Transcriptomics

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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


ABSTRACT: To monitor the global changes in gene expression of Staphylococcus aureus USA300 TCH1516 under exposure to the garlic compound Allicin by RNA-seq, cultivation was performed in shaking flasks in Luria Bertani (LB) medium in triplicate at 37C until cells have reached an optical density at 540nm 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 M Allicin stress. S. aureus cells of 3 replicate experiments were harvested before and 30 min after exposure to 300 M 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, Bblingen, 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, Bblingen, 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 MiSeq system (San Diego, CA, USA) using 75 bp read length.

INSTRUMENT(S): Illumina MiSeq

ORGANISM(S): Staphylococcus aureus subsp. aureus USA300  

SUBMITTER: Loi Vu Van   Anika Winkler   Haike Antelmann   Tobias Busche  

PROVIDER: E-MTAB-5667 | ArrayExpress | 2019-04-01

SECONDARY ACCESSION(S): ERP022653

REPOSITORIES: ArrayExpress, ENA

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