Format

Send to

Choose Destination
Genomics Proteomics Bioinformatics. 2018 Apr;16(2):136-143. doi: 10.1016/j.gpb.2018.04.003. Epub 2018 Apr 28.

GoldCLIP: Gel-omitted Ligation-dependent CLIP.

Author information

1
State Key Laboratory of Genetic Engineering, Department of Biochemistry, School of Life Sciences, Fudan University, Shanghai 200438, China; CAS Key Laboratory of RNA Biology, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China.
2
CAS Key Laboratory of RNA Biology, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China.
3
Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, Wuhan University, Wuhan 430072, China.
4
State Key Laboratory of Genetic Engineering, Department of Biochemistry, School of Life Sciences, Fudan University, Shanghai 200438, China. Electronic address: majb@fudan.edu.cn.
5
Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, Wuhan University, Wuhan 430072, China. Electronic address: yu.zhou@whu.edu.cn.
6
Cancer Research UK, Li Ka Shing Centre, University of Cambridge, Cambridge CB2 ORE, United Kingdom. Electronic address: greg.hannon@cruk.cam.ac.uk.
7
CAS Key Laboratory of RNA Biology, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China. Electronic address: yuyang@ibp.ac.cn.

Abstract

Protein-RNA interaction networks are essential to understand gene regulation control. Identifying binding sites of RNA-binding proteins (RBPs) by the UV-crosslinking and immunoprecipitation (CLIP) represents one of the most powerful methods to map protein-RNA interactions in vivo. However, the traditional CLIP protocol is technically challenging, which requires radioactive labeling and suffers from material loss during PAGE-membrane transfer procedures. Here we introduce a super-efficient CLIP method (GoldCLIP) that omits all gel purification steps. This nonisotopic method allows us to perform highly reproducible CLIP experiments with polypyrimidine tract-binding protein (PTB), a classical RBP in human cell lines. In principle, our method guarantees sequencing library constructions, providing the protein of interest can be successfully crosslinked to RNAs in living cells. GoldCLIP is readily applicable to diverse proteins to uncover their endogenous RNA targets.

KEYWORDS:

CLIP; HaloTag; PTB; RNA binding protein; UV crosslinking

PMID:
29709556
PMCID:
PMC6112358
DOI:
10.1016/j.gpb.2018.04.003
[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for Elsevier Science Icon for PubMed Central
Loading ...
Support Center