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Updated: Aug 6, 2026
A Simple, Quick, and Partially Automated Protocol for the Isolation of Single Nuclei from Frozen Mammalian Tissues for Single Nucleus Sequencing
Published on: July 28, 2023
Protocol for in vivo DNA-RNA hybrid immunoprecipitation, sequencing, and analysis from frozen mouse tissues
Hassan Massalha1, Cameron J Chee1, Julia S P Mawer1
1Cancer Research UK Cambridge Institute, University of Cambridge, Cambridge CB2 0RE, UK.
Abstract:
DNA-RNA hybrids (R-loops) form transiently on the genome and regulate cellular homeostasis. They also influence genome-editing outcomes, highlighting their therapeutic potential. Here, we present a protocol for high-resolution mapping of DNA-RNA hybrids from frozen mouse tissues, including genomic DNA extraction and isolation of digested DNA-RNA hybrids using the hybrid-specific S9.6 antibody. We detail procedures for processing hybrids for whole-genome sequencing to generate R-loop profiles. For details on the use of this protocol, please refer to Puzzo et al.1.
