|
| Status |
Public on Dec 20, 2007 |
| Title |
SP HU+ Syn rad3 080 min R2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Fission yeast cells
|
| Organism |
Schizosaccharomyces pombe |
| Characteristics |
Schizosaccharomyces pombe elutriation centrifugation synchronized (Syn) cells of rad3 mutant released to early G2 phase at 80 min treated with 8 mM Hydroxurea (HU+) .
|
| Growth protocol |
Yeast cells were cultured at 30 degree centigrade.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA of tissue samples were extracted using hot phenol methold describled in materials and metholds.
|
| Label |
Cy5
|
| Label protocol |
For fluorescence labeling of cDNAs, ~30 micro gramme total RNA was used to synthesize cDNA coupled with amino allyl-dUTP (aa-dUTP) by reverse transcriptase (Superscript-II, Invitrogen, Carlsbad, CA) according to manufacturer¡¯s instruction. cDNA was subsequently washed with Milli-Q water using microcon-YM30 (Millipore, Billerica,MA). ~1.5 micro gramme cDNA was used to couple with Cy5-fluorescence dye for 1 h in dark and purified through a spin column (Qiagen, Hilden, Germany) followed by washing on a micorcon YM-30. Hybridization was performed using EasyHyb solution (Roche) in a MUAI mixer (BioMicro systems). Hybridized slides were washed in a series of buffers containing various concentration of SSC (2x - 0.2x) with or without 1% SDS.
|
| |
|
| Channel 2 |
| Source name |
Fission yeast wildtype cells.
|
| Organism |
Schizosaccharomyces pombe |
| Characteristics |
Reference RNA was obtained by pooling equal amount of total RNA extracted from Schizosaccharomyces pombe mid-log phase cells (OD600 was around 0.3) growing at 30 degree centigrade without supplement with HU.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA of tissue samples were extracted using hot phenol methold describled in materials and metholds.
|
| Label |
Cy3
|
| Label protocol |
For fluorescence labeling of cDNAs, ~30 micro gramme total RNA was used to synthesize cDNA coupled with amino allyl-dUTP (aa-dUTP) by reverse transcriptase (Superscript-II, Invitrogen, Carlsbad, CA) according to manufacturer¡¯s instruction. cDNA was subsequently washed with Milli-Q water using microcon-YM30 (Millipore, Billerica,MA). ~1.5 micro gramme cDNA was used to couple with Cy3-fluorescence dye for 1 h in dark and purified through a spin column (Qiagen, Hilden, Germany) followed by washing on a micorcon YM-30. Hybridization was performed using EasyHyb solution (Roche) in a MUAI mixer (BioMicro systems). Hybridized slides were washed in a series of buffers containing various concentration of SSC (2x - 0.2x) with or without 1% SDS.
|
| |
|
| |
| 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 elutriation centrifugation synchroinzed cells of wildtype and mutants indicated released to early G2 phase at indicated timepoints untreated (HU-) or treated (HU+) with 8 mM Hydroxyurea vs Schizosaccharomyces pombe asynchronized mid log phase WT cells (OD600 was around 0.3) without supplement with HU.
|
| 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.
|
| |
|
| Submission date |
Feb 20, 2006 |
| Last update date |
Dec 22, 2006 |
| Contact name |
Zhaoqing Chu |
| E-mail(s) |
chuz@gis.a-star.edu.sg
|
| Phone |
65-64788128
|
| Organization name |
Genome Institute of Singapore
|
| Street address |
60, Biopolis Streat
|
| City |
Singapore |
| ZIP/Postal code |
138672 |
| Country |
Singapore |
| |
|
| Platform ID |
GPL1932 |
| Series (2) |
| GSE4283 |
HU treated cells vs wildtype untreated cells (elutriation centrifugation synchronized cells) |
| GSE4284 |
Cell Cycle-specific Expressions Are Modulated by Replication Checkpoint Kinases Rad3 and Cds1 in Fission Yeast |
|
| Data table header descriptions |
| ID_REF |
Unique ID |
| 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 |
