|
| Status |
Public on Feb 20, 2014 |
| Title |
Origin mapping WT, replicate 2, dye-swap 1 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
G2 block
|
| Organism |
Schizosaccharomyces pombe |
| Characteristics |
strain: PN292
cell type: cdc25-22
phase: G2 block
nitrogen: N/A
|
| Treatment protocol |
Cells were treated with 12 mM hydroxyurea (HU) at the time of release from restrictive temperature for cdc25-22 or at the return to nitrogen-rich conditions for wild-type -N samples. HU was added at the time of shift to 34ºC for pat1-114 cells. Cells were harvested at the time of HU addition as well as during S phase: cdc25-22, 90 minutes after return to permissive temperature; wild-type and pat1-114 -N, 4 h after addition of nitrogen; pat1-114 +N, 2h after release from M-factor arrest; pat1-114 -N/Cdc45, 5 hours after addition of nitrogen.
|
| Growth protocol |
cdc25-22: haploid cdc25-22 cells were grown in EMM4S to exponential phase at 25ºC, shifted to 36.5ºC for 4 h to arrest cells in G2, then returned to 25ºC for synchronous mitotic cycles. WT or pat1-114, -N: cells were grown in EMM4S at 25ºC, starved of nitrogen for 16 hours, then returned to nitrogen-rich conditions at 34ºC. pat1-114, +N: cells were grown in EMM4S at 25ºC, treated with M-factor for 4.5 h to arrest in G1, then washed and grown in EMM4S at 34ºC.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA extraction was performed as in Hoffman and Winston, Gene, 1987.
|
| Label |
Alexa 555
|
| Label protocol |
Genomic DNA samples were labeled using the BioPrime Plus Array CGH Indirect Genomic Labeling System (Invitrogen) with either Alexa 555 or Alexa 647 dyes.
|
| |
|
| Channel 2 |
| Source name |
G2 block - S phase
|
| Organism |
Schizosaccharomyces pombe |
| Characteristics |
strain: PN292
cell type: cdc25-22
phase: G2 block - S phase
nitrogen: N/A
|
| Treatment protocol |
Cells were treated with 12 mM hydroxyurea (HU) at the time of release from restrictive temperature for cdc25-22 or at the return to nitrogen-rich conditions for wild-type -N samples. HU was added at the time of shift to 34ºC for pat1-114 cells. Cells were harvested at the time of HU addition as well as during S phase: cdc25-22, 90 minutes after return to permissive temperature; wild-type and pat1-114 -N, 4 h after addition of nitrogen; pat1-114 +N, 2h after release from M-factor arrest; pat1-114 -N/Cdc45, 5 hours after addition of nitrogen.
|
| Growth protocol |
cdc25-22: haploid cdc25-22 cells were grown in EMM4S to exponential phase at 25ºC, shifted to 36.5ºC for 4 h to arrest cells in G2, then returned to 25ºC for synchronous mitotic cycles. WT or pat1-114, -N: cells were grown in EMM4S at 25ºC, starved of nitrogen for 16 hours, then returned to nitrogen-rich conditions at 34ºC. pat1-114, +N: cells were grown in EMM4S at 25ºC, treated with M-factor for 4.5 h to arrest in G1, then washed and grown in EMM4S at 34ºC.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA extraction was performed as in Hoffman and Winston, Gene, 1987.
|
| Label |
Alexa 647
|
| Label protocol |
Genomic DNA samples were labeled using the BioPrime Plus Array CGH Indirect Genomic Labeling System (Invitrogen) with either Alexa 555 or Alexa 647 dyes.
|
| |
|
| |
| Hybridization protocol |
Labeled samples from before and after S phase entry were added to hybridization buffer (50 mM MES pH 6.7, 500 mM NaCl, 6 mM EDTA, 0.5% Sarcosine, 30% Formamide, 1.5 ng/µl herring sperm DNA, 0.8 µg/µl yeast tRNA, 0.05µg/µl Cot1 DNA). Samples were incubated in an Agilent hybridization oven at 42ºC for 18 hours, and washes were performed for 5 min at room temperature (wash 1: 6xSSPE, 0.005% N-Lauroylsarcosine, wash 2: 0.06x SSPE).
|
| Scan protocol |
Sildes were scanned using an Agilent G2565BA scanner.
|
| Description |
Ratio of S phase to G2 block
|
| Data processing |
Agilent Feature Extraction Software was used to obtain data from scanned slides.
|
| |
|
| Submission date |
Jan 07, 2014 |
| Last update date |
Feb 20, 2014 |
| Contact name |
Pei-Yun Jenny Wu |
| E-mail(s) |
pei-yun.wu@ibgc.cnrs.fr
|
| Organization name |
Institute of Biochemistry and Cellular Genetics
|
| Lab |
CNRS UMR 5095
|
| Street address |
1 Rue Camille Saint Saens
|
| City |
Bordeaux |
| ZIP/Postal code |
33077 |
| Country |
France |
| |
|
| Platform ID |
GPL16383 |
| Series (2) |
| GSE53879 |
Replication origin mapping of mitotic and meiotic cells in different conditions |
| GSE53921 |
Replication origin selection organizes meiotic recombination |
|
