single read
Left-lobe of livers was pulverized with pestle and mortar pre-chilled in liquid nitrogen. RNA was extracted from ~20 mg of pulverized tissue using RNeasy Qiagen mini Kit (Qiagen, Valencia, CA). Concentration of extracted RNA was measured using NanoDrop 2000 (Thermo Fisher Scientific, Wilmington, DE). RNA libraries were prepared using Illumina TruSeq Stranded Total RNA kit (Illumina, San Diego, CA) with Ribo-Zero Gold rRNA removal kit (Illumina). RNA-Seq of 50 base-pair single-end reads was conducted using Illumina HiSeq 2500 instrument (Illumina) at the depth of ~50 million reads/sample. Raw reads were trimmed for any sequencing adaptors and low quality bases using Trimmomatic (Bolger, Lohse, & Usadel, 2014). Reference genomes and inferred marker founder origin for CC mice were downloaded from University of North Carolina Systems Genetics Core Facility (http://csbio.unc.edu/CCstatus/index.py?run=Pseudo). Filtered reads were mapped to each of the corresponding CC reference genome using TopHat version 2.0.3 (D. Kim, Langmead, & Salzberg, 2015). Resulting alignments were re-mapped to reference mm10 assembly coordinates using lapels (https://github.com/shunping/lapels). HTSeq (Anders, Pyl, & Huber, 2015) was used to generate raw read counts per gene using intersection-nonempty parameter to account for ambiguous read mappings. Differential gene expression tests were then performed with DESeq2 (Love, Huber, & Anders, 2014).