GSM8230781: liver, Mettl14[fl/fl]/Alb-Cre, rep2; Mus musculus; RNA-Seq
Liver tissue was collected from animals after perfusion with PBS via the portal vein to remove blood from tissue. RNA was extracted from bulk liver tissue using the Monarch total RNA miniprep kit (New England Biolabs, Ipswich, MA) after homogenization with steel beads using a TissueLyser LT bead mill (Qiagen, Venlo, The Netherlands). 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 Template Switching RT Enzyme Mix (NEB) 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 amplified libraries using a P5-containing primer and a unique multiplexed i7 indexing primer (Chromium i7 Multiplex Kit, 10X Genomics), then purified and size-selected libraries with SPRIselect (Beckman Coulter). We sequenced all pools on a NovaSeq SP v1.5 flowcell (Illumina) with 28 cycles of Read 1, 8 cycles of Read i7, and 102 cycles of Read 2.