GSM8218952: C2C12, 37C, WT, 24h, rep4; Mus musculus; RNA-Seq
RNA was harvested using TRIzol Reagent (Invitrogen) and phenol-chloroform purification. Starting from 50 ng total RNA per sample, we performed bulk RNA barcoding and sequencing (BRB-Seq) with minor modifications. We performed reverse transcription with SuperScript IV (Invitrogen) and a uniquely barcoded oligo(dT)30 primer for each sample, modified to use Illumina TruSeq Read 1 priming site instead of Nextera Read 1. Following the remainder of the standard BRB-Seq protocol, we pooled up to 24 barcoded first-strand cDNAs into a single tube, synthesized second-strand cDNA with Gubler-Hoffman nick translation, and tagmented cDNA pools with Tn5 produced in-house. We performed 14 cycles of PCR using a P5-containing primer and a unique multiplexed i7 indexing primer (Chromium i7 Multiplex Kit, 10X Genomics). We purified and size-selected libraries with 0.65X SPRIselect (Beckman Coulter) and then 0.7X SPRIselect. We sequenced all pools on a NovaSeq SP v1.5 flowcell (Illumina) with 28 cycles of Read 1, 8 cycles of Read i7, and 101 cycles of Read 2.