GSM9289624: ΔrsbX rep 2; Listeria monocytogenes; RNA-Seq
Bacterial cultures were mixed 5:1 with Stop solution (5% phenol in ethanol) and collected by centrifugation. The pellets were resuspended in Disruption solution (10% glucose, 12.5 mM Tris-HCl pH 7.6, and 5 mM EDTA) and bacteria were disrupted with 0.1 mm glass beads using a mini bead beater (BioSpec Products). RNA was isolated by mixing the lysates with TRI Reagent Solution (ThermoFisher) and chloroform, followed by centrifugation. The aqueous samples were thereafter subjected to two additional chloroform extractions. RNA was precipitated with isopropanol, the pellets were washed with 80% ethanol and dissolved in RNase-free water. RNA was treated with DNase I (Roche) and purified on RNeasy MinElute columns (Qiagen). The cDNA libraries were prepared using TruSeq Stranded mRNA kit (Illumina) following manufacturer's protocol.