|
| Status |
Public on Jul 28, 2012 |
| Title |
Mde2_rec15∆ |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
Mde2 ChIP DNA from rec15∆ cells at 4 hours after meiotic induction
|
| Organism |
Schizosaccharomyces pombe |
| Characteristics |
genotype/variation: rec15∆ (pat1-114)
culture: Meiosis 4 hours
chip antibody: FLAG M2 mouse monoclonal
chip antibody vendor: SIGMA
chip antibody lot#: 087K6002
chip antibody catalog#: F3165
|
| 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 |
To tag Mde2 with the Flag epitope at the C-terminus, we amplified the mde2+ ORF by PCR with a primer set of TM416 (5'-CCGCTCGAGGAACTGTGACAGCGTTTGAC-3') and TM417 (5'-ATAAGAATGCGGCCGCAGTACTGTAAAACTTCATCTAA-3') using wild-type genomic DNA as a template. The XhoI-NotI fragment was cloned into pTM506, which contained three copies of Flag epitope and a kanMX6 marker (pOTM194). pOTM194 was linearized at the EcoT22I site in mde2+ and used for transformation.
ChIP assay was performed as described previously (Hirota et al., 2007, PMID 17827346).
|
| 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, PMID 16793414).
|
| |
|
| Channel 2 |
| Source name |
Input DNA from mde2∆ cells at 4 hours after meiotic induction
|
| Organism |
Schizosaccharomyces pombe |
| Characteristics |
genotype/variation: mde2∆ (pat1-114)
culture: Meiosis 4 hours
chip antibody: none
|
| 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 |
To tag Rec15 with the Flag epitope at the C-terminus, we amplified the rec15+ ORF by PCR with a primer set of TM19 (5'-CCGCTCGAGCTGTTATTCGTCTTGCTGAG-3') and TM20 (5'-ATAAGAATGCGGCCGCAATAATCGTCATCTGCTAAGAA-3') using wild-type genomic DNA as a template. The PvuII-NotI fragment was then cloned into pTM506, which contained three copies of Flag epitope and a kanMX6 marker (pOTM79). pOTM79 was linearized at the HincII site in rec15+ and used for transformation.
ChIP assay was performed as described previously (Hirota et al., 2007, PMID 17827346).
|
| 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, PMID 16793414).
|
| |
|
| |
| 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 a 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, PMID 18978777).
Quantile normalization was applied to adjust differences in the signal intensity distribution across all used tiling arrays using the tilemapv2_importaffy function with the default setting. Then, MA statistics was applied to predict protein-binding regions using the tilemapv2 function with some modifications ([Method to Compute FDR] = UMS, [W] = 38, [Window Boundary] = 750, [Standardize MA Statistics] = No, [Region Boundary Cutoff, MA>] = 0.33, [Max Gap within a Region] = 750, [Max Run of Insignificant Probes within a Region] = 25, [Min Region Length] = 750, [Min No. of Significant Probes within a Region] = 25). Each IP sample was compared to the corresponding input sample.
The bar files represent signal enrichment (IP vs. Input).
|
| |
|
| Submission date |
Jun 08, 2012 |
| Last update date |
Jul 29, 2012 |
| 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 |
GPL7715 |
| Series (1) |
| GSE31846 |
Genome-wide binding site of Rec10 and Rec15 during meiosis |
|
