Briefly, the cells were crossed linked with 1% formaldehyde at ambient temperature for 10 min. The reaction was quenched by 125mM glycine for 5 min at room temperature. Cells were washed with PBS and treated with hypotonic buffer (20mM Hepes pH7.9, 10mM KCl, 1mM EDTA, 10% Glycerol and 1mM DTT with additional protease inhibitor (Roche)) to isolate nuclei. The nuclei were suspended with RIPA buffer (10 mM Tris-HCl pH 8.0, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% SDS, 0.1% sodium deoxycholate with protease inhibitor) and sonicated using Covaris S220 Focused-ultrasonicator. Fragmented chromatin was pre-cleared with protein G conjugated sepharose beads (GE). Antibodies were used to pull down the respective proteins and their associated chromatin. Washes with different concentration of NaCl were performed. The enriched protein-DNA complexes were reverse crosslinked at 65 C over night with proteinase K (NEB). DNA was purified with Qiagen MinElute kit. Sequencing library was prepared using an in-house kit, including end-repair, “A” addition and adapter ligation. The library was sequenced with illumina HiSeq 4000 for 50bp single read or 100bp pair-end, acquiring 10-30 million reads.
GEO Accession
GSM3344570