|
| Status |
Public on May 05, 2015 |
| Title |
Pink sector of pink/white sectored colony 4 of FM1468 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
FM1468 colony after 37 degree heat treatment
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
growth condition: 6 hours at 37 degrees
|
| Treatment protocol |
We observed sectored colonies of a variety of types including red/white, red/pink, and pink/white; in addition, some tri-colored colonies (white/pink/red) were observed.
|
| Growth protocol |
For our experiments, the strain FM1468 was incubated at 37C for three hours. The cells were then plated at the permissive temperature of 25C and allowed to form colonies.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
We purified single colonies derived from each sector, and prepared DNA by standard procedures (St. Charles et al., 2012).
|
| Label |
Cy5
|
| Label protocol |
The description of the labeling and hybridization protocol are copied with minor modifications from the Supplementary Information of our previous publication: DNA from these colonies was labeled with Cy5, and mixed with control DNA (DNA from the strain FM1468 not incubated at 37C) labeled with Cy3. The two samples were mixed and competitively hybridized to the SNP microarrays.
|
| |
|
| Channel 2 |
| Source name |
FM1468 colonies untreated at 25 degrees
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
growth condition: Grown at 25 degrees
|
| Treatment protocol |
We observed sectored colonies of a variety of types including red/white, red/pink, and pink/white; in addition, some tri-colored colonies (white/pink/red) were observed.
|
| Growth protocol |
For our experiments, the strain FM1468 was incubated at 37C for three hours. The cells were then plated at the permissive temperature of 25C and allowed to form colonies.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
We purified single colonies derived from each sector, and prepared DNA by standard procedures (St. Charles et al., 2012).
|
| Label |
Cy3
|
| Label protocol |
The description of the labeling and hybridization protocol are copied with minor modifications from the Supplementary Information of our previous publication: DNA from these colonies was labeled with Cy5, and mixed with control DNA (DNA from the strain FM1468 not incubated at 37C) labeled with Cy3. The two samples were mixed and competitively hybridized to the SNP microarrays.
|
| |
|
| |
| Hybridization protocol |
The hybridization reactions were prepared using an Agilent Oligo aCGH/ChIP-on-Chip Hybridization kit (5188-5220) following kit instructions. Arrays were incubated for 48 hours at 62°. Following hybridization, the arrays were washed for 5 minutes in Oligo aCGH/ChIP-on-Chip Wash Buffer 1 (Agilent 5188-5221) and 1 minute in Oligo aCGH/ChIP-on-Chip Wash Buffer 2 (Agilent 5188-5222) that was pre-warmed to 37°. The arrays were then scanned at wavelengths of 635 and 532 nm using the GenePix scanner and the GenePix Pro software using settings recommended by the manufacturer.Microarrays could be re-used approximately 4-6 times by removing the hybridized labeled DNA sequences from the oligonucleotides. Microarrays and gasket slides were stripped separately in 1x stripping buffer (10 mM potassium phosphate, pH6.6). The slides were slowly heated to the boiling point in the stripping buffer for 30-45 minutes. After stripping, they were transferred to deionized water, and then slowly removed and stored in a nitrogen cabinet. The gasket slides were centrifuged at 500 rpm to remove excess liquid. Labels on microarrays were removed prior to stripping.
|
| Scan protocol |
The data generated by GenePix Pro were exported to text files and analyzed with Microsoft Excel. Probes that were flagged by the software were deleted from the analysis.
|
| Data processing |
The ratio of the medians (635 nm/532 nm) for each probe was used for analysis, and replicate probe medians were averaged. The data was centered around one by dividing each probe median by the average of all of the probe medians in order to normalize for differences in the hybridization levels for the reference and experimental strain samples. The data were plotted separately for each haploid parental strain and, in plots showing whole chromosomes, the medians were “smoothed” by averaging over nine consecutive probes.
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| |
|
| Submission date |
May 04, 2015 |
| Last update date |
May 05, 2015 |
| Contact name |
Thomas D Petes |
| E-mail(s) |
tom.petes@duke.edu
|
| Phone |
919 684-4986
|
| Organization name |
Duke University School of Medicine
|
| Department |
Molecular Genetics and Microbiology
|
| Lab |
Petes
|
| Street address |
213 Research Drive
|
| City |
Durham |
| State/province |
NC |
| ZIP/Postal code |
27710 |
| Country |
USA |
| |
|
| Platform ID |
GPL20143 |
| Series (1) |
| GSE68530 |
The transient inactivation of the master cell cycle phosphatase Cdc14 causes genomic instability in diploid cells of Saccharomyces cerevisiae. |
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