Five mL of the C. difficile cultures were 1:1 diluted with ice cold methanol and stored overnight at 280uC. Bacterial pellets, obtained by centrifugation (20 min, 30006g, 4uC), were resuspended into 200 ml lysisbuffer (100 mM EDTA, 200 mM Tris-HCl pH 7.0, 50 mg/ml lysozyme) and incubated for 1 hr at 37uC. Tri-pure reagent (Roche) was used for the extraction of RNA according to the manufacturer's instruction with minor modifications. Briefly, 1 ml Tri-pure was added to the lysed bacterial pellets and incubated for 5 min at room temperature. Per 1 ml Tri-pure, 200 ml chloroform was added and carefully shaken by hand for 3 min, followed by an incubation of 2-5 min at room temperature. The aqueous phase was collected after centrifugation (12,0006g for 15 min at 4uC) and transferred to a fresh tube. RNA was precipitated by mixing the aqueous phase with 500 ml isopropanol, followed by an incubation of 10 min at room temperature. The precipitated RNA was collected by centrifugation (12,0006g, 10 min, 4uC) and resuspended in 100 ml DNase/ RNase free water. The RNA was re-precipitated overnight at 280uC with ammonium acetate (Fluka; 10 mM) and 3 volumes of absolute ethanol. The re-precipitated RNA was washed once with 80% ethanol and dissolved in 50 ml DNase/RNase free water. The RNA was treated twice with a TurboDNase (Ambion) according to the manufacturer's instruction followed by another Tri-pure RNA isolation. The quality and purity of the extracted RNA was assessed using a RNA nano chip on an Agilent Bioanalyzer.