UV-C Crosslinking and Immunoprecipitation

irCLIP maps protein-RNA interaction sites using less sample material, time, and increased cDNA library quality compared to previous CLIP methods (Zarnegar et al., 2016). irCLIP was designed to tackle the issues in both iCLIP and HITS-CLIP, such as reverse-transcriptase halting and short cDNA library fragments, by using on-bead nuclease digestion.

First, RNA-protein complexes are UV-crosslinked and immunoprecipitated. Next, they are digested using RNase A and RNase I to excise both ends of the protein-bound RNA strand. An IR800-biotin adapter is ligated to the 3′ end of the RNA fragment. After size selection, nitrocellulose blotting, and proteinase K digestion, the RNA strands of interest are purified. They are reverse-transcribed and immunopurified once again. The cDNA strands are circularized and PCR-amplified to produce cDNA libraries for sequencing

Other versions: iCLIP, HITS-CLIP, eCLIP


  • Nonisotopic detection of protein-RNA interactions with minimal starting material
  • On-bead nuclease digestion improves the length of RNA fragments for cDNA library prep
  • Able to accommodate small cell samples and reveal novel RBP binding sites
  • Uses thermostable reverse transcriptase at 60çC, reducing biases by melting secondary RNA structures (Martin et al., 2016)


  • Not yet adopted widely by the scientific community
  • Circularization step may introduce artifacts


Illumina Library prep and Array Kit Selector


Martin G. and Zavolan M. Redesigning CLIP for efficiency, accuracy and speed. Nat Methods. 2016;13:482-483

Haque N. and Hogg J. R. Easier, Better, Faster, Stronger: Improved Methods for RNA-Protein Interaction Studies. Mol Cell. 2016;62:650-651


Zarnegar B. J., Flynn R. A., Shen Y., Do B. T., Chang H. Y. and Khavari P. A. irCLIP platform for efficient characterization of protein-RNA interactions. Nat Methods. 2016;13:489-492