The strain was cultured on CM agar (0.6% NaNO3, 0.052% KCl, 0.052% MgSO4・7H2O, 0.152% KH2PO4, 0.1% Glucose, 0.2% Peptone, 0.1% Yeast Extract, 0.1% Casamino acid, 0.0001% Biotin, 0.0001% Pyridoxine, 0.0001% Thiamine, 0.0001% Riboflavin, 0.0001% ƿ-aminobenzoic acid, 0.0001% Nicotinic acid and trace elements).
Growth protocol
Flasks containing 150 ml complete medium were inoculated with conidia to a final concentration of 3.0 x10^6 / ml and incubated for 0, 1, 2, 4, 6, and 8hr at 37°C with constant shaking (220 rpm). Following incubation, cells were harvested using a Steritop filter (Millipore), frozen, ground with liquid nitrogen.
Extracted molecule
total RNA
Extraction protocol
RNA was purified with TRIzol (Invitrogen) and the RNeasy MiniElute Clean up kit (Qiagen) according to the manufacturers’ instructions.
Label
Cy3(IA=4hr in 940),Cy5(IB=4hr in 941)
Label protocol
cDNA synthesis and indirect Cy dye incorporation were performed using the standard J. Craig Venter Institute (J.C.V.I.) protocol (standard operating procedure [SOP] M007) with the following modifications: Six micrograms of total RNA was used in the first-strand cDNA reaction with a 3:1 aminoallyl-dUTP to dTTP ratio. Dried aminoallyl labeled cDNA was resuspened in 9 ml of 50 mM sodium carbonate (pH9.0) and added directly to the appropriate dye vial from the Amersham CyDye postlabeling reactive dye pack (RPN5661).
The strain was cultured on CM agar (0.6% NaNO3, 0.052% KCl, 0.052% MgSO4・7H2O, 0.152% KH2PO4, 0.1% Glucose, 0.2% Peptone, 0.1% Yeast Extract, 0.1% Casamino acid, 0.0001% Biotin, 0.0001% Pyridoxine, 0.0001% Thiamine, 0.0001% Riboflavin, 0.0001% ƿ-aminobenzoic acid, 0.0001% Nicotinic acid and trace elements).
Growth protocol
Flasks containing 150 ml complete medium were inoculated with conidia to a final concentration of 3.0 x10^6 / ml and incubated for 0, 1, 2, 4, 6, and 8hr at 37°C with constant shaking (220 rpm). Following incubation, cells were harvested using a Steritop filter (Millipore), frozen, ground with liquid nitrogen.
Extracted molecule
total RNA
Extraction protocol
RNA was purified with TRIzol (Invitrogen) and the RNeasy MiniElute Clean up kit (Qiagen) according to the manufacturers’ instructions.
Label
Cy5(IB=6hr in 940),Cy3(IA=6hr in 941)
Label protocol
cDNA synthesis and indirect Cy dye incorporation were performed using the standard J. Craig Venter Institute (J.C.V.I.) protocol (standard operating procedure [SOP] M007) with the following modifications: Six micrograms of total RNA was used in the first-strand cDNA reaction with a 3:1 aminoallyl-dUTP to dTTP ratio. Dried aminoallyl labeled cDNA was resuspened in 9 ml of 50 mM sodium carbonate (pH9.0) and added directly to the appropriate dye vial from the Amersham CyDye postlabeling reactive dye pack (RPN5661).
Hybridization protocol
The resulting labeled cDNA was hybridized to an A. fumigatus glass slide microarray spotted with 70-mers representing all predicted open frames (PFGRC/TIGR A. fumigatus microarray, version 3; a full description and annotation are available at Gene Expression Omnibus [accession no. GPL5346; http://www.ncbi.nlm.nih.gov/geo/ ] according to the standard J.C.V.I. protocol (SOP M0008).
Scan protocol
The slides were scanned at 10um resolution and 85% laser power using a Perkin-Elmer ScanArray microarray scanner.
Description
Analysis used isotropically expanding cell's RNA as control samples for comparison to the experimental samples taken at 6 and 8hr. Sample 12
Data processing
The resulting tagged-image format file images were imported into TIGR Spotfinder (version 3.1.1). Raw intensities were calculated for each detectable spot using the Otsu method, and quality control filtering was used to eliminate values from spots with poor morphology or low signal-to-noise ratios. The resulting intensities were then imported into TIGR MIDAS (version 2.20), and spots with values of <10,000 were removed from the data set by using the low intensity filter. Normalization was done using the LOWESS (Locifit) algorithm (global mode; 0.33 smoothing parameter), and print tip bias was addressed by using standard deviation regularization. The dye swap consistency filter was used to remove data for genes with inconsistent expression levels between dye swap replicates. Finally, the in-slide replicate function was used to average the normalized intensity values representing replicate spots on the array.
