Illumina HiSeq 1500 paired end sequencing; RNA-seq of coding RNA of Streptococcus pneumoniae D39 wild type strain before and after 30 min of exposure to sub-lethal doses of 800 µM HOCl stress during the log phase of microaerophilic grown bacteria
RNA-seq of coding RNA of Streptococcus pneumoniae D39 wild type strain before and after 30 min of exposure to sub-lethal doses of 800 µM HOCl stress during the log phase of microaerophilic grown bacteria
The S. pneumoniae D39 wild type strain was cultivated in RPMI medium and treated with various thiol-reactive compounds at an OD600 of 0.4 for 30 min. Bacteria were harvested in ice-cold killing buffer (50 mM Tris pH 7.5, 5 mM MgCl2, 20 mM NaN3), centrifuged at 4,750 rpm for 10 min at 4°C. The pellets were immediately frozen in liquid nitrogen and stored at -80°C. RNA isolation was performed using the acidic phenol-chloroform extraction protocol as described previously (1). After DNase-I treatment (Zymo Research, Germany), the RNA quality was checked by Trinean Xpose (Gentbrugge, Belgium) and the Agilent RNA Nano 6,000 kit using an Agilent 2,100 Bioanalyzer (Agilent Technologies, Böblingen, Germany). 1. Wetzstein M, Volker U, Dedio J, Lobau S, Zuber U, Schiesswohl M, Herget C, Hecker M, Schumann W. Cloning, sequencing, and molecular analysis of the dnaK locus from Bacillus subtilis. J Bacteriol 174: 3300-10, 1992. For RNA-seq transcriptomics, Ribo-Zero rRNA Removal Kit (Bacteria) from Illumina (San Diego, CA, USA) was used to remove the rRNA. TruSeq Stranded mRNA Library Prep Kit from Illumina (San Diego, CA, United States) was applied to prepare the cDNA libraries.