq-of-fresh-human-cekftctn.md new file mode 100644 index 0000000000000000000000000000000000000000..952a8c2022274eb0644f654b8c7667148531c2c3 --- /dev/null +++ b/markdown-output/single-cell-rna-sequencing-scrnaseq-of-fresh-human-cekftctn.md @@ -0,0 +1,242 @@ +```markdown +# Goal/Experiment: +The aim of this protocol is to perform Single Cell RNA sequencing (scRNAseq) on a fresh human lung cell suspension. This powerful technique allows for the evaluation of differential gene expression in single cells within a complex tissue. By targeting specific gene expressions, the protocol assists in studying the development and progression of diseases like idiopathic pulmonary fibrosis, and the senescence process in human lung cells. This protocol outlines steps for human lung tissue dissociation, cell suspension preparation, GEM generation and barcoding, cDNA amplification, and library preparation. + +# Single Cell RNA sequencing (scRNAseq) of Fresh Human Lung Cell Suspension + +## Authors: +Julian Alonso Chamuero1, Mauricio Rojas1, Ana Mora1 +1The Ohio State University + +## Abstract +Gene expression analysis is a molecular biology approach that evaluates the expression of genes in developing certain diseases and identifies possible therapeutic targets. Single-cell RNA sequencing (scRNAseq) allows evaluating gene expression in individual cells, offering insights on the development of age-related diseases and the progression of specific conditions. This protocol describes the dissociation of lung specimens, preparation of cell suspensions, and scRNAseq library generation with dual indexing. + +## Keywords +Single-cell RNA-seq, Human lung cells, TriState SenNET + +## DOI +[https://dx.doi.org/10.17504/protocols.io.4r3l20wdxv1y/v1](https://dx.doi.org/10.17504/protocols.io.4r3l20wdxv1y/v1) + +## Protocol Citation +Julian Alonso Chamuero, Mauricio Rojas, Ana Mora (2022). Single Cell RNA sequencing (scRNAseq) of fresh human lung cell suspension. protocols.io [https://dx.doi.org/10.17504/protocols.io.4r3l20wdxv1y/v1](https://dx.doi.org/10.17504/protocols.io.4r3l20wdxv1y/v1) + +## Materials + +| Material | Vendor | Catalog Number | +|---------------------------------------------------------|-----------------------|-------------------------| +| DNase I | Worthington Biochemical | LS002139 | +| Ham's F12 Nutrition Mix | Gibco - Thermo Fischer | 11765070 | +| DMEM high glucose | Gibco - Thermo Fischer | 11965118 | +| Liberase | DL Roche | 5466202001 | +| Phosphate-buffered saline (PBS) | Gibco - Thermo Fischer | 10010023 | +| Fetal Bovine Serum (FBS) | Gibco - Thermo Fischer | 16140071 | +| RBC Lysis Buffer | BioLegend | 420302 | +| Distilled Water | Thermo Fischer | 15230162 | +| Chromium Next GEM Single Cell 3′ Kit v3.1 | 10x Genomics | PN-1000268 | +| Chromium Next GEM Chip G Single Cell Kit | 10x Genomics | PN-1000127 | +| Dual Index Kit TT Set A 96 rxns | 10x Genomics | PN-1000215 | +| Nuclease-free Water | Invitrogen - Thermo Fischer | AM9937 | +| Low TE Buffer | Invitrogen - Thermo Fischer | 12090-015 | +| Buffer EB Elution Buffer | Qiagen | 1014609 | +| Ethanol Pure | Decon Labs | 3916EA | +| SPRIselect Reagent Kit | Beckman Coulter | B23317 | +| 10% Tween 20 | BIO-RAD | 1662404 | +| Glycerin (glycerol) 50% (v/v) Aqueous Solution | Ricca Chemical Company | 19086 | +| DNA LoBind Tubes 1.5 ml | Eppendorf | 022431021 | +| DNA LoBind Tubes 2.0 ml | Eppendorf | 022431048 | +| Tips LTS 200UL Filter RT-L200FLR | Rainin | 30389240 | +| Tips LTS 1ML Filter RT-L1000FLR | Rainin | 30389213 | +| Tips LTS 20UL Filter RT-L10FLR | Rainin | 30389226 | +| TempAssure PCR 8-tube strip | USA Scientific | 1402-4700 | +| 10x Vortex Adapter | 10x Genomics | 120251/330002 | +| 10x Magnetic Separator | 10x Genomics | 120250/230003 | +| Chromium Next GEM Secondary Holder | 10x Genomics | 1000142/3000332 | + +## Tissue Dissociation: + +1. **Collect lung tissue**: Collect 15 to 30 g of lung tissue. +2. **PBS Soak**: Soak the lung pieces in PBS (3x) to remove red blood cells. +3. **Dry**: Compress the tissue with a sterile gauze pad to remove excess liquid. +4. **Pleura Removal**: Carefully remove the parietal pleura. +5. **Tissue Dissection**: Dissect the tissue into 1-cm³ pieces and transfer to a 50 ml conical tube (15 g of tissue per tube) containing the enzyme cocktail (1 mg/ml Liberase, DNase I, DMEM). +6. **Digestion**: Allow the sample to digest for 2 hours at 37°C. +7. **Buffer Inactivation**: Inactivate the digestion buffer (collagenolytic and proteolytic activity) with 2 ml of cold FBS, leave on ice for 5 minutes. +8. **Serial Filtration**: Serially filter the suspension through 300-μm, 100-μm, 70-μm strainers. +9. **Centrifugation**: Centrifuge at 500 g for 7 minutes. +10. **RBC Lysis**: Remove supernatant and add 1x RBC lysis solution to the pellet. Re-suspend the pellet and incubate at 4°C for 7 minutes. +11. **PBS Addition**: Add PBS (10% FBS), centrifuge at 500 g for 7 minutes. +12. **Supernatant Removal**: Remove supernatant, re-suspend the pellet in 10 ml PBS. +13. **Straining**: Filter through 40-μm strainer one more time to remove clumped, dead cells. +14. **Cell Counting**: Count cells and check viability using an automatic cell counter, confirm under a microscope. +15. **Stock Preparation**: Prepare a vial with a cell stock concentration of 1000 cells/μl and keep on ice. +16. **Proceed to GEM Generation**: Proceed to GEM generation and barcoding step. + +## GEM Generation and Barcoding: + +1. **Prepare Master Mix**: + - Add reagents in the order listed, pipette mix 15x and centrifuge briefly. + - Prepare Master Mix as follows: + +| Reagent | 1X | 2X | 3X | 4X | 5X | 6X | 7X | 8X | +|-----------------------|-------|-------|-------|-------|-------|-------|-------|-------| +| RT Reagent B | 18.8 | 41.4 | 62.0 | 82.7 | 103.4 | 124.1 | 144.8 | 165.4 | +| Template Switch Oligo | 2.4 | 5.3 | 7.9 | 10.6 | 13.2 | 15.8 | 18.5 | 21.1 | +| Reducing Agent B | 2.0 | 4.4 | 6.6 | 8.8 | 11.0 | 13.2 | 15.4 | 17.6 | +| RT Enzyme C | 8.7 | 19.1 | 28.7 | 38.3 | 47.9 | 57.4 | 67.0 | 76.6 | +| **Total** | 31.9 | 70.2 | 105.3 | 140.4 | 175.5 | 210.5 | 245.6 | 280.7 | + +2. **GEM generation**: + - Add 31.9 μl Master Mix into each tube of a PCR 8-tube strip on ice. + - Assemble Chromium Next GEM Chip G: + +#### NOTE: Use the chip within 24 hours of removing it from the sealed bag. + +3. **Chip Loading**: + - Add 50% glycerol solution to each unused well if processing fewer than 8 samples per chip. + - For Row 1: add 70 μl to each unused well. + - For Row 2: add 50 μl to each unused well. + - For Row 3: add 45 μl to each unused well. + +4. **Prepare Master Mix + Cell Suspension**: + - Refer to Cell Suspension Volume Calculator table. + - Add the appropriate volume of nuclease-free water to the Master Mix. Pipette mix five times. Add single-cell suspension to the Master Mix for a final volume of 75 μl in each tube. + - Gently pipette mix the cell suspension before adding to the Master Mix. + - Load master mix and cell suspension into the respective chip wells using a pipette. Ensure no bubbles are introduced. + +5. **Prepare Gel Beads**: + - Keep the tubes with gel beads in the Vortex Adapter, vortex for 30 seconds. + - Centrifuge for ~5 sec and ensure no bubbles are present at the bottom. + - Place the gel bead strip back and secure the holder lid. + +6. **Load Gel Beads**: + - Puncture the seal on Gel Bead tubes. + - Aspirate 50 μl Gel Beads and dispense without bubbles into Row 2 of the chip. + +7. **Load Partitioning Oil**: + - Add 45 μl Partitioning Oil to Row 3 of the chip from a reagent reservoir. + +8. **Running the Chromium Controller**: + - Place the assembled chip with the gasket on the tray and confirm the Chromium Chip G program. The run takes approximately 18 minutes. + +## GEM-RT Incubation: + +1. **Thermal Cycling**: + - Transfer GEMs to a tube strip. + - Incubate in the thermal cycler as per the following protocol: + +| Lid Temperature | Reaction Volume | Run time | +| --------------- | --------------- | ------------- | +| 53°C | 125 μl | ~55 minutes | + +| Step | Temperature | Time | +|------|--------------|-----------| +| 1 | 53°C | 45:00 | +| 2 | 85°C | 5:00 | +| 3 | 4°C | Hold | + +2. **Storage**: + - Store the sample at 4°C for up to 72 hours or proceed to the next step. + +## Cleanup and cDNA Amplification: + +1. **Post GEM-RT Cleanup (Dynabeads)**: + +| Reagent | 1X | 2X | 3X | 4X | 5X | 6X | 7X | 8X | +|-------------------------|-----|-----|-----|-----|-----|-----|-----|-----| +| Cleanup Buffer | 182 | 400 | 601 | 801 | 1001| 1201| 1401| 1602| +| Dynabeads MyOne SILANE | 8 | 18 | 26 | 35 | 44 | 53 | 62 | 70 | +| Reducing Agent B | 5 | 11 | 17 | 22 | 28 | 33 | 39 | 44 | +| Nuclease-free Water | 5 | 11 | 17 | 22 | 28 | 33 | 39 | 44 | +| **Total** | 200 | 440 | 660 | 880 | 1100| 1320| 1540| 1760| + +2. **Clean-Up**: + - Add 125 μl Recovery Agent to each sample, wait 2 minutes. + - Firmly secure tubes, mix by inverting the tube strip 5x, centrifuge briefly. + - Remove and discard 125 μl Recovery Agent/Partitioning Oil from each tube. DO NOT aspirate aqueous samples. + +3. **Prepare Dynabeads Cleanup Mix**: + - Vortex and add 200 μl buffer to each sample. Pipette mix 10x. + - Incubate 10 minutes at room temperature with caps open. Pipette mix every 5 minutes. + - Prepare Elution Solution I. + +| Reagent | 2X | 4X | 6X | 8X | +|-----------------------|-----|-----|-----|-----| +| Buffer EB | 98 | 196 | 294 | 392 | +| 10% Tween 20 | 1 | 2 | 3 | 4 | +| Reducing Agent B | 1 | 2 | 3 | 4 | +| **Total** | 100 | 200 | 300 | 400 | + + - Incubate for an additional 10 minutes until the solution clears. + - Remove the supernatant and add ethanol to the pellet, wait 30 seconds. + +4. **Repeat Clean-Up**: + - Follow carryover process until cleanup and ethanol removal is complete. Transfer samples appropriately. + +## cDNA Amplification: + +1. **Prepare cDNA Amplification Mix**: + +| Reagent | 1X | 2X | 3X | 4X | 5X | 6X | 7X | 8X | +|-------------|-----|-----|-----|-----|-----|-----|-----|-----| +| Amplification Mix | 50 | 110 | 165 | 220 | 275 | 330 | 385 | 440 | +| cDNA Primers| 15 | 33 | 50 | 66 | 83 | 99 | 116 | 132 | +| **Total** | 65 | 143 | 215 | 286 | 358 | 429 | 501 | 572 | + +2. **Thermal Cycling**: + +| Lid Temperature | Reaction Volume | Run time | +|-----------------|-----------------|-----------| +| 105°C | 100 μl | 30-45 min | + +| Step | Temperature | Time | +|------|--------------|-----------| +| 1 | 98°C | 3:00 | +| 2 | 98°C | 0:15 | +| 3 | 63°C | 20:00 | +| 4 | 72°C | 1:00 | +| 5 | 11 Cycles* | 0:01:00 | +| 6 | 72°C | Hold | +| 7 | 4°C | Hold | + +*Target cell recovery >6,000. Store samples at 4°C for up to 72 hours. + +## cDNA Cleanup (SPRIselect): +Follow standard bead-based cleanup protocols and elution as in earlier steps. + +## 3′ Gene Expression Dual Index Library Construction + +1. **Fragmentation, End Repair & A-tailing**: + +| Lid Temperature | Reaction Volume | Run time | +|-----------------|-----------------|------------| +| 65°C | 50 μl | 30-45 min | + +| Step | Temperature | Time | +|------------------|----------------|------------| +| Pre-cool block | 4°C | Hold | +| Dehydeation/Fragmentation | Heat to 37°C | Hold | +| End Repair & A-tailing | 65°C | Hold | + +2. **Prepare Ligation Mix**: + +| Reagent | 1X | 2X | 3X | 4X | 5X | 6X | 7X | 8X | +|----------|-----|-----|-----|-----|-----|-----|-----|-----| +| Ligation Buffer | 20 | 44 | 66 | 88 | 110 | 132 | 154 | 176 | +| Adaptor Oligos | 20 | 44 | 66 | 88 | 110 | 132 | 154 | 176 | +| **Total** | 50 | 110 | 165 | 220 | 275 | 330 | 385 | 440 | + +3. **PCR and Sequencing** + +| Parameter | Value | +|----------------------|--------------------------| +| Sequencing Depth | Minimum 20,000 read pairs per cell | +| Sequencing Type | Paired-end, dual indexing | +| Read 1 | 28 cycles | +| i7 index | 10 cycles | +| i5 index | 10 cycles | +| Read 2 | 90 cycles | + +Once quantified and normalized correctly, the 3′ Gene Expression libraries can be denatured and diluted as per standard Illumina sequencing platforms protocols. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/single-nuclei-isolation-from-snap-frozen-axolotl-b-b6yqrfvw.md b/markdown-output/single-nuclei-isolation-from-snap-frozen-axolotl-b-b6yqrfvw.md new file mode 100644 index 0000000000000000000000000000000000000000..6c9397d82c6ec270dd020b50f7990b74a336658b --- /dev/null +++ b/markdown-output/single-nuclei-isolation-from-snap-frozen-axolotl-b-b6yqrfvw.md @@ -0,0 +1,111 @@ +```markdown +# Goal/Experiment: +Isolation of single nuclei incorporating EdU from snap-frozen axolotl brain tissue to generate single-nuclei gene expression libraries for downstream analysis, including single-nuclei RNA sequencing. + +# Single-Nuclei Isolation From Snap Frozen Axolotl Brain with Injected EdU +**Ashley Maynard¹, Fides Zenk¹** +¹ETHZ - ETH Zurich +_Apr 02, 2022_ + +This protocol enables isolation of single nuclei with EdU incorporation from frozen pallium dissections (from axolotl) for the purpose of generating single-nuclei gene-expression libraries following a modified protocol from 10x (Demonstrated protocol CG000365, Rev B), Div-Seq (DOI: [10.1126/science.aad7038](https://doi.org/10.1126/science.aad7038)), and EdU FACs protocol. + +In brief, we prepared and precooled wash and lysis buffers (see Materials). Lysis buffer was added to the sample and dissociated via short pulses with an electric grinder. The pestle of the grinder was washed with a wash buffer before centrifugation. Supernatant was removed and the pellet gently washed. After a final centrifugation, the supernatant was removed and the pellet was resuspended in PBS + BSA. Resulting nuclei were then assessed (count and viability) using Trypan Blue assay and counted using the automated cell counter Countess (Thermo Fisher). + +EdU staining was performed immediately using Click-iT EdU Flow Cytometry assay Kit (Thermo Fisher Scientific, #C10424), 500 µl of reaction buffer was added directly to the resuspension buffer, mixed well, and incubated at RT for 30min. 3ml of wash buffer was added to the resuspended nuclei and mixed well. Nuclei were then spun down for 5 min at 500xg (4°C), supernatant was removed, and nuclei resuspended in 500 µl PBS + 0.5% BSA with DAPI. Nuclei were immediately sorted (DAPI+/EdU+). FACS nuclei were then ready for downstream analysis. + +--- + +## Materials + +### Required Equipment +- Electric grinder with pestle + - Kimble 749521-1500 Polypropylene Pellet Pestle Only, 1.5mL Capacity (Case of 100) + - Kimble Pellet Pestles 749540-0000 Drive Unit Cordless Motor with Two AA Batteries + +### Buffers and Reagents +#### Wash/Resuspension Buffer: +| Reagent | Stock | Final | Volume | +|------------------------|----------------------|-------------|--------| +| Tri-HCL (pH 7.4) | 1 M | 10mM | 60µl | +| NaCl | 5M | 10 mM | 12µl | +| MgCl2 | 1M | 3mM | 18µl | +| BSA | 10% | 1% | 600µl | +| RNase Inhibitor | 40000U/µl | 1U/µl | 15µl | +| Nuclease-free Water | | | 5.295ml| + +*Volume will make 6mL.* + +#### Div-Seq Lysis Buffer: +| Reagent | Stock | Final | Volume | +|------------------------|----------------------|-------------|--------| +| Tri-HCL (pH 7.4) | 1 M | 10mM | 100µl | +| NaCl | 5M | 10 mM | 20µl | +| MgCl2 | 1M | 3mM | 30µl | +| Tween-20 | 10% | 0.01% | 10µl | +| NP-40 | 10% | 0.01% | 20µl | +| Digitonin | 5% | 0.001% | 2µl | +| BSA | 10% | 1% | 1000µl | +| DTT | 1M | 1mM | 10µl | +| RNase Inhibitor (M0314S) | 40000U/µl | 1U/µl | 2.5µl | +| Roche Protease Inhibitor | 100x (1 tablet/500 µl) | 1x | 100µl | +| Nuclease-free Water | | | 8.8ml | + +*Volume will make 10mL.* + +### Prepare Click-iT EdU reagents +1. To make a **10X stock solution of the Click-iT® EdU buffer additive** (Component G), add 2 mL of deionized water to the vial and mix until fully dissolved. Store at ≤ -20°C. The stock solution is stable for up to 1 year. +2. Prepare a working solution of Alexa Fluor® 647 azide (Cat. no. C10424) by adding 130 µl of DMSO to Component B and mix well. Store at ≤ -20°C. The working solution is stable for up to 1 year. +3. Prepare **1X Click-iT EdU buffer additive** by diluting the 10X stock solution 1:10 in deionized water (e.g., 2 reactions = 10µl + 90µl). + +### Prepare the Click-iT® reaction cocktail: +| Reaction component | 1x | 2x | 3x | +|-------------------------|------|------|------| +| PBS | 438 µl | 875 µl | 1.314 mL | +| CuSO4 (Component F) | 10 µl | 20 µl | 30 µl | +| Fluorescent dye azide | 2.5 µl | 5 µl | 7.5 µl | +| Reaction Buffer Additive| 50 µl | 100 µl | 150 µl | +| **Total reaction volume**| 500 µl | 1 mL | 1.5 mL | + +--- + +## Protocol + +### Buffer Preparation +1. Prepare the buffers as described in the Materials section. Store buffers at 4°C or on ice. + +### Nuclei Isolation (10m 10s) +2. Use pre-cooled buffers and store on ice. Perform isolation steps on ice, using pre-cooled micro-centrifuge at 4°C. +3. Put tissue in a cold **1.5 mL tube**. +4. Add **50 µL of lysis buffer**. +5. Using an electric grinder, grind the tissue for 10s (or 2-5 pulses depending on tissue consistency). Rinse the pestle with **150 µL wash buffer**. +6. (Optional) Check an aliquot of the nuclei using Evos or Nikon microscope. +7. Centrifuge down for 5 min at 500 x g (at 4°C). +8. (Optional) Keep the supernatant and check an aliquot using Evos or Nikon microscope. +9. Wash the pellet with **200 µL of wash buffer** (do not disturb the pellet). +10. Centrifuge down for 5 min at 500 x g (at 4°C). +11. Resuspend the pellet in **55 µL of wash/resuspension buffer**. +12. Count with trypan blue (**5 µL sample + 5 µL trypan**). + +### Stain for EdU (35m) +13. Add **500 µL Click-iT® reaction cocktail** to nuclei, mix well, and incubate at room temperature for 30 minutes protected from light. +14. Wash the nuclei once with **3 mL of 1% BSA in PBS**. +15. Centrifuge down for 5 min at 500 x g (at 4°C). +16. Remove the supernatant, dislodge the pellet, and resuspend the nuclei in **500 µL of Wash/Resuspension Buffer**. Add **5 µL DAPI** and perform FACS sorting immediately (DAPI/EdU). + +### FACS of Cells +17. Perform FACS to obtain EdU+ cells. FACS a negative control first (sample without EdU injection but also stained for EdU). From this control, gate for DAPI+ and EdU+ cells. + +--- + +Ashley Maynard, Fides Zenk 2022. Single-Nuclei Isolation from Snap Frozen Axolotl Brain with Injected EdU. [protocols.io](https://protocols.io/view/single-nuclei-isolation-from-snap-frozen-axolotl-b-b6yqrfvw). + +--- + +**Additional Information:** +- Reagents and Equipment from Thermo Fisher Scientific, Roche, and Kimble. +- Alternative Method: If specific reagents or equipment are unavailable, consider other brands or consult relevant scientific literature for substitutes. Perform small-scale validation experiments to ensure comparability of results. + +--- + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/single-nucleus-isolation-from-frozen-human-lung-ti-zu8f6zw.md b/markdown-output/single-nucleus-isolation-from-frozen-human-lung-ti-zu8f6zw.md new file mode 100644 index 0000000000000000000000000000000000000000..c52fa37f17611477ff9e8773b298dba1e1313213 --- /dev/null +++ b/markdown-output/single-nucleus-isolation-from-frozen-human-lung-ti-zu8f6zw.md @@ -0,0 +1,134 @@ +```markdown +## Goal/Experiment: +We aimed to develop a protocol for isolation of single nuclei from archival frozen human lung tissue suitable for single-cell RNA-seq using standard 10x Genomics chemistry. The protocol utilizes standard nuclei isolation buffer (Nuclei EZ buffer) supplemented with RNase inhibitor and a protease inhibitor. + +# Single-nucleus isolation from frozen human lung tissue for single-nucleus RNA-seq + +**Nikita Joshi1, Alexander Misharin1** +1Northwestern University + +Human Cell Atlas Method Development Community + +Alexander Misharin +Northwestern University + +## Abstract +We aimed to develop a protocol for isolation of the single nuclei from the archival frozen human lung tissue suitable for single-cell RNA-seq using standard 10x Genomics chemistry. The protocol utilizes standard nuclei isolation buffer (Nuclei EZ buffer) supplemented with RNase inhibitor and a protease inhibitor. + +For the homogenization step, we elected to use C Tube and GentleMACS tissue disassociation as a way to standardize the homogenization procedure. To maintain nuclei integrity we skipped washing steps and instead use large volumes of washing buffer and proceed to FACSorting immediately after lysis for nuclei purification. + +This protocol produces a good yield of nuclei and diverse libraries, with multiple cell types being detected. While the number of detected genes and UMI per nuclei is lower than for single cell RNAseq, it is sufficient for identification of the specific cell types, including rare populations. Moreover, snRNAseq allowed resolution of the cell types that are intimately integrated into the lung matrix and otherwise hard to resolve: fibroblasts and type I alveolar epithelial cells. + +We thank Oni Basu (UChicago), Luciano Martelotto (Monash University), Nicole Abreu (10x Genomics) and Sharmila Chatterjee (10x Genomics) for their advice. + +## Materials + +| Name | Catalog # | Vendor | CAS Number | RRID | +| ---- | --------- | ------ | ---------- | ---- | +| RNasin(R) Plus RNase Inhibitor, 10,000u | N2615 | Promega | - | - | +| Nuclei EZ lysis buffer | EZ PREP NUC-101 | Sigma | - | - | +| DAPI | D1306 | Thermo Fisher Scientific | - | - | +| DPBS (no Ca, no Mg) | 14190144 | Thermo Fisher | - | - | +| Albumin, Bovine Serum, 10% Aqueous Solution, Nuclease-Free | 126615-25ML | Millipore Sigma | - | - | +| cOmplete™ EDTA-free Protease Inhibitor Cocktail | 11873580001 | Sigma Aldrich | - | - | +| C Tube | 130-096-334 | Miltenyi Biotec | - | - | +| Pre-Separation Filters (30 µm) | 130-041-407 | Miltenyi Biotec | - | - | + +## Before Starting + +### Buffers + +1. **cOmplete stock (10x)** + - Nuclei EZ Prep buffer: 1000 µl + - cOmplete: 1 tablet + - Final concentration: 1 ml + +2. **Lysis buffer (1x)** + - cOmplete stock 10x: 100 µl + - Nuclei EZ Prep buffer: 875 µl + - RNasin Plus (40 U/µl): 25 µl + - Final concentration: 1 ml + +3. **Wash buffer (1x)** + - PBS: 875 µl + - BSA 10%: 100 µl + - RNasin Plus (40 U/µl): 25 µl + - Final concentration: 1 ml + +4. **Resuspension buffer (1x)** + - PBS: 974 µl + - BSA 10%: 1 µl + - RNasin Plus (40 U/µl): 12.5 µl + - Final concentration: 1 ml + +5. **Capture buffer (1x)** + - PBS: 775 µl + - BSA 10%: 200 µl + - RNasin Plus (40 U/µl): 25 µl + - Final concentration: 1 ml + +## General Protocol + +1. **Preparation** + - Prepare cOmplete stock, 10x, keep on ice. + - Prepare Lysis buffer, 2 ml per sample, keep on ice. + - Prepare Wash buffer, 4 ml per sample, keep on ice. + - Prepare Resuspension buffer, 0.3 ml per sample, keep on ice. + +2. **Tissue Handling** + - Take human lung sample from -80°C or LN2 storage, cut ~5-7 mm piece, keep on dry ice until ready. + - Place human lung sample on a small plastic weighing boat, keep on ice, let thaw almost completely (~30-60 sec). + - Using 3 ml syringe and 30G needle, inject ~1 ml of the Lysis buffer into the tissue, move the needle to distribute solution evenly (“inflate” the tissue). + - Chop with scissors into ~1.5-2.0 mm pieces (~1 min). + - Transfer chopped lung tissue and solution into C tube, add the rest of the lysis buffer, final vol 2 ml. Close C tube, invert, make sure that all small pieces are at the base, keep on ice. + +3. **Homogenization** + - Place C tube on MACS Tissue Dissociator and run `m_lung_01` program, then run `m_lung_02` program for 20 sec, stop, immediately place the tube on ice. The solution will be foamy and will contain small pieces of tissue. + - To bring foam down - briefly spin the tube in the swinging bucket rotor centrifuge (~30 sec, at 4C). + +4. **Filtration and Washing** + - Set 30 µm filter on top of the 15 ml polypropylene tube (on ice). Using wide bore tip transfer lysis buffer and remaining pieces of the lung tissue on top of the filter. + - Rinse the filter with 4 ml of Wash buffer, remove the filter, close the tube, mix by inverting. Keep on ice. + - Take 20 µl aliquot for counting: use AO/PI solution, count on Nexcelom K2 Cellometer. + +5. **Staining** + - Add 2 µl of DAPI (5 mg/ml stock) per 1 ml of the solution (~12 µl). Mix, incubate on ice for 5 min, proceed to cell sorting. + - On the sorter: make sure the UV laser is on, use 450/50 filter for DAPI. Gate on DAPI+ cells on log scale, then switch to linear and gate on G0/G1 and G2/M events, exclude subG0 events and doublets. + - Optional: SYTO RNA Select Dye can be used instead or in addition to DAPI to identify nuclei containing RNA. Acquire with the Blue (488 nm) laser and 530/50 filter. + +6. **Sorting (Option A)** + - Sort 10k events into RT mix, adjust the volume to 90 µl with H₂O, add RT enzyme, proceed to emulsion generation using 10x Chromium. + - This step is based on Luciano Martelotto's protocol: [10x Genomics Protocol](https://community.10xgenomics.com/t5/Customer-Developed-Protocols/ct-p/customer-protocols). + +7. **Sorting (Option B)** + - Sort nuclei into 200 µl of Capture buffer, use protein lo-bind 1.5 ml tubes, after sorting keep on ice. + - Pellet nuclei using swinging bucket rotor centrifuge, 300 rcf, 5 min, 4°C. + +8. **Resuspension** + - Remove supernatant add appropriate volume of Resuspension buffer (based on the number of sorted nuclei, add enough buffer to obtain 1000 nuclei/µl, adjust for 20% loss during sorting and centrifugation), let sit on ice for 1 min before gently resuspending the pellet. Filter through 30 µm filter if necessary. + - Count nuclei, adjust concentration if necessary, proceed with 10x Chromium. + +## Expected Results + +### Flow Cytometry Plots +- Typical flow cytometry plots for nuclei prep. Gating ensures separation of nuclei from debris and identification of cell cycle stages. +- Example flow cytometry data shows gating strategies for various markers (e.g., DAPI, SYTO RNA Select). + +### UMAP Plot +- UMAP plot demonstrating clustering of 4,342 nuclei into 16 clusters. +- Explanted lung from patient with systemic sclerosis-associated interstitial lung disease was frozen in liquid nitrogen and stored at -80°C for several months before processing. Library prepared using 10x Genomics V2 3’ chemistry, sequenced on Illumina HiSeq 4000, with initial processing using CellRanger package. + +## Optimizations Required +1. **Tissue Cutting** + - Starting from thick (60-100 um) cryostat sections from the frozen tissue may improve sampling of the cell types deeply integrated into the lung matrix. +2. **Lysis Buffer** + - Decreasing concentration of the EZ lysis buffer 10 times does not affect nuclei yield. Impact on nuclei quality and cell type bias needs to be tested and validated. +3. **Centrifugation Speed** + - Decreasing speed from 400 rcf to 300 rcf results in nuclei with better morphology and less "blebbing". +4. **Nozzle Size** + - Use of 70 µm nozzle instead of 100 µm nozzle will decrease the amount of ambient RNA. +5. **Washing** + - The impact of the wash vs no wash protocol on ambient RNA needs to be validated. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/slime-away-a-simple-ctab-based-high-molecular-weig-bwcwpaxe.md b/markdown-output/slime-away-a-simple-ctab-based-high-molecular-weig-bwcwpaxe.md new file mode 100644 index 0000000000000000000000000000000000000000..e92436b3452eede4e12722d660459924069d53aa --- /dev/null +++ b/markdown-output/slime-away-a-simple-ctab-based-high-molecular-weig-bwcwpaxe.md @@ -0,0 +1,152 @@ +```markdown +# Goal/Experiment: +The goal of this experiment is to present a protocol for the extraction of high-molecular-weight DNA and RNA from mucus-rich invertebrate species, particularly those recalcitrant non-model organisms that are difficult to process with commercial methods. + +# Slime away: a simple CTAB-based high molecular weight DNA and RNA extraction protocol for "difficult" invertebrates (rev20200518) + +## Authors: +- Sergio Vargas +- Cüneyt Caglar +- Gabi Büttner +- Simone Schätzle +- Fabian Deister +- Gert Wörheide + +**Department of Earth and Environmental Sciences, Paleontology & Geobiology, Ludwig-Maximilians-Universität München, Germany** + +DOI: [dx.doi.org/10.17504/protocols.io.bvccwpaxe](https://dx.doi.org/10.17504/protocols.io.bvccwpaxe) + +--- + +## Disclaimer +Before starting, please check the **Guidelines & Warnings** and the **Materials** sections. Ensure you understand all the steps and associated risks. + +This protocol is provided free of charge without warranty. Modifications might be necessary to optimize it for your group of interest. + +## Abstract +Protocols for the extraction of nucleic acids of recalcitrant non-model invertebrates are lacking. Here we present an extraction protocol for the extraction of DNA and RNA from mucus-rich invertebrate species. We have successfully used the protocol for the extraction of high-molecular-weight DNA from cnidarians and poriferans and to recover high-quality RNA from samples impossible to extract with commercial methods. + +--- + +## Keywords +- CTAB +- Invertebrates +- Difficult samples +- Non-model organisms +- Mucus +- Sponges +- Corals +- Porifera +- Cnidaria + +--- + +## Guidelines +Before starting this protocol, please read the MSDS of the reagents used and ensure you fully understand the dangers associated with each of them. + +Check with your lab manager whether you are allowed to use this protocol and use the necessary reagents. + +Wear gloves and safety goggles. Different gloves might be necessary for different chemicals. Be sure you know which type of gloves you need to wear to handle the different reagents used in this protocol. The regulations can change from country to country, **be sure you know what safety regulations apply in your locality**. + +--- + +# Materials + +1. Use 2ml microcentrifuge tubes. +2. Before starting the procedure, pre-heat the CTAB buffer in the oven to 60°C. **ALL BUFFERS** should be prepared with MilliQ water and autoclaved. + +**Optional**: +If RNA extraction will be done with the buffers, all buffers but Tris-HCl must be treated with DEPC overnight and autoclaved. + +### Table 1: Stock and working buffer concentration for DNA/RNA extraction +| Solution | Stock Concentration | Volume for 4.9 ml | +|-------------------|---------------------|-------------------| +| PVP | 10% | 1ml | +| Tris-HCl pH 8.0 | 1M | 500µl | +| EDTA pH 7.5 | 500mM | 250µl | +| NaCl | 5M | 2ml | +| H2O | - | 150µl | +| CTAB | 10% (store at 60°C) | 1ml | + +### Other solutions needed for extraction: +- β-Mercaptoethanol +- Chloroform +- 80% Ethanol +- Isopropanol +- DNase-free Water + +### Optional solutions and enzymes: +- 5M Potassium acetate (KOAc) +- RNase A + +### Safety Warnings +- **β-Mercaptoethanol** is toxic; use it only in the fume hood and be careful handling it. +- **Chloroform** is highly volatile and flammable; use it only under the fume hood and be careful handling it. Ensure the plasticware you use is resistant to chloroform. + +--- + +## Before Starting + +**Prepare the following solutions:** + +| Chemical name | Short name | Company | Cat-No. | Molar mass (g/mol) | Concentration (M) | Volume (l) | Amount (g) | +|---------------------------------------|------------|------------------|----------|--------------------|-------------------|------------|------------| +| Cetyltrimethylammoniumbromid | CTAB | Carl Roth | 9161,1 | 364.46 | 10% | 0.2 | 37.224 | +| Ethyldiamin-tetraacetic acid Disodium | EDTA | Carl Roth | 8043,2 | 372.24 | 0.5 | 0.2 | 37.224 | +| Sodium Chloride | NaCl | Carl Roth | P029.2 | 58.44 | 5 | 0.2 | 58.44 | +| Polyvinylpyrrolidon K30 | PVP | Carl Roth | 4607,1 | - | 10% | 0.2 | - | +| Potassium acetate | KoAC | Sigma Aldrich | P1190 | 98.14 | 5 | 0.2 | 98.14 | +| TRIS (hydroxymethyl)aminoethan | Tris | Sigma Aldrich | 15,456-3 | 121.14 | 1 | 0.2 | 24.228 | + +*Autoclave all solutions before using them for extraction.* + +*If the solutions will be used for RNA extraction or for co-extracting DNA and RNA from the same sample if necessary, treat all solutions (but Tris) with DEPC overnight before autoclaving.* + +--- + +## Protocol + +**Total Duration: 1h 11m** + +1. **Mix the CTAB Buffer** in a 15ml falcon tube and preheat it to 60°C before starting the extraction. +2. **Add 100 µl β-Mercaptoethanol** to the preheated CTAB Buffer under the fume-hood directly before you start with the extraction. +3. **Cut a small piece of tissue** and place it in a 2ml tube. Try to smash the tissue to make cells accessible to the buffer. + + **Optional:** + - Transfer the frozen/preserved tissue to a mortar pre-cooled with liquid nitrogen. + Best practice: let the tissue "swim" in the liquid nitrogen until the mortar is dry. The mortar should be kept cold; you can achieve this by placing the mortar in a small cool box with liquid nitrogen in it. + - Grind the tissue until you get a fine powder. + - Using a small spatula, transfer the powder to a 2mL nuclease-free microcentrifuge tube. Best practice: pre-cool the spatula and the tube by placing them in the cool box containing liquid nitrogen; this helps to keep the powder dry and cold. + +4. **Add 550 µl** of pre-heated (60°C) extraction buffer and **50 µl** Proteinase K (120 mg/ml) to the tube. +5. **Mix thoroughly until all powder** or smashed tissue is in solution. If needed, vortex lightly for ca. 1 minute. + - Incubate at least 15 minutes to overnight at 56°C with moderate mixing (~400 rpm). In general, if the tissue was ground OR smashed, after two hours the tissue is digested. It is a good practice to manually agitate the digestion approx. every 15 minutes to avoid the formation of clumps of tissue. + + **Optional** If samples have a high polysaccharide content: + - **Add 150 µl KOAc (5M)** to the tubes, mix by inversion and incubate 10 minutes at 60°C with moderate mixing. + - Centrifuge at max. speed for 15 minutes. + - Recover 600 µl supernatant trying to avoid the transfer of any pellet or viscous precipitate. + + **Optional** If you expect RNA contamination e.g., in fresh samples: + - **Add 5 µl RNase A**, mix gently by inversion and incubate at 37°C for 30 minutes. + +6. **Add one volume (600 µl) of Chloroform** to the sample and shake vigorously until the solution turns milky. +7. **Centrifuge** (full speed) at room temperature for 15 minutes. +8. **Remove the tube** carefully from the centrifuge, trying to leave the two phases undisturbed. If the phases mix, centrifuge again. Transfer 200 µl of the upper phase to a new 2ml tube. Be careful not to transfer the interphase or traces of Chloroform. +9. **Add 1 volume (400 µl)** of isopropanol to the solution containing the DNA. Mix gently by hand and incubate at room temperature for 10 minutes. You may see tiny bubbles and sometimes white flakes forming. +10. **Centrifuge** at 16°C for at least 30 minutes at full speed. +11. **Discard supernatant** (by pipetting) without disturbing the pellet. +12. **Add 1 ml ethanol** (80% (v/v)) to the DNA pellet and centrifuge at maximum speed for 15 minutes at 16°C. The pellet should be visible by now. +13. **Remove the supernatant** carefully (watch for the pellet). If wanted, you can repeat the previous step one more time. If not, proceed to 14. +14. **Centrifuge** shortly and remove any remaining EtOH with the 10 µl pipette. +15. **Leave the tube open** for about 2 minutes to dry the pellet. You can also check whether the pellet is transparent and proceed to the next step. +16. **Resuspend the DNA** in ~30 µl water. (Optional) Incubate at 37°C in the Thermomixer for 20 minutes. +17. **Load 2 µl** of the extracted DNA on a 1% Agarose gel and let the gel run at 40V for 4 to 6 hours. If high molecular weight DNA is expected, use the lambda Hind-III marker to assess the quality of the extracts. Quantify the DNA concentration using the NanoDrop: record the concentration, the 260/230 and 260/280 ratios. A good extraction should have 260/230 and 260/280 ratios above 1.8. +18. The protocol should yield high-quality DNA and RNA. Below is an example using the recalcitrant octocoral **Xenia sp.**. Without KoAc, RNA cannot be recovered on a regular basis. Also, note the quality of the DNA obtained using the protocol. + +> **Note: You can use different commercial kits in tandem to isolate either DNA or RNA and purify the extracts further.** + +![Nucleic acid extraction of the octocoral Xenia sp. using the Slime away protocol.](https://user-images.githubusercontent.com/6559126/140009411-b5be43c1-872f-46c7-beac-3058a11fbbf1.png) + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/small-volume-fungal-genomic-dna-extraction-protoco-3w9gph6.md b/markdown-output/small-volume-fungal-genomic-dna-extraction-protoco-3w9gph6.md new file mode 100644 index 0000000000000000000000000000000000000000..c0bebaf965e8d2294fa2eb2d3c481f7fbcb7e77e --- /dev/null +++ b/markdown-output/small-volume-fungal-genomic-dna-extraction-protoco-3w9gph6.md @@ -0,0 +1,122 @@ +```markdown +# Goal/Experiment: +High molecular weight DNA extraction from hyphae of filamentous fungi using a modified SDS phenol:chloroform method for Illumina genome sequencing. + +## Small Volume Fungal Genomic DNA Extraction Protocol for Illumina Genome V.1 + +### Authors +Jana M U'Ren¹, Lilly Moore¹ + +¹University of Arizona + +**DOI:** [dx.doi.org/10.17504/protocols.io.3w9gph6](https://dx.doi.org/10.17504/protocols.io.3w9gph6) + +### Overview +This protocol describes a modified SDS phenol:chloroform method for obtaining high-quality DNA from hyphae of filamentous fungi. The resulting DNA can be used for Illumina genome sequencing. Pure fungal cultures are inoculated on 60mm 2% MEA plates with a sterile cellophane overlay to minimize media carry-over and grown for 5-10 days. Mycelium is then removed with sterile forceps and scalpels and placed in a 1.5mL tube with steel beads. Flash freeze the tissue in liquid nitrogen and store at -80°C prior to extraction. + +### Notes +- Be very gentle during all mixing and pipetting steps to ensure that DNA does not get sheared. +- Phenol:chloroform is highly toxic and should only be used in a designated fume hood. All waste should be disposed of in proper containers. + +### Materials +- 100% EtOH (Ethanol) +- SDS Buffer +- Liquid Nitrogen +- Phenol:Chloroform:IAA (25:24:1) +- RNase A +- Low salt TE + +### Preparation +1. Freeze bead-beating aluminum block in -80°C for at least 1 hour. +2. Ensure you have a lab bench aliquot of 100% molecular grade ethanol, Nuclease free water, SDS Extraction buffer, etc. +3. Make fresh 70% ethanol dilution (3mL 70% EtOH x ____ samples = _____ mL 70% EtOH). + - Calculation: + - ______mL 70% EtOH needed x 0.7 = ______mL 100% EtOH + ______mL H₂O +4. Turn on 4°C refrigerated centrifuge and allow to chill. +5. Record samples to extract in notebook. +6. Record lot numbers of all reagents used in notebook. +7. Label all tubes needed for DNA extraction. +8. Turn on dry heat block and set to 65°C. + +### Protocol Steps +1. Pre-measure SDS Buffer need for all samples (e.g., 500µL SDS Buffer x 3 samples = 1,500µL) into a 5 mL tube. + - Calculation: + - 500µL SDS Buffer x _____ samples = ___________ µL of SDS Buffer pre-aliquoted. + +2. Bead beating tissue frozen in liquid nitrogen. + - Use one 15 second cycle at 1400 RPM in frozen aluminum block. + - If samples are properly homogenized, remove from bead beater and place in -20°C ice box. + - Repeat one 15-second cycle grinding until homogenization has occurred. + - Note: The samples might need to be dropped in liquid nitrogen or have liquid nitrogen poured over them to ensure the samples don’t thaw during the grinding process. + +3. Add 500µL SDS Buffer to each sample. + - Note: Vortex gently if needed to homogenize sample in SDS Buffer. + +4. Incubate at 65°C for 15 minutes, gently inverting sample 5 times every 5 minutes. + +5. Centrifuge samples at maximum speed (14,000 RPM) at 4°C for 5 minutes. + +6. Carefully remove the supernatant (~500µL) without disturbing the pellet. Place in a new, labeled 1.5mL centrifuge tube. + +7. In the chemical fume hood, add 500µL of Phenol:Chloroform:IAA (or 1:1 of supernatant to P:C:IAA) to each tube of supernatant. + +8. Invert the samples 20X to mix until "milky white." + +9. Spin tube at maximum speed at 4°C for 10 minutes. + +10. In chemical fume hood, carefully remove the aqueous layer with 200µL pipette, do not disturb the interface. Place respectively into new 1.5mL microcentrifuge tubes. + - Note: The smaller aperture of the 200µL tips makes it easier to remove the aqueous layer without disturbing the layer below. + +11. Add 0.3X volume of absolute molecular grade ethanol to each tube. This high-salt, low ethanol mixture precipitates the excess polysaccharides while gDNA remains in solution. + - Calculation: + - _________ µL supernatant x 0.3 = ____________ µL of ethanol + +12. Invert tubes 20 times to mix. + +13. Spin at maximum speed at 4°C for 15 minutes. + +14. Carefully remove the supernatant without disturbing the polysaccharide pellet. Place supernatant into a new 1.5mL centrifuge tube. + +15. Add 1.7X volume of absolute molecular grade ethanol to each tube. The gDNA can be seen as falling out of solution as long strands of gDNA. + - Calculation: + - ___________ µL supernatant x 1.7 = ____________ µL of ethanol + +16. Invert tube 20 times to mix. + +17. Spin tube at maximum speed at 4°C for 15 minutes. + +18. Carefully pour off ethanol into glass beaker. + +19. Add 1.5 mL of 70% ethanol to remove the excess salt, do not disturb the pellet. + +20. Spin the tube at maximum speed at 4°C for 1 minute. + +21. Carefully pour off ethanol into glass beaker. + +22. Add 1.5 mL of 70% ethanol to remove the excess salt, do not disturb the pellet. + +23. Spin the tube at maximum speed at 4°C for 1 minute. + +24. Carefully pour off ethanol into glass beaker. + +25. Quick spin tube with pellet to gather residual ethanol at the bottom of the tube and carefully remove with a P20 tip. + +26. Let the pellet air dry for 5 minutes at room temperature in the biosafety cabinet, taking care not to over dry. + +27. Resuspend pellet in 50µL low salt TE. + - Note: Increase volume of low salt TE of 10µL if pellet does not resuspend. + +28. Add 2 µL RNase A and place in 37°C for 1 hour. + +29. Dilute samples 1:10 and use dilution to Nanodrop and Qubit samples. Record quantification and purity values with the chart below. + +| Sample name | Nanodrop 1:10 ng/µL | Qubit 1:10 ng/µL | Qubit ng/µL | 260/280 | 260/230 | Total TE (µL) | Total DNA (µg) | +| ----------- | ------------------- | ---------------- | ----------- | ------- | ------- | ------------- | -------------- | +| | | | | | | | | +| | | | | | | | | +| | | | | | | | | +| | | | | | | | | +| | | | | | | | | + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/smart-seq2-single-cell-rna-seq-modified-method-pbgdijw.md b/markdown-output/smart-seq2-single-cell-rna-seq-modified-method-pbgdijw.md new file mode 100644 index 0000000000000000000000000000000000000000..3799afbc3cf9a5047805c9c397ca9c32f21ff7bd --- /dev/null +++ b/markdown-output/smart-seq2-single-cell-rna-seq-modified-method-pbgdijw.md @@ -0,0 +1,103 @@ +```markdown +# Goal/Experiment: +To create a comprehensive single-cell RNA-Seq library using the Smart-seq2 modified method. The method leverages the terminal transferase activity of reverse transcriptase and several other optimizations to generate high-quality full-length cDNA libraries for downstream Illumina sequencing. + +# Smart-seq2 Single-Cell RNA-Seq Modified Method +#### John J. Trombetta, David Gennert, Diana Lu, Rahul Satija, Alex K. Shalek, Aviv Regev + +## Abstract +For the past several decades, due to technical limitations, the field of transcriptomics has focused on population-level measurements that can mask significant differences between individual cells. With the advent of single-cell RNA-Seq, it is now possible to profile the responses of individual cells at unprecedented depth and thereby uncover, transcriptome-wide, the heterogeneity that exists within these populations. This unit describes a method that merges several important technologies to produce, in high-throughput, single-cell RNA-Seq libraries. + +## Introduction +The development of single-cell RNA-Seq affords new opportunities to study complex cellular systems at unprecedented resolution. This protocol takes advantage of the terminal transferase activity of the SMARTScribe reverse transcriptase in conjunction with a “template-switch” primer to create cDNAs that have PCR priming sites on both ends directly from full-length mRNA. The steps include cell lysis, RNA isolation, cDNA synthesis, PCR amplification, normalization, and sequencing library preparation. + +## Guidelines +### Oligonucleotide Primer Sequences +- **3' SMART CDS Primer IIA**: 5' AAGCAGTGGTATCAACGCAGAGTACT(30)VN +- **SMARTer II A Oligonucleotide**: 5' AAGCAGTGGTATCAACGCAGAGTACATrGrG +- **IS PCR Primer**: 5' AAGCAGTGGTATCAACGCAGAGT +- **TSO**: 5' AAGCAGTGGTATCAACGCAGAGTACATrGrG+G + +## The Protocol Workflow +### Stage I: Preparation of Single-Cell Lysates +1. **Step 1**: Prepare and distribute a mild hypotonic lysis buffer of 0.2% Triton X-100 and 2 U/μL RNase-Inhibitor into each well of a 96-well PCR plate and a 1.5 mL RNase-free tube. +2. **Step 2**: Use a FACS machine to sort a single cell into each well containing the Buffer TCL, and 10,000 cells into the 1.5 mL tube. +3. **Step 3**: Seal plate, centrifuge (800g, 1 min), freeze plate, and control on dry ice and store at -80°C. + +### Stage II: RT (Reverse Transcription) of mRNA Species +1. **Step 5**: Bring RNA-SPRI beads to room temperature, clean workbench with RNaseZap. +2. **Step 6**: Add RT primer and dNTP mix to lysate and centrifuge (800g, 1 min). +3. **Step 7**: Incubate for 3 minutes at 72°C, place on ice. +4. **Step 8-10**: Add SuperScriptII first strand buffer mix, RNase-Inhibitor, and SuperScriptII RT, mix, and carry out RT in thermal cycler (conditions: 42°C for 90 min, 50°C for 2 min, 42°C for 2 min, 70°C for 15 min, 4°C hold). + +### Stage III: Performing WTA and Post-PCR Cleanup +1. **Step 11-12**: Prepare and use KAPA HiFi HotStart ReadyMix and IS PCR primer for amplification (Thermal cycling conditions: 98°C for 3 min, 20 cycles: 98°C for 15 sec, 67°C for 20 sec, 72°C for 6 min, extension: 72°C for 5 min, hold at 4°C). +2. **Step 13-15**: Bring DNA SPRI beads to room temperature, add beads to wells, mix, and incubate. +3. **Step 16-24**: Wash beads with 80% ethanol thrice, elute with TE Buffer, measure fragment size and concentration. + +### Stage IV: Nextera XT Sequencing-Library Construction +1. **Step 28**: Arrange and label index primers using the TruSeq Index Plate Fixture. +2. **Step 29-30**: Add TD Buffer, ATM, and PCR product mix to each well, perform tagmentation (conditions: 55°C for 10 min). +3. **Step 31-34**: Add NT Buffer, mix with index primer solution, and carry out amplification (PCR conditions: 72°C for 3 min, 95°C for 30 sec, 12 cycles: 95°C for 10 sec, 50°C for 30 sec, 72°C for 60 sec, extension: 72°C for 5 min, hold at 4°C). + +### Stage V: Pooling and DNA SPRI Bead Cleanup +1. **Step 35-37**: Pool, add SPRI beads, mix, and incubate. +2. **Step 38-44**: Magnetically purify, wash thrice with 80% ethanol, dry, elute, and measure final concentration. + +## Background Information +Advances in single-cell RNA sequencing have enabled the study of cellular heterogeneity at unprecedented levels. This methodology has revolutionized insights into rare cell types, transcriptional profiles, and is highly regarded for revealing novel biology. + +## Critical Parameters and Troubleshooting +### Key Issues to Monitor +- **RNA Integrity**: Ensure high-quality RNA, work in an RNase-free environment. +- **PCR Cycles**: Optimize cycle numbers, excessive cycles can introduce bias. +- **Contamination Control**: Use proper sterility practices to avoid sample contamination. + +### Common Problems and Solutions +| Problem | Likely Cause | Solution | +|---------|--------------|----------| +| Little or no WTA yield | RNA degradation, RNA-SPRI failure | Use high-quality RNA, resuspend beads properly | +| Short WTA products | PCR-SPRI failure | Resuspend beads, repeat cleanup | +| Contamination | Cross contamination | Ensure sterile preparation and work environment | + +### Time Considerations +The entire SMARTer library construction and normalization process takes 2-3 days, with additional 1 day for Nextera XT library construction and pooling. + +## Acknowledgments +The work was supported by NIH, HHMI, and other institutional awards. + +## Materials +- **TE Buffer**: 10 mM Tris-HCl, pH 8.0, 1 mM EDTA +- **HotStart ReadyMix**: KAPA HiFi PCR kit, Kapa Biosystems +- **Superase-In**: RNase Inhibitor, Thermofisher AM2694 +- **Microseal® Seals**: BioRad Sciences +- **Buffer TCL**: Qiagen (1031576) +- **SPRI Beads**: Beckman Coulter (A63987) +- Full material list provided in the appendix. + +## Protocol Stages +### Stage I: Preparation of Single-Cell Lysates +Standard preparation and sorting procedures using appropriate lysis buffers and FACS. + +### Stage II: RT of mRNA Species +Detailed reverse transcription setup, using SuperScriptII and defined thermal cycler conditions. + +### Stage III: Performing WTA and Post-PCR Cleanup +Involves amplification with KAPA HiFi mix and SPRI bead based purification. + +### Stage IV: Nextera XT Sequencing-Library Construction +Utilizes a specific index setup and carefully defined tagmentation and amplification steps. + +### Stage V: Pooling and DNA SPRI Bead Cleanup +Processes covers bead pooling and cleanup to achieve final product ready for sequencing. + +## Literature Cited +- Eberwine J et al. (1992). Analysis of gene expression in single live neurons. +- Langmead B et al. (2009). Ultrafast alignment of short DNA sequences. +- Picelli S et al. (2013). Full-length transcriptome profiling. + +## Warnings +Refer to the SDS (Safety Data Sheet) for all safety warnings and hazard information. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/soil-organic-carbon-stocks-and-change-protocol-cnw9vfh6.md b/markdown-output/soil-organic-carbon-stocks-and-change-protocol-cnw9vfh6.md new file mode 100644 index 0000000000000000000000000000000000000000..2a80df24fb5377254a9b34c9e115acb205ae3673 --- /dev/null +++ b/markdown-output/soil-organic-carbon-stocks-and-change-protocol-cnw9vfh6.md @@ -0,0 +1,159 @@ +```markdown +Goal/Experiment: +This protocol details the methods for measuring soil organic carbon (SOC) stocks and changes. The aim is to provide precise measurements of carbon dioxide flux into and out of ecosystems to assess soil health, particularly focusing on SOC concentration, soil bulk density, and the determination of the mass of SOC accumulated in the soil. + +# Soil Organic Carbon Stocks and Change Protocol +**SCarolina Córdova¹, Curtis Dell², Mark Liebig³, Michel A Cavigelli⁴, Phil Robertson⁵** + +1. University of Nebraska, Lincoln +2. USDA-ARS-Pasture Systems Watershed Management Research Unit +3. USDA Northern Great Plains Research Laboratory +4. USDA Agricultural Research Service, Sustainable Agricultural Systems Lab (SASL), USDA GRACEnet and LTAR Network, Northeast Cover Crop Council +5. Michigan State University + +**Protocol Citation:** +SCarolina Córdova, Curtis Dell, Mark Liebig, Michel A Cavigelli, Phil Robertson 2023. Soil organic carbon stocks and change protocol. [protocols.io](https://protocols.io/view/soil-organic-carbon-stocks-and-change-protocol-cnw9vh6). + +## Abstract +The change in soil organic carbon (SOC) over time serves as an integrated indicator of carbon (C) balance in cropping systems and as an integral metric of soil health assessments that can be carried out at all LTAR locations. Determination of SOC concentration and soil bulk density allows calculation of the mass of SOC accumulated in the soil, and changes in SOC over time reflect system C balance. A dry combustion CN analyzer should be used to measure total C. + +## Guidelines + +### General Principles +SOC is a crucial attribute of soil function. Loss on ignition can estimate SOC but is not sufficiently precise for determining soil C stocks. Measurable SOC changes typically require a minimum sampling interval of 10 to 12 years, depending on local factors like existing SOC stores and rotation diversity. + +## Materials + +### Soil Sampling & Archiving +- Soil core samples (2 cm diameter, 30 cm length) +- 7 mL plastic vial +- Paper and plastic bags +- Plastic liners with caps +- Hermetic containers (e.g., mason jars) +- Labels + +### Lab Materials for SOC Concentration Analysis +- Pulverizer (e.g., SPEX SamplePrep 8530 Enclosed ShatterBox) +- Tin capsules (Elemental Microanalysis, pressed, standard weight, 8 × 5 mm) +- Forceps +- Micro spatula, scoop shaped +- Sample tray (e.g., 96-well plate, if using tin capsules) +- Electronic microbalance (resolution to 0.001 mg) +- Standards (acetanilide, atropine, or phenacetin) +- Blind standard (stored soil standard) +- CHN analyzer + +### For Carbonate Pre-Test +- Soil pH meter +- Small weigh boats or beakers, eye dropper or syringe, 1N HCl acid (~10 mL) + +### For Acid Pre-Treatment +- Microliter pipette +- Nanopure water +- Dessicator +- Concentrated hydrochloric acid (12M HCl) + +## Procedure + +### Sample Collection + +#### 1. Collect whole-profile soil samples +- **1 m depth:** Decadal intervals +- **Surface soils:** 0-25 cm or 0-30 cm at shorter intervals +- **Sampling Time:** Post-harvest (late fall/early winter) or pre-planting (early Spring) +- **Systems:** Pre-senescence in grazing systems. + +#### 1.1 Decadal One-Meter Depth Soil Sampling +Samples should be taken as intact cores in acrylic sleeves of 5 cm diameter to 1 m depth using a hydraulic probe to avoid compaction. + +**Case 1: Two- and Five-Year Rotations** +| Year | 2023 | 2024 | 2025 | 2026 | 2027 | 2028 | 2029 | 2030 | 2031 | 2032 | 2033 | +|------|------|------|------|------|------|------|------|------|------|------|------| +| Rotation | Corn | Soy | Corn | Soy | Corn | Soy | Corn | Soy | Corn | Soy | Corn | +| Sampling year | Baseline | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | +| Sampling year | X | | | | | | | | | | | + +**Case 2: Three-, Four-, and Six-Year Rotations** +| Year | 2023 | 2024 | 2025 | 2026 | 2027 | 2028 | 2029 | 2030 | 2031 | 2032 | 2033 | 2034 | 2035 | +|------|------|------|------|------|------|------|------|------|------|------|------|------|------|------| +| Rotation | Corn | Soybean | Wheat | Alfalfa | Follow | Corn | Corn | Soybean | Wheat | Alfalfa | Follow | Corn | +| Sampling year | Baseline | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | +| Sampling year | X | | | | | | | | | | | | | + +- **Plot-Scale Experiments:** At least three cores per plot, GPS-mark sampling positions, refill holes with a mix of bentonite and sand. +- **Field-Scale Experiments:** Obtain a sufficient number of soil cores, transport and store soil cores in plastic liners, or shaded containers until processing. + +#### 1.2 Surface Soil Sampling +Samples should be taken using a push core of 2 cm diameter for the same depth interval used for deep cores (0-25 or 0-30 cm). + +## Soil Processing + +1. **Pre-label paper bags** with sample information. +2. Measure and record the length of each horizon and diameter of the corer (to calculate soil volume). +3. **Core Separation:** Into multiple depth intervals (0-10 cm, 10-25 cm, etc.). +4. **Record fresh weight** of each soil layer (WWsoil). +5. Pass soil layers through a 2 mm sieve to remove gravel and plant material. +6. **Gravel measurement:** Weigh or use volume displacement. +7. **Oven dry soil samples:** + - **Option A:** 60 °C for at least 48h. + - **Option B:** 105 °C for at least 24h. + +8. **Pulverizing:** 50 g subsample to ≤250 microns with a mill (e.g., SPEX SamplePrep ShatterBox). + +### Archiving Soil Samples +Store at least 500 g to 1,000 g in hermetic containers. + +## Acid-Fumigation Pre-Treatment to Remove Carbonates +1. Use silver capsules, place 40-60 mg of soil in an open capsule with nanopure water. +2. Desiccate sample with concentrated HCl for 6-8 hours. +3. Oven dry at 60 °C and fold capsules after drying. + +## Soil Total Carbon Concentration - Using Elemental Analyzer + +1. Use dry combustion gas chromatography (e.g., Costech ECS 4010 CHNSO Analyzer). +2. Test with standards like Acetanilide or Atropine. + +### Other Equipment Options: +Elementar or Leco analyzers handle larger sample sizes and use crucibles instead of tins. + +### Testing for Inorganic Carbon: +1. If pH > 7.2, test with HCl. +2. Quantitatively measure with delta 13C isotopic analysis or acid fumigation. + +## Procedure for Soil Carbon & Nitrogen Analyses +1. **Prepare soil sample:** Grinding ~ 50 g sieved, well-mixed dry soil. +2. **Transfer** to 7 mL plastic vials. +3. **Test for Carbonates** (if necessary). + +### Preparing Analytical Sample Run +1. **Bypass Sample** and record well number. +2. **Sequence:** Bypass, blank, known standards, soil samples. + +### QA/QC Considerations +1. Re-run samples if variance among replicates >10%. + +## Calculations of Soil Organic Carbon Stocks, Change, and Rate + +### Estimation of Gravimetric Water Content +Gravimetric soil water content = (WWsoil − DWsoil ) / DWsoil + +### Estimation of Soil Carbon Concentration (%) +Report average values from replicates. Total C = total organic C (TOC) if inorganic C was removed. + +### Estimation of SOC Stocks, Change, and Rate of Change +Use spatial coordinate or equivalent soil mass methods. + +### Spatial Coordinate Method (Gifford & Roderick, 2003) +Calculate SOC stocks per depth using bulk density. + +### Equivalent Soil Mass Method (Wendt & Hauser, 2013) +Use fixed soil mass layers for consistency in comparing SOC changes. + +### Covariate Metrics +Total soil bulk density, gravel/rocks weight, and soil pH (in calcareous soils). + +## References +See references for detailed methodologies and related studies. