title: RNA-seq of GadY overexpression in EPEC bacteria in the presence and absence of CsrA
design: RNA-seq of GadY overexpression in EPEC bacteria in the presence and absence of CsrA
library_construction_protocol: EPEC ΔgadXYW and EPEC ΔgadXYW, ΔcsrA strains were transformed with pJV300 (NE9069 and NE9090)  or pZE12-gadY (NE9070 and NE9091) plasmids. Single colonies of the transformants 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). Cells were centrifuged at 4 °C and resuspended in 50 µl 10 mM Tris-HCl (pH 7.5) containing 1 mM EDTA. Lysosyme was added to 0.5 mg/ml, and samples were subjected to three freeze-thaw cycles.  RNA was extracted using TRI Reagent (Sigma) as described (Faigenbaum-Romm et al., 2020) Total expression libraries according to (Shishkin et al., 2015) were constructed with several modifications (Melamed et al., 2018)