title: Using hiCLIP to identify RNA duplexes that interact with a specific RNA-binding protein.
abstract: 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.
authors.affiliation: Department of Molecular Neuroscience, UCL Institute of Neurology, London, UK.|The Francis Crick Institute, London, UK.
authors.forename: Yoichiro
authors.initials: Y
authors.lastname: Sugimoto
authors.affiliation: The Francis Crick Institute, London, UK.|Department of Genetics, Evolution and Environment, UCL Genetics Institute, University College London, London, UK.
authors.forename: Anob M
authors.initials: AM
authors.lastname: Chakrabarti
authors.affiliation: The Francis Crick Institute, London, UK.|Department of Genetics, Evolution and Environment, UCL Genetics Institute, University College London, London, UK.|Okinawa Institute of Science and Technology Graduate University, Onna-son, Okinawa, Japan.
authors.forename: Nicholas M
authors.initials: NM
authors.lastname: Luscombe
authors.affiliation: Department of Molecular Neuroscience, UCL Institute of Neurology, London, UK.|The Francis Crick Institute, London, UK.
authors.forename: Jernej
authors.identifier: 0000-0002-2452-4277
authors.initials: J
authors.lastname: Ule
mesh_terms: D002460:Cell Line; D003101:Collodion / Q000737:chemistry; D019295:Computational Biology; D004591:Electrophoresis, Polyacrylamide Gel; D047468:Immunoprecipitation / Q000379:methods*; D008567:Membranes, Artificial; D011485:Protein Binding; D012313:RNA / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D008567:Membranes, Artificial; D016601:RNA-Binding Proteins; D012313:RNA; D003101:Collodion