|
| Status |
Public on May 27, 2015 |
| Title |
WT strain treated with glycolaldehyde first repetition |
| Sample type |
RNA |
| |
|
| Source name |
wild-type MG1655 + glycolaldehyde 10 mM
|
| Organism |
Escherichia coli |
| Characteristics |
strain: MG1655
genotype/variation: WT
agent: glycolaldehyde
|
| Treatment protocol |
WT cells were treated with 10 mM Glycolaldehyde for 30 min whenever the culture OD600nm reached ~1.
|
| Growth protocol |
M9 mineral medium containing (D)-xylose as the only carbon source (100 ml in 500 ml shake flasks) was inoculated from exponentially growing wild-type cells or synthetic cells (cultivated on xylose M9 medium) to adjust an OD of ~0.1. Cultures were incubated at 37°C under shaking until OD reached ~1. Then they were split into two 50 ml aliquots and further cultivated in 250 ml shake flasks in the presence or absence of 10 mM glycolaldehyde. After 30 min of incubation, 1 ml of the cell suspension was withdrawn and centrifuged at 1500 x g (Eppendorf 5415D) for 5 min. The supernatant was removed and the cell pellets were directly subject to RNA extraction. Experiment were repeated three times.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The RNeasy Mini Kit (QIAGEN) was used to extract RNA. Quantity and quality of the samples were determined by NanoDrop (Thermo) and Bioanalyzer (Agilent Technologies), respectively
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled RNA was prepared from 0.5 µg RNA using the One-color Low Input Quick Amp Kit (Agilent) according to the manufacturer's instructions, followed by Agilent Nano-prep RNA purification (Agilent, Santa Clara, CA). Dye incorporation and cRNA yield were checked with the NanoDrop-2000 spectrophotometer.
|
| |
|
| Hybridization protocol |
0.6 µg of Cy3-labelled cRNA (specific activity >10.0 pmol Cy3/µg cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 25 µl containing 25x Agilent fragmentation buffer and 10x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 25 µl of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent Yeast V2 Oligo Microarrays (G4813A-016322) for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent).
|
| Scan protocol |
The slides were scanned on a Tecan scanner MS200
|
| Description |
C2_1
|
| Data processing |
Data were analyzed by Feature Extraction V.11.5.1.1. RNA
|
| |
|
| Submission date |
May 27, 2015 |
| Last update date |
May 27, 2015 |
| Contact name |
Ceren Alkim |
| E-mail(s) |
[email protected]
|
| Organization name |
LISBP-INSA
|
| Street address |
135 avenue de Rangueil
|
| City |
Toulouse |
| ZIP/Postal code |
31077 |
| Country |
France |
| |
|
| Platform ID |
GPL13360 |
| Series (1) |
| GSE69265 |
Transcriptomic analysis of glycolaldehyde-treated wild type cells and a synthetic strain |
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