|
| Status |
Public on Feb 17, 2023 |
| Title |
35 rDNA strain, 40 min S phase |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
40 min S phase, HH (unreplicated) DNA, 35 copies rDNA
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
cell cycle phase: S phase
s phase time: 40 min
fraction: HH (unreplicated) DNA
|
| Treatment protocol |
Cells were released into S phase by addition of Pronase. We collected samples at intervals through the ensuing S phase (30,35,40,45,50 min for 35 rDNA strain; 25,30,35,40,45,55 min for 180 rDNA strain).
|
| Growth protocol |
We grew cells in isotopically dense (13C-glucose, 15N-ammonium sulfate) for ≥7 population doublings. We then arrested the cells in G1 using a-factor, filtered and resuspended the cells in isotopically light (12C, 14N) medium.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
DNA was extracted using glass beads and phenol/chloroform (standard smash-and-grab method). The DNA was digested with EcoRI. Heavy-heavy (HH) and heavy-light (HL) DNA were separated by ultracentrifugation in cesium chloride gradients. The gradients were drip-fractionated. HH and HL portions of the gradient were identified by slot blot hybridization. The DNA was recovered from CsCl by ethanol precipitation.
|
| Label |
Cy3
|
| Label protocol |
200 ng of HH or HL DNA were labeled with Cy3 and Cy5, respectively, using random-primed synthesis with exonuclease-deficient Klenow.
|
| |
|
| Channel 2 |
| Source name |
40 min S phase, HL (replicated) DNA, 35 copies rDNA
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
cell cycle phase: S phase
s phase time: 40 min
fraction: HL (replicated) DNA
|
| Treatment protocol |
Cells were released into S phase by addition of Pronase. We collected samples at intervals through the ensuing S phase (30,35,40,45,50 min for 35 rDNA strain; 25,30,35,40,45,55 min for 180 rDNA strain).
|
| Growth protocol |
We grew cells in isotopically dense (13C-glucose, 15N-ammonium sulfate) for ≥7 population doublings. We then arrested the cells in G1 using a-factor, filtered and resuspended the cells in isotopically light (12C, 14N) medium.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
DNA was extracted using glass beads and phenol/chloroform (standard smash-and-grab method). The DNA was digested with EcoRI. Heavy-heavy (HH) and heavy-light (HL) DNA were separated by ultracentrifugation in cesium chloride gradients. The gradients were drip-fractionated. HH and HL portions of the gradient were identified by slot blot hybridization. The DNA was recovered from CsCl by ethanol precipitation.
|
| Label |
Cy5
|
| Label protocol |
200 ng of HH or HL DNA were labeled with Cy3 and Cy5, respectively, using random-primed synthesis with exonuclease-deficient Klenow.
|
| |
|
| |
| Hybridization protocol |
Hybridization and washing were done using standard conditions specified by Agilent (using Agilent Blocking Agent and HI-rPM hybridization buffer).
|
| Scan protocol |
Arrays were scanned on an Agilent Technologies Scanner G2505C and features were extracted using protocol CGH 105 Dec08 CP in feature extractor (FE) v. 10.7.3.1
|
| Description |
HH and HL DNA were separated by CsCl density gradient centrifugation
|
| Data processing |
After extraction of hybridization intensities, we used the known % replication values from the slot blot analysis to normalize the overall % replication in each sample, with an added correction for the percentage of cells in the population that successfully completed S phase (obtained from the maximum % replication seen at the end of S phase from the slot blot analysis).
|
| |
|
| Submission date |
May 30, 2022 |
| Last update date |
Feb 19, 2023 |
| Contact name |
Mosur K Raghuraman |
| E-mail(s) |
raghu@u.washington.edu
|
| Organization name |
University of Washington
|
| Department |
Genome Sciences Box 355065
|
| Lab |
Brewer/Raghuraman
|
| Street address |
Foege Bldg, 3720 15th Ave NE
|
| City |
Seattle |
| State/province |
WA |
| ZIP/Postal code |
98195 |
| Country |
USA |
| |
|
| Platform ID |
GPL10930 |
| Series (1) |
| GSE205068 |
Ribosomal DNA replication time coordinates completion of genome replication and anaphase in yeast |
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