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
- 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
- Antibodies not specific to the target will precipitate nonspecific complexes
- Crosslinking does not cover all RNA-binding domains (Martin et al., 2016)
Illumina Library prep and Array Kit Selector
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
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;