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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, Jessica and Hafner, Markus and Landthaler, Markus and Ascano, Manuel and Farazi, Thalia and Wardle, Greg and Nusbaum, Jeff and Khorshid, Mohsen and Burger, Lukas and Zavolan, Mihaela and Tuschl, Thomas. (2014) 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. Methods in enzymology, Vol. 539. pp. 113-161.

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Official URL: http://edoc.unibas.ch/dok/A6243440

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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.
Faculties and Departments:05 Faculty of Science > Departement Biozentrum > Computational & Systems Biology > Bioinformatics (Zavolan)
UniBasel Contributors:Zavolan, Mihaela
Item Type:Article, refereed
Article Subtype:Research Article
Bibsysno:Link to catalogue
Publisher:Elsevier
ISSN:0076-6879
Note:Publication type according to Uni Basel Research Database: Journal article
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Last Modified:23 May 2014 08:33
Deposited On:23 May 2014 08:33

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