|
| Status |
Public on Dec 31, 2014 |
| Title |
CA 2 |
| Sample type |
RNA |
| |
|
| Source name |
whole fungal mycelium, wild type strain N402
|
| Organism |
Aspergillus niger |
| Characteristics |
genotype: wild type
treatment: caspofungin
time point: germlings
|
| Treatment protocol |
See growth protocol
|
| Growth protocol |
For treatment with FK506 and aureobasidin A, freshly harvested conidia (5x10^8) from strain N402 were used to inoculate 0.5 liters of FM. Cultivations were performed in BioFlo/CelliGen 115 bioreactors (New Brunswick Scientific). In brief, 250 rpm were used as agitation speed and aeration was performed via the headspace until the dissolved oxygen tension dropped to 40%. Thereafter, aeration was switched to sparger aeration. Temperature and pH were set to 30 °C and pH 3, respectively, and controlled on-line using the program NBS Biocommand. After 5 h of cultivation, AbaA (dissolved in 5 ml ethanol) or FK506 (dissolved in 5 ml DMSO) was added. 5 ml of ethanol or 5 ml of DMSO were added in the control runs. After an additional hour of cultivation, 500 ml of the culture broth were quickly harvested via filtration, and mycelial samples were immediately frozen using liquid nitrogen. In addition, samples were taken for microscopic analysis (see below) and calculation of the BI value. Cultivations of the strain lacking the rlmA gene as well as caspofungin and fempropimorph treatments of the wild-type and the corresponding control were performed in BioFlo3000 bioreactors (New Brunswick Scientific). Freshly harvested conidia (5× 10^9) were inoculated in 5 liters of FM medium and bioreactor runs were performed as described as described above. Each bioreactor run was performed twice (biological duplicate).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from homogenized mycelial samples using TRIzol reagent (Invitrogen) followed column purification including a DNAse I treatment.
|
| Label |
biotin
|
| Label protocol |
The Affymetrix One-Cycle Target Labeling Kit and Control Reagents (#900493) are used to synthesize Biotin-labeled cRNA. From each RNA sample 2 μg is used for the labeling experiments.
|
| |
|
| Hybridization protocol |
The GeneChip Hybridization, Wash and Stain Kit (#900720) is used for the for the hybridizations, washing, staining and scanning of the chips. The Affymetrixprotocols are strictly followed. The Affymetrix custom Aspergilus niger Genome Arrays are used for hybridization. To the 270 μl hybridization cocktail, 30 μl labeled material is added according the manual of the Affymetrix One Cycle Target Labeling Kit.
|
| Scan protocol |
Standard Affymetrix protocol
|
| Data processing |
RMA background correction, normalization and probe summarization steps were performed according to the default setting of the RMA package as implemented in Bioconductor.
|
| |
|
| Submission date |
Apr 02, 2014 |
| Last update date |
Dec 31, 2014 |
| Contact name |
Benjamin M. Nitsche |
| E-mail(s) |
bmnitsche@gmail.com
|
| Organization name |
Leiden University
|
| Department |
Institute of Biology
|
| Lab |
Molecular Microbiology and Biotechnology
|
| Street address |
Sylviusweg 72
|
| City |
Leiden |
| State/province |
The Netherlands |
| ZIP/Postal code |
2333 BE |
| Country |
Netherlands |
| |
|
| Platform ID |
GPL6758 |
| Series (1) |
| GSE56471 |
THE CAPACITY OF ASPERGILLUS NIGER TO SENSE AND RESPOND TO CELL WALL STRESS REQUIRES AT LEAST THREE TRANSCRIPTION FACTORS: RLMA, MSNA AND CRZA |
|