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/sop-for-populating-ebi-submission-templates-ena-bh5dj826.md b/markdown-output/sop-for-populating-ebi-submission-templates-ena-bh5dj826.md new file mode 100644 index 0000000000000000000000000000000000000000..49c140fc6bb66f54796f7e75a14dfb59c536f444 --- /dev/null +++ b/markdown-output/sop-for-populating-ebi-submission-templates-ena-bh5dj826.md @@ -0,0 +1,134 @@ +```markdown +# SOP for Populating EBI Submission Templates (ENA) + +## Goal/Experiment: +The goal of this experiment is to provide guidance on how to populate the extended PHA4GE metadata package for SARS-CoV-2 submissions, maximizing interoperability for COVID-19 surveillance. + +### Metadata Guidance for SARS-CoV-2 Submission to EBI + +This protocol helps to describe the fields needed to populate the ENA virus pathogen checklist. Follow the primary PHA4GE guidance to ensure the controlled vocabularies and ontology terms are used to populate these fields. + +#### Links to Required Specifications: +- [PHA4GE SARS-CoV-2 Metadata Specification](https://github.com/pha4ge/SARS-CoV-2-Contextual-Data-Specification) +- [PHA4GE SARS-CoV-2 EBI Submission Protocol: ENA, BioSample, and BioProject](https://www.protocols.io/private/BD4C35A5C2942B2D927E662ABC3D195) +- [PHA4GE SARS-CoV-2 EBI Assembly Submission Protocol](https://www.protocols.io/private/39BBC8E9B6F911EAA1530A58A9FEAC2A) + +### ENA Virus Pathogen Reporting + +#### 1. PHA4GE ENA Virus Pathogen Reporting Standard Checklist for Sample Metadata + +ENA submission spreadsheets are not tables with field names in the first row but specifics for each sample in subsequent rows. The spreadsheet requires particular headers: + +| Field | Example Value | +|-------------------------------------|-----------------------------------------| +| sample\_name | hCOV-sample-ENG-11 | +| tax\_id | 2697049 | +| scientific\_name | Severe acute respiratory syndrome coronavirus 2 | +| host\_age | 50 | +| organism | Severe acute respiratory syndrome coronavirus 2 | +| collection\_date | 05/05/2020 | +| geographic\_location (country and/or sea) | United Kingdom | + +#### 2. Guidelines to Populate the Sample Metadata Sheet + +These fields are required for all ENA submissions. They do not appear as part of the checklist: + +#### Description of Required Fields: +| ENA Required Fields | Definition | +|---------------------------|---------------------------------------------------------------------------| +| sample\_name | The user-provided name of the sample. | +| tax\_id | The NCBI taxon identifier for the organism being sequenced. | +| scientific\_name | The taxonomic name of the organism. | +| common\_name | The common name of the organism. | +| sample\_description | Free text description of the sample. | +| instrument\_model | Name of the sequencing instrument. | +| library\_source | Molecule type used to make the library. | +| library\_selection | Library capture method. | +| library\_strategy | Overall sequencing strategy or approach. | +| library\_layout | Single or paired. | +| file\_name | Include ALL of the files resulting from this library. (Add additional fields if there are more than two files, e.g., filename3) | + +The following table aligns PHA4GE fields with ENA Required Fields, guiding the transition between metadata formats: + +| ENA Required Fields | PHA4GE Field | PHA4GE Guidance | +|---------------------------|---------------------------------|----------------------------------------------------------------------------------------------------| +| sample\_name | specimen collector sample ID | This field can be populated by the PHA4GE field "specimen collector sample ID". | +| tax\_id | N/A | Use "2697049" as the tax\_id for SARS-CoV-2. | +| scientific\_name | organism | This field can be populated by the PHA4GE field "organism" as "Severe acute respiratory syndrome coronavirus 2". | +| common\_name | N/A | This field can be populated by the PHA4GE field "host (common name)" as "Sars-CoV-2". | +| sample\_description | N/A | See ENA SRA pick list (e.g., Illumina MiSeq, iSeq 100, GridION, MinION, PacBio Sequel II). | +| instrument\_model | N/A | See ENA SRA pick list (e.g., viral RNA, metagenomic). | +| library\_source | N/A | See ENA SRA pick list (e.g., random, PCR). | +| library\_selection | N/A | See ENA SRA pick list (e.g., WGS, RNA-Seq, Amplicon). | +| library\_strategy | N/A | See ENA SRA pick list (e.g., single, paired). | +| file\_name | r1 fastq filename | This field can be populated by the PHA4GE field "r1 fastq filename". | + +#### 3. Mapping Metadata to ENA Virus Checklist ERC000033 + +Submissions need to comply with [Checklist ERC000033](https://www.ebi.ac.uk/ena/browser/view/ERC000033). Below is the description, mapping, and requirements: + +| ENA Virus Checklist Field | ENA Definition | ENA Requirement Status | +|----------------------------|----------------------------------------------------------------------------------------------------|----------------------------------------| +| subject exposure | Exposure of the subject to infected human or animals. | optional | +| subject exposure duration | Duration of the exposure of the subject to an infected human or animal. | optional | +| type exposure | Setting of subject's exposure. | optional | +| personal protective equipment | Use of personal protective equipment like gloves, gowns during exposure. | optional | +| hospitalisation | Hospitalization details due to virus infection. | optional | +| illness duration | Number of days the illness lasted. | optional | +| illness symptoms | Symptoms reported in relation to illness. | optional | +| collection date | The date of sampling. | recommended | +| geographic location (country and/or sea) | The geographical origin of the sample. | mandatory | +| geographic location (latitude) | The geographical origin of the sample in latitude. | recommended | +| geographic location (longitude) | The geographical origin of the sample in longitude. | recommended | +| geographic location (region and locality) | The region and locality of the sample's origin. | recommended | +| sample capture status | Reason for the sample collection. | recommended | +| host disease outcome | Outcome in the host. | recommended | +| host common name | Common name of the host (e.g., human). | mandatory | +| host subject ID | Unique identifier for each subject. | mandatory | +| host age | Age of the host at the time of sampling. | recommended | +| host health state | Health status of the host at the time of sample collection. | mandatory | +| host sex | Gender or sex of the host. | mandatory | +| host scientific name | Scientific name of the host. | mandatory | +| virus identifier | Unique laboratory identifier assigned to the virus. | recommended | +| collector name | Name of the person who collected the specimen. | mandatory | +| collecting institution | Name of the institution to which the person collecting the specimen belongs. | mandatory | +| receipt date | Date on which the sample was received. | recommended | +| sample storage conditions | Conditions during which the sample was stored. | optional | +| definition for seropositive sample | The cutoff value used in seropositive determination. | recommended | +| serotype (required for a seropositive sample) | Serological variety of species based on antigenic properties. | recommended | +| isolate | Individual isolate from which the sample was obtained. | mandatory | +| strain | Name of the strain from which the sample was obtained. | optional | +| host habitat | Natural habitat of the avian or mammalian host. | recommended | +| isolation source host-associated | Name of host tissue or organ sampled for analysis. | recommended | +| host description | Descriptive information regarding the host. | optional | +| gravidity | Whether the subject is gravid. If so, reporting details. | optional | +| host behaviour | Natural behaviour of the host. | recommended | +| isolation source non-host-associated | Describes the physical/environmental source of the sample. | recommended | + +### 4. Populate ENA Run Metadata Table + +#### PRO TIPS: +1. For drastically different metadata, create a separate submission + metadata table for each case. +2. Entering fastq filenames can be easier on Mac with a direct folder-to-spreadsheet copy. +3. Develop a QA/QC step to ensure the files have correct sample names. Use tools like Excel functions for accuracy. + +#### Description of Fields in the Table: +| Field | Description | Example | +|------------------------------|----------------------------------------------------------------------------------------|-------------------------------------------| +| Sample reference | Same ID as "sample\_name" in the BioSample submission template. | UT-12345 | +| Library name | Unique ID relevant to workflow. Autogenerated ID is acceptable. | UT-12345.6 | +| Title | Short free text description for public pages. | Amplicon-based sequencing of SARS-CoV-2: UT-12345 | +| Library strategy | Overall sequencing strategy or approach. | WGS, RNA-Seq, Amplicon | +| Library source | Molecule type used to make the library. | viral RNA, metagenomic | +| Library selection | Library capture method. | random, PCR | +| instrument model | Name of the sequencing instrument. | Illumina MiSeq, iSeq 100, GridION | +| Design description | Free text methods description field. | ARTIC PCR-tiling of viral cDNA (V3), sequenced on Illumina MiSeq with DNA Flex library prep kit | +| File name | Files resulting from this library. | genome\_r1.fastq (\*must be exact) | +| Second file name | For certain file types (i.e., paired FASTQ). | genome\_r2.fastq (\*must be exact) | +| Filename3-8 | List other fastq file names. | | +| (First/Second) MD5 Checksum | MD5 checksum of the file being submitted | 07182d8b0... | + +For additional guidelines and details, refer to the [PHA4GE SARS-CoV-2 metadata specification](https://github.com/pha4ge/SARS-CoV-2-Contextual-Data-Specification). + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/southern-blotting-hf6b3re.md b/markdown-output/southern-blotting-hf6b3re.md new file mode 100644 index 0000000000000000000000000000000000000000..99cad0466fe724413eb6adb6cec165346f500e64 --- /dev/null +++ b/markdown-output/southern-blotting-hf6b3re.md @@ -0,0 +1,143 @@ +```markdown +# Goal/Experiment: +To perform Southern Blotting for the detection of specific DNA sequences in genomic DNA. + +# Southern Blotting +**Binnypreet Kaur1,2 , Drahomíra Faktorová1,2, Priscila Peña-Diaz1,2 and Julius Lukeš1,2** + +**Abstract** +Citation: Binnypreet Kaur1,2 , Drahomíra Faktorová1,2, Priscila Peña-Diaz1 and Julius Lukeš1,2 Southern Blotting. +protocols.io dx.doi.org/10.17504/protocols.io.hf6b3re +Published: 11 Jul 2018 + +## Protocol +### Step 1. +Digest 10 µg of genomic DNA overnight with desired restriction enzymes for each Southern blot probe. (10 µg per 1 well on gel) + +### Step 2. +After digestion, add 6x loading dye, incubate for 3-5 min at 55-65°C. + +### Step 3. +Run 0.75 % agarose gel (70 V **without** EtHBr) approximately 3-3.5 hours until the bromophenol reaches the end of the gel. + +### Step 4. +Stain the gel later because EtHBr influences DNA migration for 15-20 minutes. (0.5 l + 20 µl-30 µl EtHBr, the solution should slightly change color). If the gel is big, put it on a plastic bag or food film to make it easier to remove without damage. + +### Step 5. +Document gel with fluorescent marker. Place a ruler alongside the gel to estimate the distance of DNA band migration directly from the photo. + +### Step 6. +Cut the part with the gel pockets and unused areas of the gel, and trim 1 angle. + +#### Transfer +### Step 7. +Presoak the gel in 0.125 M HCl (not too old) for a maximum of 10 min at RT (depurination for more efficient transfer, pH 1; Bromophenol should turn yellow-grey; Orange G stays the same). + +### Step 8. +Rinse 3 times in dH2O, 5 min each, changing dH2O 3 times. + +### Step 9. +Denature for 30 min at RT (pH 14; Orange G turns red-brown; gel shrinks). + +### Step 10. +Rinse 3 times in dH2O, 5 min each, changing dH2O 3 times. + +### Step 11. +Neutralize for 30 min at RT (pH 7-8; Orange G turns yellow again). + +### Step 12. +Rinse 3 times in dH2O, 5 min each, changing dH2O 3 times. + +### Step 13. +Keep the gel in 20x SSC. + +### Step 14. +Cut Zeta-Probe blotting membrane to exactly the same size as the gel. Trim 1 angle of the membrane as the gel and mark 3 parts of the membrane with 2 small cuts. Put membrane in dH2O (one side at a time) and transfer the wet membrane into 20x SSC for at least 5 min. + +### Step 15. +Prepare Whatman filter paper: +- 1 filter paper should be +2 cm for each size to douse it into the 20xSSC buffer. +- 2 filter papers should have the same size as the glass. Pre-wet Whatman filter paper in 20x SSC. + +#### Blotting +### Step 16. +Invert the gel, the DNA part should be close to the membrane. + +### Step 17. +Ensure there are no bubbles between the membrane and the gel. + +### Step 18. +Cover the first layer of filter paper with food film to isolate it from napkins, ensuring there is no short-circuit between the filter paper and the membrane. + +### Step 19. +... + +### Step 20. +... + +### Step 21. +Cross-link DNA using Auto crosslink option. Membrane with DNA should face upward. + +### Step 22. +... + +### Step 23. +... + +#### Radioactive DNA Labeling (Thermo Scientific DecaLabel DNA Labeling Kit, #K0622) +### Step 24. +Use a radioactive decay calculator (Phosphorus32-alpha): +1. Add the following components into a 1.5 ml microcentrifuge tube: + - DNA template: 100 ng + - Decanucleotide in 5X Reaction Buffer: 10 µl + - Water, nuclease-free: X µl (depends on volume of [α-32 P]-dATP) + + Vortex the tube and spin down in a microcentrifuge for 3-5 s. Incubate the tube in a boiling water bath for 5-10 min and cool on ice. Spin down briefly. + +2. Add the following components to the same tube: + - Mix A: 3 µl + - [α-32 P]-dATP (minimum 16.5 µCi per reaction): X µl + - Klenow fragment, Exo- (5 u): 1 µl + +Total reaction volume should be 50 µl. Vortex the tube and spin down briefly. Incubate for 5 min at 37°C. Add 4 µl of dNTP Mix and incubate for 5 min at 37°C. Purify the probe using the Illustra G-50 column. + +#### Probe purification (Illustra G-50 columns, GE Healthcare, #28-9034-08) +### Step 25. +... + +#### Prehybridization (from Zeta-Probe membrane protocol) +### Step 26. +1. Preheat Ultrahyb at 60°C and keep it until the prehybridization step. +2. Put the blotted Zeta-Probe membrane inside a 50 ml falcon tube DNA side facing inwards. +3. Pipet 6-10 ml of hybridization solution inside the falcon tube: + - 0.5 M Na₂HPO₄, pH 7.2 + - 7% (w/v) SDS +4. Put the 50 ml falcon tube in a big glass hybridization tube. +5. Incubate in a hybridization oven at 60°C for 1 hour. + +#### Hybridization (from Zeta-Probe membrane protocol) +### Step 27. +1. Add the denatured probe. Hybridize overnight (16 hours) at 60°C with agitation. +2. Carefully pour the hybridization solution with the labeled probe into a 15 ml falcon tube. It can be reused during 1 week. Keep it at -20°C in a radioactive material container. + +**Note:** At no stage, before washing should the membranes be permitted to dry. + +#### Washing (from Zeta-Probe membrane protocol) +### Step 28. +1. Wash the membrane at 60°C: rinse 1 time (in 5 ml) and wash 2 times (in 10 ml) for 10 min each, in the following: + - 1x SSC + - 0.1% (w/v) SDS + + The first wash should be conducted at room temperature; the second and third washes should be conducted in the hybridization oven. + +2. Wash the membrane at 60°C, 2 times for 30 min each, in the following: + - 0.1x SSC + - 0.1% (w/v) SDS + + These washes should be conducted in the hybridization oven. + +3. After washing, the blotted membranes are ready for autoradiography. Put moist membrane on filter paper wetted in MQ and put them in a sealable plastic bag. +4. Keep the screen under the light for 20-30 min. Start exposure in Fuji Imaging phosphor screen (Art. No. 28956475, BAS-MS 2025, 20x25 cm). Scan phosphor screen in 4 hours in P-imager (Amersham, GE Healthcare, Typhoon 9400). + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/sparc-analysis-of-multiplexed-bead-data-using-mple-bakhict6.md b/markdown-output/sparc-analysis-of-multiplexed-bead-data-using-mple-bakhict6.md new file mode 100644 index 0000000000000000000000000000000000000000..f9129d59dd11ce468bda92a3a0cb2c39cb5feff3 --- /dev/null +++ b/markdown-output/sparc-analysis-of-multiplexed-bead-data-using-mple-bakhict6.md @@ -0,0 +1,130 @@ +```markdown +# Goal/Experiment: +The goal of this experiment is to analyze multiplexed bead data collected by flow cytometry using the MPLEX software. + +# SPARC - Analysis of Multiplexed Bead Data Using MPLEX Software + +**J Paul Robinson** +Purdue University + +## Abstract +This protocol describes the process of achieving analysis of multiplexed bead data that was collected by flow cytometry and analyzed using the MPLEX software. + +## DOI +[dx.doi.org/10.17504/protocols.io.81wgbpym1vpk/v1](https://dx.doi.org/10.17504/protocols.io.81wgbpym1vpk/v1) + +## Protocol Citation +J Paul Robinson 2022. SPARC - Analysis of multiplexed bead data using MPLEX software. **protocols.io** +[https://protocols.io/view/sparc-analysis-of-multiplexed-bead-data-using-mplex-bakhtic6](https://protocols.io/view/sparc-analysis-of-multiplexed-bead-data-using-mplex-bakhtic6) + +## Keywords +- Analysis +- Software +- Gating conditions +- Hormone assay +- Beads +- Fluorescence + +## License +This is an open-access protocol distributed under the terms of the Creative Commons Attribution License. + +## Created +Dec 17, 2019 + +## Last Modified +Aug 30, 2022 + +## Protocol Integer ID +31081 + +## Parent Protocols +- In steps of SPARC - Attune NxT Set-up for Milli-metabolic bead assay Acquisition + +## Guidelines +These assays are frequently run as "half-384 well plates". This approach is economical since cytokine and hormone kits are generally available as 96 well plate options, making per-sample costs high with standard 384 well plates. By running half-384 well plates, you essentially get 3.5 x 96 well plates from one 96 well kit while reducing sample volume requirements from 30 µL to about 8 µL. + +## Steps + +1. **Transfer the Data** + Transfer all the data files from the flow cytometer to the appropriate computer directory. + +2. **Prepare Directory for MPLEX** + Ensure the directory is available to the MPLEX software and includes the license file in the same directory as the EXE file. MPLEX is available only to academic and non-profit institutions. + +3. **Data Files in FCS Format** + Data files must be in FCS format. The instrument that collected the data should name them properly, indicating the well location (e.g., A1, B2, F12). + +4. **Identify Files by Well Position** + Files should be identified by the row and column ID of the 96 or 384 well plate with positions in the format A1, A2, etc. + +5. **Organize Files in One Directory** + All FCS files must be in the same directory. MPLEX only loads directories. + +6. **Run MPLEX** + See figure: + ![blankMPLEX.gif](path-to-image) + +7. **Select Load Icon** + Select the load icon to choose a directory containing the data files. + ![loadICON.gif](path-to-image) + +8. **Visualization** + The screen should now look like: + ![NEWdatasetMPLEX.gif](path-to-image) + +9. **Select Protocol from Options Menu** + Open the options menu and select a protocol. + ![optionsMPLEX.gif](path-to-image) + +10. **Assign GATES** + Assign GATES to the light scatter. Note that some beads may not be key populations, and these should be ignored. + ![scatterMPLEX.gif](path-to-image) + +11. **Identify Bead Populations** + Identify and assign bead populations using the manufacturer-assigned bead numbers. + ![beadmapMPLEX.gif](path-to-image) + +12. **Create Gate Regions** + To create the gate regions, use the SEMIAutomatic function or the automatic option. Adjust the population count accordingly. + ![semiautoMPLEX.gif](path-to-image) + +13. **Review Bead Locations** + Verify the bead locations against the known bead map locations. Adjust misaligned bead populations as necessary. + ![incorrectbeadgatesMPLEX.gif](path-to-image) + +14. **Reassign Bead Populations** + Drag the correct bead number to the corresponding population. + ![correctbeadMPLEX.gif](path-to-image) + +15. **Set as Default** + Select "Set As Default" to lock in the bead assignments. Save the analysis settings. + ![setdefaultMPLEX.gif](path-to-image) + +16. **Final Analysis Screen** + Ensure bead populations match the bead map data from the manufacturer. Standard curves should appear correctly. + ![finalanalysisMPLEX.gif](path-to-image) + +17. **Output Data Sets** + Use a plate map protocol to create a PLATE MAP. + ![platemapICON.gif](path-to-image) + +18. **Select Plate Map ICON** + Open the plate map and designate sample identities. This should match the data directory name. + ![platemapMOLEX.gif](path-to-image) + +19. **Data Output Methods** + Select data output method ("NIH" or "To CSV"). + ![NIH_CSVoutputMPLEX.gif](path-to-image) + +20. **Output Directories** + If using the NIH button, multiple directories are created containing all required data formats and metadata. + ![NIHdefaultMPLEX.gif](path-to-image) + +21. **Additional Information** + For more details, refer to MPLEX video tutorials: + [http://www.cyto.purdue.edu/mplex](http://www.cyto.purdue.edu/mplex) + +--- + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/sparc-bilateral-phrenic-neurophysiology-preparatio-bgfyjtpw.md b/markdown-output/sparc-bilateral-phrenic-neurophysiology-preparatio-bgfyjtpw.md new file mode 100644 index 0000000000000000000000000000000000000000..33440f3db59181ff0cfd09e5770b4c6f7a4f8985 --- /dev/null +++ b/markdown-output/sparc-bilateral-phrenic-neurophysiology-preparatio-bgfyjtpw.md @@ -0,0 +1,127 @@ +```markdown +# Goal/Experiment: +Conducting bilateral phrenic neurograms in anesthetized, paralyzed, vagotomized, and mechanically ventilated rats to test the cardiorespiratory effect of phrenic afferent stimulation approximately 12 weeks following left C2Hx. + +# SPARC Bilateral Phrenic Neurophysiology Preparation with Phrenic Afferent Stimulation - Spinal Injury Study + +**Authors**: Kristi Streeter¹, Lila Wollman¹, David Fuller¹, David Fuller¹ +¹University of Florida +**Date**: June 1, 2021 +**Protocol DOI**: [10.17504/protocols.io.bgfyjtpw](https://dx.doi.org/10.17504/protocols.io.bgfyjtpw) + +## Abstract +Protocol for conducting bilateral phrenic neurograms in anesthetized, paralyzed, vagotomized and mechanically ventilated rats to test the cardiorespiratory effect of phrenic afferent stimulation ~12 weeks following left C2Hx. + +## Keywords +- Phrenic +- Neurogram +- Neurophysiology +- Phrenic Afferent +- Stimulation +- Plasticity +- Spinal Injury + +## License +This is an open access protocol distributed under the terms of the [Creative Commons Attribution License](https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. + +## Created +May 15, 2020 + +## Last Modified +June 1, 2021 + +## Steps + +### 1. Induction of Anesthesia +1. Isoflurane induction: 2.5-3% in 100% O₂ in a closed chamber for 3-5 minutes +2. Transfer to nose cone and maintain at 3% in 100% O₂. + +### 2. Intravenous Line Placement +3. Place intravenous line into tail vein for delivery of drugs/anesthesia/fluids (flush with heparinized saline). Reduce isoflurane anesthesia to 1%. Begin urethane infusion (1.8g/kg in ddH₂O at 6ml/hr) while slowly decreasing isoflurane. Confirm adequate plane of anesthesia via toe pinch and transfer to surgical station. + +### 3. Maintain Body Temperature +4. Measure body temperature via rectal probe and maintain at 37.0 +/- 0.5°C using a heated surgical table. + +### 4. Tracheostomy and Mechanical Ventilation +5. Perform tracheostomy and initiate mechanical ventilation with the following parameters: + - Rate: 70 breaths/minute + - Tidal volume: (0.7 × body weight (in grams)) mL + - Inspired gases: 50% O₂ + a small percentage of CO₂ added to gas mixture + - Target end-tidal CO₂: ~45mmHg + - Balance N₂ + +### 5. Bilateral Vagotomy +6. Perform bilateral vagotomy. + +### 6. Femoral Arterial Line Placement +7. Place femoral arterial line to monitor arterial blood pressure and withdraw periodic blood samples (pre-fill the catheter with heparinized saline and ensure no bubbles are present in the line): + 1. Make a 1/2 inch ventral incision just distal to the groin on the inner thigh to expose the femoral artery, vein, and nerve. + 2. Gently dissect the surrounding fascia to isolate the femoral artery. + 3. Tie off the distal end of the femoral artery and place a loose suture at the proximal end. Using an alligator clamp, pull the suture to temporarily prevent blood flow at the proximal end of the femoral artery. + 4. Make a small partial incision into the wall of the femoral artery and insert the arterial catheter into the proximal portion of the artery, carefully loosening the proximal suture to enable advancement of the catheter into the vessel (~1 cm into the proximal portion of the vessel). + 5. Tie the catheter into the vessel using the proximal suture and secure in place with additional sutures as needed. + 6. Ensure no kinks in the vessel and open the stop cock at the end of the catheter to ensure unrested flow into the catheter. + 7. Flush the catheter with heparinized saline and connect it to the pressure transducer/amplifier to ensure adequate blood pressure readings. + +### 7. Phrenic Nerve Isolation +8. Isolate bilateral phrenic nerves using a dorsal approach: + 1. Using a cautery, perform a 2-inch incision just lateral to the midline and medial to the shoulder blade. + 2. Using a cautery, dissect the dorsal musculature connecting the medial border of the scapulae to the posterior trunk on both sides. + 3. Reflect the medial border of the scapulae and secure it with an alligator clip. + 4. Dissect the deep thoracic wall muscles and overlying fascia to expose the brachial plexus. Using suture, visualize the tendon running horizontal above the brachial plexus, pass 4.0 suture under the tendon twice, and place a basket stitch around the tendon. Pull the suture toward the midline of the animal and secure to open the phrenic pocket. + 5. Cut the brachial plexus distally and reflect the nerve bundle to expose the phrenic nerve. + 6. Using fine forceps, carefully isolate the phrenic nerve from the surrounding fascia without touching or pulling on the nerve. + 7. Once the nerve is fully isolated, section the nerve distally and carefully desheath the proximal end of the nerve (~1 mm). Add 0.9% saline to the phrenic pocket. + 8. Repeat to isolate the right phrenic nerve. + +### 8. Dorsal Rhizotomy +9. For groups receiving a dorsal rhizotomy: + - Cut left C3, C4, C5, and C6 dorsal rootlets: + 1. Using cautery, make a 1-inch medial incision from the base of the skull. + 2. Cut through underlying muscles to expose vertebral processes. Using cautery, separate musculature from C2 vertebrae. Using microcurette remove muscles from C3-C6 vertebrae. + 3. Using ronguers, remove the left portion of C3-C6 vertebrae. + 4. Using microscissors and fine forceps, cut and remove dura over C3-C6. + 5. Visualize and cut all dorsal rootlets from C3-C6 with microscissors. + 6. Add saline-soaked cotton above the spinal cord. + +### 9. Phrenic Nerve Recordings Setup +10. Set up phrenic nerve recordings: + 1. Place a magnetic base with L-shaped rod so that the rod runs lengthwise with the rat and above the midline. + 2. Pull and wrap suture around both tendons around the rod. + 3. Fill the phrenic cavity with 0.9% saline and suck the distal end of the proximal nerve stump into the suction electrode. The proximal end of the nerve sheath that was retracted in the prior step will form a tight seal with the tip of the suction electrode, preventing any leakage or loss of suction. The tip of the nerve should be visible within the tip of the electrode and both the inner wire and the desheathed portion of the nerve should be fully submerged in saline within the electrode. + 4. Carefully place the uninsulated tip of the outer wire against the proximal stump of the nerve proximal to the tip of the electrode and check the recording for signal quality. + +### 10. Fluid Administration +11. Following completion of the surgical procedure, administer fluids as follows: 4:1 ratio of Lactated Ringers Solution + Sodium Bicarbonate at 1.0 ml/hr. + +### 11. Paralyzing Agent Administration +12. Administer pancuronium bromide (paralytic): 1g in 1cc delivered via tail vein over a 3-minute period. + +### 12. Baseline CO₂ Parameters +13. Set baseline CO₂ parameters: Conducted no sooner than 20 minutes after pancuronium bromide delivery: + 1. Draw an initial blood sample to ensure base excess of +/- 3. + 2. Hyperinflate the lungs over 2 respiratory cycles by briefly occluding the expiratory line. + 3. Wait at least 20 minutes before starting the experimental protocol - must have "stable" baseline activity for at least 15 minutes, defined as no trend increases/decreases in nerve activity greater than 5%. + +### 13. Blood Sample for Baseline +14. Take a blood sample during baseline separated by at least 5 minutes to establish baseline blood gas values: Criteria = PaO₂ above 150 mmHg; SBE within +/- 3. Wait at least 5 minutes after blood sample before starting the experimental protocol. + +### 14. Begin Phrenic Afferent Stimulation Protocol +15. Begin phrenic afferent stimulation protocol: + 1. Set inspiratory-triggered stimulation threshold: using contralateral integrated phrenic amplitude, set horizontal cursor in the middle of the phrenic amplitude. + 2. Attach the left phrenic electrode to the stimulator. The right phrenic electrode is recorded. + 3. Establish a stimulus-response curve. Deliver the first current. All stimulation is biphasic, inspiratory triggered stimulation using a wide pulse (each phase 1.0ms). Stimulation is delivered for 20 seconds at a predetermined current. Stimulation is turned off and the output is recorded for 5 minutes. + 4. Repeat until all 4 currents have been delivered. + 5. Draw blood samples after 4 currents and adjust inspired gas concentrations to keep PaCO₂ within 2 mmHg of baseline and PaO₂ must remain above 150 mmHg at all blood draws. + 6. PaO₂ must remain above 150 mmHg at all subsequent blood draws. + 7. Deliver 30 minutes of inspiratory triggered stimulation at 90μA for 30 minutes. Take blood gas 15 minutes after the onset of stimulation. + 8. Record for 60 minutes. Take blood gases at 15, 30, and 60 minutes. + +### 15. Hypercapnia Delivery +16. At the end of the experiment, deliver hypercapnia 12% inspired CO₂ for max response. + +### 16. Analysis +17. Analysis: Bilateral phrenic activity (amplitude and frequency), blood pressure (systolic, diastolic, and mean arterial pressures), and heart rate are analyzed at baseline, during, and after each current and during hypercapnia. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/sparc-preparation-of-plasma-samples-from-rats-bamaic2e.md b/markdown-output/sparc-preparation-of-plasma-samples-from-rats-bamaic2e.md new file mode 100644 index 0000000000000000000000000000000000000000..e990d4d3ddd1cc577478701e38fb8a3b6dd7a8c9 --- /dev/null +++ b/markdown-output/sparc-preparation-of-plasma-samples-from-rats-bamaic2e.md @@ -0,0 +1,111 @@ +```markdown +# Goal/Experiment: +The objective of this protocol is to collect plasma from live rat experiments for storage at -20°C for further hormone assays. + +# SPARC - Preparation of Plasma Samples from Rats +**Author:** J Paul Robinson, Purdue University +**DOI:** [dx.doi.org/10.17504/protocols.io.261geodpol47/v1](https://dx.doi.org/10.17504/protocols.io.261geodpol47/v1) + +## Abstract +**Objective:** To collect plasma from live rat experiments for storage at -20°C for further hormone assays. + +## Keywords +rat plasma collection + +## License +This is an open access protocol distributed under the terms of the [Creative Commons Attribution License](https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. + +## Protocol Information +- **Protocol Integer ID:** 31106 +- **Created:** Dec 17, 2019 +- **Last Modified:** Aug 30, 2022 + +## Equipment and Reagents + +### Equipment +1. **Hettich Mikro 22R Refrigerated Centrifuge** + - **Vendor:** Hettich + - **Catalog Number:** 1110 + - Description: Refrigerated centrifuge capable of cooling down to 4°C. + +2. **Culex Automated Blood Collection System** + - **Vendor:** Culex + - **Model:** NxT + - Description: Automated system for blood collection from rats. + +3. **Pipettes** + - **Types:** P100, P200 + - **Vendor:** Gilson + - **Catalog Numbers:** P100 - #75874-574 + +4. **IsoFREEZE PCR Tube Chiller Rack** + - **Vendor:** RPI + - **Catalog Number:** 248002 + - Description: Freezer container for keeping tubes chilled during the collection process. + +### Reagents +1. **Protease Inhibitor Cocktail** + - **Vendor:** Sigma + - **Catalog Number:** P2714-1BTL + - Description: Used to prevent protease activity that can degrade proteins in plasma. + +2. **Sample Tubes** + - **Types:** Culex tubes (Vial Clear 8mm Crimp Rnd Bottom, 300μL Lot:0000071223 MicroLiter Wheaton Company) + - **Description:** Tubes used for blood collection and storage. + +3. **Labels** + - **Vendor:** Dymo + - **Catalog Number:** 1750283 + - Description: Labels for identifying sample tubes. + +## Protocol Steps + +### Initial Setup +1. **Centrifuge Preparation** + - Turn on the Hettich Mikro 22R refrigerated centrifuge to cool down to 4°C. + +2. **Prepare Collection Tubes** + - Add 15μL of Protease Inhibitor Cocktail to each of 5 Culex tubes. + - Firmly place caps on the tubes. Ensure they are secure and level. + +3. **Label Tubes** + - Place the plastic numbers (1-5) on each Culex tube. These are reusable and should be placed near the bottom of the tube. + +### Blood Collection +4. **Set Up Culex Machine** + - Place the Culex tubes in slots 1-5 of the Culex machine. + +5. **Label Strip Tubes** + - Ensure that labels are on the strip tubes. + +### Plasma Isolation +6. **Initial Centrifugation** + - Centrifuge the samples as they come off the Culex at 4°C, 10 minutes at 1000 x g (3000 rpm). + +7. **Plasma Extraction** + - Using the P100 pipette, set at 85μL, carefully remove as much plasma as possible and transfer to a PCR strip tube. If needed, use an additional 15μL pipette setting to remove remaining plasma without disturbing the red cell layer. + +8. **Storage During Collection** + - Keep the strip tubes in the freezer block (ISO Freeze) during the entire collection process. + +### Post-Collection Processing +9. **Repeat for All Samples** + - Repeat the plasma extraction and storage for each of the samples (S1, S2, S3, S4, S5). + +10. **Labeling and Second Centrifugation** + - Place an orange dot on the label of each collected tube using an orange sharpie. + - Change the rotor on the centrifuge for strip tubes and run program #2 (6000 rpm, 10 minutes at 4°C). + +### Plasma Aliquoting +11. **Final Plasma Transfer** + - Using the P200 set to 25μL and the P100 for additional transfers, make two aliquots of 25μL for every sample. Mark short aliquots with an "S". + +12. **Sample Transport and Storage** + - Transport aliquots using the ISO Freeze sample rack at 4°C and store them in a -20°C freezer. + - Place samples in labeled Ziploc bags in the -20°C freezer. + +13. **Mapping Samples** + - Log samples into the next plate map using the MPLEX software. Print a copy after each sample set has been added. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/sparc-rnel-bladder-january-2019-protocol-xszfnf6.md b/markdown-output/sparc-rnel-bladder-january-2019-protocol-xszfnf6.md new file mode 100644 index 0000000000000000000000000000000000000000..19211536fa4383848d285664d0173b93e70bfbd9 --- /dev/null +++ b/markdown-output/sparc-rnel-bladder-january-2019-protocol-xszfnf6.md @@ -0,0 +1,80 @@ +```markdown +# Goal/Experiment: +The goal of this experiment is to investigate lower urinary tract nerve responses to high-density epidural spinal cord stimulation. + +# SPARC RNEL Bladder January 2019 Protocol + +## Author +Max Novelli +Rehab Neural Engineering Labs, Department of Physical Medicine and Rehabilitation, University of Pittsburgh + +## Abstract +**Protocol for dataset "RNEL Bladder January 2019"** + +Lower urinary tract nerve responses to high-density epidural spinal cord stimulation (RNEL January 2019). + +## Attachments +- SPARC_RNEL_Bladder_2019_Protocol.pdf + +## DOI +[dx.doi.org/10.17504/protocols.io.xszfnf6](https://dx.doi.org/10.17504/protocols.io.xszfnf6) + +## Protocol Citation +Max Novelli 2021. SPARC RNEL Bladder January 2019 protocol. protocols.io +[dx.doi.org/10.17504/protocols.io.xszfnf6](https://dx.doi.org/10.17504/protocols.io.xszfnf6) + +## License +This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. + +## Created +Feb 05, 2019 + +## Last Modified +Mar 15, 2021 + +## Protocol Integer ID +20025 + +## Materials +- **Nerve Cuffs:** + - Micro-Leads self-closing nerve cuffs (0.5, 0.75, and 1.0 mm inner diameter). + - Ardiem nerve cuff sized for the sciatic nerve. + +- **Stimulation Array:** + - Custom 16-channel and 24-channel spinal cord arrays (Micro-Leads). + +- **Drugs:** + - Ketamine (induction) + - Isoflurane + +- **Neural Recording and Stimulation Hardware:** + - Ripple Grapevine neural interface processors + - 32 channel high-current stimulation headstage + - 32-channel recording headstage + +## Surgical Preparation +1. Anesthetize the animal and maintain physiological parameters throughout the procedure. +2. Place the animal in the supine position, insert the tracheotomy tube and place an invasive blood pressure measurement catheter. +3. Make a midline abdominal incision to expose the bladder. +4. Insert a dual-lumen catheter into the bladder dome and secure it with a purse-string suture and other sutures as necessary. +5. Identify and verify the pelvic nerve. Place a nerve cuff (MicroLeads) on the left pelvic nerve, which typically requires a 500 µm nerve cuff. +6. Close the abdominal incision in layers with suture or staples, leaving the catheter and electrode leads exposed. +7. Place the animal in a prone position and make an incision on the left side just lateral to the tail, exposing the ischioanal fossa. +8. Identify and verify the left pudendal nerve, as well as the sensory branch, deep perineal branch, and caudal rectal branch. Place appropriately sized nerve cuff electrodes on all four nerves. +9. Place a nerve cuff (Ardiem) on the sciatic nerve ipsilateral to the rest of the nerve cuffs. +10. Close the incision with suture or staples. +11. Perform a laminectomy removing the L6, L7, and S1 lamina. Mark the dorsal process location with suture prior to laminectomy. Place an epidural spinal cord array (MicroLeads) with 16 or 24 channels on the spinal cord for spinal cord stimulation. +12. Three epidural stimulation locations were tested. For the most rostral location, the array was placed such that the most rostral row of electrodes was aligned with the center L6 process. At the two more caudal locations, the array was placed such that the most caudal row of electrodes was aligned with the L7 and S1 spinous processes, respectively. +13. Connect the nerve cuff electrode contacts to the recording headstage. Connect the epidural stimulation array contacts to the stimulation headstage. + +## Electrical Stimulation +14. Stimulation is delivered using a Grapevine Neural Interface Processor through a Nano 2+Stim high-current headstage. Stimulation is controlled by the MATLAB (MathWorks) XippMex API. All pulses are asymmetric charge-balanced with a 200 µs cathodal phase, followed by a 66 µs interphase period, followed by a 400 µs anodal phase at half the current amplitude. +15. Deliver 50 stimulation pulses to all electrodes, one at a time, in a random order at a low frequency (20 Hz) and a uniform high current amplitude (typically 400-800 µA). The exact stimulation amplitude is empirically determined, using test pulse trains, and should be an amplitude that results in activation of the sciatic nerve and many or all of the other nerve cuffs. Since the ultimate goal is to identify stimulation thresholds of each nerve cuff, this high-amplitude is used to identify whether responses can be observed in the instrumented nerves. By using a low frequency in this single-amplitude survey, we observed the longest-latency responses, then added 5 ms to produce the minimum response latency of interest at a given electrode. +16. For all nerve cuff recordings, remove stimulus artifact, high-pass filter the data, and perform stimulus-triggered averaging to find the mean nerve responses to stimulus pulses. +17. Determine the presence of a compound action potential (CAP) on each nerve. Taking the stimulation-triggered averages, compare the response to baseline nerve cuff recordings without stimulation. +18. Determine the threshold stimulus current amplitude for each electrode necessary to recruit activity on each nerve. Stimulation frequencies and amplitudes can be determined using a binary search procedure. In the binary search, include electrodes where stimulation produced CAPs on the pelvic and pudendal nerves. Use a stimulation frequency determined by the minimum response latency found during the single amplitude survey. Increasing the stimulus frequency minimized the amount of time required to perform the experiments. +19. For each electrode, use a binary search algorithm to identify the minimum stimulus threshold that results in a detectable response in each instrumented nerve. 300 individual stimulus pulses are typically sufficient for averaging. +20. Stimulation was applied to monopolar electrode configurations, as well as multipolar configurations, to localize current below the electrodes stimulated. Multipolar configurations included bipolar and tripolar sets of electrodes, with bipolar configurations of two adjacent electrodes both horizontally and vertically in the electrode array, and tripolar configurations of adjacent vertical electrodes in the array. + +endofoutput +``` diff --git a/markdown-output/spatially-selective-stimulation-of-the-pig-vagus-n-cm9gu93w.md b/markdown-output/spatially-selective-stimulation-of-the-pig-vagus-n-cm9gu93w.md new file mode 100644 index 0000000000000000000000000000000000000000..65b25f85bbde4b3ef7d2e9242e24e8d642e6daae --- /dev/null +++ b/markdown-output/spatially-selective-stimulation-of-the-pig-vagus-n-cm9gu93w.md @@ -0,0 +1,123 @@ +```markdown +# Goal/Experiment: +The aim of this experiment is to investigate the selective stimulation of the pig vagus nerve to modulate target effects versus side effects using multi-contact stimulation devices. + +# Spatially Selective Stimulation of the Pig Vagus Nerve to Modulate Target Effect Versus Side Effect +*Forked from: Vagus Nerve Stimulation Evoked Electroneurography and Electromyography Recordings in Swine* + +### Authors: +- **Stephan L Blanz^1** +- **Evan N Nicolai^1** +- **Kip Ludwig^1** + +^1Department of Biomedical Engineering, University of Wisconsin-Madison, Madison, WI, United States of America + +### Contact: +- **Tech. support email:** info@neuinfo.org + +--- + +## Abstract +This protocol was used to collect data now published in the Journal of Neural Engineering, *Spatially selective stimulation of the pig vagus nerve to modulate target effect versus side effect* using the multi-contact ImThera stimulating device. [DOI: 10.1088/1741-2552/acb3fd](https://doi.org/10.1088/1741-2552/acb3fd) + +Dataset has been published to pennsieve.io and can be found at: [DOI:10.26275/efbj-8evl](https://doi.org/10.26275/efbj-8evl) + +## Guidelines + +**DISCLAIMER – FOR INFORMATIONAL PURPOSES ONLY; USE AT YOUR OWN RISK** + +The protocol content here is for informational purposes only and does not constitute legal, medical, clinical, or safety advice. Protocols.io is not peer-reviewed and may not have undergone formal approval. The information presented should not substitute for professional judgment, advice, diagnosis, or treatment. Any actions taken based on this information are at your own risk. + +--- + +## Materials + +- **Stimulation Electrode:** Six-contact ImThera cuff electrode (LivaNova, London, UK) +- **Recording Electrodes:** Made in-house, Longitudinal Intrafascicular Electrodes (LIFE) components: + - 75μm outer diameter PFA-coated platinum wire (AM-Systems, Sequim, WA) + - Suture needle (Item No. 12050–03, Fine Science Tools, Foster City, CA) + - Insulated copper extension wire with touchproof connector (441 connector with wire, Plastics1, Roanoke, VA) +- **Stimulation and Recording Devices:** Tucker Davis Technologies system (Alachua, FL; W8, IZ2MH, RZ5D, RZ6, PZ5, and S-Box units) +- **Data Analysis:** Python + +--- + +## Animal Preparation and Initial Administration of Anaesthesia + +1. Weigh pig, and perform an IM injection of a mixture of telazol (6 mg/kg) and xylazine (2 mg/kg). +2. Intubate and ventilate the pig, anesthetize using isoflurane gas (0.5-3% in room air) and IV Fentanyl (12-30 mcg/kg/hr) with anesthetic machines: + - VSA-2100 Veterinary Anesthesia Machine (Louisville, KY) + - Midmark Matrx Model 3000 ventilator (Dayton, OH) + - SurgiVet SOMNI 3 vaporizer (Plymouth, MN) +3. Supine position the pig, record heart rate via EKG 5-lead setup, pulse rate, and SpO2 via infrared (IR) probe on either the tongue or lip (AD Instruments PowerLab 8/35). + +--- + +## Invasive Blood Pressure Recording + +4. Place an invasive blood pressure catheter (Millar Inc., Houston, TX, Model #SPR-350S) into either the right or left femoral artery. + +## Cervical Vagus Nerve Preparation + +5. Perform a single incision (~9-12 cm) through the skin and superficial fat layers between the mandible and sternal notch, using a cautery. +6. Use blunt dissection to expose the carotid sheath and isolate the vagus nerve from the carotid artery. +7. Identify superior and recurrent laryngeal branches of the vagus nerve: + - Superior branch: Near the nodose ganglion, projecting towards the thyroid cartilage. + - Recurrent branch: Within the thorax, projecting towards the thyroid cartilage. +8. Expose as much of the vagus trunk as possible (~8 cm from the nodose ganglion to a more caudal location). + +--- + +## Stimulation and Recording Equipment Setup + +9. Place the six-contact ImThera cuff electrode on the right vagus nerve (VN) caudal to the nodose (~1.1 mm). +10. Secure with a 7-0 silk suture to minimize mechanical forces. +11. Record pre- and post-procedure locations to ensure cuff stability. +12. Place five LIFE devices within the VN caudally to the cuff to record ENG, ensuring the recording window is parallel to the nerve course. +13. Use Tucker Davis Technologies systems to control stimulation and record EMG and ENG signals. Ensure noise levels are below 10 microvolts peak to peak. + +### Noise Reduction: +- Use grounding techniques to minimize noise. +- Attach wires to large metal items, grounded by a banana plug. +- Remove or unplug unnecessary devices. + +--- + +## Experiments + +14. Conduct stimulation trials using a symmetric biphasic rectangular pulse (0 ms interphase delay, 0.2 ms per phase, cathodic phase first, 0.4 ms total duration) mimicking clinical parameters. +15. Stimulus delivered through one of six contacts, amplitudes from 50 to 5000 μA, with 60-second pauses between amplitudes. +16. Additional conditions for signal source confirmation: + - NMJ block (vecuronium, IV, 0.15 mg/kg bolus, 0.15 mg/kg/hour maintenance rate) + - Transection of the superior laryngeal (SL) branch + - Transection of the recurrent laryngeal (RL) branch + - Transection of the vagus trunk cranial and caudal to the cuff (double vagotomy) + +--- + +## Histological Analysis + +17. Expose the VN from the nodose ganglion to the recurrent laryngeal bifurcation for histological analysis. Mark ventral and lateral edges with tissue dye (Davidson Marking System, Bloomington, MN). +18. Fix sections in 10% neutral buffered formalin at 4 °C for 24-48 hours, embed in paraffin wax, section at 5 μm thickness, and mount on charged slides (Sakura Tissue-Tek VIP). +19. Stain with Gomori’s trichrome and image with a Motic Slide Scanner (Motic North America, Richmond, British Columbia). + +--- + +## Data Analysis + +20. Filter ENG and EMG data using in-house developed software (pyeCAP) to increase signal-to-noise ratio and remove baseline drift. + - Apply Gaussian and finite impulse response filters. + - Median filtering to eliminate spikes. +21. Average neural signals across pulse trains, segment into time-restricted windows based on conduction velocities of fiber types (Aα, Aβ, Aγ, Aδ, and B). +22. Calculate root mean square of eCAPs within each window to determine magnitude. +23. Adjust for noticeable stimulation artifacts using the Savitzky-Golay filter and a peak-detection algorithm. + +### Dose-Response Curves (DRCs) +- Generate DRCs for neural, muscle, and physiological responses. + - Analyze nerve fibers via ENG, muscle response via EMG, and HR/BP changes via EKG and arterial catheter. +- Define changes in HR/BP as maximum change from baseline during stimulation pulse train (t-3 to t-1 sec). + +--- + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/spore-based-infection-assay-on-pinus-sylvestris-se-dhxt37nn.md b/markdown-output/spore-based-infection-assay-on-pinus-sylvestris-se-dhxt37nn.md new file mode 100644 index 0000000000000000000000000000000000000000..36dc523ccc4f9060262ac07e31c23312ed292e90 --- /dev/null +++ b/markdown-output/spore-based-infection-assay-on-pinus-sylvestris-se-dhxt37nn.md @@ -0,0 +1,144 @@ +```markdown +**Goal/Experiment:** + +To assess the pathogenicity of *Diplodia sapinea* on Scots pine (*Pinus sylvestris*) seedlings through a spore-based infection assay, including inoculation, symptom evaluation, and reisolation. + +# Spore Based Infection Assay on *Pinus sylvestris* Seedlings with *Diplodia sapinea* + +## Authors +Anne Oostlander, Laura Brodde, Miriam von Bargen, Rasmus Enderle, Marco Leiterholt, Dagmar Trautmann, Malin Elfstrand, Jan Stenlid, André Fleißner + +## Institutions +1. Institute of Genetics, Technical University Braunschweig, Braunschweig, Germany +2. Department of Forest Mycology and Plant Pathology, Swedish University of Agricultural Sciences, Uppsala, Sweden +3. Institute of Forest Protection, Julius Kuehn Institute (JKI), Braunschweig + +## Date +August 01, 2024 + +## Protocol Information +- Protocol ID: 104147 +- Status: Working +- Created: July 26, 2024 +- Last Modified: August 01, 2024 +- Keywords: *Diplodia sapinea*, *Diplodia tip blight*, infection assay, *Pinus sylvestris* infection, pycnidiospores, *Sphaeropsis sapinea* + +## Abstract +This protocol describes a spore-based method for assessing *Diplodia sapinea* pathogenicity in Scots pine seedlings (*Pinus sylvestris*), including inoculation, symptom evaluation, and reisolation. + +## Materials + +### General Equipment +- Petri dishes +- *Diplodia sapinea* (e.g., ex-type strain CBS 138184) +- Light shelf with cold white daylight (e.g., Osram Lumilux 18W/865), intensity from 5000 to 6500 lux +- Cleanbench +- Centrifuge +- Sterile scalpel +- Ethanol (70%) +- Sodium hypochlorite (3% NaOCl) +- Sterile water + +### Plant Material +- Scots pine (*Pinus sylvestris*) container seedlings +- Potting soil +- Plastic pots + +### Minimal Medium (VMM) +(as described in Vogel, 1964) +- 20 ml Vogel’s solution +- 20 g sucrose +- 15 g agar +- Add 1 L water, autoclave + +### Trace Element Solution +- 50 g citric acid +- 50 g zinc sulfate (J.T. Baker) +- 10 g ammonium iron(II) sulfate +- 2.5 g copper sulfate +- 0.5 g manganese sulfate +- 0.5 g boric acid +- 0.5 g sodium molybdate +- Add 1 L water +- Chloroform (1 ml) is added as a preservative, store at room temperature + +### Vogel’s Solution +- 125 g sodium citrate +- 250 g potassium dihydrogen phosphate +- 100 g ammonium nitrate +- 10 g magnesium sulfate +- 5 g calcium chloride +- 5 ml trace element solution +- 2.5 ml biotin solution +- Add 1 L water + +### Biotin Solution +- 0.1 g biotin +- Add 1 L water +- Store at -20°C + +### 0.01 % (v/v) Tween Solution +- 0.1 ml Tween +- Add 1 L water +- Aliquot and autoclave + +## Plant Material and Greenhouse Conditions + +1. Use 2-year-old container seedlings of Scots pine (*Pinus sylvestris*). +2. Ensure seedlings have no visible symptoms and have undergone two annual cycles. +3. Apply the last fungicide treatment 8 weeks before the first inoculation. +4. Replant seedlings in plastic pots filled with potting soil. +5. Maintain plants at 20–25°C, 16 h of light per day, and high humidity (>90% RH) for 4 days after inoculation, then switch to moderate humidity (60% RH). + +## Fungal Inoculum Preparation + +6. Grow *Diplodia sapinea* on VMM (Vogels Minimal Medium) for 21 days at approximately 27°C under constant light (5000–6500 lx) to induce sporulation. +7. Harvest the spores from the plate by adding approximately 2 ml of 0.01% (v/v) Tween to the plate and rinsing the surface of the colony several times by pipetting. Repeat for a higher yield. +8. Dilute the spore suspension to 2x10⁶ spores/ml for inoculation. +9. Assess spore viability by spreading 600 μl of the spore suspension onto three VMM plates. Incubate for 7 hours at 27°C and record the germination status of 200 spores per plate. Aim for an average germination rate of 88%. +10. Confirm the absence of hyphal fragments microscopically. + +## Inoculation + +11. Wounded or not wounded plants can be inoculated. Wounding is likely to influence symptom development. +12. For wounding, use a sterile scalpel to make a 5 mm long cut down to the cambium on dormant seedlings or last year's growth segments of actively growing seedlings. Ensure the cut does not become too deep. +13. **Inoculation with Spores:** + - **Pipetting:** Apply 2 μl of spore suspension (approx. 4000 spores) directly onto the wound or unwounded stem. + - **Spraying:** Spray approximately 360 μl (approx. 720,000 spores) from a distance of 10 cm onto the wound or unwounded stem. + - **Control Treatments:** Use sterile 0.01% Tween 20 for mock inoculation. + +## Symptom Assessment + +14. Evaluate the symptoms 4- and 6-weeks post-inoculation based on classification into previously determined symptom classes, for example: + 1. No symptoms + 2. Necrotic needles, no symptoms on stem + 3. Upper third of shoot necrotic + 4. Upper two thirds necrotic + 5. Seedling dead + +## Sampling for Reisolation + +15. After six weeks, cut off and discard the shoot tip. +16. Remove an approx. 1.5 cm long piece of the stem above the wound (if applicable), remove needles, and use for reisolation. + +## Surface Sterilization + +17. Sterilize stem pieces: + 1. Immerse in 70% ethanol for 30 s. + 2. Immerse in 3% NaOCl for 60 s. + 3. Rinse twice in sterile water for 30 s each. + 4. Dry on a sterile filter paper. + +## Isolation and Identification + +18. Cut four approx. 2 mm long pieces from the center of the stem samples and place on MYP agar. Incubate for 27 days at room temperature and ambient daylight. +19. Sort isolates into morphological groups from day 4 of cultivation. +20. Extract fungal DNA from a representational number of isolates for molecular identification. +21. Use species-specific primers to identify *D. sapinea* (for example as described in Adamson et al., 2021). + +## Protocol References + +Adamson, Kalev/Laas, Marili/Blumenstein, Kathrin/Busskamp, Johanna/Langer, Gitta J./Klavina, Darta/Kaur, Anu/Maaten, Tiit/Mullett, Martin S./Müller, Michael M./Ondrušková, Emília/Padari, Allar/Pilt, Enn/Riit, Taavi/Solheim, Halvor/Soonvald, Liina/Tedersoo, Leho/Terhonen, Eeva/Drenkhan, Rein (2021). Highly clonal structure and abundance of one haplotype characterize the *Diplodia sapinea* populations in Europe and Western Asia. Journal of fungi (Basel, Switzerland) https://doi.org/10.3390/jof7080634. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/standard-operating-procedure-for-culture-identific-kvtcw6n.md b/markdown-output/standard-operating-procedure-for-culture-identific-kvtcw6n.md new file mode 100644 index 0000000000000000000000000000000000000000..94c382489892d88ca03c5e90078ddc55f78d50e5 --- /dev/null +++ b/markdown-output/standard-operating-procedure-for-culture-identific-kvtcw6n.md @@ -0,0 +1,102 @@ +```markdown +# Goal/Experiment: +To develop a standard operating procedure (SOP) for the culture identification and differentiation of Bordetella species, aimed at accurately diagnosing Bordetella pertussis, the causative agent of whooping cough. + +# Standard Operating Procedure for Culture Identification and Differentiation of Bordetella species + +## Authors: +Adria D. Lee, Pamela K. Cassiday, Lucia C. Pawloski, Kathleen M. Tatti, Monte D. Martin, Elizabeth C. Briere, M. Lucia Tondella, Stacey W. Martin, The Clinical Validation Study Group + +## Abstract +The appropriate use of clinically accurate diagnostic tests is essential for the detection of pertussis, a poorly controlled vaccine-preventable disease. The purpose of this study was to estimate the sensitivity and specificity of different diagnostic criteria including culture, multi-target polymerase chain reaction (PCR), anti-pertussis toxin IgG (IgG-PT) serology, and the use of a clinical case definition. An additional objective was to describe the optimal timing of specimen collection for the various tests. + +Clinical specimens were collected from patients with cough illness at seven locations across the United States between 2007 and 2011. Nasopharyngeal and blood specimens were collected from each patient during the enrollment visit. Patients who had been coughing for ≤2 weeks were asked to return in 2-4 weeks for collection of a second, convalescent blood specimen. Sensitivity and specificity of each diagnostic test were estimated using three methods—pertussis culture as the “gold standard,” composite reference standard analysis (CRS), and latent class analysis (LCA). + +Overall, 868 patients were enrolled and 13.6% were B. pertussis positive by at least one diagnostic test. In a sample of 545 participants with non-missing data on all four diagnostic criteria, culture was 64.0% sensitive, PCR was 90.6% sensitive, and both were 100% specific by LCA. CRS and LCA methods increased the sensitivity estimates for convalescent serology and the clinical case definition over the culture-based estimates. Culture and PCR were most sensitive when performed during the first two weeks of cough; serology was optimally sensitive after the second week of cough. + +Timing of specimen collection in relation to onset of illness should be considered when ordering diagnostic tests for pertussis. Consideration should be given to including IgG-PT serology as a confirmatory test in the Council of State and Territorial Epidemiologists (CSTE) case definition for pertussis. + +## Guidelines + +### Title +Standard Operating Procedure for Culture Identification and Differentiation of _Bordetella_ species. + +### Purpose +To describe the procedures used to identify and distinguish between _B. pertussis_, _B. parapertussis_, _B. bronchiseptica_, and _B. holmesii_. + +### Principle +_Bordetella_ species are tiny gram-negative coccobacilli occurring singly or in pairs and may exhibit a bipolar appearance. They are strict aerobes and some members of the genus are motile. _B. pertussis_ and _B. parapertussis_ are non-motile and produce no acid from carbohydrates. _B. pertussis_ will not grow well on common blood agar bases or chocolate agar, whereas _B. parapertussis_ will grow on blood agar and sometimes chocolate. Media for primary isolation consists of charcoal-based medium such as Regal-Lowe (RL) supplemented with glycerol, peptones and horse or sheep blood. The antibiotic agent cephalexin is added to reduce the growth of normal flora. _B. pertussis_ may be recovered from secretions collected from posterior nasopharynx, bronchoalveolar lavage, and transbronchial specimens. Bordetella-like organisms (BLO) isolated from clinical specimens undergo phenotypic and biochemical observation to determine correct identification. Suspected _Bordetella_ species isolates can also be phenotypically and biochemically tested to confirm identification. + +### Before Start +- **Disclaimer**: Names of vendors or manufacturers are provided as examples of suitable product sources; inclusion does not imply endorsement by the Centers for Disease Control and Prevention or the Department of Health and Human Services. +- **Reagents**: + - Regan-Lowe agar plates with cephalexin (RL+C) and without cephalexin (RL-C) + - Blood agar plates +- **Specimen Criteria**: Acceptable specimens include isolates and nasopharyngeal aspirates or swabs. Swab should be polyester (such as Dacron), rayon, or nylon. Calcium alginate and cotton swabs are not acceptable. Regan-Lowe transport medium is recommended for specimens. Amies Charcoal transports are acceptable but may decrease the probability of isolation. If only one swab is collected for both culture and PCR, the swab should be sent in Regan-Lowe transport. + +### Protocol + +#### Step 1 +Culturing the Specimen/Isolate: Plate the NP specimen/isolate on RL+C and RL-C and streak the plates for isolation. Incubate plates at 37°C for up to 10 days, checking daily. + +#### Step 2 +Phenotypic Differentiation: All phenotypic tests should be run on isolates grown on Regan-Lowe without cephalexin or blood agar plates. Growth should be no more than 3 days old for _B. pertussis_ and _B. holmesii_ or 1-2 days for _B. parapertussis_ and _B. bronchiseptica_. + +#### Step 3 +Identify any BLO on primary culture plates. RL+C will be the plate that is most useful for isolating _B. pertussis_, but the RL-C plate will be useful for isolating _B. holmesii_. + +#### Step 4 +If possible, plate a single BLO colony on RL-C and blood agar. Use the same loop for both plates. Streak the plates for isolation to make sure there are no contaminants present. + +#### Step 5 +Appearance on Regan-Lowe: +- _B. pertussis_ appears as small, pearly white colonies on Regan-Lowe agar. Pick colonies and plate them for isolation on an RL-C plate. On this second plate, _B. pertussis_ will grow faster and there should be sufficient growth for phenotypic testing after 3 days. +- _B. parapertussis_ initially appears similar to _B. pertussis_ but grows more quickly, usually within 2-3 days on primary culture. _B. parapertussis_ is not inhibited by cephalexin. The colonies will be larger than _B. pertussis_ colonies and have a slight brown pigment after 3-4 days. +- _Bordetella bronchiseptica_ grows very well on Regan-Lowe agar and blood agar after 1 day of incubation and is not inhibited by cephalexin. The colonies are much larger than _B. pertussis_ and usually have a slight brown pigment on Regan-Lowe. +- _B. holmesii_ most resembles _B. pertussis_ on Regan-Lowe agar – small, white colonies. It usually comes up in 2-3 days. Unlike _B. pertussis_, it does grow on blood agar but does not grow well on agar containing cephalexin. + +#### Step 6 +On the primary culture, if you see BLO growing on the RL-C plate but not on the RL+C plate, the isolate may be _B. holmesii_. + +#### Step 7 +If BLO grows well on blood agar, _B. pertussis_ is almost certainly ruled out. + +#### Step 8 +Gram Stain: Gram stain growth from the blood agar and/or the secondary RL-C plate +- _Bordetella_ species are very small Gram negative short rods +- Compare with a Gram stain of a known _B. pertussis_ for reference +- If the isolate is Gram positive of any shape or Gram negative cocci or long rods, discard the isolate +- If the Gram stain is correct, test the isolate by slide agglutination + +#### Step 9 +Slide Agglutination: If the isolate is negative for _B. pertussis_ and _B. parapertussis_, set up the biochemical tests (Oxidase, Nitrate reduction, Urease, Motility, HIT agar slant, MacConkey, and Citrate) on growth from the blood agar plate (BAP). + +#### Step 10 +Incubate biochemical test at 37°C for 24 hours. + +#### Step 11 +Typical biochemical results for the four species: +- _B. pertussis_ (no BAP growth, Oxidase+, Nitrate−, Urease−, Motility−, HIT- no pigment, MacConkey- no growth or fermentation, Citrate- no growth or color change) +- _B. parapertussis_ (BAP growth, Oxidase−, Nitrate−, Urease+, Motility−, HIT- brown, MacConkey- growth/no fermentation, Citrate- growth/no color change) +- _B. bronchiseptica_ (BAP growth, Oxidase+, Nitrate+, Urease+, Motility+, HIT- no pigment, MacConkey- growth/no fermentation, Citrate- growth/blue) +- _B. holmesii_ (BAP growth, Oxidase−, Nitrate−, Urease−, Motility−, HIT-brown, MacConkey- growth/no fermentation, Citrate- growth/no color change) + +#### Step 12 +Reference Values: Healthy, unexposed population is expected to be “negative.” + +#### Step 13 +Limitations: Only acceptable specimens and suspected isolates of _B. pertussis_, _B. parapertussis_, _B. holmesii_ and _B. bronchiseptica_ should be tested with these protocols. Prior antibiotic treatment and/or vaccination with an anti-pertussis vaccine may interfere with recovery of _B. pertussis_ from properly collected specimens. Failure to culture an isolate of _B. pertussis_ does not necessarily mean the patient does not have pertussis. + +#### Step 14 +Results and Interpretation: +- _B. pertussis_ identification is confirmed on the basis of _B. pertussis_ positive slide agglutination, morphological, and biochemical results. +- _B. parapertussis_ identification is confirmed on the basis of _B. parapertussis_ positive slide agglutination, morphological, and biochemical results. +- _B. bronchiseptica_ will be positive for oxidase, nitrate reduction, urease, motility, and citrate but does not produce a brown pigment on HIT agar. +- _B. holmesii_ will be negative for oxidase, nitrate, urease, and motility but does produce a brown pigment on HIT agar. + +#### Step 15 +Test Failure: If any phenotypic or biochemical test fails to create a definitive result, repeat that particular step or test. If you are still unable to get a definite result, consult the supervisor for further action. + +``` + +endofoutput \ No newline at end of file diff --git a/markdown-output/standard-operating-procedure-for-culturing-bordete-kq9cvz6.md b/markdown-output/standard-operating-procedure-for-culturing-bordete-kq9cvz6.md new file mode 100644 index 0000000000000000000000000000000000000000..ae6dad29628a99d05358e9febba0e2de992701dc --- /dev/null +++ b/markdown-output/standard-operating-procedure-for-culturing-bordete-kq9cvz6.md @@ -0,0 +1,97 @@ +```markdown +# Standard Operating Procedure for Culturing *Bordetella* Species from Nasopharyngeal Specimens + +## Goal/Experiment: +The goal of this experiment is to describe the procedures used to culture *Bordetella* species from nasopharyngeal specimens, including nasopharyngeal aspirates and swabs, in order to accurately diagnose pertussis. + +## Abstract +The use of accurate diagnostic tests is essential for detecting pertussis, a poorly controlled vaccine-preventable disease. This study aims to estimate the sensitivity and specificity of different diagnostic criteria, including culture, multi-target polymerase chain reaction (PCR), anti-pertussis toxin IgG (IgG-PT) serology, and the use of a clinical case definition. An additional objective is to describe the optimal timing of specimen collection for various tests. + +Clinical specimens were collected from patients with cough illness at seven locations across the United States between 2007 and 2011. Nasopharyngeal and blood specimens were collected from each patient during the enrollment visit. Patients with a recent cough (≤ 2 weeks) were asked to return in 2-4 weeks for a second, convalescent blood specimen. Sensitivity and specificity of each diagnostic test were estimated using three methods: pertussis culture as the "gold standard," composite reference standard analysis (CRS), and latent class analysis (LCA). + +### Findings: +- **Participants:** 868 enrolled, 13.6% *B. pertussis* positive by at least one diagnostic test. +- **Sensitivity Estimates:** Culture (64.0%), PCR (90.6%), both 100% specific by LCA. +- **Timing Considerations:** Culture and PCR most sensitive in the first two weeks of cough; serology most sensitive after the second week. + +### Recommendations: +- Specimen collection timing should consider the onset of illness. +- IgG-PT serology should be considered as a confirmatory test as per CSTE case definition for pertussis. + +## Guidelines + +### Title: +Standard Operating Procedure for Culturing *Bordetella* Species from Nasopharyngeal Specimens. + +### Purpose: +To describe the procedures used to culture *Bordetella* species from nasopharyngeal specimens, including nasopharyngeal aspirates and swabs. + +### Principle: +Regan-Lowe (RL-C) Charcoal Agar plates are used in clinical laboratories for isolating *Bordetella pertussis*, the whooping cough agent, from nasopharyngeal swabs and other pharyngeal exudate sources. This medium is enriched with cephalexin to inhibit indigenous nasopharyngeal bacteria and supports *Bordetella* growth, particularly during subcultures for additional testing. + +## Before Start + +- **Disclaimer:** Manufacturer/vendor names are examples and not endorsements by CDC or HHS. + +### Reagents: +- Regan-Lowe with Cephalexin (RL+C) +- Regan-Lowe without Cephalexin (RL-C) + +### Equipment: +- Biological Safety Cabinet (BSC) +- 37°C Incubator +- Pipette + +### Supplies: +- Inoculating Loop + +### Quality Control: +Quality control is performed using RL+C and RL-C media before diagnostic testing. QC organisms are *Bordetella pertussis* ATCC 12742 (positive control) and *Staphylococcus aureus* ATCC 29213 (negative control). + +### Specimen Criteria: +- Acceptable: Isolates, nasopharyngeal aspirates, or swabs. +- Swab Material: Polyester (e.g., Dacron), rayon, or nylon. +- Unacceptable: Calcium alginate and cotton swabs. + +#### Transport Medium: +- Recommended: Regan-Lowe transport medium. +- Acceptable but less optimal: Amies Charcoal transports. + +## Protocol + +### Step 1: +**Split Preparation Procedure (for combined culture and PCR):** +1. Use sterilized tweezers to handle the swab. +2. Remove the swab from transport medium. +3. Submerge swab tip in 500 µl of 0.85% sterile saline. +4. Cut swab shaft above micro-centrifuge tube, cap tube. + +### Step 2: +Vortex the tube and incubate for 2 minutes at room temperature. + +### Step 3: +Quickly spin the tube. + +### Step 4: +Remove 100 µl for culture plating onto RL+C and RL-C agar (50 µl/plate). + +### Step 5: +Remove 400 µl for PCR testing (200 µl for extraction) and store remaining volume at -80°C. + +### Step 6: +Inoculate plates with 50 µl of nasopharyngeal specimen using a pipette or transfer pipette. + +### Step 7: +Streak inoculum using a sterile loop in the first quadrant of the plate until the liquid is absorbed. + +### Step 8: +Incubate at 37°C with high humidity for 7-10 days. + +### Step 9: +Examine plates daily using a dissecting microscope and oblique illumination. Look for small, compact, glistening colonies characteristic of *Bordetella pertussis*. Use a quality control strain for comparison. + +### Step 10: +Pick isolated colonies of "Bordetella-like organisms" (BLO) and streak on RL-C and blood agar. Incubate 1-3 days for sufficient growth for phenotypic testing. