PAO1 treated with Iberin RNA sequencing project
Gene expression analysis (two biological replicates) was conducted by Illumina RNA sequencing (RNA-Seq tochnology). The rRNA-depleted RNA was fragmented to 200-300 bp fragments and first and second strand cDNA were synthesized, followed by end repair and adapter ligation. After a 12 cycles PCR enrichment, the quality of the libraries were assessed by Bioanalyzer. The libraries were sequenced using the Illumina HiSeq2000 platform with a paired-end protocol and read lengths of 100 nt.     The sequence reads were assembled and analyzed in RNA-Seq and expression analysis application of CLC genomics Workbench 6.0 (CLC Bio, Aarhus, Denmark). The PA01 genome (http://www.ncbi.nlm.nih.gov/nuccore/110645304) was utilized as the reference genome for the assembly. The following criteria were used to filter the unique sequence reads: minimum length fraction of 0.9, minimum similarity fraction of 0.8, and maximum number of two mismatches. Data were normalized by calculating the reads per kilobase per million mapped reads (total reads/mapped reads in millions x gene length in kb) for each gene and annotated with PseudoCAP (http://www.geneontology.org/GO.current.annotations.shtml).   T-test was performed on transformed data (0.5 was added to each number to deal with zero counts) to identify the genes with significant changes in expression (P≤0.05, Fold change ≥2.0 or ≤ -2.0) between the control and the Iberin treated samples.