Methods in enzymology, 2018, Vol.612, pp.127-145
RNA-protein interactions are at the heart of many central cellular processes, and RNA-binding proteins (RBPs) associate with virtually all RNA molecules in a cell. In bacteria, global RBPs, often in conjunction with small regulatory RNAs, affect physiology and virulence by controlling transcription, translation, and RNA decay. To understand how these regulatory proteins orchestrate global gene expression, detailed maps of their cellular RNA binding sites are required. To this end, cross-linking and immunoprecipitation followed by deep sequencing (CLIP-seq) has revolutionized RBP studies by providing knowledge about global recognition patterns of RBPs in both eukaryotic and bacterial cells. In this chapter, we provide a step-by-step protocol for global mapping of bona fide RBP binding sites using CLIP-seq in bacteria. This protocol has been successfully applied for charting the binding sites of Hfq, CsrA, and ProQ, three global regulatory RBPs in Salmonella enterica and Escherichia coli, and should be readily applicable to other RBPs and bacterial species.
Clip-Seq ; Rbp ; RNA Binding ; RNA–Protein Interaction
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