S. cerevisiae strains were cultured in YPD (1.0% yeast extract, 1.0% Bacto-peptone and 2.0% glucose)
Extracted molecule
total RNA
Extraction protocol
DNA was extracted from yeast strains grown from single colonies in YPD medium
Label
Cy5
Label protocol
Before the labeling reaction, 4 μg of DNA were treated with RNAse A (1 mg/mL, Roche Diagnostics) for 1 h at 37ºC and with Proteinase K (1 mg/ml, Quiagen) for 1.5 h at 37ºC with agitation. Phenol extracted DNA was digested with MseI or RsaI (New England Biolabs, Inc.), according to the manufacturer's instructions, and digestions were mixed. The fragmented sample was purified using the QIA-quick gel extraction kit (Qiagen, Germany), heat denatured for 5 min at 100°C, and then cooled on ice. DNA was labeled in 21-μl reaction mixtures by random priming using the BioPrime® Array CGH Genomic Labeling System (Invitrogen). Unincorporated nucleotides are removed by filtration through a Qiaquick PCR Purification column (Qiagen)
S. cerevisiae strains were cultured in YPD (1.0% yeast extract, 1.0% Bacto-peptone and 2.0% glucose)
Extracted molecule
total RNA
Extraction protocol
DNA was extracted from yeast strains grown from single colonies in YPD medium
Label
Cy3
Label protocol
Before the labeling reaction, 4 μg of DNA were treated with RNAse A (1 mg/mL, Roche Diagnostics) for 1 h at 37ºC and with Proteinase K (1 mg/ml, Quiagen) for 1.5 h at 37ºC with agitation. Phenol extracted DNA was digested with MseI or RsaI (New England Biolabs, Inc.), according to the manufacturer's instructions, and digestions were mixed. The fragmented sample was purified using the QIA-quick gel extraction kit (Qiagen, Germany), heat denatured for 5 min at 100°C, and then cooled on ice. DNA was labeled in 21-μl reaction mixtures by random priming using the BioPrime® Array CGH Genomic Labeling System (Invitrogen). Unincorporated nucleotides are removed by filtration through a Qiaquick PCR Purification column (Qiagen)
Hybridization protocol
Equal amounts of labeled DNA (1µ) were used as probes to hybridize to the PCR-amplified open reading frames (ORFs) of homoploid S. cerevisiae S288C DNA spotted onto microarrays (Eurogentec S.A., Belgium). Microarrays were pre-hybridized with 5 ml of pre-hybridization solution (3× SSC [1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate], 0.1% SDS, and 0.1 mg/ml BSA) at 42°C for 1 h. After washing slides with isopropanol and water, samples were centrifuged at 1300 rpm for 10 min at room temp. Then 10 µl of samples and 50 µl of hybridization solution (50 % (v/v) formamide, 5x SSC, 0.1% SDS and 0.1 mg/ml DNA from salmon sperm) were denatured (95ºC, 1 min) and added to the slides that were incubated for 16 h at 42ºC in a Gene Machines Hyb Chamber (Genomic Solutions). Slides were recovered and washed 5 min at 42ºC in 1 (2x SSC, 0.1% SDS), 2x10 min in solution 2 (0.1 x SSC, 0.1% SDS), 5x1min in solution 3 (0.1 x SSC) and 5 s in solution 4 (0.01 x SSC). Slides were centrifuged for 10 min at 1300 rpm.
Scan protocol
Images were acquired using GenePix 4100A (Axon Instruments, Molecular Devices Corp., USA) with a 10-µm resolution using the GenePix Pro 6.1 software (Axon Instruments, Molecular Devices Corp., USA)
Description
comparison S288c vs 102 strain
Data processing
Raw data were processed using Acuity 4.0 (Axon Instruments, Molecular Devices Corp., USA). All the slide data were normalized with a log2(ratio) average and filtered (signal of each channel > 350 and signal/noise ratio > 2.5). Poor or inconsistent signals were not considered for further analysis Hybridization signals were depicted as the log2 hybridization signal ratio (test/reference). Genomic material obtained from three biological independent replicates was used to perform hybridizations two or three times, including a dye-swap, and the genes with signal intensities ± 0.5 were considered for further analysis. FDR<0.05 was used to select significant data.
