GSM1646591: S. aureus USA300 (JE2) WT, fragmented transcripts; Staphylococcus aureus; RNA-Seq
Bacterial pellets were resuspended in a buffer containing 10 mM Tris pH7.6, 100 mM EDTA, 10% glucose and lysed with a FastPrep machine (MP Biochemicals; spee 6, 45 s, 4 °C) using Lysing Matrix B and by adding 0.5 ml acid phenol.  After centrifugation (10 min, 14,000 rpm, 4 °C) the aqueous phase was collected. 1 ml TRIzol was added, mixed and incubated at RT for 5 min. The solution was extracted twice by addition of 100 µL (in the second step 200 µL) chloroform and incubation at room temperatur for several minutes, followed by centrifugation. RNA was precipitated from the aqueous solution by addition of 500 µL 2-propanol and a 15 min incubation at room temperature.    RNA was pelleted by centrifugation for 15 min at 14,000 rpm, 4 °C. Pellets were washed with 75% ethanol, air dried and resuspended in DEPC-treated water. The cDNA libraries were constructed as previously described (Dugar et al. 2013) by vertis Biotechnology AG, Germany.