Quantification of protein enrichment at site-specific DNA double-strand breaks by chromatin immunoprecipitation in cultured human cells

Here, we present a chromatin-immunoprecipitation-based protocol to quantify the recruitment of proteins adjacent to site-specific DNA double-strand breaks (DSBs), such as proteins involved in DSB repair. We describe steps to induce DSBs in U2OS osteosarcoma cells stably expressing the restriction endonucleases FokI or AsiSI. We then detail the procedures of chromatin isolation and immunoprecipitation, followed by protein elution and quantitative-PCR-based quantification of DNA. This protocol cannot be used on DSBs generated at random loci by DNA damaging agents. For complete details on the use and execution of this protocol, please refer to Fitieh et al. (2022).1 [Display omitted] •A sensitive approach to measure protein association near DNA double-strand breaks (DSBs)•The DSBs are induced in U2OS cells stably expressing the endonucleases FokI or AsiSI•Involves ChIP and subsequent qPCR•Primers used for qPCR target genomic loci proximal to the endonuclease-generated DSBs Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Here, we present a chromatin-immunoprecipitation-based protocol to quantify the recruitment of proteins adjacent to site-specific DNA double-strand breaks (DSBs), such as proteins involved in DSB repair. We describe steps to induce DSBs in U2OS osteosarcoma cells stably expressing the restriction endonucleases FokI or AsiSI. We then detail the procedures of chromatin isolation and immunoprecipitation, followed by protein elution and quantitative-PCR-based quantification of DNA. This protocol cannot be used on DSBs generated at random loci by DNA damaging agents.