摘要
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.
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.
基金
supported in part by grants from the Ministry of Science and Technology of China(Grant No.2017YFA0504200 to YY,Grant Nos.2012CB910502 and 2011CB966304 to JM)
the National Natural Science Foundation of China(Grant Nos.91640105 and 31770875 to YY,Grant No.31230041 to JM,and Grant Nos.91640115 and 31670827 to YZ)
