期刊
NATURE METHODS
卷 8, 期 7, 页码 559-U61出版社
NATURE PORTFOLIO
DOI: 10.1038/NMETH.1608
关键词
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资金
- Swiss National Science Foundation [31003A_127307, PDAM_3_127218, PDFMP3_123123]
- Swiss Initiative in Systems Biology (Cell Plasticity)
- Swiss Cancer League [KFS 02477-08-2009]
- European Molecular Biology Organization [184-2009]
- Swiss National Science Foundation (SNF) [31003A_127307, PDFMP3_123123] Funding Source: Swiss National Science Foundation (SNF)
Cross-linking and immunoprecipitation (CLIP) is increasingly used to map transcriptome-wide binding sites of RNA-binding proteins. We developed a method for CLIP data analysis, and applied it to compare CLIP with photoactivatable ribonucleoside-enhanced CLIP (PAR-CLIP) and to uncover how differences in cross-linking and ribonuclease digestion affect the identified sites. We found only small differences in accuracies of these methods in identifying binding sites of HuR, which binds low-complexity sequences, and Argonaute 2, which has a complex binding specificity. We found that cross-link-induced mutations led 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 their binding sites sufficiently under the denaturing conditions used during the CLIP procedure, and we show that extensive digestion with sequence-specific RNases strongly biases the recovered binding sites. This bias can be substantially reduced by milder nuclease digestion conditions.
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