Hfq LiRIP-seq in Salmonella

We have developed a novel approach named LiRIP-seq to profile the global RNA-RNA interactome in Salmonella enterica. By pulse expressing T4 RNA ligase from an inducible pBAD promoter, LiRIP-seq enables in vivo proximity ligation of Hfq-bound RNAs to their interaction partners. This is followed by enrichment of ligation products (RNA chimeras) using Hfq-coIP and subsequent RNA-seq analysis. Salmonella wild type or hfq::3xFLAG tagged strains carrying the plasmid pYC582 or empty vector were grown in LB medium at 37 °C. T4 RNA ligase was induced for 30 min by addition of L-arabinose when the culture reached the indicated OD600 (0.3, 1.5, 2.0+2.5h). RNA-RNA interactomes were profiled by Hfq-coIP and subsequent RNA-seq analysis.

本研究开发了一种命名为LiRIP-seq的新型方法，用于解析肠炎沙门氏菌（Salmonella enterica）中的全局RNA-RNA互作组（RNA-RNA interactome）。该方法通过从可诱导型pBAD启动子（pBAD promoter）中脉冲式表达T4 RNA连接酶（T4 RNA ligase），可实现Hfq结合RNA与其互作伴侣的体内近端连接。随后通过Hfq共免疫沉淀（Hfq-coIP）富集连接产物（RNA嵌合体，RNA chimeras），并开展后续RNA测序（RNA-seq）分析。本研究将携带质粒pYC582或空载体的肠炎沙门氏菌野生型菌株，以及带有hfq::3xFLAG标签的菌株，接种于LB培养基中于37℃下培养。当培养物达到指定的OD600值（0.3、1.5、2.0+2.5h）时，通过添加L-阿拉伯糖诱导T4 RNA连接酶表达30分钟。最终通过Hfq-coIP与后续RNA-seq分析，完成了RNA-RNA互作组的解析。