|
| Status |
Public on May 26, 2009 |
| Title |
wild type cells treated with alpha factor, IP fraction |
| Sample type |
genomic |
| |
|
| Source name |
Chromatin-immunoprecipitated DNA
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
genotype: Wild type strain expressing Rad52-13myc
background: W303
strain: K699 R52-Myc
culture: arrested in G1 with alpha-factor
|
| Treatment protocol |
Early log phase culture was harvested and resuspended in YPAD medium at 1x10e7. Then, cells were treated with 15 ug/ml alpha-factor.
1x10e9 cells were fixed in 1% formaldehyde for 10 min at room temperature and then treated with 125 mM glycine for 5 min at room temperature. The cells were washed with 20 ml of ice-cold TBS twice, freezed with liquid nitrogen, and stored at -80C.
|
| Growth protocol |
Cells were grown to early log phase.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were suspended in lysis buffer 140 (0.1% sodium deoxycholate, 1 mM EDTA, 140 mM NaCl, 1% Triton X-100, 50 mM HEPES-KOH, pH7.5) and disrupted using Multi-Beads Shocker (YASUI KIKAI, Osaka) with 0.5-mm zirconia beads. Whole cell extract was sonicated to obtain average 500 bp genomic DNA fragments.
WCE was immunoprecipitated with anti-c-Myc (9E10) antibody sc-40 (Invitrogen) coupled to protein A Dynabeads (Invitrogen).
Chromatin immunoprecipitated sample was incubated overnight at 37C with proteinase K, incubated for 6 hrs at 65C, extracted with phenol/chloroform/isoamylalcohol, precipitated, and resuspended in 10 ul of 10 mM Tris-Cl (pH 7.6) and incubated with RNaseA.
The DNA was amplified by PCR after random priming as previously described (Katou et al., 2003, nature). The amplified DNA was digested with Dnase I to a mean size of 100 bp. After DNase I inactivation at 95ºC, fragments were labelled with indicated label protocol.
|
| Label |
Biotin
|
| Label protocol |
End labeling by terminal transferase
|
| |
|
| Hybridization protocol |
Following denaturation at 99 C for 10 minutes, cooling on ice, and spin-down. 200 micro liter of hybridization mixture was hybridized for 16 hours at 42 C on GeneChip SC3456a520015F using hybridization oven 640 (affymetrix). Each hybridization mixture contains the end-labbeled DNA, 6xSSPE, 0.005% Triton X-100, 0.1 mg/ml herring sperm DNA, oligo B2 (affymetrix) and hybridization control (affymetrix).
|
| Scan protocol |
Washing and scanning protocol (EukGE-WS1v3) provided by Affymetrix was performed automatically on an Affymetrix Fluidics station 400. Array was scanned by GeneChip Scanner 3000 7G.
|
| Description |
Distribution of Rad52-myc on chromosome III, IV, V, and the right arm of chromosome VI (wild type cells treated with alpha factor)
IP fraction used for the mapping of Rad52-13myc on chromosomes
Used baseline: WCE fraction of wild type cells treated with alpha-factor
|
| Data processing |
Data analysis was performed 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).
Primary data analysis was carried out with GeneChip Operating Software (GCOS 1.4) using default settings to obtain signal and detection p-value.
Signals from the ChIP fraction scan were compared to the WCE fraction scan with GCOS 1.4 using default settings.
|
| |
|
| Submission date |
Feb 09, 2009 |
| Last update date |
May 23, 2009 |
| Contact name |
Kazuto Kugou |
| E-mail(s) |
kkugou@kazusa.or.jp
|
| Organization name |
Kazusa DNA Research Institute
|
| Department |
Department of Frontier Research
|
| Lab |
Laboratory of Cell Engineering
|
| Street address |
2-6-7 KazusaKamatatri
|
| City |
Kisarazu |
| State/province |
Chiba |
| ZIP/Postal code |
292-0818 |
| Country |
Japan |
| |
|
| Platform ID |
GPL1280 |
| Series (1) |
| GSE14761 |
Accumulation of sumoylated Rad52 in checkpoint mutants perturbed in DNA replication |
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