Quantifying DNA End Resection at DSBs

End resection was assayed in ER-AsiSI cells that were predominantly in S/G2 phase by cell synchronization procedure mentioned earlier in the Methods section. The extent of resection adjacent to specific DSBs was measured by quantitative polymerase chain reaction (qPCR) as described previously [24 (link)]. The sequences of qPCR primers are shown in S1 Table. 20μL of genomic DNA sample (~200 ng in 1x CutSmart NEB restriction enzyme buffer) was digested or mock digested with 20 units of restriction enzymes (NmeAIII, AvaI, BsrGI, BamHI-HF or HindIII-HF; New England Biolabs) at 37°C overnight. 5 μl of digested or mock digested samples (~20 ng) were used as templates in 20 μl of qPCR reaction containing 10 μl of 2x iTaqUniversal SYBR Green Supermix, 500 nM of each primer using iCycler iQReal-Time PCR (Bio-Rad). The % ssDNA generated by resection at selected sites was determined as previously described [24 (link)]. Briefly, for each sample, a ΔCt was calculated by subtracting the Ct value of the mock-digested sample from the Ct value of the digested sample. The % ssDNA was calculated using algorithm: ssDNA% = 1/(2^{^}(ΔCt-1) + 0.5)*100 [24 (link)]. Data represent the mean of at least three independent experiments with SD values indicated by error bars.