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Transcriptome-Wide Mapping of Protein-RNA Interactions. | LitMetric

Transcriptome-Wide Mapping of Protein-RNA Interactions.

Methods Mol Biol

Department of Basic Medical Sciences, School of Medicine, Tsinghua University, Beijing, China.

Published: March 2021

RNA and RNA-binding proteins (RBPs) control multiple biological processes. The spatial and temporal arrangement of RNAs and RBPs underlies the delicate regulation of these processes. The strategy called CLIP-seq (cross-linking and immunoprecipitation) has been developed to capture endogenous protein-RNA interactions with UV cross-linking followed by immunoprecipitation. Despite the wide use of conventional CLIP-seq method in RBP study, the CLIP experiment is limited by the availability of the high-quality antibodies, potential contaminants from the co-purified RBPs, requirement of isotope manipulation, and potential loss of information during tedious experimental procedure. Here we described a modified CLIP-seq method called CLIP-seq using the FLAG-Biotin tag tandem purification. Through tandem purification and stringent wash condition, almost all the interacting RNA-binding proteins are removed; thus the indirect interacting RNAs mediated by these co-purified RBPs are also decreased. Our CLIP-seq method allows efficient detection of direct protein-bound RNAs without SDS-PAGE and membrane transfer procedure in an isotope-free and protein-specific antibody-free manner.

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Source
http://dx.doi.org/10.1007/978-1-0716-0680-3_12DOI Listing

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