Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling of Saccharomyces cerevisiae genomic DNA, nucleosomal DNA and the yeast strain to analyse diversity of eukaryotic DNA replication origins revealed by genome-wide analysis of chromatin structure

ABSTRACT: Genomic DNA from Saccharomyces cerevisiae W303-1A was isolated along with nucleosomal DNA from W303-1A and the yeast strain GAL:orc2-1. Nucleosomal DNA was isolated following a 2h release from a nocodazole block in which the medium was changed from galactose (YPAG) to glucose (YPAD).

ORGANISM(S): Saccharomyces cerevisiae

SUBMITTER: Nicolas Berbenetz

PROVIDER: E-MEXP-2369 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Purification of Nucleosomal DNA T47D-MTVL breast cancer cells carrying one stably integrated copy of the luciferase reporter gene driven by the MMTV promoter (Truss et al., 1995) were routinely grown in RPMI 1640 medium supplemented with 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin, and 100 μg/mL streptomycin. For DNA preparation, cells were plated in RPMI medium without phenol red supplemented with 10% dextran-coated charcoal-treated FBS (DCC/FBS), and 48 hr later medium was replaced by fresh medium without serum. After 1 day in serum-free conditions, cells were treated for 5 minutes at 37ºC with 2mL of buffer A (150mM sucrose, 15mM Tris pH 7.5, 60mM KCl, 15mM NaCl, 2mM CaCl2) containing 0,5 mg/mL of lysolecithin. Micrococcal nuclease digestion was performed by adding 2mL of buffer A, supplemented with 720U of MNase (Worthington) for 3 minutes at 37ºC. The reaction was stopped with 40mM EDTA. Cells were scrapped and lysated with 1% SDS, 10mM EDTA and 50mM Tris pH 8.1 buffer, during 10 minutes on ice. Samples were then treated with 0,1mg/mL RNAse A for 30 minutes at 37ºC and 0,1 mg/mL of proteinase K for 1 hour at 45ºC. DNA was purified by phenol/chloroform extractions and ethanol precipitation. The digested DNA was run on 1% agarose gel, and band corresponding to mononucleosomes was gel-extracted. Microarray hybridization Labelling was done using Bioprime genomic labeling kit (Invitrogen, cat.no. 18095-011 and 18095-012) following manufacturers instructions. Briefly, 500 ng isolated nucleosomal DNA or total genomic DNA partially digested with MNase was labeled with either dUTP-Cy5 or dUTP-Cy3 fluorescent mononucleotides (Amersham GE PA55322), respectively. 2 mg of the obtained labeled sample were co-hybridized to the Agilent customized oligo microarray in the presence of 50 mg Human Cot-1 DNA and Agilent oligo aCGH hybridization kit (cat.no. 5188-5220) with includes blocking agents for unspecific hybridization following manufacturers instructions. Hybridization was performed in a rotating oven at 65ºC for 40 hours. Arrays were washed and fluorescent images were acquired using G2565BA Microarray Scanner System (Agilent) with 100% laser power and 100% PMT settings. 16-bit TIFF images, one for each channel, were quantified using GenePix Pro 6.0 microarray analysis software (Axon Instruments Inc.). Microarray data was normalized applying a global loess algorithm using a smoothing factor of 0.2 (Yang et al. 2002) obtaining normalized log2[intensity Cy5]/[Intensity Cy3] mean values for each spot on the array.

Dataset Information

Transcription profiling of Saccharomyces cerevisiae genomic DNA, nucleosomal DNA and the yeast strain to analyse diversity of eukaryotic DNA replication origins revealed by genome-wide analysis of chromatin structure

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