strain: N402 genotype: cspA1 (wild type) tissue: mycelium age: 2 hours substrate: 25 mM D-galacturonic acid plus L-rhamnose
Treatment protocol
Mycelium was washed with MM and transferred for 2 h to 250 ml Erlenmeyer flasks containing 50 ml MM supplemented with 25 mM D-glucose, D-fructose, D-galactose, L-arabinose, D-xylose, D-mannose, D-galacturonic acid, L-rhamnose, Maltose, Sucrose, Ferulic acid or ferulic acid, or mixture of 25 mM L-rhamnose and 25 mM D-galacturonic acid, or 1% Inulin from chicory, Cellulose, Guar gum, Xyloglucan, Xylan (from beechwood), Polygalacturonic acid (from apples), Apple pectin, Galactan (from potato), Debranched 1,5-α-L-arabinan (from sugar beet), Rhamnogalacturonan I (from potato), Mannan (ivory nut), Citrus pulp, Sugar beet pulp or Soy bean hulls. The only exceptions were D-maltose cultures of N402 and ∆amyR strains that were incubated for 4 h. Mycelium was harvested by vacuum filtration, dried between towels and frozen in liquid nitrogen. While N402 liquid cultures were performed on all carbon sources listed before as well as on the mixture of L-rhamnose and D-galacturonic acid, the regulatory mutant strains ΔxlnR, ΔaraR, ΔamyR, ΔrhaR and ΔgalX were grown on D-xylose, L-arabinose, maltose, L-rhamnose and D-galactose, respectively, and L-rhamnose and D-galacturonic acid. All cultures were performed as biological duplicates.
Growth protocol
A. niger N402 wild type was pre-cultured in 1 L Erlenmeyer flasks in 250 mL complete medium (CM) supplemented with 2% D-fructose. The pre-cultures were inoculated with 10e6 spores/mL and incubated at 250 rpm for 16 h at 30°C.
Extracted molecule
total RNA
Extraction protocol
RNA was extracted using TRIzol reagent (Invitrogen) and purified using TRIzol® Plus RNA Purification Kit (Sigma-Aldrich) according to the instructions of the manufacturer.
Label
biotin
Label protocol
Biotin-labeled antisense cRNA was produced from 2 µg of total RNA with the Eukaryotic One-Cycle Target Labeling kit (Affymetrix; www.affymetrix.com).
Hybridization protocol
Following fragmentation, 10 µg of cRNA was hybridized to Affymetrix A. niger Genome GeneChips (GPL6758). GeneChips were washed and stained in an automated process.
Scan protocol
GeneChips were scanned.
Description
GalA/Rha-wt replicate 2 Wild type N402 cspA1 Gene expression data from A. niger wild type growing on D-galacturonic acid plus L-rhamnose.
Data processing
Microarray data was analyzed using the Bioconductor tool package version 2.8 (http://www.bioconductor.org/) together with house-made Perl (version .5.0) and Python (version 3.0) scripts. Probe intensities were normalized for background by the robust multi-array average (RMA) method using the R statistical language and environment. This method makes use of only perfect match (PM) probes. Normalization was processed by the quantiles algorithm. The median polish summary method was used to calculate the gene expression values.
