RNA-seq Library Preparation and Analysis

Cells were lysed with Buffer RL (Norgen) containing 10% beta-mercaptoethanol. Cell lysate was collected after addition of 300 μl 100% molecular biology grade ethanol. Total RNA was extracted using the Animal Tissue RNA Purification Kit (Norgen). RNA-seq libraries were prepared from 250 ng total RNA via polyA-selection (Dynabead mRNA Purification Kit, ThermoFisher Scientific) followed by transposase-mediated non-stranded library construction [25 (link)]. Each experimental treatment was performed in triplicate. Libraries were pooled and sequenced on an Illumina HiSeq 2000 sequencer using paired-end 50 bp reads with a 6 bp index read (Illumina, Inc., San Diego, CA). Pooled sequencing resulted in 26.5 million reads per library. Differential expression was measured using the DESeq2 program [35 (link)]. TopHat (version 1.4.1) was used to align RNA-seq paired reads to GENCODE (version 9.0) [36 (link), 37 ]. Cufflinks (version 1.3.0) and BEDTools [38 (link), 39 (link)] were used to calculate raw counts for each GENCODE transcript. For this study X and Y chromosome transcripts were omitted.