Yeast strains were cultivated in 5ml YPD (1% yeast extract, 2% peptone, 2% glucose) at 28 ºC for 24 hours.
Extracted molecule
genomic DNA
Extraction protocol
DNA was isolated according to standard procedures (Querol et al., 1992). After isolation, the DNAs were digested with HinfI (Takara Bio Inc., Japan) and purified with the High Pure PCR Product Purification Kit (Roche Molecular Biochemicals, Mannheim, Germany).
Label
Cy3,Cy5
Label protocol
Approximately 2 μg of these DNAs were labelled with fluorescently-tagged nucleotides (Amersham, Piscataway, NJ, USA), either Cy3-dCTP for the reference strain and Cy5 dCTP for the studied strains or vice-versa, using Exo-Klenow Fragment and random primer Mix (Bioprime Array CGH Kit, Invitrogen). Dye-swap hybridizations were performed for each strain. After labeling, the reactions were heat inactivated, the experimental (Cy5- or Cy3-labeled) and reference (Cy3- or Cy5-labeled) DNAs were mixed and purified away from unincorporated label using the MinElute PCR Purification Kit (QIAGEN). The degree of labeling was determined by measuring the absorbance at 260 nm and at 550 nm for Cy3- labeled DNA or at 260 nm and 650 nm for Cy5-labeled DNA. Samples with a base/dye ratio between 40 and 80 for both Cy3 and Cy5 were routinely obtained.
Yeast strains were cultivated in 5ml YPD (1% yeast extract, 2% peptone, 2% glucose) at 28 ºC for 24 hours.
Extracted molecule
genomic DNA
Extraction protocol
DNA was isolated according to standard procedures (Querol et al., 1992). After isolation, the DNAs were digested with HinfI (Takara Bio Inc., Japan) and purified with the High Pure PCR Product Purification Kit (Roche Molecular Biochemicals, Mannheim, Germany).
Label
Cy5,Cy3
Label protocol
Approximately 2 μg of these DNAs were labelled with fluorescently-tagged nucleotides (Amersham, Piscataway, NJ, USA), either Cy3-dCTP for the reference strain and Cy5 dCTP for the studied strains or vice-versa, using Exo-Klenow Fragment and random primer Mix (Bioprime Array CGH Kit, Invitrogen). Dye-swap hybridizations were performed for each strain. After labeling, the reactions were heat inactivated, the experimental (Cy5- or Cy3-labeled) and reference (Cy3- or Cy5-labeled) DNAs were mixed and purified away from unincorporated label using the MinElute PCR Purification Kit (QIAGEN). The degree of labeling was determined by measuring the absorbance at 260 nm and at 550 nm for Cy3- labeled DNA or at 260 nm and 650 nm for Cy5-labeled DNA. Samples with a base/dye ratio between 40 and 80 for both Cy3 and Cy5 were routinely obtained.
Hybridization protocol
The mixture of the required Cy3- and Cy5-labelled samples was adjusted to 60 μl with hybridization solution (50% formamide, 5X SSC, 0,1% SDS, 100 μg/ml salmon sperm DNA and water), incubated for 1 minute at 95 ºC and centrifuged at 13000 X g for 1 min. The supernatants were applied to microarrays and the hybridizations were allowed to proceed overnight at 65°C.
Scan protocol
Arrays were scanned using an Axon GenePix 4100A scanner and GenePix Pro 6.0 software.
Description
Duplicate arrays and Cy5-dCTP and Cy3-dCTP dye-swap assays.
Data processing
Using Acuity 4.0 software (Molecular Devices Corp., Union City, CA, USA), manually flagged bad spots were eliminated and the local background was subtracted before averaging the replicate features on the array. Log2 intensity ratios (M values) were then Median normalized to correct for differences in genomic DNA labelling efficiency between samples. The relative hybridization signal of each ORF was derived from the average of the two dye-swap hybridizations performed for each strain. The normalized log2 ratio (M value) was considered as a measure of the relative abundance of each ORF relatively to that of the reference strain S288C. Deviations from the 1:1 hybridization ratio were taken as indicative of changes in DNA copy number. Given that the variability usually observed between S. cerevisiae genomes (either within laboratory strains or natural isolates) is much lower than this estimate (Adams et al. 1992, Carreto et al. 2008), we interpreted statistically significant variations in hybridization signal as ORF deletions or duplications. Data imported from Acuity was manipulated and clustered, using established algorithms implemented in the software program Genesis. Average linkage clustering with centered correlation was used to generate visual representations of clusters.
