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| 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
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| 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.
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| 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.
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| Contributor(s) |
Smolka JA, Sanz LA, Hartono SR, Chedin F |
| Citation(s) |
33830170 |
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| Submission date |
Dec 11, 2019 |
| Last update date |
Jun 19, 2021 |
| Contact name |
Frederic Chedin |
| E-mail(s) |
[email protected]
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| Organization name |
UC Davis
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| Department |
MCB
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| Lab |
Chedin
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| Street address |
1 Shields Avenue
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| City |
Davis |
| State/province |
CA |
| ZIP/Postal code |
95616 |
| Country |
USA |
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| Platforms (2) |
| GPL20301 |
Illumina HiSeq 4000 (Homo sapiens) |
| GPL21290 |
Illumina HiSeq 3000 (Homo sapiens) |
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| Samples (18)
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| Relations |
| BioProject |
PRJNA594934 |
| SRA |
SRP236869 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE141833_RAW.tar |
7.1 Gb |
(http)(custom) |
TAR (of BW) |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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