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/standard-operating-procedure-for-real-time-pcr-det-kvxcw7n.md b/markdown-output/standard-operating-procedure-for-real-time-pcr-det-kvxcw7n.md new file mode 100644 index 0000000000000000000000000000000000000000..313c3b891d32844ae8436fde3bed6f33a8040ebe --- /dev/null +++ b/markdown-output/standard-operating-procedure-for-real-time-pcr-det-kvxcw7n.md @@ -0,0 +1,228 @@ +```markdown +# Goal/Experiment: +To describe the procedures used for the qualitative detection of Bordetella pertussis, B. parapertussis, and B. holmesii DNA extracted from clinical specimens or culture isolates using real-time PCR on the AB7500 platform. + +# Standard Operating Procedure for Real-time PCR Detection and Identification of Bordetella pertussis, B. parapertussis, and B. holmesii using AB7500 + +**Authors:** +Adria D. Lee, Pamela K. Cassiday, Lucia C. Pawloski, Kathleen M. Tatti, Monte D. Martin, Elizabeth C. Briere, M. Lucia Tondella, Stacey W. Martin, The Clinical Validation Study Group + +**Published:** 20 Nov 2017 + +## Abstract +The accurate use of clinically accurate diagnostic tests is essential for the detection of pertussis, a poorly controlled vaccine-preventable disease. This study aimed to estimate the sensitivity and specificity of different diagnostic criteria including culture, multi-target polymerase chain reaction (PCR), anti-pertussis toxin IgG (IgG-PT) serology, and clinical case definition. Additionally, the optimal timing of specimen collection was described. + +## Guidelines + +### Title +Standard Operating Procedure for Real-time PCR Detection and Identification of Bordetella pertussis, B. parapertussis, and B. holmesii using AB7500. + +### Purpose +To describe the procedures used for the qualitative detection of Bordetella pertussis, B. parapertussis, and B. holmesii DNA extracted from clinical specimens or culture isolates. + +### Before Start + +#### Disclaimer +Names of vendors or manufacturers are provided as examples of suitable product sources; inclusion does not imply endorsement by the Centers for Disease Control and Prevention or the Department of Health and Human Services. + +### Reagents +1. IS481 primer/probe set +2. pIS1001 primer/probe set +3. hIS1001 primer/probe set +4. ptxS1 primer/probe set +5. RNaseP primer/probe set +6. PerfeCTa® qPCR ToughMix, UNG for IS481, ptxS1 and RNaseP; Quanta Biosciences catalog number 95138-250 for 250 reactions (Storage 2-8°C for up to 6 months or −20°C for two years) +7. PCR grade water +8. B. pertussis positive control DNA for ptxS1 assay; CDC isolate A639; for IS481/pIS1001/hIS1001 assay ACF consisting of a mixture of A639 B. pertussis, F585 for B. parapertussis, and C690 for B. holmesii +9. Human genomic positive control DNA (for RNaseP assay; Applied Biosystems; catalog number 4312660; 10 ng/µl) + +### Equipment/Materials +- Microcentrifuge +- Pipettes +- AB7500 regular and software (Applied Biosystems) +- Eppendorf centrifuge 5810 or similar instrument +- Plates 96-well (Applied Biosystems; catalog #N801-0560) +- Optical caps (Applied Biosystems; catalog #4323032) +- Aerosol barrier pipette tips +- Sterile 1.5 mL microcentrifuge tubes +- Pressure sensitive film-not optical film-Falcon; catalog number 353073 +- Aluminum foil +- Bleach +- Ethanol +- DNA Away + +### Quality Control +1. Use RNaseP set as an internal positive control. +2. Ensure sterile water and blanks to avoid cross-contamination. +3. Include a Non-template control (NTC) using sterile water. +4. Include DNA extracted from positive control strains for ptxS1 assay, IS481/pIS1001/hIS1001 assay. + +### Protocol + +#### Step 1 +Aliquot undiluted primers and probe. Store for up to 6 months at -20°C. Cover probe aliquot tubes with foil or use dark colored tubes. + +#### Step 2 +- Prepare IS481 primers and probe: + - Prepare 30 µM (10X solution) concentrations of primers and 90 µM (10X solution) probe using sterile PCR grade water. + - Dispense 10X working concentrations into 10-reaction aliquots and store at -20°C. + - Prepare 3 µM working concentrations and 9 µM working concentration probe by diluting the 10X solution to 1:10 using sterile PCR grade water just before running the assay. + +#### Step 3 +Prepare pIS1001 primers and probe: Prepare 90 µM (10X solution) concentrations of primers and 30 µM (10X solution) concentrations probe using sterile PCR grade water. Dispense 10X working concentrations into 10-reaction aliquots and store at -20°C. Prepare 9 µM working concentrations of primers and 3 µM working concentration of probe by diluting the 10X solution to 1:10 using sterile PCR grade water just before running the assay. + +#### Step 4 +Prepare hIS1001 primers and probe: Prepare 30 µM (10X solution) concentrations of primers and 90 µM (10X solution) probe using sterile PCR grade water. Dispense 10X working concentrations into 10-reaction aliquots and store at -20°C. Prepare 3 µM working concentrations of primers and 9 µM working concentration probe by diluting the 10X solution to 1:10 using sterile PCR grade water just before running the assay. + +#### Step 5 +Thaw working concentrations of primers and probe and mix gently with a pipette. + +#### Step 6 +Quick spin for 5 sec. + +#### Step 7 +Add 0.84 µl per reaction of each working concentration of each primer and probe to 1.5 ml tube (PCR mix tube). + +#### Step 8 +Add 1 µl of PCR-grade water per reaction to the PCR mix tube. + +#### Step 9 +Mix PerfeCTa® qPCR ToughMix, UNG by pipetting up and down, then add 12.5 µl of the 2X master mix per reaction to the PCR mix tube. + +#### Step 10 +Mix by pipetting and quick spin. + +#### Step 11 +Dispense 23 µl of master mix to each test well. + +#### Step 12 +Add 4 µl of water and 21 µl of master mix to NTC well. + +#### Step 13 +Add 4 µl of sample DNA to the appropriate wells and close with optical caps. All specimens should be tested in duplicate. A third reaction should be performed with 1:5 dilution of the DNA extract. + +#### Step 14 +Add 4 µl of positive control dilutions and seal with optical caps. + +#### Step 15 +Briefly centrifuge a 96-well plate and confirm that reaction mixes are at the bottom of the wells, with no bubbles present. + +#### Step 16 +Place the 96-well plate in the AB7500 and close the tray. Start the run. + +#### Step 17 +**ptxS1 Primer and Probe Reagent Preparation:** + - Prepare 70 µM (10X solution) concentrations of primers and 30 µM (10X solution) probe using sterile PCR grade water. + - Dispense 10X working concentrations into 10-reaction aliquots and store at -20°C. + - Cover probe aliquot tubes with foil or use dark-colored tubes. + - Do not store aliquots in frost-free freezers and do not re-freeze thawed aliquots. + - Prepare 7 µM working concentrations of primers and 3 µM working concentration of probe by diluting the 10X solution to 1:10 using sterile PCR grade water just before running the assay. + +#### Step 18 +Thaw working concentrations of primers and probe and mix gently with a pipette. + +#### Step 19 +Quick spin for 5 sec. + +#### Step 20 +Add 2.5 µl per reaction of each working concentration of each primer and probe to a 1.5 ml tube (PCR mix tube). + +#### Step 21 +Add 1 µl of PCR-grade water per reaction to the PCR mix tube. + +#### Step 22 +Add 1 µl of PCR-grade water per reaction. + +#### Step 23 +Mix PerfeCTa® qPCR ToughMix, UNG by pipetting up and down, then add 12.5 µl of the 2X master mix per reaction to the PCR mix tube. + +#### Step 24 +Mix by pipetting and quick spin. + +#### Step 25 +Dispense 21 µl of master mix to each well. + +#### Step 26 +Add 4 µl of water to NTC well. + +#### Step 27 +Add 4 µl of sample DNA to the appropriate well and close well with an optical cap. All specimens should be tested in duplicate. A third reaction should be performed with 1:5 dilution of the DNA extract. + +#### Step 28 +Add 4 µl of the positive control to the final well and close with an optical cap. + +#### Step 29 +Briefly centrifuge a 96-well plate and confirm that reaction mixes are at the bottom of the wells, with no bubbles present. + +#### Step 30 +Place the 96-well plate in the AB7500 and close the tray. Start the run. + +#### Step 31 +**RNaseP Primer and Probe Reagent Preparation:** + - Prepare 4 µM working concentrations of primers and 1 µM working concentration of probe using sterile PCR grade water. + - Dispense working concentrations into 10-reaction aliquots and store in dark for up to 6 months at -20°C. + +#### Step 32 +Thaw working concentrations of primers and probe and mix gently with a pipette. + +#### Step 33 +Quick spin for 5 sec. + +#### Step 34 +Add 2.5 µl per reaction of each working concentration of each primer and probe to a 1.5 ml tube (PCR mix tube). + +#### Step 35 +Add 1 µl of PCR-grade water per reaction. + +#### Step 36 +Mix PerfeCTa® qPCR ToughMix, UNG by pipetting up and down, then add 12.5 µl of the 2X master mix per reaction to the PCR mix tube. + +#### Step 37 +Mix by pipetting and quick spin. + +#### Step 38 +Dispense 21 µl of master mix to each well. + +#### Step 39 +Add 4 µl of water to NTC well. + +#### Step 40 +Add 4 µl of sample DNA to the appropriate well and close wells with an optical cap. + +#### Step 41 +Add 4 µl of positive control to the final well and close with an optical cap. + +#### Step 42 +Briefly centrifuge a 96-well plate and confirm that reaction mixes are at the bottom of the wells, with no bubbles present. + +#### Step 43 +Place the 96-well plate in the AB7500 and close the tray. Start the run. + +### Interpretation +- NTC reactions should not exhibit background. +- A cycle threshold (Ct) value indicates sample contamination during the PCR setup. +- B. pertussis DNA positive control (A639) should be positive for IS481 and ptxS1 and negative for pIS1001 and hIS1001. +- Negative reactions for either IS481 or ptxS1 means: + - The control DNA is old + - The PCR was not set up correctly +- Human genomic DNA positive control should be positive for RNaseP. +- Negative results: + - Control DNA is old + - PCR was not set up correctly + - Blank swabs or sterile water should be negative + - Indicates contamination during the DNA extraction + - All specimens must be re-extracted +- Clinical specimens should be positive for RNaseP. +- Samples: + - Positive for IS481 test, positive for one of the three replicates (two reactions with undiluted DNA extract, one with DNA diluted 1:5) for ptxS1 assay: positive for B. pertussis + - Negative for IS481 assay, positive for both pIS1001 and hIS1001 assay: B. parapertussis + - Positive for IS481 assay, positive for ptxS1, negative for pIS1001: B. holmesii + - Care with high cycle IS481 positive samples, ptxS1 negative + +### Limitations +1. Presence of inhibitors might lead to false-negative results. +2. Antimicrobial treatment might affect Bordetella spp. detection. +3. RNaseP should not be performed for specimens transported in Regan Lowe transport medium containing horse blood. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/standard-operating-procedure-for-the-detection-of-kvwcw7e.md b/markdown-output/standard-operating-procedure-for-the-detection-of-kvwcw7e.md new file mode 100644 index 0000000000000000000000000000000000000000..5da0a4f04fe072c14422e8df00b1c4167a85280c --- /dev/null +++ b/markdown-output/standard-operating-procedure-for-the-detection-of-kvwcw7e.md @@ -0,0 +1,153 @@ +```markdown +# Goal/Experiment: +To describe the procedures used to estimate IgG antibodies against Pertussis Toxin (PT) in human serum by the Human, IgG Anti-Pertussis Toxin ELISA. + +# Standard Operating Procedure for the Detection of Human, IgG Anti-Pertussis Toxin + +## Authors: +Adria D. Lee, Pamela K. Cassiday, Lucia C. Pawloski, Kathleen M. Tatti, Monte D. Martin, Elizabeth C. Briere, M. Lucia Tondella, Stacey W. Martin, The Clinical Validation Study Group + +## Abstract +The appropriate use of clinically accurate diagnostic tests is essential for the detection of pertussis, a poorly controlled vaccine-preventable disease. The purpose of this study was to estimate the sensitivity and specificity of different diagnostic criteria including culture, multi-target polymerase chain reaction (PCR), anti-pertussis toxin IgG (IgG-PT) serology, and the use of a clinical case definition. An additional objective was to describe the optimal timing of specimen collection for the various tests. + +Clinical specimens were collected from patients with cough illness at seven locations across the United States between 2007 and 2011. Nasopharyngeal and blood specimens were collected from each patient during the enrollment visit. Patients who had been coughing for ≤ 2 weeks were asked to return in 2-4 weeks for collection of a second, convalescent blood specimen. Sensitivity and specificity of each diagnostic test were estimated using three methods—pertussis culture as the “gold standard,” composite reference standard analysis (CRS), and latent class analysis (LCA). + +Overall, 868 patients were enrolled and 13.6% were B. pertussis positive by at least one diagnostic test. In a sample of 545 participants with non-missing data on all four diagnostic criteria, culture was 64.0% sensitive, PCR was 90.6% sensitive, and both were 100% specific by LCA. CRS and LCA methods increased the sensitivity estimates for convalescent serology and the clinical case definition over the culture-based estimates. Culture and PCR were most sensitive when performed during the first two weeks of cough; serology was optimally sensitive after the second week of cough. + +Timing of specimen collection in relation to onset of illness should be considered when ordering diagnostic tests for pertussis. Consideration should be given to including IgG-PT serology as a confirmatory test in the Council of State and Territorial Epidemiologists (CSTE) case definition for pertussis. + +## Guidelines + +### Title: +Standard Operating Procedure for the Detection of Human, IgG Anti-Pertussis Toxin. + +### Purpose: +To describe the procedures used to estimate IgG antibodies against Pertussis Toxin (PT) in human serum by the Human, IgG Anti-Pertussis Toxin ELISA. + +### Before Start +- **Disclaimer:** Names of vendors or manufacturers are provided as examples of suitable product sources; inclusion does not imply endorsement by the Centers for Disease Control and Prevention or the Department of Health and Human Services. + +#### Reagents: +- 96 well microtiter strip assembly +- Pertussis toxin (PT) antigen, in 50% (v/v) glycerol +- Coating buffer, (0.05 M Carbonate – bicarbonate buffer, pH 9.6) +- IgG anti-PT standards (Standard) (lyophilized) A – F (15, 30, 60, 120, 240 & 480 International Units (IU)/mL) +- IgG anti-PT controls I (negative), II (26-68 IU/mL) & III (56-134 IU/mL) (Kit controls) (lyophilized) +- Assay buffer, (Phosphate buffer saline containing 4% BSA & 0.05% Tween – 20) +- Standard & Kit control diluent (Deionized water containing 0.05% Tween–20) +- 10X Wash solution concentrate, (10X PBS containing 0.5% Tween-20) +- Peroxidase labeled mouse monoclonal antihuman IgG conjugate (Conjugate) (Provided as a 1:5,000 dilution in 50% glycerol) +- TMB substrate, (3,3’,5,5’ tetramethylbenzidine in mildly acidic buffer) +- Stop solution, (1 N Hydrochloric acid) + +#### Equipment/Materials: +- Adhesive microtiter plate / strip sealers (plate sealer) +- Disposable reagent reservoirs +- Decapper +- Deionized water +- Microtiter plate or strip washer +- Microtiter reader equipped to read absorbance at 450 nm +- Software with the capability to use the four-parameter logistic (4PL) function for the analysis of absorbance data from a 96-well plate layout +- Single and multichannel micropipettes with appropriate tips +- Repeater pipettes with appropriate tips +- Class A glassware; graduated cylinders, and beakers +- Opaque box, slightly larger than microtiter plate / strip assembly +- Thermometer +- Timer +- Vortex mixer +- 50mL Conical tubes +- 5mL Test tubes +- Reservoir basins + +## Protocol + +### Step 1. +**Day 1 Plate Preparation:** Allow Coating Buffer and PT antigen to reach room temperature (18-26°C). + +### Step 2. +**Prepare coating solution:** Add PT antigen to Coating buffer and gently vortex to mix. + +### Step 3. +Dispense 100 μL of Coating solution into each well of the microtiter strip assembly. + +### Step 4. +Cover the microtiter strip assembly with a plate sealer, and incubate overnight (14 to 24 hours) at 2-8°C. + +### Step 5. +**Day 2:** Allow the Standards, Kit controls, samples, Assay buffer, Standard & Kit control diluent, Wash solution concentrate and PT coated microtiter strips to come to room temperature (18-26°C). + +### Step 6. +Dilute the Wash solution concentrate with de-ionized water in the microtiter plate / strip washer reservoir. Mix gently. + +### Step 7. +Prepare serum samples: Mix 20 μL of serum to 1980 μL of Assay buffer. Gently vortex each serum sample prior to use. + +### Step 8. +Reconstitute IgG anti-PT standards by adding 1 mL of Standard & Kit control diluent into each vial and gently vortexing. + +### Step 9. +Reconstitute IgG anti-PT controls by adding 1 mL of Standard & Kit control diluent into each vial and gently vortexing. + +### Step 10. +Wash the coated microtiter strips (coated on the previous day) three times with 300 μL of Wash solution per well. + +### Step 11. +Pipette, in triplicate, 100 μL of Standards, Kit controls, and serum samples into wells. To minimize assay drift within the plate, all standards, controls, and test samples should be dispensed into the wells within 15 minutes. + +### Step 12. +Cover the microtiter plate with a plate sealer and incubate for 2 hours ± 5 minutes at room temperature (18-26°C). + +### Step 13. +Prepare conjugate by carefully diluting room temperature Peroxidase-labeled mouse monoclonal anti-human IgG conjugate (1:5000 dilution in 50% glycerol) with Assay buffer. Gently vortex to mix. + +### Step 14. +After completion of the incubation period, wash microtiter strips three times with 300 μL of Wash solution per well. + +### Step 15. +Pipette 100 μL of Conjugate solution into all coated wells. + +### Step 16. +Seal plate and incubate 2 hours ± 5 minutes at room temperature (18-26°C). + +### Step 17. +Bring TMB substrate and Stop solutions to room temperature (18-26°C). + +### Step 18. +After completion of the incubation period, wash microtiter strips three times with 300 μL of Wash solution per well. + +### Step 19. +Pipette 100 μL of TMB substrate solution into all coated wells. + +### Step 20. +Incubate 9 - 11 minutes at room temperature (18-26°C). Avoid direct exposure to light during incubation. + +### Step 21. +Pipette 100 μL of Stop Solution into all coated wells. + +### Step 22. +Measure the absorbance at 450 nm within 30 minutes of adding Stop solution. + +### Step 23. +**Calculations**: For quantitative estimation, average optical densities of each standard is plotted against its concentration. Any automatic data processing software having a four-parameter logistic (4PL) function can be used. The reported sample value also known as concentration will be the average of the triplicate wells. + +### Step 24. +**Assay Repeat Criteria**: +- If average OD of Assay buffer wells is greater than 0.200, +- If average OD of Standard F (480 IU/mL) is less than 1.200 or greater than 3.000, +- If average OD of Kit control I (negative control) is greater than 0.400, +- If calculated value of either Kit control II or III is outside the provisional 3SD range for that control, +- If CV% for the mean OD of Standards A-F is greater than 15%, +- If correlation coefficient [r2] is less than 0.990, +- If CV% for the reported value (IU/mL) for Kit controls II and III is greater than 20%. + +### Step 25. +**Sample Repeat Criteria**: A serum sample should be retested if CV% of mean reported value (IU/mL) is greater than 25%. + +### Step 26. +**Interpretation/Results**: The final concentration (IU/mL) that is interpreted for each test sample is based on the average of two valid values calculated from two valid assays run on two separate days. + +### Step 27. +For samples with values < 15 IU/mL, if the final concentration cannot be attained from the average of two valid values due to CV%s > 25%, after three attempts, calculate the average of the three values and make note of the calculation on the assay worksheets and results. Interpretation of the final concentration is based on putative diagnostic cut-off points (Baughman et al, 2004). If the calculated concentration of the test sample is less than 49 IU/mL, the result is reported as “negative”. If the concentration is 49-93 IU/mL, the result is reported as “indeterminate”. If the concentration is ≥ 94 IU/mL, the result is reported as “positive”. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/static-glucose-stimulated-insulin-secretion-gsis-p-sp7edrn.md b/markdown-output/static-glucose-stimulated-insulin-secretion-gsis-p-sp7edrn.md new file mode 100644 index 0000000000000000000000000000000000000000..3b1c5c8566f6d15026af1ee111832b038a10b617 --- /dev/null +++ b/markdown-output/static-glucose-stimulated-insulin-secretion-gsis-p-sp7edrn.md @@ -0,0 +1,131 @@ +```markdown +## Goal/Experiment: +The goal of this experiment is to measure static glucose-stimulated insulin secretion (GSIS) in mouse islets. + +# Static Glucose-stimulated Insulin Secretion (GSIS) Protocol: Mouse Islets + +**Authors:** +- Aliya F Spigelman¹, Jocelyn E Manning Fox¹, Patrick E Macdonald¹ + ¹University of Alberta + +**Abstract:** +This protocol describes the method for static glucose-stimulated insulin secretion (GSIS) in isolated mouse islets. + +**DOI:** +[dx.doi.org/10.17504/protocols.io.sp7edrn](https://dx.doi.org/10.17504/protocols.io.sp7edrn) + +**Protocol Citation:** +Aliya F Spigelman, Jocelyn E Manning Fox, Patrick E Macdonald 2021. Static Glucose-stimulated Insulin Secretion (GSIS) Protocol: Mouse Islets. protocols.io [https://dx.doi.org/10.17504/protocols.io.sp7edrn](https://dx.doi.org/10.17504/protocols.io.sp7edrn) + +**License:** +This is an open access protocol distributed under the terms of the [Creative Commons Attribution License](https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. + +**Created:** +Aug 17, 2018 + +**Last Modified:** +May 05, 2021 + +**Protocol Integer ID:** +14815 + +## Materials + +- **BSA Sigma:** Aldrich Catalog #A7906 +- **Sodium bicarbonate Sigma:** Aldrich Catalog #S5761 +- **HEPES Fisher:** Scientific Catalog #BP310-500 +- **Penicillin-Streptomycin Gibco - Thermo:** Fisher Catalog #15140122 +- **RPMI 1640 Gibco - Thermo:** Fischer Catalog #11875 +- **FBS (Canadian Origin) Gibco - Thermo:** Fischer Catalog #12483-020 +- **Sodium Chloride Fisher:** Scientific Catalog #BP358-212 +- **Potassium Chloride Sigma:** Aldrich Catalog #P9541 +- **Calcium Chloride Sigma:** Aldrich Catalog #C4904 +- **Magnesium Chloride Hexahydrate Emd:** Millipore Catalog #MX0045 +- **D-(+)-Glucose Sigma:** Aldrich Catalog #G8270 +- **STELLUX® Chemi Rodent Insulin ELISA:** Jumbo Alpco Catalog #80-INS-MR-CH10 + +## Day Before Secretion + +1. **Mouse islets isolated as described in [Mouse Islet Isolation](#) protocol.** +2. **Pick mouse islets into Mouse Islet Culture Media until as close as possible to 100% purity.** + + **Mouse Islet Culture Media:** + + | Component | Amount | Vendor/Catalog | + | --- | --- | --- | + | RPMI 1640 (11.1mM glucose) | 500ml | Gibco 11875-119 | + | FBS Canadian Origin | 50ml | Gibco 12483-020 | + | Pen/Strep | 5ml | Gibco 15140-122 | + +3. **Culture islets overnight in incubator at 37°C, 5% CO₂.** + +## Solution Preparation + +4. **Prepare solutions as follows:** + + | Component | mM Final | per 100mL total | + |-----------|------------|---------------| + | NaCl | 115 | 5.75mL (2M) | + | KCl | 5 | 0.5mL (1M) | + | NaHCO₃ | 24 | 0.2g | + | CaCl₂ | 2.5 | 0.25mL (1M) | + | MgCl₂ | 1 | 0.1mL (1M) | + | HEPES | 10 | 1mL (1M) | + | BSA | 0.1% w/v | 0.1g | + + - Mix chemicals from the above table in milliQ water (approximately 80 mL). + - Warm KRBH solution to 37°C in an incubator with 5% CO₂ (approximately 30 min). + - Once the solution is warmed, pH KRBH solution to 7.4 with NaOH and bring to volume (100 mL). + - KRBH Solution should be kept in incubator throughout the experiment. + + **Additional Treatments:** Add glucose and/or other treatments as required. + + | Component | per 50mL total | from 1M stock | from powder | + |-----------|----------------|-------------|-------------| + | 2.8mM | 140µL | 0.025g | + | 16.7mM | 835µL | 0.150g | + + - 1M glucose stock should be made fresh on the day of the experiment. + + **Acid Ethanol:** + + | Component | Amount | + |----------------|--------| + | 95% Ethanol | 150mL | + | Acetic Acid | 47mL | + | Concentrated HCL | 3mL | + + - This solution can be made in advance. + +## Experimental Protocol + +5. **Pick islets into 35mm non-tissue cultured coated (NTCC) dish and ‘wash’ islets with 2 mL of KRBH with low glucose (2.8mM).** + +6. **Pick islets into new 35mm NTCC dish in 2 mL of low glucose KRBH and pre-incubate in incubator at 37°C, 5% CO₂ for 1 hour.** + +7. **Transfer islets into a new 35mm NTCC dish and add 2 mL of low glucose (2.8mM) KRBH and pre-incubate for 1 hour at 37°C, 5% CO₂.** + +8. **Pick 15 islets into eppendorf tubes. Each treatment group should be done in triplicate.** + - Typical control group: 2.8mM glucose for low glucose and 16.7mM glucose for high glucose. + - Number of islets can be increased/decreased depending on ELISA kit sensitivity. + +9. **Gently add 500µL of low glucose KRBH to the islets and incubate for 1 hour at 37°C, 5% CO₂. Leave tube lids open.** + +10. **Close lids, gently invert tubes, and centrifuge at 1000rpm for 1 min to pellet islets.** + +11. **Collect as much of the 500µL supernatant as possible without disturbing the pellet. Store supernatant at -20°C until insulin assay.** + +12. **Gently add 500µL of high glucose (16.7mM) KRBH to islets, and incubate for 1 hour at 37°C, 5% CO₂. Leave lids open.** + +13. **Close lids, gently invert tubes, and centrifuge at 1000rpm for 1 min to pellet islets.** + +14. **Collect as much of the 500µL supernatant as possible without disturbing the pellet. Store supernatant at -20°C until insulin assay.** + +15. **Add 500µL of acid ethanol to the islets. Store tube at -20°C until insulin assay.** + +## ELISA + +16. **Samples are assayed using ALPCO Stellux Rodent Insulin ELISA kit (Cat # 80-INS-MR-CH10).** Content samples are diluted with zero buffer 1:400. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/static-insulin-secretion-analysis-of-isolated-isle-b6vqre5w.md b/markdown-output/static-insulin-secretion-analysis-of-isolated-isle-b6vqre5w.md new file mode 100644 index 0000000000000000000000000000000000000000..598bf9d34780e67b9690c05a6af3e666f9ff3e00 --- /dev/null +++ b/markdown-output/static-insulin-secretion-analysis-of-isolated-isle-b6vqre5w.md @@ -0,0 +1,109 @@ +```markdown +# Goal/Experiment: +The goal of this experiment is to measure insulin secretion in a static, 1-hour assay from isolated pancreatic islets. This protocol is suitable for islets isolated from both rodents and humans, and can assess beta-cell function in response to glucose as well as various secretagogues. + +# Static Insulin Secretion Analysis of Isolated Islets + +**Julien Ghislain¹, Vincent Poitout², Caroline CT Tremblay², Marine Croze¹** +¹ Montreal Diabetes Research Center, CRCHUM, Montréal, QC, Canada +² Montreal Diabetes Research Center, CRCHUM, and Department of Medicine, Université de Montréal, Montréal, QC, Canada + +DOI: [10.17504/protocols.io.bp2l61dxkvqe/v1](https://dx.doi.org/10.17504/protocols.io.bp2l61dxkvqe/v1) + +## Laboratory of Vincent Poitout +Tech. support email: julien.ghislain.chum@ssss.gouv.qc.ca + +**Principal Investigator: Julien Ghislain** + +This protocol describes the steps to measure insulin secretion in a static, 1-hour assay from isolated pancreatic islets. It is suitable for islets isolated from both rodents and humans. We routinely apply this protocol to assess beta-cell function in response to glucose but it can be easily adapted to interrogate the response to a variety of secretagogues (e.g., fatty acids, hormones). Briefly, batches of 10 islets are pre-incubated in triplicate in KRB solution at 2.8 mM glucose twice for 20 min followed by incubation in either 2.8 mM or 16.7 mM glucose for 1 hour. Secreted insulin is measured in the supernatant and intracellular insulin content, after acid-alcohol extraction, by radioimmunoassay. This protocol is also suitable for assessing SST secretion; however, we recommend increasing the islet number per well from 10 to at least 20 due to the relative lower levels of SST compared to insulin. + +## Introduction + +### Terminology and Abbreviations +- **KRB**: Krebs-Ringer Bicarbonate. A buffer used to maintain pH during incubations. +- **HEPES**: A buffering agent used to maintain pH (Sigma, catalog #H6147). +- **RIA**: Radioimmunoassay, a technique to measure hormone levels. +- **SST**: Somatostatin, a peptide hormone that regulates the endocrine system. + +### Reagents and Supplies +1. **Sodium Chloride**: Fisher, Scientific Catalog #S271 +2. **Calcium Chloride Dihydrate**: Fisher, Scientific Catalog #C79 +3. **Potassium Phosphate Dibasic (KH2PO4)**: Sigma, Aldrich Catalog #P7971 +4. **Potassium Chloride**: Sigma, Aldrich Catalog #744636 +5. **Magnesium Sulfate Heptahydrate (MgSO4)**: Millipore, Sigma Catalog #63138 +6. **Sodium Bicarbonate**: Sigma, Aldrich Catalog #S6014 +7. **HEPES Buffer**: Sigma, Aldrich Catalog #H6147 +8. **D-(+)-Glucose**: Sigma, Aldrich Catalog #G-7528 +9. **Fatty Acid Free Heat Shock Bovine Serum Albumin Powder**: Equitech Bio, inc., Catalog #BAH66-0500 +10. **Ethanol (100%, Molecular Biology Grade)**: Fisher, Scientific Catalog #BP2818500 +11. **RPMI 1640 Medium**: Thermo Fisher Scientific, Catalog #11875093 +12. **FCS (Fetal Calf Serum)**: Life Technologies +13. **Rat Insulin RIA Kit**: Sigma, Aldrich Catalog #RI-13K +14. **Human Insulin-Specific RIA Kit**: Sigma, Aldrich Catalog #HI-14K + +### Preparation of KRB Solution and Plates + +1. **Prepare KRB Stocks** + - KRB stock I: weigh 27.7 g NaCl, bring to 1 L with milliQ water. + - KRB stock II: weigh 1.494 g CaCl2·2H2O, bring to 1 L with milliQ water. + - KRB stock III: weigh 0.648 g KH2PO4, bring to 1 L with milliQ water. + - KRB stock IV: weigh 1.415 g KCl, 1.17 g MgSO4·7H2O, 8.52 g NaHCO3, bring to 1 L with milliQ water. + - 1 M Glucose: weigh 18.02 g and bring to 100 mL with milliQ water. Filter sterilize using a 0.2 µm filter. + +2. **Prepare KRB Solution** + - Determine the number of static conditions for the assay. + - In a beaker, combine equal volumes of the four KRB stock solutions to achieve the desired volume. + - Add 2.38 mg/mL HEPES powder and swirl to dissolve. + - Add 1 mg/mL BSA (fatty acid-free). Cover with plastic wrap (put holes in top) and place in a 37°C incubator for > 1:00:00. + + pH standardization: + - Adjust the solution to pH 7.35 using 1 M NaOH. The solution should start at ~pH 7.2. + +### Glucose Concentration Preparation and Islet Handling + +3. **2.8 mM Glucose Condition and Islet Picking and Washing** + - Calculate the required volume of 2.8 millimolar (mM) Glucose in KRB for the two islet pre-incubations and static (1 mL/well) plus 1 mL/well and about 70 mL for the islet picking and wash steps. + - Add 2.8 µL of 1 M Glucose/mL of KRB. + +4. **16.7 mM Glucose Condition** + - Calculate the required volume of 16.7 millimolar (mM) Glucose in KRB for the static (1 mL/well) and add 16.7 µL of 1 M Glucose/mL of KRB. + +### Plate Preparation + +5. **Prepare Plates** + - Prepare islet picking plates: add 1 mL of 2.8 millimolar (mM) Glucose in KRB to three wells of a 24-well plate for each static sample. + - Prepare pre-incubation plates: add 1 mL of 2.8 millimolar (mM) Glucose in KRB to three wells of a 24-well plate for each static sample. Repeat this step for a second pre-incubation plate. Place these plates in an incubator with 5% volume CO2 at 37°C. + - Prepare static incubation plate: add 1 mL of experimental KRB to three wells of a 24-well plate for each static sample. Place these plates in the incubator with 5% volume CO2 at 37°C. + +### Islet Picking and Incubations (2 hour 40 minutes) + +6. **Islet Picking and Incubations** + - Following isolation, the islets should be allowed to recover in recovery medium (RPMI / 11.1 millimolar (mM) glucose) for 1:00:00 at 37°C. + - Wash the islets in a petri dish containing 20 mL of 2.8 millimolar (mM) Glucose in KRB. + + **Islet Transfer:** + - Pick the islets (in triplicate batches of 10) into the islet picking plate wells. + - Using a pipette transfer the islets from the picking plate to the first pre-incubation plate. Incubate at 37°C for 20:00. + - Transfer the islets to the second pre-incubation plate. Incubate at 37°C for 20:00. + - Transfer the islets to the incubation plate and incubate at 37°C for 1:00:00. + +### Media Collection and Insulin Assay (Radioimmunoassay) + +7. **Media Collection During Incubation** + - Label two 1.5 mL tubes per sample for collection of the KRB media containing secreted insulin. + - Prepare and label 1.5 mL tubes filled with 1 mL acidified ethanol (75% (v/v) ethanol / 1.5% (v/v) HCl) for insulin content analysis. + - At the end of the static incubation, collect the islets and transfer them to the tubes pre-filled with acidified ethanol. Cap the vials and store at -20°C overnight. + +8. **Centrifugation and Storage** + - Transfer the media from each well of the static incubation into a 1.5 mL tube, centrifuge at 4000 rpm, 4°C, 5:00, transfer the supernatant to a new 1.5 mL tube and store at -20°C until ready to complete the insulin assay. + - The next day, retrieve the insulin content analysis tubes, vortex and centrifuge at 4000 rpm, 4°C, 5:00. Transfer the supernatant to labelled 1.5 mL tubes. Store the insulin content samples at -20°C until ready to complete the insulin assay. + +9. **Radioimmunoassay** + - Radioimmunoassay kits are used to measure insulin levels. Several kits are available from MilliporeSigma. For the protocol please refer to the manufacturer's instruction. + +## References +1. Julien Ghislain, Vincent Poitout, Caroline CT Tremblay, Marine Croze 2022. Static insulin secretion analysis of isolated islets . protocols.io, DOI: [10.17504/protocols.io.bp2l61dxkvqe/v1](https://dx.doi.org/10.17504/protocols.io.bp2l61dxkvqe/v1) +2. Croze ML, Flisher MF, Guillaume A, Tremblay C, Noguchi GM, Granziera S, Vivot K, Castillo VC, Campbell SA, Ghislain J, Huising MO, Poitout V. "Free fatty acid receptor 4 inhibitory signaling in delta cells regulates islet hormone secretion in mice." Mol Metab. 2021 Mar;45:101166. doi: 10.1016/j.molmet.2021.101166. PMID: 33484949 + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/stellaris-rna-fish-96-well-glass-bottom-plate-prot-ekvbcw6.md b/markdown-output/stellaris-rna-fish-96-well-glass-bottom-plate-prot-ekvbcw6.md new file mode 100644 index 0000000000000000000000000000000000000000..9f58e8680c78d6053c925dc0d11ee3340f0448ee --- /dev/null +++ b/markdown-output/stellaris-rna-fish-96-well-glass-bottom-plate-prot-ekvbcw6.md @@ -0,0 +1,210 @@ +```markdown +# Goal/Experiment: +Perform Stellaris® RNA FISH in a 96-Well Glass Bottom Plate to visualize RNA molecules with high throughput. + +## Stellaris® RNA FISH 96 Well Glass Bottom Plate Protocol + +**LGC Biosearch Technologies** + +**Abstract:** +This protocol is specifically designed for high throughput applications of Stellaris in 96 well glass bottom plates. + +Citation: LGC Biosearch Technologies Stellaris® RNA FISH 96 Well Glass Bottom Plate Protocol. protocols.io dx.doi.org/10.17504/protocols.io.ekvbcw6 +Published: 23 Feb 2016 + +## Guidelines + +### Storage Guidelines + +#### Stellaris RNA FISH Probes +- Shipped dry and can be stored at +2 to +8 °C. +- Dissolved probe mix storage: + - Short-term/daily: +2 to +8 °C in the dark for up to a month with minimal freeze-thaw cycles. + - Long-term: -15 to -30 °C in the dark. + +#### Stellaris RNA FISH Hybridization Buffer +- Store at +2 to +8 °C for both short and long-term use. + +#### Stellaris RNA FISH Wash Buffer A and Wash Buffer B +- Store at room temperature for both short and long-term use. + +### Before start + +#### Reagents and Equipment + +**Reagents and Consumables:** +- TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) +- Methanol +- Glacial Acetic Acid +- 37% Formaldehyde Solution +- Ethanol for molecular biology +- 10X Phosphate Buffered Saline (PBS), RNase-free +- Nuclease-free water +- Deionized Formamide +- Stellaris RNA FISH Hybridization Buffer (LGC Biosearch Technologies Cat# SMF-HB1-10) +- Stellaris RNA FISH Wash Buffer A (LGC Biosearch Technologies Cat# SMF-WA1-60) +- Stellaris RNA FISH Wash Buffer B (LGC Biosearch Technologies Cat# SMF-WB1-20) +- 4',6-diamidino-2-phenylindole (DAPI) +- Vectashield® Mounting Medium (Vector Laboratories Cat #H-1000) +- 96-well glass bottom cell culture plates with #0 or #1 coverglass* +- Mineral Oil +- RNase free consumables such as pipette tips +- 37 °C laboratory oven + +*Note: Cell culture plate must be resistant to the fixation, wash buffers, and microscope objective immersion oil used in this protocol + +**Microscope:** +- Wide-field fluorescence microscope (e.g., Nikon Eclipse Ti or equivalent). Limited support for confocal applications. +- High numerical aperture (>1.3) and 60-100x oil-immersion objective. +- Strong light source: mercury/metal-halide lamp (or LED-based light source) +- Appropriate filter sets for fluorophores. +- Standard cooled CCD camera optimized for low-light level imaging (13 μm pixel size or less). + +### Preparation of Reagents + +**Reconstituting the dried probe stock:** + +- **ShipReady Probe Set (1 nmol):** + - Re-dissolve in 80 μL of TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) to create a 12.5 µM stock. + - Mix well, vortex, and briefly centrifuge. + +- **DesignReady or Custom Probe Set (5 nmol):** + - Re-dissolve in 400 μL of TE buffer to create a 12.5 µM stock. + - Mix well, vortex, and briefly centrifuge. + +**Standard Fixation Solution:** +- 3.7% (vol./vol.) formaldehyde in 1X PBS: + - 1 mL 37% Formaldehyde solution + - 1 mL 10X PBS, RNase-free + - 8 mL Nuclease-free water + +**Alternative Fixation Solution:** +- 3:1 Methanol-Glacial Acetic Acid: + - 7.5 mL Methanol + - 2.5 mL Glacial Acetic Acid + +**Hybridization Buffer:** +- 10% (vol./vol.) formamide in Hybridization Buffer; + - For 1 mL: + - 900 μL Stellaris RNA FISH Hybridization Buffer + - 100 μL Deionized Formamide + +**Wash Buffer A:** +- 10% (vol./vol.) formamide in 1X Wash Buffer A; + - For 10 mL: + - 2 mL Stellaris RNA FISH Wash Buffer A + - 7 mL Nuclease-free water + - 1 mL Deionized Formamide + +**Wash Buffer B:** +- Add Nuclease-free water upon first use; + - Add 88 mL Nuclease-free water to bottle and mix. + +### Nuclear Stain +- **DAPI:** Dissolved in Wash Buffer A at 5 ng/mL. + +**Mounting media:** +- Vectashield Mounting Medium from Vector Laboratories (#H-1000). + +## Materials + +- [Stellaris® RNA FISH Wash Buffer A SMF-WA1-60](https://biosearchtech.com) +- [Stellaris® RNA FISH Wash Buffer B SMF-WB1-20](https://biosearchtech.com) +- [VECTASHIELD Mounting Medium H-1000](https://vectorlabs.com) +- [Stellaris(R) RNA FISH Hybridization Buffer SMF-HB1-10](https://biosearchtech.com) + +## Protocol + +**Standard Fixation:** + +**Step 1:** +Grow cells in a 96-well glass bottom cell culture plate. + +**Step 2:** +Decant growth medium, and wash with 200 μL of 1X PBS. + +**Step 3:** +To fix cells, add 200 μL of 3.7% Formaldehyde fixation solution. + +**Step 4:** +Incubate at room temperature for 10 minutes. + +**Step 5:** +Wash with 200 μL of 1X PBS. + +**Step 6:** +Wash with 200 μL of 1X PBS again. + +**Alternative Fixation:** + +**Step 7:** +Alternative Fixation steps are an alternative to standard fixation and are not sequential. + +**Step 8:** +Grow cells in a 96-well glass bottom cell culture plate. + +**Step 9:** +Decant growth media, and wash with 200 μL of 1X PBS. + +**Step 10:** +To fix and permeabilize cells, add 200 μL of methanol-acetic acid (MeOH-AcOH) fixation solution. + +**Step 11:** +Incubate at room temperature for 10 minutes. + +**Step 12:** +Cells can be stored at +2 to +8 °C in MeOH-AcOH up to 48 hours before hybridization. + +**Hybridization in Adherent Cells:** + +**Step 13:** +If frozen, warm the reconstituted probe stock to room temperature. Mix, vortex, then centrifuge. + +**Step 14:** +To prepare Hybridization Buffer with probe: + - Add 1.5 μL probe stock to 75 μL Hybridization Buffer. + - Vortex and centrifuge. + +**Step 15:** +Decant MeOH-AcOH or 70% ethanol from wells. + +**Step 16:** +Add 200 μL of Wash Buffer A and incubate at room temperature for 2-5 minutes. + +**Step 17:** +Decant Wash Buffer A. + +**Step 18:** +Add 75 μL Hybridization Buffer containing Probe to each well. + +**Step 19:** +Incubate in the dark at 37 °C for 4 to 16 hours. +- 16 hours using Standard Fixation. +- 2 hours using Alternative Fixation. + +**Step 20:** +Aspirate Hybridization Buffer. Add 200 μL Wash Buffer A and incubate at 37 °C for 30 minutes. + +**Step 21:** +Decant Wash Buffer A, then add 200 μL DAPI nuclear stain. + +**Step 22:** +Incubate at 37 °C for 30 minutes. + +**Step 23:** +Decant DAPI stain buffer, add 200 μL Wash Buffer B, and incubate at room temperature for 2-5 minutes. + +**Step 24:** +Add 30 μL VectaShield Mounting Medium to the well, and top with 30 μL Mineral Oil. + +**Step 25:** +Proceed to Imaging. + +## Warnings + +- **Formamide** is a teratogen. Use in a chemical fume hood and consult the appropriate SDS. +- **Formaldehyde** is a known carcinogen. Use in a chemical fume hood, consulting the SDS before use. +- Let formamide warm to room temperature before opening the bottle. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/stellaris-rna-fish-simultaneous-if-fish-in-adheren-ek8bczw.md b/markdown-output/stellaris-rna-fish-simultaneous-if-fish-in-adheren-ek8bczw.md new file mode 100644 index 0000000000000000000000000000000000000000..2c9bf86c4edb8601449d7123e328c0124fcbca91 --- /dev/null +++ b/markdown-output/stellaris-rna-fish-simultaneous-if-fish-in-adheren-ek8bczw.md @@ -0,0 +1,187 @@ +``` +# Goal/Experiment: +Simultaneously label adherent cells with Immunofluorescence (IF) and RNA Fluorescence In Situ Hybridization (FISH) using Stellaris® RNA FISH protocol. + +# Stellaris® RNA FISH Simultaneous IF + FISH in Adherent Cells Protocol + +## Abstract +Stellaris RNA FISH protocol to simultaneously label adherent cells with IF and RNA FISH. + +*Citation:* LGC Biosearch Technologies Stellaris® RNA FISH Simultaneous IF + FISH in Adherent Cells Protocol. +*protocols.io* +*Published:* 24 Feb 2016 + +## Guidelines + +### Storage Guidelines + +#### Stellaris RNA FISH Probes +- The probes are shipped dry and can be stored at +2 to +8 °C in this state. +- Dissolved probe mix should be subjected to a minimum number of freeze-thaw cycles. +- For daily and short-term use of dissolved probe mix, storage at +2 to +8 °C in the dark for up to a month is recommended. +- For storage longer than a month, aliquot and freeze probes in the dark at -15 to -30 °C. + +#### Stellaris RNA FISH Hybridization Buffer +- Store at +2 to +8 °C for short-term and long-term use. + +#### Stellaris RNA FISH Wash Buffer A and Wash Buffer B +- Store at room temperature for short-term and long-term use. + +## Before Start + +### Reagents and Equipment + +#### Reagents and Consumables: +- **TE buffer** (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) +- **37% Formaldehyde Solution** +- **10X Phosphate Buffered Saline (PBS), RNase-free** +- **Nuclease-free water** +- **Deionized Formamide** +- **Ethanol for molecular biology** +- **Primary antibody** +- **Secondary antibody** +- **Stellaris RNA FISH Hybridization Buffer** (Biosearch Technologies Cat# SMF-HB1-10) +- **Stellaris RNA FISH Wash Buffer A** (Biosearch Technologies Cat# SMF-WA1-60) +- **Stellaris RNA FISH Wash Buffer B** (Biosearch Technologies Cat# SMF-WB1-20) +- **4',6-diamidino-2-phenylindole (DAPI)** +- **Vectashield® Mounting Medium** (Vector Laboratories Cat # H-1000) +- 18 mm round #1 *coverglass* +- 12-well *culture plates* +- RNase free consumables such as pipette tips + +#### Equipment: +- **Humidified chamber**: 150 mm tissue culture plate; bottom lined evenly with a flat water-saturated paper towel and a single layer of Parafilm® placed on top of the paper towel +- **Superfrost™ Plus Microscope slides** +- **37 °C laboratory oven** +- **Microscope**: + - Wide-field fluorescence microscope (e.g., Nikon Eclipse Ti or equivalent). Limited support for confocal applications. + - High numerical aperture (>1.3) and 60-100x oil-immersion objective. + - Strong light source, such as a mercury or metal-halide lamp (newer LED-based light sources may suffice). + - Filter sets appropriate for the fluorophores. + - Standard cooled CCD camera, ideal for low-light level imaging (13 µm pixel size or less). + +### Preparation of Reagents + +**Reconstituting the dried probe stock:** +- **ShipReady Probe Set (1 nmol):** Re-dissolve the dried oligonucleotide probe blend in 80 µL of TE buffer to create a probe stock of 12.5 µM. Mix well by pipetting up and down, vortex, and centrifuge briefly. +- **DesignReady or Custom Probe Set (5 nmol):** Re-dissolve the dried oligonucleotide probe blend in 400 µL of TE buffer to create a probe stock of 12.5 µM. Mix well by pipetting up and down, vortex, and centrifuge briefly. + +**Fixation Buffer:** +- Mix 1 mL 37% Formaldehyde solution, 1 mL 10X Phosphate Buffered Saline (PBS), RNase-free, and 8 mL Nuclease-free water to achieve a final composition of 3.7% (vol./vol.) formaldehyde in 1X PBS for a final volume of 10 mL. + +**Hybridization Buffer:** +- Mix 900 µL Stellaris RNA FISH Hybridization Buffer and 100 µL Deionized Formamide. The final composition is 10% (vol./vol.) formamide. +- *Note:* Do not freeze Hybridization Buffer. + +**Wash Buffer A (10 mL):** +- Mix 2 mL Stellaris RNA FISH 5X Wash Buffer A, 7 mL Nuclease-free water, and 1 mL Deionized Formamide to achieve final composition of 10% (vol./vol.) formamide. + +**Wash Buffer B:** +- Add 88 mL of Nuclease-free water to the bottle containing Stellaris RNA FISH Wash Buffer B. Mix thoroughly before use. + +**Nuclear Stain for use after hybridization:** +- DAPI prepared in Wash Buffer A at 5 ng/mL. This solution is to be used in Step 18. + +**Mounting media:** +- Use Vectashield Mounting Medium from Vector Laboratories (Cat # H-1000). + +**Note:** For best results, samples mounted with Vectashield should be imaged the same day. + +## Materials +- Stellaris® RNA FISH Wash Buffer A: [SMF-WA1-60 by Biosearch Technologies](https://www.biosearchtech.com/products/stellaris-wash-buffer-a) +- Stellaris® RNA FISH Wash Buffer B: [SMF-WB1-20 by Biosearch Technologies](https://www.biosearchtech.com/products/stellaris-wash-buffer-b) +- VECTASHIELD Mounting Medium: [H-1000 by Vector Laboratories](https://vectorlabs.com/products/vectashield-mounting-medium) +- Stellaris® RNA FISH Hybridization Buffer: [SMF-HB1-10 by Biosearch Technologies](https://www.biosearchtech.com/products/hybridization-buffer) + +## Protocol + +### Fixation for Simultaneous IF + FISH in Adherent Cells + +**Step 1.** +- This protocol has been adapted for a 12-well plate system. Adjust volumes accordingly for different systems. + +**Step 2.** +- Grow cells on 18 mm round #1 coverglass in a 12-well cell culture plate. + +**Step 3.** +- Aspirate growth medium, and wash with 1 mL of 1X PBS. + +**Step 4.** +- Add 1 mL of fixation buffer. + + - **Step 4.1.** 37% Formaldehyde solution: 1 mL\n + - **Step 4.2.** 10X Phosphate Buffered Saline (PBS), RNase-free: 1 mL\n + - **Step 4.3.** Nuclease-free water: 8 mL + +**Step 5.** +- Incubate at room temperature for 10 minutes. + +**Step 6.** +- Wash twice with 1 mL of 1X PBS. + +**Step 7.** +- To permeabilize cells, resuspend cells in 1 mL of 70% ethanol for at least 1 hour at +2 to +8 °C. Cells can be stored at +2 to +8 °C in 70% ethanol up to a week before hybridization. + +### Hybridization for Simultaneous IF + FISH in Adherent Cells + +**Step 8.** +- If frozen before using, warm the reconstituted probe solution to room temperature. Mix well by vortexing, then centrifuge briefly. +- To prepare the Hybridization Buffer containing the probe, add 1 µL of probe stock solution to 100 µL of Hybridization Buffer, then vortex and centrifuge. This creates a working probe solution of 125 nM for steps 12 and 13. + +**Step 9.** +- Aspirate the 70% ethanol off the coverglass containing adherent cells within the 12-well plate. + +**Step 10.** +- Add 1 mL of Wash Buffer A and incubate at room temperature for 2-5 minutes. + +**Step 11.** +- Assemble humidified chamber: 150 mm tissue culture plate; bottom lined evenly with a flat water-saturated paper towel and a single layer of Parafilm placed on top. This chamber prevents evaporation of the probe solution from under the coverglass. + +**Step 12.** +- Within the humidified chamber, dispense 100 µL of the Hybridization Buffer containing probe plus appropriately diluted primary antibody onto the Parafilm. + +**Step 13.** +- Gently transfer the coverglass, cells side down, onto the 100 µL drop of Hybridization Buffer containing the probe and primary antibody. + +**Step 14.** +- Cover the humidified chamber with the tissue culture lid, and seal with Parafilm. + +**Step 15.** +- Incubate in the dark at 37 °C for at least 4 hours (up to 16 hours). + +**Step 16.** +- Gently transfer the coverglass, cells side up, to a fresh 12-well plate containing 1 mL of Wash Buffer A plus appropriately diluted secondary antibody. + +**Step 17.** +- Incubate in the dark at 37 °C for 30 minutes. + +**Step 18.** +- Aspirate Wash Buffer A, then add 1 mL of Wash Buffer A consisting of 5 ng/mL DAPI plus appropriately diluted secondary antibody. + +**Step 19.** +- Incubate in the dark at 37 °C for 30 minutes. + +**Step 20.** +- Aspirate the DAPI staining buffer, then add 1 mL of Wash Buffer B. Incubate at room temperature for 2-5 minutes. + +**Step 21.** +- Add a small drop (approximately 15 µL) of Vectashield Mounting Medium onto a microscope slide and mount the coverglass onto the slide, cells side down. + +**Step 22.** +- Gently wick away excess anti-fade from the perimeter of the coverglass. + +**Step 23.** +- Seal the coverglass perimeter with clear nail polish, and allow to dry. + +**Step 24.** +- If necessary, gently wipe away any dried salt off the coverglass using water. + +**Step 25.** +- Proceed to Imaging. + +## Warnings +- WARNING! Formamide is a teratogen easily absorbed through the skin and should be used in a chemical fume hood. +- WARNING! Be sure to let the formamide warm to room temperature before opening the bottle. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/steps-for-setup-of-aws-organization-s3-data-storag-c2sayeae.md b/markdown-output/steps-for-setup-of-aws-organization-s3-data-storag-c2sayeae.md new file mode 100644 index 0000000000000000000000000000000000000000..5d2a8a81f85b7fab343873cf388bc4972c25ed72 --- /dev/null +++ b/markdown-output/steps-for-setup-of-aws-organization-s3-data-storag-c2sayeae.md @@ -0,0 +1,275 @@ +```markdown +# Goal/Experiment: +Setting up an AWS infrastructure for cloud-based data processing, storage, and computing using Python notebooks. + +# Steps for setup of AWS organization, S3 data storage, and EC2 computing for using Python notebooks V.2 + +### Authors: +- Daniel J. Pollak +- Gautam Chawla +- Andrey Andreev +- David A. Prober + +### Abstract: +With the oncoming age of big data, biologists are encountering more use cases for cloud-based computing to streamline data processing and storage. Unfortunately, cloud platforms are difficult to learn, and there are few resources geared towards biologists for demystifying them. We have developed a guide for experimental biologists to set up cloud processing on Amazon Web Services to cheaply outsource data processing and storage. Here we provide a guide on setting up a computing environment in the cloud and showcase examples of using Python and Julia programming languages. + +### Safety Warnings: +Using "cloud" computing can lead to budget overruns due to the pay-after-use nature of AWS and other providers. Consult with your home IT department on how to best manage costs and deployment of software. + +## Setting organization and budget management + +### 1. Setting up a Root account + +#### 1.1 Create "root" account for your organization, using Business account type + +![AWS Root Account Setup](https://portal.aws.amazon.com/billing/signup?type=enterprise#/account) + +#### 1.2 Register with AWS using a GMail account +- You can use the "gmail + trick" for managing multiple AWS accounts. + +#### 1.3 Enter credit card information +- Consult your department if personal credit card usage is a concern. + +### 2. Apply research credits +- Contact the entity that issued the credits (likely your IT department). + +### 3. Add users to the organization +- External accounts can be added to the organization or create accounts within the organization interface. + +![Add AWS Account](https://portal.aws.amazon.com/billing/signup?type=enterprise#/account) + +### 4. Set up password reset +- After an account is created, lab members should receive an email and use the "reset password" function to set up new passwords. + +### 5. Monthly cost budget +- Set up budget alerts and limits to manage costs on a per-account basis. + +![AWS Budget Management](https://portal.aws.amazon.com/billing/signup?type=enterprise#/account) + +### 6. Check expenditures +- Use Cost Explorer in the AWS Billing Dashboard. + +## EC2: Starting up a computing instance + +### 7. Consult tutorials +- Justin Bois's AWS setup and usage lesson ([Lesson](https://bebi103b.github.io/lessons/08/aws_setup.html)) +- Chris Albon's tutorial on Jupyter Notebooks on EC2 ([Tutorial](https://chrisalbon.com/projects/run_project_jupyter_notebooks_on_amazon_ec2/)) + +### 8. Find a new instance image +- Use Amazon Linux 2 or Ubuntu 18.04 or 20.04 (we recommend Amazon Linux 2). + +![AWS EC2 Instance Setup](https://portal.aws.amazon.com/billing/signup?type=enterprise#/account) + +### 9. Launch instance +- Choose "Launch through EC2" + +### 10. This will open a Launch wizard +- Pick configurations through 5 steps: + - Choose AMI + - Choose Instance Type + - Configure Instance + - Add Storage + - Add Tags + - Configure Security Group + +#### 10.1 Chose AMI +- This is the blueprint of the OS + +#### 10.2 Choose Instance Type +- Select virtual machine hardware + - Minimum 32 GB RAM recommended + - Number of cores (minimum 8) + +![Instance Type](https://portal.aws.amazon.com/billing/signup?type=enterprise#/account) + +#### 10.3 Configure Instance Details +- Accept defaults + +#### 10.4 Add Storage +- Allocate around 3 times the size of your dataset + +#### 10.5 Add Tags +- This is optional for better organization + +#### 10.6 Configure Security Group +- Open ports for remote connections + - Port 22 (SSH) + - Port 8888 (Jupyter Notebooks) + +![Security Group Setup](https://portal.aws.amazon.com/billing/signup?type=enterprise#/account) + +#### 10.7 Review and Launch +- Save the key and launch + +## EC2: Connecting to and using your instance + +### 11. Start/stop an instance +- If stopped, start the instance + +### 12. Use Instance ID +- View the instance summary page and select "connect" + +### 13. Using web-based terminal +- Select connect, which will open the terminal in another tab + +### 14. Using terminal/ssh +```bash +chmod 600 /path/to/key.pem +ssh -i /path/to/key.pem ec2-user@{public DNS} +``` + +## Installing dependencies + +### 15. Organize instance +- Keep it organized like a regular computer + - Create folders for downloads and git projects +```bash +mkdir Downloads +mkdir git +``` + +### 16. Install conda +- Follow Chris Albon's tutorial + - Download link: [Anaconda](https://repo.anaconda.com/archive/Anaconda3-2021.05-Linux-x86_64.sh) +```bash +wget .sh +bash .sh +``` + +### 16.1 Download suite2p environment file +- Create YAML file +```bash +wget https://raw.githubusercontent.com/MouseLand/suite2p/main/environment.yml +conda activate suite2p +``` + +#### 16.2 Install additional tools using conda +```bash +conda install -c conda-forge jupyter lab +``` + +#### 16.3 Install smart_open for s3 +```bash +pip install smart_open[s3] +``` + +### 17. If not using Amazon Linux 2, install AWS CLI +```bash +sudo apt install awscli +``` + +### 18. Install Julia +```bash +wget https://julialang-s3.julialang.org/bin/linux/x64/1.6/julia-1.6.2-linux-x86_64.tar.gz +tar zxvf julia-1.6.2-linux-x86_64.tar.gz +./julia-1.6.2/bin/julia +``` + +```julia +] add IJulia +] import Pkg; Pkg.add("Images") +] import Pkg; Pkg.add("FileIO") +``` + +## Starting Jupyter Lab + +### 19. Launch Jupyter Lab session +```bash +nohup jupyter lab --ip 0.0.0.0 --NotebookApp.max_buffer_size=75368709120 & +cat nohup.out +``` + +## S3: Uploading data via web-interface + +### 23. Create a bucket +- Ensure to block public access + +![Create S3 Bucket](https://portal.aws.amazon.com/billing/signup?type=enterprise#/account) + +### 24. Use web interface to upload data + +![Upload to S3](https://portal.aws.amazon.com/billing/signup?type=enterprise#/account) + +### 25. Command-line interface +- Use CLI for uploading large files + +## S3: Setting up access for S3 bucket from AWS/EC2 instance + +### 26. Add user’s canonical ID to the bucket’s access list +- Allow both list and write permissions + +### 27. Create a custom rule to allow downloading +- Generated using [AWS Policy Generator](https://awspolicygen.s3.amazonaws.com/) + +![Custom Rule Setup](https://portal.aws.amazon.com/billing/signup?type=enterprise#/account) + +### 28. Create custom key pair for your account + +### 29. Connect to the EC2 instance +- Use EC2 Connect or ssh + +### 30. Configure the instance using aws configure + +### 31. Test connection using boto3 library + +```python +import boto3 +s3 = boto3.resource('s3') +bucket = s3.Bucket('') +for obj in bucket.objects.all(): + print(obj.key) +``` + +## Accessing files from Python using boto3 + +```python +import os +import boto3 +import matplotlib.pyplot as plt +import numpy as np +import io +import tqdm +from smart_open import open +from suite2p import run_s2p, default_ops + +# Confirm S3 visibility and list files +s3 = boto3.resource('s3') +bucket = s3.Bucket('bucket-name') +for obj in bucket.objects.all(): + print(obj.key) + +# Download files +data_dir = "./data/" +if not os.path.isdir(data_dir): + os.makedirs(data_dir) + +filename = "sample_file.tif" + +if not os.path.isfile(data_dir + filename): + s3.Client.download_file('bucket-name', filename, data_dir + filename) + +ops = default_ops() +ops['batch_size'] = 1 +ops['data_path'] = [data_dir] +ops['tiff_list'] = [elem for elem in os.listdir(data_dir) if elem.endswith('tif')] + +db = {} +run_s2p(ops=ops, db=db) +``` + +## Creating AMI (Amazon Machine Image) from configured instance + +### 33. Save configured machine image by creating an AMI +#### 33.1 Go to image and templates -> Create image + +![Create AMI](https://portal.aws.amazon.com/billing/signup?type=enterprise#/account) + +#### 33.2 Name the image appropriately +- Ensure all data is saved + +### 34. For tutorial assistance refer to the spotlight video + +[Spotlight Video](https://protocols.io/) + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/strategic-savings-in-ligation-sequencing-a-practic-cypvxvn6.md b/markdown-output/strategic-savings-in-ligation-sequencing-a-practic-cypvxvn6.md new file mode 100644 index 0000000000000000000000000000000000000000..4130a7532d8e53dd4cbbb0b3957928046d4cc4c4 --- /dev/null +++ b/markdown-output/strategic-savings-in-ligation-sequencing-a-practic-cypvxvn6.md @@ -0,0 +1,182 @@ +```markdown +# Goal/Experiment: +The goal of this experiment is to introduce a cost-effective alternative for end repair and dA-tailing in DNA library preparation, particularly tailored for samples with an N50 of 3 kb. This method uses a homebrew end repair reagent solution, optimizing reagent quantities, and a bead-free purification method to achieve efficient adapter ligation while minimizing costs and time. + +# Strategic Savings in Ligation Sequencing: A Practical Nanopore Library Preparation Workflow + +### Jie Hao Ou, Yin-Tse Huang +1. National Chung Hsing University, Taichung +2. Kaohsiung Medical University + +**License:** This is an open access protocol distributed under the terms of the [Creative Commons Attribution License](https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. + +**Created:** Aug 15, 2023 +**Last Modified:** Nov 21, 2023 +**Protocol ID:** 86485 + +## Abstract + +This protocol introduces a cost-effective alternative for end repair and dA-tailing in DNA library preparation, particularly tailored for samples with an N50 of 3 kb. By employing a homebrew end repair reagent solution, this method replaces the use of the NEBNext® II End Repair/dA-Tailing Module recommended by Nanopore. Optimization of reagent quantities and a bead-free purification method are combined to achieve efficient adapter ligation while minimizing costs. Notably, an extended incubation time during adapter ligation enhances efficiency. This resource provides a strategic approach for researchers aiming to customize their sequencing workflows while achieving optimal results and substantial savings. + +## Guidelines + +1. **Optimized Homebrew Approach:** This protocol features a homebrew end repair reagent solution as a cost-effective alternative to the Nanopore-recommended NEBNext® Ultra™ II End Repair/dA-Tailing Module. Adhere to the specified volumes meticulously to achieve successful results with your customized reagent. +2. **Bead-Free Purification:** The protocol employs a bead-free PEG/NaCl precipitation method for efficient purification. Ensure precise centrifugation to maintain DNA pellet integrity and maximize recovery rates. +3. **Strategic Ligation Enhancement:** An extended incubation period during adapter ligation enhances efficiency. Dedicate adequate time to this step to optimize adapter ligation results. + +## Materials + +### 1. 