ENAapplication/xmlftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR106/040/SRR10669440/SRR10669440_1.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR106/032/SRR10669432/SRR10669432_1.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR106/033/SRR10669433/SRR10669433_2.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR106/031/SRR10669431/SRR10669431_1.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR106/024/SRR10669424/SRR10669424.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR106/029/SRR10669429/SRR10669429_2.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR106/039/SRR10669439/SRR10669439_1.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR106/023/SRR10669423/SRR10669423.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR106/033/SRR10669433/SRR10669433_1.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR106/034/SRR10669434/SRR10669434_2.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR106/030/SRR10669430/SRR10669430_1.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR106/040/SRR10669440/SRR10669440_2.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR106/038/SRR10669438/SRR10669438.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR106/031/SRR10669431/SRR10669431_2.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR106/027/SRR10669427/SRR10669427.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR106/032/SRR10669432/SRR10669432_2.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR106/026/SRR10669426/SRR10669426.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR106/035/SRR10669435/SRR10669435_2.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR106/034/SRR10669434/SRR10669434_1.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR106/037/SRR10669437/SRR10669437.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR106/029/SRR10669429/SRR10669429_1.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR106/030/SRR10669430/SRR10669430_2.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR106/028/SRR10669428/SRR10669428.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR106/035/SRR10669435/SRR10669435_1.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR106/039/SRR10669439/SRR10669439_2.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR106/036/SRR10669436/SRR10669436.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR106/025/SRR10669425/SRR10669425.fastq.gzprimaryOK200GenomicsChedin, MCB, UC Davishttps://www.ebi.ac.uk/ena/browser/view/PRJNA594934Homo sapiensxref:PubMed:33830170We 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. Overall design: 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.ENAfalseRecognition of cellular RNAs by the S9.6 antibody creates pervasive artifacts when imaging RNA:DNA hybridsRecognition of cellular RNAs by the S9.6 antibody creates pervasive artifacts when imaging RNA:DNA hybrids2025-09-242021-03-20PRJNA594934GSE141833960633830170