|
| Status |
Public on May 15, 2011 |
| Title |
A quantitative analysis of CLIP methods for identifying binding sites of RNA-binding proteins (mRNA-seq) |
| 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.
|
| |
|
| Overall design |
We performed duplicate mRNA-Seq experiments for cells grown in the presence of modified nucleotides or in normal medium, and following crosslinking at 365nm, 254nm or without crosslinking. In addition, we performed single mRNA-Seq experiments of cells transfected with anti-GFP or anti-HuR siRNA.
|
| |
|
| 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 |
May 15, 2019 |
| Contact name |
Jean Hausser |
| Organization name |
Weizmann Institute of Science
|
| Street address |
Herzl St.
|
| City |
Rehovot |
| ZIP/Postal code |
76100 |
| Country |
Israel |
| |
|
| Platforms (1) |
| GPL10999 |
Illumina Genome Analyzer IIx (Homo sapiens) |
|
| Samples (10)
|
|
| This SubSeries is part of SuperSeries: |
| GSE28865 |
A quantitative analysis of CLIP methods for identifying binding sites of RNA-binding proteins |
|
| Relations |
| SRA |
SRP006475 |
| BioProject |
PRJNA153961 |
Less...
More...