4X Quick Ligase Buffer + +| Reagent | Amount | +|------------------|-------------------| +| H2O | 70 mL | +| Tris-Base | 2.42 g (200 mM) | +| MgCl2 ⋅ 6H2O | 0.8 g (40 mM) | +| DTT | 0.6 g (40 mM) | +| PEG 8000 | 20 g (20% w/w) | +| HCl | Adjust pH to 7.6 | +| H2O | Bring volume up to 100 mL | + +**Notes:** Add the materials in sequential order. + +### 2. 33.5% (w/v) PEG 8000 + +### 3. NP (5M NaCl + 6.7% PEG 8000) + +| Reagent | Amount | +|------------------|-------------------| +| PEG 8000 | 3.35 g (6.7%) | +| NaCl | 14.61 g (5M) | + +### 4. 80% (v/v) Ethanol + +### 5. 99.5% Ethanol + +### 6. Elution Buffer (1X TE, pH=8.0) + +### 7. LFB Wash Buffer + +| Reagent | Amount | +|--------------------|--------| +| 33.5% (w/v) PEG 8000 | 80 µL | +| NP | 40 µL | +| H2O | 320 µL | + +### Safety Warnings + +1. **Ethanol Flammability:** Ethanol is highly flammable. Use caution when handling and storing ethanol solutions. Work in well-ventilated areas away from open flames, sparks, or heat sources. + +## Protocol + +### 1. Homebrew End Repair/dA-Tailing (50 min) + +1. **Materials:** + +| Reagent | Quantity | +|------------------------|----------------------------| +| DNA | 1600 ng (for samples with N50 of 3 kb) | +| H2O | Bring up to a volume of 68 µL | +| 4X Quick Ligase Buffer | 25 µL | +| ATP (25 mM) | 1 µL | +| dNTP (10 mM) | 5 µL | +| Taq polymerase | 0.5 µL (2.5 U) | +| T4 PNK | 0.5 µL (5 U) | + +**Note:** Due to high PEG concentration in the solution, it may be slightly viscous. Gently invert several times to ensure thorough and uniform mixing. + +2. **Incubation at 37°C for 30 min** + +**Note:** T4 PNK will add a phosphate to the 5' ends of both DNA strands. + +3. **Incubation at 65°C for 20 min** + +**Note:** T4 PNK will be inactivated, and Taq polymerase will begin repairing DNA ends and adding A-tails. + +### 2. Purification (30 min) + +4. **For the subsequent purification:** Commercially available spin-column or magnetic bead-based methods can be used. Here, to save costs, we will use the PEG/NaCl precipitation method. +5. Add 25 µL of 33.5% PEG 8000. +6. Add 15 µL of NP. + +**Note:** + - Due to the high concentration of PEG, it may be slightly viscous. Gently invert several times to ensure thorough and uniform mixing. + - T4 PNK will be inactivated and Taq polymerase will begin repairing DNA ends and adding A-tails. + +7. Centrifuge at maximum speed (at least 8000 rpm) for 30 min. +8. Carefully remove the supernatant using a pipette. + +**Note:** Most of the time, the pellet is not visible to the naked eye, so pay attention to the orientation during centrifugation. + +9. Add 200 µL of 80% ethanol. +10. Centrifuge at maximum speed (at least 8000 rpm) for 2 min. +11. Carefully remove the supernatant using a pipette. +12. Add 200 µL of 99.5% ethanol. +13. Centrifuge at maximum speed (at least 8000 rpm) for 2 min. +14. Carefully remove the supernatant using a pipette. +15. Wait for approximately 10 min to ensure complete ethanol evaporation. +16. Add 25 µL of elution buffer. + +**Note:** + Optional: Measure the DNA concentration. Normally, the DNA concentration should be >30 ng/µL. + +### 3. Adapter Ligation (30 min) + +17. **When using LNB provided by Nanopore:** + +| Reagent | Volume | +|------------------|-----------------------------------------| +| DNA | 400 ng (for samples with N50 of 3 kb) | +| H2O | Bring up to volume of 15.25 µL | +| Ligation Buffer (LNB) | 6.25 µL | +| T4 Ligase | 2.5 µL | +| Ligation Adapter | 1 µL | + +**When using homemade 4X Quick Ligase Buffer:** + +| Reagent | Volume | +|-------------------|-----------------------------------------| +| DNA | 400 ng (for samples with N50 of 3 kb) | +| H2O | Bring up to volume of 14.25 µL | +| 4X Quick Ligase Buffer | 6.25 µL | +| ATP (25 mM) | 1 µL | +| T4 Ligase | 2.5 µL | +| Ligation Adapter | 1 µL | + +**Note:** The reagent amounts used in this step are about 1/4 of the manufacturer’s recommended volume. This means that the ligation sequencing kit, originally designed for 6 uses, can now be utilized for 24 uses. This adjustment is due to utilizing a bead-free purification method later on, which significantly enhances the recovery rate. + +18. Incubate at room temperature for 1 hr. + +**Note:** + Despite the official recommendation of a 5-minute reaction, based on our experience, a 60-minute reaction significantly improves adapter ligation efficiency. + +19. Add 3 µL of NP. + +**Note:** + - Due to the high concentration of PEG in the solution, it may be slightly viscous. Gently invert several times to ensure thorough and uniform mixing. + - Normally, PEG and NaCl buffer is required for DNA precipitation. However, the LNB buffer already contains a high concentration of PEG, so adding NP alone is sufficient for DNA precipitation. For more information, refer to: https://dx.doi.org/10.17504/protocols.io.7erhjd6. + +20. Centrifuge at maximum speed (at least 8000 rpm) for at least 30 min (if a higher recovery rate is needed, centrifuging for up to one hour is also possible). + +**Note:** To prevent overheating, it is recommended to use a refrigerated centrifuge. If a refrigerated centrifuge is unavailable, it is advised to perform centrifugation in two steps of 15 minutes each, with a 10-minute interval. + +21. Carefully remove the supernatant using a pipette. + +**Note:** Most of the time, the pellet is not visible to the naked eye, so pay attention to the orientation during centrifugation. + +22. Add 200 µL of LFB wash buffer. + +**Note:** Use the LFB wash buffer instead of alcohol because the DNA now has motor proteins attached, and using alcohol could disrupt the motor proteins. + +23. Centrifuge at maximum speed (at least 8000 rpm) for 2 min. +24. Carefully remove the supernatant using a pipette. +25. Repeat the washing step twice. +26. Add 20 µL of EB buffer (provided by the Ligation Sequencing Kit). + +**Note:** + Optional: Measure the DNA concentration. Normally, the DNA concentration should be >15 ng/µL. + +--- + +**endofoutput** +``` \ No newline at end of file diff --git a/markdown-output/super-resolution-single-molecule-fish-at-the-droso-bprbmm2n.md b/markdown-output/super-resolution-single-molecule-fish-at-the-droso-bprbmm2n.md new file mode 100644 index 0000000000000000000000000000000000000000..a224fb52dd43fd64c1f39ef2f75dc510d190e42a --- /dev/null +++ b/markdown-output/super-resolution-single-molecule-fish-at-the-droso-bprbmm2n.md @@ -0,0 +1,118 @@ +```markdown +# Goal/Experiment: +Super-Resolution Single Molecule FISH at the Drosophila Neuromuscular Junction + +## Abstract +The lack of an effective, simple, and highly sensitive protocol for fluorescent in situ hybridization (FISH) at the *Drosophila* larval neuromuscular junction (NMJ) has hampered the study of mRNA biology. Here, we describe our modified single molecule FISH (smFISH) methods that work well in whole mount Drosophila NMJ preparations to quantify primary transcription and count individual cytoplasmic mRNA molecules in specimens while maintaining ultrastructural preservation. The smFISH method is suitable for high-throughput sample processing and 3D image acquisition using any conventional microscopy imaging modality and is compatible with the use of antibody co-labeling and transgenic fluorescent protein tags in axons, glia, synapses, and muscle cells. These attributes make the method particularly amenable to super-resolution imaging. With 3D Structured Illumination Microscopy (3D-SIM), which increases spatial resolution by a factor of 2 in X, Y, and Z, we acquire super-resolution information about the distribution of single molecules of mRNA in relation to co-visualized synaptic and cellular structures. Finally, we demonstrate the use of commercial and open-source software for the quality control of single transcript expression analysis, 3D-SIM data acquisition and reconstruction, as well as image archiving management and presentation. Our methods now allow the detailed mechanistic and functional analysis of sparse as well as abundant mRNAs at the NMJ in their appropriate cellular context. + +## Keywords +- smFISH +- single molecule fluorescence in situ hybridization +- structured illumination +- super-resolution imaging +- 3D-SIM +- *Drosophila melanogaster* +- larval neuromuscular junction +- mRNA localization +- synapse + +## License +This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. + +## Guidelines + +### 1. Introduction +In situ hybridization has been a mainstay of cell and developmental biology for determining where and when genes are expressed in wild-type or mutant cells and tissues... [Add introduction content here] + +### 2. Materials and Methods + +#### 2.1 smFISH Probes +- **Probes**: Stellaris® RNA FISH probes from LGC BioSearch Technologies. +- **Alternative**: Design 30−50 3’ primary amine labeled 18-mer DNA oligonucleotides. +- **Purchase**: HPLC-purified oligonucleotides (available from most manufacturers). + +#### 2.2 Larval Neuromuscular Junction Dissection +- **Equipment**: + - Dissecting microscope with light source + - 35 mm Petri dish + - Sylgard or similar elastomer + - Insect pins + - Microdissection scissors and forceps + - Saline buffer: 70 mM NaCl, 5 mM KCl, 20 mM MgCl₂, 10 mM NaHCO₃, 5 mM trehalose, pH 7.2 + +#### 2.3 Fixation and Hybridization +- **Reagents**: Flash-freeze 1 mL aliquots of deionized formamide with liquid nitrogen and store at −80 °C. Prepare reagents with DEPC-treated water and autoclave whenever possible. +- **Fix Solution**: PBS, 0.3% Triton X, 4% formaldehyde from freshly thawed aliquots of 16% EM grade PFA. +- **Wash Buffer**: 10% 20× SSC (3 M NaCl, 0.3 M sodium citrate, pH 7.0), 10% freshly thawed deionized formamide, 80% DEPC-treated water. +- **Hybridization Buffer**: 10% w/v dextran, 250 nM smFISH probe in Wash buffer. +- **Counterstain** (Optional): Use DAPI in Wash buffer. + +#### 2.4 Mounting +- **Materials**: + - Glass slide and coverslips (High Precision No. 1.5 for 3D-SIM) + - Double-sided adhesive tape + - VectaShield mounting medium + - 100 nm Tetraspek beads. + +#### 2.5 Image Acquisition +- **Equipment**: + - For conventional imaging: wide-field epifluorescence, spinning disk, or confocal microscope. + - For 3D-SIM: DeltaVision OMX, V3-Blaze with 60x 1.3 NA silicone oil immersion objective from Olympus. +- **Spherical Aberration**: Match the immersion oil refractive index or use adaptive optics approaches for deep imaging. + +#### 2.6 Image Processing and Analysis +- **Software**: + - ImageJ/FIJI with SIMCheck and FindSpot plugins + - fairSIM + - OMERO server + - Matlab with FISHQuant script + - Imaris + +#### 3. Safety Warnings +- Refer to Safety Data Sheets (SDS) for health and environmental hazards. + +### 4. Experimental Procedure + +#### 4.1 Larva Neuromuscular Junction Dissection +1. Pin the larva dorsal side up on a 35 mm Petri dish filled halfway with Sylgard. +2. Cover the larva with a few drops of saline buffer. +3. Use microdissection scissors to create a small incision at the center of the dorsal midline. +4. Carefully remove gut tissue by holding the trachea with forceps and cutting the tracheal attachments. +5. Stretch the tissue away from the midline gently. +6. (Optional) Remove the brain carefully by cutting the nerves just above the muscle tissue. + +#### 4.2 Fixation (25 min) +8. Replace the dissection buffer with fix solution and incubate with gentle rocking at room temperature for 25 minutes. +9. Remove the fix buffer and rinse 3× with PBTX. +10. (Optional) Block the tissue by incubating for 1 hour in PBTX with 1% RNAse free bovine serum albumin. + +#### 4.3 Hybridization (10 min) +12. Replace ethanol with 0.2 mL wash buffer and incubate for 10 minutes at 38 °C with gentle rocking. +13. Replace the wash buffer with 0.1 mL hybridization buffer and incubate overnight at 38 °C with gentle rocking. + +#### 4.4 Washing and Counterstaining (45 min) +14. Remove the hybridization buffer and rinse 3× with 0.2 mL wash buffer. +15. Incubate the tissue in 0.2 mL wash buffer for 45 minutes at 38 °C with gentle rocking. +16. (Optional) For counterstaining, add secondary antibodies (1:500 dilution) or DAPI. + +#### 4.5 Mounting +17. Remove the wash buffer and incubate tissue in VectaShield. +18. Position tissues dorsal side up in VectaShield on the slide. Place a coverslip on the strips of tape and seal edges with clear nail varnish. + +#### 4.6 Image Acquisition +24. Acquire optical sections of the region of interest using optimal imaging configuration. +25. Exposure times of 600–800 ms are often required for camera-based imaging systems. +26. Single transcripts appear as discrete punctae. + +### 5. Expected Results +- Transcripts appear as discrete punctae with consistent intensities. +- The method allows detailed mechanistic and functional analysis of sparse and abundant mRNAs at the NMJ. + +### 6. Acknowledgments +- Acknowledgments content here... + +### 7. References +- Femino et. al. (1998), Raj et. al. (2008), etc.... + +## End of Output +``` \ No newline at end of file diff --git a/markdown-output/supplementary-material-an-in-silico-approach-to-un-cjizukf6.md b/markdown-output/supplementary-material-an-in-silico-approach-to-un-cjizukf6.md new file mode 100644 index 0000000000000000000000000000000000000000..9db0c16566074a3d601ad02ce0ca079c5148ffbb --- /dev/null +++ b/markdown-output/supplementary-material-an-in-silico-approach-to-un-cjizukf6.md @@ -0,0 +1,200 @@ +```markdown +# Goal/Experiment: +The goal of the experiment is to understand the interaction between cardiovascular and pulmonary lymphatic dysfunction using an in silico approach. + +# Supplementary Material: An in silico approach to understanding the interaction between cardiovascular and pulmonary lymphatic dysfunction V.1 + +**DOI:** [dx.doi.org/10.17504/protocols.io.kxygx9zmwg8j/v1](https://dx.doi.org/10.17504/protocols.io.kxygx9zmwg8j/v1) + +**Author:** Kelly Burrowes +Auckland Bioengineering Institute, University of Auckland + +## Abstract + +The lung is extremely sensitive to interstitial fluid balance, yet the role of pulmonary lymphatics in lung fluid homeostasis and its interaction with cardiovascular pressures is poorly understood. In health, there is a fine balance between fluid extravasated from the pulmonary capillaries into the interstitium and the return of fluid to the circulation via the lymphatic vessels. This balance is maintained by an extremely interdependent system governed by pressures in the fluids (air and blood) and tissue (interstitium), lung motion during breathing, and the permeability of the tissues. + +Chronic elevation in left atrial pressure (LAP) due to left heart disease increases the capillary blood pressure. The consequent fluid accumulation in the delicate lung tissue increases its weight, decreases its compliance, and impairs gas exchange. This interdependent system is difficult to study experimentally. Computational modelling provides a unique perspective to analyse fluid movement in the cardiopulmonary vasculature in health and disease. We have developed an initial in silico model of pulmonary lymphatic function using an anatomically-derived structure to represent ventilation and perfusion, and underlying biophysical laws governing fluid transfer at the interstitium. This novel model was tested against increased LAP and non-cardiogenic effects (increased permeability). + +The model returned physiologically reasonable values for all applications, predicting pulmonary oedema when LAP reached 25 mmHg and with increased permeability. + +## DOI + +[dx.doi.org/10.17504/protocols.io.kxygx9zmwg8j/v1](https://dx.doi.org/10.17504/protocols.io.kxygx9zmwg8j/v1) + +## Protocol Citation + +Kelly Burrowes 2022. Supplementary Material: An in silico approach to understanding the interaction between cardiovascular and pulmonary lymphatic dysfunction. *protocols.io* [https://dx.doi.org/10.17504/protocols.io.kxygx9zmwg8j/v1](https://dx.doi.org/10.17504/protocols.io.kxygx9zmwg8j/v1) Version created by Kelly Burrowes + +## Keywords + +Pulmonary lymphatics, in silico models, cardio-pulmonary interdependence, pulmonary hypertension + +## License + +This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. + +--- + +# Protocol + +**Created:** Nov 20, 2022 +**Last Modified:** Nov 21, 2022 +**Protocol Integer ID:** 73017 + +**Materials Text:** Simulations performed on a regular desktop or laptop computer. + +--- + +## Lymphatic Model + +### 1. Estimation of Interstitial Hydrostatic Pressure (Pint) + +To represent this a sinusoidal function was used: +\[ P_{\text{int}} = A \sin(B - C) + D \] + +**Equation S1:** + +- **A**, the amplitude, is set to the difference between the maximum and minimum elastic recoil pressures for the acinus during a breath, as given by the ventilation model. This is representative of changes in thoracic pressure and unique to each acinar unit. +- The change in period, **B**, is defined by the breathing frequency, where \(f_b\) is the breathing frequency in breaths per second. + +\[ B = 2\pi f_b \] + +**Equation S2:** + +No horizontal shift is set, so **C** is equal to 0. Adding a vertical shift, **D**, enables the pressure to fluctuate between -8 (Pmin,int) and -1 mmHg (Pmax,int) for the given amplitude. The vertical shift is influenced by the interstitial saturation and can be scaled using the interstitial volume (Vint), or over interstitial capacity (Cint). + +\[ D = (P_{\text{min,int}} - P_{\text{max,int}} + A) \left( \frac{V_{\text{int}}}{C_{\text{int}}} \right)^2 + \left( -2 \cdot (P_{\text{min,int}} - P_{\text{max,int}} + A) \right) \left( \frac{V_{\text{int}}}{C_{\text{int}}} \right) + \left( P_{\text{min,int}} + \frac{A}{4} \right) \] + +**Equation S3:** + +As interstitial volume approaches its maximal capacity, the sinusoidal function moves up the y-axis, indicating greater pressures within the interstitium. Combined, the overall equation for interstitial pressure becomes: + +\[ P_{\text{int}} = A \sin \left(2\pi f_b t\right) + (P_{\text{min,int}} - P_{\text{max,int}} + A) \left( \frac{V_{\text{int}}}{C_{\text{int}}} \right)^2 + \left( -2 \cdot (P_{\text{min,int}} - P_{\text{max,int}} + A) \right) \left( \frac{V_{\text{int}}}{C_{\text{int}}} \right) + \left( P_{\text{min,int}} + \frac{A}{4} \right) \] + +**Equation S4:** +This relationship is plotted in Figure A1. + +![Figure S1: Plot of pressure versus time demonstrating the sinusoidal relationships for the hydrostatic pressure in the interstitial space (Pint, Equation S4) and hydrostatic pressure in the lymph space (Plymph, Equation S6).](assets/lynphatic_model_estimation_of_interstitial_hydrostatic_pressure.png) + +### 2. Lymphatic Hydraulic Conductivity + +Lymphatic hydraulic conductivity, \(L_{\text{plymph}}\), is estimated using the following equation: + +\[ L_{\text{plymph}} = L_{\text{pcap}} \left( \alpha_1 \left(\frac{V_{\text{int}}}{C_{\text{int}}}\right)^5 + \alpha_2 \left(\frac{V_{\text{int}}}{C_{\text{int}}}\right)^4 + \alpha_3 \left(\frac{V_{\text{int}}}{C_{\text{int}}}\right)^3 + \alpha_4 \left(\frac{V_{\text{int}}}{C_{\text{int}}}\right)^2 + \alpha_5 \left(\frac{V_{\text{int}}}{C_{\text{int}}}\right) + \alpha_6 \right) \] + +**Equation S5:** + +Coefficients \(\alpha_1\)-\(\alpha_6\) were approximated using a trial and error method using a single, representative acinar unit (selected from the middle of the right lung) to meet the following conditions: + +- Alveolar flooding occurs when left atrial pressure is 25 mmHg. +- An increase in interstitial saturation without flooding when left atrial pressure is 20 mmHg. +- Resting steady-state interstitial saturation is ~48%. +- A 50% increase or decrease in baseline saturation would return to resting steady-state values. + +This provided the coefficients displayed below in Table S1. + +| A | B | +|---|---| +| Coefficient | Value | +| \(\alpha_1\) | 846 | +| \(\alpha_2\) | -2417 | +| \(\alpha_3\) | 2389 | +| \(\alpha_4\) | -922 | +| \(\alpha_5\) | 126 | +| \(\alpha_6\) | 0 | + +**Table S1: Coefficients used for calculating lymphatic conductivity (Equation S5)** + +In reality, there is a baseline rate of conductivity that is only increased when interstitial volume reaches the threshold at which anchoring filaments begin to open initial lymphatic pores. Therefore, when interstitial saturation is 30% or less, \(L_{\text{plymph}}\) is held constant at 6.5e^-8 ml·s^-1·mmHg^-1, or 1.48 times more than capillary conductivity (see Figure S2). + +![Figure S2: Relationship between interstitial saturation and hydraulic conductivity.](assets/lymphatic_hydraulic_conductivity.png) + +### 3. Estimation of Lymphatic Hydrostatic Pressure + +The hydrostatic pressure gradient of the lymphatics was approximated using the following equation, similar to that described above in Equation S4: + +\[ P_{\text{hyd,lymph}} = \frac{A}{2} \sin \left( \left(2\pi t f_b\right) + \frac{\pi}{2} \right) + \left( P_{\text{max,lymph}} - P_{\text{min,lymph}} - A \right) \left( \frac{V_{\text{int}}}{C_{\text{int}}} \right)^2 + \left( P_{\text{min,int}} + \frac{A}{2} \right) \] + +**Equation S6:** + +Where interstitial volumes and capacities are assumed to represent the likely saturation of the lymphatics in equilibrium, in the absence of anatomical information of the initial lymphatics. The equation also includes the \(C\) value from Equation A5 that represents the horizontal offset, \(\pi/2\), which is a quarter-cycle offset. The offset enables the cyclical fluctuations in interstitial and initial lymphatic pressure to overlap, simulating the transient pressure differences between the two compartments that are hypothesized to exist. This relationship is plotted in Figure S1. + +## Perfusion Model + +The perfusion model simulates pulmonary blood supply within the pulmonary vasculature. The arterial tree is comprised of asymmetrically bifurcating elements that represent the arterial vasculature, supplying ~32,768 (2^15) vessels that feed the acinar units at the level of the terminal bronchioles in the airway tree. Within the acinar unit model, these arterioles continue to bifurcate symmetrically through 9 generations of capillaries, each connected across the alveoli to a corresponding venule creating a ladder-like structure. + +Each capillary is treated as a 'sheet' that transverses multiple alveoli, with each sheet having a specific model-calculated flow rate, blood pressure, and surface area, unique to the capillary generation of that acinar unit. + +These values are dependent on the resistance of each unit's supplying arterial pathway, the amount of expansion each unit undergoes with breathing, and the gravitational influence to establish a hydrostatic pressure gradient. + +\[ \Delta P = \frac{128 \mu L}{\pi D^4} Q + \rho g \cos \theta L \] + +**Equation S7:** + +Where: +- \( \mu \) is blood viscosity, +- \( L \) is the vessel length, +- \( D \) is the vessel diameter, +- \( Q \) is the blood flow rate, +- \( \rho \) is the density of blood in the vessel, +- \( g \) is gravitational acceleration (9.81 m/s^2), +- \( \theta \) is the angle of the vessel with respect to gravitational direction. + +This equation, alongside the conservation of mass, is solved through the full network and provides values of capillary hydrostatic pressure (Phyd,cap), capillary blood volume (Vcap), and the flow rate (Qcap) to calculate blood transit time as: + +\[ \text{transit}_{\text{Qcap}} = \frac{V_{\text{cap}}}{Q_{\text{cap}}} \] + +used in the lymphatic model. + +In addition to altering capillary pressure, modifying boundary conditions also changes the transit time of blood through a capillary bed and the capillary surface area. Whole lung capillary surface area at total lung capacity (TLC) is estimated to be 73 m², which can be divided by the number of acinar units to provide \( S_{\text{cap}} \) the surface area for an individual acinus at TLC (STLC). However, if the alveolus is not fully inflated \(S_{\text{cap}}\) needs to be scaled accordingly, based on lung compliance (C) and transpulmonary pressure (Ptp). + +Additionally, if alveolar air pressure (Palv) is greater than venous pressure (Pbv) parts of the alveolar sheet can collapse - this is the so-called 'zone 2' flow region. In this zone of flow, the available capillary surface area (Scap,zone2) is reduced by the factor described in Equation S9 as defined by Fung and Yen: + +\[ S_{\text{cap,zone2}} = S_{\text{cap}} \left( 1 - F + F e^{-\left[ \frac{P_{\text{bv}} - P_{\text{alv}}}{2 \sigma^2} \right]} \right) \] + +**Equation S9:** + +Where \( F \) is the maximum fraction of alveolar area that can be collapsed and \( \sigma \) is a constant that determines the extent of capillary collapse for a change in Pbv. + +--- + +## Ventilation Model + +### 5. Elastic Recoil Pressure + +Elastic recoil pressure was calculated for each acinus using a relationship derived by Swan et al., as shown in Eq. A4. Here, Pe is approximated from a 3D strain energy density relationship by assuming that each acinus expands isotropically. + +\[ P_e = \frac{\xi \epsilon \gamma}{2 \lambda} \left(3a + b \right) \left(\lambda^2 - 1 \right) \] + +**Equation S10:** + +Where +\[ \gamma = \frac{3}{4} (3a + b) \left(\lambda^2 - 1 \right)^2 \] + +- \(\lambda\) is the isotropic stretch from the undeformed reference volume (V0), calculated as: + +\[ \lambda = \left(\frac{V}{V_0}\right)^{1/3} \] + +Estimated strain energy density function values are \(\xi\) (2500 Pa), \(a\) (0.433) and \(b\) (-0.611). + +Maximum and minimum elastic recoil pressure values are used as a surrogate of the change in pressure within the thoracic cavity within the lymphatics model. + +--- + +## References + +1. **Taylor A., Parker J.** Pulmonary Interstitial Spaces and Lymphatics. Comprehensive Physiology 2011. +2. **Svendsen Ø., Reed R., Wiig H.** The Interstitium and Lymphatics have an Important Role in Edema Generation during Sepsis. In: Annual Update in Intensive Care and Emergency Medicine 2011, edited by Vincent J. Berlin: Springer, 2011. +3. **Sabine A., Saygili Demir C., Petrova TV.** Endothelial Cell Responses to Biomechanical Forces in Lymphatic Vessels. Antioxidants & redox signaling 25: 451-465, 2016. +4. **Zawieja D.** Contractile Physiology of Lymphatics. Lymphatic Research and Biology 7: 87-96, 2009. +5. **Clark AR., Burrowes KS., Tawhai MH.** Contribution of serial and parallel micro-perfusion to spatial variability in pulmonary inter- and intra-acinar blood flow. Journal of Applied Physiology 108: 1116-1126, 2010. +6. **Fung YC., Sobin SS.** Theory of sheet flow in lung alveoli. Journal of Applied Physiology 26: 472-488, 1969. +7. **Clark AR., Tawhai MH., Hoffman EA., Burrowes KS.** The interdependent contributions of gravitational and structural features to perfusion distribution in a multiscale model of the pulmonary circulation. Journal of Applied Physiology 110: 943-955, 2011. +8. **Gehr P., Bachofen M., Weibel ER.** The normal human lung - Ultrastructure and morphometric estimation of diffusion capacity. Respiratory Physiology and Neurobiology 32: 121-140, 1978. +9. **Fung YC., Yen RT.** A new theory of pulmonary blood flow in zone 2 condition. Journal of Applied Physiology 60: 1638-1650, 1986. +10. **Swan AJ., Clark AR., Tawhai MH.** A computational model of the topographic distribution of ventilation in healthy human lungs. J Theor Biol 300: 222-231, 2012. +11. **Tawhai MH., Nash MP., Lin CL., Hoffman EA.** Supine and prone differences in regional lung density and pleural pressure gradients in the human lung with constant shape. Journal of Applied Physiology 107: 912-920, 2009. +12. **Kowalczyk P., Kleiber M.** Modelling and numerical analysis of stresses and strains in the human lung including tissue-gas interaction. European Journal of Mechanics - A/Solids 13.3: 367-393, 1994. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/surveillance-of-antimicrobial-resistant-bacteria-c-dcqy2vxw.md b/markdown-output/surveillance-of-antimicrobial-resistant-bacteria-c-dcqy2vxw.md new file mode 100644 index 0000000000000000000000000000000000000000..e452b89880e87b446db8ca22a901b23c03381a50 --- /dev/null +++ b/markdown-output/surveillance-of-antimicrobial-resistant-bacteria-c-dcqy2vxw.md @@ -0,0 +1,158 @@ +```markdown +# Goal/Experiment: +Surveillance of antimicrobial-resistant bacteria causing community-acquired urinary tract infections in low-income countries V.3 + +## Protocol Information + +- **DOI:** [10.17504/protocols.io.kqdg3xdneg25/v3](https://dx.doi.org/10.17504/protocols.io.kqdg3xdneg25/v3) +- **Authors:** Mtebe Majigo, Stephen Mshana, Erick Komba, Nyambura Muremi, Mecky Matee +- **Version:** 3 +- **Date:** April 26, 2024 +- **License:** [Creative Commons Attribution License](https://creativecommons.org/licenses/by/4.0/) + +## Abstract + +This protocol assists in designing a sustainable surveillance program for antimicrobial resistance (AMR) in the community, targeting children above two years and adults passively presenting to lower health facilities for healthcare. The protocol includes initial laboratory preparation, recruitment and sampling over at least 12 months, and data sharing via global platforms. + +## Guidelines + +1. **Decide on target population and enrollment criteria** +2. **Selection of target laboratories, surveillance areas, bacteria, and antimicrobials** +3. **Prepare a sampling plan and sample size** +4. **Outline roles of different laboratories and agree on the surveillance plan** +5. **Laboratory procedures for antimicrobial resistance data generation** +6. **Proficiency testing and internal quality assurance** +7. **Process for data management, analysis, reporting, and sharing** + +## Materials + +- [Annex 1 - Data Collection Form.pdf](#) +- [Annex 2 - Standard Operating Procedures.pdf](#) + +## Preparations Before Beginning AMR Surveillance + +### Laboratories, Surveillance Areas, Bacteria, and Antimicrobials + +1. Identify health facilities for surveillance and appropriate laboratories for microbial analysis. +2. Identify specific bacteria and antimicrobials relevant to the region and global standards. + +### Sample Collection + +1. Design a coordinated sampling plan and sample collection form. +2. Prepare Standard Operating Procedures (SOPs) for sample collection and ensure proper storage. +3. Perform trial collection of samples and adjust for any issues. + +### Laboratory Preparation + +1. Define laboratory capacity and develop SOPs for microbial culture, identification, and antimicrobial susceptibility testing (AST). +2. Train laboratory staff on relevant procedures. + +### Stakeholders and Community Engagement + +1. Engage key stakeholders from public, private, and non-profit sectors. +2. Develop agreements with enrolled health facilities and lay down protocols for AMR data sharing and isolate transfer. + +## Target Population and Enrollment Criteria + +Sampling includes children above two years and adults from specific surveillance areas. Participants are recruited continuously over 12 months. + +Clinical classifications for UTIs include: +- **Uncomplicated UTI:** Symptoms like painful and frequent urination. +- **Complicated UTI:** Symptoms like fever and flank pain. +- **Catheter-associated UTI (CA-UTI):** Symptoms after using a urinary catheter for over two days. + +## Target Laboratories, Surveillance Areas, Bacteria, and Antimicrobials + +### Laboratories and Surveillance Areas + +1. Primary health facilities within 50 km of capable laboratories are selected. +2. Sites should represent geographic diversity. + +### Target Bacteria + +Include both Gram-negative and Gram-positive common uropathogens, principally: +- **Enterobacterales:** Escherichia coli, Klebsiella pneumoniae, etc. +- **Gram-Positive Bacteria:** Staphylococcus saprophyticus, Enterococcus spp. + +### Diagnostic and Bacterial Tests + +1. Prioritize E. coli and other common pathogens. +2. Bacterial identification through conventional and advanced methods like MALDI-TOF. +3. AST using methods such as Kirby–Bauer disk diffusion, Vitek 2, BD Phoenix, etc. + +## Sampling Plan + +### Sampling Timetable + +1. Samples should reach the laboratory during working hours or be refrigerated if delayed. +2. Schedule instructions should include clear handling, storage, and transport methods. + +### Urine Sample Collection and Handling + +1. Collect at least 10 mL of mid-stream urine (MSU) in sterile, boric acid-lined containers. +2. Collect samples between 0900 and 1200 hours. + +### Sample Transportation + +1. Transport samples to the laboratory within eight hours. +2. Refrigerate samples at 2-8°C if analysis is delayed. + +### Sample Collection Forms + +1. Complete for each sample to capture descriptive AMR data. +2. Implement unique identifiers for samples. + +### SOPs, Training, and Trialing Sample Collection + +1. Develop detailed SOPs. +2. Train staff on participation, sample handling, and reporting procedures. + +## Responsibilities of National AMR Reference and Regional/Provincial Surveillance Laboratories + +### Regional/Provincial Laboratories + +1. Conduct identification, culture, and AST. +2. Follow EUCAST/CLSI guidelines. + +### AMR Reference Laboratory + +1. Handle complex procedures and overflow samples. +2. Provide additional identification and AST using advanced methods. + +## Laboratory Procedures + +### Urine Specimen Culture + +1. Perform culture within two hours for unpreserved specimens. +2. Incubate plates and read growth for coliform determination. + +### Urinalysis + +1. Follow SOPs for urinalysis strips immersed in urine samples. + +### Identification of Isolates + +1. Identify bacteria using biochemical and semi-automated methods. + +## Proficiency Testing and Use of ATCC Strains for Internal Quality Assurance + +1. Conduct proficiency testing to ensure standard AST results. +2. Test standard ATCC strains for internal controls. + +## Data Management, Analysis, and Sharing + +1. Store data securely and limit access to authorized personnel. +2. Use WHONET and other systems for standardized reporting. + +## Protocol References + +1. Baron EJ, et al. IDSA and ASM guidelines for infectious disease diagnostics. +2. Warren JW, et al. Guidelines for UTI treatment. +3. WHO prioritization of pathogens. +4. Hay AD, et al. Development of UTI diagnosis. +5. Kupelian AS, et al. UTI symptom analysis. +6. Magiorakos AP, et al. Definitions for resistant bacteria. +7. Poulou A, et al. CLSI ESBL detection methods. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/symbiotic-dose-50-sd50-for-vibrio-fischeri-strain-cwjyxcpw.md b/markdown-output/symbiotic-dose-50-sd50-for-vibrio-fischeri-strain-cwjyxcpw.md new file mode 100644 index 0000000000000000000000000000000000000000..66f82e65c0f78c223391a3e766d7346c4e264242 --- /dev/null +++ b/markdown-output/symbiotic-dose-50-sd50-for-vibrio-fischeri-strain-cwjyxcpw.md @@ -0,0 +1,135 @@ +```markdown +# Goal/Experiment: +This protocol details symbiotic dose-50 (SD50) for *Vibrio fischeri* strain to colonize *Euprymna scolopes*. + +## Symbiotic Dose-50 (SD50) for Vibrio fischeri strain to colonize Euprymna scolopes V.2 + +**Authors:** +- ard1,2 +- ejg1,2 +- agc1,2 +- Tim I Miyashiro1,2 + +1Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA, USA; +2The One Health Microbiome Center, Huck Institutes of the Life Sciences, The Pennsylvania State University, University Park, PA, USA + +### Abstract +This protocol details symbiotic dose-50 (SD₅₀) for *Vibrio fischeri* strain to colonize *Euprymna scolopes*. + +### Materials + +#### Materials needed: +1. Culture tubes +2. LBS medium [1% (w/v) tryptone, 0.5% (w/v) yeast extract, 2% (w/v) NaCl, 50 mM Tris–HCl (pH 7.5)], with 1.5% w/v agar for solid medium +3. Shaking incubator at 28°C +4. Spectrophotometer and cuvettes +5. Plastic Tumblers (e.g., Fineline Mfr. #409-CL Savvi Serve 9 oz clear hard plastic) +6. Freshly hatched *E. scolopes* squid +7. Transfer pipets (e.g., Fisherbrand Disposable Graduated Transfer Pipettes, Catalog No. 13-711-9AM) +8. Filter-sterilized seawater (FSSW: Instant Ocean, Spectrum Brands, Blacksburg, VA) mixed according to instructions provided by manufacturer. + - Filter through 0.22-µm surfactant-free filter (e.g., Nalgene Rapid-Flow Sterile Disposable Filter Units with SFCA Membranes) +9. Microfuge tubes +10. 50-mL conical tubes +11. Vials (e.g., VWR Drosophila vials narrow #75813-162) +12. Luminometer (e.g., GloMax 20/20, Promega Corp., Madison, WI) + +### Preparation of V. fischeri Cultures + +1. For each strain of interest, initiate a starter culture by inoculating 3 mL LBS with an isolated colony. + - Incubate starter cultures **overnight (~16 h)** at 28°C, shaking at 200 RPM. + +2. Measure the OD600 of each starter culture. + - In a microfuge tube, normalize each starter culture by diluting it to an OD600 of 1.0 in fresh LBS to a final volume of 1.0 mL. + - Vortex briefly. + +3. Initiate an intermediate culture by inoculating 3 mL LBS in a fresh culture tube with 30 µL of the normalized cell suspension. + - Incubate at 28°C, shaking at 200 RPM. + +### Selection and Preparation of Juvenile E. scolopes + +4. Using transfer pipet, collect freshly hatched juvenile squid into tumblers containing 100 mL FSSW, with no more than 50 squid/tumbler. + +5. Prepare a new tumbler with 50 mL FSSW for each group. + +6. Transfer animals from the 100 mL FSSW tumblers to the new tumblers individually. + - *Note: To minimize bias, add an animal to the tumbler of a different group with each transfer*. + +### Preparation of Inoculums + +7. For each strain, when the turbidity of culture is OD600 = 0.8-1.0, transfer culture volume equivalent to 1 mL of OD600 = 1.0 to a microfuge tube. + +8. Concentrate cells by centrifugation: + 1. Centrifuge at 5000 x g for 2:30. Then, remove 0.9 mL supernatant, add 0.9 mL FSSW, and resuspend the pellet. + 2. Repeat the procedure as in 8.1. + +9. Prepare a serial dilution by transferring 100 µL of the cell suspension described in Step 8 into 0.9 mL FSSW in a microfuge tube (10-1 dilution). + - Continue ten-fold dilutions until the desired dilution range has been achieved. + - *Note: Three-fold dilutions can be used instead for greater resolution*. + +10. Prepare a control for an apo-symbiotic group by transferring 1 mL FSSW to a microfuge tube. + +11. For each group, transfer 100 µL from the corresponding microfuge tube into a 50-mL conical tube containing 50 mL FSSW and invert several times to mix. + +### Inoculation Phase + +12. To initiate the inoculation phase, pour the cell suspension into the corresponding tumbler to bring the total volume to 100 mL. + - Repeat for the control described in Step 10. + +13. Sample tumblers by plating 100 µL onto solid LBS medium in triplicate and incubate the plates at 28°C **overnight**. + - *Note: For high inoculum levels, dilution may be necessary to obtain countable CFUs. At low inoculum levels, use the known dilution factor from more concentrated inoculums to estimate the corresponding abundance of V. fischeri*. + +14. After 3.5 hours, wash the animals by serially transferring them as a group into a tumbler containing 100 mL FSSW twice, with 5-minute intervals between transfers. + +15. Transfer animals into vials containing 4 mL FSSW, with one animal per vial. + +16. Store animals in a room that has a 12-h day/12-h night light cycle. + +### Measurement of Bioluminescence + +17. After 16-18 hours, transfer animals to clean vials containing 4 mL FSSW. + +18. Using a luminometer, measure the luminescence emitted by each sample. + +### Euthanasia and Storage of Animals + +19. To initiate the anesthesia step, transfer each animal with seawater (total volume of 0.5 mL) to a microfuge tube and place on ice. + +20. After 5 minutes, add 0.5 mL cold 6% ethanol/FSSW to each microfuge tube and keep on ice. + +21. After 15 minutes, remove the liquid volume from the tube and store the anesthetized animal at -80°C, completing euthanasia. + +### Scoring of Bioluminescence + +22. Use the luminescence measurements of the apo-symbiotic group to determine the 99.9th percentile, above which animals are considered to be bioluminescent. + +23. Score each animal as symbiotic or non-symbiotic by comparing the corresponding luminescence measurement with the bioluminescence cutoff defined in Step 22. + +### Determining Inoculum Levels + +24. Count CFU on the inoculum plates generated in Step 13. Also verify that no CFU are present on the apo-symbiotic control plates. + +25. Calculate the concentration of CFUs in each inoculum cell suspension described in Step 9 by dividing the CFU counts by the volume plated (in mL) and multiplying by the dilution factor, if any. + +### Calculation of SD50 + +26. For each strain, generate a table with the number of symbiotic and non-symbiotic animals at each inoculum concentration, with rows arranged in order of highest to lowest concentration. + +27. Prepare two additional columns containing adjusted counts for: + 1. Animals that could be assumed to be symbiotic at higher inoculums. + 2. Animals that could be assumed to be non-symbiotic at lower inoculums. + +28. Calculate the adjusted percent of symbiotic animals at each inoculum by dividing the adjusted counts of symbiotic animals by the total adjusted animal counts in the corresponding row. + +29. Calculate the SD50 using the following equation: + ```markdown + SD50 = 10^[log(DF^(X) + log(c)] + + Where, + X = [(50% - a)/(b - a)] + a = Adjusted percent symbiotic below 50% closest to 50% + b = Adjusted percent symbiotic above 50% closest to 50% + c = Inoculum concentration of the adjusted percent colonized below 50% closest to 50% + DF = Dilution factor (fold-change difference between groups in the experiment) + ``` +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/synchronized-spinal-nerve-and-dorsal-root-ganglia-b83sryne.md b/markdown-output/synchronized-spinal-nerve-and-dorsal-root-ganglia-b83sryne.md new file mode 100644 index 0000000000000000000000000000000000000000..a2e3cd75f9878613af0547d9d6614b0575078d8f --- /dev/null +++ b/markdown-output/synchronized-spinal-nerve-and-dorsal-root-ganglia-b83sryne.md @@ -0,0 +1,111 @@ +```markdown +# Goal/Experiment: +The goal of this experiment is to assess the instantaneous effect of dorsal root ganglia (DRG) stimulation on the transmission of electrically evoked action potentials from the spinal nerve via an ex vivo preparation with mouse L6 spinal nerve (SN), DRG, and dorsal root (DR) in continuity (SN-DRG-DR preparation). + +# Synchronized Spinal Nerve and Dorsal Root Ganglia Stimulation + +### Authors +- Longtu Chen +- Bin Feng + +**Department of Biomedical Engineering, University of Connecticut, Storrs, CT, United States** + +**Publication Date:** June 2, 2022 + +**DOI:** [dx.doi.org/10.17504/protocols.io.36wgq7b4ovk5/v1](https://dx.doi.org/10.17504/protocols.io.36wgq7b4ovk5/v1) + +## Abstract +Assessing the instantaneous effect of dorsal root ganglia (DRG) stimulation on the transmission of electrically evoked action potentials from the spinal nerve via the ex vivo preparation with mouse L6 spinal nerve (SN), DRG, and dorsal root (DR) in continuity, i.e., the SN-DRG-DR preparation. Synchronized spinal nerve and DRG stimulation reveal a progressive increase in conduction delay by DRG stimulation, suggesting an activity-dependent slowing in blocked fibers. Midrange frequencies (50-500 Hz) are more efficient at blocking transmission than lower or higher frequencies. + +## Materials and Equipment + +### Animals +- **C57BL/6 mice**: 8-16 weeks old, 25-35 g, of either sex (taconic.com, Germantown, NJ). + +### Reagents +- **Isoflurane** (Hospira Inc., Lake Forest, IL) +- **Ketamine/Xylazine cocktail** (100/10 mg per kg body weight) + +### Solutions +- **Oxygenated Krebs Solution** (95% O₂, 5% CO₂, ice-cold): + - 117.9 mM NaCl + - 4.7 mM KCl + - 25 mM NaHCO₃ + - 1.3 mM NaH₂PO₄ + - 1.2 mM MgSO₄ + - 2.5 mM CaCl₂ + - 11.1 mM D-glucose + +### Equipment +- **TDT system** (PZ5-32, RZ5D, IZ2H, Tucker-Davis Technologies [TDT], Alachua, FL) +- **Needle electrode** (FHC, platinum-iridium) +- **Suction electrode** (quartz glass capillary tip diameter ~300 µm) +- **Custom-built recording electrodes** +- **Custom-built perfusion chamber** +- **Stimulus isolator** (A365; World Precision Instruments, New Haven, CT) +- **Software**: + - SigmaPlot v11.0 (Systat software, Inc., San Jose, CA) + - MATLAB v2022 (Mathworks Inc., Natick, MA) + +## Protocol + +### Ex vivo spinal nerve-dorsal root ganglia-dorsal root (SN-DRG-DR) preparation + +1. **Anesthesia**: + - C57BL/6 mice (8-16 weeks, 25-35 g, either sex) were anesthetized by isoflurane inhalation followed by intraperitoneal and intramuscular injection of a ketamine/xylazine cocktail (100/10 mg per kg body weight). + +2. **Euthanasia and Perfusion**: + - Mice were euthanized by exsanguination from the right atrium, followed by transcardiac perfusion from the left ventricle with oxygenated ice-cold Krebs solution. + +3. **Dorsal Pediculectomy**: + - Performed to expose the spinal cord and DRG from T12 to S1 segments. + +4. **Dissection Chamber**: + - The exsanguinated mouse carcass was placed in a dissection chamber circulated with oxygenated ice-cold Krebs solution. + +5. **Tissue Transfer**: + - The SN, DRG, and attached DR were dissected and transferred to a custom-built chamber with tissue and recording compartments. + +6. **Perfusion**: + - The SN and L6 DRG were placed in a tissue chamber perfused with oxygenated Krebs solution at 30°C. + - The DR was placed in the recording chamber filled with mineral oil. + +7. **Single-Fiber Recordings**: + - L6 DR was split into fine filaments (∼10 μm) for single-fiber recordings using a custom-built microwire electrode array. + +### Determine DRG Stimulation Location and Amplitude + +8. **Neural Transmission**: + - Transmission from the SN to the DR was evoked via electrical stimulation of the SN using a suction electrode and a stimulus isolator (A365; World Precision Instruments, New Haven, CT). + +9. **Electrode Stimulation**: + - A blunt-tipped needle electrode (FHC, platinum-iridium) delivered biphasic constant-current stimulation to the L6 DRG using an IZ2H stimulator (Tucker-Davis Technologies Inc, Alachua, FL). + +10. **Threshold Amplitude**: + - Determined by stimulating the DRG at 0.5 Hz, identifying identical waveforms but different conduction delays in single-fiber recordings. + +11. **Current Amplitude Locations**: + - DRG stimulation was applied at 3 locations along the DRG's length to determine the minimal current amplitude that evoked 3 to 5 action potentials per 10 pulses at 0.5 Hz. + +12. **Stimulus Intensities**: + - Set to either subthreshold or suprathreshold, corresponding to 70-80% and 120-150% of the threshold current amplitude, respectively. + +### Synchronized SN and DRG Stimulation + +13. **Baseline Recording**: + - Conduct spinal nerve stimulation for 20 seconds (10 stimulations at 0.5 Hz). Simultaneously record the dorsal root's action potentials. + +14. **Synchronized Protocol**: + - Follow with a synchronized SN and DRG stimulation protocol. Use train frequencies for DRG and SN of 0.5 Hz with selected pulse frequencies (10, 50, 100, 500, 1000 Hz). + +15. **Post-Stimulation Recording**: + - Record spinal nerve stimulation immediately for 20 seconds post-DRG stimulation (10 stimulations at 0.5 Hz). + +16. **Recovery Recording**: + - After 15-30 minutes, conduct another spinal nerve stimulation for 20 seconds (10 stimulations at 0.5 Hz). + +## References +- Longtu Chen, Bin Feng. Synchronized spinal nerve and dorsal root ganglia stimulation. protocols.io. [https://dx.doi.org/10.17504/protocols.io.36wgq7b4ovk5/v1](https://dx.doi.org/10.17504/protocols.io.36wgq7b4ovk5/v1) + +## endofoutput +``` \ No newline at end of file diff --git a/markdown-output/testimony-on-a-successful-lab-protocol-to-disrupt-cw9sxh6e.md b/markdown-output/testimony-on-a-successful-lab-protocol-to-disrupt-cw9sxh6e.md new file mode 100644 index 0000000000000000000000000000000000000000..1d72559f903f6e40fa52a4bae6703db0b4923a6c --- /dev/null +++ b/markdown-output/testimony-on-a-successful-lab-protocol-to-disrupt-cw9sxh6e.md @@ -0,0 +1,118 @@ +```markdown +# Goal/Experiment: +The objective of this experiment is to document and verify a successful lab protocol to disrupt the cell wall of *Chlorella vulgaris* microalgae, thereby enabling increased bioavailability of microalgae compounds in monogastric animal diets. This is achieved through enzymatic treatment, primarily using Carbohydrate-Active enZymes (CAZymes). + +## Testimony on a Successful Lab Protocol to Disrupt *Chlorella vulgaris* Microalgae Cell Wall V.2 +**PLOS One** | **Peer-reviewed method** + +**Authors:** +- Diogo Coelho¹ +- Paula A. Lopes¹ +- José A. M. Prates¹ + +¹ CIISA - Centro de Investigação Interdisciplinar em Sanidade Animal and Laboratório Associado para Ciência Animal e Veterinária (AL4AnimalS), Faculdade de Medicina Veterinária, Universidade de Lisboa, Avenida da Universidade Técnica, Pólo Universitário do Alto da Ajuda, 1300-477 Lisboa, Portugal + +![Diogo Coelho](path/to/diogo_coelho_image) + +**Version 2 | July 14, 2023** + +**DOI:** [dx.doi.org/10.17504/protocols.io.dm6gpb9z5lzp/v2](https://dx.doi.org/10.17504/protocols.io.dm6gpb9z5lzp/v2) +**External link:** [https://doi.org/10.1371/journal.pone.0268565](https://doi.org/10.1371/journal.pone.0268565) + +**Protocol Citation:** +Diogo Coelho, Paula A. Lopes, José A. M. Prates 2023. Testimony on a successful lab protocol to disrupt *Chlorella vulgaris* microalga cell wall. **protocols.io**. Version created by Diogo Coelho + +**Manuscript Citation:** +Lopes PA, Coelho D, Prates JAM (2022) Testimony on a successful lab protocol to disrupt *Chlorella vulgaris* microalga cell wall. **PLOS ONE 17(5)**: e0268565. [https://doi.org/10.1371/journal.pone.0268565](https://doi.org/10.1371/journal.pone.0268565) + +**License:** +This is an open access protocol distributed under the terms of the **Creative Commons Attribution License**, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. + +**Protocol status:** Working protocol + +**Created:** July 14, 2023 +**Last Modified:** July 14, 2023 +**Protocol integer ID:** 85010 + +### ABSTRACT +Over the last decades, microalgae have gained prominence due to their potential in innovative mass production as a source for multiple processes. However, most species, including *Chlorella vulgaris*, have a complex cell wall that presents challenges for digestion and extraction of nutrients. This protocol focuses on disrupting the *C. vulgaris* cell wall using specific enzymes (CAZymes), thus improving the bioavailability of its compounds in monogastric diets. + +### Key Steps: +1. Microalgae cultivation with available pre-selected CAZymes. +2. Screening functional enzymes. +3. Determining reducing sugars via 3,5-dinitrosalicylic acid (DNSA)-based method. +4. Testing enzyme synergies and characterizing enzymatic properties. +5. Conducting a thorough assessment for breakdown quantification. + +Beyond animal feed, this protocol has potential applications in other biotechnological fields. + +### Construction of the CAZymes Bank and Production of *Chlorella vulgaris* Suspension. + +**1.** Preparation of *C. vulgaris* suspension (20 mg/mL): Resuspend the microalgae powder in PBS buffer and mix using a vortex. + +**2.** Prewash the *C. vulgaris* suspension: + - Incubate in an orbital shaker at 37ºC, 150 rpm for 30 minutes. + - Centrifuge at 4000 g for 30 minutes. + - Discard the supernatant and resuspend the pellet in fresh PBS buffer (20 mg/mL) using a vortex. + +**3.** Transfer 1 mL of *C. vulgaris* suspension to each well of a 24-well plate. Add the respective enzyme to a final concentration of 20 μg/mL, or 50 μL PBS for controls. Incubations should be performed in triplicate. + +**4.** Seal and incubate in an orbital shaker at 37ºC, 60 rpm, overnight. + +**5.** Centrifuge at 4000 g for 15 minutes. Recovery the supernatant to an Eppendorf tube. + +**6.** Boil samples for 5 minutes; centrifuge at 10000 g for 5 minutes. + +**7.** Recover supernatant for reducing sugars determination. + +### Reducing Sugars Release Determination (DNSA Method) + +**8.** Add to a new Eppendorf 600 μL of the supernatant (stage 7) and 600 μL of DNSA reagent (Note 1). + + - **Note 1:** DNSA reagent: 1% 3,5-Dinitrosalicylic acid; 0.2% phenol; 1% sodium hydroxide. Add 200 μL of sodium sulfite (5%) and 2 μL of glucose (20%) per 20 mL of DNSA reagent. + +**9.** Mix vigorously and incubate in boiling water at 100ºC for 15 minutes. + +**10.** Place eppendorfs on ice for 5 minutes. + +**11.** Measure absorbance (λ = 570 nm). + +**12.** Calculate reducing sugars using a glucose calibration curve (Miller, 1959). + +### Incubation of *C. vulgaris* Suspension with the Enzyme Mix + +**13.** Repeat stages 1 to 7, but add the enzyme mix at stage 3. Final enzyme concentration in the mix is 20 μg/mL in a 1:1:1:1 ratio. + +### Biochemical Characterization of Enzymes + +**14.** **Thermostability analysis:** Incubate enzymes at 30°C, 37°C, 40°C to 80°C for 30 minutes each. Cool on ice, centrifuge, and quantify protein. + +**15.** **Proteolysis resistance:** Place 200 μL of each enzyme (1 g/L) in eppendorfs. Incubate with porcine pancreatin (5 g/L) at 37 °C. Remove and analyze samples by 14% SDS-PAGE gels. + +### Analysis on the Supernatant + +**16.** Repeat stages 8 to 12 for reducing sugars release determination. + +**17.** **Fatty Acids Analysis:** Lyophilize 1 mL of supernatants for 24 hours. Use the Folch method (1957) for lipid extraction, esterify fatty acids, and analyze methyl esters (FAME). + +**18.** **Oligosaccharides Quantification:** Perform using HPLC with Dionex CarboPac PA10 column and ECD. Inject 10 μL of supernatants using standard glucose calibrations. + +**19.** **Protein Quantification:** Apply the Kjeldahl method (AOAC, 2000). + +**20.** **Pigments Quantification:** Follow Hynstova et al., 2018, for chlorophylls and carotenoids. + +### Analysis on the Residue + +**20.** After centrifugation (stage 5), recover the microalgae residue. + +**21.** **Fatty Acids Analysis on Residue:** Lyophilize 10 mg residues for 24 hours. Repeat stages 18 to 20. + +**22.** **Cell Counting:** Resuspend 10 mg residue in PBS buffer, add to Neubauer chamber, and count under microscope. + +**23.** **Fluorescence Intensity Quantification:** Stain homogenate with Calcofluor White and KOH, observe under fluorescence microscope, and quantify with Image J software. + +## Supplementary Information +- Spotlight video featuring extra context and tips. