title: Optimization of PAR-CLIP for transcriptome-wide identification of binding sites of RNA-binding proteins.
abstract: Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation (PAR-CLIP) in combination with next-generation sequencing is a powerful method for identifying endogenous targets of RNA-binding proteins (RBPs). Depending on the characteristics of each RBP, key steps in the PAR-CLIP procedure must be optimized. Here we present a comprehensive step-by-step PAR-CLIP protocol with detailed explanations of the critical steps. Furthermore, we report the application of a new PAR-CLIP data analysis pipeline to three distinct RBPs targeting different annotation categories of cellular RNAs.
authors.affiliation: Laboratory of RNA Molecular Biology, Howard Hughes Medical Institute, The Rockefeller University, 1230 York Avenue, New York, NY 10065, USA.
authors.forename: Aitor
authors.initials: A
authors.lastname: Garzia
authors.affiliation: Laboratory of RNA Molecular Biology, Howard Hughes Medical Institute, The Rockefeller University, 1230 York Avenue, New York, NY 10065, USA.
authors.forename: Cindy
authors.initials: C
authors.lastname: Meyer
authors.affiliation: Laboratory of RNA Molecular Biology, Howard Hughes Medical Institute, The Rockefeller University, 1230 York Avenue, New York, NY 10065, USA.
authors.forename: Pavel
authors.initials: P
authors.lastname: Morozov
authors.affiliation: Laboratory of RNA Molecular Biology, Howard Hughes Medical Institute, The Rockefeller University, 1230 York Avenue, New York, NY 10065, USA.
authors.forename: Marcin
authors.initials: M
authors.lastname: Sajek
authors.affiliation: Laboratory of RNA Molecular Biology, Howard Hughes Medical Institute, The Rockefeller University, 1230 York Avenue, New York, NY 10065, USA. Electronic address: ttuschl@rockefeller.edu.
authors.forename: Thomas
authors.initials: T
authors.lastname: Tuschl
mesh_terms: D000906:Antibodies / Q000737:chemistry; D001483:Base Sequence; D001665:Binding Sites; D045744:Cell Line, Tumor; D051860:Fragile X Messenger Ribonucleoprotein 1 / Q000235:genetics / Q000378:metabolism; D015723:Gene Library; D057809:HEK293 Cells; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D047468:Immunoprecipitation / Q000379:methods*; D009690:Nucleic Acid Conformation; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism; D010761:Phosphorus Radioisotopes; D011485:Protein Binding; D012313:RNA / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D034702:RNA-Binding Protein FUS / Q000235:genetics / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D012260:Ribonucleases / Q000737:chemistry; D016415:Sequence Alignment; D017423:Sequence Analysis, RNA / Q000379:methods*; D013891:Thiouridine / Q000737:chemistry / Q000378:metabolism*; D014466:Ultraviolet Rays
chemical_list: D000906:Antibodies; D051860:Fragile X Messenger Ribonucleoprotein 1; D010750:Phosphoproteins; D010761:Phosphorus Radioisotopes; D012313:RNA; D034702:RNA-Binding Protein FUS; D016601:RNA-Binding Proteins; D012260:Ribonucleases; D013891:Thiouridine; C497683:FMR1 protein, human; C000599550:FUS protein, human; C574674:La protein, human
keywords: Next-generation sequencing; Photocrosslinking; Ribonucleoprotein analysis; UV RNA-protein crosslinking