|
| Status |
Public on Jun 27, 2017 |
| Title |
YYy310.9-G1-15J-17R3 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
cell culture of strain YYy310.9-G1-15J-17R3
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
strain: YYy310.9-G1-15J-17R3
|
| Treatment protocol |
Yeast cells were collected after culture in YPD medium for 20 hours.
|
| Growth protocol |
Yeast cells were grown in YPD medium at 30 degree.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Approximately 55 mg of the pelleted culture was resuspended in 500 ml of a melted agarose solution (0.5% low melt agarose, 100 mM EDTA pH 7.5 at approximately 42°), and then 20 μl of a 25 mg/ml Zymolase solution was added. This cell-agarose mixture was distributed into five plug molds and allowed to solidify. The plugs were then incubated in 700 μl of an EDTA/Tris solution (500 mM EDTA, 10 mM Tris, pH 7.5) for 5 hours at 37°. We then add 400 μl of a sarcosyl/proteinase K solution (5% sarcosyl, 5 mg/ml proteinase K, 500 mM EDTA pH 7.5) to the tubes containing the plugs, and incubate at 50° for 12 hours. DNA was isolated from three plugs per strain using the Fermentas Life Sciences GeneJet Gel Extraction Kit. Following the addition of the “Binding” buffer from the kit, the samples were incubated at room temperature (~25°) until the agarose had melted and were then incubated on ice for 5 minutes. These samples were sonicated using a BioRupter sonicater for two 15-minute sessions of 30-second high pulses, followed by 30 seconds without sonication, resulting in DNA fragments of about 200-300 bp. Following sonication, the samples were treated according to the kit protocol.
|
| Label |
Cy5
|
| Label protocol |
The DNA was labeled using either Cy3-dUTP (control) or Cy5-dUTP (samples) using Invitrogen Bioprime Array CGH Genome Labeling Module using the kit protocol.
|
| |
|
| Channel 2 |
| Source name |
cell culture of JSC24-2
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
reference strain: JSC24-2
|
| Treatment protocol |
Yeast cells were collected after culture in YPD medium for 20 hours.
|
| Growth protocol |
Yeast cells were grown in YPD medium at 30 degree.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Approximately 55 mg of the pelleted culture was resuspended in 500 ml of a melted agarose solution (0.5% low melt agarose, 100 mM EDTA pH 7.5 at approximately 42°), and then 20 μl of a 25 mg/ml Zymolase solution was added. This cell-agarose mixture was distributed into five plug molds and allowed to solidify. The plugs were then incubated in 700 μl of an EDTA/Tris solution (500 mM EDTA, 10 mM Tris, pH 7.5) for 5 hours at 37°. We then add 400 μl of a sarcosyl/proteinase K solution (5% sarcosyl, 5 mg/ml proteinase K, 500 mM EDTA pH 7.5) to the tubes containing the plugs, and incubate at 50° for 12 hours. DNA was isolated from three plugs per strain using the Fermentas Life Sciences GeneJet Gel Extraction Kit. Following the addition of the “Binding” buffer from the kit, the samples were incubated at room temperature (~25°) until the agarose had melted and were then incubated on ice for 5 minutes. These samples were sonicated using a BioRupter sonicater for two 15-minute sessions of 30-second high pulses, followed by 30 seconds without sonication, resulting in DNA fragments of about 200-300 bp. Following sonication, the samples were treated according to the kit protocol.
|
| Label |
Cy3
|
| Label protocol |
The DNA was labeled using either Cy3-dUTP (control) or Cy5-dUTP (samples) using Invitrogen Bioprime Array CGH Genome Labeling Module using the kit protocol.
|
| |
|
| |
| 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, pre-warmed to 37C).
|
| Scan protocol |
We acquired the microarray images with a GenePix 4000B scanner and analyzed the images with the GenePix Pro 6.0 software
|
| Description |
Genotype of YYy310.9-G1-15J-17R3
|
| Data processing |
The data generated by GenePix Pro were exported to text files and analyzed with R. 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 ratio of the entire probe set.
Ratio of the medians (635 nm/532 nm).
|
| |
|
| Submission date |
Jun 26, 2017 |
| Last update date |
Jan 23, 2018 |
| Contact name |
Yi Yin |
| E-mail(s) |
yy224@uw.edu
|
| Organization name |
University of Washington
|
| Department |
Genome Sciences
|
| Lab |
Shendure Lab
|
| Street address |
3720 15th Ave NE
|
| City |
Seattle |
| ZIP/Postal code |
98195 |
| Country |
USA |
| |
|
| Platform ID |
GPL20144 |
| Series (1) |
| GSE100497 |
High-resolution mapping of heteroduplex DNA formed in UV-induced and spontaneous mitotic recombination events in yeast |
|
