RNA interactome profiling in hypervirulent Klebsiella pneumoniae

To map the Hfq-mediated RNA regulatory network in HvKP, we harnessed the LiRIP-seq approach recently established in our lab, which enabled global profiling of RNA-RNA interactomes in live bacteria. Transformation of the pBAD-t4rnl1 plasmid into Klebsiella allowed us to express T4 RNA ligase 1 for proximity-ligation of RNAs in vivo, followed by co-immunoprecipitation (coIP) to enrich the Hfq-bound RNA transcripts (singleton reads) and ligated RNA fragments (chimeras: sRNA-mRNA, sRNA-sRNA, etc). Overall design: LiRIP-seq was carried out in triplicate in cells at early stationary phase of growth in LB (OD600 of 3.0), with a monoclonal anti-FLAG antibody for IP and mouse IgG as a negative control. The resulting RNA samples were purified and subjected to Illumina paired-end sequencing.

为解析高毒力肺炎克雷伯菌（Hypervirulent Klebsiella pneumoniae, HvKP）中Hfq介导的RNA调控网络，本研究采用本课题组近期建立的LiRIP-seq技术，该技术可实现活细菌体内RNA-RNA互作组的全局图谱分析。将pBAD-t4rnl1质粒转化至肺炎克雷伯菌中，可表达T4 RNA连接酶1以在活体内介导RNA的邻近连接，随后通过免疫共沉淀（co-immunoprecipitation, coIP）富集结合Hfq的RNA转录本（单片段读段，singleton reads）以及连接形成的RNA嵌合片段（嵌合片段包括小RNA（small RNA, sRNA）-信使RNA（messenger RNA, mRNA）、小RNA-小RNA等类型）。
实验整体设计：在LB培养基中生长至早期稳定期（OD600值为3.0）的细菌细胞内，设置3次生物学重复开展LiRIP-seq实验；免疫共沉淀步骤采用抗FLAG单克隆抗体，以小鼠免疫球蛋白G（mouse IgG）作为阴性对照。获取的RNA样品经纯化后，进行Illumina双端测序（Illumina paired-end sequencing）。