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

ABSTRACT

The S9.6 antibody is broadly used to detect RNADNA hybrids but has significant affinity for double-stranded RNA. The impact of this off-target RNA binding activity has not been thoroughly investigated, especially in the context of immunofluorescence microscopy. We report that S9.6 immunofluorescence signal observed in fixed human cells arises predominantly from ribosomal RNA, not RNADNA hybrids. S9.6 staining was unchanged by pretreatment with the RNADNA hybrid-specific nuclease RNase H1, despite verification in situ that S9.6 recognized RNADNA hybrids and that RNase H1 was active. S9.6 staining was, however, significantly sensitive to RNase T1, which specifically degrades RNA. Additional imaging and biochemical data indicate that the prominent cytoplasmic and nucleolar S9.6 signal primarily derives from ribosomal RNA. Importantly, genome-wide maps obtained by DNA sequencing after S9.6-mediated DNARNA immunoprecipitation (DRIP) are RNase H1 sensitive and RNase T1 insensitive. Altogether, these data demonstrate that imaging using S9.6 is subject to pervasive artifacts without pretreatments and controls that mitigate its promiscuous recognition of cellular RNAs.