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Sample GSM2140560 Query DataSets for GSM2140560
Status Public on Jan 01, 2017
Title PPY10
Sample type genomic
 
Channel 1
Source name CENP-A ChIP DNA from PPY10 cells
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.
Label Cy5
Label protocol Standard NimbleGen protocol.
 
Channel 2
Source name Input DNA from PPY10 cells
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.
Label Cy3
Label protocol Standard NimbleGen protocol.
 
 
Hybridization protocol Manufacturer's NimbleScan Protocol.
Scan protocol Manufacturer's NimbleScan Protocol.
Description ChIP-chip PPY10 cells for CENP-A
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).
 
Submission date May 02, 2016
Last update date Jan 01, 2017
Contact name Stefania Purgato
E-mail(s) stefania.purgato2@unibo.it
Organization name University of Bologna
Department Pharmacy and Biotechnology
Street address via Selmi 3
City Bologna
ZIP/Postal code 40126
Country Italy
 
Platform ID GPL21736
Series (1)
GSE81003 ChIP-chip from four orangutan cells with CENP-A

Supplementary file Size Download File type/resource
GSM2140560_PPY10_532.pair.gz 5.9 Mb (ftp)(http) PAIR
GSM2140560_PPY10_635.pair.gz 5.9 Mb (ftp)(http) PAIR
GSM2140560_PPY10_ratio.gff.gz 4.5 Mb (ftp)(http) GFF
Processed data provided as supplementary file

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