Chromatin fractionation
HeLa cells were treated with 500 μM phleomycin for 1 hour, washed with ice cold PBS, scraped into PBS and the chromatin fractionation was performed as described 55, 56. Briefly, cells were resuspended in buffer A (10 mM Hepes-KOH pH7.9, 10 mM KCl, 1.5 MgCl2, 340 mM Sucrose, 10% glycerol, 1 mM DTT, protease inhibitors) and Triton X-100 was added to final concentration 0.1 %. After 5 min incubation on ice, nuclei were spun down at 1300 × g for 4 min. Pelleted nuclei were washed with buffer A, resuspended in buffer B (3 mM EDTA, 0.2 mM EGTA, 1 mM DTT, protease inhibitors) and lysed for 20 min on ice before centrifugation at 1700 × g for 5 min. Supernatant (nuclear soluble fraction) was saved, and pellet (chromatin fraction) was washed with buffer B, resuspended in urea buffer (9 M urea, 50 mM Tris-HCl, pH 7.3) and sonicated.