Illumina HiSeq 1500 paired end sequencing; Monitoring global differential gene expression in context of protein thiol-oxidation and protein S-mycothiolation in Mycobacterium smegmatis str. MC2 155 under hypochlorite stress.
Monitoring global differential gene expression in context of protein thiol-oxidation and protein S-mycothiolation in Mycobacterium smegmatis str. MC2 155 under hypochlorite stress.
For stress experiments Mycobacterium smegmatis mc2155 wild type strain was grown in modified Hartmans-de Bont minimal medium (HdB)  (finally per liter: EDTA [0.01 g], MgCl2x6H2O [0.1 g], CaCl2x2H2O [1 mg], NaMoO4x2H2O [0.2 mg], CoCl2x6H2O [0.4 mg] , MnCl2x2H2O [1 mg] , ZnSO4x7H2O [2 mg] , FeSO4x7H2O [5 mg] , CuSO4x5H2O [0.2 mg]; complemented with 15 mM (NH4)2SO4, 0.05% Tween80, 27.4 mM glycerol, 50 mM MOPS; 8.90 mM K2HPO4 and 7.08 mM NaH2PO4; pH 7.0) at 37°C and vigorous agitation. 15% sodium hypochlorite (NaOCl) was purchased from Sigma Aldrich. After harvesting control samples cultures were exposed to 1 mM NaOCl  at an optical density at 500 nm (OD500) of 0.4 – 0.45 in the early log phase. Briefly, M. smegmatis mc2155 cells of 3 replicate experiments were harvested before and 30 min after exposure to 1 mM NaOCl and disrupted in 3 mM EDTA/ 200 mM NaCl lysis buffer with a Precellys24 Ribolyzer. RNA isolation was performed using 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, Boblingen, Germany). Samples contaminated with DNA were treated with DNase (Qiagen), cleaned as described above and rechecked by Xpose and Agilent Bioanalyzer.  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, Boblingen, Germany).
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