|
| Status |
Public on Feb 13, 2010 |
| Title |
coli_coldstress_timepoint3_rep1 |
| Sample type |
RNA |
| |
|
| Source name |
Escherichia coli_stress experiment
|
| Organism |
Escherichia coli str. K-12 substr. MG1655 |
| Characteristics |
treatment group: cold stress
treatment time: T3: 10 min after stress
|
| Treatment protocol |
Oxidative stress 200 ug/ml of 30% pre-warmed hydrogen peroxide (Fluka) was added to 150 ml constantly stirred (330 rpm) cultures kept in 1000 ml flasks. Cold stress Cultures were transferred from 37oC into an ice cold water bath in order to lower the temperature, while stirring, to 16oC in less than 2 min, heat stress Cultures were transferred from 37oC to a 50oC water bath. While stirring, the temperature of each culture was raised to 45oC in less than 2 min. The constantly stirring (330rpm) cultures were then transferred to a 45oC water bath to maintain this temperature glucose lactose shift Carbon source concentrations of 0.15% lactose and 0.05% glucose were used (150 ml culture in 1000 ml flasks, 330 rpm stirring).
|
| Growth protocol |
All cultures were grown aerobically in a thermostatically controlled 37oC culture room. Cultures (150ml culture volume) were stirred by magnetic stirrers at 330 rpm (Thermo Scientific Variomag Multipoint 6in) 1000ml Erlenmeyer flask. Starting cultures were inoculated from a single colony and grown overnight. Each experimental culture was then inoculated from such an overnight culture at a dilution of 1:20 into 150 ml fresh MOPS minimal medium in a 1000 ml flask. The minimal medium used for all experiments was a modification of MOPS (morpholinopropane sulfonate) minimal medium obtained from Teknova, CA (product number M2006) which contains 86 mM NaCl, 9.5 mM NH4Cl, 5 mM K2HPO4 and 0.2% glucose.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted using the Qiagen RNeasy Mini Kit (74104) and mechanical cell disruption with glass beads but without enzymatic lysis. This was carried out in the Qiagen RNeasy kit lysis RLT buffer with beta-mercaptethanol, according to the manufacturer’s recommendations. Mechanical cell disruption was completed through shaking for five min using a Retsch mill (Retsch MM200) on maximum speed. RNA was subsequently cleaned on-column with an additional DNase treatment (Qiagen 79254). The quality of extracted RNA was determined with an Agilent 2100 bioanalyzer having used an Agilent RNA 6000 Nano Kit according to the manufacturer’s recommendations.
|
| Label |
Cy3
|
| Label protocol |
The labelling and hybridisation of cDNA microarrays was performed by the out-sourced service provider imaGene GmbH (Berlin, Germany) and was based on Agilent technology.
|
| |
|
| Hybridization protocol |
The labelling and hybridisation of cDNA microarrays was performed by the out-sourced service provider imaGene GmbH (Berlin, Germany) and was based on Agilent technology.
|
| Scan protocol |
The scanning of cDNA microarrays was performed by the out-sourced service provider imaGene GmbH (Berlin, Germany) and was based on Agilent technology.
|
| Description |
US45103052_251684410019_S01_GE1-v5_95_Feb07_1_3_cold1_3c.txt
|
| Data processing |
For further analyses the processed signal intensities of all coding regions and RNA genes were extracted and used. Variance stabilization and normalization of the extracted intensities were performed with the vsn packages of the R software environment (R Development Core Team, 2007) and back-transformed to normal intensity scale. For each probeset, e.g. all probes representing for example, a single coding gene, outliers were removed by boxplot statistics and the outlier-removed probe intensities were averaged in a robust way by computing the Tukey biweight.
|
| |
|
| Submission date |
Feb 12, 2010 |
| Last update date |
May 20, 2011 |
| Contact name |
Szymon Jozefczuk |
| E-mail(s) |
jozefczuk@mpimp-golm.mpg.de
|
| Organization name |
Max Planck Institute
|
| Street address |
Am Mühlenberg 1
|
| City |
Potsdam |
| ZIP/Postal code |
14476 |
| Country |
Germany |
| |
|
| Platform ID |
GPL10041 |
| Series (1) |
| GSE20305 |
Time resolved response of E. coli to 5 different stress conditions |
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