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Sample GSM6204754 Query DataSets for GSM6204754
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) [email protected]
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

Data table header descriptions
ID_REF
VALUE Values shown are normalized % replication at the indicated probe locations, calculated as 100*(HL/2)/((HL/2) + HH)). For loess-smoothed % replication values at evenly-spaced chromosomal coordinates, see the associated 35_rDNA_Replication_Smoothed and 180_rDNA_Replication_Smoothed files. For a detailed description of this protocol, see Peng et al. (2014), Methods Mol Biol 1170:477-99 (PMCID: PMC4338859).

Data table
ID_REF VALUE
A_75_P01000003 16.07
A_75_P01000016 19.56
A_75_P01000071 16.67
A_75_P01000148 20.24
A_75_P01000219 7.92
A_75_P01000274 24.41
A_75_P01000289 24.13
A_75_P01000310 27.23
A_75_P01000338 25.68
A_75_P01000360 24.25
A_75_P01000376 27.12
A_75_P01000394 26.68
A_75_P01000410 24.32
A_75_P01000426 26.70
A_75_P01000446 25.01
A_75_P01000473 23.67
A_75_P01000507 27.85
A_75_P01000523 25.68
A_75_P01000549 24.06
A_75_P01000563 27.45

Total number of rows: 41480

Table truncated, full table size 846 Kbytes.




Supplementary file Size Download File type/resource
GSM6204754_35_rDNA_tp40_251481011817_201611041215_S01.txt.gz 4.6 Mb (ftp)(http) TXT
Processed data included within Sample table

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