strain: N402 carbon source: xylose nitrogen source: 4xNH4Cl nitrogen concentration: 282.4 mM ph of the medium: pH5 time point relative to carbon source depletion: -3.5h
Treatment protocol
Samples were collected either around the time point carbon source depleted or a considerable time (~24 h) after carbon souce depletion. The amount of sample collected depended on the concentration of biomass atthat time point. Collected samples were quenched immediately in methanol at -45 ºC as described previously (Pieterse B., R. H. Jellema, M. J. van der Werf. 2006. Quenching of microbial samples for increased reliability of microarray data. J. Microbiol. Methods 64:207-216.) and centrifuged at -20 ºC to remove supernatant. Biomass was frozen into liquid nitrogen and stored at -80 ºC until further use.
Growth protocol
Fermentor cultures were grown in minimal medium (MM) at a constant temperature of 30 ± 0.5 ºC and with differing combinations of carbon source (either 277,5 mM glucose or 333,0 mM xylose), nitrogen source (NH4Cl or NaNO3) and nitrogen concentration (4x: 282·4 mM; 8x: 564·8 mM), and pH (pH4 or pH5) of the medium (M. Braaksma, A.K. Smilde, M.J. van der Werf, P.J. Punt, submitted for publication). Fermentor inoculum was pre-cultured in baffled 500 ml Erlenmeyer flasks containing 100 ml MM (pH6.5) supplemented with the carbon source and nitrogen source concentrations corresponding to fermentor conditions. These flasks were inoculated with 10^6 spores per liter and incubated in a rotary shaker at 125 rpm until approximately half of the available carbon source was consumed. Fermentor cultures were carried out in 6.6-liter fermentors (New Brunswick Scientific) with 5.0 liters of MM. The fermentors were inoculated with 4% (weight/vol) pre-culture. To prevent foaming, 1% (vol/vol) Struktol J-673 antifoam was added to the medium and additional antifoam was added during cultivation when necessary. Each fermentor was sparged with 75 liters/h of air with the stirrer speed set at 400 rpm at the start of the cultivation. When dissolved oxygen tension levels dropped below 20%, the stirrer speed was automatically increased to maintain oxygen tension at 20% or until the maximum of 1000 rpm was reached. The pH was controlled by automatic addition of 8 M KOH or 1.5 M H3PO4. The culture was allowed to grow for at least twice the time it took to consume all carbon source.
Extracted molecule
total RNA
Extraction protocol
Frozen mycelium was ground using a mortar and pestle, followed by a trizol-chloroform extraction using the Qiagen Rneasy mini columns according to the “yeast protocol”.
Label
biotin
Label protocol
Extracted total RNA was processed using the Bioarray High Yield RNA transcript labeling kit (Enzo). 15 ug of cRNA was used for labeling.
Hybridization protocol
Washing, staining, and hybridization was done according to the Hybridization, Wash, and Stain kit (Affymetrix) according to the manufacturers' recommendations.
Scan protocol
Arrays were scanned on an Agilent G2500A Gene Array scanner. Pixel value was 3 um, wavelength 570 nm.
Description
the time indicated at the sample title indicates the time point each sample was collected prior to (-xh) or after (+xh) carbon souce depletion.
Data processing
Raw signal intensities, detection p-values, and present/absent calls were derived using Microarray Suite Software version MAS5 (Affymetrix).
