|
| Status |
Public on Jan 30, 2006 |
| Title |
S. cerevisiae rad53 +HU 2hr |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
rad53 G1 phase
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
ssDNA in rad53 G1 phase
|
| Treatment protocol |
Log phase cells were arrested in G1 phase by 200 nM alpha factor until the unbudded cells reach over 95%
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
genomic DNA extracted as described on http://fangman-brewer.genetics.washington.edu/DNA_prep.html
|
| Label |
Cy5
|
| Label protocol |
Five micrograms of genomic DNA were digested with EcoRI for 3 hours at 37oC, followed by ethanol precipitation. The DNA was then used as template for random primed labeling at 37oC for 2.5 hours. The reaction contains 50 mM Tris-HCl, pH 6.8; 5 mM MgCl2; 10 mM -mercaptoethanol; 300 ug/ml random hexamers; 0.12 mM of each dATP, dCTP, and dGTP; 0.06 mM dTTP; 1 mM Tris-HCl, pH 8.0; 3 ul of Cy5-conjugated dUTP (Amersham) and 50 units of exonuclease deficient Klenow. The labeling reaction was performed WITHOUT denaturing the template DNA. The labeled DNA was purified through a sephadex G50 column.
|
| |
|
| Channel 2 |
| Source name |
rad53 +HU 2hr
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
ssDNA in rad53 +HU 2hr S phase
|
| Treatment protocol |
Alpha factor arrested cells were released by addition of pronase at 0.02 mg/ml into media containing 200 mM HU. Cells were collected at 1hr post release.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
genomic DNA extracted as described on http://fangman-brewer.genetics.washington.edu/DNA_prep.html
|
| Label |
Cy3
|
| Label protocol |
Five micrograms of genomic DNA were digested with EcoRI for 3 hours at 37oC, followed by ethanol precipitation. The DNA was then used as template for random primed labeling. The reaction contains 50 mM Tris-HCl, pH 6.8; 5 mM MgCl2; 10 mM -mercaptoethanol; 300 ug/ml random hexamers; 0.12 mM of each dATP, dCTP, and dGTP; 0.06 mM dTTP; 1 mM Tris-HCl, pH 8.0; 3 ul of Cy3-conjugated dUTP (Amersham) and 50 units of exonuclease deficient Klenow. The labeling reaction was performed WITHOUT denaturing the template DNA. The labeled DNA was purified through a sephadex G50 column.
|
| |
|
| |
| Description |
S. cerevisiae rad53 in HM14-3a background (MATa bar1 trp1-289 leu2-3,112 ura3-52::URA3 his6 RAD53::rad53K227A(KanMX4)) cells
|
| Data processing |
Background subtracted median values from the two channels were used to calculate the ratio of Cy3:Cy5 as the raw ratio of ssDNA (S/G1) (in table below). The raw ratio was then normalized by the total amount of ssDNA in both the G1 and S phase samples by slot blotting and hybridization experiments (Table 1 in GSE4099). The raw normalized ratios of ssDNA for all genomic locations were then smoothed over a 4kb window by Fourier transformation (Table 2 in GSE4099).
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| |
|
| Submission date |
Jan 24, 2006 |
| Last update date |
Jan 30, 2006 |
| Contact name |
Bonita J Brewer |
| E-mail(s) |
bbrewer@gs.washington.edu
|
| Phone |
(206) 685-2870
|
| Fax |
(206) 543-0754
|
| Organization name |
University of Washington
|
| Department |
Genome Sciences
|
| Lab |
Brewer/Raghuraman
|
| Street address |
Box 357730
|
| City |
Seattle |
| State/province |
WA |
| ZIP/Postal code |
98195 |
| Country |
USA |
| |
|
| Platform ID |
GPL3388 |
| Series (1) |
| GSE4099 |
Single stranded DNA formation during S phase in the presence of hydroxyurea in S. cerevisiae and S. pombe |
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