Protocol for in vivo DNA-RNA hybrid immunoprecipitation sequencing and analysis from frozen mammalian tissues

DNA-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. For complete details on the use and execution of this protocol, please refer to Puzzo, Crossley et al1. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=188 SRC="FIGDIR/small/716701v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@3d8f86org.highwire.dtl.DTLVardef@199cd84org.highwire.dtl.DTLVardef@83c51eorg.highwire.dtl.DTLVardef@1024332_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOgraphical abstractC_FLOATNO C_FIG