Next Generation Sequencing Facilitates Quantitative Analysis of control of Pseudomonas aeruginosa PAO1 and farnesol-treated P. aeruginosa PAO1 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 PAO1 (RNA-seq) to transcriptome profiling of farnesol-treated P. aeruginosa PAO1 and to evaluate protocols for optimal high-throughput data analysis. Methods:LB medium (50 mL) was inoculated with exponential growth phase P. aeruginosa PAO1 at a concentration of 108 CFU/mL. Farnesol was then added at a concentration of either 0 (control) or 0.56 mg/mL, 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 farnesol 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: Our RNA-seq results showed that less than 100 genes of P. aeruginosa PAO1 were differentially expressed following farnesol treatment. We found that about 1.7% of all detected genes (96 of 5554 genes) were more than two-fold differentially expressed following farnesol treatment. Conclusions: Overall design: The mRNA profiles of P. aeruginosa PAO1 (ck) and farnesol-treated P. aeruginosa PAO1 (sample1) were generated by deep sequencing, in triplicate, using Illumina Hiseq 2000.