title: RNA-seq analysis of MG1655 Escherichia coli (Wild-type, and mutants lacking the glucose metabolism regulator TmaR) experiencing short- & long-term N starvation and following treatment with rifampicin during long-term N starvation in Gutnick minimal media
design: RNA-seq analysis of MG1655 Escherichia coli (Wild-type, and mutants lacking the glucose metabolism regulator TmaR) experiencing short- & long-term N starvation and following treatment with rifampicin during long-term N starvation in Gutnick minimal media
library_construction_protocol: 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. 