High-Throughput Sequencing of CLIP cDNA Library

Crosslinking and immunoprecipitation (CLIP), with the use of RNase T1 trimming, was first described by Ule et al (Ule et al., 2005). and later applied to high-throughput sequencing to map protein-RNA binding sites in vivo (Licatalosi et al., 2008) (Chi et al., 2009). This approach is similar to RIP-Seq but uses crosslinking to stabilize the protein-RNA complexes.

In HITS-CLIP, RNA-protein complexes are UV-crosslinked and immunoprecipitated. The protein-RNA complexes are treated with RNase T1, followed by proteinase K. RNA is extracted and reverse-transcribed to cDNA. Deep sequencing of the cDNA provides single-base resolution mapping of protein binding sites on RNA.

An improvement on the HITS-CLIP protocol was published by Gillen et al., which reduces artifacts from mispriming occurences (Gillen et al., 2016).

Other versions: iCLIP, irCLIP, eCLIP, miCLIP


  • Crosslinking stabilizes the protein-target binding
  • UV crosslinking can be carried out in vivo
  • Provides low background and higher resolution of binding site, due to RNase digestion
  • No prior knowledge of the RNA is required
  • Genome-wide RNA screen


  • Over-representation of the RT complement due to mispriming (Gillen et al., 2016)
  • Antibodies not specific to the target may precipitate nonspecific complexes.
  • UV crosslinking is not efficient and requires close protein-RNA interactions
  • Artifacts may be introduced during the crosslinking process


Illumina Library prep and Array Kit Selector


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