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

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Pappas G, Gram HH, Bartek J, Galanos P

STAR Protoc 6 (4) 104157 [2025-12-19; online 2025-10-22]

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.

PubMed 41134671

DOI 10.1016/j.xpro.2025.104157

Crossref 10.1016/j.xpro.2025.104157

pmc: PMC12590427
pii: S2666-1667(25)00563-5