biostudies-arrayexpressRahul JagadeesanEscherichia coli str. K-12 substr. MG1655https://www.ebi.ac.uk/biostudies/studies/E-MTAB-15747Bacteria experience concurrent stresses in nature; here we tested how temperature shifts modulate antibiotic responses in Escherichia coli. We profiled RNA-seq of Escherichia coli K-12 MG1655 grown in LB (37 °C, 250 rpm) and exposed to cold shock (15 °C), heat shock (42 °C), antibiotics (norfloxacin 0.10 μg/mL, ofloxacin 0.5 μg/mL, rifampicin 2.5 μg/mL), and combinations (temperature + antibiotic). Cultures were sampled at mid-exponential phase; growth was monitored and RNA was harvested 160 min after stress. The Conditions include: Standard, cold shock (CS), heat shock (HS), NOR, OFL, RIF, NOR+CS, OFL+CS, RIF+CS, NOR+HS, OFL+HS, RIF+HS (3 biological replicates each). Libraries were prepared from total RNA using bacterial rRNA depletion and sequenced as paired-end reads.biostudies-arrayexpressNucleic Acid Extraction - Total RNA was extracted using RNeasy kit (Qiagen).Growth Protocol - Growth rates were measured by spectrophotometry (BioTek Synergy HTX Multi-Mode Microplate Reader). From a glycerol stock (-80 °C), cells were streaked on LB agar plates (2%) and incubated at 37 °C, overnight. Next, a single colony was picked, inoculated in LB medium and incubated at 30°C overnight with shaking. Overnight cultures were diluted into fresh medium to an optical density of 600 nm (O.D.600) of 0.03 and incubated for growth by shaking at 250 rpm at 37°C. We used Luria-Bertani (LB) medium with 10 g peptone, 10 g NaCl, and 5 g yeast extract in 1000 ml distilled water and the antibiotics kanamycin and chloramphenicol from Sigma Aldrich, U.S.A. Cells were grown to mid-exponential phase and then subjected to stress conditions: norfloxacin (0.10 μg/mL), rifampicin (2.5 μg/mL), ofloxacin (0.5 μg/mL), cold shock (15 °C), heat shock (42 °C), and the combinations norfloxacin + cold shock, ofloxacin + cold shock, rifampicin + cold shock, norfloxacin + heat shock, ofloxacin + heat shock, and rifampicin + heat shock. The growth is monitored until 160 min and the samples were then collected for RNAseq.Library Construction - RNA-seq libraries were constructed according to the Illumina's protocol.Sample Treatment - Cells from 3 independent biological replicates of E. coli MG1655 were treated with RNA protect bacteria reagent (Qiagen, Germany), to prevent degradation of RNA.Sample Collection - The samples from 3 independent biological repeats were collected 160 minutes after inducing the stress.Sequencing - Ribosomal RNA depletion was performed using Ribo-Zero Gold Kits (Bacteria probe) (Illumina, San Diego, CA, USA), while the RNA sequencing library was prepared using the NEBNext Ultra RNA Library Prep Kit. The sequencing libraries were multiplexed and clustered on one lane of a Flowcell, which was loaded on an Illumina HiSeq 4000 instrument (after the thermal shift) or on an Illumina NovaSeq 6000 instrument (after adding the antibiotic). In both instruments, the samples were sequenced using a single-index 2x150 Paired-End (PE) configuration. Image analysis and base calling were conducted by the HiSeq Control Software (Illumina HiSeq) and by the NovaSeq Control Software v1.7 (Illumina NovaSeq). The raw sequence data (.bcl files) were converted into “fastq” files and de-multiplexed using Illumina bsl2fastq v.2.20. One mismatch was allowed in the index sequence identification.OrganizationMINSEQE ScoreAssays and DataProcessed DataMAGE-TAB FilesData Transformation - featureCounts from Rsubread R package (v.2.8.2) was used to calculate unique gene hit counts. Genes with less than 5 counts in more than 3 samples, and genes whose mean counts are less than 10, were removed. Gene hit counts were used for subsequent differential expression analysis, using DESeq2 R package (v.1.34.0) to compare gene expression between samples and calculate log2 of fold changes (LFC) using Wald tests (function nbinomWaldTest). P-values were adjusted for multiple hypotheses testing (Benjamini–Hochberg, BH procedure)Sequence Alignment - RNA sequencing reads were trimmed to remove possible adapter sequences and nucleotides with poor quality with Trimmomatic v.0.39. Trimmed reads were mapped to the reference genome (E. coli MG1655, NC_000913.3) using using the Bowtie2 aligner v.2.3.5.1MetabolomicsUnknownTranscriptomicsGenomicsProteomicsIllumina NovaSeq 6000RNA-seq of total RNAEscherichia coli str. K-12 substr. MG1655Vatsala ChauhanSuchintak DashRahul JagadeesanAndre RibeirofalseRNA-seq of E. coli MG1655 under temperature shocks, antibiotics, and combined treatmentsBacteria experience concurrent stresses in nature; here we tested how temperature shifts modulate antibiotic responses in Escherichia coli. We profiled RNA-seq of Escherichia coli K-12 MG1655 grown in LB (37 °C, 250 rpm) and exposed to cold shock (15 °C), heat shock (42 °C), antibiotics (norfloxacin 0.10 μg/mL, ofloxacin 0.5 μg/mL, rifampicin 2.5 μg/mL), and combinations (temperature + antibiotic). Cultures were sampled at mid-exponential phase; growth was monitored and RNA was harvested 160 min after stress. The Conditions include: Standard, cold shock (CS), heat shock (HS), NOR, OFL, RIF, NOR+CS, OFL+CS, RIF+CS, NOR+HS, OFL+HS, RIF+HS (3 biological replicates each). Libraries were prepared from total RNA using bacterial rRNA depletion and sequenced as paired-end reads.2026-09-22T00:00:00Z2026-09-22T08:52:34.648Z2025-10-16T10:44:44.199ZE-MTAB-15747ERP182282EFO_0002944EFO_0004170EFO_0009653EFO_0003789EFO_0004917EFO_0005518EFO_0003816EFO_0004184EFO_0003969