Mycelia were harvested from cultures growing on wheat straw, lactose, and glucose, respectively, for 50 (wheat straw)and 28 hrs (lactose and glucose), respectively.
Growth protocol
Cultures were grown in 250ml of Mandels Andreotti (MA) medium (per litre: 1.4g (NH4)2SO4, 2.0g KH2PO4, 0.3g MgSO4*7H2O, 0.3g CaCl2*2H2O, 0.3g urea, 1g peptone (casein), 5mg FeSO4*7H2O, 1.6mg MnSO4*H2O, 1.4mg ZnSO4*7H2O and 2mg CoCl2*2H2O) with 10g/l glucose monohydrate, lactose monohydrate or pretreated wheat straw (dry basis) as the sole carbon source and inoculated with 106 conidia ml-1 of spores. Furthermore, 0.5g l-1 of tween 80 were added in the case of lactose cultures and the pH of wheat straw media was adjusted to 4.8 with 1M KOH. All cultivations were performed in a rotary shaker at 28°C and 250rpm. Biomass samples for total RNA extraction or measurement of biomass were withdrawn at appropriate time points as stated below. Cultures for the qPCR analyses were pregrown for 24h in glycerol containing (10 g l-1) MA medium and equal portions of the harvested and washed mycelium were aseptically replaced into MA medium, again containing either 10 g l-1 glucose monohydrate, lactose monohydrate or pretreated wheat straw (dry basis) as the sole carbon source, but this time devoid of urea or peptone.
Extracted molecule
total RNA
Extraction protocol
Total RNAs from glucose and lactose cultures were extracted using TRIzol® reagent (Invitrogen Life Technologies, Carlsbad, CA, USA), according to the manufacturer's instructions, and then purified. For isolation and purification of total RNA from wheat sraw cultures, the RNeasy Plant Mini Kit and the RNeasy MinElute Kit (both Quiagen, Hilden, Germany) respectively were used according to the manufacturer’s instructions.
Label
Cy3
Label protocol
cDNA synthesis, labelling and hybridization was performed by Roche NimbleGen (Roche-NimbleGen, Inc., Madison, WI, USA) with a high density oligonucleotide (HDO) microarray using 60-mer probes representing the 9.129 genes of T. reesei.
Hybridization protocol
Hybridization was performed by NimbleGen Systems Inc., Madison, WI, USA following their standard operating protocol. See www.nimblegen.com.
Scan protocol
Scanning was performed by NimbleGen Systems Inc., Madison, WI USA, following their standard operating protocol. See www.nimblegen.com.
Description
lactose
Data processing
The raw data (.pair file) was subjected to RMA (Robust Multi-Array Analysis; Irizarry et al. Biostatistics 4(2):249), quantile normalization (Bolstad et al. Bioinformatics 19(2):185), and background correction as implemented in the NimbleScan software package, version 2.4.27 (Roche NimbleGen, Inc.). Microarray scanning, data acquisition and identification of probe sets showing a significant difference (p=0.05) in expression level between the different conditions were performed essentially as described by Metz et al. (2011) Eukaryot Cell 10:1527-1735.
