|
| Status |
Public on Nov 21, 2017 |
| Title |
H2O2_20 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
cell culture of strain HOS20
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
ploidy: diploid strain
background strain: JSC25-1 (also called W303-1AⅹYJM789)
|
| Treatment protocol |
To allow accumulation of recombination events, 30 JSC25-1-derived isolated colonies were independently treated with H2O2 (100 mM) for 20 passages. In each passage, cells were treated with 100 mM H2O2 for 1 h, washed, and then plated on YPD medium to form colonies. After 20 sequential treatments of H2O2, 30 JSC25-1-derived mutants (named as HOS1-30) from the last treatment were examined by whole genome SNP microarrays.
|
| 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
ploidy: diploid strain
|
| Treatment protocol |
To allow accumulation of recombination events, 30 JSC25-1-derived isolated colonies were independently treated with H2O2 (100 mM) for 20 passages. In each passage, cells were treated with 100 mM H2O2 for 1 h, washed, and then plated on YPD medium to form colonies. After 20 sequential treatments of H2O2, 30 JSC25-1-derived mutants (named as HOS1-30) from the last treatment were examined by whole genome SNP microarrays.
|
| 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.
|
| 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.
|
| |
|
| Submission date |
Nov 20, 2017 |
| Last update date |
Jan 23, 2018 |
| 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) |
| GSE107178 |
Effects of oxidative stress on mitotic recombination and genomic stability in yeast after 20 passages |
|
