Protocol for in vivo DNA-RNA hybrid immunoprecipitation sequencing and analysis from frozen mammalian tissues
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Protocol for in vivo DNA-RNA hybrid immunoprecipitation sequencing and analysis from frozen mammalian tissues
Massalha, H.; Chee, C. J.; Mawer, J. S. P.; Puzzo, F.; Crossley, M. P.
AbstractDNA-RNA hybrids (R-loops) form transiently on the genome and regulate cellular homeostasis. They also influence genome editing outcomes, highlighting their therapeutic potential in vivo. This protocol enables high-resolution mapping of DNA-RNA hybrids directly from frozen mouse tissues. Following tissue homogenisation and lysis, genomic DNA is extracted, digested and DNA-RNA hybrids are isolated using the hybrid-specific S9.6 monoclonal antibody. The purified hybrids are then processed for whole-genome sequencing to generate R-loop profiles.