Setdb1-mediated H3K9 methylation is enriched on the inactive X and plays a role in its epigenetic silencing

Purpose: The aim of this study was to profile the transcriptome of differentiating embryonic stem cells (ES) with nonsilencing shRNA mediated knockdown and Setdb1 geneTrap heterozygous cells with Setdb1 shRNA mediated knockdown. Methods: Female Setdb1+/+ Xist?A/+ ES cells were induced to differentiate and RNA sampled on day 0, day 3 and day 5 of differentiation. RNAseq libraries were prepared with TruSeq RNA sample preparation v2 kit. Libraries were pooled and sequenced on the Illumina HiSeq 2000 platform for 100 bp single-end reads. Image analysis was performed in real time by the HiSeq Control Software (HCS) v1.4.8 and Real Time Analysis (RTA) v1.12.4.2, running on the instrument computer. Real-time base calling on the HiSeq instrument computer was performed with the RTA software. Illumina CASAVA1.8 pipeline was used to generate the sequence data. Overall design: Total RNA was extracted and purified from each cell type and their transcriptomes analysed by RNA-Seq.