Transcriptome-Wide Mapping of Protein–RNA Interactions

Abstract

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 ^FbioCLIP-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 ^FbioCLIP-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.