|
Status |
Public on May 31, 2018 |
Title |
rad3∆_Replicate3_dye1 |
Sample type |
genomic |
|
|
Channel 1 |
Source name |
cdc25-22 rad3∆
|
Organism |
Schizosaccharomyces pombe |
Characteristics |
strain: cdc25-22 rad3∆; unreplicated DNA
|
Treatment protocol |
Cells were treated with 6mM HU 5 minutes after release from restrictive temperature. Unreplicated DNA samples were taken at this time.
|
Growth protocol |
S. pombe cells were grown in EMM6S to exponential phase at 25˚C and synchronized using a temperature shift (blocked 4h at 36.5˚C and shifted at 25˚C for release).
|
Extracted molecule |
genomic DNA |
Extraction protocol |
Genomic DNA extraction was performed as in Hoffman and Winston, Gene, 1987, and samples were purified using the Qiagen Genomic Tip-20.
|
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/Alexa 647 dyes or Cy3/Cy5 dyes (Amersham CyDye Post-Labelling Reactive Dye pack).
|
|
|
Channel 2 |
Source name |
cdc25-22 rad3∆
|
Organism |
Schizosaccharomyces pombe |
Characteristics |
strain: cdc25-22 rad3∆; S phase DNA
|
Treatment protocol |
Cells were treated with 6mM HU 5 minutes after release from restrictive temperature. S phase DNA samples were taken after a time when cells would normally have completed S phase in the absence of HU.
|
Growth protocol |
S. pombe cells were grown in EMM6S to exponential phase at 25˚C and synchronized using a temperature shift (blocked 4h at 36.5˚C and shifted at 25˚C for release).
|
Extracted molecule |
genomic DNA |
Extraction protocol |
Genomic DNA extraction was performed as in Hoffman and Winston, Gene, 1987, and samples were purified using the Qiagen Genomic Tip-20.
|
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/Alexa 647 dyes or Cy3/Cy5 dyes (Amersham CyDye Post-Labelling Reactive Dye pack).
|
|
|
|
Hybridization protocol |
Labeled samples from before and after S phase entry were quantified and 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 to 20 hours, and washes were performed for 5 min at room temperature (2X wash 1: 6xSSPE, 0.005% N-Lauroylsarcosine, 1X wash 2: 0.06x SSPE).
|
Scan protocol |
Scanned on an Agilent G2505C scanner.
|
Data processing |
Agilent Feature Extraction Software (v 10.7.3.1)
1) pre-Value column: the ratios of S phase vs. unreplicated DNA, 2) Value column: pre-Value is baseline corrected as part of the protocol for origin identification described in the paper.
BASELINE CORRECTION:To obtain origin efficiencies, the lowest ratios, which represent non-replicated DNA, were adjusted to be centered on a value of 1. This resulted in a correction of 0.25 for all datasets
|
|
|
Submission date |
May 02, 2017 |
Last update date |
May 31, 2018 |
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 |
GPL16235 |
Series (2) |
GSE98444 |
Replication origin mapping in cdc25-22 and cdc25-22 rad3∆ in S. pombe |
GSE98462 |
Genome-wide profiles of replication initiation, single-stranded DNA accumulation, and Rad52 binding in cells exposed to replication stress |
|