Stat3 inhibitor

Naïve hESCs Elf1 2iLIF grown on matrigel were treated with 100µM of STAT3 inhibitor (Selleckchem) for 6h or 24h and analyzed for methylation marks by Western blot and ChIP Seq. For ChIP-seq analysis, cells were crosslinked and chromatin processed as previously described^{67 (link)} with minor modifications. Briefly, cells were harvested with accutase and crosslinked in suspension with 1% formaldehyde solution for 10min at room temperature. Reaction was quenched with glycine and crosslinked cells were rinsed with ice-cold PBS. Nuclei were isolated and chromatin sonicated using a Covaris E210 to approximately 200–500bp size range. ChIP-seq was conducted as previously described^{67 (link)} with minor modifications. Briefly, magnetic Dynabeads were incubated overnight rotating at 4C with antibody against H3K27me3 (Active Motif, cat # 39155). Sonicated chromatin from approximately 200 thousand cells was added to the bead-bound-antibodies and incubated at 4C rotating overnight. Beads were washed and bound chromatin was eluted from beads and reverse crosslinked overnight. Purified DNA was prepared for next-generation sequencing via end repair, A-tailing, ligation of custom Y-adapters and PCR amplification to generate final DNA library following gel size selection.