title: Illumina MiSeq paired end sequencing; The global  effect of the garlic compound Allicin on the changes in the transcriptome in Bacillus subtilis and Staphylococcus aureus USA300 analysed by RNA-seq of coding RNA
design: The global  effect of the garlic compound Allicin on the changes in the transcriptome in Bacillus subtilis and Staphylococcus aureus USA300 analysed by RNA-seq of coding RNA
library_construction_protocol: To monitor the global changes in gene expression of Staphylococcus aureus USA300 TCH1516 under Allicin stress by RNA-seq, cultivation was performed in Luria Bertani (LB) medium in triplicate at 37°C until cells have reached an optical density at 540 nm of 2.0. Cells were harvested by centrifugation, washed with Belitsky minimal medium (BMM) and adapted to BMM for one hour before exposure to 300 µM Allicin stress. S. aureus cells of 3 replicate experiments were harvested before and 30 min after exposure to 300 µM Allicin S. aureus cells were harvested before and 30 min after exposure to 300 µM Allicin 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.
attributes.tag: Experimental Factor: stimulus
attributes.value: none