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| Status |
Public on Jan 01, 2017 |
| Title |
PPY17 |
| Sample type |
genomic |
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| Channel 1 |
| Source name |
CENP-A ChIP DNA from PPY17 cells
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| Organism |
Pongo pygmaeus |
| Characteristics |
cell type: lymphoblastoid cells
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
2x10E6 cells were harvested and washed in PBS. The cells were lysed in 5ml of Buffer I (85 mM KCl, 5.5% sucrose, 10 mM Tris pH 7.6, 0.5 mM Spermidine, 0.2 mM EDTA, 250 uM PMSF) and 5 ml of Buffer II (85 mM KCl, 5.5% sucrose, 10 mM Tris pH 7.6, 0.5 mM Spermidine, 0.2 mM EDTA, 250 uM PMSF, 0.1% NP-40). Nuclei were washed and resuspended in Buffer III (85 mM KCl, 5.5% sucrose, 10 mM Tris pH 7.6, 1.5 mM CaCl2, 3 mM MgCl2, 250 uM PMSF). Nuclei were digested with MNase (60 units/ml) and RNase A (3ug/ml) for 20 minutes at room temperature. Digestion was stopped adding EDTA (10mM final concentration). The supernatant containing the first soluble fraction of chromatin was recovered by centrifugation. After over-night incubation of chromatin in 5 mM NaCl buffer (10 mM Tris pH 7.5, 0.5 mM EDTA, 0.5 mM EGTA, 250 uM PMSF, 0.05% NP-40, 5 mM NaCl) on ice, the supernatant containing the second soluble fraction of chromatin was recovered. The chromatin was diluted in 50 mM NaCl buffer (10 mM Tris pH 7.5, 0.5 mM EDTA, 0.5 mM EGTA, 250 uM PMSF, 0.05% NP-40, 50 mM NaCl) and pre-cleared with protein A sepharose beads (pre-absorbed with salmon sperm DNA and BSA) for 1 h at 4C. The chromatin supernatant was then incubated overnight with 5 µg of anti-CENP-A antibody at 4C. After incubation with protein A sepharose beads (50 µl) for 4 h at 4C, the immune complexes were collected by centrifugation and washed with the following buffers each for 15 min at 4C: 140 mM NaCl buffer (10 mM Tris pH 7.5, 0.5 mM EDTA, 0.5 mM EGTA, 250 uM PMSF, 0.05% NP-40, 140 mM NaCl) and 200 mM NaCl buffer (10 mM Tris pH 7.5, 0.5 mM EDTA, 0.5 mM EGTA, 250 uM PMSF, 0.05% NP-40, 200 mM NaCl). The protein-DNA complexes were eluted from the beads in 400 µl elution buffer (10 mM Tris pH 7.5, 0.5 mM EDTA, 0.5 mM EGTA, 250 uM PMSF, 0.05% NP-40, 50 mM NaCl, 1% SDS) at room temperature for 30 min. Genomic DNA was isolated from the precipitated material as well as from the sheared chromatin input using Qiagen PCR Purification kit and was amplified using the Sigma-Aldrich Whole Genome Amplification kit.
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| Label |
Cy5
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| Label protocol |
Standard NimbleGen protocol.
