title: GSM5047301: orfN*; Pseudomonas aeruginosa; RNA-Seq
library_construction_protocol: Ten milliliters of each culture was centrifuged at 7,000 rpm and 4°C for 20 min. Five hundred seventy microliters of TE buffer (10 mM Tris-HCl, 1 mM EDTA [pH 8.0]) and 30 μl 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 μl of buffer RLT (Qiagen) was added, and samples were sonicated twice for 5 to 10 s (constant frequency, 0.45 Hz). Then, 1,410 μl 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). RNAs obtained from three independent cultures of each strain were pooled and 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