U2OS-DRGFP cells, which carrying chromosomal integrated single-copy of I-SceI site, were used to detect protein recruitment to I-SceI endonuclease-induced DSB sites as described previously (24 (link)). Briefly, 24 h after transfection of indicated siRNA, cells were transfected with pCBASceI plasmids to introduce DSBs. Twenty four hours after pCBASceI transfection, ChIP assay was performed using Pierce Agarose ChIP Kit (Thermo Scientific, USA) and ZNF830 (HPA027211, Sigma) or CtIP (79809, Novus Biologicals) antibodies according to the manufacturer's instruction. The ZNF830 or CtIP associated DNA was detected by PCR using the primer at the I-SceI site. The primer sequence was: forward, 5′-TAC AGC TCC TGG GCA ACG TG-3′; reverse, 5′-TCC TGC TCC TGG GCT TCT CG-3′. The PCR program was: 95 °C for 5 min, 1 cycle; 95 °C for 45 s, 56 °C for 30 s and 72 °C for 30 s, 35 cycles; 72 °C for 10 min, 1 cycle. The amplified products were analyzed on a 1.2% agarose gel.