|
| Status |
Public on Dec 10, 2018 |
| Title |
Ecoli_MG1655_T0_rep2_re-analysis |
| Sample type |
RNA |
| |
|
| Source name |
Ecoli_MG1655_T0
|
| Organism |
Escherichia coli str. K-12 substr. MG1655 |
| Characteristics |
genotype/variation: wild type
sampling time point: before rifampicin addition
repetition: repetition 2
|
| 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.
|
| Growth protocol |
The laboratory strain E. coli K12 MG1655 and two mutant strains E. coli MG1655_rnr) and E. coli MG1655_pnp) 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 |
SAMPLE 2
the cells were in stationary phase, the sampling was performed just before rifampicin addition. It is the second of three biological replicates used in this experiment, each from separate cultures.
|
| Data processing |
Raw probe intensities ( .pair files three replicates for each strain) were processed and analyzed with R computing environment using the affy and limma package of Bioconductor. Raw probe intensities were processed and analyzed with the R computing environment using the affy and limma packages of Bioconductor. Raw data were submitted to a RMA-based background correction [Irizarry et al, 2003, Biostatistics 4(2): 249-264]. After background correction, intra-replicate quantile normalization was performed for each strain. A set of probes in the background for which the ranks were roughly invariant across all nine 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. Intensity values were multiplied by the total RNA extraction yield (in µg total RNA per mg of dry cell weight) to provide the mRNA concentration value in arbitrary units per mg of dry cell weight.
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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) |
| GSE116577 |
Exoribonucleases coordinate degradation and expression of mRNAs in Escherichia coli at the stationary phase [transcriptome] |
| GSE116652 |
Exoribonucleases coordinate degradation and expression of mRNAs in Escherichia coli at the stationary phase |
|
| Relations |
| Reanalysis of |
GSM3243096 |
