Optimization of PAR-CLIP for transcriptome-wide identification of binding sites of RNA-binding proteins.

Aitor Garzia, Cindy Meyer, Pavel Morozov, Marcin Sajek, Thomas Tuschl

Journal: Methods (San Diego, Calif.) 2018;118-119():24-40

PMID: 27765618

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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.

Address: Laboratory of RNA Molecular Biology, Howard Hughes Medical Institute, The Rockefeller University, 1230 York Avenue, New York, NY 10065, USA.; Laboratory of RNA Molecular Biology, Howard Hughes Medical Institute, The Rockefeller University, 1230 York Avenue, New York, NY 10065, USA. Electronic address: [email protected].

DOI: 10.1016/j.ymeth.2016.10.007

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MeSH Terms: Antibodies, Base Sequence, Binding Sites, Cell Line, Tumor, Fragile X Mental Retardation Protein, Gene Library, HEK293 Cells, High-Throughput Nucleotide Sequencing, Humans, Immunoprecipitation, Nucleic Acid Conformation, Phosphoproteins, Phosphorus Radioisotopes, Protein Binding, RNA, RNA-Binding Protein FUS, RNA-Binding Proteins, Ribonucleases, Sequence Alignment, Sequence Analysis, RNA, Thiouridine, Ultraviolet Rays