title: Illumina MiSeq paired end sequencing; RNA-seq of coding RNA reveals an oxidative stress response induced by the plant-derived naphthoquinone lapachol in Staphylococcus aureus
design: RNA-seq of coding RNA reveals an oxidative stress response induced by the plant-derived naphthoquinone lapachol in Staphylococcus aureus
library_construction_protocol: S. aureus COL grown in triplicates in RPMI medium and subjected to 300 µM lapachol for 30 min or untreated after cells have reached an optical density at 500 nm of 0.5 were harvested by fast centrifugation an freezing liquid nitrogen. S. aureus COL grown in triplicates in RPMI medium until the cells have reached an optical density at 500 nm of 0.5 and then subjected to 300 µM lapachol for 30 min or remained untreated  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.