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/testing-safety-mechanism-bkuvkww6.md b/markdown-output/testing-safety-mechanism-bkuvkww6.md new file mode 100644 index 0000000000000000000000000000000000000000..f95bacefd13638b2a5dddea5b67258aa351a46f2 --- /dev/null +++ b/markdown-output/testing-safety-mechanism-bkuvkww6.md @@ -0,0 +1,93 @@ +```markdown +# Goal/Experiment: +In order to test the efficiency of the kill switch mechanism, we plan on performing the following experiments. + +# Testing Safety Mechanism + +**Author**: Andreea S +**Institution**: University of Groningen +**Date**: Oct 24, 2020 + +DOI: [dx.doi.org/10.17504/protocols.io.bkuvkw6](https://dx.doi.org/10.17504/protocols.io.bkuvkw6) + +iGEM Groningen 2020 + + + +## Abstract + +In order to test the efficiency of the kill switch mechanism, we plan on performing the following experiments: + +### Test Amino Acid Autotrophy in vitro +An overnight culture will be used to inoculate a test culture. The knockout strains will be grown in a medium containing the test amino acid and then transferred to a minimal medium lacking the specific amino acid. Samples of 0.5 mL will be removed every 30 minutes to test the optical density (OD). + +### Testing Solanine Dependency in vitro +A test culture will be inoculated from the overnight culture. The cells will be grown in LB supplemented with solanine (CONCCCCCC). The cells will be collected, washed, and resuspended in LB lacking solanine. Samples of 0.5 mL will be removed every 30 minutes in order to test OD. + +### Testing Dependency in Soil +The GFP-mutant strains (amino acid auxotrophs and solanine dependent mutants) will be inoculated at the root of the potato plants. The potato plants will be grown and described as above. + +## Protocol + +### Test Safety Mechanisms in the Lab + +1. Plate mutants on minimal media supplemented with the auxotrophy amino acid or solanine. +2. Pick one colony and inoculate 5 mL of *Bacillus mycoides*. Grow overnight at **200 rpm**, **30°C**. +3. The next day, inoculate a working culture (30 mL media + 1% overnight culture). +4. Grow at **200 rpm, 30°C** until OD600nm is 0.5. +5. Collect cells by centrifugation at **4000 x g, 4°C, 10 min**. +6. Wash 5 times with growth media (without amino acid). _This step may need adjustment. The purpose is to eliminate the dependency molecule from the growth medium._ +7. Resuspend in 30 mL minimal media. +8. Divide culture into 2 x 15 mL cultures. Supplement one of them with the required amino acid. +9. Incubate both samples at **200 rpm, 30°C**. +10. Every **30 min**, take a 0.5 mL sample and check OD600. +11. _Expected Result_: The OD of the sample with amino acid will increase exponentially. The other one will remain 0.5. + +### Test Safety Mechanism in Soil + +12. Grow potato plants in a 40 cm diameter pot. +13. Inoculate 5 mL of a GFP-labeled *B. mycoides* (auxotroph) culture in exponential growing phase (OD600nm = 0.5). For a negative control, use GFP-labeled *B. mycoides*. _Refer protocol for fluorescent labeling Bacillus mycoides._ + +#### Routine Culture of Bacillus Strains + +13.1. Routinely culture *Bacillus* strains in Luria-Bertani (LB) medium at 30°C with aeration at 200 rpm. + +#### Preparation of B. mycoides Strain Aliquots for Electroporation + +13.3. Prepare the B. mycoides strain aliquots for electroporation. +13.3.1. Cultivate the bacterial strain overnight in LB broth at 30°C and 180 rpm. +13.3.2. Transfer 1 mL of the overnight culture into 100 mL of LB medium (with 2% [wt/wt] glycine) and incubate it at 30°C and 180 rpm until optical density at 600 nm is 0.4 to 0.7. +13.3.3. Centrifuge the cells and wash the pellets with increasing concentrations of ice-cold glycerol (2.5%, 5%, and 10%). Resuspend this pellet in precooled electroporation buffer (10% glycerol) and shock freeze in liquid nitrogen. + +13.4. Add the library vector DNA in an amount of 2 µg to the cells, and perform electroporation. The settings for electroporation are 2.0 kV, 25 µF and 200 Ω in a 2-mm cuvette using a Bio Rad Gen Pulser II electroporation system (Bio-Rad). +13.5. Add 1 mL of LB medium and grow the cells for 2 hours at 30°C and 150 rpm for recovery and then plate on LB-Cm4 agar. +13.6. After 24 hours of growth at 30°C, harvest the colonies from the plates and pool in LB medium. +13.7. Store the libraries at -80°C as 15% glycerol stocks. +13.8. Inoculate the B. mycoides strain mKate2mut library in 50 mL of LB-Cm4 and grow at pH 7 or pH 6 to an OD600 nm of 0.3-0.6. +13.9. B. mycoides has been seen to show extensive cell-chaining and hence a mild sonication step of 4 rounds of 3 X 10 pulses of 1s with an amplitude of 30% can be applied to disassemble the aggregated cells. +13.10. Sort the cells on a flow cytometer at 20 psi using a 70 Micromolar (µM) nozzle at a flow rate of 1.0 with the highest sort precision mode (0– 32.0 sort purity mask). +13.11. Using a sequential gating strategy with FCS height versus widths, followed by SCC height versus width, cellular debris, and chained cells can be excluded. +13.12. To separate the brightest variants choose a cutoff of 3% of the brightest event in the first round of cell sorting and 0.3% of the brightest events in the second round of sorting with the light scatter parameters. +13.13. After FACS sorting, plate the final fluids containing bright cells on LB-Cm4 plates and grow them overnight at 30°C. +13.14. Observe the colonies using a fluorescence microscope. Keep the filter setting for GFP as excitation at 460/480 nm and emission at 495/540 nm with a 485 nm dichromatic mirror; for RFP, the filter setting can be kept as excitation at 545/580 nm and emission at 610 nm with a 600 nm dichromatic mirror. +13.15. Capture the images on a camera and calculate the intensity of single-cell with Image J software. +13.16. Calculate the total cell fluorescence. The formula is: Corrected total cell fluorescence (CTCF) = Integrated Density – (Area of selected cell x Mean fluorescence of background readings). + +### Soil Sample Collection + +14. Take soil samples of control and mutants from: + - As close to the root as possible + - 10 cm distance from root + - 20 cm distance from root + - 30 cm distance from root + - 40 cm distance from root + - 50 cm distance from root + +## License +This is an open access protocol distributed under the terms of the [Creative Commons Attribution License](https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. + +## Citation +Andreea S (10/24/2020). Testing safety mechanism. [https://dx.doi.org/10.17504/protocols.io.bkuvkw6](https://dx.doi.org/10.17504/protocols.io.bkuvkw6) +``` + +endofoutput \ No newline at end of file diff --git a/markdown-output/testing-the-effect-of-paraquat-on-c-elegans-behavi-cgesttee.md b/markdown-output/testing-the-effect-of-paraquat-on-c-elegans-behavi-cgesttee.md new file mode 100644 index 0000000000000000000000000000000000000000..be7ec00dc956b2cfbf67cdfa84af1b3cbcb47fe9 --- /dev/null +++ b/markdown-output/testing-the-effect-of-paraquat-on-c-elegans-behavi-cgesttee.md @@ -0,0 +1,133 @@ +```markdown +Goal/Experiment: +The objective is to test the effect of paraquat on Caenorhabditis elegans behavior when cultured on Keio E. coli mutants using 6-well plates. + +# Testing the Effect of Paraquat on C. elegans Behavior When on Keio E. coli Mutants (6-well Plates) + +**Saul Moore** +Imperial College London, MRC London Institute of Medical Sciences (LMS) + +DOI: [dx.doi.org/10.17504/protocols.io.6qpvr4e6zgmk/v1](dx.doi.org/10.17504/protocols.io.6qpvr4e6zgmk/v1) + +## Abstract +This protocol details the procedure for screening candidate behavior-modifying E. coli BW25113 single-gene deletion mutants from the 'Keio Collection' to investigate their effects on Caenorhabditis elegans behavior in combination with the herbicide, paraquat dichloride. + +## Disclaimer +**FOR INFORMATIONAL PURPOSES ONLY; USE AT YOUR OWN RISK** + +## License +This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. + +## Materials + +### For Bacterial Culture +- 500 mL LB +- 50 mL Erlenmeyer flasks + +### For Worm Maintenance and Imaging Plates +- 1 L NGM agar (for ingredients, see protocol for making NGM agar) +- 60 mm Petri plates ('maintenance plates') +- 90 mm Petri plates ('nursery plates') +- 6-well flat-bottom plates ('imaging plates') +- Paraquat dichloride hydrate, PESTANAL® (analytical standard, Sigma-Aldrich, CAS: 75365-73-0) +- KIMTECH Science lint-free precision wipes + +### Safety Warnings +Paraquat dichloride is poisonous and will absorb quickly into your skin causing toxic reactions. Wear gloves and a lab coat when handling this chemical. + +## Protocol Details + +### Preparing NGM Agar + Pouring Plates +1. **Materials Preparation**: + - 6-well plates (imaging plates) + - 15 mL Falcon tubes + - 50 mL Erlenmeyer flasks + - 90 mm Petri plates (maintenance plates) + - 150 mm Petri plates (nursery plates) + +2. **NGM Agar Preparation**: + - Make 1L NGM agar following the protocol for making normal NGM for imaging plates ([Protocol Link](https://dx.doi.org/10.17504/protocols.io.6bhhbaj6)). + +3. **Pouring Plates**: + - Pour 15 mL NGM agar into each 60 mm maintenance plate, and 35 mL NGM agar into each 90 mm nursery plate. + - Keep the remaining agar warm at 65°C for pouring into 6-well imaging plates. + +4. **6-well Plates Preparation**: + - Using the Integra ViaFill, dispense 4 mL NGM agar into each well following the protocol for dispensing agar into multiwell plates ([Protocol Link](https://dx.doi.org/10.17504/protocols.io.6bhhbaj6)). + - Leave the plates to cool and solidify for approximately 1 hour. + - Measure the weight of 3 imaging plates and record the average weight. + - Dry the imaging plates under a hood until they lose 3-5% of their original plate weight. + - Store the imaging plates upside-down at 4°C until use. + +### Preparing Worms +9. **Inoculation**: + - Inoculate 10 mL LB broth with E. coli BW25113 and grow overnight ([Protocol Link](https://dx.doi.org/10.17504/protocols.io.6eahbaj6)). + +10. **Incubation**: + - Incubate at 37°C, shaking at 200 rpm. +11. **Preparation**: + - After incubation, place BW culture in a 4°C fridge until seeding. + +12. **Seeding and Synchronization**: + - Seed maintenance plates with 250 µL of BW25113 culture. + - Pick 30 N2 Bristol C. elegans onto maintenance plates and incubate at 20°C. + - After 24 hours, remove adults leaving eggs to hatch into L1 larvae. +13. **Preparation of Culture**: + - Inoculate another 10 mL LB broth with BW25113 for overnight culture. + - Seed nursery plates with approximately 1 mL of fresh BW25113 culture. +14. **Washing and Synchronous Development**: + - Wash worms into two 15 mL Falcon tubes and perform egg prep ([Protocol Link](https://dx.doi.org/10.17504/protocols.io.6bahbaj6)). + - Wash L1 larvae and re-feed onto BW-seeded nursery plates dispensing around 500 worms per plate. + - Incubate at 20°C for 68 hours. + +### Preparing Bacteria +24. **Inoculation**: + - Fill 2 Erlenmeyer flasks with 25 mL LB each. Add 50µg/mL Kanamycin to one. + +25. **Stock Plates**: + - Retrieve Keio frozen stock plates from -80°C to partially thaw. + - Inoculate the strains for antioxidant testing into the flasks ([Protocol Link](https://dx.doi.org/10.17504/protocols.io.5omhbaj6)). + +27. **Culturing**: + - Incubate overnight at 37°C, 200 rpm. + +### Seeding Imaging Plates +30. **Preparation**: + - Remove plates from 4°C storage, dry to 3-5% weight loss. + - Seed each well with 30 µL of bacterial culture. + - Dry under laminar flow hood for 20 minutes. + +33. **Growth**: + - Incubate at 25°C for 8 hours then store at 4°C until use. + +### Adding Paraquat +35. **Preparation**: + - Dry seeded plates for 30 minutes under laminar flow hood. + - Prepare 50 and 100 mM paraquat solutions. + +37. **Application**: + - Dispense 40 µL of paraquat solution to each well (0.5 and 1 mM final concentration). + - Dry plates for 30 minutes and record weight. + +### Picking Worms and Hydra Tracking +41. **Setup**: + - Ensure imaging cave air conditioning and empty dehumidifier waste tray. + - Remove worms from 20°C incubator. + +43. **Handling**: + - Pick 10 Day 1 worms into each well, incubate at 20°C before tracking. + +44. **Hydra Tracking**: + - Acclimate plates for tracking in imaging cave, ensuring removal of condensation. + - Record worm behavior using the Hydra rig for 15 minutes. + +47. **Post-Tracking**: + - Discard plates in a biological waste bin. + +48. **Saving Data**: + - Ensure all videos are saved: `'/Volumes/behavenom$/Documentation/Protocols/analysis/tracking-checklist-20210210.docx'`. + +--- + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/testo-edge-x-reviews-benefits-that-boosts-testoste-ccz9sx96.md b/markdown-output/testo-edge-x-reviews-benefits-that-boosts-testoste-ccz9sx96.md new file mode 100644 index 0000000000000000000000000000000000000000..e0152e421b6ccf8d17b40592479ba3dee9de980a --- /dev/null +++ b/markdown-output/testo-edge-x-reviews-benefits-that-boosts-testoste-ccz9sx96.md @@ -0,0 +1,107 @@ +```markdown +# Goal/Experiment: +The primary goal of the experiment is to review the benefits of Testo Edge X, a supplement purported to boost testosterone levels and increase lean muscle mass. + +# Testo Edge X: Reviews Benefits, that boosts testosterone levels, increases lean muscle mass + +## Abstract +Testo Edge X is a unique tool for improving male sexual activity and endurance. This product is an innovative development from experts. They made this dietary supplement with all the requirements of the male body and the most recent research. The huge number of different ingredients identified within the framework of such thorough research. These natural components have a particular impact on the sexual health of men. That is why manufacturers chose the best innovative ingredients to formulate considered pills. + +## Document Information +- DOI: [dx.doi.org/10.17504/protocols.io.dm6gpbkb8lzp/v1](https://dx.doi.org/10.17504/protocols.io.dm6gpbkb8lzp/v1) +- License: This is an open-access document distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. +- Created: Jul 09, 2022 +- Last Modified: Jul 09, 2022 +- Document Integer ID: 66337 + +## Abstract +### Citation: +leesopers Testo Edge X: Reviews Benefits, that boosts testosterone levels, increases lean muscle mass [DOI](https://dx.doi.org/10.17504/protocols.io.dm6gpbkb8lzp/v1) + +## Product Information +- **Name**: [Testo Edge X](www.testoedgex.com) +- **Main Benefits**: Booster Health +- **Composition**: Natural Organic Compound +- **Side-Effects**: NA +- **Rating**: ⭐⭐⭐⭐⭐ +- **Supplement Type**: Miracle Pills +- **Availability**: Online (Exclusive Offers on Official Website) +- **Where to Buy**: [www.testoedgex.com](www.testoedgex.com) + +## Introduction + +### Initial Description: +Testo Edge X is a unique tool for improving male sexual activity and endurance. This product is an innovative development from experts. They made this dietary supplement with all the requirements of the male body and the most recent research. The huge number of different ingredients identified within the framework of such thorough research. + +### Natural Components: +These natural components have a particular impact on the sexual health of men. Manufacturers chose the best innovative ingredients to formulate considered pills. Organic ingredients make Testo Edge X a safe product with a reliable and effective action. Ingredients in the formula of this capsule aim to improve the working of the male body from both external and internal perspectives. + +### Effects on External and Internal Parameters: +- **External Parameters**: + - Directed primarily at increasing muscle. + - Regular use may lead to a larger penis size and harder, more defined muscles. + +- **Internal Parameters**: + - An increase in the level of male hormones. + - Improved blood circulation in the veins of the user's body. + +### Specific Impact: +This process specifically targets the penis, resulting in significant enhancements. Testo Edge X successfully increases the level of free testosterone, improving the male physique by providing strength, muscles, and an active sex life. + +## Detailed Information and Mechanism + +### Ingredients of Testo Edge X: +Testo Edge X aims to increase the penis size and create conditions for permanent active circulation in the male body. The product development involves only qualified ingredients that have significant properties to address male potency, libido, and related issues. + +- **Ingredients**: + - **Tongkat Ali**: + - Known for enhancing libido and improving testosterone levels. + - **Saw Palmetto**: + - Supports prostate health and balances testosterone levels. + - **Sarsaparilla**: + - Contains steroids and saponins beneficial for hormonal modulation. + - **Extract Wild Yam**: + - Provides diosgenin, which may act as a natural alternative to anabolic steroids. + - **Epimedium**: + - Also known as Horny Goat Weed, supports sexual function and libido. + +### Additional Benefits: +These natural components are derived from South America, Europe, and China maximizing the efficiency of the supplement that includes certain aphrodisiac herbs. + +## Effects of Ingredients: +- **Fenugreek**: + - Enhances sexual function and libido. + - Provides antioxidants and vitamins. +- **Tribulus Terrestris**: + - Known for improving sexual desire and muscle mass growth. + +### Mechanism of Action: +- The pill provides a multi-action supplement approach to improve erectile function, increase sex hormones, and improve blood circulation. +- The formula also includes anti-oxidants that enhance muscle endurance and concentration. +- Regular usage can reduce stress levels and improve mental well-being. +- Ingredients contribute to better reproductive health and increased sperm quality. + +## Benefits of Using Testo Edge X: +1. **Increases Testosterone**: + - Enhances muscle strength and endurance. +2. **Improves Sexual Health**: + - Better erection quality and duration. +3. **Enhances Libido**: + - Supplements containing natural aphrodisiacs and testosterone boosters. +4. **Improves Overall Health**: + - Contains ingredients beneficial for general health and vitality. +5. **Safe and Natural**: + - Made entirely of herbal extracts and organic compounds. + +### Results and Recommendations: +- **Effectiveness**: Users report significant improvements in sexual health, stamina, and overall well-being. +- **Usage**: One pill per day, preferably in the morning. Overdose may lead to adverse effects. +- **Long-term Use**: Beneficial effects often persist, enhancing long-term sexual health and energy levels. + +### How to Purchase: +Testo Edge X supplements are available online. Visit the official website or trusted e-commerce platforms for purchase. + +[Order Testo Edge X For Only ($39.95/Bottle) (With 60 Days Money-Back Guarantee)](https://www.example.com) + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/tetraselmis-transformation-by-microprojectile-bomb-hjtb4nn.md b/markdown-output/tetraselmis-transformation-by-microprojectile-bomb-hjtb4nn.md new file mode 100644 index 0000000000000000000000000000000000000000..ae33ec1ad3a36d4252593c26ec0836d278e2075e --- /dev/null +++ b/markdown-output/tetraselmis-transformation-by-microprojectile-bomb-hjtb4nn.md @@ -0,0 +1,120 @@ +```markdown +# Tetraselmis transformation by microprojectile bombardment + +**Fulei Luan, Xiaoxue Wen, Patrick Beardslee and Heriberto Cerutti** + +**Published:** 04 Apr 2017 + +## Goal/Experiment: +Procedure for the transformation of the nuclear genome of *Tetraselmis striata* by microprojectile bombardment. + +## Abstract +Procedure for the transformation of the nuclear genome of _Tetraselmis striata_ by microprojectile bombardment. + +## Guidelines +The overall bombardment protocol has been modified from that used for chloroplast transformation of _Chlamydomonas reinhardtii_ (Boynton and Gillham, 1993). + +## References +1. Boynton J. E. and Gillham N. W. (1993) Chloroplast transformation in Chlamydomonas. Meth. Enzymol. 217: 510-536. +2. Guillard R. R. L. and Ryther J. H. (1962) Studies of marine planktonic diatoms. I. Cyclotella nana Hustedt and Detonula confervacea Cleve. Can. J. Microbiol. 8: 229-239. + +## Protocol + +### Step 1. Materials + +- Sterile liquid f/2 medium (permissive for the target strain, without any selective agent). + **Note:** This will be used to wash the cells by centrifugation and for cell resuspension. The concentration of the original 'f medium' (Guillard and Ryther, 1962) has been reduced by half. + +- Sterile centrifugation bottles and tubes. + +- Petri plates (10-cm diameter) of solid f/2 medium (1.5% agar). + +- Sterile deionized water. + +- Linearized plasmid DNA with an appropriate selectable marker (at 1.0 µg DNA/µL). + **Example:** _(Rps12PRO:Bar, conferring resistance to glufosinate, restriction enzyme digested overnight and purified by phenol/chloroform extraction)_ + +- 2.5 M CaCl₂, sterile + **Note:** Sterilize by filtration. + +- 0.1 M Spermine free base, sterile + **Note:** Freshly prepared and filter sterilized. + +- Macrocarriers for Helium gun, sterile. + **Note:** Sterilize by dipping in isopropanol and allowing to dry on sterile filter paper placed in a laminar flow hood. + +- Stopping screens for Helium gun, sterile. + **Note:** Sterilize by autoclaving. + +- Sterile microcentrifuge tubes and tips. + +- Sterile filter papers and forceps. + +- Recovery liquid growth medium, sterile. + **Note:** Eight-mL aliquots of f/2 medium in 50-mL capped, sterile polypropylene tubes. + +### Step 2. +Grow _Tetraselmis striata_ (UTEX B 2565 or related strain) to a density of 5 x 10⁵ cells/mL (mid-logarithmic phase) in liquid f/2 medium under continuous illumination (150 µmol m² s⁻¹ photosynthetically active radiation) on an orbital shaker (190 rpm) at 23°C and ambient levels of CO₂. Approximately 1.0 L of culture at mid-logarithmic phase is needed for 24 bombardments. + +### Step 3. +Collect cells by centrifugation in sterile centrifugation bottles at room temperature (4,500g x 5 min). Discard supernatant. + +### Step 4. +Resuspend cells in 1/30 the initial volume of f/2 medium and pool into a single sterile centrifugation tube. + +### Step 5. +Collect cells by centrifugation at room temperature (4,500g x 5 min). Discard supernatant. + +### Step 6. +Resuspend cells in 6.0 mL f/2 medium (8.5 x 10⁷ cells/mL). Count a 1/100 dilution with a hemocytometer under the microscope. Adjust the volume to obtain a concentration of 8.0 x 10⁷ cells/mL. + +### Step 7. +Plate 250 µL of cell suspension evenly on a f/2-agar plate (10-cm diameter Petri plate). Allow the liquid to dry (protect from light to avoid phototactic movements of the cells). +**Note:** This procedure yields a uniform monolayer of cells on the surface of the solid f/2 medium. + +### Step 8. +While plates are drying prepare the microprojectiles for bombardment. A 20-mg aliquot of gold particles (0.6 µm in diameter, Bio-Rad) is added to a 1.5-mL microcentrifuge tube with 200 µL absolute ethanol, vortexed for 1 to 2 min and spun in a microcentrifuge for 30 sec. The pellet is then washed twice with 300 µL sterile deionized water. The pellet is finally resuspended in 250 µL sterile deionized water. If necessary, sonicate briefly to achieve uniform resuspension. + +### Step 9. +In a sterile 1.5-mL microcentrifuge tube, add in order: +- 50 µL gold particles resuspended in water +- 5 µL transforming DNA (1.0 µg DNA/µL) +- 50 µL CaCl₂ (2.5 M) +- 20 µL Spermine free base (0.1 M) + +### Step 10. +Vortex for at least 3 min at room temperature and allow the tube to sit at room temperature for an additional 5 min, spin in a microcentrifuge for 10 sec and remove as much supernatant as possible. + +### Step 11. +Wash the pelleted particles with 250 µL of absolute ethanol, finger flick several times to resuspend, vortex briefly, and spin again. + +### Step 12. +Resuspend the pellet in 50 µL of absolute ethanol. Finger flick and vortex for 5 min to make sure to obtain uniform resuspension. +**Note:** These particles are enough for four bombardments. + +### Step 13. +In a laminar flow hood, pipet 12 µL of the resuspended particles in the center of each macrocarrier and allow to dry at room temperature. +**Note:** Make sure that particles are well resuspended, by finger flicking or brief sonication, before dispensing on the macrocarriers. + +### Step 14. +For the PDS-1000/He Particle Delivery System (Bio-Rad), dip a 1350- or 1550-psi rupture disk in isopropanol and immediately install it in the retaining cup. Place the stopping screen and the macrocarrier launch assembly in the first slot from the top of the chamber. Place the plate of target cells (without the lid) in the third or fourth slot from the top of the chamber. Close the chamber, pull a vacuum of 20 in. Hg and bombard cells as described in the operation procedure for the PDS-1000/He system. +**Note:** Negative controls are bombarded with gold particles coated with plasmid DNA lacking a selectable marker. Parameters that can be optimized for different strains include: Helium pressure (rupture disks), vacuum in the chamber, and distance from the macrocarrier holder to the target plate. + +### Step 15. +One to three hours after bombardment, resuspend the cells from each plate by adding 2.0 mL of f/2 medium and loosening the cell lawn by rubbing the surface of the agar with a glass spreader. Transfer the resuspended cells, under sterile conditions, to a tube containing 8 mL of f/2 medium and allow them to recover under dim lights for 18 h. + +### Step 16. +Add glufosinate to each recovery tube, to a final concentration of 15 µg/mL, and incubate cells for 7 days under standard culture conditions (see step 2). + +### Step 17. +Pellet cells by centrifugation, resuspend the pellet in fresh f/2 medium containing 30 µg/mL glufosinate and incubate cells for an additional 7 days under standard culture conditions. + +### Step 18. +After this selection in liquid medium, transfer cells to a sterile centrifuge tube (avoiding dead cells sticking to the walls of the tube), pellet cells by centrifugation and resuspend the pellet in 1.0 mL of f/2 medium. Spread 250 µL-aliquots of resuspended cells onto each of four selective plates (f/2 medium containing 110 µg/mL glufosinate, 1.5% agar). Seal plates with Parafilm and incubate for 4 weeks under continuous illumination (150 µmol m² s⁻¹ photosynthetically active radiation). + +### Step 19. +Transfer colonies appearing on the selective plates (individually, with a sterile toothpick) to fresh plates with higher concentration of the herbicide (f/2 medium containing 150 µg/mL glufosinate, 1.5% agar) and incubate as before. Examine incorporation of the transforming DNA, by PCR and/or Southern hybridization, in the colonies surviving the second round of selection. + +``` +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/the-black-identity-hair-product-use-and-breast-can-4a8gshw.md b/markdown-output/the-black-identity-hair-product-use-and-breast-can-4a8gshw.md new file mode 100644 index 0000000000000000000000000000000000000000..103a5fb976afd4602cf992939b38a83d8b7ccf5a --- /dev/null +++ b/markdown-output/the-black-identity-hair-product-use-and-breast-can-4a8gshw.md @@ -0,0 +1,185 @@ +```markdown +# Goal/Experiment: +To develop and validate the Black Identity, Hair Product Use, and Breast Cancer Scale (BHBS) in a diverse sample of Black women to measure social and cultural constructs associated with Black women's hair product use and perceived breast cancer risk. + +## The Black Identity, Hair Product Use, and Breast Cancer Scale + +**Authors:** +- Dede Teteh +- Marissa Ericson +- Sabine Monice +- Lenna Dawkins-Moultin +- Nasim Bahadorani +- Phyllis Clark +- Eudora Mitchell +- Lindsey S. Treviño +- Adana Llanos +- Rick Kittles +- Suzanne Montgomery + +### Affiliations: +1. City of Hope Comprehensive Cancer Center +2. University of Southern California +3. Loma Linda University +4. California State University-Northridge +5. Healthy Heritage Movement +6. Quinn Community Outreach Corporation +7. Rutgers School of Public Health and Cancer Institute of New Jersey + +### Publication: +Teteh D, Ericson M, Monice S, Dawkins-Moultin L, Bahadorani N, Clark P, Mitchell E, Treviño LS, Llanos A, Kittles R, Montgomery S (2019) The Black identity, hair product use, and breast cancer scale. PLoS ONE 14(12): e0225305. doi: [10.1371/journal.pone.0225305](https://doi.org/10.1371/journal.pone.0225305) + +### Abstract: +Across the African Diaspora, hair is synonymous with identity. As such, Black women use a variety of hair products, which often contain more endocrine-disrupting chemicals than those used by women of other races. An emerging body of research indicates a link between chemicals in hair products and breast cancer, but there is no validated instrument that measures constructs related to hair, identity, and breast health. The objective of this study was to develop and validate the Black Identity, Hair Product Use, and Breast Cancer Scale (BHBS) in a diverse sample of Black women to measure the social and cultural constructs associated with Black women's hair product use and perceived breast cancer risk. + +### Methods: +Participants completed a 27-item scale that queried perceptions of identity, hair products, and breast cancer risk. Principal Component Analyses (PCA) were conducted to establish the underlying factor structure, and confirmatory factor analysis (CFA) was used to determine model fit. + +### Results: +Participants (n = 185) were African American (73%), African, and Caribbean Black women (27%) aged 29 to 64. PCA yielded two factors that accounted for 61% of total variance. Five items measuring sociocultural perspectives about hair and identity loaded on Factor 1 and accounted for 32% of total variance (α = 0.82; 95% CI = 0.77-0.86). Six items assessing perceived breast cancer risk related to hair product use loaded on Factor 2 and accounted for 29% of total variance (α = 0.82, 95% CI = 0.74-0.86). CFA confirmed the two-factor structure (Root Mean Square Error of Approximation=0.03; Comparative Fit Index = 0.91; Tucker Lewis Index = 0.88). + +### Conclusions: +The BHBS is a valid measure of social and cultural constructs associated with Black women's hair product use and perceived breast cancer risk. This scale is useful for studies that assess cultural norms in the context of breast cancer risk for Black women. + +--- + +## Guidelines +Questions provided by type of participants: stylist, young women, women with or without a history of breast cancer. + +### Qualitative Study Focus Group and Key Informant Interview Questions + +#### 1. Participant type: Young women. + +1. **How important are hairstyles for women like you? How important is your hair to you?** + - a. What is the meaning of hair in black culture as you see it? + - i. Does that differ from what you see somewhat older women? If so, how? + - ii. How does dating fit into this? +2. **How do you most often carry your hair?** + - a. Do you change it a lot? + - i. How? +3. **What made you choose your current hairstyle?** + - a. Was it for financial reasons - explain? + - b. Trends? How you see others doing their hair + - c. Is there pressure to look a certain way—explain? +4. **If you do your own hair:** + - a. Do you feel that you know enough about hair products to style your own hair? + - i. What do you usually do? + - ii. Where do you get your information about hair styles, what products to use? + - iii. Do you feel you know enough about hair to style your own hair? +5. **What are some of the popular styles today?** + - a. How do you learn about them, including what products to use? + - b. Are they different from what your mom or older women you know have worn or wear today? +6. **If ever, how has the issue of BC come up for you in the past?** + - a. What are some of the causes of breast cancer? + - b. What are your thoughts on the link between hair and cancer? +7. **If you found out a product was harmful to your health, would you stop using it?** + - a. Why or why not? (benefits/barriers) + - b. How do you see others reacting to such changes? Men? Women? + - c. What would make you stop using a particular hair product completely? +8. **How could we reach young women like you to talk about this use?** + - a. How do you think they would react? + +*Exit questions:* Is there any information about hair or health that you would like to see be more available in the community? Do you have any additional comments or questions? + +--- + +#### 2. Participant type: Women with history of breast cancer. + +1. **When someone brings up the topic of hair, what do you think about?** +2. **What do you think are the most popular hairstyles right now?** + - a. How important is it for a woman to have a certain hairstyle? + - b. When do hairstyles matter or not matter? + - c. What do people mean when they say someone has a "natural" hairstyle? Are these popular? + - d. How much time does AA/B women spend doing their hair or getting their hair done? +3. **What do you think are some of the most common hair products that AA/B women use?** + - a. What do you think about the products that are said to be "natural"? +4. **How old were you when you first started doing your own hair?** + - a. What age were you when you first started using products in your hair? + - b. Before you did your own hair, who did it for you? + - c. What kinds of styles did they give you? What kinds of products did they use? +5. **What does it mean to have "healthy" hair?** + - a. What does the general term of "health" mean to you? + - b. What does it mean to have "good" or "bad" health? + - c. What do you think are some of the causes of good or bad health? +6. **What has been your experience with breast cancer?** + - a. Testing, diagnosis, treatment +7. **Have you ever heard that hair products or ingredients in beauty products might be harmful to people's health?** + - a. If so, what health problems and what hair products + - b. Have you heard they might affect a woman's risk of breast cancer? +8. **If you found out certain ingredients in hair products were harmful to your health, do you think you would avoid using these products?** + - a. What if they were found in a product that you used often? +9. **Do you think that if most women found out that certain ingredients in hair products were harmful, that they would stop using those products?** + - a. Would they share this information with other women they know? +10. **If we found out that some hair products or ingredients are harmful to women's health, what do you think would be a good way to get the word out?** + +*Exit questions:* Is there any information about hair or health that you would like to see be more available in the community? Do you have any additional comments or questions? + +--- + +#### 3. Participant type: Women without a known history of breast cancer. + +1. **When someone brings up the topic of hair, what do you think about?** +2. **What do you think are the most popular hairstyles right now?** + - a. How important is it for a woman to have a certain hairstyle? + - b. When do hairstyles matter or not matter? + - c. What do people mean when they say someone has a "natural" hairstyle? Are these popular? + - d. How much time does AA/B women spend doing their hair or getting their hair done? +3. **What do you think are some of the most common hair products that AA/B women use?** + - a. On a regular basis + - b. Experiment with + - c. How do you decide what products to use? +4. **How old were you when you first started doing your own hair?** + - a. What age were you when you first started using products in your hair? + - b. Before you did your own hair, who did it for you? + - c. What kinds of styles did they give you? What kinds of products did they use? +5. **What does it mean to have "healthy" hair?** + - a. What does the general term "health" mean to you? + - b. What do you think are some of the causes of good or bad health? +6. **What do you know about breast cancer?** + - a. As a disease + - b. In your community? What concerns have you heard? + - c. Have you ever done anything regarding breast cancer for yourself or others? +7. **Have you ever heard that hair products or ingredients in beauty products might be harmful to people's health?** + - a. If so, what health problems and what hair products + - b. Have you heard they might affect a woman's risk of breast cancer? +8. **If you found out certain ingredients in hair products were harmful to your health, do you think you would avoid using these products?** + - a. What if they were found in a product that you used often? +9. **Do you think that if most women found out that certain ingredients in hair products were harmful, that they would stop using those products?** + - a. Would they share this information with other women they know? +10. **If we found out that some hair products or ingredients are harmful to women's health, what do you think would be a good way to get the word out?** + +*Exit questions:* Is there any information about hair or health that you would like to see be more available in the community? Do you have any additional comments or questions? + +--- + +## Glossary of Terms and Reagents + +### Endocrine-Disrupting Chemicals (EDCs) +- **Definition:** Chemicals that can interfere with endocrine (or hormonal) systems. +- **Function:** They may cause cancerous tumors, birth defects, and other developmental disorders. + +### Principal Component Analysis (PCA) +- **Definition:** A statistical procedure that uses orthogonal transformation to convert observations of possibly correlated variables into a set of values of linearly uncorrelated variables called principal components. +- **Function:** Used to reduce the dimensionality of a data set while retaining most of the variation in the data. + +### Confirmatory Factor Analysis (CFA) +- **Definition:** A type of structural equation modeling used to test if measurements of a construct are consistent with a researcher's understanding of the nature of that construct (or factor). +- **Function:** Used to test the validity of measurements in research. + +## Alternative Methods/Supplies + +Some products or reagents might be difficult to find or expensive. Here are some alternatives: + +1. **Hair Product Usage:** + - Look for natural and organic alternatives that do not contain harmful chemicals. Examples include products with coconut oil, shea butter, and aloe vera. + +2. **Statistical Software:** + - If access to high-end statistical software for PCA and CFA is limited, consider using open-source alternatives such as R or Python packages like `FactoMineR` and `Lavaan`. + +3. **Sensitive Self-report Scales:** + - While developing new scales is essential, validated and widely used existing scales can be adapted to measure the constructs relevant to the study population. + +--- + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/the-monocyte-derived-dendritic-cell-modc-assay-an-cp5avq2e.md b/markdown-output/the-monocyte-derived-dendritic-cell-modc-assay-an-cp5avq2e.md new file mode 100644 index 0000000000000000000000000000000000000000..fc0070644e69c826a2e2dba0697cda63cbe28274 --- /dev/null +++ b/markdown-output/the-monocyte-derived-dendritic-cell-modc-assay-an-cp5avq2e.md @@ -0,0 +1,131 @@ +```markdown +# Goal/Experiment: +To develop and optimize an in vitro assay for testing the immunogenicity of potential regenerative medicine cellular therapies by employing the monocyte-derived dendritic cell (MoDC) assay. + +# The Monocyte-Derived Dendritic Cell (MoDC) Assay – An In Vitro Assay for Testing the Immunogenicity of Cellular Therapies V.2 + +**Authors**: +- Annabel J Curle +- Sarah Howlett +- Joanne Jones + +**Affiliation**: +University of Cambridge + +**Version**: 2 +**Date**: Feb 27, 2023 + +**Abstract**: +The monocyte-derived dendritic cell (MoDC) assay can be used to test, in vitro, the immunogenicity of potential regenerative medicine cellular therapies. Similar to a mixed lymphocyte reaction (MLR), cells of interest (COI) are co-cultured with T cells, but with the addition of induced professional antigen-presenting cells (APCs; moDCs) to ensure optimal T cell activation and to improve the limited sensitivity of the MLR-like assay. + +Autologous CD14+ monocytes and T cells are isolated from PBMCs. Monocytes are differentiated to mature dendritic cells over 7 days, then co-cultured with T cells and cells of interest for 5 days. Recommended readouts of the assay are ELISA (for IFNγ/TNFα release) and flow cytometry (for T cell proliferation and CD25 expression). + +Positive control wells of allogeneic moDC/T cells and bead-activated T cells are utilized. Biological replicates can be run in parallel. Well-well variation is minimal, but samples can be run in technical duplicates if desired. + +--- + +## Materials + +### Cell Preparations: +- PBMC isolation materials +- MACS equipment and LS tubes +- CD14+ microbeads for MACS (Miltenyi CD14+ microbeads) +- PanT cell isolation kit for MACS (Miltenyi PanT cell isolation kit) +- 96 well tissue culture plates +- Cell proliferation dye (eBioscience™ Cell Proliferation Dye eFluor™ 450) +- Cell freezing media (FBS + 10% DMSO) + +### moDC Differentiation: +- RPMI +- Penicillin/streptomycin +- Human AB serum +- rhIL-4 +- rhGM-CSF +- LPS +- PBS + +### Co-Culture: +- Cell of interest media +- T cell activation beads (Gibco Dynabeads™ Human T-Activator CD3/CD28 for T Cell Expansion and Activation) + +### Readouts: +- ELISA materials +- Flow cytometry materials + +--- + +## Before Start Instructions +- Fresh blood must be used each time as monocytes do not survive the freeze/thaw process +- Optimization experiments must be performed to ensure all cell types survive without compromise to survival, proliferation, and differentiation state in the 1:1 mix of medias (T cell/moDC media: cell of interest media) + +--- + +## Protocol + +### Cell Preparations and moDC Differentiation - Day 0-7 + +1. **Make PBMCs using the preferred method from whole blood** +2. **Take whole PBMCs and enrich for CD14+ cells using magnetic-activated cell sorting (MACS) with CD14+ microbeads** +3. **Count monocytes, then seed in a 96 well plate at 150,000 cells/well in 200 µl RPMI + 10% P/S + 5% human AB serum (HAB) + 50 ng/ml rhIL-4 and 50 ng/ml rhGM-CSF (day 0)** + Incubate in 37°C incubator for 7 days refreshing half media on day 3 and 6. + - **Media Refresh (Day 3 & 6)**: Each media refresh should be done with double concentration IL-4 and GM-CSF to account for the half media change. + - Remove 100 µl media, add 100 µl RPMI + 10% P/S + 5% HAB + 100 ng/ml rhIL-4 and 100 ng/ml rhGM-CSF + - **Day 6 media change with activation**: Remove 100 µl media, add 100 µl RPMI + 10% P/S + 5% HAB + 100 ng/ml rhIL-4 and 100 ng/ml rhGM-CSF + 200 ng/ml LPS + 1. **Optional**: + - **Collect monocytes (day 0)**, immature moDCs (day 6), and mature moDCs (day 7) to check purity of MACS enrichment by flow cytometry: + - **Monocytes**: CD14+ + - **Immature DCs**: CD14+, CD80+, CD86+, HLA-ABC+, HLA-DR+ + - **Mature DCs**: CD14+, CD80+, CD86+, HLA-ABC+, HLA-DR+, CD83+ +4. **Using the CD14- population, enrich for T cells using MACS with a PanT negative selection kit** + 1. **If specific T cell population is required, perform MACS or FACS (fluorescence-activated cell sorting) for the required population** + 1. **Optional**: Collect PanT to check the purity of MACS enrichment by flow cytometry. +5. **Stain T cells with proliferation dye as per preferred protocol** +6. **Count then freeze the stained-T cells in FBS + 10% DMSO for the duration of the monocyte-DC differentiation** +7. **Prepare your "cells of interest" (COI) ready to have enough for 150,000 cells per well on day 7** + +--- + +### Seeding the Immunogenicity Assay - Day 7 (Assay Day 0) + +8. **Thaw proliferation dyed-T cells as per preferred protocol** +9. **Remove all media from moDCs and wash once with PBS (moDCs are adherent but aspirate gently to avoid disturbing cells)** +10. **Experimental wells**: + - **Seed 150,000 autologous T cells per well** in 150 µl RPMI + 10% P/S + 5% HAB + - **Seed 150,000 COI per well** with 150 µl media specific to cells of interest (+ ROCKi if required) + - Final: 1:1:1 ratio of moDC:T cell:COI in 300 µl of 50:50 media (RPMI: COI media) + 1. **Note**: Optimization will be required to ensure both cell types survive and proliferate as expected in the 1:1 media mix. +11. **Positive control wells**: + - **Allogeneic T cell:moDC**: + - Seed 150,000 allogeneic T cells into moDC wells in 150 µl RPMI + 10% P/S + 5% HAB + - Add 150 µl media specific to cells of interest + - **Polyclonally activated T cells**: + - Seed 150,000 T cells into an empty well (no moDC) in 150 µl RPMI + 10% P/S + 5% HAB + - Add activation beads at recommended ratio in 150 µl media specific to cells of interest + +--- + +### Immunogenicity Assay - Day 7-12 (Assay Day 0-5) + +12. **Incubate co-culture for 5 days**, refreshing half media every 48 hours (day 2 and day 4). + - Each change: Remove 150 µl media and add 150 µl prepared 1:1 media + +--- + +### Readouts - Day 12 (Assay Day 5) + +13. **For cytokine assays**: + - **Collect 150 µl supernatant per well** into a V-bottom 96 well plate + 1. Centrifuge supernatants at 1500 x g for 10 minutes then re-collect supernatants avoiding any pellet/debris that have collected. + 2. Freeze supernatants at -80°C until use. + - Perform ELISA or luminex cytokine assays including IFNγ and TNFα as readouts of immunogenicity. +14. **For flow cytometry**: + - **Collect remaining supernatants** containing non-adherent T cells by agitating gently, washing the well 1-2 times with the supernatant. + - **Note**: moDCs are adherent so will not be collected, consider that COI may be adherent or non-adherent (it is okay if some are collected as they will be gated out during flow cytometry). + 1. Perform flow cytometry immediately, including a minimum panel of: CD3, CD4, and CD8 (T cell markers), CD25 (activated T cell marker), and live/dead stain. + - **Note**: Cells are already pre-stained with proliferation dye. + - Activated T cells (CD25+, proliferation dye low/negative) are used as a readout of immunogenicity of COI + +--- + +**End of Output** +``` \ No newline at end of file diff --git a/markdown-output/time-course-live-imaging-of-maize-and-sorghum-prot-cf2jtqcn.md b/markdown-output/time-course-live-imaging-of-maize-and-sorghum-prot-cf2jtqcn.md new file mode 100644 index 0000000000000000000000000000000000000000..c3a7d82000610149cf8cb3fdcaa09ef8075f27f7 --- /dev/null +++ b/markdown-output/time-course-live-imaging-of-maize-and-sorghum-prot-cf2jtqcn.md @@ -0,0 +1,219 @@ +```markdown +# Goal/Experiment: +The primary goal of this experiment is to perform time-course live imaging of maize and sorghum protoplasts. Through the isolation, transfection, and immobilization of protoplasts, the time-course live imaging provides insights into cellular processes like gene expression, cell wall regeneration, and dynamic cellular responses to different stimuli. This protocol aims to streamline protoplast handling and imaging with clear methods, reagents, and troubleshooting notes. + +# Time-course Live Imaging of Maize and Sorghum Protoplasts + +*Rachel Baschieri, Thai Dao, Samuel Leiboff - Oregon State University* + +DOI: [dx.doi.org/10.17504/protocols.io.4r3l27me3g1y/v1](https://dx.doi.org/10.17504/protocols.io.4r3l27me3g1y/v1) + +## Abstract +A protoplast is a living plant, fungal, or bacterial cell with the cell wall removed. Protoplasts offer a simplified system for studies of gene expression compared to more complex whole-plant systems and are promising for the interrogation of cellular physiology, metabolism, and responses to stimuli. Performing live imaging of protoplast experiments in a time-course experiment provides more information about how these cellular processes progress over time. Protoplast time-course live imaging studies have previously explored intracellular auxin localization, auxin's effect on gene expression, and other cellular processes like cell wall regeneration and cell division. + +Though time-course live imaging of protoplasts is a useful tool, it is also a skill that can be difficult to acquire. Practice and troubleshooting of various protoplast isolation, transfection, and immobilization protocols can be time-consuming and may deter many from performing experiments with protoplasts. This protocol combines protoplast isolation, transfection, and immobilization methods to form a cohesive protoplast time-course live imaging protocol with notes and modifications from our troubleshooting. + +This protocol was developed for time-course live imaging of maize and sorghum protoplasts. The isolation and transfection steps are based on methods published with a few modifications, including the addition of MES buffer to PEG-calcium transfection solution, bovine serum albumin (BSA) to the incubation solution, and ampicillin to decrease bacterial growth. The alginate immobilization steps were also adapted from existing methods. + +## Keywords +- Time-course live imaging +- Protoplast +- Maize +- Sorghum +- Plant transfection + +## Materials and Reagents + +### Enzyme Solution - pH 5.7 +| Stock | Amount for 10mL | +|-----------------------------|-----------------| +| 0.1M KCL | 1ml | +| 0.1M MES | 800 uL | +| 1M CaCl2 | 10 uL | +| 1M mannitol | 5 ml | +| Sterile ddH2O | 3.19 ml | +| Cellulase RS | 0.06 g | +| Maceroenzyme R-10 | 0.01 g | +| BSA | 0.01 g | +| Polyvinylpyrrolidone K30 | 0.01 g | + +### W5 Solution - pH 5.7 +| Stock | 15mL | 20mL | 30mL | +|----------------|-------|-------|-------| +| NaCl | 0.134 g | 0.180 g | 0.270 g | +| 1M CaCl2 | 1.875 mL | 2.5 mL | 3.75 mL | +| 0.1M KCl | 750 uL | 1 mL | 1.5 mL | +| 0.1M MES | 300 uL | 400 uL | 600 uL | +| Sterile ddH2O | 12.075 mL | 16.1 mL | 24.15 mL | + +### Suspension Solution - pH 5.7 +| Stock | 1 mL | 5 mL | 15 mL | +|----------------|---------|---------|-------| +| 1M mannitol | 400 uL | 2 mL | 6 mL | +| 1M CaCl2 | 20 uL | 100 uL | 300 uL| +| 0.1M MES | 50 uL | 250 uL | 750 uL| +| Sterile ddH2O | 530 uL | 2.65 mL | 7.95 mL| + +### PEG Solution - pH 5.7 +| Stock | 1 mL | 3 mL | +|-----------------|---------|-----------| +| 0.1M MES | 500 uL | 1.5 mL | +| 1M CaCl2 | 100 uL | 300 uL | +| 1M mannitol | 400 uL | 1.2 mL | +| PEG 4000 | 0.4 g | 1.2 g | + +### Incubation Solution - pH 5.7 +| Stock | 3 mL | 6 mL | 10 mL | 15 mL | +|-----------------|--------|-------|-------|--------| +| 1M mannitol | 1.5 mL | 3 mL | 5 mL | 7.5 mL | +| 0.1M KCl | 120 uL | 240 uL| 400 uL| 600 uL | +| 0.1M MES | 120 uL | 240 uL| 400 uL| 600 uL | +| Sterile ddH2O | 1.26 mL| 2.52 mL| 4.2 mL| 6.3 mL | +| BSA | 0.03 g | 0.06 g| 0.1 g |0.15 g | +| Ampicillin (100 mg/mL) | 1.5 uL | 3 uL | 5 uL | 7.5 uL| + +### MMM - pH 5.7 +| Reagent | Amount to Make 100 mL | +|-----------------------------|------------------------| +| MgCl2 Hexahydrate | 102 mg | +| MgSO4 Heptahydrate | 125 mg | +| Mannitol | 8.5 g | +| MES | 195.2 mg | + +### Alg-A - pH 5.7 +| Reagent | Amount to Make 100 mL | +|-----------------------------|------------------------| +| MgCl2 Hexahydrate | 102 mg | +| MgSO4 Heptahydrate | 125 mg | +| Mannitol | 8.5 g | +| MES | 195.2 mg | +| Alginic acid | 1.2 g | + +### 2M NH4-Succinate +| Reagent | Amount to Make 200 mL | +|---------------|------------------------| +| NH4Cl | 21.2 g | +| KOH | 44.8 g | +| Succinic acid | 47.2 g | + +### FPCN - pH 5.7 +| Reagent | Amount to Make 50 mL | +|----------------------------|--------------------------| +| KNO3 | 0.0506 g | +| CaCl2 dihydrate | 0.032 g | +| MgSO4 Heptahydrate | 0.0185 g | +| KH2PO4 | 0.0085 g | +| MS | 0.2202 g | +| NH4-succinate | 500 uL | +| Inositol | 0.01 g | +| Sucrose | 1 g | +| Glucose | 4 g | +| MES | 0.0976 g | +| 10 mM Biotin | 0.2 uL | +| 100 mM Thiamine-HCl | 1.5 uL | +| 100 mM Nicotinic Acid | 8 uL | + +## Day 1 - Protoplast Isolation and Transfection + +1. **Prepare enzyme solution.** + +2. **Harvest 10-14 day old seedlings and submerge in 5% bleach 1% Tween solution for 1 min** + - Remove and rinse seedlings thoroughly in autoclaved tap water 6x. + - Pat seedlings dry with autoclaved Kimwipes. + +3. **Fill a glass petri dish with the filter sterilized enzyme solution, and set aside.** + +4. **Cut green leaf tissue into \~1 mm strips** + - Transfer immediately to the glass petri dish with enzyme solution. + +5. **Incubate 4 hours in dark at RT with 40 RPM shaking.** + +6. **Prepare W5 solution, suspension solution, PEG solution, and incubation solution.** + +7. **Add 10 mL W5 solution** + - Gently swirl to mix, and shake an additional 1 hour at 80 RPM. + +8. **Filter the W5 and enzyme solution mixture** through 100 or 70 um mesh into 50 mL tube. + +9. **Centrifuge at 1000-1200 RPM for 5 min** to collect protoplasts. + +10. **Use a pipette to remove the supernatant** + - Resuspend the protoplast pellet in 600 uL suspension solution (1 mL for maize). + +11. **Use 9 uL to count with a hemocytometer.** + +### Fluorescein Diacetate (FDA) Viability Stain +- *FDA stain to check viability of protoplasts:* + - 81.2 uL 1M mannitol + - 43.6 uL diH2O + - 2.5 uL FDA + +**Mix 10 uL protoplast suspension with 5 uL FDA stain, incubate RT 2 min, load on hemocytometer.** + +12. **Reduce protoplast concentration to \~2-4 x 10^5 cells/mL for transfection.** + +13. **In a 1.5 mL tube add 20-40 ug plasmid DNA and 200 uL of protoplast suspension.** + - Let sit RT 5 min. + +14. **Add 220 uL PEG solution**, mix immediately by gentle tube inversion. + - Incubate 15 min at 28°C. + +15. **Quickly add 800 uL W5 solution** + - Gently invert the tube 2x. + +16. **Collect protoplasts by centrifuge 3 min at 1000 RPM.** + - Remove PEG and W5 supernatant. + +17. **Suspend collected protoplasts in 1 mL incubation buffer** + - Incubate in dark at 28°C overnight with gentle shaking (\~20 RPM). + +18. **Ensure MMM, Alg-A, and FPCN solutions are prepared for Day 2.** + - MMM and Alg-A solutions may be stored at 4°C for 6 months or longer. + - FPCN may be stored at 20°C for 6 months or longer. + +## Day 2 - Protoplast Immobilization in Well-Plate + +19. **Coat the bottom surface of the slide-well with 50 ug/mL Poly-L-lysine.** + - Sit for 30 min-1 hour, remove Poly-L-lysine, allow to air-dry. + +20. **Centrifuge transformed protoplasts after overnight incubation at 1000 RPM for 3 min.** + +21. **Remove the incubation buffer supernatant.** + - Resuspend the protoplast pellet in 100 uL MMM and 100 uL Alg-A solution. Mix gently. + +22. **Pipette a large droplet of protoplast solution onto Poly-L-lysine coated slide well.** + - 8-well chamber, 50 uL protoplast droplet in each well. + +23. **Allow the alginate droplet to sit for 10-15 minutes at RT** + - Protoplasts settle to the surface. + +24. **Gently pipette 0.5-1 uL droplets of W5 solution** + - Coat the surface of the droplet with W5. + +25. **Allow W5 coated protoplast-alginate droplet to sit for 15 minutes RT.** + +26. **Pipette equivalent volume of W5 into the well containing the droplet.** + - Incubate for 45 min. + +27. **Remove W5 and incubate droplet in FPCN for 15 min.** + - This is wash 1. + +28. **Remove FPCN and replace with fresh FPCN, incubate 15 min.** + - This is wash 2. + +29. **Remove FPCN and incubate with final FPCN or treatment solution.** + - The alginate-embedded protoplasts are ready to be imaged. + +## References +1. Middleton, A. M., et al. (2018). Data-driven modeling of intracellular auxin fluxes... +2. Maurel, C., et al. (1994). Alterations of auxin perception in rolB-transformed... +3. Wu, S. & Gallagher, K. L. (2014). The movement of the non-cell-autonomous... +4. Kuki, H., et al. (2017). Quantitative confocal imaging method for analyzing... +5. Xu, M., et al. (2021). Stochastic gene expression drives mesophyll protoplast regeneration... +6. Meng, R., et al. (2020). An efficient sorghum protoplast assay... +7. Ohshima, M. & Toyama, S. (1989). Studies on Culture... +8. Yoo, S. D. Cho, Y. H., & Sheen, J. (2007). Arabidopsis mesophyll protoplasts... +9. Dovzhenko, A., et al. (1998). Thin-alginate-layer technique for protoplast culture of tobacco... + +__endofoutput__ +``` \ No newline at end of file diff --git a/markdown-output/time-resolved-fret-in-384-well-plate-format-to-ide-bsw8nfhw.md b/markdown-output/time-resolved-fret-in-384-well-plate-format-to-ide-bsw8nfhw.md new file mode 100644 index 0000000000000000000000000000000000000000..58449f11c2ff074aef08e55a72ef338fb4bed660 --- /dev/null +++ b/markdown-output/time-resolved-fret-in-384-well-plate-format-to-ide-bsw8nfhw.md @@ -0,0 +1,130 @@ +```markdown +# Goal/Experiment: +To identify small molecular modifiers of mutant Huntingtin (mHTT) conformational inflexibility using Time-resolved FRET (TR-FRET) in a 384-well plate format. + +# Time-resolved FRET in 384-well Plate Format to Identify Small Molecular Modifiers of Mutant Huntingtin Conformational Inflexibility + +**Johannes H Wilbertz¹, Julia Frappier¹, Barbara Calamini¹** + +¹Sanofi Strasbourg R&D Center + +DOI: [10.17504/protocols.io.bsw8nfhw](https://dx.doi.org/10.17504/protocols.io.bsw8nfhw) + +## Abstract + +### Background +Huntingtin (HTT), if mutated, is the key driver of the neurodegenerative Huntington's disease (HD). Wild-type HTT (wtHTT) and excessive poly-glutamine containing mutant HTT (mHTT) differ in the intramolecular flexibility of their N-terminal region. The loss of flexibility and conformational change in mHTT is thought to drive protein aggregation and decrease protein-protein interactions with other critical neuronal proteins. Decreased conformational flexibility of mHTT likely results in neurodegeneration. It has been demonstrated that post-translational modifications (PTMs), such as N-terminal amino acid phosphorylation, can rescue mHTT inflexibility. + +### Methodology +Time-resolved FRET (TR-FRET) using the two HTT N-terminus-specific antibodies 2B7 (donor, labeled with terbium cryptate (Tb)) and MW1 (acceptor, labeled with D2) can detect intramolecular conformational changes of HTT. Different temperature stimuli can be used to measure changes: flexible wtHTT shows a high degree of flexibility and low FRET signal at 20°C, while at 4°C, intramolecular flexibility is reduced and the FRET signal increases. TR-FRET helps in screening small molecules for their effects on mHTT conformational rescue. + +### Anticipated Outcome +Compounds that restore mHTT conformational flexibility should lead to a 4°C/20°C FRET signal ratio of > 1. This effect could be due to the direct binding of a small molecule to mHTT or via indirect influences on PTMs, such as stimulation of a kinase or inhibition of a phosphatase. + +## Materials + +- GM04691 fibroblasts (affected donor, passage #10), provided by Evotec, Storage: Liquid nitrogen +- GM04729 fibroblasts (healthy donor, passage #9), provided by Evotec, Storage: Liquid nitrogen +- 384-well assay plate, white, flat bottom, cell culture treated, Greiner 781080, Lot: E19043HU +- DMEM + Glutamax, Gibco 105566-016, Lot: 2026753, Storage: +4°C +- FBS, Gibco 16000-044, Lot: Z, Storage: -20°C +- Pen/Strep 100X, Gibco 15140-122, Lot: 209152, Storage: -20°C, Stock: 10000 U/ml +- PBS (-), Gibco 14190-094, Lot: 2026787, Storage: +4°C +- Trypsin 0.25%, Gibco 25200-056, Lot: 2029691, Storage: +4°C +- Trizma base, Sigma T1503, Lot: SCBX6926, Storage: +4°C +- Trizma HCl, Sigma T3253, Lot: SCBX6980, Storage: +4°C +- Igepal (CA-630), Sigma I8896-50ML, Lot: MKCC9036, Storage: +4°C +- Bovine Serum Albumine (BSA), Sigma A7030-500G, Lot: SCBS2014V, Storage: +4°C +- Protease & phosphatase inhibitor cocktail, Thermo Scientific 1861281, Lot: TH271623A, Storage: +4°C +- DMSO, Sigma D5879-500ML, Lot: 05K61073, Storage: RT +- Antibody MW1, DSHB-AB_528290, Lot: 10ea9/1/17-1mg/ml, Storage: -20°C, purified and labelled with D2 by Cisbio, Lot: 190329, Storage: -80°C +- Antibody 2B7-Tb, CHDI Foundation, purified by Sanofi Biologicals, purified and labelled with Terbium cryptate by Cisbio, Lot: 190513, Storage: -80°C +- Compound library of choice, ideally pre-diluted in DMSO. This protocol has been optimized for screening up to 4,000 compounds. + +## Protocol + +### 1. Seed Cells +1. Seed 10,000 cells/well (50 μl, GM04691 fibroblasts) in DMEM + 15% FBS + PenStrep in Greiner 384-well plates according to the anticipated plating scheme. Do not seed cells in the left-most column 1. Later, this column will serve to calculate the FRET background signal. When seeding multiple plates, use a Multidrop cell dispenser and centrifuge plates briefly to ensure all liquid is at the bottom of the well. + +### 1.1 Reagent Preparation - Growth Medium +| Reagent | Volume | Final Concentration | +| ------- | ------ | ------------------- | +| DMEM+Glutamax | 500 ml | N/A | +| FBS (100%) | 89 ml | 15% | +| Penicillin/Streptomycin 100X | 5.9 ml | 1X | + +### 2. Incubate Plates +1. Incubate plates for 24 hours at 37°C and 5% CO₂. + +### 3. Treat Cells +1. If possible, use robotic liquid handling for the next steps. Treat cells with compounds or DMSO for 24 hours (3 μM of each library compound, 3 μl per well). 3 plates were treated with DMSO only and 6 plates treated with library compounds in duplicate. + +### 4. Aspirate Medium +1. Aspirate 40 μl medium (rest: 10 μl). + +### 5. Lyse Cells +1. Lyse cells with 10 μl TR-FRET lysis buffer (2X concentrated). + +### 5.1 Reagent Preparation - TR-FRET Lysis Buffer (100 ml total) +| Reagent | Volume | Final Concentration | +| ------- | ------ | ------------------- | +| 100mM Tris, pH7.4 | 10 ml | 10 mM | +| 5M NaCl | 3 ml | 150 mM | +| H₂O | 85 ml | N/A | +| Protease/phosphatase inhibitor | 2 ml (add just before use) | 2% | +| Igepal | 0.6 ml (add just before use) | 0.6% | + +### 6. Incubate Lysis +1. Incubate plates for 1 hour at room temperature. + +### 7. Add Antibodies +1. Add 10 μl of 2B7-Tb/MW1-D2 in TR-FRET antibody buffer (3X concentrated) to reach the desired concentration in the wells. See the antibody dilution table for details. + +### 7.1 Reagent Preparation - TR-FRET Antibody Buffer (65 ml total) +| Reagent | Volume | Final Concentration | +| ------- | ------ | ------------------- | +| 100mM Tris, pH7.4 | 6.5 ml | 10 mM | +| 5M NaCl | 1.95 ml | 150 mM | +| H₂O | 56.55 ml | N/A | +| Igepal | 0.195 ml (add just before use) | 0.25% | +| BSA (add just before use, prepared as 10X for ease of use) | 0.065 g (add just before use) | 0.1% | +| Antibodies | Add just before use (see table below) | + +### 7.2 Reagent Preparation - Dilution of Antibodies in TR-FRET Antibody Buffer +| Antibody | Stock Antibody Concentration (ng/ul) | Desired Antibody Concentration (3X) (ng/ul) | Total Required Volume (ul) | Amount of Stock to Add to Required Volume (ul) | Obtained Concentration | Final Concentration in Well (10ul + 20ul) | +| -------- | ----------------------------------- | ----------------------------------------- | ------------------------- | --------------------------------------------- | ----------------------- | ----------------------------------------- | +| 2B7-Tb | 245 | 0.075 | 65,000 | 19.90 | 3X | 1X | +| MW1-D2 | 753 | 0.75 | 65,000 | 64.74 | 3X | 1X | + +### 8. Incubate Overnight +1. To prevent evaporation, seal plates with Thermoplast adherent foil and incubate at 20°C overnight for 16 hours. Ensure the temperature does not exceed 22°C during incubation. + +### 9. Read Plates +1. Read plates on a PheraStar plate reader (200 flashes, 60 – 460 μs integration window, focal height: 8.9 mm). Export the data as a .csv file. + +### 10. Incubate at 4°C +1. Incubate plates for 2 hours at 4°C. + +### 11. Repeat Readings +1. Repeat the reading on the PheraStar plate reader using identical settings. Export the data as a .csv file. + +### 12. Data Analysis - Step 1 +1. Aggregate the multiple results files (one for each temperature per plate). The provided KNIME workflow can be used to perform this task. + +### 13. Data Analysis - Step 2 +1. Calculate the 665nm/620nm ratio (FRET efficiency) for both temperatures (4°C & 20°C) for all individual wells. + +### 14. Data Analysis - Step 3 +1. Calculate the mean 4°C/20°C ratio for all cells which did not contain cells to determine the background FRET signal. + +### 15. Data Analysis - Step 4 +1. Subtract the mean 4°C/20°C background FRET signal from all DMSO- and compound-containing wells. + +### 16. Data Analysis - Step 5 +1. Identify active compounds: For all DMSO-only containing wells, calculate the median and standard deviation of the 4°C/20°C background-corrected FRET signal. Active compounds should have a 4°C/20°C background-corrected FRET signal higher than the DMSO median + three times the DMSO standard deviation. + +### 17. Repeat Screening +1. Ideally, the screen is repeated on a different day. Compounds that are identified twice as active compound are considered as hit compounds. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/tissue-procurement-normal-colon-6y9hfz6.md b/markdown-output/tissue-procurement-normal-colon-6y9hfz6.md new file mode 100644 index 0000000000000000000000000000000000000000..495015302f8fb0b827617acf2e0cface961a90cf --- /dev/null +++ b/markdown-output/tissue-procurement-normal-colon-6y9hfz6.md @@ -0,0 +1,95 @@ +```markdown +# Goal/Experiment: +The goal of this experiment is to procure and preserve normal colon tissue. The tissue is to be collected from a surgical pathology laboratory to ensure a quality specimen that can be used for diagnostic purposes and research. Various preservation methods, including paraffin blocks and snap-frozen samples, are to be applied. + +# Tissue Procurement: Normal Colon + +**Authors:** +- Kerry Wiles1 +- Madison Tyler + +1Cooperative Human Tissue Network Western Division at Vanderbilt University Medical Center + +**DOI:** [dx.doi.org/10.17504/protocols.io.6y9hfz6](dx.doi.org/10.17504/protocols.io.6y9hfz6) +**Protocol Citation:** +Kerry Wiles 2021. Tissue Procurement: Normal Colon. *protocols.io*. [https://dx.doi.org/10.17504/protocols.io.6y9hfz6](https://dx.doi.org/10.17504/protocols.io.6y9hfz6) + +**License:** +This protocol is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. + +**Created:** Aug 30, 2019 +**Last Modified:** Feb 18, 2021 +**Protocol Integer ID:** 27939 + +## Abstract +Tissues are procured from a variety of different anatomic sites, each with their unique qualities to be considered during collection. Anatomic site-specific standard operating procedures (SOPs) are required to address these unique qualities and assist with investigator-specific requests. This protocol for the collection of normal (NL) colon applies for normal adjacent tissue (NAT) in cancerous colons and also for histologically normal diverticulitis colon tissue. + +## Before Starting +**Reference to other CHTN SOPs or Policies:** +- Tissue Procurement: Cryopreservation with OCT Compound ([dx.doi.org/10.17504/protocols.io.6y7hfnz](dx.doi.org/10.17504/protocols.io.6y7hfnz)) +- Tissue Procurement: Fixation with 10% NBF ([dx.doi.org/10.17504/protocols.io.6y4hfwy](dx.doi.org/10.17504/protocols.io.6y4hfwy)) + +## Procedure + +1. **Identify the tissue to be collected** in the surgical pathology laboratory. Note that this protocol for the collection of normal (NL) colon applies for normal adjacent tissue (NAT) in cancerous colons and also for histologically normal diverticulitis colon tissue. + +2. **Prepare the colon**: + - The length of the colon should be opened and properly cleaned of withheld matter by a resident pathologist or PA before collection of tissue. + - Note that colon specimens are often very large and often require relatively small amounts of tissue for diagnostic purposes. + + ![Norma Colon in Surgical Pathology](link_to_image1) + *Figure 1. Normal colon in surgical pathology. Omental adipose tissue (top) surrounds mucosal surface (overlying the muscularis).