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Methods Mol Biol. 2017;1648:177-200. doi: 10.1007/978-1-4939-7204-3_14.

Robust, Cost-Effective Profiling of RNA Binding Protein Targets with Single-end Enhanced Crosslinking and Immunoprecipitation (seCLIP).

Van Nostrand EL1,2,3, Nguyen TB1,2,3, Gelboin-Burkhart C1,2,3, Wang R1,2,3, Blue SM1,2,3, Pratt GA1,2,3,4, Louie AL1,2,3, Yeo GW5,6,7,8,9,10,11.

Author information

1
Department of Cellular and Molecular Medicine, University of California at San Diego, La Jolla, CA, USA.
2
Stem Cell Program, University of California at San Diego, La Jolla, CA, USA.
3
Institute for Genomic Medicine, University of California at San Diego, La Jolla, CA, USA.
4
Bioinformatics and Systems Biology Graduate Program, University of California at San Diego, La Jolla, CA, USA.
5
Department of Cellular and Molecular Medicine, University of California at San Diego, La Jolla, CA, USA. geneyeo@ucsd.edu.
6
Stem Cell Program, University of California at San Diego, La Jolla, CA, USA. geneyeo@ucsd.edu.
7
Institute for Genomic Medicine, University of California at San Diego, La Jolla, CA, USA. geneyeo@ucsd.edu.
8
Bioinformatics and Systems Biology Graduate Program, University of California at San Diego, La Jolla, CA, USA. geneyeo@ucsd.edu.
9
Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore. geneyeo@ucsd.edu.
10
Molecular Engineering Laboratory, A*STAR, Singapore, Singapore. geneyeo@ucsd.edu.
11
Sanford Consortium for Regenerative Medicine, University of California at San Diego, 2880 Torrey Pines Scenic Dr., La Jolla, CA, 92037, USA. geneyeo@ucsd.edu.

Abstract

Profiling of RNA binding protein targets in vivo provides critical insights into the mechanistic roles they play in regulating RNA processing. The enhanced crosslinking and immunoprecipitation (eCLIP) methodology provides a framework for robust, reproducible identification of transcriptome-wide protein-RNA interactions, with dramatically improved efficiency over previous methods. Here we provide a step-by-step description of the eCLIP method, along with insights into optimal performance of critical steps in the protocol. In particular, we describe improvements to the adaptor strategy that enables single-end enhanced CLIP (seCLIP), which removes the requirement for paired-end sequencing of eCLIP libraries. Further, we describe the observation of contaminating RNA present in standard nitrocellulose membrane suppliers, and present options with significantly reduced contamination for sensitive applications. These notes further refine the eCLIP methodology, simplifying robust RNA binding protein studies for all users.

KEYWORDS:

CLIP; CLIP-seq; RNA binding protein; RNA genomics; eCLIP; seCLIP; seCLIP-seq

PMID:
28766298
PMCID:
PMC5991800
DOI:
10.1007/978-1-4939-7204-3_14
[Indexed for MEDLINE]
Free PMC Article

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