|
| Status |
Public on Jul 31, 2014 |
| Title |
Aspergillus nidulans grown in suberin media, for two days, biological rep. 1 |
| Sample type |
RNA |
| |
|
| Source name |
Aspergillus nidulans mycelia after growth in suberin media for two days
|
| Organism |
Aspergillus nidulans |
| Characteristics |
tissue: mycelia
|
| Treatment protocol |
After two days of incubation the exhausted glucose media were replaced by fresh media containing 0.1% w/v of suberin and kept for an additional period of two, four or six days. At each time point the mycelia were recovered and preserved at -80 °C . Cultures grown in minimal media containing glucose for two days were herein regarded as the control.
|
| Growth protocol |
Fungal cultures with a density of A. nidulans (FGSC A4) conidia of 1.00E+5 per mL (5mL six-well plates, minimal media containing 1% w/v glucose) were incubated (27 ºC, dark, 90 rpm) for the establishment of a mycelia mat against the ground of the wells.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from fungal mycelia (grounded to powder using mortar and pestle in liquid nitrogen) using the RNeasy Plant Mini Kit (QIAGEN) and further purified following a standard ethanol precipitation.
|
| Label |
biotin
|
| Label protocol |
RNA was processed for use on Affymetrix custom dual species FungiANC arrays, according to the manufacturer’s GeneChip 3’ IVT Express kit user manual. Briefly, 100 ng of total RNA containing spiked in Poly-A RNA controls was used in a reverse transcription reaction (GeneChip 3’ IVT Express Kit; Affymetrix) to generate first-strand cDNA. After second-strand synthesis, double-stranded cDNA was used in a 16h in vitro transcription (IVT) reaction to generate aRNA (GeneChip 3’ IVT Express Kit; Affymetrix). Size distribution of the aRNA and fragmented aRNA, respectively, was assessed using an Agilent 2100 Bioanalyzer with a RNA 6000 Nano Assay.
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| |
|
| Hybridization protocol |
A total of 200µl of the hybridisation mixture containing 10µg of fragmented cRNA was hybridised on arrays for 16 hours at 45 °C. Standard post hybridisation washes and double-stain protocols (FS450_0001) were used on an Affymetrix GeneChip Fluidics Station 450, in conjunction with the GeneChip Hybridisation Wash and Stain Kit (Affymetrix).
|
| Scan protocol |
Arrays were scanned on an Affymetrix GeneChip scanner 3000 7G.
|
| Description |
Two days in glucose media, plus two days in suberin media
Gene expression data from Aspergillus nidulans grown in glucose media for two days, biological rep. 1
|
| Data processing |
Subsequent analysis was carried out with DNA-Chip Analyzer (dChip) 2010 (http://www.dchip.org) applying a probeset mask file considering only the A. nidulans probes on the array representing 9674 transcripts. The arrays were normalized to a baseline array with median CEL intensity by applying an Invariant Set Normalization Method. Normalized CEL intensities of the 12 arrays were used to obtain model-based gene expression indices based on a PM (Perfect Match)-only model.
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| |
|
| Submission date |
Jan 27, 2014 |
| Last update date |
Jul 31, 2014 |
| Contact name |
Cristina Silva Pereira |
| E-mail(s) |
spereira@itqb.unl.pt
|
| Organization name |
Instituto de Tecnologia Química e Biológica (ITQB)
|
| Lab |
Applied and Environmental Mycology Laboratory (AEM)
|
| Street address |
Rua da Quinta Grande nº6
|
| City |
Lisboa |
| ZIP/Postal code |
2780-156 Oeiras |
| Country |
Portugal |
| |
|
| Platform ID |
GPL18227 |
| Series (1) |
| GSE54427 |
Suberin degradation by Aspergillus nidulans |
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