* + +3. **Section the colon**: + - The section of colon taken should exhibit both the mucosa and the muscularis. + - Should be large enough to yield tissue for all desired methods of collection (snap-frozen in liquid nitrogen, frozen in OCT, fixation in paraffin, fresh shipping, etc.) + - Should be free of significant necrosis. + - If NAT, it should be free of all non-normal tissue. + + ![Tissue Section Given to CHTN](link_to_image2) + *Figure 2. Tissue section given to CHTN.* + +4. **Transport the tissue**: + - Once the tissue has been procured and transported to the CHTN laboratory, it can then be prepared for preservation. + - Colon samples will often have visible fat attached, which should be (unless otherwise requested) trimmed off and disposed of. + +5. **Quality Assurance (QA) Section**: + - Before portioning out the tissue, make sure to cut away a small QA section. + - This should be excised from the center of the colon sample, should represent both the mucosa and the muscularis, and should also be free of adipose tissue. + + ![QA Section and Colon Section](link_to_image3) + *Figure 3. Muscle layer and mucosa of tissue section given to CHTN.* + +6. **Portion out the tissue**: + - Utilize the CHTN databases in determining how the collected tissue should be portioned out (i.e., what dimensions, weights, collection methods, etc.), to best serve CHTN's investigators most efficiently and effectively. + +7. **Divide the tissue**: + - Unless other specifications take priority, normal colon should generally be divided up into equal numbers of paraffin blocks, frozen OCT samples, snap-frozen full-cut samples (i.e., displaying both mucosa and muscularis), and snap-frozen mucosa-only samples. + - If using NAT colon, be sure to cut away any inked tissue before preservation. + +8. **Produce mucosa-only samples**: + - Shear a short length of the lining away from the muscularis using dissection scissors or a razor blade—a delicate but achievable process. + + ![MD Sample](link_to_image4) + *Figure 4. Sheared mucosa away from muscularis using dissection scissors or a razor blade.* + +9. **Snap-freeze samples**: + - While snap-frozen samples of full-cut colon should weigh ~0.25g, it may be difficult to consistently produce ~0.25g snap-frozen samples of mucosa-only colon. + - Those clamshells that only contain mucosa should be labeled as such. + - If no indication is made, then it is assumed that the clamshell contains a full-cut colon sample. + - Additionally, when it applies, include "mucosa-only" in the descriptors of the tissue acquisition page of the CHTN database. + + ![Labeled Clamshells](link_to_image5) + *Figure 5. Labeled clamshells for a full-cut section (left) and a mucosa-only section (right).* + +10. **Paraffin block samples**: + - Paraffin block samples should be cut in long, thin (less than 0.5cm-thick) strips, in a manner as to show both the mucosa and the muscularis of the colon. + + ![QA Block and Paraffin Blocks](link_to_image6) + *Figure 6. QA block sample (left) and paraffin block samples (right) mounted on cassettes.* + +11. **OCT fixation**: + - Similarly, normal colon samples frozen in OCT should display both the mucosa and the muscularis (unless otherwise requested) and follow the general protocol for OCT fixation. + +**endofoutput** +``` \ No newline at end of file diff --git a/markdown-output/tomogram-reconstruction-and-sub-tomogram-averaging-df323qqe.md b/markdown-output/tomogram-reconstruction-and-sub-tomogram-averaging-df323qqe.md new file mode 100644 index 0000000000000000000000000000000000000000..ae266ece29b03c6994339f492aa89bfc3ebfb4e1 --- /dev/null +++ b/markdown-output/tomogram-reconstruction-and-sub-tomogram-averaging-df323qqe.md @@ -0,0 +1,139 @@ +```markdown +# Goal/Experiment: +The goal of this protocol is to perform tomogram reconstruction and sub-tomogram averaging to obtain full-length, auto-inhibited LRRK2 filaments on microtubules. + +# Tomogram Reconstruction and Sub-Tomogram Averaging to Obtain Full-Length, Auto-inhibited LRRK2 Filaments on Microtubules V.2 + +DOI: [dx.doi.org/10.17504/protocols.io.n92ld89zxv5b/v2](https://dx.doi.org/10.17504/protocols.io.n92ld89zxv5b/v2) + +Authors: Siyu Chen, Josh Hutchings, Digvijay Singh + +## Abstract +This protocol describes all the data analysis steps after obtaining the LRRK2 on microtubule dataset. + +## Materials and Software +- **Warp**: Used for the pre-processing of tomograms before sub-tomogram analysis. + - OS: Windows 10 + - Developer: Dmitriy Tegunov +- **IMOD**: Used for aligning tilt series in eTomo using patch tracking. + - OS: Linux + - Developer: University of Colorado Boulder +- **Dynamo**: Used for sub-tomogram extraction and analysis. + - OS: Linux + - Developer: CU Boulder +- **Chimerax**: Used for visualization and microtubule tracing. + - OS: Mac + - Developer: UCSF +- **Relion**: Used for refinement of sub-tomograms. + - OS: Linux + - Developer: MRC +- **ISOLDE**: Used to analyze and resolve clashing and obvious wrong fitting with torsion and distances restrained. + - OS: Mac + - Developer: Altos Labs + +## Protocol Steps + +### Tilt Series Alignment and Tomogram Reconstruction + +1. **Use Warp Software** + - Perform motion correction, CTF estimation, and gain correction. + - Set pixel size, dimensions, binning, CTF searching range, and number of frames. + - Save averaged .tif files in the 'average' folder under the data folder. + +2. **Generate Tilt Stacks** + - Go to 'tomostar' page, click 'Import Tilt Series from IMOD'. + - Select data folder and metadata folder, and create stacks for IMOD. + +3. **Align Tilt Series in IMOD** + - Align all tilt series in eTomo using patch tracking. + +4. **Batch Processing** + - Use command line run 'etomo' and click 'Batch Tomograms' to process all tilt series. + - Choose cryoSample.doc template for 'System template'. + +5. **Set Dataset Parameters** + - In the 'Dataset Values' tab: + - Bin 2 or 4 for coarse alignment. + - Use patch tracking with patch size of 170,170 or 340,340. + - Tomograph thickness 1500 - 2500 pixels. + - Pixel size and tilt axis rotation angle as per data collection parameters. + - Patch overlap 0.33,0.33 (percentage). + - Fix magnification, tilt angle, rotation, and beam tile alignment. + - Adjust smoothing and other parameters as needed. + +6. **Run Batch Job** + - Run the batch job and optionally perform tomogram reconstruction. + +7. **Log File Analysis** + - Check the log file for residual error mean (in nm), keeping it <2 nm. + +8. **eTomo Alignment** + - Use eTomo alignment data for tomogram reconstruction in Warp. + +9. **Estimate 3D CTF** + - Use weighted back-projection at 10 Å/pixel. + - Visualize and trace microtubules using de-convoluted tomograms. + +### Microtubule Tracing and LRRK2 Sub-Tomogram Picking + +10. **Manual Tracing in IMOD** + - Use IMOD to trace the backbone of microtubules. + +11. **Extraction in Dynamo** + - Import the trajectories into Dynamo and use the function `dmodels.filamentWithTorsion()` for re-sampling. + - Randomize azimuth angle using `drandomize_azimuth()`. + - Crop sub-tomograms using `dtcrop()`. + - Calculate averages using `daverage()`, box size was 48. + +12. **Align Picked Sub-Tomograms** + - Use `dpktt.exclusionPerVolume()` to distribute coordinates. + +13. **Pick Sub-Tomograms in Rings** + - Use `dmodels.filamentRings()`. + +14. **Manually Scan Microtubules** + - Use the same set of parameters for 10 iterations without initial reference. + +15. **Align Sub-Tomograms** + - Use the reference sub-tomograms and apply azimuth correction. + +16. **Center Picked Sub-Tomograms** + - Use `dynamo_align()`, `dynamo_align0()`, and manual re-sampling when required. + +### Sub-Tomogram Analysis and 3D Model Reconstruction of LRRK2 + +20. **Extract Sub-Tomograms in Warp** + - Use the "Reconstruct Sub-tomograms" function with binning by 2 (4.322 Å/pixel) and a box size of 96. + +21. **Refine Sub-Tomograms in Relion3** + - Refine using C1 symmetry first without a mask, then perform focused refinement. + +22. **Detailed Refinement** + - Extract sub-tomograms utilizing nested star files in Relion4 and apply optimal CTF refinement. + +23. **3D Classification** + - Set regularisation parameter T = 1 instead of default 4, resolution E-step to 20 Å. + +24. **Mask LRRK2 Copies** + - Expand the stack by 2-fold and include all copies for alignment. + +25. **Unbin and Perform Auto-Refinement** + - Refine the stack to 3.21 Å/pixel for better resolution. + +### Model Building +36. **Use Published Models**: + - PDB: 7LHW as main reference for building LRRK2 model on microtubules. + +### Geometric Analysis +40. **Perform Sub-Boxing and Refinement Steps in Dynamo** + +41. **Align for Tubular Reference** + - Use `dynamo_align()` and `dynamo_subboxing_table()`. + +45. **Calculate Helical Angle and Plot Data** + - Plot and analyze to ensure LRRK2 dimers are counted and aligned correctly. + +**End of Protocol** +``` +[endofoutput] +``` \ No newline at end of file diff --git a/markdown-output/total-rna-purification-from-plasma-or-serum-isolat-f5hbq36.md b/markdown-output/total-rna-purification-from-plasma-or-serum-isolat-f5hbq36.md new file mode 100644 index 0000000000000000000000000000000000000000..112838258fb4de4341190bfe68c0d9a627703677 --- /dev/null +++ b/markdown-output/total-rna-purification-from-plasma-or-serum-isolat-f5hbq36.md @@ -0,0 +1,145 @@ +```markdown +# Goal/Experiment: +RNA Purification from Plasma or Serum using the ISOLATE II Biofluids RNA Kit to isolate RNA from non-coagulated, fresh whole blood samples for further downstream applications such as real-time PCR or microarrays. + +# Total RNA Purification from Plasma or Serum (ISOLATE II Biofluids RNA Kit) + +## Abstract +Protocol for RNA Purification from Plasma or Serum, using the ISOLATE II Biofluids RNA Kit. This protocol includes the lysate preparation procedure. + +*Citation: Bioline Total RNA Purification from Plasma or Serum (ISOLATE II Biofluids RNA Kit). protocols.io dx.doi.org/10.17504/protocols.io.f5hbq36* + +*Published: 12 Dec 2016* + +## Guidelines + +*Before you start:* + +- Plasma or serum of all human and animal subjects is considered potentially infectious. All necessary precautions recommended by the appropriate authorities in the country of use should be taken when working with plasma or serum. +- We recommend the use of this kit to isolate RNA from plasma or serum prepared by a standard protocol from non-coagulated, fresh whole blood using EDTA or sodium citrate as the anti-coagulant. +- Plasma prepared from fresh blood using heparin as an anti-coagulant is not suitable for use with this protocol. +- Due to the relatively low DNA content in plasma, the Genomic DNA Removal Column is not necessary for this protocol. +- It is recommended that no more than 200 µL of plasma or serum is used in order to prevent clogging of the column. +- Yields of RNA from plasma and serum is highly variable. In general, the expected yield could vary from 1 to 100 ng per 100 µL plasma or serum used. In addition, the expected A260/A280 ratio as well as the A260/A230 ratio will be lower (<1.8) than the normal acceptable range from other cells or tissues. Nonetheless, these isolated RNA can be effectively used in different downstream applications such as real-time PCR or microarrays. +- Avoid multiple freeze-thaw cycles of the plasma or serum sample. Aliquot out the appropriate volume for usage prior to freezing. +- It is important to work quickly during this procedure. + +Please review the Guidelines under [Genomic DNA removal and total RNA purification from all types of lysate](dx.doi.org/10.17504/protocols.io.f5hbq36) for other important details. + +## Before start + +## Materials + +- ISOLATE II Biofluids RNA Kit BIO-52086 by Bioline + +## Protocol + +### Lysate Preparation from Plasma/Serum + +**Step 1.** +Transfer up to 200 µL of plasma or serum to a 1.5mL RNase-free microcentrifuge tube (not supplied). + +**Step 2.** +Add 300 µL of Lysis Buffer RX to every 100 µL of plasma or serum. + +**Step 3.** +Mix by vortexing for 10s. + - **Duration:** 00:00:10 + +**Step 4. (Optional)** +Add 0.7µL of 0.8µg/µL MS2 RNA per sample. + + > Note: The use of MS2 RNA can increase the consistency of downstream applications such as real-time PCR- and RT-PCR. However, the use of MS2 RNA is not recommended for applications involving global gene expression analysis such as microarrays or sequencing. + +**Step 5.** +Add 400 µL of 96-100% ethanol to every 400 µL of lysate (equivalent to every 100µL plasma or serum used). + +**Step 6.** +Mix by vortexing for 10s. + - **Duration:** 00:00:10 + +### Binding RNA to Column + +**Step 7.** +Assemble an ISOLATE II RNA Column (black ring) with a provided Collection Tube. + +**Step 8.** +Apply up to 600µL of the ethanolic lysate onto the column and centrifuge for 1 min at ≥ 3,500 x g. + - **Duration:** 00:01:00 + + > Note: Ensure the entire lysate volume has passed through into the Collection Tube by inspecting the column. If the entire lysate volume has not passed through, spin for an additional minute at 14,000 x g. + +**Step 9.** +Discard the flow-through. Reassemble the spin column with its Collection Tube. + +**Step 10.** +Depending on the lysate volume, repeat steps 8 and 9 as required. + +### RNA Column Wash + +**Step 11.** +Apply 400 µL of 96-100% ethanol to the column and centrifuge for 1 min at 14,000 x g. (wash 1/3) + - **Duration:** 00:01:00 + +**Step 12.** +Discard the flow-through and reassemble the spin column with its Collection Tube. (wash 2/3) + +**Step 13.** +Apply 400 µL of 96-100% ethanol to the column and centrifuge for 1 min at 14,000 x g. (wash 2/3) + - **Duration:** 00:01:00 + + > Note: Ensure the entire wash buffer volume has passed through into the Collection Tube by inspecting the column. If the entire wash buffer volume has not passed through, spin for an additional minute at 14,000 x g. + +**Step 14.** +Discard the flow-through and reassemble the spin column with its Collection Tube. (wash 2/3) + +**Step 15.** +Wash column a third time by adding 400µL of 96-100% ethanol and centrifuge for 1 min at 14,000 x g. (wash 3/3) + - **Duration:** 00:01:00 + +**Step 16.** +Discard the flow-through and reassemble the spin column with its Collection Tube. (wash 3/3) + +**Step 17.** +Spin the column for 2 min at 14,000 x g in order to dry the column thoroughly. Discard the Collection Tube. + - **Duration:** 00:02:00 + +### RNA Elution + +**Step 18.** +Place the column into a fresh 1.7mL Elution Tube. + +**Step 19.** +Add 50µL of RNA Elution Buffer to the column. + +**Step 20.** +Centrifuge for 2 min at 200 x g. + - **Duration:** 00:02:00 + +**Step 21.** +Centrifuge for 1 min at 14,000 x g. Note the volume eluted from the column. If the entire 50 µL has not been eluted, spin the column at 14,000 x g for an additional minute to elute the RNA. + - **Duration:** 00:01:00 + + > Note: For maximum RNA recovery, it is recommended to apply a second volume of RNA Elution Buffer and elute into the same microcentrifuge tube (repeat steps 19-21). Alternatively, re-apply the first eluate onto the column and re-elute into the same microcentrifuge tube (for higher concentration). + +### Storage of RNA + +**Step 22.** +The isolated RNA can be stored at -20°C for a few days or at -80°C (recommended) for long-term storage. + +## Warnings + +- Plasma or serum of all human and animal subjects is considered potentially infectious. All necessary precautions recommended by the appropriate authorities in the country of use should be taken when working with plasma or serum. + +- When working with chemicals, always wear a suitable lab coat, gloves, and safety glasses. + +- Lysis Buffer RX contains guanidinium thiocyanate. This chemical is harmful in liquid form when in contact with skin or ingested. If the solution is allowed to dry, the powder is harmful if inhaled. + + > CAUTION: Do not add bleach directly to solutions or sample preparation waste containing guanidinium salts. Reactive compounds and toxic gases can form. In the case of spillage, clean the affected area with a suitable laboratory detergent and water. + +For detailed information, please consult the material data safety sheet (MSDS) available on our website at [www.bioline.com](https://www.bioline.com). + +Biofluids derived from all human and animal sources are considered potentially infectious. All necessary precautions recommended by the appropriate authorities in the country of use should be taken when working with biofluids. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/total-soluble-sugar-quantification-from-ethanolic-b2nsqdee.md b/markdown-output/total-soluble-sugar-quantification-from-ethanolic-b2nsqdee.md new file mode 100644 index 0000000000000000000000000000000000000000..f10a7ea339a91510e3876ee198190daa3c573e14 --- /dev/null +++ b/markdown-output/total-soluble-sugar-quantification-from-ethanolic-b2nsqdee.md @@ -0,0 +1,110 @@ +```markdown +# Goal/Experiment: +Quantification of total soluble sugars (as glucose) in plant tissue extracts via the sulfuric phenol method adapted for 96 well plates. + +# Total Soluble Sugar Quantification from Ethanolic Plant Extracts + +## Authors +- Lynn Doran +- Amanda P. De Souza + +### Realizing Increased Photosynthetic Efficiency (RIPE) +Burgess Lab UIUC + +DOI: [10.17504/protocols.io.b2nsqdee](https://dx.doi.org/10.17504/protocols.io.b2nsqdee) + +## Funding +Realizing Increased Photosynthetic Efficiency (RIPE) is funded by the Bill & Melinda Gates Foundation, Foundation for Food and Agriculture Research, and the U.K. Foreign Commonwealth & Development Office. +Grant ID: OPP1172157 + +## Protocol Reference +Masuko T, Minami A, Iwasaki N, Majima T, Nishimura S, Lee YC. Carbohydrate analysis by a phenol-sulfuric acid method in microplate format. Anal Biochem. 2005 Apr 1;339(1):69-72. doi:10.1016/j.ab.2004.12.001. PMID: 15766712. [Link to Article](https://pubmed.ncbi.nlm.nih.gov/15766712/) + +## Protocol Date +- December 06, 2021 + +## Summary +Quantification involves preparing a standard curve, extracting sugars, and measuring absorbance to estimate glucose equivalents. + +## Materials and Equipment + +### Reagents +- **Sulfuric Acid**: Merck/Millipore brand, ACS grade +- **Phenol 5% w/v**: 5 g phenol diluted up to 100 mL with MilliQ water (freshly prepared daily) +- **Glucose Solution (1 mg/mL)**: Sigma G6918-100 mL Lot SLCD7032 +- **MilliQ or Distilled Water** + +### Materials +- Pipette Tips +- 96-Well Plates (Polypropylene or PTFE; polystyrene should be avoided due to chemical interference) + +### Equipment +- Single Channel Pipette +- Multi-Channel Pipette +- Repeat Pipette +- Analytical Balance +- Water Bath +- Ice Bucket +- Chemical Fume Hood +- UV-Vis Plate Reader + +## Safety +- Use chemical fume hoods for procedures involving sulfuric acid and phenol. +- Refer to safety modules such as "Laboratory Safety," "Chemical Safety- An Introduction," and "Chemical Spills" Division of Research Safety training before handling hazardous materials. + +## Procedure + +### Preparation of Glucose Standards +1. Prepare glucose standards in microcentrifuge tubes. + - Pipette appropriate amounts of 1 mg/mL glucose standard and distilled water into each labeled tube. + +| Glucose (µg) per 50 µL well | Amount of 1 mg/mL Glucose (µL) | Amount of Distilled Water (µL) | +|-----------------------------|-------------------------------|-------------------------------| +| 5 | 20 | 180 | +| 10 | 40 | 160 | +| 15 | 60 | 140 | +| 20 | 80 | 120 | +| 25 | 100 | 100 | + +2. Pipette 50 µL of each prepared glucose standard in triplicate into the assigned wells. + +### Sample Preparation +3. Extract and purify soluble sugars from plant tissue as described in [Extraction of Non-Structural Carbohydrates](https://dx.doi.org/10.17504/protocols.io.b2nsqdee). +4. Pipette 10-30 µL of each sample extract in triplicate into the assigned wells. Record the amount added. + - Adjust sample volumes to fall within the absorbance range of 5-25 µg glucose standards. +5. Add distilled water to bring the final volume in each well to 50 µL. + +### Assay +6. Move the plate to the chemical fume hood. +7. Add 150 µL of sulfuric acid to each well inside the fume hood. + - Use a multi-channel or repeat pipette for efficiency. +8. Immediately after, add 30 µL of phenol (5%) into each well. + - Be cautious of the heat produced. Wear PPE and maintain sampling within the fume hood. + +### Incubation +9. Incubate the plate by floating it in a 90°C water bath for 5 minutes. +10. Place the plate in an ice bath to cool. +11. Once cooled, read absorbance at 490 nm on a UV-VIS spectrophotometer. + +### Additional Assay Plates +12. Maintain consistency with the spectrophotometer lamp. Include a blank (0 µg glucose standard) on every plate. + +### Calculations +14. Normalize absorbances to zero using 0 µg glucose standard per plate. +15. Calculate total soluble sugar as glucose for each sample using averaged normalized standard curve absorbances for technical replicates. +16. Divide total soluble sugar (µg) by the volume of total sample extract loaded to obtain concentration. +17. Multiply soluble sugar concentration by the dilution factor of the extract. +18. Divide the total soluble sugar concentration by the initial weight of ground tissue used in extraction to report µg Total Soluble Sugars (as glucose) per mg plant tissue. + +## End of Protocol +--- + +## Notes +- Accurate pipetting and sample handling are crucial for reliable results. +- Be consistent with sample preparation across multiple assays to obtain reproducible data. + +## References +- Doran, L., De Souza, A.P. 2021. Total Soluble Sugar Quantification from Ethanolic Plant Extracts. protocols.io. DOI: [10.17504/protocols.io.b2nsqdee](https://dx.doi.org/10.17504/protocols.io.b2nsqdee). + +endofoutput +``` diff --git a/markdown-output/toto-1-nucleic-acid-labeling-protocol-nkfdctn.md b/markdown-output/toto-1-nucleic-acid-labeling-protocol-nkfdctn.md new file mode 100644 index 0000000000000000000000000000000000000000..edb9efe411fd714c7532fa14135784a11d683161 --- /dev/null +++ b/markdown-output/toto-1-nucleic-acid-labeling-protocol-nkfdctn.md @@ -0,0 +1,127 @@ +```markdown +# Goal/Experiment: +The goal of this experiment is to use the TOTO-1 iodide labeling protocol to enable visualization of nucleic acid loading efficiency. This allows for the verification that nucleic acid constructs are entering cells while introducing additional cleanup steps to decrease background staining. This assay is convenient for use in conjunction with transfection optimization. + +# TOTO-1 Nucleic Acid Labeling Protocol + +### April Woods, G Jason Smith + +## Abstract + +A modified TOTO-1 iodide labeling protocol to enable visualization of nucleic acid loading efficiency, as a means to verify that nucleic acid constructs are entering the cells. With additional cleanup steps to decrease background staining. A convenient assay to use in conjunction with transfection optimization. (Modified from Golzio, M., Teissie, J. & Rols, M.P. 2002. Direct visualization at the single-cell level of electrically mediated gene delivery. Proc Natl Acad Sci USA 99, 1292–1297.) + +## Citation +April Woods, G Jason Smith TOTO-1 nucleic acid labeling protocol. protocols.io dx.doi.org/10.17504/protocols.io.nkfdfctn + +## Published +03 Mar 2018 + +## Guidelines + +### DNA Copy Number Calculation and Dye Binding Ratio + +| Parameter | Description | +|--------------------------|---------------------------------------| +| **DNA Size, bp** | Enter value | +| **DNA Conc, µg/µL** | Enter value | +| **DNA Abundance, copies/µL** | =((DNA Conc, µg/µL) * 6.023 * 1E+23) / (DNA Size, bp * 1,000,000 * 650) | +| **bp per vol** | =(DNA Abundance, copies/µL) * (DNA Size, bp) | +| **Binding Target** | 5 bp/dye | +| **Dye molecules needed** | =(bp per vol) / (Binding Target) | +| **Dye Conc, M** | Enter value | +| **molecules/L** | =Dye Conc, M * 6.023E+23 | +| **Dye Volume Needed** | =(Dye molecules needed / molecules per L) * 1,000,000 µL Dye per µL DNA | + +## Materials + +- **TOTO™-1 Iodide (514/533)** - 1 mM Solution in DMSO [T3600 by Thermo Fisher Scientific](https://www.thermofisher.com/order/catalog/product/T3600) +- **Microcon-30kDa Centrifugal Filter Unit with Ultracel-30 membrane** [MRCF0R030 by Emd Millipore](https://www.emdmillipore.com/US/en/product/Microcon-30kDa-Centrifugal-Filter-Unit,MM_NF-MRCF0R030) +- **Sheared Salmon Sperm DNA 10mg/mL** [AM9680 by Thermo Fisher Scientific](https://www.thermofisher.com/order/catalog/product/AM9680) + +## Protocol + +### Preparing the Millipore Column + +#### Step 1. +Hydrate Microcon-30 kDa centrifugal filter columns at least 1h to overnight with nuclease-free sterile water. + +#### Step 2. +Spin columns at 3000xg for 20 minutes, repeat as many times as needed until all water has been pushed through column to collection tube (in older columns, this may require extended spin time. Column must be fully hydrated before proceeding). + +### Labeling Nucleic Acids with TOTO-1 Dye + +#### Step 3. +On ice, aliquot sufficient volume of DNA or RNA for experiments, want at least 1 µg/µL conc. + +#### Step 4. +Add TOTO-1 dye from stock (1 mg/mL, in DMSO) to desired concentration (see guidelines for dye concentration calculation). + +#### Step 5. +Mix well by gentle pipetting. + +#### Step 6. +Shield from light and incubate on ice for 1 hour + +### Clean up Dye Labeled Nucleic Acids + +#### Step 7. +Transfer TOTO-1 labeled nucleic acid mixture to Millipore spin filter column. + +#### Step 8. +Centrifuge for 20 minutes at 3,000xg in microcentrifuge. If filter intact, there should be ca. 20 µL remaining in filter cup. + +#### Step 9. +Add 200 µL TE pH8 to filter cup and centrifuge as in step 8. Repeat if background fluorescence is an issue in procedural blanks. + +#### Step 10. +The stained nucleic acid is retained in filter cup of the hydrated spin column in ca 20 µL of TE and will be visible. The eluate and collection tube can be discarded. + +#### Step 11. +Resuspend filter retentate in TE buffer, adjust volume added to yield [DNA] ≥ 1 µg/µl. Gently pipette up and down around the filter to collect all the dye bound DNA. + +### Proceed to Transfection + +#### Step 12. +Add dye bound nucleic material to cells prepared for electroporation (in electroporation buffer). + +Incubate on ice for 10 minutes. + +### Electroporation + +#### Step 13. +Electroporation steps (follow preferred electroporation protocol) + +#### Step 14. +Allow electroporated cells to incubate at room temperature for 10 minutes in cuvette. This will allow nucleic acid adhered to extracellular wall to enter cellular pores generated during electroporation. + +#### Step 15. +Visualize aliquots (drops) 10 min post electroporation in the same buffer. Border of cells should show signal on GFP channel but background staining will be high due to residual TOTO-1:DNA complex in solution. *(This is an optional QC step to view staining progress).* + +#### Step 16. +Gently transfer electroporated cells from cuvette to 30 mL falcon tube in addition of 10 mL sterile seawater for wash post electroporation. Spin at 3,000xg for no more than 5 minutes to minimize extracellular adhesion loss. The wash step will remove unbound stained nucleic material from the media. + +Remove supernatant and gently resuspend cell pellet in 0.5mL sterile seawater (adjust final volume based on the desired density of your cell sample). + +### Image Cells + +#### Step 17. +TOTO-1 nucleic acid complex fluorescence overlaps with standard FITC / GFP filter sets (e.g. GFP Light Cube on our Thermo-Fisher EVOS FL inverted microscope). + +## Tips and Tricks + +### Step 18. +Filtering transfection samples onto black filters (Isopore HTBP01300, 0.4um pore or equivalent) followed by seawater rinses provides a low noise, high contrast background for TOTO-1 nucleic acid imaging (see below, however, this does not hold true for the Cy5 light cube used to stimulate chlorophyll fluorescence). + +### Step 19. +Carrier DNA (e.g Salmon sperm DNA, Thermo-Fisher AM9680 at 1-10µg/mL) may be added to electroporation step to aid in transfection of nucleic material. + +### Step 20. +TOTO-1 DNA accumulates along raphe with strong intrafrustule labeling in proximity to the nuclear compartment in *Pseudo-nitzschia multiseries* cells. + +### Step 21. +![Image description](https://www.protocols.io/p/124876) + +Intracellular labeling is diffuse and not apparent in chloroplasts. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/transfection-hepg2-m6jc9cn.md b/markdown-output/transfection-hepg2-m6jc9cn.md new file mode 100644 index 0000000000000000000000000000000000000000..6c8aa3ee7fbc3bbbe260876c4b053b547213ff9a --- /dev/null +++ b/markdown-output/transfection-hepg2-m6jc9cn.md @@ -0,0 +1,118 @@ +```markdown +# Goal/Experiment: +Transfection of HepG2 cells using Lipofectamine™ 2000 to deliver nucleic acids (DNA and RNA) and achieve gene expression or knockdown. + +# Transfection HepG2 Version 2 + +## IGEM AFCM-EGYPT + +### Abstract +**Lipofectamine™ 2000 Transfection Protocol** + +### Description +Lipofectamine™ 2000 is a proprietary formulation for the transfection of nucleic acids (DNA and RNA) into eukaryotic cells providing the following advantages: +- Highest transfection efficiency in many cell types and formats (e.g., 96-well). +- Nucleic acid-Lipofectamine™ 2000 complexes can be added directly to cells in culture medium, in the presence or absence of serum. +- It is not necessary to remove complexes or change/add medium after transfection, but complexes may be removed after 4-6 hours. + +Refer to the Cell Lines database at [Invitrogen](http://www.invitrogen.com) for a list of cell types successfully transfected. + +*Citation*: IGEM AFCM-EGYPT Transfection HepG2. [protocols.io](dx.doi.org/10.17504/protocols.io.m6jc9cn) +*Published*: 09 Feb 2018 + +--- + +## Protocol + +### Step 1. +One day before transfection, plate cells in 500 μl of growth medium without antibiotics such that they will be 30-50% confluent at the time of transfection. + +*Note*: Transfecting cells at a lower density allows a longer interval between transfection and assay time and minimizes the loss of cell viability due to cell overgrowth. + +### Step 2. +For each transfection sample, prepare oligomer-Lipofectamine™ 2000 complexes as follows: +- Dilute 20 pmol Stealth™ RNAi or siRNA oligomer in 50 μl Opti-MEM® I Reduced Serum Medium without serum (resulting concentration of RNAi is 40 nM). Mix gently. +- Mix Lipofectamine™ 2000 gently before use, then dilute 1 μl in 50 μl Opti-MEM® I Reduced Serum Medium. Mix gently and incubate for 5 minutes at room temperature. + +*Note*: Proceed to Step 3 within 25 minutes. + +After the 5-minute incubation, combine the diluted oligomer with the diluted Lipofectamine™ 2000. Mix gently and incubate for 20 minutes at room temperature (solution may appear cloudy). + +### Step 3. +- Add the oligomer-Lipofectamine™ 2000 complexes to each well containing cells and medium. +- Mix gently by rocking the plate back and forth. +- Incubate the cells at 37°C in a CO2 incubator for 24-96 hours until you are ready to assay for gene knockdown. +- Medium may be changed after 4-6 hours. + +*Optimizing Stealth™ RNAi or siRNA Transfection:* +To obtain the highest transfection efficiency and low non-specific effects, optimize transfection conditions by varying RNA and Lipofectamine™ 2000 concentrations. + +- Test 10-50 pmol RNA and 0.5-1.5 μl Lipofectamine™ 2000 for 24-well format. +- Depending on the nature of the target gene, transfecting cells at higher densities may also be considered. + +### Step 4. +Adherent cells: One day before transfection, plate 0.5-2 x 10^5 cells in 500 μl of growth medium without antibiotics so that cells will be 90-95% confluent at the time of transfection. +Suspension cells: Just prior to preparing complexes, plate 4-8 x 10^5 cells in μl of growth medium without antibiotics. + +### Step 5. +For each transfection sample, prepare complexes as follows: +- Dilute DNA in 50 μl of Opti-MEM® I Reduced Serum Medium without serum (or other medium without serum). Mix gently. +- Mix Lipofectamine™ 2000 gently before use, then dilute the appropriate amount in 50 μl of Opti-MEM® I Medium. Incubate for 5 minutes at room temperature. + +*Note*: Proceed to Step 6 within 25 minutes. + +After the 5-minute incubation, combine the diluted DNA with diluted Lipofectamine™ 2000 (total volume = 100 μl). Mix gently and incubate for 20 minutes at room temperature (solution may appear cloudy). + +*Note*: Complexes are stable for 6 hours at room temperature. + +### Step 6. +- Add the 100 μl of complexes to each well containing cells and medium. +- Mix gently by rocking the plate back and forth. + +### Step 7. +- Incubate cells at 37°C in a CO2 incubator for 18-48 hours prior to testing for transgene expression. +- Medium may be changed after 4-6 hours. + +### Step 8. +Surface areas may vary depending on the manufacturer. + +### Step 9. +For stable cell lines: +- Passage cells at a 1:10 (or higher dilution) into fresh growth medium 24 hours after transfection. +- Add selective medium (if desired) the following day. + +### Step 10. +*Scaling Up or Down Transfections:* +To transfect cells in different tissue culture formats, vary the amounts of Lipofectamine™ 2000, nucleic acid, cells, and medium used in proportion to the relative surface area, as shown in the table. With automated, high-throughput systems, a complexing volume of 50 μl is recommended for transfections in 96-well plates. + +*Note*: You may perform rapid 96-well plate transfections by placing diluted reagents directly into the transfection mix. Prepare complexes in the plate and directly add cells at twice the cell density as in the basic protocol in a 100 μl volume. Cells will adhere as usual in the presence of complexes. + +### Step 11. +Culture shared reagents: + +| **DNA Transfection** | **RNAi Transfection** | +|----------------------|-----------------------| +| Lipofectamine™ 2000 RNA | Lipofectamine™ 2000 | +| 0.8 μg 0.5 μl* | 20 pmol 1.0 μl* | +| 2 cm2 500 μl | 2 x 50 μl 0. 8 μg 2.0 μl 20 pmol 1.0 μl* | +| 4 cm2 1 ml | 2 x 100 μl 1.6 μg 4.0 μl* | +| 12-well | 6 cm2 2 ml 2 x 250 μl 4.0 μg 10 μl* | +| 100 pmol | 5.0 μl/mL | +| 20 cm2 5 ml | 2 x 0.5 ml 8.0 μg 20 pmol 10 μl 10-cm | +| 60 cm2 15 ml | 2 x 1.5 ml 24 μg 60 pmol 30 μl | + +\* Add Lipofectamine™ 2000 ratios ranging from 1:0.5 to 1:5. + +### Step 12. +Volumes of dilution medium in Step 2a & 2b of DNA or RNAi transfection protocols. + +*Purchaser Notification*: This product is covered by one or more Limited Use Label Licenses (see the Invitrogencatalog or our website, [www.invitrogen.com](http://www.invitrogen.com)). By using this product you accept the terms and conditions of all applicable Limited Use Label Licenses. + +*Limited Use Label License No. 27*: Lipofectamine™ 2000 Reagent +*Limited Use Label License No. 173*: Inhibition of Gene Expression by Double-Stranded RNA +*Limited Use Label License No. 196*: Stealth™ RNAi ©2000-2005 Invitrogen Corporation. All rights reserved. + +--- + +*Endofoutput* +``` \ No newline at end of file diff --git a/markdown-output/transformation-of-diplonema-papillatum-by-electrop-c7i9zkh6.md b/markdown-output/transformation-of-diplonema-papillatum-by-electrop-c7i9zkh6.md new file mode 100644 index 0000000000000000000000000000000000000000..f377f219b4c3f7615feff684ab086392a0832b56 --- /dev/null +++ b/markdown-output/transformation-of-diplonema-papillatum-by-electrop-c7i9zkh6.md @@ -0,0 +1,121 @@ +```markdown +# Goal/Experiment: +The goal of this experiment is the transformation of **Diplonema papillatum** by electroporation using a "home-made" transformation buffer. + +# Transformation of Diplonema papillatum by Electroporation V.3 + +## Authors: +- Matus Valach +- Gertraud Burger + +Université de Montréal, Montreal, Quebec, Canada + +## Abstract +Variant protocol for the transformation of *Diplonema papillatum* by electroporation using a "home-made" transformation buffer. The procedure was devised based on previously published protocols by Kaur et al. (DOI: 10.1111/1462-2920.14041) and Dyer et al. (DOI: 10.3791/54342). For additional details, see also Faktorová et al. (DOI: 10.1111/1462-2920.15130). + +## Materials + +### Reagents +- Sodium Phosphate monobasic +- Glucose +- KCl +- CaCl2 +- MgCl2 +- BSA (Bovine Serum Albumin) +- Sucrose +- HEPES +- EDTA +- Inosine triphosphate +- G418 (geneticin sulfate) - Bioshop Catalog #GEN418 + +## Before Start Instructions +Perform a simple test of antibiotic resistance of wild-type cells in the chosen culture conditions, e.g., temperature (16 °C vs 20 °C vs 27 °C), medium composition, or antibiotic supplier: + +1. Into a 24-well plate, distribute 1.5 mL medium per well. +2. Add the antibiotic at several different concentrations (e.g., for G418, choose 0, 50, 75, 100, 150, and 200 µg/mL). +3. This arrangement (6 columns, each with a different antibiotic concentration) allows to perform three WT replicates together with one positive control resistant to the antibiotic of choice. +4. Inoculate 1–5×10^5 cells per well and let the cells grow for 3–4 days. +5. Examine the extent of growth. +6. The lowest antibiotic concentration at which the WT cells do not grow is then used for the selection. + +For example, when cultivating *Diplonema papillatum* in a horse serum-based medium and using G418 (Bioshop, potency min. 650 µg/mg), 100 µg/mL is the threshold value at 20 °C, but >125 µg/mL is needed for efficient selection at 16 °C. + +## Protocol Steps + +### 1. Prepare the Transformation (Cytomix-like) Buffer + +| Component | Final Concentration | +|-------------------------------|----------------------| +| HEPES pH 7.5 | 25 mM | +| KCl | 25 mM | +| CaCl2 | 0.15 mM | +| NaH2PO4 pH 7.5 | 10 mM | +| MgCl2 | 2.5 mM | +| EDTA | 1 mM | +| Glucose | 30 mM (0.5%) | +| Sucrose | 145 mM (4.35%) | +| Bovine Serum Albumin (BSA) | 0.1 mg/mL | +| Inosine Triphosphate (ITP) | 1 mM | + +**Note:** +The addition of ITP (or hypoxanthine) is optional (alternatively, ATP can be used). If preparing a large volume of the buffer, make aliquots and store them at -70 °C until further use. + +### 2. Inoculate Diplonema Cells +Inoculate *Diplonema* cells at 1–2×105 /mL into 100 mL OSS medium supplemented with 0.05% tryptone and let them grow for 2–3 days. + +**Note:** +Cell density is ~5 times higher when cultivating *Diplonema* cells in a medium containing 0.05% tryptone compared to a medium without such supplementation. This improves survival after the pulse and is especially useful for *high-voltage* conditions (see below), which seems to favor homologous integration. + +### 3. Harvest the Cells +Harvest the cells while they are in the late exponential phase (optimal density 8×10^6–2×10^7 cells/mL). Wash twice with OS (i.e., medium without the serum) and aliquot the cells into tubes, keeping them on ice. + +### 4. Resuspend the Pellet +Resuspend the pellet in ice-cold 200 µL transformation buffer (see the recipe above), immediately centrifuge (4 °C, 1,000×g, 2 min), and discard the supernatant. + +### 5. Add DNA and Resuspend +Resuspend the pellet in ice-cold 100 µL transformation buffer supplemented with 1–4 µg linearized DNA (e.g., a PCR product or a restriction fragment of a plasmid). + +**Note:** +Optimally, add the DNA in a volume of 5 µL or less. To the negative control, add the same volume of the buffer used to solubilize the linearized DNA (e.g., 10 mM Tris pH 8.0). + +### 6. Transfer Cells to the Cuvette +Immediately transfer the cell suspension into an electroporation cuvette (0.2 mm), which has been pre-cooled on ice. + +### 7. Apply the Pulse +Wipe the cuvette to remove moisture, quickly transfer the cuvette into an electroporation apparatus (e.g., Gene Pulser Xcell from Bio-Rad), and apply the pulse. + +**Pulse parameters:** +- 1,500 V, 0.4 ms (also referred to here at "high voltage"); or +- 140 V, 1,400 µF ("low voltage"). + +**Note:** +Cell line selection is more straightforward and clear-cut for the option 1 (high voltage) and we observed that a higher proportion of transformants had the construct integrated at the intended locus (~60%), but the number of independent cell lines is limited (up to 5 independent cell lines have been obtained, but usually only about 2). In contrast, cell survival is much more substantial in the option 2 (low voltage) and may be preferred when numerous clones are required (up to 45 independent cell lines have been obtained). However, as indicated above, transformants tend to integrate the construct at a non-homologous location much more prominently. + +### 8. Resuspend After Pulse +Immediately after the pulse, put the cuvette back on ice, add 1 mL cold (5–10 °C) OSS, and resuspend the cells. + +### 9. Distribute into Plates +Transfer the cell suspension into a well of a 24-well (or 48-well) plate. Distribute the pulsed cell suspension into 24 or 48 wells: + +- 24 wells: 1-2 mL per well +- 48 wells: 0.8 to 1 mL per well + +Incubate for 5–8 hours without selection. + +### 10. Prepare Antibiotic Medium +Prepare OSS with the antibiotic of choice at a concentration that is double of the selection concentration (e.g., 200 µg/mL G418 if the final selection concentration is to be 100 µg/mL). To each well with pulsed cell suspension in OSS, add an equal volume of this medium. + +**Note:** +Make sure that the final concentration of the antibiotic is as determined by the resistance test. Optionally, keep a single well without the antibiotic to keep track of the recovery of the pulsed cells. + +### 11. Incubate +Let the cells grow for ~2 days. Observe the cells in the plates under a microscope to check their growth. If there is visible growth (i.e., cells swimming in the 'column'), transfer an aliquot of these swimming cells into a new plate with a 1.5–2× higher concentration of the antibiotic. Continue growth for an additional 5–9 days. + +**Note:** +Passaging the cells through a medium with a higher concentration of the antibiotic ensures that only truly resistant clones (i.e., those expressing the antibiotic resistance conferring gene at a sufficient level) are selected. + +### 12. Clone Expansion +If a well population is a mixture of cells containing a wild-type allele and a correctly integrated DNA construct, perform 10× serial dilutions of cells from each selection well into a new plate with fresh medium (if using G418, usually at 100–150 µg/mL) to ensure that truly independent cell lines are selected. This phase may take up to 3 weeks in total. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/trapping-and-blood-sampling-small-mammals-in-semi-wnxfdfn.md b/markdown-output/trapping-and-blood-sampling-small-mammals-in-semi-wnxfdfn.md new file mode 100644 index 0000000000000000000000000000000000000000..f0fc3d6b73b7418dbe6d68183a9123dd29360791 --- /dev/null +++ b/markdown-output/trapping-and-blood-sampling-small-mammals-in-semi-wnxfdfn.md @@ -0,0 +1,120 @@ +```markdown +# Trapping and Blood-Sampling Small Mammals in Semi-Arid Environments + +## Goal/Experiment: +The goal of this experiment is to provide a comprehensive guide for trapping and blood-sampling small terrestrial mammals in semi-arid environments. This includes detailed information on materials needed, procedures to follow, and recommendations for ensuring animal welfare and data accuracy. + +### Authors: +Pedro E. Cattan1, Carezza Botto-Mahan2, Juana P. Correa3, Antonella Bacigalupo1, Berenice Cornejo-Villar1 + +**Affiliations:** +1. Departamento de Ciencias Biológicas Animales, Facultad de Ciencias Veterinarias y Pecuarias, Universidad de Chile, Santiago, Región Metropolitana, Chile +2. Departamento de Ciencias Ecológicas, Facultad de Ciencias, Universidad de Chile, Santiago, Región Metropolitana, Chile +3. Departamento de Ciencias Biológicas Animales, Facultad de Ciencias Veterinarias y Pecuarias, Universidad de Chile, Santiago, Región Metropolitana, Chile + +**External Link:** +[https://doi.org/10.1371/journal.pntd.0007170](https://doi.org/10.1371/journal.pntd.0007170) + +**Protocol Status: Working** + +## Materials + +### Trapping +- **Gloves**: Thick gloves (leather, rubber, or textile plus nitrile gloves) +- **Wire-mesh traps**: Rodentrap®, Sherman™, Tomahawk™, or Havahart™ live-traps +- **Baits**: Rolled oats or another adequate attractant +- **Marking material**: Masking tape, flags, flagging tape, markers, etc. +- **Bedding material**: Hydrophilic cotton +- **Shelter material**: Plastic or paper +- **GPS or mobile phone** with geo-positioning software +- **Personal protection equipment** + +### Blood-sampling +- **Field laboratory**: Tent or isolated room +- **Anesthesia equipment**: Isoflurane, ketamine, xylazine; portable anesthesia machine or induction chamber +- **Weighing implements**: Portable balance or spring scale +- **Shaving implements**: Electric shaver, scissors +- **Sanitization**: 70% ethanol +- **Needles**: 21 G, 23 G, 25 G +- **Syringes**: 1 ml, 3 ml +- **Cryovials or microcentrifuge tubes**: 0.6 ml, 1.6 ml, 2 ml +- **Anticoagulants and blood preservatives**: 6M Guanidine-HCl 0.2M EDTA +- **Optional**: Warming pad + +### Marking +- **Ear tags**: Uniquely coded mouse ear tags +- **Tag applicator** + +## Procedures + +### Trapping + +1. **Prevent contamination**: + - Wear disposable gloves, a whole-body disposable suit (e.g., Tyvek™), filtered mask, or respirator if diseases are suspected. + +2. **Visual inspection**: + - Inspect areas for signs of small mammals (urine marks, droppings, hair, burrows). + +3. **Trap setup**: + - Handle traps with thick gloves. + - Code each trap uniquely. + - Incorporate bait and bedding material. + - Place traps in a horizontal, stable position in sheltered areas. + +4. **Site marking**: + - Mark the site with a flag or flag tape and note the trap code and geo-coordinates. + +5. **Activation**: + - Activate traps during the target species' maximum activity period (overnight for nocturnal, during resting time for diurnal). + - Monitor frequently to avoid thermal stress. Check every 2-3 hours. + +6. **Animal inspection**: + - Inspect traps for captured animals, releasing non-target species gently. + +7. **Handling trap-happy animals**: + - Remove bait from traps without captures; fold and place in less accessible areas. + +8. **Transport**: + - Transport trapped mammals in a container, ensuring they are not exposed to extreme conditions. + +### Blood-sampling + +9. **Protective equipment**: + - Always wear protective clothing, goggles, and gloves. + +10. **Anesthesia administration**: + - Use isoflurane, ketamine, or xylazine. Refer to dosage and handling guidelines: + [UMT SOP](https://www.umt.edu/research/LAR/sops/SOPodendropsisoflurane.php) + +11. **Blood withdrawal**: + - Collect a minimum of 0.2 ml of peripheral venous blood (1% body weight). + - Use ethanol to clean clipped regions after shaving. + +12. **Anatomical sampling sites**: + - **Ototomys spp., Rattus spp., Abrocoma bennetti**: Saphenous vein (19 or 21G needle). + - **Phyllotis darwini, Mus musculus**: Submandibular zone (21G needle). + - **Thylamys elegans, Oligoryzomys longicaudatus, Abrothrix spp.**: Jugular vein (1 ml 25 G syringe). + - **Oryctolagus cuniculus, Lepus europaeus**: Ear vein (1 ml 25 G syringe). + +13. **Blood sample handling**: + - Mix blood with anticoagulants. + - Store samples immediately in cryovials at room temperature for DNA preservation or centrifuge for serum. + +### Mark and Release + +14. **Individual marking**: + - Use uniquely coded ear tags on both ears. + - Other methods may include hair clipping or identification chips. + +15. **Recovery and release**: + - Transport and release fully recovered animals back to their exact capture point. + +## References + +1. Joslin JO. Blood collection techniques in exotic small mammals. J Exot Pet Med. 2009;18(2):117-139. +2. Avila HA, et al. Polymerase chain reaction amplification of Trypanosoma cruzi kinetoplast minicircle DNA. Mol Biochem Parasitol. 1991;48(2):211-221. + +--- + +_endofoutput_ +``` \ No newline at end of file diff --git a/markdown-output/trpb-passaging-protocol-dgh23t8e.md b/markdown-output/trpb-passaging-protocol-dgh23t8e.md new file mode 100644 index 0000000000000000000000000000000000000000..e6efd8fbd2a224c71bd35f5d891854839681584c --- /dev/null +++ b/markdown-output/trpb-passaging-protocol-dgh23t8e.md @@ -0,0 +1,72 @@ +```markdown +# Goal/Experiment: +Passaging the evolving TrpB populations using the OrthoRep system for continuous directed evolution. These instructions are directed toward maintaining and monitoring the growth of TrpB populations to ensure successful directed evolution. + +# TrpB Passaging Protocol + +**DOI:** [dx.doi.org/10.17504/protocols.io.6qpvr8p3plmk/v1](https://dx.doi.org/10.17504/protocols.io.6qpvr8p3plmk/v1) + +**Author:** Sparrow +**Affiliation:** UIUC + +**Protocol Citation:** is Sparrow 2024. TrpB passaging protocol. *protocols.io*. [dx.doi.org/10.17504/protocols.io.6qpvr8p3plmk/v1](https://dx.doi.org/10.17504/protocols.io.6qpvr8p3plmk/v1) + +**License:** This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. + +**Protocol Status:** Working +**Created:** July 01, 2024 +**Last Modified:** July 08, 2024 +**Protocol Integer ID:** 102682 + +## Disclaimer +**DISCLAIMER – FOR INFORMATIONAL PURPOSES ONLY; USE AT YOUR OWN RISK** + +The protocol content here is for informational purposes only and does not constitute legal, medical, clinical, or safety advice, or otherwise; content added to *protocols.io* is not peer-reviewed and may not have undergone a formal approval of any kind. The information presented in this protocol should not substitute for independent professional judgment, advice, diagnosis, or treatment. Any action you take or refrain from taking using or relying upon the information presented here is strictly at your own risk. You agree that neither the Company nor any of the authors, contributors, administrators, or anyone else associated with *protocols.io*, can be held responsible for your use of the information contained in or linked to this protocol or any of our Sites/Apps and Services. + +## Abstract +Instructions for passaging the evolving TrpB populations using the OrthoRep system for continuous directed evolution. Protocol developed from Rix et al., 2020 and optimized in Burgess lab. Special thanks to Vincent Hu for his aid in troubleshooting. + +## Growth Conditions +1. TrpB evolution-ready strains should be grown in SC-UL with shaking at 220 RPM at 30°C. + - **First growth:** 100 micromolar (µM) Trp. + - **Subsequent growths:** 37 micromolar (µM) Trp. + - Note: It may be a good idea to passage the cells in the same condition for a few times regardless of final OD to help prevent crashes. + +## Passages +2. After ~2 days of growth, the cultures will be saturated, either by excessive growth or tryptophan limitation. + - If cultures are visibly saturated by excessive growth before the 2 days, passaging can be done then. + +3. Aliquot 1 mL of culture into: + - A spectrophotometer cuvette. + - A clear 1.5 mL tube. + - A screw-cap centrifuge tube. + +4. Add 1 mL 50% glycerol to the screw cap tube containing culture, label it, and store at -80°C. + - Note: For strains with poor growth (OD < 0.5), resuspend in 0.5 mL YPD, then add 0.5 mL 50% glycerol. This helps with recovery by concentrating the cells and putting them in friendlier media. + +5. Centrifuge the 1.5 mL tubes for 1 minute at 16000 x g. + - Inspect the pellet visually for contamination (e.g., brown/yellow/green pellet indicates contamination; discard the tubes). + +6. Measure the OD600 of each strain and record it. + - If the strain is above 1.0, passage to a slightly lower tryptophan concentration. + - Trp concentration sequence: 37 > 25 > 20 > 15 > 10 > 7.5 > 5 > 0 works well. + - Note down the Trp concentration used to graph out evolution trajectories. + +7. To passage, prepare a 50 mL Falcon tube containing 10 mL of SC-UL media for each population. + - Don't forget to add His and Glucose to the stock of media. + - Note: Include required additives: + - Filter-sterilized Indole (final concentration = 400 micromolar (µM)). Solubility in water is 16 mM (250 µL for a 10 mL tube). + - Filter-sterilized Tryptophan to the final concentration if needed (e.g., for a final 5 micromolar (µM) concentration, add 5 µL of a 10 mM stock). + - For the wild-type control, add filter-sterilized 100X Uracil (strain is URA-). + +8. After the required additives, add 100 µL of the previous passage to the tube (1:100) and gently invert to mix. + - Label the tubes as appropriate. + - Place tubes to grow in the conditions specified. + +## End of Evolution +9. Once a culture grows in 0 µM Trp, passage it in 0 µM Trp (including indole) 6 additional times to saturate the variant. + - After the 6th passage, make 2 glycerol stocks (one for the evolution box and one for the main yeast box). + - Proceed to gDNA isolation, PCR amplification of p1, and Sanger Sequencing. + +**endofoutput** +``` \ No newline at end of file diff --git a/markdown-output/u-251mg-spheroid-generation-using-hanging-drop-met-btstnnen.md b/markdown-output/u-251mg-spheroid-generation-using-hanging-drop-met-btstnnen.md new file mode 100644 index 0000000000000000000000000000000000000000..0ce140bc9eecddc317b87df461fb2a133cdf0abf --- /dev/null +++ b/markdown-output/u-251mg-spheroid-generation-using-hanging-drop-met-btstnnen.md @@ -0,0 +1,112 @@ +```markdown +# Goal/Experiment: +To develop a protocol for in vitro generation of U-251MG spheroids using the hanging drop method, aimed at mimicking in vivo physiological tissue characteristics for drug testing and analysis. + +# U-251MG Spheroid Generation Using Hanging Drop Method Protocol +**Authors:** Lara J Carroll, Brijesh Tiwari, James F Curtin, Janith Wanigasekara +**Institution:** Technological University Dublin +**DOI:** [10.17504/protocols.io.btstnnen](https://dx.doi.org/10.17504/protocols.io.btstnnen) +**Date:** May 05, 2021 + +## Abstract +The use of 3D cell culture has become a significant approach in developing cellular models that can closely mimic physiological tissues. U-251MG spheroids are grown using the hanging drop method, providing uniform size, low cost, comfort in handling, and suitability for short-term culture. This method applies the Perfecta3D® hanging drop plate, simplifying spheroid formation, testing, and analysis. + +## Advantages of Hanging Drop Method +- Produces uniform-sized spheroids +- Low cost +- Easy to handle +- Suitable for short-term culture + +## Disadvantages +- Medium change, different drug treatment at different time points are labor-intensive + +![Fig. 1: Hanging Drop Plate Technique](image_link_1) + +![Fig. 2: Hanging Drop Plate](image_link_2) + +![Fig. 3: U-251MG Tumorsphere Formation](image_link_3) + +## Materials +- **Mammalian cells (U-251 MG Human glioblastoma astrocytoma cells)** +- **HDP1096 Perfecta3D® 96-Well Hanging Drop Plate (Sigma-Aldrich)** +- **Complete medium:** Dulbecco's Modified Eagle Medium (DMEM) + 10% Fetal Bovine Serum (FBS) + 1% Penicillin-Streptomycin (Sigma-Aldrich) +- **1X Phosphate-buffered saline (PBS)** (Sigma-Aldrich) +- **Trypsin-EDTA solution** (Sigma-Aldrich) +- **Multi-channel micropipette and tips** +- **100-1000µl pipette and pipette tips** +- **Bright-field microscope** +- **Humidified incubator** +- **Centrifuge with swinging bucket rotor** +- **Centrifuge tubes** +- **Cell counter or cytometer** +- **Sterile reagent reservoirs** + +## Safety Warnings +- Laboratory coat and gloves must be worn at all times +- Use laminar flow hood when working with cultures + +## Protocol +1. **Cell Seeding** + - Culture U-251 MG cells in 100mm or 150mm dishes until they reach 80-90% confluence. + - Wash cells with 1X PBS solution. + +2. **Cell Dissociation** + - Incubate cells for 4 minutes at 37°C with Trypsin-EDTA solution (0.25% w/v). + - Inactivate trypsin by re-suspending in full growth medium pre-warmed at 37°C. + +3. **Cell Suspension** + - Centrifuge cell suspension for 5 minutes at 300g. + - Re-suspend the cell-pellet in 1ml of full growth medium. + - Pipette cell suspension up and down to obtain uniform single cell suspension. + +4. **Preparation of Plate** + - Add sterile, room temperature PBS to the reservoirs located on the plate's peripheral rim and tray which are divided into sections. + - Prevent drying out by pipetting 2ml per section and 1ml per tray reservoir section. + + *Note: Avoid tilting the plate to prevent spillage and contamination.* + +5. **Cell Suspension in Plate** + - Prepare 10ml stock solution of U-251MG cell suspension in full growth medium. + - Target concentration: 2.5 x 10^5 cells/ml to achieve 5000 U-251MG cells per 20µl hanging drop. + - Thoroughly mix the cell suspension. + +6. **Creating Hanging Drops** + - Carefully pipette 20-50µl of cell suspension into the center of each well from the top side of the plate. + - Use a multi-channel pipette and ensure only 2-4 drops per access point. + +7. **Incubation** + - Place the lid on the plate. + - Insert into a humidified incubator at 37°C and 5% CO2. + - Monitor formation for a minimum of 4 days using the bright-field microscope. + + *Note: Exercise caution when transporting plates to prevent shift or fall of spheroids.