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| Status |
Public on Jun 07, 2022 |
| Title |
RNAP_0.25X_3 |
| Sample type |
SRA |
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| Source name |
E.coli
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| Organism |
Escherichia coli str. K-12 substr. MG1655 |
| Characteristics |
intracellular rnap concentration: RNAP_0.25X
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| Treatment protocol |
Cells from 3 independent biological replicates of MG1655 in each modified LB medium richness were treated with RNA protect bacteria reagent (Qiagen, Germany), to prevent degradation of RNA.
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| Growth protocol |
From glycerol stocks (at - 80 ºC), cells were streaked on lysogeny broth (LB) agar plates with appropriate antibiotics and kept at 37 ºC overnight. Next, a single colony was picked and inoculated into fresh LB medium and kept at 30 ºC overnight, with appropriate antibiotics and aeration at 250 rpm. From overnight cultures (ONC), cells were diluted to 1:1000 in tailored LB media (see below), with antibiotics, and incubated at 37 ºC with aeration and allowed to grow until reaching the mid-log phase (with optical density at 600 nm (OD600) ≈ 0.4). Using this protocol, to attain cells with different intracellular RNAP concentration, starting from LB, we used tailored various media differing in richness, denoted as ‘LB1.0x', ‘LB0.75x, ‘LB0.5x’, ‘LB0.25x’ specifically. Their composition for 100 ml (pH of 7.0) are, respectively: (LB1.0x) 1 g tryptone, 0.5 g yeast extract and 1 g NaCl; (LB0.75x) 0.75 g tryptone, 0.375 g yeast extract and 1 g NaCl; (LB0.5x) 0.5 g tryptone, 0.25 g yeast extract and 1 g NaCl; and (LB0.25x) 0.25 g tryptone, 0.125 g yeast extract and 1 g NaCl.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using RNeasy kit (Qiagen).
RNA-seq libraries were constructed according to the Illumina's protocol.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina MiSeq |
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| Data processing |
RNA sequencing reads were trimmed to remove possible adapter sequences and nucleotides with poor quality with Trimmomatic v.0.36
Trimmed reads were then mapped to the reference genome, E. coli MG1655 (NC_000913.3), using the Bowtie2 v.2.3.5.1 aligner, which outputs BAM files
featureCounts from the Rsubread R package (v.1.34.7) was used to calculate unique gene hit counts
Gene hit counts was then used for the subsequent differential expression analysis. For this, we used the DESeq2 R package (v.1.24.0) to compare gene expression between groups of samples and calculate p-values and log2 of fold changes (LFC) using Wald tests (function nbinomWaldTest). Genes with less than 5 counts in more than 3 samples, and genes whose mean counts are less than 10 were removed from further analysis.
P-values were adjusted for multiple hypotheses testing (Benjamini–Hochberg, BH procedure)
Genome_build: Escherichia coli str. K-12 substr. MG1655 (NC_000913.3)
Supplementary_files_format_and_content: tab-delimited .csv file containing raw counts
Supplementary_files_format_and_content: tab-delimited .csv file containing normalized raw counts
Supplementary_files_format_and_content: tab-delimited .csv file containing results from differential expression analysis
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| Submission date |
Jun 16, 2021 |
| Last update date |
Jun 07, 2022 |
| Contact name |
Andre Sanches Ribeiro |
| E-mail(s) |
[email protected]
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| Organization name |
Tampere University
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| Department |
Faculty of Medicine and Health Technology
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| Lab |
Laboratory of Biosystem Dynamics
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| Street address |
Arvo Ylpön katu 34
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| City |
Tampere |
| State/province |
Pirkanmaa |
| ZIP/Postal code |
33520 |
| Country |
Finland |
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| Platform ID |
GPL17439 |
| Series (2) |
| GSE178278 |
The transcription factor network of E. coli steers global responses to shifts in RNAP concentration |
| GSE178281 |
The transcription factor network of E. coli steers global responses to shifts in RNAP concentration |
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| Relations |
| BioSample |
SAMN19718736 |
| SRA |
SRX11156758 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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