Global mRNA decay in B. cereus
Global mRNA decay in B. cereus
14_D_t0
B. cereus strains ATCC 10987 and ATCC 14579 were grown to mid-exponential phase (OD = 0.60) in Luria-Bertani (LB) broth (500 ml) at 30C, 250 rpm. Rifampicin (10 ml) suspended in 5% DMSO/95% LB medium was added to a final concentration of 150 ug/ml for transcriptional arrest. Samples (20 ml) for mRNA isolation were drawn at 0, 2.5, 5, 10 and 20 minutes following Rifampicin addition, immediately suspended in an equal volume of methanol/phenol (50:1), and incubated 3-5 minutes at room temperature, before harvesting of cells by centrifugation (4000 x g, 4C, 5 min). Cell pellets were snap-frozen in liquid nitrogen and stored at -80C. For RNA isolation, cell pellets were suspended in RLT buffer (Qiagen, Germany) and lysed using 0.1 um glass beads in a Precellys 24 instrument at 5800 rpm for 30 sec x 2 (bertin Technologies, France). RNA was isolated using the RNeasy Mini kit (Qiagen, Germany) according to manufacturers protocol, including the optional on-column DNase treatment. Isolated RNA was treated with Turbo DNase (Ambion, US) and subjected to clean-up using the RNeasy Mini kit. Ribosomal RNA (rRNA) was depleted using one (B-series) or two (C and D-series) rounds of treatment with the Microbe Express kit (Ambion, US) following manufacturers instructions.