GSM9130603: WT vehicle 3; Staphylococcus aureus subsp. aureus USA300_FPR3757; RNA-Seq
To extract RNA from frozen S. aureus cell pellets were washed with TE buffer (pH 8.0) and re-suspended in a Tris-HCl mixture with 1 mg/mL lysostaphin (pH 7.5) to lyse for 1 hour at 37°C. RNA was extracted using 2 mL of TRI Reagent (Sigma) and 500 μL of BCP (Sigma). After mixing, the aqueous layer was added to an equal volume of ice-cold isopropanol, and the samples were then incubated overnight at -20C. Samples were pelleted at 4C for 20 minutes at 11 000 × g, resuspended in 500 μL of ice-cold 75% ethanol and pelleted again. Pellets were dried and resuspended in 86 μL of nuclease-free ddH2O before treatment with Invitrogen Turbo DNase for 1 h RNA samples were processed by the Microbial Genome Sequencing Center in Pittsburgh, PA. Ribosomal RNA (rRNA) was depleted (Qiagen FastSelect5S/16S/23S) and cDNA libraries were generated (Qiagen Total Stranded RNA). cDNA libraries were sequenced using NextSeq 550 using a 75bp x 8bp x 8bp setup