Approximately 1-2 X 107 cells were fixed by addition of 1% formaldehyde to cell media for 10 min at RT with gentle agitation. Formaldehyde was quenched by addition of glycine to 0.125 M with gentle agitation for 5 min at RT. Cells were pelleted at 2000xg, washed twice in PBS, and resuspended in 0.8 mL ice–cold cell lysis buffer (5 mM PIPES pH 8, 85 mM KCl, 0.5% NP-40) supplemented with Complete protease inhibitors (Roche), incubated on ice 10 min pelleted by centrifugation at 2000 xg for 5 min at 4°C. Next, the supernatant was removed and pellets were resuspended in 1 mL nuclear lysis buffer (50 mM Tris-HCl pH 8, 10 mM EDTA.Na2, 1% SDS) and incubated for 10 min at 4°C. Afterwards, 0.5 mL of IP dilution buffer was added (16.7 mM Tris-HCl pH 8, 1.2 mM EDTA, 167 mM NaCl, 1.1% Triton X-100, 0.01% SDS) and chromatin was fragmented to an average size of 300 bp by using PicoBioruptor (Diagenode) using 10 cycles of 30 s on plus 30 sec off, maximum output. Samples were centrifuged at max speed for 10 min at 4°C, and the supernatant (sheared chromatin) was saved at -80°C. Chromatin was diluted to 1:5 with IP buffer, and the assayed antibody in addition to 50 μL of prewashed protein A/G 50% slurry was added and rotated overnight at 4°C. The next day, beads were washed as follows: -- 3X Low salt IP dilution buffer (20 mM Tris-HCl pH 8, 2 mM EDTA.Na2, 150 mM NaCl, 1% Triton X-100, 0.1% SDS). -- 3X High salt IP dilution buffer (20 mM Tris-HCl pH 8, 2 mM EDTA.Na2, 500 mM NaCl, 1% Triton X-100, 0.1% SDS). -- 2X times LiCl buffer (10 mM Tris-HCl pH 8, 1 mM EDTA.Na2, 0.25M LiCl, 1% NP-40, 1% DOC). Chromatin was eluted twice with 200 μL of elution buffer (500 μL of 1M NaHCO3, 250μL of 20% SDS, 4.25 mL dH2O) for 30 min at 65°C each and further incubated overnight at 65°C with 38 μL of decrosslinking solution (20 μL of 5M NaCl, 8 μL of 0.5M EDTA, 10 μL of 1M Tris-HCl pH 8). After de-crosslinking, samples were treated with Proteinase K for 2 h at 50°C and then combined with 1 vol of phenol/chloroform/isoamyl alcohol (25:24:1), vortexed 15 s, and centrifuged for 5 min at 10,000 xg . The top layer was transferred to a new tube, and the procedure was repeated using 1 vol chloroform. The top layer was collected and subsequently precipitated with 0.1 vol of 3M NaOAc pH 5.2 and 2.5 vol of 100% ethanol supplemented with 2 μL of Glycoblue (Ambion). After incubating 30 min at -80°C, samples were centrifuged 20 min at 4°C at 10,000 xg. Pellets were washed with 70% ethanol and centrifuged 5 min at 4°C at 10,000 xg. Pellets were air dried at RT prior to resuspension in 10 μL of dH2O. Samples for ChIP-seq were prepared according to the manufacturer’s protocol with Clontech or TruSeq adapters (Illumina).