Next Generation Sequencing Facilitates Quantitative Analysis of control of Pseudomonas aeruginosa PAS81 and LVFX-treated P. aeruginosa PAS81 Transcriptomes

Purpose: Next-generation sequencing (NGS) has revolutionized systems-based analysis of cellular pathways. The goals of this study are to compare transcriptome profiling of control of P. aeruginosa PAS81 (RNA-seq) to transcriptome profiling of LVFX-treated P. aeruginosa PAS81 and to evaluate protocols for optimal high-throughput data analysis. Methods:LB medium (50 mL) was inoculated with exponential growth phase P. aeruginosa PAS81 at a concentration of 1.0E8 CFU/mL. Levofloxacin was then added at a concentration of either 0 (control) or 0.125 μg/mL, respectively, in triplicate. All six experiment groups were incubated in a water bath shaker at 37 ºC with a shaking rate of 180 rpm for 5 h. Cells were then sampled and centrifuged from the three control groups and three LVFX treatment groups, respectively. The cell precipitates were separately snap-frozen at -80ºC. Total RNA was isolated from cells using Trizol (Life Technologies, USA) according to the manufacturer’s protocol. Results: The transcriptome sequencing data of control and LVFX-treated groups were compared and analyzed in the form of the following: control vs LVFX. The details of the differentially expressed genes among the two groups showed that the amount of differential expressed genes between control group and LVFX-treated group was high (total 5581, up-regulated 241, down-regulated 16). The mRNA profiles of P. aeruginosa PAS81 (control) and LVFX-treated P. aeruginosa PAS81 were generated by deep sequencing, in triplicate, using Illumina Higseq 2000.