|
| Status |
Public on Mar 03, 2016 |
| Title |
DZ12-highgal_5 (DZHG5) |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
DZ12-derived isolates
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
ploidy: Diploid
background: W3031-AxYJM789
strain: diploid strain DZ12
growth protocol: one cycle of growth on high-gal medium
|
| Treatment protocol |
The expression of POL3 were downregulated using either low-gal medium or YPD medium.
|
| Growth protocol |
Yeast cells were cultured in low-gal (2% yeast extract, 2% peptone and 3% raffinose, 0.005% galactose) or high-gal medium (2% yeast extract, 2% peptone and 3% raffinose, 0.05% galactose) to extract DNA
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA were 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 JSC24) labeled with Cy3. The two samples were mixed and competitively hybridized to the SNP microarrays.
|
| |
|
| Channel 2 |
| Source name |
Control strain JSC24
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
ploidy: Diploid
background: W303-1AxYJM789
strain: control strain JSC24
|
| Treatment protocol |
The expression of POL3 were downregulated using either low-gal medium or YPD medium.
|
| Growth protocol |
Yeast cells were cultured in low-gal (2% yeast extract, 2% peptone and 3% raffinose, 0.005% galactose) or high-gal medium (2% yeast extract, 2% peptone and 3% raffinose, 0.05% galactose) to extract DNA
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA were 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 JSC24) 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 24 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 as .gpr files and analyzed with a homemade software pipeline. Probes that were flagged by the software were deleted from the analysis.
|
| Description |
DZ12-derived isolates from one cycle growth on high-gal medium
|
| 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. In the labels of the oligonucleotides “SF”, “SR”, “YF” and “YR” refer to the orientation and origin of the sequences used for the oligonucleotides on the microarrays. “S” and “Y” indicate whether the oligonucleotide contains sequences identical to the reference S288c genome (closely related to W303-1A) or the YJM789 genome, respectively. “F” and “R” indicate whether the orientation (5’ to 3’) is identical to the orientation in SGD (“F” standing for forward) or opposite to that shown in SGD (“R” for reverse). Thus, “F” and “R” have the sequences of the complementary Watson and Crick strands at each position. For all oligonucleotides for a particular SNP, the number used to label the coordinate is the coordinate of the first base of the oligonucleotide in the “F” orientation. In addition, the oligonucleotides for the YJM789-derived sequences were labeled with the same coordinate as the S288c-derived sequences. The sequence information for the YJM789 sequences was from SGD based on the paper by (Wei et al., 2007. PNAS 104:12825-12830). The Whole genome arrays were completed with Agilent platform with Design ID Agilent-027438 was used. Sectored colonies were mapped using Agilent platforsm with Design IDs Agilent-031671, which contains probes specific only to chromosome IV, and Agilent-047217, which contains many probes on chromsome IV, but few probes near the telomere and centromere of all chromosomes.
|
| |
|
| Submission date |
Mar 02, 2016 |
| Last update date |
Mar 03, 2016 |
| Contact name |
Daoqiong Zheng |
| E-mail(s) |
zhengdaoqiong@163.com
|
| Phone |
86 571 88206636
|
| Organization name |
College of Life Sciences, Zhejiang University
|
| Department |
Institute of Microbiology
|
| Street address |
866 Yuhangtang Road
|
| City |
Hangzhou |
| State/province |
Zhejiang |
| ZIP/Postal code |
310058 |
| Country |
China |
| |
|
| Platform ID |
GPL20144 |
| Series (1) |
| GSE78856 |
Genomic instability induced by lowered expression of POL3 in yeast |
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