Protocol for image-based monitoring of de novo RNA synthesis at DNA double-strand breaks in human cell lines
Year: 2025
Type: article
Abstract: 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 • Protocol for quantification of nascent transcription at DSBs • Guidance on BrUTP incorporation assay and site-specific UV-A laser ablation • Steps for immunochemical staining of DSBs and incorporated BrUTP • Instructions for analysis of de novo RNA synthesis on DSBs Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. 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.
Source: STAR Protocols
Authors: Georgios Pappas, Helena Hagner Gram, Jiri Bartek, Panagiotis Galanos
Language: English
Topic: DNA Repair Mechanisms
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: gold
Estimated APC paid: $1,550
OpenAlex ID: W4415554459