|
| Status |
Public on Mar 06, 2009 |
| Title |
cells at 90 minutes in HU after G2/M arrest; biol rep 2 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
cells in S phase, after 90 minutes in hydroxyurea
|
| Organism |
Schizosaccharomyces pombe |
| Characteristics |
genotype: h- [Msmt-0] cdc25-22 leu1-32 his7-366 leu1::pFS181(leu1 adh1:hENT1) pJL218 (his7 adh1:tk)
|
| Treatment protocol |
Cells were treated with 0.01% sodium azide and harvested by centrifugation, followed by washing with 50 mM EDTA and TE buffer.
|
| Growth protocol |
cdc25-22 cells were synchronized by growing in minimal medium plus supplements (EMM4S) at 25ºC to 1-2x106 cells/ml, shifting to 36.5ºC to block cells in late G2, followed by release at 25ºC. Treatments for specific samples are indicated.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA was prepared according to Hoffman and Winston, 1987 and purified using the Qiagen Genomic DNA kit. After sonication to 300-400 bp average length, 4-5 µg DNA was mixed with 40 µl BrdU antibody (anti-mouse, BD Biosciences) followed Protein G beads (GE Healthcare). Washes and elution were performed according to Martens and Winston, 2002.
|
| Label |
biotin
|
| Label protocol |
Prior to labeling, IP DNA was amplified by blunting using T4 DNA polymerase, followed by linker ligation and PCR amplification for 22 cycles and purification on a Qiagen PCR purification column.). 2 µg of DNA was fragmented to 50-100 bp in One-Phor-All Buffer Plus (GE Healthcare) using Genechip fragmentation reagent (Affymetrix) and biotin labeled using the BioArray Terminal Labeling Kit (Enzo Life Sciences).
|
| |
|
| Channel 2 |
| Source name |
non-replicating control_071306
|
| Organism |
Schizosaccharomyces pombe |
| Characteristics |
genotype: h- [Msmt-0] cdc25-22 leu1-32 his7-366 leu1::pFS181(leu1 adh1:hENT1) pJL218 (his7 adh1:tk)
dataset: "0"
|
| Treatment protocol |
Cells were treated with 0.01% sodium azide and harvested by centrifugation, followed by washing with 50 mM EDTA and TE buffer.
|
| Growth protocol |
cdc25-22 cells were synchronized by growing in minimal medium plus supplements (EMM4S) at 25ºC to 1-2x106 cells/ml, shifting to 36.5ºC to block cells in late G2, followed by release at 25ºC. Treatments for specific samples are indicated.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA was prepared according to Hoffman and Winston, 1987 and purified using the Qiagen Genomic DNA kit. After sonication to 300-400 bp average length, 4-5 µg DNA was mixed with 40 µl BrdU antibody (anti-mouse, BD Biosciences) followed Protein G beads (GE Healthcare). Washes and elution were performed according to Martens and Winston, 2002.
|
| Label |
biotin
|
| Label protocol |
Prior to labeling, IP DNA was amplified by blunting using T4 DNA polymerase, followed by linker ligation and PCR amplification for 22 cycles and purification on a Qiagen PCR purification column.). 2 µg of DNA was fragmented to 50-100 bp in One-Phor-All Buffer Plus (GE Healthcare) using Genechip fragmentation reagent (Affymetrix) and biotin labeled using the BioArray Terminal Labeling Kit (Enzo Life Sciences).
|
| |
|
| |
| Hybridization protocol |
Labeled DNA was hybridized to Affymetrix GeneChip S. pombe Tiling 1.0FR Arrays using standard protocols (20 hours at 45 ºC). After hybridization, GeneChips were stained with streptavidin-phycoerythrin, followed by an antibody solution (anti-streptavidin) and a second streptavidin-phycoerythrin solution, with all liquid handling performed by a GeneChip Fluidics Station 450.
|
| Scan protocol |
GeneChips were scanned with the Affymetrix GeneChip Scanner 3000.
|
| Description |
90_2: Cells synchronized at G2/M for 3h 45min were released into medium containing hydroxyurea at 25ºC for 90 minutes.
|
| Data processing |
The data were analyzed using the Affymetrix Tiling Array Software using default settings with linear output.
Chromosomal datasets were obtained by taking a ratio to the "0" dataset for normalization; all probes which returned a value of "1" were discarded. After averaging by the geometric mean for a sliding windows of 200 adjacent probes, we determined extrapolated values for every 500 bp. Data were then normalized to the median value of the dataset for each chromosome.
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| |
|
| Submission date |
Feb 17, 2009 |
| Last update date |
Feb 17, 2009 |
| Contact name |
Pei-Yun Jenny Wu |
| E-mail(s) |
pwu@rockefeller.edu
|
| Organization name |
Rockefeller University
|
| Street address |
1230 York Avenue
|
| City |
New York |
| State/province |
NY |
| ZIP/Postal code |
10021 |
| Country |
USA |
| |
|
| Platform ID |
GPL7715 |
| Series (1) |
| GSE14864 |
Establishing the program of origin firing during S phase in fission yeast |
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