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| Status |
Public on Dec 10, 2014 |
| Title |
RutC30_2 T0vsT1 |
| Sample type |
RNA |
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| Channel 1 |
| Source name |
RutC30_T0h
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| Organism |
Trichoderma reesei |
| Characteristics |
strain: RutC30
time: T0h
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| Growth protocol |
Frozen spores were used to inoculate a Fernbach flask containing 250 ml of the culture medium described previously (Gilbert HJ (2010) The biochemistry and structural biology of plant cell wall deconstruction. Plant physiology 153: 444–455). Cultivation was carried out at 30°C with stirring at 150 rpm. After 72 h, the medium containing mycelia was used as an inoculum for bioreactor culture.Cellulase was produced in a 4-liter bioreactor with a two-step cultivation procedure. Strains were first grown at 28°C in 2 liters of a medium containing 30 g·liter−1 of glucose as a carbon source and pH regulated at 4.8 with 5.5 M NH3. The airflow was adjusted at 0.5 volume per volume per min (VVM), and initial stirring was set at 400 rpm. This parameter was gradually increased to maintain partial O2 pressure (pO2) above 40% oxygen saturation. When the initial glucose was close to depletion (<20% of initial glucose content), the fed-batch phase was initiated. During this second step, a 250-g·liter−1carbon source solution (lactose) was injected at a rate of 0.98 g·h−1. Samples were collected periodically to determine the biomass, carbon, and protein concentrations
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| 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.
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| Label |
Cy3
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| 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.
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| Channel 2 |
| Source name |
RutC30_T1h
|
| Organism |
Trichoderma reesei |
| Characteristics |
strain: RutC30
time: T1h
|
| Growth protocol |
Frozen spores were used to inoculate a Fernbach flask containing 250 ml of the culture medium described previously (Gilbert HJ (2010) The biochemistry and structural biology of plant cell wall deconstruction. Plant physiology 153: 444–455). Cultivation was carried out at 30°C with stirring at 150 rpm. After 72 h, the medium containing mycelia was used as an inoculum for bioreactor culture.Cellulase was produced in a 4-liter bioreactor with a two-step cultivation procedure. Strains were first grown at 28°C in 2 liters of a medium containing 30 g·liter−1 of glucose as a carbon source and pH regulated at 4.8 with 5.5 M NH3. The airflow was adjusted at 0.5 volume per volume per min (VVM), and initial stirring was set at 400 rpm. This parameter was gradually increased to maintain partial O2 pressure (pO2) above 40% oxygen saturation. When the initial glucose was close to depletion (<20% of initial glucose content), the fed-batch phase was initiated. During this second step, a 250-g·liter−1carbon source solution (lactose) was injected at a rate of 0.98 g·h−1. Samples were collected periodically to determine the biomass, carbon, and protein concentrations
|
| 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.
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| 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.
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| Scan protocol |
Microarrays of the GPL9076 plateform were scanned using the Agilent DNA microarray Scanner model G2567AA at 5 microns resolution using the extended dynamic range (XDR) feature. Microarrays of the GPL19095 plateform were scanned using Genepix 4000B (Molecular Devices, Sunnyvale, CA, USA) and the resulting image files analyzed by GenePix Pro 6.1 software (Axon).
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| Description |
RutC30 T0h vs T1h - Replicate 2
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| Data processing |
For Microarray GPL9076, spot and background intensities were extracted with the Feature Extraction (FE, v9.5.3) software (Agilent) using the GE2-v4_95_Feb07 default protocol. Subsequent flagging was done according to the GenePix Pro software (Molecular Devices Syunnyvale, CA, USA) nomenclature, including four levels of flags (good (100), bad (-100), not found (-50), moderate (0)). FE software normalized data (Local background was subtracted and LOWESS normalization). For Microarray GPL19095, 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 |
Aug 28, 2014 |
| Last update date |
Dec 10, 2014 |
| Contact name |
Frederique Bidard |
| E-mail(s) |
frederique.bidard-michelot@ifpen.fr
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| Organization name |
IFPEN
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| Department |
Biotechnology
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| Street address |
1-4 avenue de Bois Préau
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| City |
Rueil Malmaison |
| ZIP/Postal code |
92852 |
| Country |
France |
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| Platform ID |
GPL9076 |
| Series (1) |
| GSE60908 |
Kinetic transcriptome study reveals an essentially intact cellulase induction system in a cellulase hyper-producer Trichoderma reesei strain |
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| Supplementary file |
Size |
Download |
File type/resource |
| GSM1493449_lame38_rep_T0_vs_T1_1h_950750.gpr.gz |
23.6 Mb |
(ftp)(http) |
GPR |
| Processed data are available on Series record |
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