GSM6757933: mES cells, WT2, 2i, input; Mus musculus; ChIP-Seq
Mouse ESCs and LCLs were crosslinked with 1% formaldehyde for 10min at room temperature (RT) and the reaction was quenched by adding glycine at a final concentration of 0.125M for 5min at RT. Fixed cells were washed and adherent cells were harvested with phosphate-buffered saline (PBS). Cell nuclei were isolated using a cell lysis buffer (5mM Pipes pH8.0, 85mM KCl, 0.5% NP40, Complete protease inhibitor cocktail) for 20min, at 4°C. Nuclei were then pelleted by centrifugation and lysed in a nuclei lysis buffer (50mM Tris HCl pH8.1, 150mM NaCl, 10mM EDTA, 1% SDS, Complete protease inhibitor cocktail) for 20min, 4°C. Chromatin was subjected to sonication with a Bioruptor (Diagenode) yielding genomic DNA fragments with a bulk size of 150- 300 bp. ChIP-seq libraries were prepared using the MicroPlex Library Preparation Kit v2 (Diagenode) according to the manufacturer's protocol. After adapters ligation, fragments were amplified with Illumina primers for 10 cycles. Libraries were purified using AMPure XP beads protocol (Beckman Coulter) and quantified using the Qubit fluorometer (Thermo Fisher Scientific). Libraries' size distribution was assessed using the Bioanalyzer High Sensitivity DNA chip (Agilent Technologies). Libraries were normalized and pooled at 2nM and spike-in PhiX Control v3 (Illumina) was added.