Total RNA was isolated from mES cells using the TRI Reagent (Sigma) following the protocol provided by the manufacturer. Contaminating genomic DNA was removed by incubating 10 µg RNA with 10 U DNaseI (Thermo Scientific) in Reaction Buffer for 30 min at 37 °C. The reaction was stopped by adding 10 µl of 50 mM EDTA after which RNA was extracted using a phenol-choloroform purification and ethanol precipitation. Total RNA was depleted of ribosomal RNA using Ribo-Zero rRNA Removal Kit (Epicentre/Illumina, Human/Mouse/Rat) and library preparation was completed using NEBNext® Ultra Directional RNA Library Prep Kit for Illumina® (New England Biolabs), both following manufacturer's instructions. Libraries were amplified (12 cycles) on a Tetrad (Bio-Rad). Equivalent amounts of individual libraries were pooled together. Paired end sequencing was performed using a Hiseq2000 51 bp platform (v3 SBS chemistry), generating a raw read count of >40 million reads per sample.
GEO Accession
GSM3772813