iCLIP: protein-RNA interactions at nucleotide resolution

Methods. 2014 Feb;65(3):274-87. doi: 10.1016/j.ymeth.2013.10.011. Epub 2013 Oct 25.


RNA-binding proteins (RBPs) are key players in the post-transcriptional regulation of gene expression. Precise knowledge about their binding sites is therefore critical to unravel their molecular function and to understand their role in development and disease. Individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) identifies protein-RNA crosslink sites on a genome-wide scale. The high resolution and specificity of this method are achieved by an intramolecular cDNA circularization step that enables analysis of cDNAs that truncated at the protein-RNA crosslink sites. Here, we describe the improved iCLIP protocol and discuss critical optimization and control experiments that are required when applying the method to new RBPs.

Keywords: High-throughput sequencing; Post-transcriptional regulation; Protein–RNA interaction; RNA; RNA-binding protein; UV crosslinking and immunoprecipitation (CLIP); iCLIP.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Binding Sites
  • DNA, Circular / chemistry
  • DNA, Circular / genetics
  • Gene Expression Regulation
  • Gene Library*
  • HeLa Cells
  • High-Throughput Nucleotide Sequencing
  • Humans
  • Immunoprecipitation / methods*
  • Protein Binding
  • RNA / chemistry*
  • RNA / genetics
  • RNA-Binding Proteins / chemistry*
  • RNA-Binding Proteins / genetics
  • Ultraviolet Rays


  • DNA, Circular
  • RNA-Binding Proteins
  • RNA