GSM7091151: Bl6, day3, macrophages, replicate 2; Mus musculus; RNA-Seq
Total RNA was extracted using TRIzol Reagent (Invitrogen) according to manufacturer's protocol. First-strand reverse transcription and template switching was performed using an Oligo(dT) primer (dT30VN-ME-A), a locked nucleic acid-containing TSO (NotI-TSO), and Superscript IV reverse transcriptase (Invitrogen, # 18090050). PCR preamplification of cDNA was performed with IS PCR and Tn5ME-A-aHic using 2X KAPA PCR mix (Kapa Biosystem, KK2602) followed by cleanup using SPRISelect beads (Beckman Coulter, REF B23319). DNA was digested by NotI-HF (NEB, #R3189L), tagmented using Tn5 assembled with adaptors Tn5ME-A/Tn5MErev and Tn5ME-B/Tn5MErev. A Zymo DNA clean and concentrator kit (Zymo, R1014 ) was utilized (PMID: 24385147). Library PCR was performed using unique combinations of Nextera-PCR i5/i7 primers. DNA fragments between 400-600bp were selected by gel extraction using Zymoclean Gel DNA recovery kit (Zymo, #D4002).