Illumina HiSeq 1500 paired end sequencing; RNAseq of Staphylococcus aureus wildtype and ppGpp0 mutant reveals rolen of alarmon (p)ppGpp confering tolerance to oxidative stress and antibiotics during the stationary phase in Staphylococcus aureus
RNAseq of Staphylococcus aureus wildtype and ppGpp0 mutant reveals rolen of alarmon (p)ppGpp confering tolerance to oxidative stress and antibiotics during the stationary phase in Staphylococcus aureus
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 0.05 (log phase) and OD500 of 1.2 (stationary phase). S. aureus cells were harvested before by fast centrifugation. For RNA isolation, S. aureus USA300JE2 and the ppGpp0 mutant were cultivated in RPMI medium in 3 biological replicates and harvested at an OD500 of 0.05 (log phase) and OD500 of 1.2 (stationary phase) as described in previous studies (12) - Fritsch VN, Loi VV, Busche T, Sommer A, Tedin K, Nurnberg DJ, Kalinowski J, Bernhardt J, Fulde M, Antelmann H. 2019. The MarR-Type Repressor MhqR Confers Quinone and Antimicrobial Resistance in Staphylococcus aureus. Antioxid Redox Signal 31:1235-1252.  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.
Experimental Factor: genotype
ppGpp0 mutant (PLoS Pathog 8:e1003016.)
Experimental Factor: growth condition
OD 1.2 stationary phase