|
| Status |
Public on Dec 10, 2018 |
| Title |
Ecoli_MG1655(Δpnp)_T12_rep3 |
| Sample type |
RNA |
| |
|
| Source name |
Ecoli_MG1655(Δpnp)_T12
|
| Organism |
Escherichia coli str. K-12 substr. MG1655 |
| Characteristics |
genotype/variation: {delta}pnp
sampling time point: 12 minutes after rifampicin addition
repetition: repetition 3
|
| Treatment protocol |
Three hours after the stationary phase of growth was reached, samples were collected for transcriptomic analysis; this time point was also the reference time point (T0) for the half-life determination procedure. Subsequently, rifampicin (500 µg.mL-1) was added to inhibit the initiation of transcription, and cells were harvested at three different time points after this addition. Cultures were performed in triplicate. Samples were taken at either at 0’, 2’, 8’ and 15’, or 0’, 5’, 10’ and 18’ or 0’, 3’, 12’ and 30’ min after rifampicin addition. Samples were immediately frozen in liquid nitrogen upon collection.
|
| Growth protocol |
The laboratory strain E. coli K12 MG1655 and two mutant strains E. coli MG1655(Δrnr) and E. coli MG1655(Δpnp) were grown in baffled flasks in LB medium at 37 °C and 180 rpm. Initial pH was set to 7. All cultures were inoculated at OD 0.1 from overnight pre-cultures performed in similar conditions. Biomass was estimated from absorbance at 600 nm. Cultures were performed in triplicate.
|
| 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. Total RNA extraction profiles were checked to be similar for all the three tested strains.
|
| 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 |
the cells were in stationary phase, the sampling was performed 12 minutes after rifampicin addition. It is the third of three biological replicates used in this experiment, each from separate cultures.
SAMPLE 34
|
| 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. Only a normalization between arrays according to an invariant probeset intensities was performed. For each strain, a set of probes in the background for which the ranks were roughly invariant across all twelve arrays was selected. The median value of the invariant probe set intensities in each condition was used as a scaling factor for normalization between the three strains. 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.
After normalization, in each array, transcript-specific intensity was computed as the median value of the 16 targeting probe intensities.
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| |
|
| Submission date |
Jul 03, 2018 |
| Last update date |
Dec 10, 2018 |
| 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 |
GPL14649 |
| Series (2) |
| GSE116576 |
Exoribonucleases coordinate degradation and expression of mRNAs in Escherichia coli at the stationary phase [stabilome] |
| GSE116652 |
Exoribonucleases coordinate degradation and expression of mRNAs in Escherichia coli at the stationary phase |
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