|
| Status |
Public on May 29, 2009 |
| Title |
Rec8-FLAG distribution on chromosome VI at meiosis 5 hr |
| Sample type |
genomic |
| |
|
| Source name |
Chromatin-immunoprecipitated DNA
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
SK1 background strain expressing FLAG tagged Rec8 protein
Strain: RKD1317
Culture: 5 hrs after transfer to sporulation medium
|
| Treatment protocol |
100 ml culture (2x10e9 cells) was taken at 5 hrs in sporulation medium.
The culture was 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 40 ml of ice-cold TBS twice, freezed with liquid nitrogen, and stored at -80C.
|
| Growth protocol |
Cells were grown on a YPD plate for 2 days at 30C. The cells were grown to a density of 4.0x10e7 cells/ml at 30C in SPS presporulation medium (0.5% yeast extract, 1% peptone, 0.17% yeast nitrogen base w/o amino acid and ammonium sulfate, 1% potassium acetate, 0.5% ammonium sulfate, usual level of amino acids, 50 mM potassium phthalate buffer pH 5.0, and antifoam). The cells were suspended in sporulation medium (1% potasium acetate, 1/5 level of amino acids, and polypropylene glycol) to a density of 2x10e7 cells/ml, and incubated at 30C for sporulation.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
The cells were 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.
The WCE was immunoprecipitated with anti-FLAG monoclonal antibody M2 (Sigma-Aldrich Co., St Louis, MO) coupled to Dynabeads (Invitrogen, protein A Dynabeads).
The 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 TE 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-N6-ddATP
|
| Label protocol |
End labeling by terminal transferase
|
| |
|
| Hybridization protocol |
Following denaturation at 100 C for 10 minutes, cooling, and spin-down, 150 micro liter of hybridization mixuter was hybridized for 16 hours at 42 C on GeneChip rikDACF 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 (mini-WS1v2) provided by Affymetrix was performed automatically on a fluidics station (GeneChip fluidics station 400, Affymetrix). Arrays were scanned by Hewlett-Packard GeneArray Scanner at an emission wavelength of 560 nm at 7.5 microM resolution.
|
| Description |
ChIP-chip data used for the mapping of Rec8 distribution on chromosome VI at meiosis 5 hr
Used baseline: WCE fraction (REC8-FLAG cells at meiosis 2 hr, chromosome VI)
|
| Data processing |
Data analyses were 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 analyses were carried out with GeneChip Operating Software (GCOS 1.4) using default settings to obtain signal and detection p-value.
To discriminate significant signals for binding to DNA, signals from the ChIP fraction scan were compared to the WCE fraction scan with GCOS 1.4 using default settings.
|
| |
|
| Submission date |
Jul 09, 2007 |
| Last update date |
Aug 14, 2011 |
| Contact name |
Kazuto Kugou |
| E-mail(s) |
[email protected]
|
| 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 |
GPL347 |
| Series (1) |
| GSE8422 |
Rec8 guides canonical Spo11 distribution along yeast meiotic chromosomes |
|
