Chromatin immunoprecipitation (ChIP) and ER-AsiSI resection assay

HELQ-KI SFB U2OS cells expressing ER-AsiSI (75,76), in which the restriction enzyme AsiSI is fused to the estrogen receptor hormone-binding domain, were treated with 300 nM 4-OHT for 4 h to induce DSBs. Then, a ChIP assay was performed as previously described (67,70) using 200 μg chromatin immunoprecipitated with IgG and FLAG antibodies (2 μg). The immunoprecipitated DNA and input DNA were analyzed by qPCR using Taq pro Universal SYBR qPCR Master Mix (Vazyme). The following primers were used for qPCR, as previously described (76): DSB-F 5-GATTGGCTATGGGTGTGGAC and DSB-R 5-CATCCTTGCAAACCAGTCCT. The IP efficiency was calculated as the percentage of the input DNA immunoprecipitated. ER-AsiSI resection assay was performed as previously described (65,76).