|
Status |
Public on Jan 30, 2006 |
Title |
S. pombe WT |
Sample type |
genomic |
|
|
Channel 1 |
Source name |
WT G1 phase
|
Organism |
Schizosaccharomyces pombe |
Characteristics |
ssDNA in WT G1 phase
|
Treatment protocol |
Log phase cells were arrested in G1 phase by nitrogen starvation
|
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 |
WT +HU early S phase
|
Organism |
Schizosaccharomyces pombe |
Characteristics |
ssDNA in WT in early S phase in HU
|
Treatment protocol |
Log phase cells were arrested in early S phase by treatment of 12 mM HU for 3 hours at 30oC
|
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. pombe WT cells (h-)
|
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 3 in GSE4099). The raw normalized ratios of ssDNA for all genomic locations were then smoothed over a 4kb window by Fourier transformation (Table 4 in GSE4099).
|
|
|
Submission date |
Jan 30, 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 |
GPL3398 |
Series (1) |
GSE4099 |
Single stranded DNA formation during S phase in the presence of hydroxyurea in S. cerevisiae and S. pombe |
|