GSM4670383: Scramble, anti-BMI1, line 1299, replica 1; Homo sapiens; ChIP-Seq
RNA-Seq:Total RNA was extracted with RNA/DNA/Protein Purification Plus kit (Norgen) following manufacturer's instruction. ChIP-Seq: BMI1, CHD7, H3K4me3 and H3K27me3 ChIPs were performed using ChIP-IT High Sensitivity kit (Active Motif) following manufacturer's instructions. Briefly, 1x106 cells were fixed with formaldehyde-based fixing solution for 15 minutes at room temperature and lysed with provided lysis solution supplemented with protease inhibitors. Next, nuclei pellets were lysed and chromatin sonicated with Bioruptor Plus sonication device (Diagenode) to obtain DNA fragments within the recommended 200-1200bp range. 25µg or 10µg of sheared chromatin was then incubated with 4µg of antibody against BMI1 (BMI1, clone AF27, Active Motif), CHD7 (ab117522, abcam), H3K27me3 (Diagenode) or H3K4me3 (Diagenode) overnight at 4°C with rotation. Following incubation with Protein G agarose beads, bound chromatin was washed, eluted and purified following manufacturer's protocols. Validation by qPCR-ChIP on target genes was done before proceeding to sequencing. RNA-Seq: Total RNA was extracted with RNA/DNA/Protein Purification Plus kit (Norgen), polyA mRNA was selected and library prepared using the NEBNext Ultra II Directional RNA Library Prep kit. Libraries were size selected, multiplexed and 75bp paired-end sequences were obtained with HiSeq4000 (Illumina). ChIP-Seq: ChIPed DNA was end-repaired, A-tailed and adapter-ligated before size selection and amplification. The obtained libraries were QC'ed and multiplexed before 75 bp paired end sequencing on HiSeq4000 (Illumina).
GEO Accession
GSM4670383