 |
 |
GEO help: Mouse over screen elements for information. |
|
| 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 |
| E-mail(s) |
flchedin@ucdavis.edu
|
| 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)
|
|
| 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 |
|
|
|
|
 |
Less...
More...