Small RNAs (sRNAs) are major post-transcriptional regulators of gene expression in bacteria. To enable transcriptome-wide mapping of bacterial sRNA–target pairs, we developed RIL-seq (RNA interaction by ligation and sequencing). RIL-seq is an experimental–computational methodology for capturing sRNA–target interactions in vivo that takes advantage of the mutual binding of the sRNA and target RNA molecules to the RNA chaperone protein Hfq. The experimental part of the protocol involves co-immunoprecipitation of Hfq and bound RNAs, ligation of RNAs, library preparation and sequencing. The computational pipeline maps the sequenced fragments to the genome, reveals chimeric fragments (fragments comprising two ligated independent fragments) and determines statistically significant overrepresented chimeric fragments as interacting RNAs. The statistical filter is aimed at reducing the number of spurious interactions resulting from ligation of random neighboring RNA fragments, thus increasing the reliability of the determined sRNA–target pairs. A major advantage of RIL-seq is that it does not require overexpression of sRNAs; instead, it simultaneously captures the in vivo targets of all sRNAs in the native state of the cell. Application of RIL-seq to bacteria grown under different conditions provides distinctive snapshots of the sRNA interactome and sheds light on the dynamics and rewiring of the post-transcriptional regulatory network. As RIL-seq needs no prior information about the sRNA and target sequences, it can identify novel sRNAs, along with their targets. It can be adapted to detect protein-mediated RNA–RNA interactions in any bacterium with a sequenced genome. The experimental part of the RIL-seq protocol takes 7–9 d and the computational analysis takes ∼2 d.
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This study was supported by European Research Council Advanced Grant 322920, the I-CORE Programs of the Planning and Budgeting Committee and The Israel Science Foundation (grants 1796/12 and 41/1), and the Israel Science Foundation, administered by the Israeli Academy for Sciences and Humanities (grant 1411/13). We thank Y. Gatt, S. Pearl-Mizrahi, A. Zhang and H. Zhang for their useful comments.
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