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Series GSE141833 Query DataSets for GSE141833
Status Public on Mar 17, 2021
Title Recognition of cellular RNAs by the S9.6 antibody creates pervasive artifacts when imaging RNA:DNA hybrids
Organism Homo sapiens
Experiment type Other
Summary 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.
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.
Contributor(s) Smolka JA, Sanz LA, Hartono SR, Chedin F
Citation(s) 33830170
Submission date Dec 11, 2019
Last update date Jun 19, 2021
Contact name Frederic Chedin
Organization name UC Davis
Department MCB
Lab Chedin
Street address 1 Shields Avenue
City Davis
State/province CA
ZIP/Postal code 95616
Country USA
Platforms (2)
GPL20301 Illumina HiSeq 4000 (Homo sapiens)
GPL21290 Illumina HiSeq 3000 (Homo sapiens)
Samples (18)
GSM4213949 RNase T1 (DG3G)
GSM4213950 RNase III (DG3H)
GSM4213951 RNase T1+III (DG3I)
BioProject PRJNA594934
SRA SRP236869

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Supplementary file Size Download File type/resource
GSE141833_RAW.tar 7.1 Gb (http)(custom) TAR (of BW)
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Raw data are available in SRA
Processed data provided as supplementary file

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