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| Status |
Public on Oct 03, 2019 |
| Title |
TL-Om1_shEZH1_H3K27me3 |
| Sample type |
genomic |
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| Channel 1 |
| Source name |
H3K27me3 ChIP DNA from TL-Om1_shEZH1
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| Organism |
Homo sapiens |
| Characteristics |
cell type: ATL cell line
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
1×10E7 cells were cross-linked in fixing solution. After 15 min incubation at room temperature, cross-linking was stopped by adding 1.375 M glycine. The cells were centrifuged for 5 min, 1,400 × g at 4°C. The supernatant was discarded and the pellet was washed two times in PBS. After the washes, cell pellets were resuspended in LB1 (50 mM Hepes-KOH (pH 7.5), 140 mM NaCl, 1 mM EDTA, 10% Glycerol, 0.5% IGEPAL CA-630, 0.25% Triton X-100) and incubated for 10 min on ice. After centrifugation for 5 min, 1,400 × g at 4°C, the pellet was resuspended in LB2 (10 mM Tris-HCl (pH 8.0), 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA) for 10 min at room temperature. After centrifugation for 5 min, 1,400 × g at 4°C, the nuclei were resuspended in micrococcal nuclease digestion buffer and incubated in 37°C to digest chromatin to the appropriate size fragments. Digestion was stopped by adding 0.5 M EDTA. After centrifugation, nuclei pellets were resuspended in LB3 (10 mM Tris-HCl (pH 8.0), 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% Sodium Deoxycholate, 0.5% N-Lauroylsarcosine) and sonicated for 10 min. After sonication and 10 min high-speed centrifugation, sheared chromatin was recovered in the supernatant.
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| Label |
Cy5
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| Label protocol |
Labeled DNA was prepared from 2.6 μg of amplified DNA using SureTag DNA Labeling Kit according to the manufacturer's instructions.
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| Channel 2 |
| Source name |
Input DNA from TL-Om1_shEZH1
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| Organism |
Homo sapiens |
| Characteristics |
cell type: ATL cell line
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
1×10E7 cells were cross-linked in fixing solution. After 15 min incubation at room temperature, cross-linking was stopped by adding 1.375 M glycine. The cells were centrifuged for 5 min, 1,400 × g at 4°C. The supernatant was discarded and the pellet was washed two times in PBS. After the washes, cell pellets were resuspended in LB1 (50 mM Hepes-KOH (pH 7.5), 140 mM NaCl, 1 mM EDTA, 10% Glycerol, 0.5% IGEPAL CA-630, 0.25% Triton X-100) and incubated for 10 min on ice. After centrifugation for 5 min, 1,400 × g at 4°C, the pellet was resuspended in LB2 (10 mM Tris-HCl (pH 8.0), 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA) for 10 min at room temperature. After centrifugation for 5 min, 1,400 × g at 4°C, the nuclei were resuspended in micrococcal nuclease digestion buffer and incubated in 37°C to digest chromatin to the appropriate size fragments. Digestion was stopped by adding 0.5 M EDTA. After centrifugation, nuclei pellets were resuspended in LB3 (10 mM Tris-HCl (pH 8.0), 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% Sodium Deoxycholate, 0.5% N-Lauroylsarcosine) and sonicated for 10 min. After sonication and 10 min high-speed centrifugation, sheared chromatin was recovered in the supernatant.
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| Label |
Cy3
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| Label protocol |
Labeled DNA was prepared from 2.6 μg of amplified DNA using SureTag DNA Labeling Kit according to the manufacturer's instructions.
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| Hybridization protocol |
Hybridization was performed at 65°C for 40h in a rotating Agilent hybridization oven. After hybridization, microarrays were washed at room temperature with Agilent Oligo aCGH/ChIP-on-chip Wash Buffer 1 for 5min and with Agilent Oligo aCGH/ChIP-on-chip Wash Buffer 2 for 5min at 37°C.
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| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner C using the two color scan setting for 2x400K microarray slides (Scan Area: 61x21.6 mm, Scan resolution: 3 um, Dye channel: red and green, Tiff: 16bit, XDR ratio: NoXDR)
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| Data processing |
The scanned images were analyzed with Feature Extraction Software 10.7 using default parameters (protocol: ChIP_107_Sep09) to obtain background subtracted and spatially detrended Processed Signal intensities.
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| Submission date |
Oct 02, 2019 |
| Last update date |
Oct 03, 2019 |
| Contact name |
Makoto Yamagishi |
| E-mail(s) |
[email protected]
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| Organization name |
The University of Tokyo
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| Lab |
Lab. Tumor Cell Biology
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| Street address |
4-6-1, Shirokanedai
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| City |
Minato-ku |
| State/province |
Tokyo |
| ZIP/Postal code |
108-8639 |
| Country |
Japan |
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| Platform ID |
GPL19784 |
| Series (1) |
| GSE138342 |
Epigenetic pattern after EZH1,2 inhibition in lymohoma cells |
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| Supplementary file |
Size |
Download |
File type/resource |
| GSM4105781_TL-Om1_shEZH1_H3K27me3.txt.gz |
43.4 Mb |
(ftp)(http) |
TXT |
| Processed data are available on Series record |
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