RNA-seq of GadY overexpression in EPEC bacteria in the presence and absence of CsrA
In order to capture the regulation achieved directly by GadY independently of its effect on gadX and potentially gadW, we constructed an EPEC strain with deletions of gadX, gadY, and gadW (?gadXYW). Subsequently, to investigate the role of CsrA in GadY-mediated regulation, we constructed a ?csrA,?gadXYW double mutant. We then introduced a plasmid expressing GadY under an inducible promoter (pGadY) or an empty control vector (pJV300) into these strains. Next, the four strains (i.e. ?gadXYW/pJV300 (NE9069); ?gadXYW/pGadY; ?csrA,?gadXYW/pJV300; and ?csrA, ?gadXYW/pGadY were grown overnight in LB at 37°C without shaking. Cultures were diluted 100-fold in fresh DMEM, re-grown without shaking at 37°C at 5% CO2 to exponential phase (OD600 = 0.2) and induced with IPTG (1mM, 2h). RNA was extracted and sequencing libraries were condtructed.