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.
- Spitzer J Howard Hughes Medical Institute, Laboratory of RNA Molecular Biology, The Rockefeller University, New York, NY, USA; These authors contributed equally to this work.
- Hafner M Howard Hughes Medical Institute, Laboratory of RNA Molecular Biology, The Rockefeller University, New York, NY, USA; These authors contributed equally to this work.
- Landthaler M Present address: Berlin Institute for Medical Systems Biology, Max-Delbruck-Center for Molecular Medicine, Berlin, Germany.
- Ascano M Howard Hughes Medical Institute, Laboratory of RNA Molecular Biology, The Rockefeller University, New York, NY, USA.
- Farazi T Howard Hughes Medical Institute, Laboratory of RNA Molecular Biology, The Rockefeller University, New York, NY, USA.
- Wardle G Howard Hughes Medical Institute, Laboratory of RNA Molecular Biology, The Rockefeller University, New York, NY, USA.
- Nusbaum J Howard Hughes Medical Institute, Laboratory of RNA Molecular Biology, The Rockefeller University, New York, NY, USA.
- Khorshid M Biozentrum der Universität Basel and Swiss Institute of Bioinformatics (SIB), Basel, Switzerland.
- Burger L Biozentrum der Universität Basel and Swiss Institute of Bioinformatics (SIB), Basel, Switzerland.
- Zavolan M Biozentrum der Universität Basel and Swiss Institute of Bioinformatics (SIB), Basel, Switzerland.
- Tuschl T Howard Hughes Medical Institute, Laboratory of RNA Molecular Biology, The Rockefeller University, New York, NY, USA. Electronic address: ttuschl@rockefeller.edu.
- 2014-03-04
Published in:
- Methods in enzymology. - 2014
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
Animals
Binding Sites
Cells, Cultured
Endopeptidase K
Humans
Immunoprecipitation
Photochemical Processes
Proteolysis
RNA
RNA-Binding Proteins
Transcriptome
Ultraviolet Rays
English
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.
- Language
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- English
- Open access status
- green
- Identifiers
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- DOI 10.1016/B978-0-12-420120-0.00008-6
- PMID 24581442
- Persistent URL
- https://sonar.ch/global/documents/53536
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