Protocol for image-based monitoring of de novo RNA synthesis at DNA double-strand breaks in human cell lines

DNA double-strand breaks (DSBs) halt canonical transcription and simultaneously trigger local non-canonical RNA synthesis. Despite its significance, existing approaches to monitor this process are limited. Here, we present a protocol to monitor and quantify nascent transcription on DSBs in the nucleus. We describe steps for employing UV-A laser to induce site-specific DSBs. We then detail procedures for supplementation of the ablated cells with 5-bromouridine 5'-triphosphate (BrUTP) to label nascent RNA. For complete details on the use and execution of this protocol, please refer to Pappas et al.¹.