Isolation and Characterization of RNA-DNA Hybrids

Non-crosslinked HeLa cells were lysed in 85 mM KCl, 5 mM PIPES (pH 8.0), and 0.5% NP-40 for 10 min on ice. Pelleted nuclei were resuspended in RSB buffer (10 mM Tris-HCl pH 7.5, 200 mM NaCl, 2.5 mM MgCl2) with 0.2% sodium deoxycholate [NaDOC], 0.1% SDS, 0.05% sodium lauroyl sarcosinate [Na sarkosyl] and 0.5% Triton X-100, and extracts were sonicated for 10 min (Diagenode Bioruptor). Extracts were then diluted 1:4 in RSB with 0.5% Triton X-100 (RSB + T) and subjected to IP with the S9.6 antibody, bound to protein A dynabeads (Invitrogen), and preblocked with 0.5% BSA/PBS for 2 hr. CBP80 and IgG2a antibodies were used as control. RNase A (PureLink, Invitrogen) was added during IP at 0.1 ng RNase A per microgram genomic DNA. Beads were washed 4x with RSB + T; 2x with RSB; and eluted either in 2x LDS (Invitrogen), 100 mM DTT for 10 min at 70°C (for SDS-PAGE), or 1% SDS and 0.1 M NaHCO₃ for 30 min at room temperature (for RNA/DNA hybrid slot blot). Where indicated, nuclear extracts were treated with 1 U/μL benzonase (Sigma) for 30 min at 37°C before IP. Sequences and preparation of double-stranded competitors were described by Phillips et al. (2013) (link) and Rigby et al. (2014 (link)). For MS analysis, eluted samples were processed by filter-aided sample preparation (FASP) with trypsin (Wiśniewski et al., 2009 (link)). Table S1 provides the list of proteins that make up the RNA/DNA interactome.