E-MTAB-7218
The role of the quinone-sensing regulator MhqR in Staphylococcus aureus analysed by RNA-seq of coding RNA
Using RNAseq transcriptomics, we monitored the global changes in gene expression of Staphylococcus aureus COL and the ?mhqR mutant after exposure to 45 µM methylhydroquinone (MHQ) stress. 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 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 (San Diego, CA, USA) using 70 bp read length.
Using RNAseq transcriptomics, we monitored the global changes in gene expression of Staphylococcus aureus COL and the ?mhqR mutant after exposure to 45 µM methylhydroquinone (MHQ) stress. 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 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 (San Diego, CA, USA) using 70 bp read length.