|
| Status |
Public on Aug 26, 2011 |
| Title |
BrdUIP_hsk1ts_mrc1∆_30ºC_60minHU |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
BrdU IPed from BrdU-incorporated DNA
|
| Organism |
Schizosaccharomyces pombe |
| Characteristics |
cell type: hsk1-89, mrc1∆
cell cycle: Early S-phase in HU at 60min after release from metaphase arrst
sample type: BrdU
antibody: Anti-bromodeoxyuridine (MBL)
|
| Treatment protocol |
The stains were treated with 200µM BrdU and 25mM Hydroxyurea after Metaphase arrest.
|
| Growth protocol |
The stains were grown until exponential phase in YES medium and arrest at Metaphase at 20ºC for 5hr or 8hr. The arrested cells were released into subsequential phase at 30ºC for 60min.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Growing nda3-KM311 cells (1.0 x 10E9 cells) expressing Thymidine Kinase under control by Adh1 promoter, mrc1∆, and hsk1ts were arrested at metaphase and release in the presence of 200µM BrdU and 25mM HU at 30C for 60min. BrdU was incorporated into nascent DNA during the sub-sequential S-phase. The genomic DNA was extracted and purified by Qiagen Genomic DNA Buffer Set and Genomic-tip. BrdU-incorporated DNA after shearing and denature was immuno-precipitated by anti-bromodeoxyuridine (MBL) and purified by QIAquick PCR Purification Kit (QIAGEN). The catalog Number, Clone and Subclass of Anti-Bromodeoxyuridine (MBL) antibody are MI-11-3, 2B1 and mouse IgG1 respectively.
The purified Input, BrdUIP DNA were amplified by IVT as described previously (Liu, C. et al., 2003, BMC Genomics).
|
| Label |
Biotin
|
| Label protocol |
About 10µg amplified DNA was fragmented to 50bp with DNase I, and the ends of the fragments were labeled with biotin-6N-ddATP by terminal transferase.
|
| |
|
| Channel 2 |
| Source name |
Input DNA from Metaphase arrested cells
|
| Organism |
Schizosaccharomyces pombe |
| Characteristics |
sample type: input
|
| Treatment protocol |
The stains were treated with 200µM BrdU and 25mM Hydroxyurea after Metaphase arrest.
|
| Growth protocol |
The stains were grown until exponential phase in YES medium and arrest at Metaphase at 20ºC for 5hr or 8hr. The arrested cells were released into subsequential phase at 30ºC for 60min.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
The genomic DNA was extracted and purified from the arrested at M-phase in nda-KM311 cells. The genomic DNA was sheared by sonication.
The purified Input, BrdUIP DNA were amplified by IVT as described previously (Liu, C. et al., 2003, BMC Genomics).
|
| Label |
Biotin
|
| Label protocol |
About 10µg amplified DNA was fragmented to 50bp with DNase I, and the ends of the fragments were labeled with biotin-6N-ddATP by terminal transferase.
|
| |
|
| |
| Hybridization protocol |
6µg of DNA was hybridized to Affymetrix S. pombe Tiling 1.0FR Array using Affymetrix recommend component. The arrays were hybridized for 16 hours at 42ºC at 60 rpm using an Affymetrix hybridization oven.
|
| Scan protocol |
Standard protocol by Affymetrix
|
| Data processing |
For the discrimination of positive and negative signals for the binding, we compared ChIP fraction with Input fraction by using three criteria as follows. First, the reliability of strength of signal was judged by detection p-value of each locus (p-value lower than 0.025 was considered as significant). Secondly, reliability of binding ratio was judged by change p-value (p-value lower than 0.001 was considered as significant). Thirdly, clusters consisted of at least three contiguous loci which filled above two criteria were selected as significantly enriched locus, because it is apparent that a single site of protein-DNA interaction or BrdU-incorporation region will result in immuno-precipitation of DNA fragments that hybridized not only to the locus of the actual binding site but also to its neighbors. This third criterion is very unique, make the data highly reliable and can be only applicable to the chip data obtained by our high-resolution tiling array.
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| |
|
| Submission date |
Aug 25, 2011 |
| Last update date |
Aug 26, 2011 |
| Contact name |
Yutaka Kanoh |
| E-mail(s) |
kanou-yt@igakuken.or.jp
|
| Organization name |
Tokyo Metropolitan Institute of Medical Science
|
| Department |
Genome Medicine
|
| Lab |
Genome Dynamics Projec
|
| Street address |
2-1-6 Kamikitazawa, Setagaya-ku
|
| City |
Tokyo |
| ZIP/Postal code |
156-8506 |
| Country |
Japan |
| |
|
| Platform ID |
GPL7715 |
| Series (1) |
| GSE31650 |
Multiple bypass pathways of Hsk1 kinase essential for initiation of DNA replication in fission yeast |
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