Trizol solution

Real-time quantitative PCR was performed to further analyze the transcription levels of hla, agrA, Panton-Valentine leucocidin (PVL), phenol-soluble modulin alpha (psmα) and seven serine protease A (sspA) in the presence of various concentrations of 2,3-DHKV. A culture of S. aureus with an initial OD₆₀₀ of 0.3 was added with 2,3-DHKV (0–16 μg/ml) or DMSO, and was shaken and incubated until the OD₆₀₀ was 2.5. After centrifugation at 12,000 rpm for 1 min, the supernatant was discarded. The bacteria were subsequently collected and total RNA from the co-cultures was extracted with TRIzol solution (Tiangen, Beijing, China). Subsequently, 1 μg of purified RNA was used to generate cDNA using the HiScript^® II 1st Strand cDNA Synthesis Kit (#R211, Vazyme, Nanjing, China) and stored at-20°C. Next, qPCR was performed with the following cycle parameters: 95°C for 30 s, 40 cycles of 95°C for 5 s and 60°C for 30 s and 72°C for 30 s on an ABI 7900HT Real-time PCR system. The 16sRNA was used as an internal reference gene. The 2^−ΔΔCT method was used to calculate the relative transcript levels of the gene hla, agrA, PVL and psmα. Table 1 lists the primers for these genes.

ADSCs were harvested, and total RNA was extracted using TRIzol solution (TIANGEN, China). The concentration of RNA was measured using Nanodrop 2000 and evaluated using Agilent 2100 (LabChip GX). mRNA was purified using mRNA Capture Beads (Vazyme, China), reverse transcribed into cDNA using the Script RT reagent kit (Vazyme, China), and then synthesized into dsDNA. The dsDNA library was constructed using VAHTS Universal V6 RNA-seq Library Prep Kit (Vazyme) and purified using VIHTS DNA Clean Beads (Vazyme) according to the manufacturer’s instructions. The qualified dsDNA samples were sequenced using Illumina Nova Seq 6000 platform (San Diego, USA). Raw sequencing reads were mapped to GRCh38 assembly of the human genome using Tophat2, version 2.0.10 [33 (link)]. Fragments per kilobase of exon model per million mapped reads (FPKM) were calculated using Stringtie and normalized with TMM [34 (link)–36 (link)]. Differentially expressed genes (DEGs) were analyzed using the DESeq 2 package, version 1.10.1 [37 (link)]. Genes with log-fold change > 1.5 and false discovery rate (FDR) < 0.05 were considered significant transcriptomic changes. This sequencing dataset is available at GEO: GSE222263.