|
| Status |
Public on Aug 25, 2013 |
| Title |
PglA-RacA(G18V).2h.rep2 |
| Sample type |
RNA |
| |
|
| Source name |
whole fungal mycelium, exponential growth phase, 2 hours after induction of racA G18V mutant allele expression
|
| Organism |
Aspergillus niger |
| Characteristics |
genotype: PglA-RacA(G18V)
time point: 2h
|
| Growth protocol |
Maltose-limited batch cultivation of the racA deletion mutant was initiated by inoculation of 5 L (kg) ammonium based minimal medium with conidial suspension to give 10 to the power of 9 conidia L per L. Maltose was sterilized separately from the MM and final concentration was 0.8% (w/v). Temperature was 30 °C and pH 3, kept constant by computer controlled addition of 2 M NaOH or 1 M HCl. Acidification of the culture broth was used as an indirect growth measurement (Iversen et al., 1994). Submerged cultivation was performed with 6.6 L BioFlo3000 bioreactors (New Brunswick Scientific, NJ, USA). A more detailed description of the fermentation medium and cultivation is given in Jorgensen et al. (2010). Batch cultivation for PglaA-RacAG18V or PglaA-RacA were run similarly as the maltose-limited batch cultivations of the racA deletion mutant cultures except that 0.75% xylose was used as a initial carbon source instead of maltose. When the exponential growth phase was over (indicated by sharp rise of dissolved oxygen tension as well as the pH value), 0.75% maltose was added to induce expression of PglaA-RacAG18V or PglaA-RacA. Samples for the analysis of morphological properties, biomass formation, protein yield and RNA were taken every hour.
|
| 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 |
Nov 13, 2012 |
| Last update date |
Aug 25, 2013 |
| 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) |
| GSE42258 |
The transcriptomic signature of RacA activation and inactivation provides new insights into the morphogenetic network of Aspergillus niger |
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