Livers were rinsed with PBS and processed for RNA extraction using RNeasy mini kit (Qiagen). Total RNA quality was verified using Thermo Scientific NanoDrop and Agilent Bioanalyzer. RNA was processed for RNA-Seq at the Genomics Resources Core Facility, Weill Cornell Medical College, New York, NY. Briefly, cDNA conversion and library preparation were performed using the TrueSeq v2 Illumina library preparation kit, following manufacturers recommended protocols. Libraries were multiplexed 9 per lane and sequenced on 8 lanes of an Illumina HiSeq 4000 instrument. While the original and agreed upon design called for 8 lanes of sequencing in one run, the core facility sequenced these lanes in three separate runs, sequencing a subset of lanes twice (batches B and C), but not others (batch A).
Alignments of reads to the transcriptome were performed with Kallisto version 0.44.0. We aligned reads to the mouse transcriptome downloaded from Ensembl (Mus_musculus.GRCm38.cdna.all.fa.gz). Alignment was run on a MacOS laptop with 16GB of memory, using the NextflowWorkbench to orchestrate alignment and count aggregation for the analyzed samples. RNA-Seq transcript counts were analyzed with analysis scripts written with the MetaR languages.