Monitoring global differential gene expression in context of protein thiol-oxidation and protein S-mycothiolation in Mycobacterium smegmatis str. MC2 155 under hypochlorite stress.

Mycobacterium smegmatis mc2 155 wild type strain was grown in triplicate in modified Hartmans-de Bont minimal medium (HdB) at 37°C and vigorous agitation. 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. M. smegmatis mc2 155 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. 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, Boblingen, 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 HiSeq 1500 and MiSeq system (San Diego, CA, USA) using 75 bp read length.