eCLIP

eCLIP

Method Category: Transcriptome > RNA-Protein Interactions

Description: 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