* + +8. **Media Exchange** + - Daily exchange required to maintain nutrient levels and osmolality. + - Add 5µl of fresh media into each hanging drop via the access holes of the plate. + +9. **Transfer of Tumorspheres** + - Once formed, transfer tumorspheres to low attachment plates/pre-coated wells. + - Add 50µl of fresh media into each hole. + +## References +1. [Hikage et al.](https://academic.oup.com/endo/article/160/1/20/5150427) +2. [Moraes et al.](https://www.sciencedirect.com/science/article/pii/S0171933520300340) +3. [Sigma-Aldrich Protocol](https://www.sigmaaldrich.com/technical-documents/protocols/biology/perfecta3d-hdp1096-media-exchange-protocol.html) + +## Licensing +This protocol is distributed under the terms of the [Creative Commons Attribution License](https://creativecommons.org/licenses/by/4.0/). + +**Citation:** +Lara J Carroll, Brijesh Tiwari, James F Curtin, Janith Wanigasekara (2021). U-251MG Spheroid Generation Using Hanging Drop Method Protocol. [dx.doi.org/10.17504/protocols.io.btstnnen](https://dx.doi.org/10.17504/protocols.io.btstnnen) + +**Ownership History:** +- Mar 30, 2021 by Lara Carroll +- May 05, 2021 by James Curtin +- Technological University Dublin + +Created Mar 30, 2021 +Last Modified May 05, 2021 + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/u-mass-hyperinsulinemic-euglycemic-clamp-xzefp3e.md b/markdown-output/u-mass-hyperinsulinemic-euglycemic-clamp-xzefp3e.md new file mode 100644 index 0000000000000000000000000000000000000000..a2ba11fe7b842eb509263b282fd7630dec6a83f1 --- /dev/null +++ b/markdown-output/u-mass-hyperinsulinemic-euglycemic-clamp-xzefp3e.md @@ -0,0 +1,129 @@ +```markdown +# Goal/Experiment: +The goal of this experiment is to assess insulin sensitivity using the hyperinsulinemic-euglycemic clamp method in mice. This technique is widely used for measuring insulin action on glucose utilization in both clinical and basic science research. By incorporating radioactively-labeled glucose, this method enables the measurement of glucose metabolism in individual organs of awake mice, critical for understanding insulin resistance and obesity. + +## U Mass - Hyperinsulinemic-Euglycemic Clamp Protocol + +**Researcher:** Jason Kim[^1] +**Institution:** University of Massachusetts +**Published Date:** May 10, 2019 +**Protocol DOI:** [dx.doi.org/10.17504/protocols.io.xzfep3e](dx.doi.org/10.17504/protocols.io.xzfep3e) +**External Link:** [MMPC Protocol](https://mmpc.org/shared/document.aspx?id=136&docType=Protocol) + +### Summary: +Hyperinsulinemic-euglycemic clamp is the gold-standard method to assess insulin sensitivity. This technique involves continuous infusion of insulin to raise plasma insulin levels while concomitantly infusing glucose to maintain euglycemia. This setup allows for the measurement of insulin-stimulated glucose uptake and metabolism in various tissues, particularly in the context of insulin resistance and type 2 diabetes. + +### Materials: +| **Name** | **Catalog #** | **Vendor** | **CAS Number** | **RRID** | +|------------------------------------------------------|------------------------|-------------------------------------|----------------------|-------------------------| +| HelixMark Standard Silicone Tubing | 0.012" ID / 0.025" OD | Helix Medical, Inc. | | | +| [3-3H] D-glucose | NET331C005MC | Perkin Elmer | | | +| [2-1-14C] Deoxy-D-glucos | NEC49500IMC | Perkin Elmer | | | +| Pentobarbital | NDC76478-501-50 | Oak Pharmaceuticals, Inc. | | | +| Microdialysis pumps | CMA 402 | CMA/Microdialysis | | | +| Analox GM7 Micro-stat Rapid Multi-assay Analyser | GM7 | Analox | | | +| Insulin | Regular human insulin, U-100 | Novolin | | | +| 20% Dextrose Injection USP | NDC0409-7935-19 | Hospira(Pfizer) | | | +| 0.9% Sodium Chloride Injection USP | NDC0264-4001-55 | B.Braun Medical Inc. | | | +| 1 ml tuberculin syringes | REF 309659 | BD Biosciences | | | +| Microhematocrit capillary tubes | 22-362-566 | Fisher Scientific | | | +| Heparin-coated blue polyethylene open-top tubes | 652825 | Beckman Coulter | | | +| Microcentrifuge tubes (1.5 ml) | C-2170 | Denville Scientific Inc. | | | + +### Detailed Protocol: +1. **Surgical Preparation:** + - Perform survival surgery to establish a chronic indwelling catheter 5-6 days prior to the experiment for intravenous infusion. Refer to MMPC Protocol M1023: Surgery-jugular vein cannulation. + +2. **Fasting:** + - Fast the mice overnight (~15 hours) or for 5 hours before the start of the experiment. + +3. **Mouse Placement:** + - Place the mouse in a rat-size restrainer with its tail tape-tethered at one end. + +4. **Catheter Preparation:** + - Expose and flush the intravenous catheter using a saline solution. Connect the catheter to the CMA Microdialysis infusion pump. + +5. **Acclimation:** + - During the 2-hour acclimation period, infuse D-[3-3H] glucose at 0.05 µCi/min to measure the basal rate of whole-body glucose turnover. + +6. **Basal Parameter Collection:** + - Collect a plasma sample (30 µl) for the measurement of plasma glucose, insulin, and [3-3H] glucose concentrations at the end of the acclimation period. + +7. **Insulin Infusion:** + - Start a 2-hour hyperinsulinemic-euglycemic clamp with a primed (150 mU/kg body weight) and continuous infusion of human insulin at 2.5 mU/kg/min to increase plasma insulin levels. + +8. **Euglycemic Maintenance:** + - Infuse 20% dextrose at variable rates to maintain euglycemia throughout the 2-hour clamp. + +9. **Whole Body Glucose Turnover:** + - Estimate insulin-stimulated whole-body glucose turnover rates using continuous infusion of [3-3H] glucose at 0.1 µCi/min throughout the clamp. + +10. **Plasma Sampling:** + - Collect plasma samples (10 µl) at 20, 40, 60, 70, 90, 100, 110, and 120 minutes to measure plasma glucose concentrations. + +11. **Adjust Glucose Infusions:** + - Adjust glucose infusion rates based on instantaneous glucose levels to maintain euglycemia. + +12. **Organ-Specific Glucose Uptake:** + - Administer a bolus injection of 10 µCi of 2-deoxy-D-[1-14C] glucose at 75 minutes to estimate insulin-stimulated glucose uptake in individual organs. + +13. **Sample Processing:** + - Centrifuge plasma samples for 5 minutes at 12,000g (~14,000 rpm), label samples, and prepare for scintillation counts. + +14. **End of Experiment Plasma Sample:** + - Collect an additional plasma sample at the end of the clamp (120 minutes). + +15. **Animal Sacrifice and Tissue Collection:** + - Anesthetize mice using pentobarbital, quickly dissect, and collect tissues from hindlimbs, white and brown adipose tissues, liver, and heart for biochemical analysis. + +16. **Freezing and Storage:** + - Rapidly freeze-clamp tissues in liquid nitrogen and store in -80°C freezer. + +17. **Radiolabel Assay:** + - Measure plasma samples for [3-3H] D-glucose, 2-deoxy-D-[1-14C] glucose, and H2O content using a Beckman Coulter Scintillation Counter. + +18. **Glycolysis Calculation:** + - Calculate H2O concentration differences between Dry and Non-Dry samples to estimate whole-body glycolysis. + +19. **Glucose Uptake Measurement:** + - Refer to MMPC Protocol M1003 for organ-specific glucose uptake. + +20. **Hepatic Glucose Production (HGP):** + - Determine basal and insulin-stimulated HGP from plasma glucose concentrations at designated time points. + +21. **Whole-Body Glycolysis:** + - Use linear regression of H2O concentration over time to calculate whole-body glycolysis rates. + +22. **Glycogen Synthesis Estimate:** + - Subtract whole-body glycolysis rates from whole-body glucose turnover to estimate glycogen plus lipid synthesis. + +### Supplementary Information: + +#### Professional/Scientific Terms and Reagents: +1. **Hyperinsulinemic-Euglycemic Clamp:** + A method for measuring insulin sensitivity by infusing insulin to elevate plasma levels and glucose to maintain stable blood glucose (euglycemia). + +2. **[3-3H] D-Glucose and [2-14C] Deoxy-D-Glucose:** + Radioactively-labeled glucose isotopes used to trace and measure glucose metabolism. + +3. **Insulin:** + A hormone used to elevate plasma insulin levels during the clamp to assess insulin sensitivity. + +4. **Microdialysis Pumps:** + Devices used to deliver continuous and precise fluid infusions necessary for the clamp procedure. + +#### Alternative Methods: +- **Surgery-Jugular Vein Cannulation:** + Different protocols or anesthesia methods can be used for catheter insertion to minimize variability. + +- **Radioactive Glucose Isotopes:** + Non-radioactive glucose analogs can sometimes be used with appropriate detection methods in less stringent studies. + +### References: +- **Hosira:** [RRID:SCR_003985](https://scicrunch.org/resolver/SCR_003985) +- **Fisher Scientific:** [RRID:SCR_008452](https://scicrunch.org/resolver/SCR_008452) +- **BD Biosciences:** [RRID:SCR_013311](https://scicrunch.org/resolver/SCR_013311) +- **Beckman Coulter:** [RRID:SCR_008940](https://scicrunch.org/resolver/SCR_008940) + +endofoutput +``` diff --git a/markdown-output/u-michigan-podocyte-counting-and-density-analysis-565g9g6.md b/markdown-output/u-michigan-podocyte-counting-and-density-analysis-565g9g6.md new file mode 100644 index 0000000000000000000000000000000000000000..0543d93e55f2c6f7141c6739d432eb1a7fc71457 --- /dev/null +++ b/markdown-output/u-michigan-podocyte-counting-and-density-analysis-565g9g6.md @@ -0,0 +1,137 @@ +```markdown +# U Michigan - Podocyte Counting And Density Analysis V.2 + +**Authors:** Jeff Hodgin, Jharna Saha +**Institution:** University of Michigan - Ann Arbor +**Date:** August 07, 2019 +**Provided by:** Mouse Metabolic Phenotyping Centers + +## Goal/Experiment: + +The goal of this experiment is to stain and count podocyte nuclei in mouse glomeruli for diabetic nephropathy study. + +## Summary: +This protocol is used to stain and count podocyte nuclei in mouse glomeruli for diabetic nephropathy study. + +**External Link:** [Protocol](https://mmpc.org/shared/document.aspx?id=322&docType=Protocol) + +## Materials + +| Name | Catalog # | Vendor | +|------------------------------------------|---------------------|-------------------| +| MACH3 Rabbit HRP-Polymer Detection Kit | M3R531L | Biocare Medical | +| Da Vinci Green Diluent | PD900L | Biocare Medical | +| Peroxidase 1 | PX968H | Biocare Medical | +| Betazoid DAB-CHROMOGENK Kit | BDB2004L | Biocare Medical | +| Tachas Auto Hematoxylin | NMHHEM | Biocare Medical | +| Background Sniper | BS966 | Biocare Medical | +| Xylene | X3P-1GAL | Fisher Scientific | +| Ethanol (EtOH) 200 Proof | 2701 | Decon-Laboratories Inc | +| ImmEdge hydrophobic barrier pap pen | H-4000 | Vector Laboratories | +| Humidified chamber | Customized by lab personnel | | +| Mounting Medium | 8312-4 | Thermo Scientific | +| Cover Glass | 12-542-B | Fisher Scientific | +| PT Link Module | A80400012 | Thermo Scientific | +| Universal Imaging Metamorph Imaging System| | Molecular Devices | +| Scientific grade digital color CCD camera| | RT SLIDER DIAGNOSTIC| +| Perfusion-fixed paraffin embedded sections| | | +| Microscope and Lense | Leica DM IRB | Leica Microsystems| + +## Reagent Preparation: + +### Reagent 1: 20x TBST Wash Buffer (working concentration 1x) + +**Reagents and Materials:** +- Tris-base +- Sodium chloride +- Tween-20 +- HCl +- Distilled water + +**Procedure:** +- Tris-base: 48.4g +- Sodium chloride: 160g +- Tween-20: 10 ml +- Mix to dissolve +- Adjust pH 7.4 with concentrated hydrochloric acid (HCl) before adding Tween-20 +- Distilled water up to 1L +- Store at room temperature + +**Note:** +- Biocare Medical, RRID:SCR-013549 +- Thermo Fisher Scientific/ Fisher Scientific, RRID:SCR_008452 +- Vector Laboratories, RRID:SCR_000821 +- Vector Laboratories Cat# H-4000, RRID:AB_2336517 +- Leica Microsystems, RRID:SCR_008960 + +## Protocol 1: +### Podocyte Nuclei Staining with Wilms'Tumor-1 (WT-1) Protein: + +1. Bake slides for 10 minutes at 60°C if it is not baked previously +2. Wash 2x in xylene for 5 minutes +3. Wash 2x in 100% EtOH for 3 minutes +4. Wash 1x in 95% EtOH for 3 minutes +5. Wash 1x in 70% EtOH for 3 minutes +6. Rinse with diH₂O +7. Place in PT Link containing 1x antigen retrieval reagent, pH 9, incubate at 97°C for 20 minutes, and wait until machine temp reaches 70°C. +8. Wash in TBST for 5 minutes +9. Use Pap Pen to encircle tissue +10. Block with Peroxidase 1 for 5 minutes +11. Wash 2x in 1x TBST for 2 minutes +12. Block with Background Sniper for 5 minutes +13. Wash 2x in TBST for 2 minutes +14. Dilute WT-1 antibody in Da Vinci Green diluent (1:200) +15. Incubate for 2 hours at room temperature in a humidified chamber. +16. Wash 2x in TBST for 2 minutes +17. Incubate with Mach 3 Rabbit Probe for 20 minutes +18. Wash 2x in TBST for 2 minutes +19. Incubate with Mach 3 Rabbit HRP Polymer for 20 minutes +20. Wash 2x in TBST for 3 minutes +21. Develop with Betazoid DAB-CHROMOGENK for 5 minutes, place slide under the microscope to check staining intensity +22. Stop reaction in diH₂O +23. Counterstain with Tacha's Hematoxylin for 3 minutes +24. Rinse in diH₂O to check staining intensity – reapply hematoxylin if not dark enough +25. Rinse in TBST to blue +26. Wash with diH₂O +27. Wash in 70% EtOH for 2 minutes +28. Wash in 95% EtOH for 2 minutes +29. Wash 2x in 100% EtOH for 2 minutes +30. Wash in xylene for 8 minutes +31. Mount slides with mounting medium - 1 drop +32. Insert cover glass carefully, avoid bubbles + +## Protocol 2: +### Podocyte Counting and Density Analysis: + +**Pre-Operating Instructions:** +- Camera and Microscope should be calibrated and values loaded into MetaMorph® Program. + +**Procedure:** +1. Photograph 50 consecutive glomerular cross-sections moving systematically from outer cortex to inner cortex and back so as to provide an equal sample from all cortical regions. Use phase contrast which gives podocyte nuclei golden color that makes them easier to count. + +2. From these photomicrographs (which contain 1-3 glomeruli per photograph in about 30 photographs), measure the glomerular area by using Metamorph Image Analysis Software (version 6.1) and count the podocytes in 50 sequential glomerular cross-sections at two thicknesses (3 and 9 microns). + +3. Measure the glomerular area by using Metamorph software (camera and microscope should be calibrated and values should be loaded into Metamorph program before outlining the glomerular tuft area). Click on the desktop icon (metamorph software icon) to open the metamorph program, and then open the images. From the menu bar, select Measure, then select Calibrate Distance. A calibration window will appear. Highlight the 40x calibration and then click on Apply. Use Polygon tool from menu bar for outlining the glomerular tuft area. From the menu bar, select Measure and then Region Measurement to the measurement of the glomerular tuft area. + +4. Use a systematic method (large size cut-off) which helps to count WT-1 positive podocyte nuclei but eliminates false counting of granules. From the menu bar, choose Measure, then Manually Count Objects. Select the number 6. Use this size restriction method to check if the number 6 from the Metamorph Image Analysis System fits within the nuclear profile. If so, count it. If the number 6 was larger than the WT-1 positive area, do not count it. + +**Data Analysis:** +1. Count the podocyte numbers (P) and measure the glomerular tuft area (GA) from 100 consecutive cross-sections per animal (50 from thick and 50 from thin section) then calculate the average for each set of 50. + +2. Divide the average podocyte number (P) by the average glomerular area (GA) to get podocyte number per glomerular area (P/GA) for both 3 and 9 micron sections. + +3. The difference between the P/GA of the thick and the P/GA of the thin sections yielded the P/GA Δ which is directly related to the actual difference in section thickness (6.3um). + +4. Calculate the average glomerular volume per podocyte (GV/P) by dividing the actual section thickness of 6.3 by the P/GA Δ. + +5. Determine the glomerular volume by using the Weibel formula. First, calculate the glomerular radius of both thick and thin sections by assuming circular cross-sections using formula: + \( \text{radius R} = (\text{GA}/\pi)^{1/2} \) + and then calculate the average radius (Rav) that yielded the average maximum radius: + \( R_{\text{max}} = 4R_{\text{av}}/\pi \). + Then calculate average glomerular volume as + \( GV = 4/3\pi (R_{\text{max}})^3 \). + Then divide the average glomerular volume by the average volume per podocyte (GV/P) to get the podocyte number per glomerulus: + \( P = GV/(GV/P) \). + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/ultra-deep-atac-seq-for-sorted-neurons-dcaj2scn.md b/markdown-output/ultra-deep-atac-seq-for-sorted-neurons-dcaj2scn.md new file mode 100644 index 0000000000000000000000000000000000000000..72013761d5564c75bbbc4d4f3427a3816c4add16 --- /dev/null +++ b/markdown-output/ultra-deep-atac-seq-for-sorted-neurons-dcaj2scn.md @@ -0,0 +1,176 @@ +```markdown +Goal/Experiment: +Isolation of neurons from frozen post-mortem human brain tissue and preparation of ATAC-seq libraries for ultra-deep sequencing and somatic mutation detection. + +# Ultra-Deep ATAC-Seq for Sorted Neurons + +**DOI**: [10.17504/protocols.io.eq2lyw35rvx9/v1](https://dx.doi.org/10.17504/protocols.io.eq2lyw35rvx9/v1) + +**Authors**: Andrea Kriz, Alisa Mo +**Institution**: Boston Children's Hospital +**Protocol status**: Working + +*Protocol Citation*: Andrea Kriz, Alisa Mo 2024. Ultra-deep ATAC-seq for sorted neurons. protocols.io doi: [10.17504/protocols.io.eq2lyw35rvx9/v1](https://dx.doi.org/10.17504/protocols.io.eq2lyw35rvx9/v1) + +**License**: This protocol is distributed under the Creative Commons Attribution License. + +**Created**: April 17, 2024 +**Last Modified**: April 23, 2024 + +## Abstract +Isolation of neurons from frozen post-mortem human brain tissue and preparation of ATAC-seq libraries for ultra-deep sequencing and somatic mutation detection. + +## Protocol Materials + +- **Tagmentase (Unloaded)** Diagenode Catalog #C01070010-20 +- **Tagmentation Buffer (2X)** Diagenode Catalog #C01019043-5000 +- **Anti-NeuN Antibody, clone A60, Alexa Fluor®488 conjugated**, Merck MilliporeSigma (Sigma-Aldrich) Catalog #MAB377X +- **DAPI Solution (1 mg/mL)** Thermo Fisher Scientific Catalog #62248 +- **DNA Clean & Concentrator-5 (capped)** Zymo Research Catalog #D4014 +- **Nextera XT Index Kit v2 Set B (96 indexes, 384 samples)**, Illumina, Inc. Catalog #FC-131-2002 +- **NEBNext High-Fidelity 2X PCR Master Mix - 250 rxns** New England Biolabs Catalog #M0541L + +## Protocol + +### Isolation of Nuclei from Adult Human Brain Tissue (modified from Allen Human Brain Tissue PF0291) + +#### Reagent List: +- **Tagmentase (Unloaded)** Diagenode Catalog #C01070010-20 +- **Tagmentation Buffer (2X)** Diagenode Catalog #C01019043-5000 +- **Anti-NeuN Antibody, clone A60, Alexa Fluor®488 conjugated** Merck MilliporeSigma (Sigma-Aldrich) Catalog #MAB377X +- **DAPI Solution (1 mg/mL)** Thermo Fisher Scientific Catalog #62248 +- **DNA Clean & Concentrator-5 (capped)** Zymo Research Catalog #D4014 +- **Nextera XT Index Kit v2 Set B (96 indexes, 384 samples)** Illumina, Inc. Catalog #FC-131-2002 +- **NEBNext High-Fidelity 2X PCR Master Mix - 250 rxns** New England Biolabs Catalog #M0541L + +#### Buffer Preparation: +**Nuclei Isolation Media (NIM):** + +| Formula (in milliQ water) | Ingredients | For 1.5 mL (1 ATAC-seq brain) | For 5 mL (3 ATAC-seq brains) | For 20 mL | +|----------------------------|-------------|------------------------------:|----------------------------:|-----------:| +| 0.25M Sucrose | Sucrose | 0.13 g | 0.43 g | 1.72 g | +| 25mM KCl | 2M KCl | 18.8 µL | 62.7 µL | 250.8 µL | +| 5mM MgCl2 | 1M MgCl2 | 7.5 µL | 25 µL | 100 µL | +| 10mM Tris-HCl, pH 8 | 1M Tris-HCl | 15 µL | 50 µL | 200 µL | +| 0.1% Triton X-100 | 10% Triton X-100 | 15 µL | 50 µL | 200 µL | +| Water | Water | ~1410 µL | ~4.7 mL | 18.8 mL | + +Filter through 0.22 µM filter and store at 4°C. Can keep for ~1-2 weeks. + +**Blocking buffer:** + +| Formula (in milliQ water) | Ingredients | For 5 mL | For 10 mL | For 20 mL | +|----------------|-------------|-----------:|----------:|----------:| +| Water | Water | 4.3 mL | 8.6 mL | 17.2 mL | +| 1X PBS | 10X PBS | 500 µL | 1000 µL | 2000 µL | +| 0.8% BSA | 20% BSA | 200 µL | 400 µL | 800 µL | + +Store at 4°C. Can keep for ~1-2 weeks. + +**1X Tagmentation buffer w/ BSA for ATAC-seq:** + +| Ingredients | For 4 ATAC-seq reactions | For 6 ATAC-seq reactions | +|---------------------|-------------------------:|--------------------------:| +| 2X Tagmentation buffer (Diagenode) | 220 µL | 330 µL | +| 5% digitonin | 0.88 µL | 1.3 µL | +| 10% Tween-20 | 4.4 µL | 6.6 µL | +| PBS | 145 µL | 218 µL | +| 20% BSA | 22 µL | 33 µL | +| Water | 25.5 µL | 38.3 µL | + +Place at 4°C until ready to use. + +#### Procedure: + +1. **Load Tn5**: + - Mix 1 µL of annealed oligo A/oligo Rev with 1 µL of the annealed oligo B/oligo Rev as per Diagenode instructions. + - Add 2 µL of Diagenode Tagmentase. + - Incubate at RT for 30 min. + - Add 2 µL glycerol. Place at -20°C until ready for use. Can store for a few weeks. + +2. **Tissue Preparation**: + - Chill tweezers, scalpel, and dissecting plate (cell culture plate) on dry ice. Chill dounce homogenizers with B pestle on ice. Ensure swinging-bucket centrifuge is cooled to 4°C. + - Spray down all surfaces and pipettes with 70% Ethanol and DNA Away. + - **Homogenization buffer (NIM + additives)**: 5mL NIM + 5 µL 1M DTT (1 mM final concentration) + Mini Protease inhibitor (1/2 tablet) + 25 µL 100 mM spermidine (0.5 mM final concentration). + - Scrape ~10mg frozen brain tissue onto cell culture plate, either on top of dry ice or in a cryostat at -20°C. Add 1.5 mL Homogenization buffer to tissue. Add tissue/buffer mix to Dounce homogenizer. + - Dounce 15-20 strokes with B pestle. If the tissue is large, use more homogenization buffer (use up to 5 mL) and dounce with A pestle followed by B pestle. + - Filter through 40 µM filter into Eppendorf tube. + - Spin in 4°C swinging-bucket centrifuge for 10min @ 900xg. + +3. While spin is going on, clean homogenizers by rinsing thoroughly with ddH2O, and then soaking them in 20% bleach for at least 20 minutes. + +4. **Immunostaining buffer**: + - Preparation: Blocking buffer + 1:1000 dilution NeuN-488. + +5. Remove supernatant. Do not disturb the pellet. +6. Add 1mL Blocking buffer + NeuN. Resuspend pellet gently with pipette. Rotate end-to-end in the cold room for 15-20 minutes. +7. Spin in 4°C swinging-bucket centrifuge for 5min @ 400xg. +8. **DAPI Solution**: + - First, dilute stock DAPI (1mg/mL) 1:15 in blocking buffer. Then, add 1 µL of the diluted DAPI solution to 1 mL blocking buffer. + +9. Remove supernatant from the pellet. +10. Add 1mL Blocking buffer + DAPI and again resuspend. Strain through 40 µM filter into a new Eppendorf tube immediately before nuclei sorting. +11. Prepare tubes for ATAC-seq: Add 60 µL of 1X Tagmentation buffer into each tube for ATAC-seq (2 tubes per brain). + +### ATAC-seq (modified from Omni-ATAC protocol (Corces et al., 2017)) + +1. Sort 10,000 NeuN+ nuclei into 60 µL of tagmentation buffer per ATAC-seq (2 replicates per brain sample). Proceed immediately after sorting to ATAC-seq. +2. After sorting, spin each tube in 4°C swinging bucket centrifuge at 500xg for 5 minutes. +3. Remove ~75 µL of supernatant leaving ~10 µL at bottom. Tap tube gently to mix the pellet. +4. Add 45 µL of 1X Tagmentation buffer. Do not mix. +5. Add 1 µL of loaded Tn5. Pipette gently 3-4 times and tap to mix. +6. Incubate at 37°C for 30 minutes in the Thermomixer without mixing. Gently tap the tube at ~10 minutes and at ~20 minutes into the incubation to mix. +7. Stop tagmentation reaction by adding 250 µL of DNA binding buffer from Zymo Clean and Concentrator 5 kit. Add to the sample tube, pipette 6x or vortex until homogeneous. +8. Follow Zymo kit instructions to purify DNA: + - Add solution to spin-column tube. Spin at 10,000xg for 30 seconds. + - Wash x 200 µL wash buffer. Spin at 10,000xg for 30 seconds. + - Repeat wash x 200 µL wash buffer. Spin at 10,000xg for 30 seconds. + - Transfer spin-column tube into Eppendorf tube. Add 20 µL of water. Let sit for 1 minute. Then spin at 10,000xg for 1 minute. + +9. **Prepare PCR Reaction on ice**: + - 2.5 µL i5 primer + - 2.5 µL i7 primer + - 25 µL NEBNext High-Fidelity 2X PCR Master Mix, thaw on ice and invert until homogeneous. + - 20 µL purified tagmented DNA + +10. **PCR Amplification**: + 1. 72°C x 5 minutes. + 2. 98°C x 30 seconds. + 3. 98°C x 10 seconds. + 4. 63°C x 30 seconds. + 5. 72°C x 1 minute. + 6. Loop back to step (3) 8 times (9 cycles total). + 7. 72°C x 1 minute. + 8. 4°C hold. + +11. Clean the library using Zymo clean and concentrator 5 kit. Add 250 µL of DNA binding buffer to PCR reaction and follow Zymo kit directions to purify. +10. Elute in 40-50 µL 0.1X TE (or Zymo elution buffer). Let sit for 1 minute, spin for 1 minute. +11. Run a 1:4 dilution of library on HS5000 Tapestation. + +### Expected Result +Library traces from frozen brain tissues are quite variable. In general, a nucleosomal ladder should be apparent with a sub-nucleosomal peak around ~170bp (keep in mind the size of the adapters is around ~100bp, so this corresponds to an insert size of ~70bp), mono-nucleosomal peak around ~300bp, di-nucleosomal peak around ~450bp, tri-nucleosomal peak. +The strength of the nucleosome ladder relative to larger molecular weight fragments can differ greatly between samples (and even between replicates). We have had success sequencing ATAC libraries with traces seen below. Note that for brain samples, the mono-nucleosomal peak should be larger than the sub-nucleosomal peak. An overly large sub-nucleosomal peak can indicate degradation of chromatin structure. + +**Representative Traces of Libraries**: + +- ATAC library with very apparent nucleosomal ladder +![Nucleosomal Ladder](image1.png) + +- ATAC library with a greater quantity of large molecular weight fragments relative to nucleosomal ladder +![Large Fragments](image2.png) + +- ATAC library with a nucleosomal ladder as well as a comparable quantity of large molecular weight fragments +![Ladder and Large Fragments](image3.png) + +Store libraries at -20°C. + +## Protocol References + +**Corces MR, Trevino AE, Hamilton EG, Greenside PG, Sinnott-Armstrong NA, Vesuna S, Satpathy AT, Rubin AJ, Montine KS, Wu B, Kathiria A, Cho SW, Mumbach MR, Carter AC, Kasowski M, Orloff LA, Risca VI, Kundaje A, Khavari PA, Montine TJ, Greenleaf WJ, Chang HY** (2017). An improved ATAC-seq protocol reduces background and enables interrogation of frozen tissues. [Nat Methods](https://doi.org/10.1038/nmeth.4396). + +**LeinU01 BRAIN grant**. Isolation of Nuclei from Adult Human Brain Tissue for 10x Genomics Platform. protocols.io. [Protocol](https://protocols.io/view/isolation-of-nuclei-from-adult-human-brain-tissue-y6rfzd6). + +**Matevossian A, Akbarian S** (2008). Neuronal nuclei isolation from human postmortem brain tissue. [Journal](https://doi.org/10.3791/914). + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/ultrasound-96-probe-device-protocol-for-cancer-cel-b2eeqbbe.md b/markdown-output/ultrasound-96-probe-device-protocol-for-cancer-cel-b2eeqbbe.md new file mode 100644 index 0000000000000000000000000000000000000000..ceb9d1476de65eb28d77532ca0be8960a4982c08 --- /dev/null +++ b/markdown-output/ultrasound-96-probe-device-protocol-for-cancer-cel-b2eeqbbe.md @@ -0,0 +1,79 @@ +```markdown +# Goal/Experiment: +To utilize an ultrasound probe device to trigger the release of liposomes in glioblastoma cells, which can be applied for cancer treatment by enhancing chemotherapeutic delivery. + +# Ultrasound 96 Probe Device Protocol for Cancer Cell Treatment + +**Aisling Field1, Brijeshtiwar2, James F. Curtin1, Julie Rose Mae Mondala1, Janith Wanigasekara1** + +1Technological University Dublin, 2Teagasc Food Research Centre Dublin + +**Technological University Dublin** + +**TU Dublin** + +## Abstract + +Ultrasound is a sound wave with frequencies ranging between 20 kHz and 20 MHz. It is able to temporarily and repeatedly open the blood-brain barrier (BBB) safely and enhance chemotherapeutic delivery without adverse effects (Deprez et al., 2021). This novel technique in drug delivery benefits from the powerful ability of ultrasound to produce cavitation activity. + +**Cavitation** is the generation and activity of gas-filled bubbles in a medium exposed to ultrasound. As the pressure wave passes through the media, gas bubbles expand at low pressure and contract at high pressure. This leads to oscillation which produces a circulating fluid flow known as microstreaming around the bubble with velocities and shear rates proportional to the amplitude of the oscillation. At high amplitudes, the associated shear forces can cut open liposomes (Wanigasekara et al., 2021). + +Vesicles denser than the surrounding liquid are drawn into the shear field surrounding an oscillating bubble. If the shear stress is greater than the strength of the vesicle, it will burst and spill its contents. In a liposome, the vesicle will reform, often at a smaller size than before meeting the shear field. Hence, some interior liquid must be released during the breakdown (Pitt et al., 2004). + +This protocol describes the use of an ultrasound probe to trigger the release of liposomes in glioblastoma cells using the following parameters: +- **Time**: 3 min, +- **Pulse**: 59/01, +- **Amplitude**: 20%. + +The Sonics VCX 750 ultrasonic liquid processor is used for this experiment. It is an easy and reliable technique, suitable for drug delivery and other applications such as oncology. + +## Materials + +- VCX 750 ultrasonic liquid processor +- 96 probe system +- 96 well plate +- Retort stand +- Laboratory jack +- Sonics - Vibra cell power unit +- 96-well seed plate +- Nunclon™ Sphera™ 96-Well plate +- Incubator +- Fresh media +- Dimethyl sulfoxide (DMSO) (20%) + +## Safety Warnings + +- Laboratory coat and gloves must be worn at all times. +- Ear protection must be worn due to the high-frequency sound level of the VCX 750 ultrasonic liquid processor. +- Maintain a safe distance between the operator and the VCX 750 while in operation. +- Signage should notify others that an ultrasound device is in operation. + +## Procedure + +1. **Preparation:** Before starting the ultrasound probe system, ensure all parts of the system/device are free from mechanical damage and the probe is connected tightly. + +2. **Setup and Preliminary Checks:** The 96-probe system, designed to fit into the 96 well plate for cancer cells, must be secured via a retort stand and laboratory jack. Ensure the jack positions the cells correctly for the ultrasound application. + +3. **Power Unit Configuration:** Connect the Sonics - Vibra cell power unit to the ultrasound 96 probe system. Set the parameters: + - Treatment time: **3 min** + - Pulse: **59 seconds on** / **1 second off** + - Amplitude: **20%** + +4. **Cell Culture Preparation:** + - **2D Cell Culture Assays**: Seed cells in 100 µl of media in a 96-well seed plate. + - **3D Cell Culture Assays**: Seed cells in 200 µl of media in a Nunclon™ Sphera™ 96-Well plate. Leave 2D cells to adhere overnight at 37°C in a humidified atmosphere at a density of 2 x 10^3 cells/well or 1 x 10^4 cells/well for 96 hours post-treatment and 24 hours post-treatment incubation. + +5. **Media Addition for Ultrasound Treatment:** On the day of ultrasound treatment, remove the culture media from the plates and add 100 µl fresh media to each plate ensuring the probe tips are immersed. + +6. **Placement and Treatment:** Place the plate (without lid) in the lab jack’s center and move it into the 96 probe system, maintaining 2mm distance from the probe tip. Start the device. + +7. **Post-Treatment and Analysis:** Following treatment, remove sample plates from the probe system and incubate at 37°C with 5% CO₂. DMSO (20%) serves as a positive control. Post-treatment, ultrasound-treated cells/tumorspheres are ready for further analysis. + +## References + +[Deprez et al., 2021](https://dx.doi.org/10.17504/protocols.io.e6nwkpdwvmk/v1) +[Pitt et al., 2004](https://doi.org/10.1517/17425247.1.1.37) +[Wanigasekara et al., 2021](https://doi.org/10.1016/j.trcan.2021.07.004) + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/university-of-helsinki-and-natural-resources-insti-bp7umrnw.md b/markdown-output/university-of-helsinki-and-natural-resources-insti-bp7umrnw.md new file mode 100644 index 0000000000000000000000000000000000000000..7e3630efe6db2f4f228122b6dd7d2fc718335926 --- /dev/null +++ b/markdown-output/university-of-helsinki-and-natural-resources-insti-bp7umrnw.md @@ -0,0 +1,103 @@ +```markdown +# Goal/Experiment: +The goal of this experiment is to extract DNA and perform multiplex PCR genotyping of 17 microsatellites for Atlantic salmon (Salmo salar L.). + +# University of Helsinki and Natural Resources Institute Finland (Luke) Protocol for DNA Extraction and Multiplex PCR Genotyping of 17 Microsatellites for Atlantic Salmon (Salmo salar L.) + +**Authors**: Jarmo Koskiniemi, Marja-Liisa Koljonen, Tuomas Leinonen +**Affiliations**: +- University of Helsinki +- Natural Resources Institute Finland (Luke) +**DOI**: [10.17504/protocols.io.bp7umrnw](dx.doi.org/10.17504/protocols.io.bp7umrnw) + +## Abstract +In this protocol, we describe laboratory methods for DNA extraction and multiplex genotyping of Atlantic salmon with microsatellite markers. The protocol has been used in several studies at the University of Helsinki and the Natural Resources Institute Finland (Luke). Publications from these studies are listed in the attachment. + +## Keywords +Atlantic salmon, Salmo salar, DNA extraction, multiplex PCR, genotyping, microsatellite, genetic variation + +## License +This protocol is distributed under the terms of the [Creative Commons Attribution License (CC BY 4.0)](https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. + +## Materials +- **DNeasy Blood & Tissue Kit (250)**, Qiagen Catalog #69506 +- **DNeasy 96 Blood & Tissue Kit (12)**, Qiagen Catalog #69582 +- **Type-it Microsatellite PCR Kit (200)**, Qiagen Catalog #206243 +- **Type-it Microsatellite PCR Kit (2000)**, Qiagen Catalog #206246 + +## Steps + +1. **DNA Extraction** + - DNA is extracted from dried scales or fin fins or other tissues preserved in alcohol, frozen or fresh, or from eggs. The extractions are done using Qiagen DNeasy or DNeasy 96 Blood & Tissue Kits with the kit manual's 'Animal Tissues' protocols with a few modifications for the egg samples. + +2. **Sample Preparation** + - Use usually only 1 scale, or if very small, 2-3 scales are used. From the tissue samples, a small piece (max. 10 mg) is cut and the pieces from samples in alcohol are kept overnight in open tubes to let the alcohol evaporate. + +3. **Egg Sample Processing** + - The eggs are used as whole. When needed, the volume of the ATL-buffer and proteinase K mixture is increased from the volume suggested in the kit manual so that the volume of the mixture is always at least 4 times the volume of the egg. The eggs are punctured with sharp tweezers. If the volumes of the ATL-buffer and proteinase K are increased, also the volume of AL-buffer and alcohol mixture is also increased so that it is 2.05 times the volume of the ATL-buffer and proteinase K mixture. + +4. **PCR Preparation** + - The PCRs are done using Qiagen Type-it Microsatellite Kit. The kit manual's 'Optimized cycling protocol for multiplex PCR amplification of microsatellites' is used with the annealing temperature of 56˚C, but with modifications on the reaction volumes. When the samples are fresh or have been kept frozen or in alcohol for max. 1 year, 10 µl reactions are used. For max. 1 year old dried scales, 15 µl reactions are used. If samples are kept frozen or in alcohol for more than 1 year or if the dried scales are older than 1 year, 25 µl reactions are used. The extracted DNA is usually used without dilution. When the samples are very old, the extracted DNA is concentrated to 1/10 of the original volume by keeping the DNA in open tubes at room temperature. For the 10 µl reaction, 5 µl of kit's master mix and 3 µl of extracted DNA are used. For the 15 µl reaction, these volumes are multiplied by 1.5, and for 25 µl reactions by 2.5. + +5. **Genotyping** + - 17 microsatellite loci are analyzed in three multiplex-reactions. The multiplexes, primer sequences, primer concentrations, dyes, loci names in references and GenBank, and references for each locus and GenBank accession numbers are: + + **Table 1: Primer Sequences and Concentrations** + + | Locus | Multiplex | Forward primer sequence (5'-3') | Reverse primer sequence (5'-3') | + |-------------|-----------|----------------------------------|----------------------------------------| + | Ss4l4 | MP1 | CTTTTTGGAATTTAAGGTTTC | CAAACAAACTACATAAACAGC | + | SSA171 | MP2 | GAGGTGCTGGGGTTTTTAT | TTATTATTCACAAAGGGGTAAAA | + | SSA197 | MP2 | GGGTGAGTAGGAGGGACTTG | TGCAGGAGTTAAGGATAAC | + | SSA202 | MP2 | CTTGGAATTAGGAATTAGC | TTCAATGTTAATATTGGGTGTG | + | SSA289 | MP2 | CTTTAACAAAGACGAGAGCT | TCTAACTCGATTATGTCGTGTG | + | SSA407 | MP1 | TGTGTAGGAAGTGTGGC | CACTCGTGTTACTGTTGTGATTG | + | SSA85 | MP2 | AGGGTGGTCCCTCACAGTAC | ACCCTGGGTTACTTTTGGT | + | SSAD157 | MP1 | ATGCAAATGGACTTTTAGTG | GGTCAAGGCCTGAGGAGAGA | + | SSOSL417 | MP1 | TTGTAGATTATGTTCGGCCAT | GGTTACTGAGCCGATTGAGGA | + | SSOSL438 | MP2 | GACACACAAAACAAGGCAC | TATGCTAGTCTTTCTTACTGTT | + | SSOSL85 | MP2 | TTGGAATTTATATTTGTA | ATAACTTTCCTTCTCAAGT | + | SSsp1605 | MP7 | CGCAGGCAGAAGTGGGATC | CTCATTTTTGCCTTTTAGTGCC | + | SSsp2201 | MP7 | TTAAGTGGAGTGGAGGGC | CGGGAGACCAAAACTACCAT | + | SSsp2210 | MP3 | AAGTTAGTCTCGAACACTCTC | AACAGCCCCTTATTCCAACTTGATT | + | SSsp2216 | MP1 | GCCCGAACAGGATAAACAACC | CCCACAGAGACAGTTACACCC | + | SSSp3016 | MP1 | CACAGGGCTAAGGAGGTCA | GATTCTTATATATCTTTATCCCCATT | + | SSSpG7 | MP3 | CTTGGTCCCCTTTCTACGAACC | TGACCGTGCTGTGTCCTTG | + + **Table 2: Primer Concentrations and Dyes** + + | Locus | Primer concentration (µM) | Dye | Orig. Locus Name | Reference | GenBank Accession no. | + |------------|----------------------------|------|------------------|----------------|-----------------------| + | Ssa14 | 0.20 | PET | Ssa14 | R1 | L48596.1 | + | SSA171 | 0.05 | NED | SSA171 | R2 | U43693.1 | + | SSA197 | 0.03 | VIC | SSA197 | R2 | U43694.1 | + | SSA202 | 0.10 | PET | SSA202 | R2 | U43695.1 | + | SSA289 | 1.35 | VIC | SSA289 | R1 | | + | SSA407 | 0.25 | VIC | SSA407UOS | R3 | AJA402724.1 | + | SSA85 | 0.06 | NED | SSA85 | R2 | U43692.1 | + | SSAD157 | 0.20 | 6FAM | SSAD157 | R4 | AF525204.1 | + | SSOSL417 | 0.10 | PET | SSOSL417 | R5 | Z48598.1 | + | SSOSL438 | 0.15 | 6FAM | SSOSL438 | R6 | Z49134.1 | + | SSsp1605 | 0.07 | PET | SSsp1605 | R7 | AY081812.1 | + | SSsp2201 | 0.07 | NED | SSsp2201 | R7 | AY081807.1 | + | SSsp2210 | 0.05 | 6FAM | SSsp2210 | R7 | AY081808.1 | + | SSsp2216 | 0.04 | 6FAM | SSsp2216 | R7 | AY081811.1 | + | SSSp3016 | 0.10 | NED | SSSp3016 | R8 | AY372820.1 | + | SSSpG7 | 0.20 | VIC | SSSpG7 | R7 | AY081813.2 | + + **References for Loci** + + - **R1**: McConnell SK, O'Reilly P, Hamilton L, Wright JM, Bentzen P (1995). **Polymorphic microsatellite loci from Atlantic salmon (Salmo salar)**: genetic differentiation of North American and European populations. Canadian Journal of Fisheries and Aquatic Sciences 52:1863-1872. + - **R2**: O'Reilly P, Hamilton LC, McConnell SK, Wright JM (1996). **Rapid analysis of genetic variation in Atlantic salmon (Salmo salar) by PCR multiplexing of dinucleotide and tetranucleotide microsatellites**. Canadian Journal of Fisheries and Aquatic Sciences 5:2292-2298. + - **R3**: Cairney M, Taggart JB, Hoyheim B (2000). **Characterization of microsatellite and minisatellite loci in Atlantic salmon (Salmo salar L.) and cross-species amplification in other salmonids**. Molecular Ecology 9:2175-2178. + - **R4**: King TL, Eackles MS, Letcher BH (2005). **Microsatellite DNA markers for the study of Atlantic salmon (Salmo salar)**: rehabilitation, population structure, and mixed-fishery analyses. Mol. Ecol. Notes 5:130-132. + - **R5**: Slettan A, Olsaker I, Lie Ø (1995). **Polymorphic Atlantic salmon, Salmo salar, microsatellites at the SSOSL25, SSOSL85, SSOSL311, SSOSL417 loci**. Animal Genetics 26:277-285. + - **R6**: Slettan A, Olsaker I, Lie Ø (1996). **Polymorphic Atlantic salmon, Salmo salar, microsatellites at the SSOSL438, SSOSL439 and SSOSL444 loci**. Animal Genetics 27:57-64. + - **R7**: Paterson S, Piertney SB, Knox D, Gilbey J, Verspoor E (2004). **Characterization and PCR multiplexing of novel highly variable tetranucleotide Atlantic salmon (Salmo salar L.) microsatellites**. Molecular Ecology Notes 4:160-162. + - **R8**: Paterson S, Verspoor E, Knox D (2003). Unpublished. + +6. **Microsatellite Detection** + - Microsatellite genotypes are detected with an Applied Biosystems ABI 3130 automated DNA sequencer and analyzed with GeneMapper analysis software v5.0, with the size standard of Applied Biosystems GeneScan 500 LIZ. Automatic outputs are checked for errors and corrected manually. + +endofoutput +``` diff --git a/markdown-output/unix-and-bioinformatics-eptbdnn.md b/markdown-output/unix-and-bioinformatics-eptbdnn.md new file mode 100644 index 0000000000000000000000000000000000000000..012fd372b97157206c45765eb86be48eb14e647c --- /dev/null +++ b/markdown-output/unix-and-bioinformatics-eptbdnn.md @@ -0,0 +1,692 @@ +```markdown +# Goal/Experiment: +This document provides instructions and descriptions of various Unix commands beneficial for bioinformatics applications. + +# Unix and Bioinformatics + +### Abstract +This protocol details the use of various Unix commands commonly used in bioinformatics. + +**Citation:** Benjamin Tully, Unix and Bioinformatics. *protocols.io* +**DOI:** [10.17504/protocols.io.eptbdnn](https://dx.doi.org/10.17504/protocols.io.eptbdnn) +**Published:** 25 Jul 2016 + +--- + +## Guidelines + +### Unix Commands +```plaintext +pwd rm grep tail install +ls '>' sed cut +cd cat nano top +mkdir '<' history screen +touch '|' $PATH ssh +cp sort less df +mv uniq head rsync/scp +``` + +--- + +## Protocol + +### The Start + +#### Step 1 +Open a terminal window. + +#### Step 2 +Use `ls` to list items in the current directory. + +```shell +ls +``` +*Lists items in the current directory.* + +#### Step 3 +Many commands have additional options that can be set by a `-` + +```shell +ls -a # lists all files/directories, including hidden files('.') +ls -l # lists in long format +ls -lt # lists in long format, ordered by date last modified +``` + +*Expected Results:* + +![Expected Results](img/unix-protocol-3.png) + +### Directory System + +#### Step 4 +Change directory: + +```shell +cd ecogeo/ +``` + +#### Step 5 +List the contents of the current directory. + +#### Step 6 +Move into the directory called `Part1_Unix` + +#### Step 7 +Show the current directory path: + +```shell +pwd +``` +*Prints the path to the current directory.* + +*Expected Results:* +```plaintext +/home/c-debi/ecogeo/unix +``` + +#### Step 8 +Move to the root directory. + +```shell +cd / +``` + +#### Step 9 +Change directory through the following steps: + +```shell +cd home +cd c-debi +cd ecogeo +cd unix +``` + +List contents in-between steps. + +#### Step 10 +Change directory to one step: + +```shell +cd /home/c-debi/ecogeo/unix/data +``` + +#### Step 11 +Move back in the path directory: + +```shell +cd .. +pwd +``` +*Expected Results:* +```plaintext +/home/c-debi/ecogeo/unix +``` + +#### Step 12 +Step back up to the `c-debi` directory. + +#### Step 13 +Change directory to `BioinfPrograms` + +#### Step 14 +List contents + +#### Step 15 +Change directory to `unix` + +#### Step 16 +Make a directory named "storage": + +```shell +mkdir storage +``` + +#### Step 17 +List the contents of the directory + +### Manipulating Files + +#### Step 18 +Move into the storage directory + +#### Step 19 +The `touch` command allows you to create a blank file of the input name. + +```shell +touch temp.txt +``` + +#### Step 20 +The `cp` command allows you to copy a file and move a copy of a file to a directory. + +```shell +cp temp.txt newtemp.txt +cp temp.txt ../ +``` + +#### Step 21 +The `mv` (move) command "destroys" the original and places the content elsewhere. + +```shell +mv newtemp.txt oldtemp.txt +mv oldtemp.txt /home/c-debi/ecogeo/unix/data +``` + +#### Step 22 +Using copy: + +```shell +cp temp.txt newtemp.txt +cp temp.txt ../ +``` + +#### Step 23 +Change directory up a level. + +#### Step 24 +List contents. + +#### Step 25 +Change directory to `storage` + +#### Step 26 +Utilize move command: + +```shell +mv newtemp.txt oldtemp.txt +mv oldtemp.txt /home/c-debi/ecogeo/unix/data +``` + +#### Step 27 +List current working directory. + +#### Step 28 +Remove a file permanently: + +```shell +rm oldtemp.txt +``` + +#### Step 29 +Change directory to `storage` + +#### Step 30 +Remove `temp.txt` + +#### Step 31 +Change directory to `unix` + +#### Step 32 +Remove the storage directory: + +```shell +rm -r storage +``` + +#### Step 33 +Create a directory called `bestdirectoryever`. + +Change directory to `bestdirectoryever`. + +Create a file called `glam.txt`. + +Change `glam.txt` to `formerGlam.txt`. + +Remove `formerGlam.txt`. + +Change directory to `unix`. + +Remove `bestdirectoryever`. + +#### Step 34 +Change the directory to `data`. + +#### Step 35 +List contents. + +#### Step 36 +Remove oldtemp.txt + +#### Step 37 +Work with FASTA files: +``` +group12_contigs.fasta +group20_contigs.fasta +group24_contigs.fasta +``` + +**FASTA Files:** +- Specific format: + > Header line, contains ID and information. + > ATGATAGCTAGCAGCAGCTA[...] 80bp and then a newline. + +### Looking at the Contents of a File + +#### Step 38 +`head` allows you to view the first 10 lines of a file. + +```shell +head [filename] +``` +*Defaults to display the first 10 lines.* + +#### Step 39 +`tail` allows you to view the last 10 lines of a file. + +```shell +tail [filename] +``` +*Defaults to display the last 10 lines.* + +#### Step 40 +`less` allows you to scroll through a file using arrow keys, the spacebar for page advancing, `b` to reverse page, and `q` to quit. + +```shell +less [filename] +``` + +#### Step 41 +Use `head` to display the first 10 lines of `group12_contigs.fasta`. + +- Display the first 5 lines with: + +```shell +head -n 5 group12_contigs.fasta +``` + +#### Step 42 +`grep` - a file pattern searcher. + +```shell +grep [pattern] [file] +``` + +#### Step 43 +`wc` - counts the number of words, lines, and characters. + +#### Step 44 +Use `grep` on `group12_contigs.fasta`. + +```shell +grep ">" group12_contigs.fasta +``` +*Prints all matches of `>` in the file.* + +#### Step 45 +Combine `grep` with `wc` using the pipe ('|') symbol. + +```shell +grep ">" group12_contigs.fasta | wc +``` + +#### Step 46 +Repeat the above command with the `-l` option added to `wc`. + +#### Step 47 +Use the same technique to determine the number of sequences in `group20_contigs.fasta`. + +#### Step 48 +Count the number of matches to "47" in `group12_contigs.fasta` using `grep`. + +```shell +grep ">" group12_contigs.fasta | grep 47 +``` + +#### Step 49 +Redirecting output to a file: + +```shell +grep ">" group12_contigs.fasta > group12_ids +``` + +#### Step 50 +Examine the contents of `group12_ids`: + +```shell +cat group12_ids +``` +*With a single input, this prints the file contents.* + +#### Step 51 +Using `cat` to redirect output: + +```shell +cat group12_ids_with_47 > temp1_ids +cp group12_ids_with_47 temp2_ids +``` + +#### Step 52 +Check if `temp1_ids` equals `temp2_ids` with: + +```shell +diff temp1_ids temp2_ids +``` + +#### Step 53 +Concatenate files `cat`: + +```shell +cat temp1_ids temp2_ids > duplicate_ids +``` + +#### Step 54 +Check the contents of `duplicate_ids` using `less` or `cat`. + +#### Step 55 +Extract the IDs from `group20_contigs.fasta` that contain the number "51": + +```shell +grep 51 group20_contigs.fasta +``` + +#### Step 56 +Concatenate the new IDs into `duplicate_ids` by creating `multiple_ids`: + +#### Step 57 +`uniq` command to remove duplicates or identify lines with 1 or multiple occurrences: + +```shell +uniq +``` + +#### Step 58 +`sort` command to sort lines in a file alphanumerically: + +```shell +sort +``` + +#### Step 59 +Compare `multiple_ids` before and after using `uniq`: + +```shell +uniq multiple_ids +``` + +#### Step 60 +Identify why there was no change: + +*Note: `uniq` can only identify duplicates on adjacent lines.* + +#### Step 61 +Sort and remove duplicates: + +```shell +sort multiple_ids | uniq > clean_ids +``` + +#### Step 62 +Clear all files with a 'temp' in the title: + +```shell +rm temp* +``` + +#### Step 63 +Compare `temp1_ids` and `temp2_ids`: + +```shell +sort multiple_ids | uniq -d > temp1_ids # identifies duplicates +sort multiple_ids | uniq -u > temp2_ids # identifies unique entries +``` + +#### Step 64 +Note that: +`` temp1_ids = group12_ids_with_47 + temp2_ids = group20_ids_with_51 +`` + +#### Step 65 +Remove all present files with `temp` in the title. + +#### Step 66 +Use `sed` to modify files based on issued commands: + +```shell +sed [command] +``` + +#### Step 67 +Create a list of sequence IDs without the ‘>’: + +```shell +sed 's/>//' clean_ids > newclean_ids +``` + +### Step 68 +Substitute uppercase 'C' for lowercase 'c': + +```shell +sed 's/C/c/' newclean_ids +``` + +#### Step 69 +`seqmagick` a wrapper that utilizes Biopython to manipulate and change FASTA files. Requires Biopython. URL: [Seqmagick](http://fhcrc.github.io/seqmagick/) + +--- + +### Looking at the Contents of a File + +#### Step 70 +Discuss utilizing: +- `convert` - produce a modified new file +- `mogrify` - change the input file +- `info` - present information of files in a directory + +### Step 71 +Execute Seqmagick convert: + +```shell +seqmagick convert --include-from-file newclean_ids group12_contigs.fasta newgroup12_contigs.fasta +``` + +#### Step 72 +Count the sequence in `newgroup12_contigs.fasta` using `grep '>':` + +```shell +seqmagick extract-ids newgroup12_contigs.fasta | wc +seqmagick info *fasta +``` + +#### Step 73 +Store the output of `seqmagick info` into a new file, `fasta_info`. + +```shell +cut -f 2-4 fasta_info +``` + +### Additional Tools + +#### Step 74 +`history` - prints a sequential list of all commands in the current session. +```shell +history +``` + +#### Step 75 +`echo $PATH` - lists the directories for which the OS is checking for commands and data. + +```shell +echo $PATH +``` + +#### Step 76 +`nano` - an in-window text editor. + +```shell +nano fasta_info +``` +Use `Ctrl+X` to close out, `Y` to save. + +Create a new file: + +```shell +nano [new_file] +``` + +#### Step 77 +Simple bash scripts to bundle commands together. View `simplebashscript`. + +#### Step 78 +`chmod` - change file modes. + +```shell +chmod 755 simplebashscript +``` + +#### Step 79 +Convert plain text file to an executable text file. + +```shell + ./simplebashscript +``` + +--- + +### Accessing a Server + +#### Step 80 +Login from a terminal: + +```shell +ssh -l USERNAME SERVERNAME.WEBADDRESS.EDU +ssh -l btully kuat.usc.edu +``` + +#### Step 81 +Using `top`: + +```shell +top +``` +*Produces a table of server users, CPU usage, and memory/RAM utilization.* + +#### Step 82 +Producing human-readable output of storage: + +```shell +df -h +du -h +``` + +#### Step 83 +`screen` - creates an additional instance of the shell unaffected by interruptions: + +```shell +screen +``` + +#### Step 84 +Detach from screen instance: + +`Ctrl+A`, `Ctrl+D` + +```shell +screen -ls +screen -r XXXX.pts-1.cdebi-VirtualBox +``` +*To terminate screen session (type `exit` in the screen, same for server logout)* + +#### Step 85 +Kill a detached screen: + +```shell +screen -S XXXX.pts-1.cdebi-VirtualBox -X quit +``` + +#### Step 86 +Using `scp` for secure copy: + +```shell +scp filename.fasta btully@kuat.usc.edu://directory/destination +``` + +#### Step 87 +`rsync` - transfers and synchronizes files, maintaining transfers if the connection is lost: + +```shell +rsync filename.fasta btully@kuat.usc.edu://directory/destination +``` + +--- + +### Installations + +#### Step 88 +Easy installations from: + +1. Program manager: `pip`, `apt-get`, `macports` +2. Executables: `mothur`, `Trimmomatic` +3. Source + +--- + +#### Step 89 +Hard installations due to annotated dependencies or prerequisites. + +#### Step 90 +Install AMOS (a software for developing assembly tools) from: + +[AMOS Getting Started](http://amos.sourceforge.net/wiki/index.php/AMOS_Getting_Started). + +#### Step 91 +Install IDBA: iterative De Bruijn Graph De Novo Assembler. + +#### Step 92 +Change directory to: + +```shell +cd /home/c-debi/Downloads +``` + +#### Step 93 +Move compressed IDBA file to `BioinfPrograms`: + +```shell +mv idba-1.1.1.tar.gz /home/c-debi/BioinfPrograms +``` + +#### Step 94 +Uncompress file: + +```shell +tar zxvf idba-1.1.1.tar.gz +``` + +#### Step 95 +Change directory to `/idba-1.1.1` + +#### Step 96 +Examine README contents + +#### Step 97 +Modify values in `sequence.h` directory `/idba-1.1.1/src/sequence/` + +#### Step 98 +Change back to `/idba-1.1.1` directory + +```shell +cd /idba-1.1.1 +``` + +#### Step 99 +Complete the installation: + +```shell +./configure +make +``` + +#### Step 100 +Access programs by linking to `/usr/local/bin`. + +```shell +sudo ln -s /home/c-debi/BioinfPrograms/idba-1.1.1/bin/idba /usr/local/bin/ +``` + +#### Step 101 +Repeat for other files. + +```markdown +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/upregulating-trnas-in-mammalian-cells-through-tran-hetb3en.md b/markdown-output/upregulating-trnas-in-mammalian-cells-through-tran-hetb3en.md new file mode 100644 index 0000000000000000000000000000000000000000..97e9ea9aa0ee6d5d521599dd8d04e9baca531517 --- /dev/null +++ b/markdown-output/upregulating-trnas-in-mammalian-cells-through-tran-hetb3en.md @@ -0,0 +1,139 @@ +```markdown +# Goal/Experiment: +The aim of this experiment is to upregulate tRNAs in mammalian cells by transfecting the cells with in vitro transcribed tRNAs. This protocol specifically describes the transfection of mammalian cells with in vitro transcribed, unmodified, non-charged tRNAs. + +# Upregulating tRNAs in Mammalian Cells through Transfection of In Vitro Transcribed tRNAs + +**Authors**: Sebastian Kirchner, Robert Rauscher, Andreas Czech, Zoya Ignatova + +## Abstract +This protocol describes the transfection of mammalian cells with in vitro transcribed, unmodified, non-charged tRNAs. This method is used to raise intrinsic tRNA concentrations in HeLa and N2a cells. Furthermore, tRNAs synthesized as described in this protocol have been shown to be translation competent. + +## References +1. Kirchner et al. Alteration of Protein Function by a Silent Polymorphism Linked to tRNA Abundance. *PLoS Biology*. 2017, in press. +2. Girstmair et al. Depletion of Cognate Charged Transfer RNA Causes Translational Frameshifting within the Expanded CAG Stretch in Huntingtin. *Cell Reports*. 2013, 3(1):148-59. DOI: [10.1016/j.celrep.2012.12.019](http://dx.doi.org/10.1016/j.celrep.2012.12.019) + +## Before start + +### Considerations for tDNA Oligo Design: +1. Single stranded tDNA oligonucleotides encoding the desired tRNA sequence and the T7 promoter sequence (5'-TAATACGACTCACTATAG-3') are annealed and the resulting 5' overhangs are subsequently filled-up to create a linear dsDNA molecule. This dsDNA molecule then serves as a template for the subsequent tRNA transcription. + +2. The length of the overlapping parts between 5' and 3' tDNA oligonucleotides should be approximately 20 nucleotides. + +3. Example of human tRNA^(Thr)(UGC) tDNA oligonucleotide: + **Full-length tRNA^(Thr)(UGC)** + ```plaintext + GGCGCGGTGGCCAAAGTGGTAAAGGCTCGGTCTCGTAAACCGGAAGATCACGGGTTCGAACCCGCTCGTGCCCTcca + ``` + **T7 Promoter** + ```plaintext + TAATACGACTCACTATAGGGCGCGGTGGCCAAAGTGGTAAAGGCTCGGTCTCGTAAACCGGAAG + ATCACGGGTTCGAACCCGCTCGTGCCCTcca + ``` + **FWD tDNA Oligonucleotide** + ```plaintext + TAATACGACTCACTATAGGGCGCGGTGGCCAAAGTGGTAAAGGCTCGGTCTCGTAAACC + ``` + **REV tDNA Oligonucleotide (reverse complement)** + ```plaintext + tggAGGCACGACGCGGGTTCGAACCCGTGACTTTCGGTTTCACGACGACGCCT + ``` + +--- + +## Protocol + +### Generation of tDNA Template + +#### Step 1. +Combine the following for annealing of overlapping single stranded tDNA oligonucleotides: +- 9.6 μl forward ssDNA oligonucleotide (100 μM) +- 9.6 μl reverse ssDNA oligonucleotide (100 μM) +- 4 μl Tris-HCl (200 mM, pH 7.5) +- 16.8 μl H₂O + +#### Step 2. +Incubate at 95°C for 2 minutes, followed by 3 minutes at 22°C. Store on ice until further use. + +#### Step 3. +Prepare Reverse Transcription master mix on ice as follows: +- 40 μl 5x Reverse transcription buffer (ThermoFisher, #EP0441) +- 8 μl dNTPs (10 mM) +- 4 μl RevertAid H Minus reverse transcriptase (200 U/μl; ThermoFisher, #EP0441) +- 108 μl H₂O + +#### Step 4. +To fill-in 5' single-stranded tDNA overhangs to form blunt ends, incubate the mixture for 40 minutes at 37°C. + +### Generation of tDNA Template + +#### Step 5. +For tDNA purification, add one volume Crush & Soak buffer and two volumes phenol/chloroform/IAA, vortex for 30 seconds, and centrifuge for 5 minutes at 21,000 x g (4°C). + +Crush & Soak buffer: +- 50 mM KOAc +- 200 mM KCl + - Adjust pH to 7.0, filter sterilize, aliquot and store at -20°C. + +#### Step 6. +Recover the upper aqueous phase and precipitate tDNA with 2.7 Vol 100% EtOH for 30 minutes at -80°C. + +#### Step 7. +Pellet tDNA for 40 minutes at 21,000 x g (4°C) and remove supernatant. + +#### Step 8. +Resuspend tDNA pellet in 50 μl nuclease-free H₂O and determine concentration. Store tDNA at -20°C. + +### In Vitro tRNA Transcription + +#### Step 9. +Combine 2 μg tDNA and 25 μl transcription mix, and add nuclease-free H₂O to a final volume of 50 μl. For preparative tRNA synthesis (e.g., for tRNA transfection) transcription should be scaled up to 500 μl reactions. + +#### Step 10. +Incubate for 7 hours (or overnight) at 37°C. + +#### Step 11. +Purify tRNAs on a denaturing 10% TBE-PAGE. In case larger transcription reactions have been prepared, distribute transcription mixtures into several slots of the polyacrylamide gel. Visualize by UV-shadowing, cut tRNA bands, and elute with Crush & Soak buffer overnight. + +#### Step 12. +Remove gel particles by centrifugation (5 minutes, 4°C, top speed), precipitate with one volume 100% isopropanol at -20°C for 30 minutes, and pellet tRNA by centrifugation (21,000 x g, 4°C, 40 minutes). + +#### Step 13. +Wash tRNAs once with 80% ethanol and resuspend tRNAs in 30 μl nuclease-free H₂O. Store tRNAs at -80°C until use. + +### Refolding and Storage of In Vitro Transcribed tRNAs + +#### Step 14. +For refolding, denature tRNAs at 95°C for 2 minutes, place at 22°C for 3 minutes, and incubate for further 5 minutes at 37°C. tRNAs should be stored at -80°C. + +### Transfection of In Vitro Transcribed tRNAs + +#### Step 15. +Seed cells (e.g., 200,000 HeLa cells) into a 3.5 cm cell culture dish in DMEM (Dulbecco's Modified Eagle Medium, PAN-Biotech, #P04-03500; supplemented with 10% FCS and 2 mM L-glutamine) 24 hours prior to transfection and incubate at 37°C in a humidified atmosphere with 5% CO₂. + +#### Step 16. +On the day of transfection (cells should have reached 70-80% confluency), thaw refolded tRNAs on ice. For each 3.5 cm cell culture dish, add tRNAs to 50 μl opti-MEM (ThermoFisher, #31985062) (Tube1). Add 5 μl Lipofectamin 2000 Transfection Reagent (ThermoFisher, #11668027) to 50 μl opti-MEM (Tube2). + +#### Step 17. +Vortex Tube1 and Tube2 for 10 seconds, spin briefly, and incubate 5 minutes at 22°C separately. + +#### Step 18. +Add the content of Tube2 to Tube1, vortex for 10 seconds, spin briefly and incubate 30 minutes at 22°C. + +#### Step 19. +While incubating, exchange the culture medium with 1.9 ml fresh DMEM (supplemented with 10% FCS and 2 mM L-glutamine). + +#### Step 20. +Once incubation is completed, add tRNA-Lipofectamin mixture dropwise to cells and incubate for 4 hours at 37°C in a humidified atmosphere with 5% CO₂. + +#### Step 21. +Replace the medium with fresh medium and incubate for further 20 hours at 37°C in a humidified atmosphere with 5% CO₂. Cells can then be further manipulated as necessary. + +--- + +**Note**: tRNA transfection efficiency should be analyzed by qRT-PCR or Northern blotting. Examplary results can be seen in Kirchner et al., PLoS Biology, 2017 and Girstmair et al., Cell Reports, 2013. + +--- + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/useful-methods-4-stock-cultivation-of-duckweed-b56qq9dw.md b/markdown-output/useful-methods-4-stock-cultivation-of-duckweed-b56qq9dw.md new file mode 100644 index 0000000000000000000000000000000000000000..6c426b98c5f7a822d5e6d7eea6f11aeee45fdb0b --- /dev/null +++ b/markdown-output/useful-methods-4-stock-cultivation-of-duckweed-b56qq9dw.md @@ -0,0 +1,121 @@ +```markdown +# Goal/Experiment: +The goal of this experiment is to establish a protocol for the stock cultivation of duckweed. This involves using specific environmental conditions and nutrient media to maintain and propagate duckweed cultures, particularly for long-term preservation and controlled growth rates. + +# Useful Methods 4: Stock Cultivation of Duckweed + +**Authors**: K. Sowjanya Sree1, Klaus-J. Appenroth2 +1Amity Institute of Biotechnology, Amity University, UP, Noida, India +2University of Jena, Plant Physiology, Dornburger Str. 159, 07743 Jena, Germany + +**Reviewed by**: Chris Carlson, University of Toronto + +**Date Published**: April 14, 2023 + +## Abstract + +This protocol details the stock cultivation of duckweed. It includes instructions from the International Steering Committee on Duckweed Research and Application (ISCDRA) Newsletter. + +## Guidelines + +The guidelines in this protocol are drawn from standardized methods for optimal cultivation of duckweed. The aim is to reduce the rate of growth to sustain long-term cultivation. + +## Materials + +- Agar-based medium +- Nutrient medium +- KH₂PO₄ (Potassium dihydrogen phosphate) +- Glucose +- Sucrose +- 100 ml Erlenmeyer flask +- Petri dishes +- Parafilm + +## Methods + +### 1. Temperature Control + +- **Optimal growth temperature**: 25°C (DF 3, 59-62 (2015)) + - **Stock cultivation temperature**: 18°C, tolerable down to 15°C for most species + - Clones from tropical climates: Cannot tolerate 5°C for long durations + +### 2. Light Intensity + +- **Optimal intensity**: 100 µmol m-2 s-1 + - **Stock cultivation intensity**: 30 µmol m-2 s-1 + - Duckweed can tolerate low light intensity when temperature is also reduced + +### 3. Water Availability + +- Replace liquid medium with an Agar-based medium (GELRITE as an alternative) + - Use N-medium (DF 3, 182-183 (2015)) + +### 4. Nutrient Concentration + +- KH₂PO₄ concentration: Increase to 1 mM + - Note: At lower concentrations, survival of plants on Agar is short + +### 5. Agar Addition + +- Add solid Agar to nutrient medium (0.9% concentration; 0.7% for sensitive clones; GELRITE at 0.45%) + +### 6. Agar Preparation + +- Prepare 1 L Agar suspension + - Heat in microwave to 80°C + - Pour 50 ml to 75 ml into each 100 ml Erlenmeyer flask + - Secure with cotton wool stoppers + +### 7. Autoclaving + +- Sterilize the flasks via autoclaving + - Enables long-term stock cultures (4-5 months) + +### 8. Stock Collection + +- Use standard glass test tubes with 5 ml Agar medium + - Close with cotton wool stoppers + - Optional: Use sterile plastic Petri dishes (9 cm or smaller) + +### 9. Sterilizing Petri Dishes + +- Autoclave the Agar medium + - Pour into sterile Petri dishes in a laminar flow box + +### 10. Inoculation and Sealing + +- Inoculate plants + - Seal Petri dishes with parafilm + +### 11. Sterility Control + +- Add low molecular weight carbohydrates to control sterility + - **Glucose**: 50 mM (sometimes 25 mM for regenerating plants) + - **Sucrose**: 25 mM + - Fructose is not recommended + +### 12. Spirodela Species Growth + +- Species of Spirodela grow in layers, upper layers lose contact with Agar and die + - Use liquid medium without sugar for these species + - Conduct sterility tests + +### 13. Long-Term Preservation + +- **Species**: S. polyrrhiza + - Preserve using turions (storage organs) + +### 14. Turion Storage + +- Store turions at 5°C in darkness for several years + +### 15. Turion Formation + +- Inoculate plants in liquid medium with low phosphate (60 µM) + - Reduce phosphate to 2 µM to induce turion formation + - Addition of 50 mM glucose accelerates turion yield + +--- + +**endofoutput** +``` diff --git a/markdown-output/useful-methods-cefotaxime-a-useful-antibiotic-for-b6i8rchw.md b/markdown-output/useful-methods-cefotaxime-a-useful-antibiotic-for-b6i8rchw.md new file mode 100644 index 0000000000000000000000000000000000000000..336e931d48d5700ccff857a90df83f92545b0289 --- /dev/null +++ b/markdown-output/useful-methods-cefotaxime-a-useful-antibiotic-for-b6i8rchw.md @@ -0,0 +1,80 @@ +```markdown +# Goal/Experiment: +To develop an efficient method for managing duckweed cultures using the antibiotic cefotaxime, which helps in the removal of difficult bacteria and suppresses reinfection in gnotobiotic duckweed plants. + +# Useful Methods: Cefotaxime - A Useful Antibiotic for Duckweed Culture Management + +### Authors +Eric Lam1, Kenneth Acosta1 +1Rutgers State University of New Jersey and the Rutgers Duckweed Stock Cooperative, Department of Plant Biology, 59 Dudley Road, New Brunswick, NJ 08901, USA + +### Abstract +This protocol describes cefotaxime as a useful antibiotic for duckweed culture management. It contains protocols from the International Steering Committee on Duckweed Research and Application (ISCDRA) Newsletter. + +### Guidelines + +#### Background +Biology is complicated! Despite the simplicity of duckweed plants, managing a living collection of over 800 clones (or strains) in the Rutgers Duckweed Stock Cooperative (RDSC) is complex. Maintaining consistent and reliable germplasm stock for duckweed requires them to be free from bacterial or fungal endophytes. + +#### Problem +Some strains and species of duckweed are particularly resistant to purging their resident microbes. This resistance can cause the duckweed tissues to display slow growth and death in later stages due to infection by bacteria or fungal endophytes. + +#### Solution +Cefotaxime is a useful antibiotic agent to manage duckweed strains. Employing various concentrations of sodium hypochlorite (active ingredient in bleach) for sterilization can be difficult. The antibiotic cefotaxime facilitates this process by suppressing bacterial growth and reinfection in sterilized duckweed tissues. + +### Characteristics of Cefotaxime + +- **Chemical Structure**: Cefotaxime is a β-lactam antibiotic related to penicillin. +- **Antibacterial Activity**: It can inhibit both Gram-negative and Gram-positive bacteria. Not effective against Pseudomonas and Enterococcus species. +- **Mechanism of Action**: Inhibits bacterial cell wall biosynthesis, leading to bacterial lysis and inhibits cell division in various organisms. +- **Toxicity**: Very low toxicity in vascular plants, extensively used in plant tissue culture. + +### Application in Duckweed Cultures + +- **Concentration**: Typically applied at 100 mg/L to infected duckweed tissues. +- **Medium Storage**: Cultures maintained on mediums consisting of: + - 0.5X Schenk and Hildebrandt (SH) salts + - Cefotaxime + - 