eCLIP

eCLIP

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. 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.

Pros:

• High-throughput mapping of protein-RNA binding sites
• Barcoded adapters significantly reduce PCR duplicate reads and improves throughput
• Improved ligation efficiency by ~1000 fold
• Avoids usage of radioactive markers

Cons:

• Antibodies not specific to target will precipitate nonspecific complexes
• Cross-linking does not cover all RNA-binding domains¹

• eCLIP: Van Nostrand E. L., Pratt G. A., Shishkin A. A., Gelboin-Burkhart C., Fang M. Y., et al. (2016) Robust transcriptome-wide discovery of RNA-binding protein binding sites with enhanced CLIP (eCLIP). Nat Methods
• 1. Martin G. and Zavolan M. (2016) Redesigning CLIP for efficiency, accuracy and speed. Nat Methods 13: 482-483

1. 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
2. Bangru S. and Kalsotra A. Advances in analyzing RNA diversity in eukaryotic transcriptomes: peering through the Omics lens. F1000Research. 2016;5:2668
3. 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
4. Sundararaman B., Zhan L., Blue S. M., et al. Resources for the Comprehensive Discovery of Functional RNA Elements. Mol Cell. 2016;61:903-913

1. Conway Anne E., Van Nostrand Eric L., Pratt Gabriel A., Aigner S., Wilbert Melissa L., et al. (2016) Enhanced CLIP Uncovers IMP Protein-RNA Targets in Human Pluripotent Stem Cells Important for Cell Adhesion and Survival. Cell Reports