The structure of RNA molecules has a critical role in regulating gene expression, largely through influencing their interactions with RNA-binding proteins (RBPs). RNA hybrid and individual-nucleotide resolution UV cross-linking and immunoprecipitation (hiCLIP) is a transcriptome-wide method of monitoring these interactions by identifying RNA duplexes bound by a specific RBP. The hiCLIP protocol consists of the following steps: in vivo cross-linking of RBPs to their bound RNAs; partial RNA digestion and purification of RNA duplexes interacting with the specific RBP using immunoprecipitation; ligation of the two arms of RNA duplexes via a linker; reverse transcription; cDNA library amplification; and finally high-throughput DNA sequencing. Mapping of the sequenced arms to a reference transcriptome identifies the exact locations of duplexes. hiCLIP data can directly identify all types of RNA duplexes bound by RBPs, including those that are challenging to predict computationally, such as intermolecular and long-range intramolecular duplexes. Moreover, the use of an adaptor that links the two arms of the RNA duplex permits hiCLIP to unambiguously identify the duplexes. Here we describe in detail the procedure for a hiCLIP experiment and the subsequent streamlined data analysis with an R package, 'hiclipr' (https://github.com/luslab/hiclipr/). Preparation of the library for high-throughput DNA sequencing takes ∼7 d and the basic bioinformatic pipeline takes 1 d.
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We thank F. Agostini (Luscombe laboratory) for helpful advice on the 'hiclipr' package; C. Militti (Ule laboratory) for valuable comments on the manuscript; F. Lee and I. Ruiz de los Mozos (Ule laboratory) for testing the 'hiclipr' package; and all the members of the Ule and Luscombe laboratories for providing fruitful discussions throughout the study. The hiCLIP project was supported by funding from the European Research Council (617837-Translate) to J.U., a Wellcome Trust Joint Investigator Award to J.U. and N.M.L. (103760/Z/14/Z), the Nakajima Foundation Fellowship and an MRC Centenary Early Career Award to Y.S., a Wellcome Trust PhD Training Fellowship for Clinicians to A.M.C., and the Francis Crick Institute, which receives its core funding from Cancer Research UK (FC001002), the UK Medical Research Council (FC001002), and the Wellcome Trust (FC001002).
The authors declare no competing financial interests.
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Sugimoto, Y., Chakrabarti, A., Luscombe, N. et al. Using hiCLIP to identify RNA duplexes that interact with a specific RNA-binding protein. Nat Protoc 12, 611–637 (2017) doi:10.1038/nprot.2016.188
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