title: PAR-CLIP (Photoactivatable Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation): a step-by-step protocol to the transcriptome-wide identification of binding sites of RNA-binding proteins.
abstract: We recently developed a protocol for the transcriptome-wide isolation of RNA recognition elements readily applicable to any protein or ribonucleoprotein complex directly contacting RNA (including RNA helicases, polymerases, or nucleases) expressed in cell culture models either naturally or ectopically (Hafner et al., 2010). Briefly, immunoprecipitation of the RNA-binding protein of interest is followed by isolation of the crosslinked and coimmunoprecipitated RNA. In the course of lysate preparation and immunoprecipitation, the mRNAs are partially degraded using Ribonuclease T1. The isolated crosslinked RNA fragments are converted into a cDNA library and deep-sequenced using Solexa technology (see Explanatory Chapter: Next Generation Sequencing). By introducing photoreactive nucleosides that generate characteristic sequence changes upon crosslinking (see below), our protocol allows one to separate RNA segments bound by the protein of interest from the background un-crosslinked RNAs.
authors.affiliation: Howard Hughes Medical Institute, Laboratory of RNA Molecular Biology, The Rockefeller University, New York, NY, USA; These authors contributed equally to this work.
authors.forename: Jessica
authors.initials: J
authors.lastname: Spitzer
authors.affiliation: Howard Hughes Medical Institute, Laboratory of RNA Molecular Biology, The Rockefeller University, New York, NY, USA; These authors contributed equally to this work.
authors.forename: Markus
authors.initials: M
authors.lastname: Hafner
authors.affiliation: Present address: Berlin Institute for Medical Systems Biology, Max-Delbruck-Center for Molecular Medicine, Berlin, Germany.
authors.forename: Markus
authors.initials: M
authors.lastname: Landthaler
authors.affiliation: Howard Hughes Medical Institute, Laboratory of RNA Molecular Biology, The Rockefeller University, New York, NY, USA.
authors.forename: Manuel
authors.initials: M
authors.lastname: Ascano
authors.affiliation: Howard Hughes Medical Institute, Laboratory of RNA Molecular Biology, The Rockefeller University, New York, NY, USA.
authors.forename: Thalia
authors.initials: T
authors.lastname: Farazi
authors.affiliation: Howard Hughes Medical Institute, Laboratory of RNA Molecular Biology, The Rockefeller University, New York, NY, USA.
authors.forename: Greg
authors.initials: G
authors.lastname: Wardle
authors.affiliation: Howard Hughes Medical Institute, Laboratory of RNA Molecular Biology, The Rockefeller University, New York, NY, USA.
authors.forename: Jeff
authors.initials: J
authors.lastname: Nusbaum
authors.affiliation: Biozentrum der Universität Basel and Swiss Institute of Bioinformatics (SIB), Basel, Switzerland.
authors.forename: Mohsen
authors.initials: M
authors.lastname: Khorshid
authors.affiliation: Biozentrum der Universität Basel and Swiss Institute of Bioinformatics (SIB), Basel, Switzerland.
authors.forename: Lukas
authors.initials: L
authors.lastname: Burger
authors.affiliation: Biozentrum der Universität Basel and Swiss Institute of Bioinformatics (SIB), Basel, Switzerland.
authors.forename: Mihaela
authors.initials: M
authors.lastname: Zavolan
authors.affiliation: Howard Hughes Medical Institute, Laboratory of RNA Molecular Biology, The Rockefeller University, New York, NY, USA. Electronic address: ttuschl@rockefeller.edu.
authors.forename: Thomas
authors.initials: T
authors.lastname: Tuschl
mesh_terms: D000818:Animals; D001665:Binding Sites; D002478:Cells, Cultured; D019286:Endopeptidase K / Q000737:chemistry; D006801:Humans; D047468:Immunoprecipitation; D055668:Photochemical Processes; D059748:Proteolysis; D012313:RNA / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D059467:Transcriptome; D014466:Ultraviolet Rays
chemical_list: D016601:RNA-Binding Proteins; D012313:RNA; D019286:Endopeptidase K
keywords: 3′- and 5′-adapter ligation; Cell lysate preparation; Crosslinking and immunoprecipitation (CLIP) protocol; Immunoprecipitation and second RNase T1 digestion; PCR amplification of cDNA library; Posttranscriptional regulation (PTR); Protein G magnetic beads; Proteinase K digestion; UV crosslinking of 4-thiouridine-labeled cells; cDNA library preparation; cDNA library preparation/reverse transcription