RNA-Seq Library Preparation and Analysis

RNA-Seq experiments were performed as previously described [29 (link)]. Briefly, RNA was extracted from sorted cells using the automated Maxwell 16 magnetic particle processor and a Maxwell 16 LEV simply RNA kit (Promega). Polyadenylated RNAs were isolated using NEBNext magnetic oligo d(T)₂₅ beads (New England BioLabs), individually bar-coded, and next generation sequencing expression libraries were prepared using the NEBNext mRNA Library Prep Reagent Set for Illumina (New England BioLabs). Each library was diluted to a final concentration of 12.5nM and pooled equimolar prior to clustering. Paired-End (PE) sequencing (25 million, 50-bp, paired-end reads) was performed using a 200 cycle TruSeq SBS HS v3 kit (Illumina) on an Illumina HiSeq2000 sequencer. Image analysis and base calling were performed using the standard Illumina Pipeline consisting of Real time Analysis (RTA) v1.13. Raw reads were de-multiplexed using a bcl2fastq conversion software v1.8.3 (Illumina) with default settings. Paired-end reads were mapped against the human reference genome (GRCh37) using TopHat v2.0.0. Human gene models and annotations were obtained from ENSEMBL v63 (June 2011). Gene expression quantification was conducted on the gene level using Subread v1.4.6 counting mapped paired-reads to obtain fragment counts per gene (see S2 Text for additional details).