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.

中文翻译：

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使用RIL-seq绘制细菌中的小RNA相互作用组。

小RNA（sRNA）是细菌中基因表达的主要转录后调节因子。为实现细菌sRNA-靶标对的转录组全图谱，我们开发了RIL-seq（通过连接和测序进行RNA相互作用）。RIL-seq是一种用于在体内捕获sRNA-靶标相互作用的实验计算方法，该方法利用sRNA和靶标RNA分子与RNA伴侣蛋白Hfq的相互结合的优势。该方案的实验部分涉及Hfq和结合的RNA的共免疫沉淀，RNA的连接，文库制备和测序。计算流水线将测序的片段映射到基因组，揭示嵌合片段（包含两个连接的独立片段的片段），并将统计上显着过量的嵌合片段确定为相互作用的RNA。统计过滤器旨在减少由于随机相邻RNA片段的连接而导致的虚假相互作用的数量，从而提高确定的sRNA-靶标对的可靠性。RIL-seq的一个主要优点是它不需要sRNA的过表达。相反，它同时捕获处于细胞天然状态的所有sRNA的体内靶标。RIL-seq在不同条件下生长的细菌上的应用提供了sRNA相互作用组的独特快照，并揭示了转录后调控网络的动态和重新布线。由于RIL-seq不需要有关sRNA和靶序列的任何先验信息，因此它可以识别新型sRNA及其靶标。它可以适用于检测具有序列化基因组的任何细菌中蛋白质介导的RNA-RNA相互作用。