|
| Status |
Public on Aug 14, 2007 |
| Title |
SP HU syn WT harvested at 005 min Bio_2 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
Fission yeast cells
|
| Organism |
Schizosaccharomyces pombe |
| Characteristics |
Schizosaccharomyces pombe wild type (WT) cells were synchronized by treating the cells with 8 mM Hydroxyurea (HU+) for 3 hrs, and the cells were released in HU free media and harvested after 5 min.
|
| Growth protocol |
Yeast cells were cultured at 30 degree centigrade.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
DNA of the cells were extracted as describled in materials and metholds.
|
| Label |
Cy5
|
| Label protocol |
Sheared DNA was random-priming labeled with amino-allyl-dUTP (aa-dUTP) using the BioPrime DNA Labeling kit (Invitrogen Corporation, Carlsbad, CA) according the manufacture¡¯s instruction. Labeled DNA was extracted with phenol/chloroform/isoamyl alcohol and precipitated with ethanol or siopropanol. The labeled DNA was then coupled with cyanine dyes Cy5 (Amersham, Buckinghamshire, UK)
|
| |
|
| Channel 2 |
| Source name |
Fission yeast arrested cells with 8 mM Hydroxyurea for 3 hrs.
|
| Organism |
Schizosaccharomyces pombe |
| Characteristics |
Reference DNA was obtained by extracting the DNA from the cells treated with 8 mM HU for 3 hrs.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
DNA of the cells were extracted as describled in materials and metholds.
|
| Label |
Cy3
|
| Label protocol |
Sheared DNA was random-priming labeled with amino-allyl-dUTP (aa-dUTP) using the BioPrime DNA Labeling kit (Invitrogen Corporation, Carlsbad, CA) according the manufacture¡¯s instruction. Labeled DNA was extracted with phenol/chloroform/isoamyl alcohol and precipitated with ethanol or siopropanol. The labeled DNA was then coupled with cyanine dyes Cy3 (Amersham, Buckinghamshire, UK)
|
| |
|
| |
| Hybridization protocol |
Samples were mixed with DIG Easy Hyb buffer (Roche) and Herring Sperm DNA (Invitrogen) before applying to MAUI mixer-slide assembly. The slides were hybridized overnight for 16 hours at 42¡ãC under MAUI system (BioMicro). Hybridized slides were washed consecutively in 2¡Á SSC/0.1% SDS for 1 min, 1¡Á SSC for 4 min, 0.2¡Á SSC for 4 min, and 0.05¡Á SSC for 1 min, and then spin-dried before scanning.
|
| Scan protocol |
Fluorescent array images were collected for both Cy3 and Cy5 with a GenePix 4000A fluorescent scanner and image intensity data were extracted and analyzed with GenePix Pro 4.0 analysis software.
|
| Description |
Schizosaccharomyces pombe cells synchronized with 8 mM HU for 3 hrs and released in HU free media at 0 time points vs Schizosaccharomyces pombe cells synchronized with 8 mM HU for 3 hrs.
|
| Data processing |
Fluorescent array images were collected for both Cy3 and Cy5 with a GenePix 4000A fluorescent scanner and image intensity data were extracted and analyzed with GenePix Pro 4.0 analysis software. After background correction and removal of flagged values, features with low intensity (F/B<2 at either 635 or 532 channel) were removed. Meidans of log base 2 expression ratios were given in the data table.
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| |
|
| Submission date |
Feb 07, 2007 |
| Last update date |
Aug 14, 2007 |
| Contact name |
Majid Eshaghi |
| E-mail(s) |
meshaghi@hotmail.com
|
| Organization name |
Genome Institute of Singapore
|
| Department |
Bilogical Investigation
|
| Lab |
System Biology
|
| Street address |
Biopolis
|
| City |
Singapore |
| State/province |
Singapore |
| ZIP/Postal code |
138672 |
| Country |
Singapore |
| |
|
| Platform ID |
GPL1932 |
| Series (1) |
| GSE6977 |
Genome-wide Profiling of DNA Replication Timing in pombe. |
|
| Data table header descriptions |
| ID_REF |
|
| VALUE |
same as UNF_VALUE but with flagged values removed |
| CH1_Median |
CH1 (F635) median fluorescence intensity |
| CH1_BKD |
CH1 (B635) background median fluorescence intensity |
| CH2_Median |
CH2 (F532) median fluorescence intensity |
| CH2_BKD |
CH2 (B532) background median fluorescence intensity |
| CH1_Median - CH1_BKD |
Channel 1 median signal - absolute intensity |
| CH2_Median - CH2_BKD |
Channel 2 median signal - absolute intensity |
| Flags |
Denotes which features met our filtering criterion. A negative value means that the feature did not have at least 60% of its pixels greater than two standard deviations over the background intensity. |
| 2Fold_F/B |
Denotes which features met our filtering criterion. Zero value means that the feature whose intensity did not pass either F635/B635>=2 or F532/B532>=2, that is, low-intensity features. |
| UNF_VALUE |
Median of log2 ratio defined by CH1/ CH2 |
