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| Status |
Public on May 15, 2011 |
| Title |
A quantitative analysis of CLIP methods for identifying binding sites of RNA-binding proteins (CLIP) |
| Organism |
Homo sapiens |
| Experiment type |
Expression profiling by high throughput sequencing
|
| Summary |
Crosslinking and immunoprecipitation (CLIP) is increasingly used to map transcriptome-wide binding sites of RNA-binding proteins (RBPs). We developed a method for CLIP data analysis and applied it to compare 254 nm CLIP with PAR-CLIP, which involves crosslinking of photoreactive nucleotides with 365 nm UV light. We found small differences in the accuracy of these methods in identifying binding sites of HuR, a protein that binds low-complexity sequences and Argonaute 2, which has a complex binding specificity. We show that crosslink-induced mutations lead to single-nucleotide resolution for both PAR-CLIP and CLIP. Our results confirm the expectation from original CLIP publications that RNA-binding proteins do not protect sufficiently their sites under the denaturing conditions used during the CLIP procedure, and we show that extensive digestion with sequence-specific ribonucleases strongly biases the set of recovered binding sites. We finally show that this bias can be substantially reduced by milder nuclease digestion conditions.
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| Overall design |
We performed duplicate experiments for each variant of the CLIP protocol (CLIP, PAR-CLIP), each protein (HuR, Ago2), and enzymatic digestion (complete T1 digestion, mild MNase digestion). In addition, we performed a single PAR-CLIP experiment with mild T1 digestion.
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| Contributor(s) |
Kishore S, Jaskiewicz L, Burger L, Hausser J, Khorshid M, Zavolan M |
| Citation(s) |
21572407 |
| Submission date |
Apr 26, 2011 |
| Last update date |
Jul 17, 2019 |
| Contact name |
Jean Hausser |
| Organization name |
Weizmann Institute of Science
|
| Street address |
Herzl St.
|
| City |
Rehovot |
| ZIP/Postal code |
76100 |
| Country |
Israel |
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| Platforms (1) |
| GPL10999 |
Illumina Genome Analyzer IIx (Homo sapiens) |
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| Samples (13)
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| This SubSeries is part of SuperSeries: |
| GSE28865 |
A quantitative analysis of CLIP methods for identifying binding sites of RNA-binding proteins |
|
| Relations |
| SRA |
SRP006474 |
| BioProject |
PRJNA153959 |
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