|
| Status |
Public on Nov 15, 2017 |
| Title |
H2O2_20_32R |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
cell culture of JSC25-derived strain HO-20-32R
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
strain: H2O2_20_32R
|
| Treatment protocol |
JSC25 cells were grown in 7 mL YPD overnight to reach an OD600 of 0.2. Yeast cells (5×107) were collected by centrifugation and arrested in G1 in 1 mL YPD containing 2.5 μg α-factor at 300. After two hours, H2O2 was added into the cell culture. Cell culture was incubated at 300 for another hour. Yeast cells were then washed twice to remove α-factor and H2O2 before plating. Yeast cells were cultured at 300 for 3 days and sectored colonies were selected for whole genome SNP microarray analysis.
|
| Growth protocol |
Yeast cells were grown in YPD medium at 30 degree.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA were prepared by standard procedures (St. Charles et al., 2012)
|
| Label |
cy5
|
| Label protocol |
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 |
cell culture of control strain JSC24
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
strain: JSC24
|
| Treatment protocol |
JSC25 cells were grown in 7 mL YPD overnight to reach an OD600 of 0.2. Yeast cells (5×107) were collected by centrifugation and arrested in G1 in 1 mL YPD containing 2.5 μg α-factor at 300. After two hours, H2O2 was added into the cell culture. Cell culture was incubated at 300 for another hour. Yeast cells were then washed twice to remove α-factor and H2O2 before plating. Yeast cells were cultured at 300 for 3 days and sectored colonies were selected for whole genome SNP microarray analysis.
|
| Growth protocol |
Yeast cells were grown in YPD medium at 30 degree.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA were prepared by standard procedures (St. Charles et al., 2012)
|
| Label |
Cy3
|
| Label protocol |
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 |
JSC24 and JSC25 have similar genotypes. Both of these two strains were constructed by crossing PSL2 (isogenic with W303-1A) and PSL5 (isogenic with YJM789). The MATα gene was replaced by NAT marker in JSC24, while was replaced by HYG marker in JSC25. JSC25 is heterozygous for all SNPs that distinguish W303-1A and YJM789 except for a small region (SGD coordinates 369892-373127) on chromosome XV that is homozygous for YJM789-derived SNPs. JSC24 has no homologous regions on its genome.
|
| Data processing |
The data generated by GenePix Pro were exported to text files and analyzed with Microsoft Excel. The 635 nm/532 nm ratio was analyzed for each oligonucleotide. The average value of the median ratios of the control probes was calculated and used to normalize the ratios of the experimental probes to a value of 1 by dividing each probe ratio by the average control probe ratio. 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.
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| |
|
| Submission date |
Nov 13, 2017 |
| Last update date |
Aug 10, 2018 |
| Contact name |
Daoqiong Zheng |
| E-mail(s) |
[email protected]
|
| 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) |
| GSE106816 |
Effects of oxidative stress on mitotic recombination and genomic stability in yeast |
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