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| Channel 2 |
| Source name |
Input DNA from PPY17 cells
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| Organism |
Pongo pygmaeus |
| Characteristics |
cell type: lymphoblastoid cells
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
2x10E6 cells were harvested and washed in PBS. The cells were lysed in 5ml of Buffer I (85 mM KCl, 5.5% sucrose, 10 mM Tris pH 7.6, 0.5 mM Spermidine, 0.2 mM EDTA, 250 uM PMSF) and 5 ml of Buffer II (85 mM KCl, 5.5% sucrose, 10 mM Tris pH 7.6, 0.5 mM Spermidine, 0.2 mM EDTA, 250 uM PMSF, 0.1% NP-40). Nuclei were washed and resuspended in Buffer III (85 mM KCl, 5.5% sucrose, 10 mM Tris pH 7.6, 1.5 mM CaCl2, 3 mM MgCl2, 250 uM PMSF). Nuclei were digested with MNase (60 units/ml) and RNase A (3ug/ml) for 20 minutes at room temperature. Digestion was stopped adding EDTA (10mM final concentration). The supernatant containing the first soluble fraction of chromatin was recovered by centrifugation. After over-night incubation of chromatin in 5 mM NaCl buffer (10 mM Tris pH 7.5, 0.5 mM EDTA, 0.5 mM EGTA, 250 uM PMSF, 0.05% NP-40, 5 mM NaCl) on ice, the supernatant containing the second soluble fraction of chromatin was recovered. The chromatin was diluted in 50 mM NaCl buffer (10 mM Tris pH 7.5, 0.5 mM EDTA, 0.5 mM EGTA, 250 uM PMSF, 0.05% NP-40, 50 mM NaCl) and pre-cleared with protein A sepharose beads (pre-absorbed with salmon sperm DNA and BSA) for 1 h at 4C. The chromatin supernatant was then incubated overnight with 5 µg of anti-CENP-A antibody at 4C. After incubation with protein A sepharose beads (50 µl) for 4 h at 4C, the immune complexes were collected by centrifugation and washed with the following buffers each for 15 min at 4C: 140 mM NaCl buffer (10 mM Tris pH 7.5, 0.5 mM EDTA, 0.5 mM EGTA, 250 uM PMSF, 0.05% NP-40, 140 mM NaCl) and 200 mM NaCl buffer (10 mM Tris pH 7.5, 0.5 mM EDTA, 0.5 mM EGTA, 250 uM PMSF, 0.05% NP-40, 200 mM NaCl). The protein-DNA complexes were eluted from the beads in 400 µl elution buffer (10 mM Tris pH 7.5, 0.5 mM EDTA, 0.5 mM EGTA, 250 uM PMSF, 0.05% NP-40, 50 mM NaCl, 1% SDS) at room temperature for 30 min. Genomic DNA was isolated from the precipitated material as well as from the sheared chromatin input using Qiagen PCR Purification kit and was amplified using the Sigma-Aldrich Whole Genome Amplification kit.
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| Label |
Cy3
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| Label protocol |
Standard NimbleGen protocol.
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| Hybridization protocol |
Manufacturer's NimbleScan Protocol.
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| Scan protocol |
Manufacturer's NimbleScan Protocol.
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| Description |
ChIP-chip PPY17 cells for CENP-A
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| Data processing |
Each feature on the array has a corresopnding log2-ratio that is provided in the GFF files. This is the ratio of the input singals for the experimental and test samples were were co-hybridized to the array. The log2-ratio is computed and scaled to center the ratio data round zero. Scaling is performed by subtracting the bi-weight mean for the log2-ratio values of all features on the array from each log2-ratio values.
Normalized log-ratios ch1/ch2 (q-spline normalization).
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| Submission date |
May 02, 2016 |
| Last update date |
Jan 01, 2017 |
| Contact name |
Stefania Purgato |
| E-mail(s) |
stefania.purgato2@unibo.it
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| Organization name |
University of Bologna
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| Department |
Pharmacy and Biotechnology
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| Street address |
via Selmi 3
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| City |
Bologna |
| ZIP/Postal code |
40126 |
| Country |
Italy |
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| Platform ID |
GPL21737 |
| Series (1) |
| GSE81003 |
ChIP-chip from four orangutan cells with CENP-A |
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| Supplementary file |
Size |
Download |
File type/resource |
| GSM2140563_PPY17_532.pair.gz |
11.0 Mb |
(ftp)(http) |
PAIR |
| GSM2140563_PPY17_635.pair.gz |
10.9 Mb |
(ftp)(http) |
PAIR |
| GSM2140563_PPY17_ratio.gff.gz |
8.6 Mb |
(ftp)(http) |
GFF |
| Processed data provided as supplementary file |
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