|
| Status |
Public on Dec 13, 2011 |
| Title |
Mcm4ChIP_rif1∆_60minHU |
| Sample type |
genomic |
| |
|
| Source name |
Flag ChIPed from the strain expressing 3Flag-fused Mcm4 in rif1∆
|
| Organism |
Schizosaccharomyces pombe |
| Characteristics |
cell cycle: Early S-phase in HU at 60min after release from metaphase arrest
molecule tag: FLAG
antibody: Monoclonal ANTI-FLAG M2 antibody(Sigma), Subclass; IgG1, Clone; M2, Cat No.; F1804
|
| Treatment protocol |
The stains were treated with 200µM BrdU and 25mM Hydroxyurea after Metaphase arrest.
|
| Growth protocol |
Protocol 1: The stains were grown to early exponential phase in YES medium and arrested at Metaphase at 20ºC for 5h. The arrested cells were released into subsequential phase at 30ºC for 60min.
Protocol 2: The stains were grown to early exponential phase in YES medium at 25ºC and arrested at late G2-phase at 36ºC for 3h. The arrested cells were released into subsequential phase at 25ºC for 90min and then the cells were around G1/S boundary.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
The cells (1.0 x 10E9 cells) which were synchronized at appropriate cell cycle were cross-linked with 1% formaldehyde for 15 minutes and were homogenized by Multi-beads Shocker (Yasui-kikai Co., Osaka). DNA was sheared to 600 bp by sonication. Immunoprecipitation was performed by using monoclonal anti-FLAG M2 antibody (SIGMA). After incubation of lysate with antibody and protein G beads (Dynal) for 4 h, beads were washed 6 times with the lysis buffer. Co-immunoprecipitated DNA was reverse-crosslinked and purified by QIAquick PCR Purification Kit (QIAGEN).
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 |
Dec 12, 2011 |
| Last update date |
Dec 13, 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) |
| GSE34369 |
Rif1 is a global regulator of timing of replication origin firing in fission yeast |
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