Staphylococcus aureus ATCC 29213 cells (OD600∼0.4) were treated with 10 × MIC IBG for 4 h. Then bacteria was collected and preserved with RNA protect (Qiagen, United States). Total RNA of each sample was extracted using TRIzol Reagent (Invitrogen)/RNeasy Mini Kit (Qiagen). Control samples were collected from an antibiotic-free culture. RNA sequencing was conducted by High-Throughput Sequencing Facility at the GENWIZ lnc (Jiangsu, China). Raw sequence data were underwent quality control using Cutadapt (V1.9.1) and FastQC (V0.10.1). Clean data were aligned with the reference genome of S. aureus NCTC 8325 (NCBI accession number NC_007795.1) via software Bowtie2 (v2.1.0) and the gene expression level were estimated by HTSeq (v0.6.1p1). The calculation of fragment per kilobase of exon per million fragments mapped (FPKM) for all genes were performed through Cufflinks (v2.2.1) software. Differential expression genes (DEGs) were screened out by using the DESeq Bioconductor package and defined as those with a change in expression of >twofold and a corresponding false discovery rate (FDR) of <0.05. The gene ontology terms and functional pathways were annotated via Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genome (KEGG), respectively. Cell-PLoc 2.0 was used to analyze the subcellular localization of DEGs (Chou and Shen, 2010).

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