Yield D - RNA integrity: method/instrument E NA RIN/RQI or Cq of 3’ and 5’ E NA transcripts Electrophoresis traces D - Inhibition testing Followed the instruments’ E NA (Cq dilutions, or other) protocol Reverse transcription Mix RNA, primer, dNTP, buffer, DTT, RNase out, Complete reaction conditions E + and superscript III RT. Heat mixture to 50 for 50 min and °C 70 for 15 min °C Amount of RNA and reaction Reaction volume of 10 with μl E + volume 50 ng total RNA Priming oligonucleotide (if using E + 2 pM each GSP) and concentration SuperScript III Reverse Reverse transcriptase and E + Transcriptase (Invitrogen, concentration Carlsbad, CA, USA) 50 for 50 min and 70 for °C °C Temperature and time E + 15 min Manufacturer of reagents and REF 18080-044 / Qty: 10,000 U D + catalogue numbers (200 U/ul) Cqs with and without reverse D - transcription cDNA was directly used without Storage conditions of cDNA D - storage qPCR target information Gene symbol E + and hcaT cysG Genebank, hcaT Sequence accession number E + (2665363..2666502); (4141215..4142588) cysG 1220 bp and 545 bp apart from Location of amplicon D + 5’ end of and gene cysG hcaT Amplicon length E + 86~105 bp In silico specificity screen E + BLAST (BLAST, and so on) Pseudogenes, retropseudogenes, D + No homolog or other homologs? Sequence alignment D + PrimerBLAST Min G homodimer, -29.8 hcaT: Secondary structure analysis of D + kcal; Min monodimer, -10.9 G amplicon kcal
Yield D - RNA integrity: method/instrument E NA RIN/RQI or Cq of 3’ and 5’ transcripts E NA Electrophoresis traces D - Inhibition testing (Cq dilutions, or other) E NA Followed the instruments’ protocol Reverse transcription Complete reaction conditions E + Mix RNA, primer, dNTP, buffer, DTT, RNase out, and superscript III RT. Heat mixture to 50 °C for 50 min and 70 °C for 15 min Amount of RNA and reaction volume E + Reaction volume of 10 μl with 50 ng total RNA Priming oligonucleotide (if using GSP) and concentration E + 2 pM each Reverse transcriptase and concentration E + SuperScript III Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA) Temperature and time E + 50 °C for 50 min and 70 °C for 15 min Manufacturer of reagents and catalogue numbers D + REF 18080-044 / Qty: 10,000 U (200 U/ul) Cqs with and without reverse transcription D - Storage conditions of cDNA D - cDNA was directly used without storage qPCR target information Gene symbol E + hcaT and cysG Sequence accession number E + Genebank, hcaT (2665363..2666502); cysG (4141215..4142588) Location of amplicon D + 1220 bp and 545 bp apart from 5’ end of cysG and hcaT gene Amplicon length E + 86~105 bp In silico specificity screen (BLAST, and so on) E + BLAST Pseudogenes, retropseudogenes, or other homologs? D + No homolog Sequence alignment D + PrimerBLAST Secondary structure analysis of amplicon D + hcaT: Min G homodimer, -29.8 kcal; Min G monodimer, -10.9 kcal