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| Status |
Public on Jul 08, 2016 |
| Title |
RAD51_flower_phs1_ChIPSeq |
| Sample type |
SRA |
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| Source name |
Flower, phs1 mutant, RAD51 ChIP
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| Organism |
Zea mays |
| Characteristics |
inbred line: B73
genotype/variation: phs1 mutant
tissue: flower
chip antibody: ZmRAD51 (custom made)
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Flowers at the zygotene stage of meiotic prophase I were collected from at least twenty maize tassels, fixed in 1% formaldehyde for 10 min, and then quenched in 0.125mM glycine for 5 min.
1.5g of fixed flowers were ground into fine power and homogenized in Chromatin Extraction Buffer A (10 mM Tris-HCl pH 8.0, 0.4 mM sucrose, 10 mM MgCl2, 1 mM PMSF, 5 mM β-mercaptoethanol, 1 tablet of cOmplete Protease Inhibitor Cocktail (Roche, Penzberg, Germany)) for 20 min at 4°C with gentle shaking. The suspension was filtered into a new 50 ml conical tube through 2 layers of Miracloth (EMD Millipore, Billerica, MA, USA), placed in a plastic funnel and centrifuged at 4,000 rpm for 20 min at 4°C. The pellet was resuspended in 1 ml of extraction buffer B (10 mM Tris-HCl pH 8.0, 0.25 M sucrose, 10 mM MgCl2, 1% Triton X-100, 1 mM PMSF, 5 mM β-mercaptoethanol, 1 μg/ml of cOmplete Protease Inhibitor Cocktail) and centrifuged at 14,000 rpm for 10 min. The pellet was resuspended in 500 μl of Extraction Buffer C (10 mM Tris-HCl pH 8.0, 1.7 M sucrose, 2 mM MgCl2, 0.15% Triton X-100, 1 mM PMSF, 5 mM β-mercaptoethanol, 1 μg/ml cOmplete Protease Inhibitor Cocktail), placed on top of 500 μl of Extraction Buffer C cushion and centrifuged at 14,000 rpm for 1 h at 4°C. The nuclei pellet was resuspended in 500 μl Nuclei Lysis Buffer (50 mM Tris–HCl pH 8.0, 10 mM EDTA, 1% SDS, 1 mM PMSF, 1 μg/ml cOmplete Protease Inhibitor Cocktail) to release chromatin.
The resulting chromatin was sonicated into fragments of average length of 500-1000 bp using eight sonicator pulses, five seconds each, and centrifuged at 14,000 rpm for 5 min at 4°C. The supernatant was diluted 10 fold with Dilution Buffer (16.7 mM Tris–HCl pH 8.0, 1.2 mM EDTA, 167 mM NaCl, 1.1% Triton X-100, 1 mM PMSF, 1 μg/ml cOmplete Protease Inhibitor Cocktail) and pre-cleared with Dynabeads Protein A (Invitrogen, Carlsbad, CA, USA). 10 μl of the pre-cleared chromatin was saved as chromatin input control sample. The remaining chromatin was incubated with antibodies overnight at 4°C, and then captured by Dynabeads Protein A. After incubation, beads were washed 5 times in the following buffers: (1) Low Salt Wash Buffer (20 mM Tris-HCl pH 8.0, 2 mM EDTA, 150 mM NaCl, 0.1% SDS, 1% Triton X-100), (2) High Salt Wash Buffer (20 mM Tris-HCl pH 8.0, 2 mM EDTA, 500 mM NaCl, 0.1% SDS, 1% Triton X-100), (3) LiCl Buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA, 250 mM LiCl, 1% NP-40, 1% sodium deoxycholate), and (4) TE Buffer, twice (10 mM Tris-HCl pH 8.0, 1 mM EDTA). Chromatin was eluted from beads with 200 μl Elution Buffer (50 mM Tris-HCl pH 8.0, 10 mM EDTA, 200 mM NaCl, 1% SDS), treated with RNase for 2h at 37°C, deproteinized with Proteinase K for 2h at 45°C, and decrosslinked at 65°C for 8 h. DNA was purified using MinElute PCR Purification Columns (Qiagen, Hilden, Germany). DNA concentration was quantified using Quant-IT dsDNA HS Assay Kit (Invitrogen).
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
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| Data processing |
Basecalls performed using CASAVA version 1.8.
ChIP-seq reads were aligned to the B73 reference genome sequence (release AGPv2) using the Burrows-Wheeler Aligner (BWA).
Data were filtered based on unique mapping.
Peaks were called with the following parameters: bandwidth = 800 bp, shift size = 400 bp.
Genome_build: B73 (release AGPv2)
Supplementary_files_format_and_content: Bed file was generated to represent peaks positions.
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| Submission date |
Mar 07, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Wojciech Pawlowski |
| E-mail(s) |
mw729@cornell.edu
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| Organization name |
Cornell University
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| Department |
Plant Breeding and Genetics
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| Street address |
Bradfield Hall, Room 403
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| City |
Ithaca |
| State/province |
NY |
| ZIP/Postal code |
14853 |
| Country |
USA |
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| Platform ID |
GPL15463 |
| Series (2) |
| GSE55701 |
Genomic features shaping the landscape of meiotic double-strand break hotspots in maize [ChIP-Seq] |
| GSE84369 |
Genomic features shaping the landscape of meiotic double-strand break hotspots in maize |
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| Relations |
| BioSample |
SAMN02677624 |
| SRA |
SRX483000 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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