HITS-CLIP/CLIP-Seq/PTB-Seq

HITS-CLIP/CLIP-Seq/PTB-Seq RNA

Crosslinking and immunoprecipitation (CLIP), with the use of RNase T1 trimming, was first described by Ule et al. ¹ and later applied to high-throughput sequencing to map protein-RNA binding sites in vivo.² 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.³

Other versions: iCLIP, irCLIP, eCLIP, miCLIP

Pros:

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

Cons:

Over-representation of the RT complement due to mispriming⁴
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

Cook K. B., Hughes T. R. and Morris Q. D. High-throughput characterization of protein-RNA interactions. Brief Funct Genomics. 2015;14:74-89