0.1% (W/V) sucrose + +### Dilution-by-Division Approach for Recalcitrant Infections +1. **Isolation**: Spot bleach-treated duckweed fronds onto SH plates containing cefotaxime. +2. **Growth**: After 2-3 weeks, transfer new clusters to fresh SH-cef plates. +3. **Subculture**: Repeat transfer until new clusters form. +4. **Validation**: Check for bacterial presence on LB and TSB agar plates. + +### Important Notes +- Cefotaxime stability decreases by ~20% after 5 days at 25°C. +- Transfer to fresh plates is necessary every 2-3 weeks to maintain efficacy. + +### Summary +This discussion topic aims to assist duckweed researchers and application specialists in managing their duckweed culture collections using cefotaxime. A detailed protocol for including cefotaxime in culturing media is provided. + +## Protocols + +### Preparation of Cefotaxime Stock Solution + +1. Add 1 g of cefotaxime (GoldBio; Catalog # C-104) to 10 mL sterile H₂O. Dissolve completely. +2. Filter sterilize the solution using a 0.22 µm syringe filter. +3. Aliquot into 1-mL centrifuge tubes. +4. Store at -20°C until use. + +### Preparing Agar Media With Cefotaxime + +1. Autoclave agar media at 122°C for 30 minutes. +2. Let agar media cool until it is warm to the touch. +3. Thaw cefotaxime stock solution. +4. Add 500 µL cefotaxime stock solution (100 mg/mL stock; 1,000x) to 500 mL agar media for a final concentration of 100 mg/L. +5. Pour plates; leave overnight in a laminar flow hood to solidify and dry. +6. Store plates the following day at 4°C until use. + +### Preparing Liquid Media With Cefotaxime + +1. Thaw cefotaxime stock solution. +2. Add 500 µL cefotaxime stock solution (100 mg/mL stock; 1,000x) to 500 mL liquid media for a final concentration of 100 mg/L. + +### References +1. Behin S, Punitha ISR, and Krishnan S (2012) Physical and Chemical Stability Studies on Cefotaxime and its Dosage Forms by Stability Indicating HPTLC Method. Int. J. Pharma. Chem. and Biol. Sci. 2(4): 517-523. ISSN: 2249-9504 + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/using-tracefinder-and-excel-software-to-evaluate-a-czajx2cn.md b/markdown-output/using-tracefinder-and-excel-software-to-evaluate-a-czajx2cn.md new file mode 100644 index 0000000000000000000000000000000000000000..9a85f0bcb7b0866513367c5618c74b6e4a4a2480 --- /dev/null +++ b/markdown-output/using-tracefinder-and-excel-software-to-evaluate-a-czajx2cn.md @@ -0,0 +1,185 @@ +```markdown +# Goal/Experiment: +To evaluate and report multi-analyte targeted LC-MS data acquired on a ThermoScientific Exploris 240 Orbitrap using TraceFinder and Excel software. + +## Using TraceFinder and Excel Software to Evaluate and Report Multi-Analyte Targeted LC-MS Data Acquired on a ThermoScientific Exploris 240 Orbitrap + +### Authors: +- Margaux Billen1,2 +- Scott G Denham2 +- Joanna P Simpson2 +- Natalie ZM Homer2 + +1 KU Leuven; 2 University of Edinburgh + +### Section: Clinical Mass Spectrometry + +### Abstract +In our lab, we focus on targeted analysis and have found that acquiring LC-HRMS data using Xcalibur software and assessing the data using TraceFinder software is the most robust and flexible approach. + +This protocol describes our approach to evaluating LC-HRMS data that has been collected in Xcalibur software, on LC-HRMS instruments including ThermoScientific Exploris mass spectrometers. It describes how to build a processing method in TraceFinder and use it to evaluate a full scan analysis data set acquired in Xcalibur software. + +The LC-HRMS data set must contain a calibration curve and unknowns analyzed as a batch using the same Acquisition method in Xcalibur. The results from TraceFinder are then transferred to Microsoft Excel to summarize the calculated amounts of the analytes of interest in the samples as a final result. + +### Guidelines +- Use methods that have well-defined calibration protocols, method details (masses, analyte retention times), and well-defined calibration ranges and known units for the calibration ranges. +- TraceFinder is only successful if the calibration range is well-defined, the calibration units are known, and the volume of extracted sample is recorded. +- QCs used across multiple batches are best included in each batch and give confidence to the results. + +## Materials +1. **Xcalibur Data Acquisition and Interpretation Software 4.5 SP1** (ThermoScientific) +2. **TraceFinder 5.1 Software License** (ThermoScientific) +3. **Excel Software Package** (Microsoft) +4. Details of the method used to acquire data in Xcalibur - including retention times, names of analytes, calibration ranges and calibration units, and volume (or mass) of sample extracted. + +## Before Start Instructions +Acquire a dataset in Xcalibur and use the raw file in TraceFinder. + +## Creating a Compound Database in TraceFinder + +1. In TraceFinder, navigate to the 'Method Development' tab in the bottom left corner of the screen. + +2. Select 'Compound Database' on the left-hand side. + +![Compound Database Screen](image_path_here) + +3. Go to File > New Small Molecule Compound Database + - A new screen pops up where you can give a name to your new compound database. + +4. **Build your compound database** as seen in this example: + +![Compound Database](image_path_here) + + - Enter each individual compound that should be analyzed + all internal standards in the method and enter the chemical formula, the polarity, the adduct, and the expected retention time for each of the compounds. + - Select each individual compound in the Tree View Pane on the left-hand side and fill in the 'Compound Details Pane' on the bottom. + + **For Internal Standards (ISTD):** + - Select 'Internal Standard' for compound type in the lower tab and fill in the ISTD concentration as 1. + + **For the Analytes:** + - Select 'Analyte' for compound type in the lower tab. + +5. Save the compound database. + - Go to File > Save Compound Database. + +## Creating a Processing Method in TraceFinder + +6. Go to the 'Method View' tab on the left-hand side. + - Select File > New > Master method... + +7. The following screen will pop up: + +![Method Templates](image_path_here) + + - Select the option 'Quan by Selecting Compounds from CDB' and click on 'OK'. + +8. The following screen will pop up: + +![CDB](image_path_here) + - Using the browse function, select your prepared compound library. + - Tick the 'Select All' and 'Associate Raw Datafile' boxes. + - Select a datafile from a mid-high standard on the calibration curve. + - Use the settings as displayed in the screenshot above. + - Click on 'Add Selected Compounds to Method and Associate Raw File'. + +9. Save your Master Method by selecting File > Save as... + +10. Navigate to the 'Compounds' tab on the left-hand side. The screen should look like this: + +![Compounds Tab](image_path_here) + + - Here, select the individual compound and ensure correctness of the chemical formula, polarity, adduct, and retention time. + - Assign internal standard for each analyte. + +11. Navigate to the 'Detection' tab on top: + + ![Detection Tab](image_path_here) + + - Alter parameters for peak detection, including retention times, windows, algorithms, etc. + +12. Navigate to the 'Calibration' tab on top: + + ![Calibration Tab](image_path_here) + + - Check and enter the calibration details. + - Choose 'Linear' as curve type with '1/X' weighting. + +13. Navigate to the 'Calibration levels' tab on top. + + ![Calibration Levels Tab](image_path_here) + + - Enter names and specifics for all calibration levels. + - Associate concentrations for each analyte. + - Save the Master Method via File > Save. + +## Using TraceFinder to Process Xcalibur Acquired LC-MS Data + +15. In TraceFinder, navigate to the 'Analysis' tab in the bottom left corner of the screen. + +16. Select File > New > Batch + - A screen pops up where you can save your new batch. + +![New Batch](image_path_here) + + - Select a folder, name the batch, choose the Master Method, and click 'Create'. + +17. Go to Batch > Import Samples + +![Import Samples](image_path_here) + + - Browse for the SLD file of the dataset to process. + - Remove the 'Unknown' labeled sample. + - Select the correct calibration level for each standard. + +18. Map raw files to samples: + - Right-click on a sample and select 'Map raw files to samples...' + - Select all raw files in the file explorer. + - Confirm that files now link to respective samples in the batch. + +![Mapping Raw Files](image_path_here) + +19. Submit the batch for processing: + - Click the appropriate icon at the top of the screen: + +![Submit Batch](image_path_here) + + - Confirm settings and click 'OK'. + - The 'sample status' should turn green upon successful processing. + +## Using TraceFinder to Evaluate Xcalibur Acquired LC-MS Data + +20. In TraceFinder, go to the 'Compound View' tab on the left-hand side. + +![Compound View](image_path_here) + +21. Evaluate each analyte in every sample: + - Ensure correct peak integration and retention times. + - Verify calibration curves for accuracy (<20%) and a regression coefficient R>0.99 with at least 6 points per curve. + - Save the results via File > Save... + +## Transferring TraceFinder Alphanumeric Data into Excel to Summarize Results + +23. Go to File > Export data to > CSV or Excel... + +![Exporting Data](image_path_here) + +24. A screen pops up with these options: + - Select 'Excel' as file format. + - Select 'Multiple Worksheets'. + - Choose 'Export selected columns and rows only'. + +25. Open the exported Excel file. Create a new tab called 'Summary'. + +26. Copy and paste sample names and analyte concentrations into the Summary tab: + - Repeat this for all analytes. + +27. Calculate concentration results (ng/mL): + - Add a column for extraction volume. + - Calculate ng/mL by dividing the 'calculated amount' by the volume (uL) and multiply by 1000. + +28. Save the final excel file. + +--- + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/vaginal-lipidomics-of-women-with-vulvovaginal-cand-rpud5nw.md b/markdown-output/vaginal-lipidomics-of-women-with-vulvovaginal-cand-rpud5nw.md new file mode 100644 index 0000000000000000000000000000000000000000..94a876ceaa3b74a4e07a54baf549d88af67f742f --- /dev/null +++ b/markdown-output/vaginal-lipidomics-of-women-with-vulvovaginal-cand-rpud5nw.md @@ -0,0 +1,80 @@ +```markdown +# Goal/Experiment: +To characterize the lipid profile in vaginal discharge of women with vulvovaginal candidiasis, cytolytic vaginosis, or no vaginal infection or dysbiosis using non-targeted Liquid Chromatography-Mass Spectrometry (LC-MS). + +--- + +## Vaginal Lipidomics of Women with Vulvovaginal Candidiasis and Cytolytic Vaginosis: A Non-Targeted LC-MS Pilot Study + +### Authors +José Marcos Sanches, Paulo César Giraldo, Rose Amaral, Marcos Nogueira Eberlin, Lygia de Azevedo Marques, Isabel Migliorini, Marcel Nakahira, Michel Jan Marinus Bieleveld, Michelle Garcia Discacciati + +### Abstract +**Objective:** To characterize the lipid profile in vaginal discharge of women with vulvovaginal candidiasis, cytolytic vaginosis, or no vaginal infection or dysbiosis. + +**Design:** Cross-sectional study. + +**Setting:** Genital Infections Ambulatory, Department of Tocogynecology, University of Campinas, Campinas, São Paulo–Brazil. + +**Sample:** Twenty-four women were included in this study: eight with vulvovaginal candidiasis, eight with cytolytic vaginosis and eight with no vaginal infections or dysbiosis (control group). + +**Methods:** The lipid profile in vaginal discharge from different study groups was determined by liquid chromatography-mass spectrometry and further analyzed with MetaboAnalyst 3.0 platform. + +**Main Outcome Measures:** Vaginal lipids concentration and its correlation with vulvovaginal candidiasis and cytolytic vaginosis. + +**Results:** PCA, PLS-DA, and hierarchical clustering analyses indicated 38 potential lipid biomarkers for the different groups, correlating with oxidative stress, inflammation, apoptosis, and integrity of the vaginal epithelial tissue. + +**Conclusions:** Lipids related to oxidative stress and apoptosis were found in higher concentrations in women with vulvovaginal candidiasis and cytolytic vaginosis, while lipids related to epithelial tissue integrity were more pronounced in the control group. + +### Guidelines +- **Reliable Diagnosis of Bacterial Vaginosis:** + - Nugent RP, Krohn MA, Hillier SL. Reliability of diagnosing bacterial vaginosis is improved by standardized method of gram stain interpretation. *J Clin Microbiol* 1991;29(2):297-301 + - Amsel R, Totten PA, Spiegel CA, et al. Nonspecific vaginitis. Diagnostic criteria and microbial and epidemiologic associations. *Am J Med* 1983;74(1):14-22. +- **XCMS Online:** + - Tautenhahn R, Patti GJ, Rinehart D, Siuzdak G. XCMS Online: a web-based platform to process untargeted metabolomic data. *Anal Chem* 2012;84(11):5035-9. +- **MetaboAnalyst 3.0:** + - Xia J, Sinelnikov IV, Han B, Wishart DS. MetaboAnalyst 3.0--making metabolomics more meaningful. *Nucleic Acids Res* 2015;43(W1):W251-7. +- **Useful Online Resources:** + - http://www.hmdb.ca/metabolites. + - http://www.lipidmaps.org/. + +### Materials +- Chloroform by Contributed by users +- Methanol by Sigma Aldrich + +### Protocol + +#### Define the Groups +**Step 1:** +Define the groups by clinical and microbiological findings (gram stain). + +#### Sample Collection +**Step 2:** +Collect two samples of the vaginal wall by sterile Dacron swabs in a Falcon (15mL) tube and store immediately at -80ºC, until processing. + +#### Lipid Extraction +**Step 3:** +Randomize the samples and resuspend in 1 mL of 1:2 CHCl₃:MeOH solution, followed by the addition of 0.33 mL of CHCl₃ and 0.33 mL of deionized water. The extraction was made in 15-mL glass tubes. The solution was stirred for 5 minutes, followed by centrifugation at 13,000 rpm for 5 minutes. The supernatant was discarded, and the bottom layer containing the lipid fraction was transferred to 1.5-mL glass tubes. All samples were dried using SpeedVac for 30 minutes at 30°C and kept frozen at -80°C until the date of analysis. + +#### Data Acquisition +**Step 4:** +Lipid chromatographic separation was performed by ultra-high performance liquid chromatography (UHPLC) Agilent 1290 Infinity system. Chromatographic elution was performed on Kinetex C18 column (4.6 mm x 50 mm x 2.6 µm). For the positive ion mode, mobile phase A solvent was 0.1% formic acid and phase B was methanol; for the negative ion mode, phase A solvent was 5 mM Ammonium Acetate and phase B was methanol. The gradient started at 5% of phase B and changed linearly to 95% within 15 minutes. + +#### Mass Spectrometry +**Step 5:** +Mass spectra of samples in positive and negative ion modes were obtained using a hybrid mass spectrometer with QTOF 6550 mass analyzer (Agilent). Instrumental parameters of the electrospray ionization source were set for both positive and negative ion modes. Spectra were acquired in centroid mode and the mass range used was 50-1700 Da. + +#### Data Processing +**Step 6:** +The raw data was converted to the mzData format using the MassHunter Qualitative software. After conversion, files were imported into XCMS online for peak detection, alignment, retention time correction, and other pre-processing steps. Data was normalized, scaled, and converted into an Excel table. Exploratory multivariate data analysis was done via PCA, PLS-DA, and hierarchical clustering via MetaboAnalyst 3.0 platform. + +**Step 7 - Step 11:** +Continue normalizing and preprocessing the data for comprehensive understanding and interpretation. + +### Warnings +For the samples of vaginal content use just the sterile swabs (without PBS or any conservative medium) and immediately stored in dry 10-mL tubes at -80ºC, until processing. + +--- + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/validation-of-manual-blood-culture-bottles-bzexp3fn.md b/markdown-output/validation-of-manual-blood-culture-bottles-bzexp3fn.md new file mode 100644 index 0000000000000000000000000000000000000000..61306aa7c20503d229e63e2522271d91c78fa376 --- /dev/null +++ b/markdown-output/validation-of-manual-blood-culture-bottles-bzexp3fn.md @@ -0,0 +1,76 @@ +```markdown +# Goal/Experiment: +To evaluate the performance of manual blood culture bottles in comparison with an automated system using simulated blood cultures. + +# Validation of Manual Blood Culture Bottles V.2 + +**Authors:** +Sien Ombelet1,2, Liselotte Hardy1, Jan Jacobs1,2 + +*Affiliations:* +1Institute of Tropical Medicine, +2Department of Microbiology, Immunology & Transplantation, KU Leuven + +## Introduction +The use of equipment-free "manual" blood cultures remains prevalent in low-resource settings where automated systems are often inaccessible. The quality of available manual blood culture bottles is typically unquantified. This protocol aims to provide a method for evaluating manual blood culture bottles using simulated blood cultures. + +## Materials + +### Devices +- Vortex +- Densitometer +- Incubator +- Roller-mixer (optional) +- Pipettes (100 µL - 1000 µL) +- Pipet controller (for 10 mL glass pipettes) + +### Consumables +- Human or horse blood +- Sterile saline +- 50 mL tubes +- 10 mL tubes +- 10 µL loops +- 10 mL syringes +- Blood culture bottles +- 10 mL glass pipettes +- 300 µL pipette tips +- Columbia agar 5% sheep blood + +## Protocol + +### Spiking of the Blood (Human or Horse) +1. Use frozen (−80°C) clinical or ATCC strains for spiking. +2. Plate strains from the freezer on blood agar; incubate 18–24 hours at 35°C (use CO₂ incubator for fastidious organisms). +3. Homogenize blood using a roller-mixer (optional). +4. Prepare 0.5 McFarland suspension (1.5 × 10⁸ CFU/mL) in sterile saline and dilute to a final concentration of approximately 375 CFU/mL. Validate with colony count on three blood agar plates. +5. Add 1 mL of this suspension to 25 mL of blood for pediatric bottles, final concentration ~15 CFU/mL. +6. Inoculate three bottles per type with 2 mL spiked blood, target ~30 CFU/bottle. +7. For adult bottles, add 1 mL suspension to 50 mL blood, aim for ~7 CFU/mL. Ensure equal volumes in all bottles to avoid bias. +8. For automated BCB, inoculate with 10 mL spiked blood, target ~70 CFU/bottle. +9. Incubate manual BCB at 35°C for 7 days. + +### Processing of Incubated BCB +10. Inspect bottles twice daily for visual signs of growth. +11. Use standardized detection methods like a lightbox for better visibility. +12. Record time and date of visual growth, noting the growth type (hemolysis, turbidity, puff balls, pellicle/film, gas). +13. For biphasic bottles, note growth in agar (single colonies, confluent growth, etc.). +14. Continue incubation until both broth and agar show growth or end after 7 days if no growth is observed. +15. Perform a blind subculture on blood agar after 24 hours (D1). +16. Perform subcultures at signs of positivity; if blind subculture is negative, re-culture. +17. If no growth by day 7, perform terminal subculture on blood agar. + +### Vacuum Testing (Optional) +18. Test 10 bottles of each type in at least 2-3 batches to measure vacuum strength: +19. **Needle and Syringe Method:** + 1. Connect a 21-gauge needle to a 10 mL syringe with distilled water. + 2. Inject through bottle septum without pressure and allow bottle to fill. + 3. Measure vacuum by the volume of water aspirated. +20. **Butterfly Needle Method:** + 1. Use a 21-gauge butterfly needle and a 100 mL measuring glass with distilled water. + 2. Insert needle into septum, measure water uptake in the cylinder. + 3. Calculate the difference between start and stop volume for vacuum strength. + +--- + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/visualization-of-a-low-concentration-and-molecular-cedqta5w.md b/markdown-output/visualization-of-a-low-concentration-and-molecular-cedqta5w.md new file mode 100644 index 0000000000000000000000000000000000000000..58a34c414f73e35f6f71e7c356033b62c7854fc3 --- /dev/null +++ b/markdown-output/visualization-of-a-low-concentration-and-molecular-cedqta5w.md @@ -0,0 +1,85 @@ +```markdown +# Goal/Experiment: +Visualization of Low Concentration and Molecular Weight DNA Using DNA Gel Stain + +## Visualization of a Low Concentration and Molecular Weight DNA (DNA Gel Stain) V.2 + +**Authors**: Nadine Mowoh, Stephane Fadanka, Shalo Minette +**Affiliation**: Beneficial Bio, Mboalab + +**DOI**: [10.17504/protocols.io.5jyI89x46v2w/v2](https://dx.doi.org/10.17504/protocols.io.5jyI89x46v2w/v2) +**Version 2 Created by**: Nadine Mowoh +**Creation Date**: July 27, 2022 +**Last Modified**: July 28, 2022 + +### Abstract +After PCR amplification of DNA, agarose gel electrophoresis is run to separate DNA based on their size. The agarose gel consists of microscopic pores that act as a molecular sieve, separating molecules by charge, size, and shape. Agarose is isolated from seaweed genera Gelidium and Gracilaria and consists of repeated agarobiose (L- and D-galactose) subunits. Most gels range between 0.5%-2% concentration of agarose. + +This protocol describes the use of BenBio DNA gel stain for the visualization of low molecular weight and low concentration DNA with agarose gel electrophoresis, serving as an alternative to EtBr-based DNA gel stains. + +### Keywords +Quality control test of DNA gel stain + +### License +This is an open access protocol distributed under the terms of the [Creative Commons Attribution License](https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. + +## Materials Text + +### Reagents +- 10000x TO-DMSO gel stain stock (test sample) +- DNA amplicon _(e.g., 50 or 100bp DNA ladder)_ +- 10x TBE buffer +- EtBr-based gel stain (positive control or standard) + +### Materials and Equipment +- Glass beaker +- Micropipette and tips +- Microwave +- Gel casting tray +- Well comb +- UV transilluminator +- Voltage source (electrophoresis unit) + +### Safety Warnings +- If using a UV transilluminator to visualize, care must be taken to avoid UV exposure. +- A safer alternative is the blue light transilluminator. +- Handle Ethidium Bromide (EtBr) carefully as it is a known mutagen. + +### Before Starting +Ensure all materials, equipment, and chemicals to be used for this experiment are all in place before starting. + +## Protocol Steps + +### Visualization of a Low Concentration and Molecular Weight DNA + +#### 1. Test for Visualization +The test for visualization of a low concentration and molecular weight DNA is applicable for DNA gel stains, electrophoresis buffers, and DNA loading dye. The visualization tests can be slightly modified to suit a particular product. + +The described protocol confirms a product's ability to allow visualization of low molecular weight DNA and low concentration DNA. + +#### 2. Confirming Visualization of a Low Molecular Weight DNA +- Prepare a 2% agarose gel as described in [this protocol](https://dx.doi.org/10.17504/protocols.io.5jyI89x46v2w/v2). +- Use TO-DMSO DNA gel stain as "test DNA gel stain" and EtBr based DNA gel stain as "positive control or standard DNA gel stain." + + **Note**: Prepare two gels and run them simultaneously if possible. Otherwise, run one gel to completion before starting the next. + +- Load the gels by pipetting 3 to 5 µL of the sample into the wells: + - **Test Gel Stain**: Using a DNA ladder with tracking dye. + - **Control Gel Stain**: Using a DNA ladder with tracking dye. + +- Connect electrophoresis units to a power pack and run the gels simultaneously under the same conditions (80-100 Volts for 15-20 minutes). + +- Transfer the gel onto a UV transilluminator to visualize and separate the targeted DNA bands. + +#### 3. Confirming Visualization of Low Concentration DNA +- Make 1:5 serial dilutions of the 100bp DNA ladder or any DNA amplicon: + - Load different dilutions as amplicons to load a gel well. + + **Note**: Prepare and run gels simulating the process after the completion of each prior gel. + +- Repeat the steps described in loading a gel using different dilutions of the DNA. +- Connect the electrophoretic unit to the power source and run through (80-100 Volts for 15-20 minutes). +- Transfer the gel onto a UV transilluminator to identify and visualize the bands indicating the highest dilution of DNA detected by the gel stain. + +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/vta-surgery-protocol-db642rgw.md b/markdown-output/vta-surgery-protocol-db642rgw.md new file mode 100644 index 0000000000000000000000000000000000000000..d0f31ddb8e26ed2ebc239555c8e46da7980d2bd0 --- /dev/null +++ b/markdown-output/vta-surgery-protocol-db642rgw.md @@ -0,0 +1,155 @@ +``` +Goal/Experiment: +To conduct VTA surgery procedures which involve infecting VTA dopamine neurons with virus and implanting various cannulas and fibers intracranially. + +# VTA Surgery Protocol + +**Author:** +Sasha Burwell +Duke University +ASAP Collaborative Research Network + +**Date Created:** +April 3, 2024 + +**Last Modified:** +April 15, 2024 + +**Protocol Status:** +Working + +**DOI:** [dx.doi.org/10.17504/protocols.io.yxmvmezxng3p/v1](https://dx.doi.org/10.17504/protocols.io.yxmvmezxng3p/v1) + +--- + +## Abstract +This protocol details the VTA surgery procedures used to infect VTA dopamine neurons with virus and implant various cannulas and fibers intracranially. + +--- + +## Setup + +1. **Preparation:** + - Wipe down the surgical area and stereotax (Kopf Model 1900) with 100% ethanol. + +2. **Sterile Surface Preparation:** + - Surgical tools (scissors, fine forceps, Graefe forceps) + - Surgical eyespears + - Sterile cotton gauze pads + - Sterile cotton-tipped applicators + - Scalpel + - Marking tool for the skull (pen or fine-tip needle) + +3. **Center the Stereotax:** + - Place the Centering Height Gauge (Kopf Model 1900-51) on the stereotax base, rotate it to the 'plus' position. + - Attach the Centering Scope (Kopf Model 1915) to the stereotax arm. + - Center the scope crosshairs on the tool. + - Zero the X and Y coordinates on the Digital Display Console. + +4. **Calibrate the Stereotaxic Alignment Indicator (Kopf Model 1905):** + - Attach the Alignment Indicator to the stereotax arm. + - Rotate the Centering Gauge 45 degrees to an ‘X’ orientation. + - Ensure both dials hit zero at the same time when lowering the arm in the Z direction. + +5. **Prepare Narishige Injection Pump (MO-10):** + - Prepare the injection needle by cutting the sealed tip off a pulled capillary (Drummond Scientific Company Wiretrol II, pulled with Sutter Instrument P-97). + - Backfill the injection needle with red mineral oil, avoiding air bubbles. + - Loosen the hex screw on the injection pump slightly. + - Slide the needle over the metal injection plunger and gently tighten the hex screw. + +6. **Prepare Virus and Implants:** + - Dilute virus in hyperosmotic PBS to desired titer. + +--- + +## Begin Surgery + +7. **Calculate and Prepare Buprenorphine SR:** + - Weigh the mouse and calculate the amount of Buprenorphine SR for injection. + +8. **Prepare Isoflurane Chamber:** + - Oxygen flow rate to 1 mL, with 3% isoflurane. + - Place mouse in chamber after preloading for 1-2 minutes. + +9. **Ensure Mouse is Anesthetized:** + - Confirm ~1 breath per second, no tail pinch response. + - Adjust isoflurane to 2% and oxygen to 0.6 mL. + +10. **Position Mouse in Ear Bars:** + - Secure bite bar in mouse’s mouth. + - Position and tighten nose cone. + +11. **Prepare the Surgical Area:** + - Apply Puralube to whiskers + - Apply Nair to the scalp using a cotton-tipped applicator, against the grain of the fur, and avoid eyes. + - After 30 seconds, wipe off Nair. + - Sterilize the cleared skin with betadine and 70% ethanol alternately. + +12. **Administer Buprenorphine SR:** + - Inject Buprenorphine SR subcutaneously. + +13. **Prepare the Skull:** + - Remove the scalp using forceps and scissors, exposing the skull. + - Clean the exposed skull with hydrogen peroxide. + +14. **Apply Bupivacaine 0.25%:** + - To the skin around the edges of the incision. + +--- + +### Electrode Setup: + +15. **Drilling Craniotomies:** + - Insert small screws for grounding. + - Drill craniotomies using a carbide drill bit. + +16. **Leveling and Marking Skull:** + - Use Centering Scope and Alignment Indicator to mark bregma. + - Level skull and mark drilling coordinates. + +17. **Virus Injection:** + - Load the Narishige injection needle with virus. + - Move the needle to coordinate -3.2 and zero 'Z'. + - Inject virus slowly into designated site. + +18. **Provide Saline with Dextrose:** + - Every hour, give 1mL of sterile saline with 5% dextrose subcutaneously. + +19. **Suture and End Surgery:** + - Suture the skin if not adding implants, using Vetbond as necessary. + +--- + +### Implant Setup: + +20. **Prepare Skull for Implants:** + - Scratch skull in crosshair pattern. + - Apply Optibond and set with UV light. + - Re-moisten skin with Bupivacaine. + +21. **Electrode/Cannula Implant:** + - Clean electrode/cannula, attach to the stereotax arm, and lower into the skull. + - Secure with dental cement and superglue. + +--- + +### Complete Implant: + +22. **Finalize Implant Setup:** + - Give another saline with dextrose if needed. + - Fill headbar with dental cement, ensuring skull is covered. + - Add necessary accessories (dust cap, headcap) post cement drying. + +--- + +### End Surgery: + +23. **Recovery:** + - Turn off isoflurane, keep mouse under oxygen until waking. + - Weigh mouse post-surgery and place in cage to recover. + - Perform post-operative care over the following days. + +--- + +[endofoutput] +``` \ No newline at end of file diff --git a/markdown-output/well-tempered-metadynamics-protocol-b5fyq3pw.md b/markdown-output/well-tempered-metadynamics-protocol-b5fyq3pw.md new file mode 100644 index 0000000000000000000000000000000000000000..c81fc06c0bf6c04c5ce33be923f4847af538a8b8 --- /dev/null +++ b/markdown-output/well-tempered-metadynamics-protocol-b5fyq3pw.md @@ -0,0 +1,111 @@ +```markdown +# Goal/Experiment: + +The goal of this protocol is to run well-tempered metadynamics simulations using the Desmond software to analyze the free-energy surfaces (FES) of chosen variable sets and understand the potential energy landscapes of various molecular systems. + +# Well-Tempered Metadynamics Protocol V.2 + +**Authors:** +- Vidya Niranjan, Akshay Uttarkar + R V College of Engineering + Centre of Excellence in Computational Genomics (Vidya Lab) + +**DOI:** +[dx.doi.org/10.17504/protocols.io.b5fyq3pw](https://dx.doi.org/10.17504/protocols.io.b5fyq3pw) + +## Overview +Metadynamics is a computational technique where the potential energy landscape of certain variables (collective variables) is explored by periodically adding repulsive Gaussian potentials. This process modifies the potential energy surface, helping the system to overcome energy barriers and sample different configurations, ultimately leading to the flattening of the free-energy surface (FES). The protocol described here uses the well-tempered metadynamics module of Desmond for running simulations and analyzing the results. + +## Step-by-Step Protocol + +### 1. Protein Preparation +1. Import the crystal structure into the Maestro GUI. +2. Use the Protein Preparation Wizard panel: + - Add hydrogen atoms. + - Patch end groups. + - Add missing side chains and loops. + - Assign protonation states of histidine, aspartate, and glutamate at pH 7.045. + - Optimize polar hydrogen orientations in crystal water molecules and proteins. + +**Note:** Problems related to the structure can be viewed in the "Diagnostics Tab". + +### 2. Simulation System Setup +#### Solvation +1. Solvate the complex in an orthorhombic box with dimensions 10 Å larger than the total size of the receptor complex. +2. Click on `minimize volume` to obtain the optimal box dimensions. + +#### Ions +1. Place ions based on the requirement of the simulation environment (neutral, acidic, or basic). + - Solvents will consist of water molecules and neutralizing agents such as sodium (Na) or chloride (Cl). + +### 3. Preparation of Metadynamics Input Files +1. Load the solvated complex into the Metadynamics panel. +2. Define the CV (collective variable) distance using either of the following methods: + - Use the `Define` mode in the Maestro GUI: `Select > Define > Molecule list`. + - Use the `Selection` mode: `Select > More Objects > Your Selection`. + + **Parameter Settings:** + - The height and width of the Gaussian must be set. + - The height-to-interval ratio should be roughly 0.25 to 0.33. + - Interval for Gaussian 0.09 picoseconds (ps). + - Simulation temperature: 310 K. + - Pressure: 1.01325 bar. + - Simulation time: 25-50 nanoseconds (ns). + +3. Click on `Write file` to generate three input files (`cfg`, `msj`, and `cms`) and one command file (`sh`). + +### 4. Changes in Parameters for Well-Tempered Metadynamics +1. Add the bias factor keyword `kTemp` with a value of 2.4 (corresponds to 1200 K) to the meta block of the `.msj` file: + ``` + meta = { + ktemp = 2.4 + } + ``` +2. Suggestions for choosing `kTemp`: + - \(3 \, \text{kcal/mol} \rightarrow 1.7 \, \text{kTemp}\) + - \(6 \, \text{kcal/mol} \rightarrow 3.4 \, \text{kTemp}\) + - \(10 \, \text{kcal/mol} \rightarrow 5.6 \, \text{kTemp}\) + - \(15 \, \text{kcal/mol} \rightarrow 8.4 \, \text{kTemp}\) + - \(20 \, \text{kcal/mol} \rightarrow 11.2 \, \text{kTemp}\) + +### 5. Multisim Run +1. Execute multiple stages as instructed in the `.msj` file: + - Brownian dynamics of the NVT ensemble at 10 K with small timesteps. + - Berendsen dynamics of the NVT ensemble at 10 K. + - Berendsen dynamics of the NPT ensemble at 10 K. + - Berendsen dynamics of the NPT ensemble at 310 K. + - Martyna-Tobias-Klein dynamics of the NPT ensemble at 310 K without restraints. + +**Outputs:** +- `kerseq` file, recording all Gaussian functions infused. +- Trajectory file. +- Other output files. + +### 6. Metadynamics Analysis +1. Open the Metadynamics analysis panel from Maestro GUI. +2. Input the `"-out.cms"` file and run the analysis. +3. Save the plot. A `.fes` file is generated automatically containing corresponding FES values for each CV. + +--- + +## Professional Terms and Reagents + +- **Gaussian Potential**: A bell-shaped potential used to smoothen the energy landscape in metadynamics. +- **Protonation States**: Refers to the addition or removal of protons (hydrogen ions) from an atom, affecting its chemical properties. +- **Orthorhombic Box**: A type of cuboid simulation box with rectangular faces, used here for containing the solvated molecular complex. +- **Neutralizing Agents**: Chemicals like Na (sodium) or Cl (chloride) ions that are added to maintain charge neutrality in the system. + +--- + +## Alternative Methods + +1. **Protein Preparation Wizard Alternatives**: + - Use the `PDBFixer` tool for preparing protein structures by adding missing atoms and residues. + +2. **Simulation Software**: + - Alternative metadynamics packages include `PLUMED`, which can be used in combination with molecular dynamics engines like `GROMACS`. + +--- + +**End_of_output** +``` \ No newline at end of file diff --git a/markdown-output/western-blot-to-evaluate-infection-level-incurred-mjac4ie.md b/markdown-output/western-blot-to-evaluate-infection-level-incurred-mjac4ie.md new file mode 100644 index 0000000000000000000000000000000000000000..9234f5ed1dc82271f32f40e6ed3932bff832ecff --- /dev/null +++ b/markdown-output/western-blot-to-evaluate-infection-level-incurred-mjac4ie.md @@ -0,0 +1,160 @@ +```markdown +# Goal/Experiment: +To evaluate how newly-developed ECMO reduces infection levels by controlling and stabilizing the overall hemodynamic status, assessed using a Western blot technique. + +# Western Blot to Evaluate Infection Level Incurred by ECMO (Pilot Study) + +### Hyunsuk Frank Roh, Jung Mogg Kim + +## Abstract +ECMO inevitably increases the risk of infection over time; therefore, lowering infections acquired during ECMO should be further investigated and developed accordingly (Bizzarro, 2011). The degree of dysfunction by ECMO is evaluated using several methods, namely: ultrastructural changes in gut epithelium by TEM, plasma lipopolysaccharide (LPS) levels of bacterial products after receiving ECMO, etc. In particular, bacterial lipoteichoic acid (LTA) antigens in plasma can be measured over time by Western blot laboratory technique in association with the loss of gut barrier function below. The purpose of this pilot study is to evaluate how newly-developed ECMO reduces infection levels by controlling and stabilizing the overall hemodynamic status, based on the outcome of a Western blot. + +Citation: Hyunsuk Frank Roh, Jung Mogg Kim Western Blot to Evaluate Infection Level Incurred by ECMO (Pilot Study). protocols.io dx.doi.org/10.17504/protocols.io.mjac4ie Published: 11 Jan 2018 + +## Protocol + +### 1. Cell Culture (SubCulture + Cell Counting) + +**Step 1.** + +**Media:** RPMI1640 to provide nutrition required for cell growth and to neutralize the trypsin. +- Warm the media at 37°C for 30 min +- Prepare the trypsin (with EDTA) and PBS + +1. Suck away the media from the flask and then wash the cells (HT-29) with PBS (T-25 = 5mL, T-75=10mL) +2. Treat the cells with 1.0 mL of trypsin (with EDTA) and incubate the flask at 37°C for 5 min to separate the cells from the flask floor. +3. Prepare the materials (6 dishes, one 1.5mL tube for cell counting, 1 flask for cell subculture) +4. After the incubation, add 4.5mL media to the previous flask and take 5mL of the cells in the cube into the 15mL tube for centrifugation for 3 min at 1000 RPM. +5. After centrifugation, remove the media (in case of cell counting, re-suspend the cells with 1mL of media, take 50uL and then proceed with cell counting). +6. Afterwards, add 2mL of media, do pipetting up and down. +7. For culturing, place 6.0*10^5 cells/disk on each dish and a new flask for subculture. Then, add media up to 5mL in total to each dish and T-25 (when using T-75 flask, add media up to 10mL in total). + +Note: Although it is the standard method to count the cells in a flask per a protocol using lattice, it is also somewhat acceptable to get a ball-park number for cell numbers under the microscope. + +### 2. Cell Stimulation and Protein Extraction + +**Step 2.** + +1. Treat the HT-29 cells with 6μM of TNF-α or LPS (50ng/mL) (in each dish for the purpose of cell stimulation and incubate for 0h, 1/6h, 1/2h, 1h, and 2h each, accordingly). +2. Prepare ice in a styrofoam box. +3. Discard the media and wash cells with 5 mL of PBS. +4. Harvest cells (in 1.5mL of PBS) and centrifuge at 3000-5000 RPM for 10 min at 4°C. +5. Remove PBS and add 5080µL of lysis buffer based upon the number of centrifuged cells. +6. Maintain constant agitation (or vortexing of the tube every 5 min) for 30 min at 4°C or ice box. +7. Centrifuge at 13200 RPM for 15 min at 4°C. +8. Transfer the supernatant of each tube into its corresponding new 1.5mL tube. + +### 3. Western Blotting + +**Step 3.** + +Similar to Southern blotting, Western blotting employs gel electrophoresis to separate materials, performs a membrane transfer procedure, and then detects to locate the specific biological product in need. Western blotting detects protein instead of DNA segments and uses antibody instead of complementary DNA segment probe; it also employs acrylamide gel instead of agar gel. + +#### Making a Running Gel (10% Running Gel) + +**Step 4.** + +1. Prepare the electrophoresis kit and fill the chamber with DDW for detecting any possible fluid leakage from the kit. +2. Prepare a 50mL tube for running gel and a 15mL tube for stacking gel. +3. Make mixtures according to the instruction (both running gel and stacking gel), by adding reagents for an acrylamide gel polymerization reaction in the order specified by the manufacturer-specific guidelines: + + (a) **10% Running Gel Solution (20mL)** + - 7.9mL DDH2O + - 6.7mL 30% Acrylamide Mix + - 5mL 1.5M Tris pH8.8 + - 0.2mL 10% SDS + - (Add the followings immediately before use): + - 0.2mL 10% APS + - 0.008mL TEMED + + (b) **5% Running Gel Solution (6mL)** + - 4.1mL DDH2O + - 1.8mL 30% Acrylamide Mix + - 0.75mL 1.0M Tris pH6.8 + - 0.06mL 10% SDS + - (Add the followings immediately before use): + - 0.06mL 10% APS + - 0.006mL TEMED + +4. Prepare the 10% Running Gel Solution per the aforementioned protocol (Note that APS & TEMED must be added immediately before adding the solution to the cassette). +5. Place dry short plate in front of spacer plate in the casting frame with the arrows on spacer plate facing up. +6. Add 250uL of isopropanol to the top of the running gel to even out the surface of running gel and leave it to harden. + +#### Making a Stacking Gel + +**Step 5.** + +1. Wash isopropanol on top of running gel of the kit. +2. Check whether the running gel has been polymerized. If so, remove the isopropanol layer on top of the running gel. +3. Dry out the chamber and add sequentially APS and TEMED to the gel mixtures. +4. After gently stirring the solution, carefully fill the stacking gel on top of the hardened running gel and install a comb between the two glass plates (The comb should be carefully and obliquely slid down into the running gel between the two plates in order to prevent the formation of a bubble in between the comb and gel by inserting the comb too quickly into the gel.) +5. After the gel gets hardened, very carefully remove the comb. +6. Place the sample in wells and proceed to electrophoresis. + +#### Sample Protein Quantification + +**Step 6.** + +1. Mix and vortex 530𝜇L Bradford (5X) solution and 530*4 𝜇L of DW; +2. Load 200𝜇L of the mixture (0, 1, 2, 3, 4, 5)𝜇L of BSA (1 mg/mL), and 1𝜇L of samples in quantification kit; pipette up and down to mix the protein with Bradford mix; then, remove bubbles with a needle; +3. Perform the Quantification accordingly; + + :: On the 96 Well-Plate, load 200𝜇L of the mixture each, and then add the following accordingly: + - 0𝜇L BSA + 1𝜇L BSA + 2𝜇L BSA + - 3𝜇L BSA + 4𝜇L BSA + 5𝜇L BSA + - 1𝜇L sample I + 1𝜇L sample II + 1𝜇L sample III + - 1𝜇L sample IV + 1𝜇L sample V + +#### Sample Preparation + +**Step 7.** + +1. Add 5x sample buffer, lysis buffer, and the calculated amount of sample into 600𝜇L tubes, according to the quantification calculation; +2. Place tubes in the boiled water for 10 min; +3. Cool the tubes in the ice. + +#### Gel Electrophoresis + +**Step 8.** +1. make 1X EP buffer (100mL 5X EP buffer + 400mL DW water); +2. Wash the glass with DW, carefully remove the combs, and remove the bubbles with DW; +3. Assemble electrophoresis kits and pour 1X EP buffer between the plates; +4. Pour 1X EP buffer into the kit (check whether 1X EP buffer leaks out into the tank); Load samples and run electrophoresis; +5. Make 1X transfer buffer (100mL 10X transfer buffer + 200mL EtOH + 700mL DW) immediately and store at -20°C, while running electrophoresis; +6. When electrophoresis is almost finished, activate PVDF membrane by immersing it in MeOH for 1 minute. + +#### Transfer + +**Step 9.** + +1. Prepare transfer kits, tray, and 1X transfer buffer; +2. Place the following materials in the order of bottom to top (Make sure that the concaved surface of the PVDF membrane faces the gel.): + - Plate – Sponge – Filter paper – Gel – Membrane – Filter Paper – Sponge – Plate +3. Mount the plates into the tank and fill the tank with transfer buffer; +4. Run the kit at 100V for 80-90 min; +5. After transfer, cut the membrane into appropriate sizes. + +#### ImmunoBlot + +**Step 10.** + +1. Add Ponceau solution (5𝜇L of Ponceau + 6mL of DW) to check the bands with bare eyes; +2. Wash Ponceau solution with DW and TBST (Tris Buffered Saline with Tween) several times; +3. Block the membrane with 5% of skim milk for 1 hr; +4. Wash the membrane with 10mL of TBST for 5 minutes, 3 times in total; +5. Add 1.5𝜇L first antibody diluted in 6mL of 5% BSA (1:4000) and incubate for 2hr; +6. Rinse 3 times with 10mL of TBST and incubate for 5 min each time; +7. Add 1𝜇L of second antibody diluted in 6mL of 5% BSA (1:6000) and incubate for 1 hr; +8. Rinse 3 times with 10mL of TBST and incubate for 5 min each time. + +#### Development + +**Step 11.** + +1. Prepare the developing cassette and ECL kit; +2. Place the membrane of the cassette; then, rinse the membrane with DW and TBST; +3. Cautiously remove the wash buffer around the membrane with a paper towel; then, treat the membrane with ECL solution (750mL of white bottle + 750mL of brown bottle of West-Q chemiluminescent Substrate Kit); +4. Proceed with developing. + +## endofoutput +``` \ No newline at end of file diff --git a/markdown-output/wet-mount-method-for-enumeration-of-aquatic-viruse-c8pzvm.md b/markdown-output/wet-mount-method-for-enumeration-of-aquatic-viruse-c8pzvm.md new file mode 100644 index 0000000000000000000000000000000000000000..26d95bc316daa6ab77be038dfb37f637c630b371 --- /dev/null +++ b/markdown-output/wet-mount-method-for-enumeration-of-aquatic-viruse-c8pzvm.md @@ -0,0 +1,222 @@ +```markdown +# Wet-mount Method for Enumeration of Aquatic Viruses + +## Goal/Experiment: +To develop an accurate, low-cost alternative method for the enumeration of aquatic viruses using a wet-mount preparation and epifluorescence microscopy. + +### Authors: +B.R. Cunningham, J.R. Brum, S.M. Schwenck, M.B. Sullivan, S.G. John + +### Abstract +**Purpose:** This method for the enumeration of aquatic viruses is a low-cost alternative to the commonly used filter-mount method. Briefly, fluorescently-stained samples are wet-mounted directly onto slides for epifluorescence microscopy after an optional chemical flocculation concentration step used for samples with anticipated virus concentrations of <5×10⁷ viruses mL⁻¹ (samples with >5×10⁷ viruses mL⁻¹ do not require this concentration step prior to analysis). Virus concentration in the wet-mounted sample is determined from the ratio of viruses to microsphere beads, which are added at a known concentration. + +This wet-mount method for enumerating viruses is significantly less expensive than the filter-mount method (i.e., the cost of microsphere beads per sample is ~500-fold lower than the cost of one filter per sample) and is appropriate for rapid, precise, and accurate enumeration of aquatic viruses over a wide range of viral concentrations encountered in field and laboratory samples. The only limitation of this method is that samples with virus concentrations ≤1×10⁶ viruses mL⁻¹ cannot be enumerated, as the abundance of viruses is too low for efficient enumeration. + +![Figure 1. Overview of the wet-mount method for enumeration of aquatic viruses.](https://i.imgur.com/5UEqn1S.png) + +Citation: B.R. Cunningham, J.R. Brum, S.M. Schwenck, M.B. Sullivan, S.G. John Wet-mount Method for Enumeration of Aquatic Viruses. +[protocols.io](dx.doi.org/10.17504/protocols.io.c8pzvm) + +Published: 07 Jan 2016 + +## Guidelines + +### Optional Viral Concentration Method +Samples with <5×10⁷ viruses mL⁻¹ must be concentrated using the iron chloride flocculation method adapted from John et al. (1) prior to the enumeration of viruses with the wet-mount method. The steps for this procedure are displayed in Figure 1A. + +#### Materials Required: +- 1.5 mL microcentrifuge tubes +- Microcentrifuge +- Vortexer +- Phosphate Buffered Saline (PBS) +- Iron chloride (FeCl₃•6H₂O) +- Ultrapure water +- Magnesium EDTA (Mg2EDTA) +- Ascorbic acid +- Sodium hydroxide (NaOH) + +#### Reagent Preparation: +- [Iron Chloride Solution](#iron-chloride-solution) +- [Ascorbic-EDTA Buffer](#ascorbic-edta-buffer) + +### Virus Counting Procedure +This part of the protocol starts with either 10 µL of unconcentrated sample (if the anticipated virus concentration is >5×10⁷ viruses mL⁻¹ ) or 10 µL of concentrated, resuspended sample (see above). The steps for this procedure are displayed in Figure 1B. + +#### Materials Required: +- Epifluorescence microscope (1000X magnification and ~495 nm excitation) +- Vortexer +- 1.5 mL microcentrifuge tubes +- Isopropanol +- Glass slides (25x75 mm) +- Cover slips (24x60 mm) +- Microsphere silica beads (Bangs Laboratories, 2.34 µm diameter, conc. 7.845x10⁹ beads/mL, Cat. # SS04N/4186, Inv. # L060320A) +- Glycerol +- Phosphate Buffered Saline (PBS) +- SYBR Gold (Invitrogen) +- Kimwipes +- Ascorbic acid (if analyzing unconcentrated samples) + +#### Reagent Preparation: +- [SYBR Gold Working Stock](#sybr-gold-working-stock) +- [Working Bead Solution](#working-bead-solution) +- [Ascorbic Acid Antifade Solution](#ascorbic-acid-antifade-solution) + +## Protocol + +### Optional Viral Concentration Method +**Step 1:** +Place 1 mL of sample into a 1.5 mL microcentrifuge tube. + +**Step 2:** +Add 1 µL Iron Chloride Solution and vortex to mix. + +- **Amount:** 1 µL additional info: +> [Iron Chloride Solution](#iron-chloride-solution) + +**Step 2.1:** +Dissolve FeCl₃•6H₂O into ultrapure water to form a solution of 10 g Fe L⁻¹ (i.e., 0.484 g FeCl₃•6H₂O dissolved in 10 mL of ultrapure water). + +**Step 3:** +Centrifuge the sample at 14K RCF for 20 minutes. + +- **Duration:** 00:20:00 + +**Step 4:** +Remove supernatant using a pipette, leaving a small, undisturbed pellet of Fe oxyhydroxides behind (Figure 2). + +![Figure 2. Fe oxyhydroxide pellet after removal of supernatant.](https://i.imgur.com/vOPxPbZ.png) + +> **Notes:** +> James Thornton Jr, 28 Jul 2015 +> Note: Removing all of the supernatant is not critical, as a tiny bit remaining will not affect results. + +**Step 5:** +Dissolve the pellet in 10 µL of Ascorbic-EDTA Buffer, creating a 100-fold concentration of the original sample. Vortex and then pipette up and down to ensure complete dissolution. + +- **Amount:** 10 µL additional info: +> [Ascorbic-EDTA Buffer](#ascorbic-edta-buffer) + +**Step 5.1:** +Combine equal parts of 0.4 M Mg2EDTA and 0.8 M ascorbic acid, adjust with 10 N NaOH to reach a pH of 6-7. + +**Step 5.2:** +Prepare fresh within 48 hours of use. + +**Step 6:** +Vortex and then pipette up and down to ensure complete dissolution. + +### Virus Counting Procedure +**Step 7:** +Combine 10 µL sample (concentrated or unconcentrated) and 2 µL SYBR Gold Working Stock, vortex to mix, and place in dark for 15 minutes. + +- **Amount:** 2 µL additional info: +- **Duration:** 00:15:00 +> [SYBR Gold Working Stock](#sybr-gold-working-stock) + +**Step 7.1:** +Dilute SYBR Gold (Invitrogen; 10,000X stock) into PBS (phosphate buffered saline) to prepare 1000X solution. Prepare fresh daily. Store in dark between uses. + +**Step 8:** +If the sample is unconcentrated, add 1 µL of Ascorbic Acid Antifade Solution. + +- **Amount:** 1 µL additional info: +> [Ascorbic Acid Antifade Solution](#ascorbic-acid-antifade-solution) + +**Step 8.1:** +Dissolve 0.1 g ascorbic acid into 1 mL PBS, creating a 10% (wt/vol) solution. Prepare fresh daily. + +**Step 9:** +Add 5 µL of glycerol to the stained sample and vortex to mix. + +**Step 10:** +Add 2 µL Working Bead Solution to the sample. + +- **Amount:** 2 µL additional info: +> [Working Bead Solution](#working-bead-solution) + +> **Notes:** +> James Thornton Jr, 28 Jul 2015 +> Note: The Working Bead Solution must be thoroughly mixed by vortexing prior to adding it to the sample so that the appropriate number of beads are added to the sample. + +**Step 10.1:** +Dilute stock bead solution 10-fold into PBS to obtain a concentration of 10⁸ beads mL⁻¹; store at 4°C. + +**Step 11:** +Clean glass slides and cover slips with isopropanol and Kimwipes. + +**Step 12:** +Thoroughly mix the sample/bead mixture by pipetting up and down, then immediately pipette 10 µL of it onto a glass microscope slide. Place a coverslip over the mixture and avoid trapping air under the coverslip. + +**Step 13:** +Place a coverslip over the mixture and avoid trapping air under the coverslip. + +**Step 14:** +View viruses under 495 nm excitation at 1000X magnification using an epifluorescence microscope. Count the number of viruses in one defined field of view. + +**Step 15:** +Once complete, switch off the excitation and turn on the white light of the microscope to count the beads in the same field of view (Figure 3). + +![Figure 3. Epifluorescence image of a wet-mounted sample.](https://i.imgur.com/Etl8d6e.png) + +**Step 16:** +Repeat Step 14 & 15 by counting viruses and beads in multiple fields until at least 100 of each have been counted. + +**Step 17:** +The concentration of viruses can then be determined with the following equation: + +\[ +c_{\text{virus}} = \frac{n_{\text{virus}} \times v_{\text{beads}} \times c_{\text{beads}}}{n_{\text{beads}} \times v_{\text{sample}}} +\] + +> **Notes:** +> James Thornton Jr, 31 Jul 2015 +> where: +> - \(c_{\text{virus}}\) = concentration of viruses (viruses mL⁻¹) +> - \(n_{\text{virus}}\) = total number of viruses counted in all fields +> - \(n_{\text{beads}}\) = total number of beads counted in all fields +> - \(v_{\text{beads}}\) = volume of Working Bead Solution added +> - \(v_{\text{sample}}\) = volume of sample used (if the sample has been concentrated with iron chloride flocculation, use the pre-concentration sample volume here) +> - \(c_{\text{beads}}\) = concentration of beads in Working Bead Solution (beads mL⁻¹) + +**Step 18:** +Prepared samples can be stored at -20°C either in the microcentrifuge tube (i.e., after completing Step 4) or after mounted on slides (i.e., after completing Steps 12&13) with no significant change in the calculated virus concentration. + +> **Notes:** +> James Thornton Jr, 28 Jul 2015 +> Note: These storage conditions have currently been validated for a time period of 7 days. + +### Related References +1. John SG, Mendez CB, Deng L, Poulos B, Kauffman AKM, Kern S, Brum J, Polz MF, Boyle EA, Sullivan MB (2011) A simple and efficient method for concentration of ocean viruses by chemical flocculation. Environmental Microbiology Reports, 3:195-202. doi:10.1111/j.1758-2229.2010.00208.x + +## Reagent Preparation + +### Iron Chloride Solution +**Step 2.1:** +Dissolve FeCl₃•6H₂O into ultrapure water to form a solution of 10 g Fe L⁻¹ (i.e., 0.484 g FeCl₃•6H₂O dissolved in 10 mL of ultrapure water). +Contact: VERVE Team + +### Ascorbic-EDTA Buffer +**Step 5.1:** +Combine equal parts of 0.4 M Mg2EDTA and 0.8 M ascorbic acid, adjust with 10 N NaOH to reach a pH of 6-7. +**Step 5.2:** +Prepare fresh within 48 hours of use. +Contact: VERVE Team + +### SYBR Gold Working Stock +**Step 7.1:** +Dilute SYBR Gold (Invitrogen; 10,000X stock) into PBS (phosphate buffered saline) to prepare 1000X solution. Prepare fresh daily. Store in dark between uses. +Contact: VERVE Team + +### Ascorbic Acid Antifade Solution +**Step 8.1:** +Dissolve 0.1 g ascorbic acid into 1 mL PBS, creating a 10% (wt/vol) solution. Prepare fresh daily. +Contact: VERVE Team + +### Working Bead Solution +**Step 10.1:** +Dilute stock bead solution 10-fold into PBS to obtain a concentration of 10⁸ beads mL⁻¹; store at 4°C. +Contact: VERVE Team + +``` +endofoutput +``` \ No newline at end of file diff --git a/markdown-output/whole-cell-patch-clamp-recordings-from-striatal-ch-ctszwnf6.md b/markdown-output/whole-cell-patch-clamp-recordings-from-striatal-ch-ctszwnf6.md new file mode 100644 index 0000000000000000000000000000000000000000..e1abde5689e880c14c9e54219338a8aaef22a3f7 --- /dev/null +++ b/markdown-output/whole-cell-patch-clamp-recordings-from-striatal-ch-ctszwnf6.md @@ -0,0 +1,146 @@ +```markdown +# Goal/Experiment: +Perform whole-cell patch-clamp recordings of striatal cholinergic interneurons (ChIs) previously labeled with the fluorophore mCherry from acute, ex vivo mouse brain slices. + +# Whole-cell Patch-Clamp Recordings from Striatal Cholinergic Interneurons in ex vivo Mouse Brain Slices + +Jeffrey Stedehouder^1,2,3^, Stephanie J Cragg^1,2,3^ + +^1^ Department of Physiology, Anatomy and Genetics, University of Oxford, OX1 3PT, UK +^2^ Oxford Parkinson’s Disease Centre, University of Oxford, Oxford, United Kingdom +^3^ Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD, 20815 + +- DOI: [dx.doi.org/10.17504/protocols.io.3by4ljo5zlo5/v1](https://dx.doi.org/10.17504/protocols.io.3by4ljo5zlo5/v1) + +## Abstract +This protocol describes the steps to perform whole-cell patch-clamp recordings of striatal cholinergic interneurons (ChIs) previously labeled with the fluorophore mCherry from acute, ex vivo mouse brain slices. + +## Guidelines +The high quality of whole-cell patch-clamp recordings critically depends on four, partially interrelated, variables: + +1. **Solution Preparation**: All solutions have to be made fresh in clear, rinsed glassware with careful inclusion of all ingredients and measured amounts. +2. **Slice Quality**: The brain must be removed from the skull quickly without causing any damage, transferred to an ice-cold solution, and cut into 300 μm coronal slices with minimal manipulation. +3. **Cell Selection**: Choose healthy-looking cells that are not bloated, damaged, too deep, or obscured by other cells. +4. **Giga-Seal Quality**: High giga-seal (>GΩ) should be attained before breaking in, followed by proper access (low MΩ) of the cell. + +## Materials + +### Reagents +- AAV5-hSyn-DIO-mCherry ([ETH Zurich Viral Vector Facility](https://www.vvf.ethz.ch)) or equivalent +- Salts +- Sodium pentobarbital + +### Equipment +- VT1200S Vibrating blade microtome (Leica) +- Multiclamp 700B amplifier (Molecular Devices Corp.) +- Digidata 1440A acquisition board (Molecular Devices Corp.) +- P-1000 Horizontal Pipette Puller (Sutter Instruments) + +### Mouse Lines +- Heterozygous ChAT-Cre mice ([The Jackson Laboratory](https://www.jax.org)) or equivalent + +## Solution Preparations + +### NMDG-based Cutting Solution (~310 mOsm, pH 7.4) + +| Reagent | Concentration | +|------------------------------- | ------------- | +| N-methyl-d-glucamine (NMDG) | 93 mM | +| HCl | 93 mM | +| NaHCO₃ | 30 mM | +| D-glucose | 25 mM | +| HEPES Buffer | 20 mM | +| Na-ascorbate | 5 mM | +| Thiourea | 2 mM | +| MgCl₂ | 7 mM | +| Na-pyruvate | 3 mM | +| KCl | 5 mM | +| NaH₂PO₄ | 1.25 mM | +| CaCl₂ | 0.5 mM | + +Adjust pH to 7.4 with HCl. Prepare fresh on the morning of the experiment. + +### Artificial Cerebrospinal Fluid (aCSF) + +| Reagent | Concentration | +|-------------|---------------| +| NaCl | 127 mM | +| NaHCO₃ | 25 mM | +| D-glucose | 25 mM | +| KCl | 2.5 mM | +| NaH₂PO₄ | 1.25 mM | +| MgSO₄ | 5 mM | +| CaCl₂ | 1.6 mM | + +Prepare fresh on the morning of the experiment. + +### Intracellular Solution (~290 mOsm, 7.4 pH) + +| Reagent | Concentration | +|--------------------|---------------| +| K-gluconate | 120 mM | +| KCl | 10 mM | +| HEPES | 10 mM | +| K-phosphocreatine | 10 mM | +| ATP-Mg | 4 mM | +| GTP | 0.4 mM | + +Adjust pH to 7.4 with KOH. Store aliquots in a -20°C freezer. Thaw and filter through a 20 μM filter before use. + +### Software +- Python +- Clampex 10.0 (Molecular Devices Corp.) +- Linlab + +### Safety Warnings +- Blades! +- Acids! +- Sharps! + +### Ethics Statement +Experiments involving animals must be conducted according to internationally-accepted standards and have prior approval from an Institutional Animal Care and Use Committee (IACUC) or equivalent ethics committee(s). + +## Protocol + +### 1. Injection of mCherry Virus to Label Cholinergic Interneurons + +1. Inject AAV5-hSyn-DIO-mCherry (~1.3 x 10^13^ genome copies/mL) into the Caudate Putamen (CPu) or Nucleus Accumbens core (NAc) of heterozygous adult (8-16 weeks old) ChAT-cre male or female mice. + +### 2. Preparation of Ex Vivo Mouse Brain Slices + +2. At 3-5 weeks post-injection, induce anesthesia using i.p. sodium pentobarbital (200 mg/kg). +3. Decapitate the mouse and remove the brain quickly in ice-cold, NMDG-based cutting solution oxygenated with 95% O₂/5% CO₂. +4. Slice striatum into 300 μm coronal slices using a vibrating blade microtome (Leica VT1200S). +5. Incubate slices in a heat bath at 34°C for 15 minutes in NMDG-based cutting solution. +6. Transfer slices carefully to artificial cerebrospinal fluid (aCSF) and incubate at 34°C for another 15 minutes. +7. Allow slices to recover at room temperature for at least 1 hour in aCSF oxygenated with (95% O₂/5% CO₂). Use slices within ~6 hours from cutting. + +### 3. Whole-Cell Patch-Clamp Recordings + +8. Place a coronal slice in a recording chamber perfused with aCSF oxygenated with 95% O₂/5% CO₂ at 32-33°C at ~3 ml/min. Weigh the slice down with a harp or metal clips. +9. Use the DIC mode on a microscope at 40x to identify the region of interest based on gross landmarks. +10. Turn off the brightfield illumination. Use a red wavelength LED or laser to locate mCherry+ somata keeping exposure brief to prevent phototoxicity. +11. Focus over the soma of this cell, ensuring it looks healthy and patchable. +12. Pull a glass pipette (1.5 mm OD x 0.86 mm IK x 100 mm L; Harvard Apparatus) using a P1000 Horizontal Pipette Puller (Sutter Instruments). +13. Backfill the glass pipette with ~10-15 μl of freshly thawed intracellular solution. Avoid bubbles near the tip. +14. Load the pipette on the headstage and place it near the slice in aCSF with resistance ranging from 3–5 MΩ. Log the exact pipette resistance in your lab journal. +15. Perform whole-cell recordings from mCherry-positive neuronal somata. Record access resistance, capacitance, and 'resting' membrane potential. Fully compensate for bridge balance and capacitance. +16. Run stimulation protocols tailored for the experiment, such as current clamp steps from -100 pA to +200 pA. +17. Perform additional protocols, keeping an eye on changes in 'resting' membrane potential, holding potential, or series resistance. + +### Data Analysis + +18. Transfer data for offline analysis using custom Python software. +19. Determine physiological characteristics from voltage responses to square-wave current pulses of 750 ms duration ranging from -200 pA to +300 pA. +20. Determine input resistance by the slope of the linear regression through the voltage-current curve from -200 pA to 0 pA. +21. Determine Sag from the voltage difference between the lowest response and the steady-state response at the last 100 ms. +22. Determine single action potential (AP) characteristics from the first elicited action potential. +23. Calculate 'Resting' membrane potential as the average membrane voltage during the 500 ms interval before current injection. +24. Define AP threshold from the inflection point at the foot of the upstroke where the first derivative exceeded 10 mV/ms. +25. Define AP amplitude as the voltage difference between the threshold and peak voltage. +26. Measure AP half-width at half of the peak amplitude. +27. Measure after-hyperpolarizing potential (AHP) amplitude as the peak hyperpolarizing deflection following AP. +28. 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