GSE139071
Transcriptome Comparison of staphylococcus aureus phoU Homologies: Genes Deletion strain and the Parent Strain in stationary phase (12 h)
Purpose: The goals of this study are to compare the wild type and phoU1 or phoU2 gene knock out transcriptome profiling (RNA-seq)  to analysis  gene expression change Methods: Samples was prepared according to the Illumina RNA Sequencing Sample Preparation Guide. In brief, three biological replicates for each of the S. aureus and derivative strains were treated with RNase-free DNase I (Takara) to remove the genomic DNA. BioAnalyzer 2100 system used to evaluate the RNA quality. Samples were treated with RiboZero rRNA removal kit (gram-positive organisms) to remove ribosomal RNA. Fragmented RNA was reverse transcribed using random primers. The cDNA library included the fragment size (200–300 bp) were prepared by the mRNA-Seq Sample Prep kit and verified on a BioAnalyzer 2100 system. Then, the fragment size is amplified by Illumina cBot and sequenced by Illumina HiSeq 2500. Quality control involves discards of rRNA reads, sequencing adapters, short-fragment and other low-quality reads. The remaining reads were multi-mapped to the genome of S. aureus USA500 2395 at the NCBI website with Bowtie2 software. BED Tools software was used to count the transcript expression levels. Per Kilobase of Gene Per Million Mapped Reads (RPKM) reported RNA-seq gene expression values. Integrated Genomics Viewer was used to visualize the date. DEGseq software was used to quantify differential expression of different transcripts. Significant differences in expression ratios were defined at least a 2.0-fold or 0.5-fold change in transcript level. Results:Transcriptome analysis revealed that 573 or 285 genes were differentially expressed by at least 2.0-fold in the ?phoU1 or ?phoU2 mutant's vs the wild type. Genes involved in carbon and pyruvate metabolism were changed, and virulence genes and virulence regulatory genes were downregulated Conclusions:  In conclusion, both PhoU1 and PhoU2 in S. aureus regulate virulence and persister via the metabolism. Overall design: RNA profiles of  ?phoU1 or ?phoU2 mutant and the wild type grown for 12h were generated by deep sequencing, in triplicate, using Illumina HiSeq 2500.