Biotin-labeled antisense cRNA was generated by labeling 20 or 2 μg of total RNA with a BioArray high-yield RNA transcription labeling kit (ENZO) or an Affymetrix eukaryotic one-cycle target labeling and control reagent package, respectively. The quality of the cRNA was checked using the Agilent 2100 bioanalyzer. The labeled cRNA was hybridized to Affymetrix A. niger GeneChips (Affymetrix, Santa Clara, CA). The coding sequence of the annotated genome of CBS513.88 (Pel et al. 2007) was taken as the sequence template. Oligonucleotide probes were designed with 600-bp fragments, starting from the 3’ end of the gene. The probe sets consist of 12 pairs (match and mismatch) of 25-bp oligonucleotide probes, which are scattered across the chip. Arrays were hybridized with three independently obtained RNA samples of the peripheries of 5-day-old sandwiched cultures grown on sugar beet pulp.
Growth protocol
A. niger N402 (cspA1) (Bos et al. 1988) was grown at 30°C under constant light in water-saturated air. Colonies were grown as a sandwiched culture (Levin et al. 2007) in 9-cm petri dishes in a 0.2-mm thin layer of 1.25% agarose in between two perforated polycarbonate membranes (diameter, 76 mm; pore size, 0.1 m; Osmonics, GE Water Technologies, Trevose, PA) placed on top of solidified (1.5% agar) minimal medium (7) with 1% sugar beet pulp as a carbon source or in liquid minimum medium contained in a ring plate. Sandwiched cultures were inoculated with 1.5 l of spore suspension (108 spores l-1) and harvested after 6 days of growth. A ring plate consists of a polycarbonate disc (9 cm in diameter, 1.2 cm thick) with six ring-shaped wells. The inner two rings are collectively called ring 1 because of their small volume, and the outer ring is called ring 5. The wells are separated by 0.1 cm and are 0.5 cm deep and 0.5 cm wide.
Extracted molecule
total RNA
Extraction protocol
Mycelium was ground using a microdismembrator (B. Braun GmBh, Melsungen, Germany), and RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA) according to the instructions of the manufacturer. The RNA was purified using a Nucleospin RNA cleanup kit (Macherey-Nagel GmBh, Düren, Germany). The concentration of RNA was measured at A260. The quality of the RNA was analyzed with an Agilent 2100 bioanalyzer, using an RNA6000 LabChip kit (Agilent Technology, Palo Alto, CA).
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 ug of cRNA was hybridized to Affymetrix A. niger Genome Gene chips (GPL6758). GeneChips were washed and stained in an automated process.
Scan protocol
GeneChips were scanned.
Description
Gene expression data from A.niger wild type grow on sugar beet pulp for 5 days
Data processing
Affymetrix CEL-data files were preprocessed by using the statistical language and environment R (R Development Core Team, 2007) version 2.6.1. The probe intensities were normalized for background by using the robust multiarray average method (Irizarry, 2003) by using only perfect match (PM) probes. Normalization was performed subsequently by using the quantiles algorithm (Bolstad, 2003). Gene expression values were calculated from the PM probes with the medianpolish summary method (Irizarry, 2003). All statistical preprocessing methods were used by invoking them through the affy package (Gautier, 2004).
