Illumina MiSeq paired end sequencing; Analysis of the Hfq regulon in Pseudomonas fluorescens SBW25.
Analysis of the Hfq regulon in Pseudomonas fluorescens SBW25.
Bacterial cultures were grown at 28°C in defined M9 medium supplemented with 0.4% pyruvate and 0.4% casamino acids. Tetracycline was used at 12.5 μg/ml. Total RNA was extracted from 50ml cultures grown in M9 medium supplemented with 0.4% pyruvate and 0.4% casamino acids. 30 ml of 60% RNAlater (in PBS) was added to each tube, and sealed tubes were vortexed and centrifuged for 10 min at 4°C. The pellets were resuspended in 1x PBS and chilled β-mercaptoethanol RT solution, and lysed by mechanical disruption. Finally, RNA was purified from the lysate by column capture using an RNeasy Mini Kit (Qiagen). Purified RNA was subjected to an additional DNase treatment (TurboTM DNase, Ambion), and RNA quantification was performed with an ND-1000 Spectrophotometer. mRNA libraries were prepared by the sequencing contractor using the TruSeq Stranded mRNA Library Prep Kit from Illumina.