|
| Status |
Public on May 12, 2020 |
| Title |
Ecoli_P2_T0.5_kinetic2 |
| Sample type |
RNA |
| |
|
| Source name |
E. coli in P2 phase, Sampling 0.5 minute after rifampicin addition in kinetic 2
|
| Organism |
Escherichia coli str. K-12 substr. MG1655 |
| Characteristics |
sampling time, repetition: Sampling 0.5 minute after rifampicin addition in kinetic 2
|
| Treatment protocol |
At glucose exhaustion, during acetate consumption and during carbon starvation, samples were collected for the reference time point (T0) for the half-life determination procedure. Subsequently, rifampicin (500 µg.mL-1) was added to inhibit transcription initiation, and cells were harvested at different time points after this addition. Cultures were performed in independent biological triplicates. Samples were immediately frozen in liquid nitrogen upon collection.
|
| Growth protocol |
The laboratory strain E. coli K12 MG1655 was grown in bioreactors in M9 glucose (3g/l) at 37 °C. The pH was set to 7. All cultures were inoculated at OD 0.2 from overnight pre-cultures performed in similar conditions. Biomass was estimated from absorbance at 600 nm. Each culture was repeated three times to provide independent biological kinetics.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
After thawing and centrifugation steps, total RNA was extracted with TRIZOL® Reagent (Ambion) according to the manufacturer’s instructions. DNA contamination was eliminated with Turbo DNase kit (Ambion). Total RNA concentration and integrity were measured using a Nanodrop® spectrophotometer and Agilent BioAnalyzer, respectively.
|
| Label |
Cy3
|
| Label protocol |
According to manufacturer instructions, 1 µg of cDNA was labeled using one color DNA labeling kit
|
| |
|
| Hybridization protocol |
According to manufacturer instructions, 2 µg of labeled cDNA were hybridized onto E. coli K-12 gene expression arrays (Nimblegen, Roche) during 17 h at 42 °C.
|
| Scan protocol |
Arrays were later washed and scanned using MS200 Microarray Scanner (Nimblegen, Roche). Pictures were analyzed with DEVA 1.2.1 software.
|
| Description |
Cells were in P2 phase (glucose exhaustion) the sampling was performed 0.5 min after rifampicin addition. It is the second kinetics out of 3 for this phase
|
| Data processing |
Raw probe intensities ( .pair files three replicates for each strain before and after transcription arrest by rifampicin addition) were processed and analyzed with R computing environment using the affy and limma package of Bioconductor. Twelve arrays (three reference T0 samples, and nine time points after addition of rifampicin) were used for P2 and P4 and eleven arrays (three reference T0 samples, and eight time points after addition of rifampicin) were used for P3. Only a normalization between arrays according to an invariant probeset intensities was performed. For each growth condition, a set of probes in the background for which the ranks were roughly invariant across all arrays was selected. The median value of the invariant probe set intensities was used as a scaling factor for normalization between the growth conditions. After normalization, the intensity of a transcript was calculated by a RMA-summarization procedure [Irizarry et al, 2003, Biostatistics 4(2): 249-264] within each condition.
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| |
|
| Submission date |
Jan 27, 2020 |
| Last update date |
May 12, 2020 |
| Contact name |
Laurence Girbal |
| E-mail(s) |
girbal@insa-toulouse.fr
|
| Phone |
33 5 61 55 97 24
|
| Organization name |
TBI
|
| Street address |
135 avenue de rangueil
|
| City |
Toulouse |
| ZIP/Postal code |
31077 |
| Country |
France |
| |
|
| Platform ID |
GPL25406 |
| Series (1) |
| GSE144315 |
Genome-wide stabilization of mRNA during E. coli growth from "feast to famine" (stabilome) |
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