RNA isolation, RNA-seq library preparation, and sequencing

RNA-seq libraries were prepared from isolated total RNA, and were sequenced as previously described¹⁶. Briefly, total cellular RNA was purified from cell pellets using the TRIzol Reagent (Life Technologies) according to the manufacturer’s instructions. RNA-seq libraries were generated from 1 μg of total RNA from duplicated or triplicated samples (2 or 3 cell cultures) per condition using the TruSeq LT RNA Library Preparation Kit v2 (Illumina) following the manufacturer’s protocol. An Agilent 2100 BioAnalyzer and DNA1000 kit (Agilent) were used to quantify amplified cDNA and to control the quality of the libraries. Illumina HiSeq2500 was used to perform 100-cycle single-read sequencing. Image processing and sequence extraction were performed using the standard cloud-based Illumina pipeline in BaseSpace. The experimenter was blinded to treatment.