Experimental approach to analyze the formation and processing of DSB1 and DSB2 in U-2OS-SEC (stably expressing Cas9) cells. Specific oligonucleotides were designed to quantify ssDNA at the indicated distance from the DSBs (Table S1). The two DSBs are induced simultaneously by transfection with sgRNA1 and sgRNA2. Genomic DNA is extracted and digested with given restriction enzymes at specific distances from the DSB. If the target sequence has been converted into ssDNA by the resection started at the DSB, it cannot be digested by the restriction enzyme, and the DNA will be amplified by the ddPCR. A region on chromosome 22 that is not cut by these enzymes served as control. Values indicating the percentage of cutting efficiency, exposed ssDNA, and resected DSB were calculated as described previously.26
