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| Status |
Public on Aug 06, 2014 |
| Title |
EARSHOOT_LIGHT_REP1 |
| Sample type |
genomic |
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| Channel 1 |
| Source name |
nucleosomal DNA from lightly-digested earshoot nuclei
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| Organism |
Zea mays |
| Characteristics |
cultviar: B73
tissue: immature ear
mnase concentration: 2 U/mL Mnase
buffer: BFA (15mM PIPES-NaOH pH 6.8, 0.32mM sorbitol, 80mM KCl 20mM, NaCl 0.5mM, EGTA 2.0mM, EDTA 1mM, DTT 0.15mM spermine, 0.05mM spermidine)
fraction: nucleosomal DNA
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| Treatment protocol |
light Mnase digestion
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Ten grams of seedling tissue, or 1g of immature ear tissue, were ground under liquid nitrogen with a mortar and pestle and crosslinked by stirring for 10 minutes in 10 mL ice-cold nuclear isolation buffer with 1% formaldehyde. Fixation was stopped with 125 mM Glycine for 5 minutes. Tissue was pelleted at 2,000 x g for 10 min at 4ºC in a swinging-bucket centrifuge, then the tissue pellet was resuspended in 25 mL nucleus isolation buffer with 1% hexylene glycol and 1% Triton X-100. After 5 minutes of gentle rotary shaking, nuclear suspensions were filtered through 2 layers of miracloth (Calbiochem) and nuclei were pelleted at 2,000 x g for 10 min at 4ºC. Nuclei were washed once with 10 mL nuclear isolation buffer, and resuspended in 10 mL MNase digestion buffer (50 mM Tris-HCl pH 7.5, 320 mM sucrose, 4 mM MgCl2, 1 mM CaCl2). Nuclei were sequentially filtered through 150, 100, 50 and 30 µm CellTric® filters (Partec), pelleted at 2,000 x g for 10 min at 4ºC, resuspended in 1 ml of MNase Digestion Buffer, flash frozen in liquid nitrogen, and stored at -80C until use. 100 uL nuclei were digested with MNase at 37 C for 5 minutes and stopped with 20 mM EGTA. 100 uL diH20 was added to each sample and nuclei were decrosslinked with 1% SDS and 100 ug/mL Proteinase K overnight at 65 ºC. DNA was phenol-chloroform extracted, ethanol precipitated, and resuspended in diH20. Digested DNA were treated with 40 ug/mL RNase A and run in a 1% agarose gel. 100-200-bp DNA fragments were excised and gel extracted with the Qiaex II gel extraction kit (Qiagen) following the manufacturer’s instructions.
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| Label |
cy3
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| Label protocol |
Per manufacturer instructions for NimbleGen dual-color Labeling kit
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| Channel 2 |
| Source name |
bare genomic DNA from earshoot nuclei
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| Organism |
Zea mays |
| Characteristics |
tissue: immature ear
fraction: bare genomic DNA
cultviar: B73
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Ten grams of seedling tissue, or 1g of immature ear tissue, were ground under liquid nitrogen with a mortar and pestle and crosslinked by stirring for 10 minutes in 10 mL ice-cold nuclear isolation buffer with 1% formaldehyde. Fixation was stopped with 125 mM Glycine for 5 minutes. Tissue was pelleted at 2,000 x g for 10 min at 4ºC in a swinging-bucket centrifuge, then the tissue pellet was resuspended in 25 mL nucleus isolation buffer with 1% hexylene glycol and 1% Triton X-100. After 5 minutes of gentle rotary shaking, nuclear suspensions were filtered through 2 layers of miracloth (Calbiochem) and nuclei were pelleted at 2,000 x g for 10 min at 4ºC. Nuclei were washed once with 10 mL nuclear isolation buffer, and resuspended in 10 mL MNase digestion buffer (50 mM Tris-HCl pH 7.5, 320 mM sucrose, 4 mM MgCl2, 1 mM CaCl2). Nuclei were sequentially filtered through 150, 100, 50 and 30 µm CellTric® filters (Partec), pelleted at 2,000 x g for 10 min at 4ºC, resuspended in 1 ml of MNase Digestion Buffer, flash frozen in liquid nitrogen, and stored at -80C until use. 100 uL nuclei were digested with MNase at 37 C for 5 minutes and stopped with 20 mM EGTA. 100 uL diH20 was added to each sample and nuclei were decrosslinked with 1% SDS and 100 ug/mL Proteinase K overnight at 65 ºC. DNA was phenol-chloroform extracted, ethanol precipitated, and resuspended in diH20. Digested DNA were treated with 40 ug/mL RNase A and run in a 1% agarose gel. 100-200-bp DNA fragments were excised and gel extracted with the Qiaex II gel extraction kit (Qiagen) following the manufacturer’s instructions.
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| Label |
cy5
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| Label protocol |
Per manufacturer instructions for NimbleGen dual-color Labeling kit
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| Hybridization protocol |
Per manufacturer instructions for NimbleGen dual-color Arrays
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| Scan protocol |
Per manufacturer instructions for NimbleGen dual-color Arrays
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| Data processing |
Generation of probe-level log2 ratios (Cy3/Cy5) was performed with NimbleScan 2.6 (Roche). Probe-level log2 (Cy3/Cy5) ratios were quantile-normalized using the R statistical computing software.
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| Submission date |
Aug 05, 2014 |
| Last update date |
Aug 06, 2014 |
| Contact name |
Daniel Vera |
| E-mail(s) |
dvera@bio.fsu.edu
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| Organization name |
Florida State University
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| Department |
Biological Science
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| Street address |
319 Stadium Drive
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| City |
Tallahassee |
| State/province |
FL |
| ZIP/Postal code |
32306-4295 |
| Country |
USA |
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| Platform ID |
GPL19042 |
| Series (2) |
| GSE60090 |
MNase-chip of maize B73 immature ears and seedlings |
| GSE60092 |
MNase-chip of maize B73 |
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| Supplementary file |
Size |
Download |
File type/resource |
| GSM1464863_EARSHOOT_LIGHT_REP1_2012-11-08_532.pair.gz |
2.5 Mb |
(ftp)(http) |
PAIR |
| GSM1464863_EARSHOOT_LIGHT_REP1_2012-11-08_635.pair.gz |
2.5 Mb |
(ftp)(http) |
PAIR |
| Processed data are available on Series record |
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