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.
Keywords:
Next-generation sequencing; Photocrosslinking; Ribonucleoprotein analysis; UV RNA-protein crosslinking.
Copyright © 2016 Elsevier Inc. All rights reserved.
Publication types
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Review
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Research Support, Non-U.S. Gov't
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Research Support, N.I.H., Extramural
MeSH terms
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Antibodies / chemistry
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Base Sequence
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Binding Sites
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Cell Line, Tumor
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Fragile X Mental Retardation Protein / genetics
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Fragile X Mental Retardation Protein / metabolism
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Gene Library
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HEK293 Cells
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High-Throughput Nucleotide Sequencing / methods*
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Humans
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Immunoprecipitation / methods*
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Nucleic Acid Conformation
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Phosphoproteins / genetics
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Phosphoproteins / metabolism
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Phosphorus Radioisotopes
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Protein Binding
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RNA / chemistry*
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RNA / genetics
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RNA / metabolism
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RNA-Binding Protein FUS / genetics
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RNA-Binding Protein FUS / metabolism
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RNA-Binding Proteins / genetics*
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RNA-Binding Proteins / metabolism
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Ribonucleases / chemistry
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Sequence Alignment
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Sequence Analysis, RNA / methods*
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Thiouridine / chemistry
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Thiouridine / metabolism*
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Ultraviolet Rays
Substances
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Antibodies
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FMR1 protein, human
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FUS protein, human
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La protein, human
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Phosphoproteins
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Phosphorus Radioisotopes
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RNA-Binding Protein FUS
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RNA-Binding Proteins
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Fragile X Mental Retardation Protein
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Thiouridine
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RNA
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Ribonucleases