biostudies-arrayexpressJosh McQuailEscherichia coli K-12https://www.ebi.ac.uk/biostudies/studies/E-MTAB-14999RNA-seq was carried out to compare the transcriptomes of wild-type BW25113 E coli with mutants lacking either the glucose metabolism regulator TmaR or the ubiquitous RNA chaperone Hfq, in bacteria that had been exposed to long-term (24hr) nitrogen starvation in Gutnick minimal media. All strains also contain the empty plasmid pBAD18, as this was initial performed alongside a larger RNA-seq study - as such the WT and Hfq mutant are previously available in E-MTAB-14054. TmaR is also required for the formation of Hfq foci, a novel sub-cellular feature that forms during long-term nitrogen starvation. The aim of this work was to use the mutant lacking TmaR to understand the functional role of the Hfq foci during long-term nitrogen starvation.biostudies-arrayexpressSample Collection - 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.Growth Protocol - 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.Nucleic Acid Extraction - 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.Sequencing - The cDNA was sequenced on the Illumina HiSeq 2000 system.Library Construction - 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).OrganizationMINSEQE ScoreAssays and DataProcessed DataMAGE-TAB FilesData Transformation - RNA-seq reads were mapped to the E. coli K-12 MG1655 (U00096.3) genome. Reads that mapped uniquely were used for further analysis. The number of reads mapping to each gene was calculated and matrix of read counts was generated. The matrix was analysed using the DESeq2 BioConductor package for differential gene expression analysis. Genes with ≤10 reads mapped to them were excluded from analysis.UnknownTranscriptomicsGenomicsProteomicsIllumina HiSeq 2000RNA-seq of total RNAEscherichia coli K-12Josh McQuailSivaramesh WigneshwerarajfalseRNA-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 mediaRNA-seq was carried out to compare the transcriptomes of wild-type BW25113 E coli with mutants lacking either the glucose metabolism regulator TmaR or the ubiquitous RNA chaperone Hfq, in bacteria that had been exposed to long-term (24hr) nitrogen starvation in Gutnick minimal media. All strains also contain the empty plasmid pBAD18, as this was initial performed alongside a larger RNA-seq study - as such the WT and Hfq mutant are previously available in E-MTAB-14054. TmaR is also required for the formation of Hfq foci, a novel sub-cellular feature that forms during long-term nitrogen starvation. The aim of this work was to use the mutant lacking TmaR to understand the functional role of the Hfq foci during long-term nitrogen starvation.2025-12-31T00:00:00Z2025-12-31T02:02:14.871Z2025-04-04T09:12:53.575ZE-MTAB-14999ERP171194EFO_0002944EFO_0004170EFO_0009653EFO_0003789EFO_0005518EFO_0003816EFO_0004184