|
| Status |
Public on Apr 10, 2008 |
| Title |
INO80 chromatin remodeling complex promotes the restart of DNA replication_03Ino80Myc_random1_ip |
| Sample type |
genomic |
| |
|
| Source name |
Chromatin-immunoprecipitated DNA
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
W303 background cells expressing Myc-tagged Ino80
Strain: GA3248
|
| Treatment protocol |
2x10e9 cells from 200ml culture were fixed by 1% formaldehyde for 15min at 30C.
The cells were washed with sterilized water. The cells were washed with ice-cold TBS, freezed in liquid nitrogen, and stored at -80C.
|
| Growth protocol |
Cells were grown on a YPD plate for 2 days at 30C. The cells were grown in 200 ml of YPD to 1x10e7 cells/ml at 30C.
|
| 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-Myc monoclonal antibody 9E11 (abcam, ab56) coupled to Dynabeads (Invitrogen, protein A Dynabeads).
The chromatin immunoprecipitated sample was incubated overnight at 65ºC, incubated with proteinase K, 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-11-ddATP
|
| Label protocol |
End labeling by terminal transferase
|
| |
|
| Hybridization protocol |
Following denaturation at 100 C for 10 minutes, cooling, and spin-down, 200 micro liter of hybridization mixuter 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 a fluidics station (GeneChip fluidics station 400, Affymetrix). Arrays were scanned by GeneChip Scanner 3000 7G.
|
| Description |
ChIP-chip data used for the mapping of Ino80 distribution on chromosome III-VIR
Used baseline: WCE fraction of randumly cultured Ino80Myc expression cells for Ino80Myc ChIP
|
| 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.
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| |
|
| Submission date |
Oct 24, 2007 |
| Last update date |
Aug 14, 2011 |
| Contact name |
Yukako Oma |
| E-mail(s) |
labharata@gmail.com
|
| Organization name |
Tohoku University
|
| Department |
Graduate School of Agricultural Science
|
| Lab |
Molecular Biology
|
| Street address |
Tsutsumidori-Amamiyamachi 1-1, Aoba-ku
|
| City |
Sendai |
| ZIP/Postal code |
981-8555 |
| Country |
Japan |
| |
|
| Platform ID |
GPL1280 |
| Series (2) |
| GSE9420 |
Genome-wide localization of Ino80 on chromosome in Saccharomyces cerevisiae |
| GSE9421 |
Ino80 chromatin remodeling complex promotes recovery of stalled replication forks |
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