RNA-seq analysis of BW25113 Escherichia coli (Wild-type, and mutants lacking the glucose metabolism regulator TmaR or the RNA Chaperone Hfq) experiencing long-term nitrogen starvation in Gutnick minimal media
Three biological replicates of each strain were taken and mixed with a phenol:ethanol (1:19) solution at a ratio of 9:1 (culture:solution) before harvesting the bacteria immediately by centrifugation. Bacteria were grown in Gutnick minimal medium according to (Figueira et al., Sci Rep, 2015), where the sole source of nitrogen is NH4Cl; overnight cultures were grown in medium containing 10 mM NH4Cl and N-limiting growth curves were carried out in medium containing 3 mM NH4Cl. Cultures were grown as in (Figueira et al., Sci Rep, 2015) and sampled at N-24, where bacteria were subjected to 24 hours of N starvation. Pellets were resuspended in RNA extraction solution (18 mM EDTA, 0.025% SDS, 1% 2-mercaptoethanol, 95% formamide) and lysed at 95°C for 10min. Cell debris was pelleted by centrifugated. RNA was precipitated by addition of 1/10 volume of 3M sodium acetate (pH 5.2) and 3 volumes of 100% ethanol, followed by incubated at -80°C for at least an hour. RNA was washed with 75% ethanol and resuspended in water. Samples were then poly (A)-tailed followed by treatment with Tobacco Acid Pyrophosphatase (TAP, Epicentre). First strand cDNA synthesis was performed using oligo (dT)-adapter primer and M-MLV reverse transcriptase. Resulting cDNAs were PCR amplified. The primers used for PCR amplification were designed for TruSeq sequencing according to the manufacturer's guidelines (Illumina).