Transcriptome Comparison of staphylococcus aureus phoU Homologies: Genes Deletion strain and the Parent Strain in stationary phase (12 h). 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 ΔphoU1 or ΔphoU2 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 ΔphoU1 or ΔphoU2 mutant and the wild type grown for 12h were generated by deep sequencing, in triplicate, using Illumina HiSeq 2500.

研究目的：本研究旨在对比野生型与phoU1、phoU2基因敲除菌株的转录组测序（RNA-seq）数据，以分析基因表达变化情况。
实验方法：样品制备参照Illumina RNA测序样品制备指南完成。具体步骤如下：每株金黄色葡萄球菌（S. aureus）及其衍生菌株均设置3次生物学重复，采用无RNase活性的DNase I（Takara）处理以去除基因组DNA；使用BioAnalyzer 2100系统评估RNA质量；通过RiboZero核糖体RNA去除试剂盒（革兰氏阳性菌专用）去除核糖体RNA；将片段化后的RNA采用随机引物进行反转录；利用mRNA-Seq样品制备试剂盒制备片段大小为200–300 bp的cDNA文库，并在BioAnalyzer 2100系统上完成文库验证；随后通过Illumina cBot扩增文库片段，再采用Illumina HiSeq 2500进行测序。
质量控制环节包括剔除rRNA读数、测序接头、短片段读数及其他低质量读数。将剩余读数通过Bowtie2软件比对至NCBI数据库中公布的S. aureus USA500 2395基因组序列；使用BEDTools软件统计转录本的表达水平；以每百万比对读数每千碱基基因长度的读取数（Reads Per Kilobase of Gene Per Million Mapped Reads，RPKM）作为RNA-seq基因表达量数值；使用整合基因组浏览器（Integrated Genomics Viewer）可视化测序数据；采用DEGseq软件定量分析不同转录本的差异表达情况。将转录水平至少2.0倍或0.5倍的变化定义为表达差异显著。
实验结果：转录组分析表明，相较于野生型菌株，ΔphoU1或ΔphoU2突变株中分别有573个、285个基因发生至少2.0倍的差异表达。参与碳代谢与丙酮酸代谢的基因表达发生改变，毒力基因及毒力调控基因均呈现下调趋势。
研究结论：综上，金黄色葡萄球菌中的PhoU1与PhoU2均可通过代谢途径调控菌株毒力与持留菌形成。
整体实验设计：对培养12h的ΔphoU1、ΔphoU2突变株及野生型菌株进行转录组深度测序，每组设置3次生物学重复，测序平台为Illumina HiSeq 2500。