title: RNA-Seq of Pseudomonas aeruginosa PAO1 wild type: exponential phase
design: Ten milliliters of each culture was centrifuged at 7,000 rpm and 4C for 20 min. Five hundred seventy microliters of TE buffer (10 mM Tris-HCl, 1 mM EDTA [pH 8.0]) and 30 l of lysozyme (Sigma) from a 20 mg/ml stock, for a final concentration of 1 mg/ml, were added to each sample. Afterwards, the samples were mixed by vortexing for 10 s and were incubated at room temperature for at least 10 min with regular vortexing. A volume of 2,100 l of buffer RLT (Qiagen) was added, and samples were sonicated twice for 5 to 10 s (constant frequency, 0.45 Hz). Then, 1,410 l of ethanol was added and the protocol continued using the RNeasy minikit (Qiagen), according to the manufacturer's instructions. In order to eliminate any residual DNA, a DNase I (Qiagen) treatment was carried out, according to the manufacturer's instructions. A second treatment was performed using Turbo DNA-free (Ambion). RNA-sequencing (RNA-seq) was performed at the Next Generation Sequencing Service of the Centre for Research in Agricultural Genomics by using paired end format lectures (2x75bp) in an Ion PGM Sequencer