GSM7731025: SRF bound chromatin of OPC_R1; Rattus norvegicus; ChIP-Seq
Rat OPCs were allowed to proliferate for 6 days to reach 40 million cells in multiple 15cm plates. Half of the plates were fixed at the OPC stage while the rest were switched to differentiation medium and fixed on day 3 of differentiation. Replicates in this experiment were independent OPC preps. Chromatin immunoprecipitation was performed following iDeal ChIP-seq kit for Transcription Factors (Diagenode, Cat. No. C01010055) using 7 cycles for chromatin shearing on a Bioruptor Pico sonicator (Diagenode, Cat. No. B01060001). Prior to sonication, each sample was split to 300ul reactions (containing each roughly 4 million cells) in 1.5 ml Bioruptor Microtubes (Cat. No. C30010016), sonicated, spun 16,000g for 10 min and 270ul of supernatant was collected to a new tube and stored in -80 deg. One aliquot of each sample was used for immunoprecipitation with 5ul of Rabbit-anti-SRF antibody that was validated for ChIP (SRF (D71A9) XP® Rabbit mAb, #5147) and an additional OPC and oligodendrocyte sample from each group for immunoprecipitation with an IgG control antibody (provided in the kit). Libraries for next-generation sequencing were prepared using MicroPlex Library Preparation Kit v3 x48 rxns (C05010001, Diagenode) using 12 PCR cycles on a 96-plate thermal cycler (Biorad) and purified without size selection. Library quantity and quality was assessed using a Bioanalyzer (Agilent) and Qubit. Libraries were pooled and sequenced on a Nextseq550 sequencer (Illumina) using single end 75bp for Read 1 and 8bp for index 1 and 8bp for Index 2 with a high output 75bp kit (20024906, Illumina). In pilot studies, Chromatin Shearing Optimization kit - Low SDS (iDeal Kit for TFs) (Cat. No.C01020013) was used to determine optimal number of sonication shearing cycles to yield 200-300 bp fragments.