RNA-seq of coding RNA of Staphylococcus aureus treated with the antimicrobial coating AgXX reveals strong thiol-specific stress response and protein S-bacillithiolation in Staphylococcus aureus

Using RNAseq transcriptomics, we monitored the global changes in gene expression of Staphylococcus aureus USA300 TCH1516 after exposure to the antimicrobial coating AgXX373 (Largetec GmbH, Berlin). Strain cultivation was performed in RPMI1640 medium (Lonza) under vigorous agitation at 37°C in triplicate until cells have reached an optical density at 500?nm of 0.5. S. aureus cells were harvested before and 30 min after exposure to 4.5 µg/ml AgXX373 and disrupted in 3 mM EDTA/ 200 mM NaCl lysis buffer with a Precellys24 Ribolyzer. RNA isolation was performed using the phenol-chloroform-isoamylalcohol approach. After precipitation with 3 M sodium acetate and isopropanol, the total RNA was washed with cold ethanol and the pellet was solved in sterile water. Initially RNA quality was checked by Trinean Xpose (Gentbrugge,Belgium) and Agilent RNA Nano 6000 kit on Agilent 2100 Bioanalyzer (Agilent Technologies, Böblingen, Germany). Samples contaminated with DNA were treated with DNase (Qiagen), cleaned as described above and rechecked by Xpose and Agilent Bioanalyzer. Finally RNA was free of DNA with an RNA Integrity Number (RIN) > 9 and rRNA Ratio [23s / 16s] > 1.5. Ribo-Zero rRNA Removal Kit (Bacteria) from Illumina (San Diego, CA, USA) was used to remove the ribosomal RNA molecules from the isolated total RNA. Removal of rRNA was checked by Agilent RNA Pico 6000 kit on Agilent 2100 Bioanalyzer (Agilent Technologies, Böblingen, Germany). RNA was free of detectable rRNA. TruSeq Stranded mRNA Library Prep Kit from Illumina (San Diego, CA, USA) was used to prepare cDNA libraries. The resulting cDNAs were sequenced paired end on an Illumina HiSeq 1500 system using 70 bp read length.