GSM3463698: input Müller glia DNA Exp2; Mus musculus; ChIP-Seq
Freshly isolated cells 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 25,000 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.