Protocol for imaging-based quantification of RNAPII clearance during transcription-coupled DNA repair

Abstract

Elongating RNA polymerase II (RNAPII) stalls at transcription-blocking lesions in the DNA template strand and is removed by transcription-coupled DNA repair (TCR) factors. Here, we present a protocol to measure RNAPII clearance during TCR at sites of localized UV-induced DNA damage in adherent cells. We describe how to induce local UV damage and visualize the damaged area and chromatin-bound RNAPII levels using immunofluorescence staining. This approach can quantify the clearance of DNA damage-stalled RNAPII and its dependence on TCR factors. For complete details on the use and execution of this protocol, please refer to van den Heuvel et al.1