|
| Status |
Public on Mar 31, 2014 |
| Title |
Rec8_4h_IP |
| Sample type |
genomic |
| |
|
| Source name |
Rec8-HA ChIP at 4h after meiotic induction
|
| Organism |
Schizosaccharomyces pombe |
| Characteristics |
strain: wild type (pat1-114)
culture: 4h after transfer to EMM containing nitrogen (0.05% NH4Cl) at 34ºC
|
| Treatment protocol |
The cells were fixed in 1% formaldehyde for 15 min at room temperature and then incubated on ice for 45 min. The cells were washed with ice-cold PBS twice, freezed with liquid nitrogen, and stored at -80ºC.
|
| Growth protocol |
For the synchronous induction of meiosis, the pat1-114 mutant strain was cultured in EMM medium containing nitrogen (0.5% NH4Cl) at 25ºC. This culture was washed and re-suspended in EMM medium without nitrogen and starved for 20 h at 25ºC to arrest the cell cycle in G1. The cells were then released into EMM containing nitrogen (0.05% NH4Cl) at 34ºC to allow for synchronous progression into meiosis.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
The cells were disrupted using Multi-Beads Shocker (YASUI KIKAI, Osaka) with 0.5-mm zirconia beads. Cell lysates were sonicated to obtain average 500 bp of genomic DNA fragments. Whole cell extracts (WCEs) were subjected to immunoprecipitation with anti-HA antibody 16B12 (Covance) coupled to Dynabeads protein A (Invitrogen). A small amount of WCEs was used as input sample. The chromatin immunoprecipitated sample and input sample were incubated overnight at 75ºC, incubated for 3 hours at 55ºC with proteinase K, and DNA was purified using QIAquick Spin Purification Kit (QIAGEN).
|
| Label |
biotin
|
| Label protocol |
Immunoprecipitated DNA and input DNA were amplified and end-labeled with the IVT (in vitro transcription). The amplification method is as described elsewhere (Katou et al., 2006).
|
| |
|
| Hybridization protocol |
16 h at 45C using a hybridization oven 640 (affymetrix) as described in the Affymetrix Chromatin Immunoprecipitation Assay Protocol (P/N 702238 Rev. 3)
|
| Scan protocol |
Fluidics station 400 protocol Midi-euk2.v3 (affymetrix) was performed to wash and stain arrays. The arrays were scanned on GeneChip Scanner 3000 7G (affymetrix).
|
| Data processing |
Quantile normalization and calculation of MA statistics (bar file) were performed with CisGenome v2 (Ji et al., 2008).
|
| |
|
| Submission date |
Dec 02, 2013 |
| Last update date |
Mar 31, 2014 |
| Contact name |
Masaru Ito |
| Organization name |
Osaka University
|
| Street address |
Yamadaoka 3-2
|
| City |
Suita |
| State/province |
Osaka |
| ZIP/Postal code |
565-0871 |
| Country |
Japan |
| |
|
| Platform ID |
GPL7715 |
| Series (2) |
| GSE52858 |
Meiotic recombination cold spots in chromosomal cohesion sites [ChIP-chip] |
| GSE52863 |
Meiotic recombination cold spots in chromosomal cohesion sites |
|
