eCLIP
Enhanced Cross-linking Immunoprecipitation
eCLIP maps the binding sites of RBPs on their target RNAs using a modified individual nucleotide resolution CLIP (iCLIP) protocol, improving efficiency and decreasing execution complexity (Van Nostrand et al., 2016). The hallmark of this method is the ligation of barcoded single-stranded DNA adapters, which reduce amplification bias significantly.
First, RNA and the protein of interest are UV-crosslinked, followed by cell lysis and RNase I digestion. Next, the protein-RNA complexes are immunoprecipitated and ligated to an RNA adapter on the 3′ end of the target RNA. The bound protein is removed by proteinase K digestion, and the RNA is reverse-transcribed. The resulting cDNA is ligated to single-stranded DNA adapters on the 3′ end that contain either an N5 or N10 sequence to serve as unique identifiers against PCR duplicates. Finally, the paired-end cDNA fragments are amplified and sequenced.
Similar methods: iCLIP, irCLIP, HITS-CLIP
Advantages:
- High-throughput mapping of protein-RNA binding sites
- Barcoded adapters significantly reduce PCR duplicate reads and improve throughput
- Improved ligation efficiency by ~1000-fold
- Avoids usage of radioactive markers
Disadvantages:
- Antibodies not specific to the target will precipitate nonspecific complexes
- Crosslinking does not cover all RNA-binding domains (Martin et al., 2016)
Reagents:
Illumina Library prep and Array Kit Selector
Reviews:
Marchese D., de Groot N. S., Lorenzo Gotor N., Livi C. M. and Tartaglia G. G. Advances in the characterization of RNA-binding proteins. Wiley Interdiscip Rev RNA. 2016;7:793-810
Bangru S. and Kalsotra A. Advances in analyzing RNA diversity in eukaryotic transcriptomes: peering through the Omics lens. F1000Research. 2016;5:2668
Manning K. S. and Cooper T. A. The roles of RNA processing in translating genotype to phenotype. Nat Rev Mol Cell Biol. 2017;18:102-114
Sundararaman B., Zhan L., Blue S. M., et al. Resources for the Comprehensive Discovery of Functional RNA Elements. Mol Cell. 2016;61:903-913
References:
Conway Anne E., Van Nostrand Eric L., Pratt Gabriel A., et al. Enhanced CLIP Uncovers IMP Protein-RNA Targets in Human Pluripotent Stem Cells Important for Cell Adhesion and Survival. Cell Reports. 2016;
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History: eCLIP
Revision by sbrumpton on 2017-06-21 07:50:24 - Show/Hide
Enhanced Cross-linking Immunoprecipitation
eCLIP maps the binding sites of RBPs on their target RNAs using a modified individual nucleotide resolution CLIP (iCLIP) protocol, improving efficiency and decreasing execution complexity (Van Nostrand et al., 2016). The hallmark of this method is the ligation of barcoded single-stranded DNA adapters, which reduce amplification bias significantly.
First, RNA and the protein of interest are UV-crosslinked, followed by cell lysis and RNase I digestion. Next, the protein-RNA complexes are immunoprecipitated and ligated to an RNA adapter on the 3' end of the target RNA. The bound protein is removed by proteinase K digestion, and the RNA is reverse-transcribed. The resulting cDNA is ligated to single-stranded DNA adapters on the 3' end that contain either an N5 or N10 sequence to serve as unique identifiers against PCR duplicates. Finally, the paired-end cDNA fragments are amplified and sequenced.
Similar methods: iCLIP, irCLIP, HITS-CLIP
Advantages:- High-throughput mapping of protein-RNA binding sites
- Barcoded adapters significantly reduce PCR duplicate reads and improve throughput
- Improved ligation efficiency by ~1000-fold
- Avoids usage of radioactive markers
Disadvantages:- Antibodies not specific to the target will precipitate nonspecific complexes
- Crosslinking does not cover all RNA-binding domains (Martin et al., 2016)
Reagents:Illumina Library prep and Array Kit SelectorReviews:Marchese D., de Groot N. S., Lorenzo Gotor N., Livi C. M. and Tartaglia G. G. Advances in the characterization of RNA-binding proteins. Wiley Interdiscip Rev RNA. 2016;7:793-810Bangru S. and Kalsotra A. Advances in analyzing RNA diversity in eukaryotic transcriptomes: peering through the Omics lens. F1000Research. 2016;5:2668Manning K. S. and Cooper T. A. The roles of RNA processing in translating genotype to phenotype. Nat Rev Mol Cell Biol. 2017;18:102-114Sundararaman B., Zhan L., Blue S. M., et al. Resources for the Comprehensive Discovery of Functional RNA Elements. Mol Cell. 2016;61:903-913References:Conway Anne E., Van Nostrand Eric L., Pratt Gabriel A., et al. Enhanced CLIP Uncovers IMP Protein-RNA Targets in Human Pluripotent Stem Cells Important for Cell Adhesion and Survival. Cell Reports. 2016;