Wild type strain EP155 grown on PDA with cellophane overlay (sample B).
Biomaterial provider
Hillman lab
Treatment protocol
Wild type strain EP155
Growth protocol
Grow at 22-24 C with a 12-h light-dark cycle at 1300-1600 lx.
Extracted molecule
total RNA
Extraction protocol
Cultures used for RNA isolation were grown on PDA-cellophane for 6 days and harvested by freezing the mycelia in liquid nitrogen and then immediately grinding the mycelia into a fine powder by using a mortar and pestle. The powder was resuspended in RNA extraction buffer (200 mM NaCl, 100 mM Tris-Cl [pH 8.0], 4 mM EDTA [pH 8.0], 2% sodium dodecyl sulfate [SDS], 2 mM dithiothreitol) at a ratio of 4 ml of buffer per g of mycelia. This step was followed sequentially by extraction with equal volumes of water-saturated phenol, acid phenol-chloroform-isoamyl alcohol (25:24:1) (pH 4.5), and chloroform. Single-stranded RNA was precipitated on ice for 2 h by the addition of LiCl to a final concentration of 2 M. The single-stranded RNA precipitate was collected by centrifugation, resuspended in 2 ml of double-distilled H2O, reprecipitated by the addition of 2.5 volumes of ice-cold ethanol and 0.1 volume of 3 M sodium acetate (pH 5.2), and incubated for 30 min at -20°C. The RNA was collected by centrifugation, washed with 2 ml of ice-cold 75% ethanol, dried, and treated with 2 U of RQ1 DNase (Promega) in 0.5 ml of 20 mM Tris (pH 8.0)-20 mM MgCl2 in the presence of 40 U of RNasin (Promega) for 1 h at 37°C. Following phenol-chloroform and chloroform extractions, the RNA was precipitated with ethanol and resuspended in 100 µl of double-distilled H2O.
Label
Cy3
Label protocol
Fluorescence-labeled cDNA probes were prepared from total RNA (25 μg per probe) by the direct incorporation of Cy3-UTP using a CyScribe first-strand cDNA labeling kit (Amersham pharmacia) primed with oligo (dT) according to the manufacturer’s instructions. Unincorporated nucleotides were removed with a Microcon-30 spin column.
Reovirus MyRV1-Cp9B21 infected strain EP155 grown on PDA with cellophane overlay (sample B).
Biomaterial provider
Hillman lab
Treatment protocol
Reovirus MyRV1-Cp9B21 infection
Growth protocol
Grow at 22-24 C with a 12-h light-dark cycle at 1300-1600 lx.
Extracted molecule
total RNA
Extraction protocol
Cultures used for RNA isolation were grown on PDA-cellophane for 6 days and harvested by freezing the mycelia in liquid nitrogen and then immediately grinding the mycelia into a fine powder by using a mortar and pestle. The powder was resuspended in RNA extraction buffer (200 mM NaCl, 100 mM Tris-Cl [pH 8.0], 4 mM EDTA [pH 8.0], 2% sodium dodecyl sulfate [SDS], 2 mM dithiothreitol) at a ratio of 4 ml of buffer per g of mycelia. This step was followed sequentially by extraction with equal volumes of water-saturated phenol, acid phenol-chloroform-isoamyl alcohol (25:24:1) (pH 4.5), and chloroform. Single-stranded RNA was precipitated on ice for 2 h by the addition of LiCl to a final concentration of 2 M. The single-stranded RNA precipitate was collected by centrifugation, resuspended in 2 ml of double-distilled H2O, reprecipitated by the addition of 2.5 volumes of ice-cold ethanol and 0.1 volume of 3 M sodium acetate (pH 5.2), and incubated for 30 min at -20°C. The RNA was collected by centrifugation, washed with 2 ml of ice-cold 75% ethanol, dried, and treated with 2 U of RQ1 DNase (Promega) in 0.5 ml of 20 mM Tris (pH 8.0)-20 mM MgCl2 in the presence of 40 U of RNasin (Promega) for 1 h at 37°C. Following phenol-chloroform and chloroform extractions, the RNA was precipitated with ethanol and resuspended in 100 µl of double-distilled H2O.
Label
Cy5
Label protocol
Fluorescence-labeled cDNA probes were prepared from total RNA (25 μg per probe) by the direct incorporation of Cy5-UTP using a CyScribe first-strand cDNA labeling kit (Amersham pharmacia) primed with oligo (dT) according to the manufacturer’s instructions. Unincorporated nucleotides were removed with a Microcon-30 spin column.
Hybridization protocol
Printed slides were prepared for hybridization by the addition of 30 μl of prehybridization solution, which contained 50% formamide, 6X SSPE (1X SSPE is 0.15 M NaCl, 0.001 M NaH2PO4 and 0.001 M EDTA), 0.5% SDS, 5X Denhardt’s solution, and 100 μg of salmon sperm DNA/ml, to the arrayed surface of a glass slike (covered with a coverslip to evenly distribute the prehybridization solution). The slide was incubated for 30 min at 42 C in a hybridization chamber. Fluorescence-labeled probes were dried and resuspended in 20 μl of hybridization solution, which contained 50% formamide, 6x SSPE, 0.5% SDS, 5x Denhardt's solution, blocking solution [2 µg of poly(dA), 4 µg of yeast tRNA, 10 µg of salmon sperm DNA)], 14 µl of master mix solution (70% formamide, 3x Denhardt's solution, 0.7% SDS), and 6 µl of 20x SSPE. The resuspended probes were heated to 100°C for 2 min, vortexed, and collected by a brief spin in a microcentrifuge. The probes were applied to the arrayed surface, covered with a coverslip, and placed in a hybridization chamber overnight at 42°C.
Scan protocol
Hybridized slides were washed in each of three solutions (solution 1 is 1x SSC- 0.1% SDS, solution 2 is 2x SSC- 0.1% SDS, and solution 3 is 2x SSC) for 10 min, spun dry, scanned in both Cy3 and Cy5 channels with an Affymetrix 418 Scanner at a 10-µm resolution and a 70% photomultiplier tube value, and exported as 16-bit TIFF images for analysis.
Description
NA
Data processing
Integrated pixel intensity values for each spot were calculated using TIGR Spotfinder software (TIGR, Rockville, MD; http://www.tigr.org/software) and saved in tab-delimited format for analysis in Mathematica 5.1 (Wolfram Research Inc., Champaign, IL; http://www.wolfram.com). All hybridization data among two sets of dye-swap experiments (representing a total of 4 datasets) were processed according to the following pipeline: (Step 1) Each hybridization dataset was individually processed using a locally weighted linear regression (Lowess) algorithm (smoothing factor = 0.15) to remove systemic dye bias present in each group of spots within a single metarow, metacolumn block of the microarray chip. (Step 2) Following the intra-slide normalization routine described above, all lowess-normalized datasets were loaded simultaneously to rescale each spot through the use of a Z-transformation. Specifically, the global log2 (cy3/cy5) mean and standard deviations were calculated across all four datasets simultaneously and then used to rescale each spot in all four normalized datasets using Equation 1: the rescaled log2 (cy3/cy5) value of an individual spot = [the spot’s own log2 (cy3/cy5) value – global mean of all log2 (cy3/cy5) values] / by the global standard deviation of all log2 (cy3/cy5) values.
