DNA end resection converts broken ends of double-stranded DNA (dsDNA) to 3′-single-stranded DNA (3′-ssDNA). The extent of resection regulates DNA double-strand break (DSB) repair pathway choice and thereby genomic stability. Here, we characterize an optimized immunofluorescence (IF) microscopy-based protocol for measuring ssDNA in mammalian cells by labeling genomic DNA with 5-bromo-2′-deoxyuridine (BrdU). BrdU foci can be detected under non-denaturing conditions by anti-BrdU antibody, providing an accurate and reliable readout of DNA end resection in most mammalian cell lines. For complete details on the use and execution of this protocol, please refer to Kilgas et al. (2021).
This work was funded by the MRC Program grant MC_PC 12001/1 and MC_UU_00001/1 to K.R., and S.K. was supported by the MRC Oxford Institute of Radiation Oncology (OIRO) Cancer Research UK (CRUK) Studentship. We thank Dr. Rhodri Wilson from the microscopy imaging core (University of Oxford, Department of Oncology) for his technical advice and assistance. Graphical abstract was created with BioRender.
Data Availability StatementThis study did not generate any unique datasets or code.
- Cell Biology
- Cell-based Assays
- Molecular Biology