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| Status |
Public on Mar 26, 2024 |
| Title |
Ciprofloxacin (12 ng/mL) 25 min post-treatment; BR 2 |
| Sample type |
SRA |
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| Source name |
K12 MG1655
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| Organism |
Escherichia coli str. K-12 substr. MG1655 |
| Characteristics |
strain: K12 MG1655
genotype: WT
treatment: Ciprofloxacin 12 ng/mL
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| Treatment protocol |
At OD600 = 0.2, E. coli was treated with 12 ng/mL ciprofloxacin.
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| Growth protocol |
The E. coli pre-culture was diluted to OD600 = 0.0125. E. coli was grown in cation-adjusted Mueller-Hinton broth in 1 1L Applikon bioreactors as batch culitvation. Stirring was adjusted between 200 and 600 rpm to maintain a dissolved oxygen concentration above 40%. The aeration rate was kept at 500 mL min-1. pH 7 was maintained by automatic titration of 3 M HCl and 4 M NaOH.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Transcriptomics was sampled according to the protocol provided with RNeasy Mini kit (Qiagen). The sample volume was based on OD600 at each sampling timepoint to ensure 3.35 x 10^8 cells per sample. The sample was immediately added to 2 volumes of RNA protect and vortexed. After minimum 5 min, the samples were centrifuged (10 min, max rcf). The supernatant was discarded, and the pellet was frozen in N2 (l). RNA was isolated using RNeasy Mini Kit (Qiagen), according to manufacturer's protocol 4 (Enzymatic lysis and Proteinase K digestion of bacteria) and protocol 7 (Purification of total RNA) with on-column Dnase (Qiagen) digestion. The RNA concentration in each sample was measured using NanoDrop-1000 spectrophotometer. Isolated RNA was sent to the Genomics Core Facility at NTNU for further processing and sequencing.
RNA sequencing libraries were prepared using the QIAseq FastSelect 5S/16S/23S kit (Qiagen) for rRNA removal and the QIAseq stranded RNA Lib kit (Qiagen) for library construction, according to the manufacturer's instructions. Briefly, 500 ng total RNA was used as starting material. Removal of ribosomal RNA (rRNA) was conducted by a combined heat fragmentation (89°C for 7 min) and FastSelect hybridization protocol (75-4°C ramping process) where the FastSelect reagent inhibited reverse transcription of bacterial rRNA. Next, purification was conducted using QIAseq Beads followed by a first-strand synthesis using a RNase H- Reverse Transcriptase (RT) in combination with random primers, a second-strand synthesis, end-repair, A-addition, and adapter ligation. The second-strand synthesis was performed using 5’phosphorylated random primers which enable subsequent strand-specific ligation. DNA fragments were further enriched by CleanStart library amplification (15 cycles of PCR reaction). Finally, the libraries were purified using the QIAseq Beads, quantitated by qPCR using Collibri Library Quantification Kit (Thermo Fisher Scientific), and validated using Perkin Elmer DNA 1K/12K/Hi Sensitivity Assay LabChip on a Labchip GX instrument (Perkin Elmer, USA). The size range of the DNA fragments were measured to be in the range of 270 to 570 bp and peaked around 355 bp. Prior to sequencing, libraries were normalized and pooled to 2.3 pM and subjected to clustering on three NextSeq 500 HO flowcells (Illumina, USA).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Data processing |
FASTQ files were generated using bcl2fastq2 Conversion Software v 2.20.0.422. Processing of the sequence data was done with the ProkSeq v 2.0 program using Docker, with the E. coli K-12 substr. MG1655 reference genome ASM584v2 (RefSeq: GCF_000005845.2) and corresponding reference transcriptome. ProkSeq is a docker based, full RNA-Seq pipeline for prokaryotes, which includes quality assessment, alignment, gene counting, and analysis. The count tables countFile.csv, countFileNucleotideAvgCount.csv and countFile_TPM_CPM.tsv were exported from ProkSeq and used for further analysis.
The sample counts were were analyzed in R using the quasi-likelihood test in EdgeR v 3.36.0 from Bioconductor. Poorly expressed genes were filtered out, and library size was normalized using trimmed mean of M-value (TMM). Differentially expressed genes were defined with FDR < 0.05 and presented as LFC compared to the control.
Assembly: ASM584v2 (RefSeq: GCF_000005845.2)
Supplementary files format and content: The processed data file (countFile.csv) contains the count for each sample of each identified gene.
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| Submission date |
Dec 08, 2023 |
| Last update date |
Mar 26, 2024 |
| Contact name |
Amanda Holstad Singleton |
| E-mail(s) |
amanda.singleton@ntnu.no
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| Organization name |
Norwegian University of Science and Technology
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| Department |
Department of Clinical and Molecular Medicine
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| Street address |
Erling Skjalgsons gate 1
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| City |
Trondheim |
| ZIP/Postal code |
7491 |
| Country |
Norway |
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| Platform ID |
GPL21117 |
| Series (1) |
| GSE249682 |
SOS Genes Are Rapidly Induced While Mutagenesis Is Temporally Regulated by Changes in Protein Activation and Nucleotide Pools After a Sub-lethal Dose of Ciprofloxacin in Escherichia coli |
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| Relations |
| BioSample |
SAMN38727565 |
| SRA |
SRX22828107 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
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