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

Abstract

Summary: 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 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Keywords

• Cell Biology
• Molecular Biology
• Chromatin immunoprecipitation (ChIP)
• Protein Biochemistry