Enterococcus Grad-seq and KhpB RIP-seq

We investigated the RNA-protein interactome of Enterococcus faecalis V583 and Enterococcus faecium Aus0004 by native gradient fractionation of complexes coupled to RNA-sequencing. Whole bacterial cell lysates were analysed by size and density in a glycerol gradient. At native conditions, RNA-protein complexes stay intact and sediment as a whole. Sedimentation profiles of individual RNAs appear correlated in case of interaction in a complex. The profile of KhpB caught our attention and we determined its RNA interactome by immunoprecipitation that suggests a role at the post-transcriptional level, binding notably several tRNAs, sRNAs, and 3'UTRs. Overall design: Cellular lysate was analysed by gradient fractionation into 20 fractions plus the Pellet (P) for E. faecalis and faecium. Subsequently, RNA samples were prepared and sequenced to obtain sedimentation profiles of RNAs. For the RNA-immunoprecipitation of KhpB, we raised two antibodies (Ab1, Ab2) against E. faecalis KhpB, precipitated KhpB from E. faecalis lysate, and sequenced RNA interaction partners, the pre-immune-serum (ctrl1,ctrl2) was used as a negative control.