GSM3406790: WT RNAseq S1; Streptococcus salivarius; RNA-Seq
S. salivarius WT, ∆scuR, ∆sarF, scuR-ST++ or sarF-ST++ strains were pre-cultured overnight in CDMG at 37°C. They were resuspended in 50 ml of fresh pre-warmed CDMG to a final OD600 of 0.05 and grown for approximately 2 h 30 min (OD600 = 0.3) at 37°C. We then supplemented the medium with either DMSO (negative control) or 1 µM sComS and incubated for 30 min at 37°C. Cells were harvested by centrifugation (10 min; 4,050 × g), the supernatant were discarded and the cell pellets were frozen with liquid nitrogen. Finally, RNA was extracted using the RiboPure bacteria kit (Ambion-Life Technologies) and the protocol provided by the manufacturer, with protocol changes to cell lysis and RNA precipitation. For lysis, cells were resuspended in RNAwiz buffer (Ambion-Life Technologies) supplemented with Zirconia beads and shaked for 40 sec (4 times) in a fastPrep homogenizer device (MP biomedicals). For RNA precipitation, a 1.25-ethanol volume (instead of 0.5) was added to partially purified RNAs. Total RNA was checked for quality on a RNA Nano chip (Agilent Technologies) and concentration was measured using Ribogreen assay (Life Technologies). rRNA depletion was performed on 2 µg total RNA with the Ribo-Zero rRNA removal kit for Gram-positive bacteria (Illumina) according to manufacturer's instructions. Total stranded mRNA libraries were prepped with the NEBNext Ultra Directional RNA Library Prep kit for Illumina (New England Biolabs). Library PCR was executed for 15 cycles. Quality of the libraries was evaluated with the use of a High sensitivity DNA chip (Agilent Technologies) and concentrations were determined through qPCR according to Illumina protocol. Both Libraries were equimolar pooled for sequencing on a NextSeq 500 high-throughput run with 76bp single reads. 2.3 pM of the library pool was loaded on the flowcell with a Phix spike-in of 5%.