Escherichia coli K-12
Protocols: 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).
strain
BW25113
isolate
not applicable
environmental stress
Long-term nitrogen starvation
organism
Escherichia coli K-12
organism
Escherichia coli K-12
growth condition
Gutnick minimal medium with 3 millimolar NH4Cl
collection date
not collected
scientific_name
Escherichia coli K-12
organism part
whole organism
genotype
WT+pBAD18
geographic location (country and/or sea)
not collected