|
| Status |
Public on Jun 01, 2011 |
| Title |
QM9414 strain under carbon catabolite repression versus under carbon catabolite derepression dye switch |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Total RNA extracted from T reesei QM9414 strain under carbon catabolite derepression.
|
| Organism |
Trichoderma reesei |
| Characteristics |
strain: QM9141
condition: Carbon Catabolite Derepression
|
| Treatment protocol |
Chemostat cultures with D-glucose as the limiting carbon source at 0.025 gram/hour which have previously been shown to lead to CCR derepression (Karaffa et al. 2006)
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Fungal mycelia were harvested by filtration, washed with distilled cold water, frozen and ground under liquid nitrogen. For extraction of total RNA purification kits (SV Total RNA Isolation System, Promega) were used according to the manufacturer’s protocol.
|
| Label |
Cy3
|
| Label protocol |
1 µg RNA are retro transcribed using Superscript III retro transcriptase. The cDNA are purified using QiaQuick column (Quiagen) and incubated 1 hour at room temperature in presence of 0,05M NaBicarbonate and NHS-ester Cy.The coupling is stoped by addition of hydroxylamine to a final concentration of 0,6M. Labeled cDNA are purified using QiaQuick column.
|
| |
|
| Channel 2 |
| Source name |
Total RNA extracted from T reesei QM9414 strain under carbon catabolite repression.
|
| Organism |
Trichoderma reesei |
| Characteristics |
strain: QM9141
condition: Carbon Catabolite Repression
|
| Treatment protocol |
Chemostat cultures with D-glucose as the limiting carbon source at 0.07 gram/hour which have previously been shown to lead to CCR repression (Karaffa et al. 2006)
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Fungal mycelia were harvested by filtration, washed with distilled cold water, frozen and ground under liquid nitrogen. For extraction of total RNA purification kits (SV Total RNA Isolation System, Promega) were used according to the manufacturer’s protocol.
|
| Label |
Cy5
|
| Label protocol |
1 µg RNA are retro transcribed using Superscript III retro transcriptase. The cDNA are purified using QiaQuick column (Quiagen) and incubated 1 hour at room temperature in presence of 0,05M NaBicarbonate and NHS-ester Cy.The coupling is stoped by addition of hydroxylamine to a final concentration of 0,6M. Labeled cDNA are purified using QiaQuick column.
|
| |
|
| |
| Hybridization protocol |
1.5µg of labelled cDNA is mixed with one volume of 2X GE buffer (Agilent) and 10% of blocking agent. The hybridization sample volume is dispense on the selected microarray and incubated overnight at 65°C in a hybridization oven (Agilent). The array slides are washed 1 minute in "wash 1 buffer" at room temperature and one minute in "wash 2 buffer" at 37°C. The array slides are then air dryed and ready for scaning.
|
| Scan protocol |
The hybridized microarrays were scanned using Genepix 4000B (Molecular Devices, Sunnyvale, CA, USA) and the resulting image files analyzed by GenePix Pro 6.1 software (Axon).
|
| Data processing |
For each GenePix output file, two filters were applied, one to clear out flagged spots and another one to discard saturating spots where the median foreground intensity was greater than 60,000 in one of the two channels. The resulting median foreground intensities were normalized, without background signal subtraction, using a global Lowess correction followed by a print-tip median normalization step (Lemoine et al., 2006).
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| |
|
| Submission date |
Mar 25, 2010 |
| Last update date |
Jun 01, 2011 |
| Contact name |
Stéphane LE CROM |
| Organization name |
École normale supérieure
|
| Department |
Biology Institute - IBENS
|
| Lab |
Genomic platform
|
| Street address |
46 rue d'Ulm
|
| City |
Paris |
| ZIP/Postal code |
75013 |
| Country |
France |
| |
|
| Platform ID |
GPL9076 |
| Series (1) |
| GSE21072 |
The CRE1 carbon catabolite repressor of the fungus Trichoderma reesei: a master regulator of carbon assimilation |
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