GSM2057849: Pasteurella multocida VP161 hfq mutant replicate 1; Pasteurella multocida; RNA-Seq
The P. multocida strains AL2521 (VP161 hfq mutant) and VP161 (wild-type) were each grown in BHI broth to mid-exponential growth phase (OD600 = 0.6, approximately 1.0 x 10^9 CFU/ml). To each 25 ml culture, 2.5 ml of ice-cold killing buffer (containing 0.05 M Tris-HCl [pH 7.5], 15 mg/ml sodium azide and 0.6 mg/ml chloramphenicol) was added and the mixed sample cooled on ice before centrifugation at 9,000 x g for 10 min (4°C). Pelleted cells were then resuspended in 1 ml RNAlater Stabilisation reagent (Qiagen) and incubated for 5 min at room temperature before centrifugation at 5,000 x g for 10 min. Total RNA was purified using the RNeasy minikit (Qiagen) with DNase treatment according to the manufacturer’s instructions. RNA samples were quantified and analysed for DNA contamination using the Qubit kit (Invitrogen) and visualised by gel electrophoresis. The construction of the cDNA library from purified RNA samples was performed using the Truseq RNA Preparation Kit (Illumina). Ribosomal RNA (rRNA) was removed from the samples using the Ribozero kit (Epicentre).
GEO Accession
GSM2057849