|
| Status |
Public on Apr 29, 2017 |
| Title |
SJR4317-sectored_6W |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
45-minute DSB induction
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
ploidy: diploid (W303 x YJM789)
dsb on chromosome iv: Yes
|
| Treatment protocol |
45- or 90-minute of galactose treatment to induce I-SceI cleavage on chromosome IV.
|
| Growth protocol |
Prior to I-SceI induction, yeast cells were grown overnight at 30°C in 5 ml of non-selective medium containing 1% yeast extract, 2% bacto-peptone and 2% raffinose (YEPR). Following overnight growth, cells were then diluted to an OD of 0.4 in fresh YEPR medium and split into two parallel cultures. When cells reached an OD of 0.8-1, galactose (2% final concentration) was added to one culture of each pair and cells were incubated for an additional 45 or 90 minutes. Following induction for the specified time, yeast cells were pelleted and resuspended in water. Approximately 500 cells/plate were spread on synthetic dextrose medium supplemented with uracil and all amino acids except arginine. Adenine was additionally present at 10 μg/ml, a limiting amount that allows red color development of Ade- cells. Plates were incubated at 30°C overnight and at room temperature for additional 2 days before being moved to 4°C to allow red pigment development.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells in each half of a sectored colony were purified by streaking on YEP-glucose medium, and a single colony from each was used for DNA isolation. Genomic DNA was extracted in agarose plugs and then sheared to 200-400 bp by sonication.
|
| Label |
Cy5
|
| Label protocol |
DNA from each sector was labeled with Cy5-dUTP and mixed with control parental DNA labeled with Cy3-dUTP.
|
| |
|
| Channel 2 |
| Source name |
uninduced parental control SJR4317
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
ploidy: diploid (W303 x YJM789)
dsb on chromosome iv: No
|
| Treatment protocol |
45- or 90-minute of galactose treatment to induce I-SceI cleavage on chromosome IV.
|
| Growth protocol |
Prior to I-SceI induction, yeast cells were grown overnight at 30°C in 5 ml of non-selective medium containing 1% yeast extract, 2% bacto-peptone and 2% raffinose (YEPR). Following overnight growth, cells were then diluted to an OD of 0.4 in fresh YEPR medium and split into two parallel cultures. When cells reached an OD of 0.8-1, galactose (2% final concentration) was added to one culture of each pair and cells were incubated for an additional 45 or 90 minutes. Following induction for the specified time, yeast cells were pelleted and resuspended in water. Approximately 500 cells/plate were spread on synthetic dextrose medium supplemented with uracil and all amino acids except arginine. Adenine was additionally present at 10 μg/ml, a limiting amount that allows red color development of Ade- cells. Plates were incubated at 30°C overnight and at room temperature for additional 2 days before being moved to 4°C to allow red pigment development.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells in each half of a sectored colony were purified by streaking on YEP-glucose medium, and a single colony from each was used for DNA isolation. Genomic DNA was extracted in agarose plugs and then sheared to 200-400 bp by sonication.
|
| Label |
Cy3
|
| Label protocol |
DNA from each sector was labeled with Cy5-dUTP and mixed with control parental DNA labeled with Cy3-dUTP.
|
| |
|
| |
| Hybridization protocol |
Mixed labeled DNA was competitively hybridized to a SNP microarray, and the ratio of Cy5 to Cy3 hybridization to each SNP was determined. Each SNP was represented by four 25-nt oligonucleotides that corresponded to the Watson and Crick strands of each homolog.
|
| Scan protocol |
Scanned on an Agilent scanner (GenePix 4000B).
|
| Description |
SJR4317 isolate#2 45-minute galactose induction, white sector6
|
| Data processing |
The ratio of the medians (635 nm/532 nm) for each probe was used for analysis, and replicate probe medians were averaged (St. Charles et al., 2012).
|
| |
|
| Submission date |
Apr 28, 2017 |
| Last update date |
Jan 23, 2018 |
| Contact name |
Yee Fang Hum |
| E-mail(s) |
yfhum728@gmail.com
|
| Phone |
9196845891
|
| Organization name |
Duke University
|
| Department |
Molecular Genetics and Microbiology
|
| Lab |
Jinks-Robertson
|
| Street address |
381 CARL Building
|
| City |
Durham |
| State/province |
NC |
| ZIP/Postal code |
27710 |
| Country |
USA |
| |
|
| Platform ID |
GPL21553 |
| Series (1) |
| GSE98349 |
Mapping of mitotic gene conversion tracts associated with repair of a defined double-strand break (DSB) using SNP microarray |
|
