strain: N402 developmental stage: conidia germinated for 6h
Growth protocol
A. niger strain N402, a cpsA1 derivative of A. niger N400 (Bos et al. 1988) was grown on Aspergillus complete medium (ACM) (containing per litre: NaNO3 6g, KCl, 0.52 g; MgSO4.7H2O, 0.52 g; KH2PO4, 1.52 g; Na2B4O7.10H2O, 0.008 mg; CuSO4.5H2O, 0.16 mg; FePO4.H2O, 0.16 mg; MnSO4.4H2O, 0.16 mg; NaMoO4.2H2O, 0.16 mg; ZnSO4, 1.6 mg, Bacto casamino acids, 1g, yeast extract, 1g, Bacto peptone, 2g, glucose, 10g, vitamins: p-aminobenzoic acid, 4mg, thiamine HCl, 0.5mg, D-biotin, 0.02mg, nicotinic acid, 1mg, pyridoxine hydrochloride, 2.5mg, choline chloride, 0.014g, riboflavin, 1mg, agar 20g where applicable ) for 6 days at 28oC to develop mature conidia. Conidia were harvested by washing the agar slopes with a 0.01% (w/v) Tween 80 solution. The conidial suspension was filtered through sterile synthetic wool and conidia were counted using a haemocytometer. Conidia (104/ml) were germinated in liquid ACM media for 1, 2, 4 and 6 hours at 28˚C, in 2L conical flasks containing 1000 ml of medium, shaken at 150rpm. Germinated conidia were recovered by filtration into 0.5 ml RNA extraction buffer (0.6M NaCl, 0.2M sodium acetate, 0.1M EDTA, 4% w/v SDS) and snap frozen in liquid nitrogen. Frozen dormant or germinated conidia were mixed with 0.5ml glass beads and disintegrated in a Sartorius dismembranator (4 min, 2000rpm).
Extracted molecule
total RNA
Extraction protocol
RNA was extracted using the TRIzol reagent protocol (Invitrogen) according to manufacturer’s instructions, followed by an additional clean-up using RNeasy columns (Qiagen) including the on-column DNAase treatment step.
Label
biotin
Label protocol
GeneChip® IVT Express Kit
Hybridization protocol
GeneChip® Hybridization, Wash, and Stain Kit
Scan protocol
standard Affymetrix protocol
Description
gene expression data from conidia germinated for 6h in liquid ACM media
Data processing
GeneSpring GX 11 (Agilent Technologies, Inc), Data were normalized using the RMA algorithm. Raw intensity signal values were normalized per chip to the 75th percentile and baseline transformation to the median of all samples (time points) was used.
