Recognition of cellular RNAs by the S9.6 antibody creates pervasive artifacts when imaging RNA:DNA hybrids

We showed that the majority of the signal observed in S9.6 immunofluorescence microscopy images of fixed human cells arises from RNA, not RNA:DNA hybrids. S9.6 staining is quantitatively unchanged by pre-treatment with the human RNA:DNA hybrid-specific nuclease, RNase H1, despite experimental verification that the enzyme was active in situ. S9.6 staining was, however, significantly sensitive to pre-treatments with RNase T1, and in some cases RNase III, two ribonucleases that specifically degrade single-stranded and double-stranded RNA, respectively. In contrast, genome-wide maps obtained by high-throughput DNA sequencing after S9.6-mediated DNA:RNA Immunoprecipitation (DRIP) are RNase H1-sensitive and RNase T1- and RNase III-insensitive. Altogether, these data demonstrate that the S9.6 antibody, though capable of recognizing RNA:DNA hybrids in situ and in vitro, suffers from a lack of specificity that precludes reliable imaging of RNA:DNA hybrids and renders associated imaging data inconclusive in the absence of controls for its promiscuous recognition of cellular RNAs. Characterization of RNA:DNA hybrid structures in NTERA-2 D1 (NT2) cells using sDRIP-seq on samples treated with RNase H1, RNase III, and/or RNase T1.