Denature at 70°C for 2 minutes and then transfer immediately to ice. Add 13.6 µL of ligation master mix: 10× NEB Ligase 1 Buffer 2 µl DMSO (100%) 1.8 µl ATP (100 mM) 0.2 µl PEG 8000 (50%) 8 µl Murine RNase Inhibitor 0.3 µl T4 RNA Ligase 1 (30 U / µL) 1.3 µl Total 13.6 µl Close cap and mix by shaking vigorously and spinning down tubes many times. Incubate at room temperature for 1.5 hours. Clean RNA: Add 15 µL SILANE beads in 61 µL RLT. Mix well. Add 65 µL 100% ethanol. Mix well and wait 2 minutes. Wash twice by resuspending beads in 70% ethanol. Dry for 5-10 minutes. Elute in 14.3 µL H2O. Reverse Transcription Strand cDNA – To each sample, add 10 pmol (0.5 µL of 20 µM stock) AR17 primer. Denature at 70°C for 2 minutes and then immediately transfer to ice. Add 6 µL of reverse transcription master mix: 10× AffinityScript RT Buffer 2 µl 100 mM DTT 2 µl 100 mM dNTPs (25 mM each) 0.8 µl Murine RNase Inhibitor 0.4 µl AffinityScript Reverse Transcriptase Enzyme 0.8 µl Total 6 µl Quickly mix by pipet and transfer to a preheated 55°C incubator. Incubate at 55°C for 5 minutes, 54°C for 50 minutes, then 4°C for 1 minute. Immediately remove samples from thermal cycler. Digest excess RT primers by adding 3 µL of ExoSAP-IT. Mix and incubate at 37°C for 12 minutes. Probe Removal Take 20 µL of Streptavidin C1 magnetic beads per sample. Wash in 1× C1 Binding Buffer (10 mM Tris pH 7.5, 250 mM LiCl, 20 mM EDTA, 0.1% Triton X-100). Aliquot out beads into an empty strip tube, place on magnet, then remove supernatant. Add 2.5 µL of 10× C1 Binding Buffer to each sample, then transfer the samples to the washed beads and mix.
Denature at 70°C for 2 minutes and then transfer immediately to ice. Add 13.6 µL of ligation master mix: 10× NEB Ligase 1 Buffer 2 µl DMSO (100%) 1.8 µl ATP (100 mM) 0.2 µl PEG 8000 (50%) 8 µl Murine RNase Inhibitor 0.3 µl T4 RNA Ligase 1 (30 U / µL) 1.3 µl Total 13.6 µl Close cap and mix by shaking vigorously and spinning down tubes many times. Incubate at room temperature for 1.5 hours. Clean RNA: Add 15 µL SILANE beads in 61 µL RLT. Mix well. Add 65 µL 100% ethanol. Mix well and wait 2 minutes. Wash twice by resuspending beads in 70% ethanol. Dry for 5-10 minutes. Elute in 14.3 µL H2O. Reverse Transcription – Strand cDNA To each sample, add 10 pmol (0.5 µL of 20 µM stock) AR17 primer. Denature at 70°C for 2 minutes and then immediately transfer to ice. Add 6 µL of reverse transcription master mix: 10× AffinityScript RT Buffer 2 µl 100 mM DTT 2 µl 100 mM dNTPs (25 mM each) 0.8 µl Murine RNase Inhibitor 0.4 µl AffinityScript Reverse Transcriptase Enzyme 0.8 µl Total 6 µl Quickly mix by pipet and transfer to a preheated 55°C incubator. Incubate at 55°C for 5 minutes, 54°C for 50 minutes, then 4°C for 1 minute. Immediately remove samples from thermal cycler. Digest excess RT primers by adding 3 µL of ExoSAP-IT. Mix and incubate at 37°C for 12 minutes. Probe Removal Take 20 µL of Streptavidin C1 magnetic beads per sample. Wash in 1× C1 Binding Buffer (10 mM Tris pH 7.5, 250 mM LiCl, 20 mM EDTA, 0.1% Triton X-100). Aliquot out beads into an empty strip tube, place on magnet, then remove supernatant. Add 2.5 µL of 10× C1 Binding Buffer to each sample, then transfer the samples to the washed beads and mix.
rna-seq.docx