GSM8497428: Set 1 WT F; Staphylococcus aureus; RNA-Seq
Bacterial pellets were incubated in lysis buffer (50 μM Tris-EDTA pH 7.5, 8 U ml−1mutanolysin (Sigma-Aldrich), 0.018 mg ml−1lysostaphin (Sigma-Aldrich), 0.05 mg ml−1lysozyme (Sigma-Aldrich)) at 37 °C for 45 min and TRK lysis buffer (Omega Bio-tek) was added. After 10 min at room temperature, 70% ethanol was added, and samples were transferred to E.Z.N.A. RNA isolation columns (Omega Bio-tek). RNA was isolated following the manufacturer's instructions and treated with DNase using the DNA-free DNA removal kit (Invitrogen). The RNA was precipitated with 0.1 volume 3 M sodium acetate (Thermo Fisher, S209) and 3 volumes of 100% ethanol, recovered by centrifugation, and washed with ice cold 70% ethanol. A ribosomal RNA-depleted cDNA library was prepared according to the manufacturer's instructions using the Universal Prokaryotic RNA-Seq Prokaryotic AnyDeplete kit (NuGEN, 0363-32) and sequenced with Illumina HiSeq.