GSE66522
Setdb1-mediated H3K9 methylation is enriched on the inactive X and plays a role in its epigenetic silencing
Purpose: The aim of this study is (1) to identify the chromatin occupancy of the epigenetic regulator Setdb1 in mouse embryonic fibroblasts (MEF); (2) to profile key epigenetic marks H3K9me2, H3K9me3 and H3K27me3; utilizing wildtype cells with nonsilencing shRNA mediated knockdown and Setdb1 geneTrap heterozygous cells with Setdb1 shRNA mediated knockdown. Methods: Chromatin immunoprecipitation for Setdb1, H3K9me2, H3K9me3 and H3K27me3 was performed essentially as in (Nelson et al. 2006). Briefly, nuclei were isolated from formaldehyde crosslinked MEFs and chromatin was fragmented by sonication. Chromatin immunoprecipitation was performed with corresponding antibodies for Setdb1, H3K9me2, H3K9me3 and H3K27me3. DNA was extracted from the immunoprecipitated fraction following reverse-crosslinking. Isolated DNA was used to generate sequencing libraries with Illumina''s TruSeq DNA Sample Preparation Kit according to manufacturer''s instruction. 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: Chromatin occupancy of the epigenetic regulator Setdb1, H3K9me2, H3K9me3 and H3K27me3 in mouse embryonic fibroblasts (MEFs) with wildtype MEFs and nonsilencing shRNA mediated knockdown or Setdb1 geneTrap heterozygous MEFs with Setdb1 shRNA mediated knockdown was determined by Setdb1, H3K9me2, H3K9me3 and H3K27me3 ChIP-seq, respectively.