BioSample

Protocols: Strain cultivation was performed in RPMI1640 medium (Lonza) under vigorous agitation in triplicate at 37°C 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 45 µM MHQ Staphylococcus aureus COL and the ∆mhqR mutant were cultivated in RPMI1640 medium (Lonza) under vigorous agitation in triplicate at 37°C 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 45 µM MHQ. 30 min treatment with 45 µM methylhydroquinone (MHQ) or none treatment S. aureus cells were harvested before and 30 min after exposure to 45 µM MHQ 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. RNA free of DNA with an RNA Integrity Number (RIN) > 9 and rRNA Ratio [23s / 16s] > 1.5 was used to prepare cDNA-sequencing libraries. 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.