strains were grown in YPD (1% Yeast Extract, 2% Peptone, 2% glucose) overnight at 30€™C in shaking.
Extracted molecule
genomic DNA
Extraction protocol
genomic DNA was extracted from over-night strains cultures with the phenol-chloroform protocol. DNA quality was checked through gel electrophoresis and photometric measurement.
Label
Cy5
Label protocol
The genomic DNA extracted was concentrated using the filter Microcon YM-30 (Millipore) and fragmented by sonication. The labeling of the DNA was performed, at 37°C for 2 hours, using the BioPrime DNA labeling Sytem (Invitrogen, Life Technologies), but using 100mM dNTP set (Invitrogen, Life Technologies) instead of the biotinilated nucleotides present in the kit, as well as the fluorescent nucleotides Cy3-dCTP and Cy5-dCTP of Amersham, GE Healthcare. Once finished the reaction, the DNA was purified with the Microcon YM-30 and the appropriate pairs of samples labeled with the different fluorochromes were combined in a single eppendorf in 450µL of TE (Tris-EDTA), pH 8.0 (Sigma-Aldrich). We used Human cot-1 DNA (Invitrogen, Life Technologies) to block non-specific hybridization, and Yeast tRNA (Invitrogen, Life Technologies) as coprecipitant.
strains were grown in YPD (1% Yeast Extract, 2% Peptone, 2% glucose) overnight at 30€™C in shaking.
Extracted molecule
genomic DNA
Extraction protocol
genomic DNA was extracted from over-night strains cultures with the phenol-chloroform protocol. DNA quality was checked through gel electrophoresis and photometric measurement.
Label
Cy3
Label protocol
The genomic DNA extracted was concentrated using the filter Microcon YM-30 (Millipore) and fragmented by sonication. The labeling of the DNA was performed, at 37°C for 2 hours, using the BioPrime DNA labeling Sytem (Invitrogen, Life Technologies), but using 100mM dNTP set (Invitrogen, Life Technologies) instead of the biotinilated nucleotides present in the kit, as well as the fluorescent nucleotides Cy3-dCTP and Cy5-dCTP of Amersham, GE Healthcare. Once finished the reaction, the DNA was purified with the Microcon YM-30 and the appropriate pairs of samples labeled with the different fluorochromes were combined in a single eppendorf in 450µL of TE (Tris-EDTA), pH 8.0 (Sigma-Aldrich). We used Human cot-1 DNA (Invitrogen, Life Technologies) to block non-specific hybridization, and Yeast tRNA (Invitrogen, Life Technologies) as coprecipitant.
Hybridization protocol
Hybridization took place at 65 °C for 16 h.
Scan protocol
Fluorescent DNA bound to the microarray was detected with a GenePix 4000B microarray scanner (Axon Instruments), using the GenePixPro6.1 software package to quantify microarray fluorescence.
Description
Arsenic-sensitive strain
Data processing
Data were normalized using the limma R package and DEGs (Differentially Expressed Genes) were identified with the RankProduct R package
