NextSeq 500 sequencing; mRNA transcriptional profiles in Streptococcus thermophilus wild-type strain LMD-9 and 3 mutants deleted for the pptAB, eep and amiCDE genes.
mRNA transcriptional profiles in Streptococcus thermophilus wild-type strain LMD-9 and 3 mutants deleted for the pptAB, eep and amiCDE genes.
The four Streptococcus thermophilus strains were grown in chemically defined medium (PMID: 11851809) at 42°C, and samples were taken from the cultures at the end of the exponential growth phase (OD600 = 1). Cells were harvested via fast centrifugation (at 13,400 g for 15 sec); the supernatants were removed; and the bacterial pellets were frozen in liquid nitrogen and stored at -80°C. Cells were lysed in phenol-chloroform 5:1 (v/v) with a FastPrep® FT120 (Thermo Savant, USA). Two cycles of cell disruption were performed, both times at 6.5 m/sec for 45 sec. Cellular debris was pelleted by centrifugation (16,000 × g, 10 min, 4°C) and the RNA-containing aqueous supernatants were collected. Total RNA extraction was then performed using the Direct-Zol™ RNA MiniPrep kit (ZymoResearch, USA) according to the manufacturer instructions. Contaminant genomic DNA was removed with the DNA-free™ DNA Removal kit (Invitrogen, USA).  The total transcript libraries were created using a Stranded mRNA Library Prep Kit (Illumina). We followed the manufacturer's instructions, except in the initial steps (i.e., up until RNA fragmentation), where polyA purification was replaced by rRNA depletion using a Ribo-Zero Kit (Illumina). Quality control was carried out on the libraries using a Bioanalyzer HS DNA Kit (Agilent) and a dsDNA HS Assay Kit (Thermo Fisher Scientific, USA).
Experimental Factor: genotype
delta comR delta amiCDE