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). 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.