GSE131228
Next Generation Sequencing Facilitates Quantitative Analysis of Wild Type and non-antibiotic pharmaceuticals stressed E. coli K-12 LE392, P. putida KT2440, and RP4 plasmid Transcriptomes [experiment 2]
The goals of this study are to use Next-generation sequencing (NGS) to detect bacterial mRNA profiles of E. coli K-12 LE392, P. putida KT2440 and IncPa RP4 plasmid, and their mRNA response under the exposure of CuO NPs and Cu2+. The concentrations were 5 µmol/L for CuO NPs and Cu2+. The group without dosing CuO NPs or Cu2+ was the control group. Each concentration was conducted in triplicate. By comparing the mRNA profiles of experimental groups and control group, the effects of these CuO NPs and Cu2+ on transcriptional levels can be revealed. Illumina HiSeq 2500 was applied. The NGS QC toolkit (version 2.3.3) was used to treat the raw sequence reads to trim the 3'-end residual adaptors and primers, and the ambiguous characters in the reads were removed. Then, the sequence reads consisting of at least 85% bases were progressively trimmed at the 3'-ends until a quality value = 20 were kept. Downstream analyses were performed using the generated clean reads of no shorter than 75 bp. The clean reads of each sample were aligned to the E. coli reference genome (NC_000913), P.putida reference genome (NC_002947), and IncPa plasmid reference genome (NC_00) using SeqAlto (version 0.5). Cufflinks (version 2.2.1) was used to calculate the strand-specific coverage for each gene, and to analyze the differential expression in triplicate bacterial cell cultures. The statistical analyses and visualization were conducted using CummeRbund package in R (http://compbio.mit.edu/cummeRbund/). Gene expression was calculated as fragments per kilobase of a gene per million mapped reads (FPKM, a normalized value generated from the frequency of detection and the length of a given gene. Overall design: Bacterial mRNA profiles of wild-type E. coli K-12 LE392, P. putida KT2440, and IncPa RP4 plasmid in the pressure of CuO NPs or Cu2+ at 5 µmol/L  for 2 hours, bacteria and plasmid without CuO NPs or Cu2+ are the control group, in triplicate, using Illumina HiSeq 2500