Next Generation Sequencing Facilitates Quantitative Analysis of control of Pseudomonas aeruginosa PAO1 and farnesol-treated P. aeruginosa PAO1 Transcriptomes. 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.

研究目的：下一代测序（Next-generation sequencing, NGS）已彻底革新了细胞通路的系统性分析。本研究旨在对比铜绿假单胞菌PAO1（Pseudomonas aeruginosa PAO1）对照组的转录组测序（RNA-seq）分析结果与法尼醇（farnesol）处理组的转录组谱，并优化适用于最优高通量数据分析的实验方案。
实验方法：将浓度为10^8 CFU/mL的指数生长期铜绿假单胞菌PAO1接种至50 mL LB培养基（LB medium）中，随后分别添加浓度为0（对照组）或0.56 mg/mL的法尼醇，每组设置3次生物学重复。将全部6个实验组置于37℃水浴摇床中，以180 rpm的转速振荡培养5小时。随后分别从3个对照组与3个法尼醇处理组中取样并离心收集细胞，将细胞沉淀分别置于-80℃快速冷冻。采用Trizol试剂（Life Technologies公司，美国）按照制造商说明书提取细胞总RNA。
实验结果：本研究的RNA-seq结果显示，经法尼醇处理后，铜绿假单胞菌PAO1的差异表达基因不足100个。我们发现，在所有检测到的5554个基因中，约1.7%（共96个）在法尼醇处理后呈现出2倍以上的差异表达。
研究结论：整体实验设计：本研究采用Illumina Hiseq 2000平台进行深度测序，设置3次生物学重复，分别获取了铜绿假单胞菌PAO1对照组（ck）与法尼醇处理组（sample1）的mRNA表达谱。