organism: Escherichia coli str. K-12 substr. MG1655
description: Protocols: Three biological replicates of each strain/timepoint/time-post-treatment 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 cultures were carried out in medium containing 3 mM NH4Cl. Cultures were grown as in (Figueira et al., Sci Rep, 2015) and sampled at N-3 & N-24, where bacteria were subjected to 3 & 24 hours of N starvation respectively. Further samples were taken following treatment with rifampicin, as described in treatment protocol. Note that N-24 and T=0 are the same samples. Cultures from N-24 were treated with 100ug/ml of rifampicin and, samples were taken prior to treatment (T=0), and following 15 & and 60 minutes (T=15 & T=60) of incubation. Note that N-24 and T=0 are the same samples. 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 purified with PureLink RNA Mini Kit extraction columns (Invitrogen, 12183018A) largely in accordance with the manufacturer's protocol for Total Transcriptome Isolation except with a final ethanol concentration of 66% to increase the yield of smaller RNA species. Ribo-depleted RNA samples were first fragmented using ultrasound (4 pulses of 30 s at 4°C). Then, an oligonucleotide adapter was ligated to the 3' end of the RNA molecules. First-strand cDNA synthesis was performed using M-MLV reverse transcriptase with the 3' adapter as primer. After purification, the 5' Illumina TruSeq sequencing adapter was ligated to the 3' end of the antisense cDNA. The resulting cDNA was PCR-amplified using a high-fidelity DNA polymerase and the barcoded TruSeq-libraries were pooled in approximately equimolar amounts.
attributes.tag: ENA first public
attributes.value: 2025-09-23
attributes.tag: INSDC center name
attributes.value: Department of Infectious Diseases, Imperial College London
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-15469:WT T=60_R2
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: collection date
attributes.value: not collected
attributes.tag: environmental stress
attributes.value: Nitrogen Starvation
attributes.tag: genotype
attributes.value: wild type genotype
attributes.tag: geographic location (country and/or sea)
attributes.value: not collected
attributes.tag: growth condition
attributes.value: Long-term N starvation
attributes.tag: isolate
attributes.value: not applicable
attributes.tag: sample name
attributes.value: E-MTAB-15469:WT T=60_R2
attributes.tag: scientific_name
attributes.value: Escherichia coli K-12
attributes.tag: strain
attributes.value: K-12
attributes.tag: substrain
attributes.value: MG1655