GSM3405385: H3K9me3_ChIPSeq_RPE Exp1; Mus musculus; ChIP-Seq
Freshly isolated RPE were cross-linked in 1% formaldehyde with 1X PBS. To stop the cross linking reaction, glycine was added to a final concentration of 0.125M. The chromatin was prepared using the True MicroChIP Kit (Diagenode Cat# C01010130). Chromatin was sheared using the Bioruptor® Pico sonication device (Diagenode Cat# B01060001). ChIP was performed using IP-Star® Compact Automated System (Diagenode Cat# B03000002) following the protocol of the aforementioned kit. Chromatin corresponding to 14,000-28,000 cells was immunoprecipitated using the antibodies. Libraries were prepared from input and ChIP'd DNA (500 pg) using the MicroPlex Library Preparation Kit v2 (12 indices) (Diagenode Cat# C05010013). Library amplification is assessed using the High Sensitivity NGS Fragment Analysis Kit (DNF-474) on a Fragment Analyzer™ (Advanced Analytical Technologies, Inc.). Libraries were then purified using Agencourt® AMPure® XP (Beckman Coulter) and quantified using Qubit™ dsDNA HS Assay Kit (Thermo Fisher Scientific, Q32854). Finally their fragment size was analyzed by High Sensitivity NGS Fragment Analysis Kit (DNF-474) on a Fragment Analyzer™ (Advanced Analytical Technologies, Inc.). When the proportion of fragments >500bp was too high, libraries were subjected to a double size selection using Agencourt® AMPure® XP (Beckman Coulter). Libraries were pooled and sequenced on an Illumina HiSeq 4000 with single-end reads of 50bp lengths, running HiSeq Control Software HD version 3.4.0.38.