Nascent chromatin capture assay protocol

The nascent chromatin capture assay was performed as described previously (48 (link)) with slight modifications. Cells were treated with a mixture of biotin-16-dUTP and biotin-16-dCTP (0.5 μM each, Jena Bioscience) in hypotonic buffer (50 mM KCl, 10 mM HEPES) for 5 min and followed by another 5 min dUTP/dCTP treatment in regular DMEM medium (For etoposide treated samples, etoposide was added 20 min prior dUTP/dCTP treatment and kept until cells were fixed). Cells were then fixed by 0.2% PFA for 5 min at room temperature and quenched by co-incubation with 200 mM glycine for 1 min. Cells were resuspended in TNT-300 buffer (25 mM Tris–HCl pH 7.4, 300 mM NaCl, 1% Triton-X100) together with protease and phosphatase inhibitors (Sigma, 1:1000) and sonified (30% amplitude, 45 s on/15 s off for 10 min) before incubated with 10 μl of Streptavidin Magnetic Beads (50% slurry, NEB) at room temperature 45 min on a rotating wheel. Beads were collected by centrifugation and washed three times with the TNT-300 buffer. Samples were denatured by incubation with the 4× Laemmli loading buffer at 95°C for 10 min and analyzed by immunoblotting.