|
| Status |
Public on Dec 31, 2020 |
| Title |
hpr1Δ_37C_25 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
hpr1Δ_G1
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
strain: BY4741 hpr1<delta>
growth phase: G1 control
|
| Growth protocol |
Cells grown exponentially at 30°C were synchronized in G1 by 3 µM α factor. Fifteen minutes prior to the release into S phase by 0.3 mg/ml pronase addition, half of the cells were shifted to 37°C and used for ssDNA mapping. As control (for flow cytometry analysis) the other half of the culture were maintained at 30°C and released into S phase at 30°C. Following the release into S phase, cells were sampled at 10, 15, 20 and 25 min and subjected to ssDNA labeling.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
ssDNA-labeled chromosomal DNA was extracted from agarose plugs by beta-agarase digestion and sonicated in a Bioruptor to reduce DNA fragment size to an average of 500 bp.
|
| Label |
Cy5
|
| Label protocol |
Agarose-embedded chromosomal DNA samples were differentially labeled for ssDNA by random-primed DNA synthesis by Klenow (Exo-), with Cy5- or Cy3-conjugated dUTP. All procedures were performed as previously described (Feng et al., G3 (Bethesda) 2011 Oct;1(5):327-35. doi: 10.1534/g3.111.000554).
|
| |
|
| Channel 2 |
| Source name |
hpr1Δ_37C_25
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
strain: BY4741 hpr1<delta>
growth phase: S
time point: 25 min post shift to 37C
|
| Growth protocol |
Cells grown exponentially at 30°C were synchronized in G1 by 3 µM α factor. Fifteen minutes prior to the release into S phase by 0.3 mg/ml pronase addition, half of the cells were shifted to 37°C and used for ssDNA mapping. As control (for flow cytometry analysis) the other half of the culture were maintained at 30°C and released into S phase at 30°C. Following the release into S phase, cells were sampled at 10, 15, 20 and 25 min and subjected to ssDNA labeling.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
ssDNA-labeled chromosomal DNA was extracted from agarose plugs by beta-agarase digestion and sonicated in a Bioruptor to reduce DNA fragment size to an average of 500 bp.
|
| Label |
Cy3
|
| Label protocol |
Agarose-embedded chromosomal DNA samples were differentially labeled for ssDNA by random-primed DNA synthesis by Klenow (Exo-), with Cy5- or Cy3-conjugated dUTP. All procedures were performed as previously described (Feng et al., G3 (Bethesda) 2011 Oct;1(5):327-35. doi: 10.1534/g3.111.000554).
|
| |
|
| |
| Hybridization protocol |
In each experiment a G1 control sample and an S phase sample (released into S phase at 37C for 10, 15, 20 and 25 min) were paired and the DNA cohybridized to Agilent G4493A Yeast Whole Genome ChIP-on-Chip 4x44k microarrays.
|
| Scan protocol |
Scanned on an Agilent G2565B scanner.
|
| Description |
hpr1_37C_25min
BY4741 hpr1Δ
ssDNA
|
| Data processing |
Images were quantified using Agilent Feature Extraction Software (version A.8.5.1.1).
Agilent Feature Extraction Software (v 9.5.1) was used for background subtraction and LOWESS normalization. Chromosome coordinates (kb) for each probe were extracted.
|
| |
|
| Submission date |
Jun 04, 2020 |
| Last update date |
Jan 01, 2021 |
| Contact name |
Wenyi Feng |
| E-mail(s) |
[email protected]
|
| Phone |
3154648701
|
| Organization name |
SUNY Upstate Medical University
|
| Department |
Biochemistry and Molecular Biology
|
| Lab |
Wenyi Feng
|
| Street address |
750 East Adams Street
|
| City |
Syracuse |
| State/province |
NY |
| ZIP/Postal code |
13210 |
| Country |
USA |
| |
|
| Platform ID |
GPL10930 |
| Series (1) |
| GSE151849 |
Centromeric R loops contribute to defects in kinetochore assembly and chromosomal instability |
|
