|
| Status |
Public on Mar 26, 2011 |
| Title |
Mcm4ChIP_hsk1ts_240minHU |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
Flag ChIPed from the strain expressing 3Flag-fused Mcm4 and hsk1-89
|
| Organism |
Schizosaccharomyces pombe |
| Characteristics |
cell cycle: Early S-phase in HU at 60min after release from metaphase arrst
chip target: Mcm4 and hsk1-89
antibody: FLAG M2 mouse monoclonal (Sigma, Cat No:F-1804)
|
| 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 and ChIPed 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 |
antibody: none
|
| 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 and ChIPed 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 |
As previously described in Methods in Enzymology, Elsevier Life Sciences (CA), vol.409,chapter 23,389-410 in “DNA Repair” (edited by J.Campbell 2006)
All Samples (ch1) were compared to the same input DNA control CEL file linked to the Series record.
|
| |
|
| Submission date |
Mar 25, 2011 |
| Last update date |
Mar 28, 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) |
| GSE28182 |
Mrc1 marks early-firing origins and coordinates timing and efficiency of initiation in fission yeast |
|
