|
| Status |
Public on Nov 30, 2015 |
| Title |
human Vs human Sample 3 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
NA15510
|
| Organism |
Homo sapiens |
| Characteristics |
45 kb lcr copy number: 4
sample characteristics: subjects without NPHP1
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
DNA was isolated from peripheral blood with a Puregene kit (Gentra Systems), following the manufacturer’s protocol
|
| Label |
Cy5
|
| Label protocol |
0.9μg genomic DNA from proband (test sample) and control (NA10851) were digested with AluI and RsaI (Promega) for 2 hours at 37°C. Digestion product was run on 1% agarose gel for verification. Labeling of the digestion product was performed using BioPrime Array CGH Genomic Labelling Module (Life Technologies) following the manufacturer’s protocol. Cy5-dCTP and Cy3-dCTP from Cyanine Smart Pack dCTP (PerkinElmer) were used for proband and control digestion product, respectively. Labeling reaction was performed for 2 hours at 37°C. Labeling product underwent purification process using Amicon Ultra 0.5 Centrifugal Filter (30 kDa, Millipore). Labeling efficiency was quantitated using a NanoDrop ND-1000 UV-VIS spectrophotometer.
|
| |
|
| Channel 2 |
| Source name |
NA10851
|
| Organism |
Homo sapiens |
| Characteristics |
45 kb lcr copy number: 4
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
DNA was isolated from peripheral blood with a Puregene kit (Gentra Systems), following the manufacturer’s protocol
|
| Label |
Cy3
|
| Label protocol |
0.9μg genomic DNA from proband (test sample) and control (NA10851) were digested with AluI and RsaI (Promega) for 2 hours at 37°C. Digestion product was run on 1% agarose gel for verification. Labeling of the digestion product was performed using BioPrime Array CGH Genomic Labelling Module (Life Technologies) following the manufacturer’s protocol. Cy5-dCTP and Cy3-dCTP from Cyanine Smart Pack dCTP (PerkinElmer) were used for proband and control digestion product, respectively. Labeling reaction was performed for 2 hours at 37°C. Labeling product underwent purification process using Amicon Ultra 0.5 Centrifugal Filter (30 kDa, Millipore). Labeling efficiency was quantitated using a NanoDrop ND-1000 UV-VIS spectrophotometer.
|
| |
|
| |
| Hybridization protocol |
Labeled proband and control samples were combined in a mass ratio of 1:1 with addition of 2μg human Cot-1 DNA (Life Technologies), 10X Agilent Blocking Agent (Agilent Technologies) and 2X Hi-RPM Hybridisation Buffer (Agilent Technologies). The mixture was incubated at 95°C for 3 min and then at 37°C for 30 min for pre-hybridization. Afterwards, the mixture was hybridized onto the array placed in a hybridization chamber (Agilent Technologies) in a rotating over (Agilent Technologies) for at least 40 hours at 65°C.
|
| Scan protocol |
Washing step after hybridization were performed following the manufacturer’s protocol (Agilent Oligonucleotide Array-Based CGH for Genomic DNA Analysis version 7.2, Agilent Technologies) with Agilent Oligo CGH Wash Buffer 1 and 2 (Agilent Technologies), Acetonitrile (Sigma-Aldrich), and Stabilization and Drying Solution (Agilent Technologies). The dried slides were scanned on Agilent Microarray Scanner (Agilent Technologies) for image acquisition.
|
| Description |
human-human array CGH
|
| Data processing |
The images after scanning were subsequently processed by Feature Extraction Software version 11.5 (Agilent Technologies) to generate FE files, which were subsequently imported into Agilent Genomic Workbench version 7.0 (Agilent Technologies) for copy number variation (CNV) analysis.
The copy number was defined by normalized log2 ratio of Cy5/Cy3 fluorescence signal of each probe. Human reference sequence GRCh37/hg19 assembly was used to define the genomic coordinated of detected CNVs.
|
| |
|
| Submission date |
Oct 13, 2015 |
| Last update date |
Nov 30, 2015 |
| Contact name |
Bo Yuan |
| E-mail(s) |
yuanbo1007@gmail.com
|
| Organization name |
Baylor College of Medicine
|
| Street address |
One Baylor Plaza
|
| City |
Houston |
| ZIP/Postal code |
77030 |
| Country |
USA |
| |
|
| Platform ID |
GPL21019 |
| Series (1) |
| GSE73962 |
Comparative genomic analyses of the human NPHP1 locus reveal complex genomic architecture and its regional evolution in primates |
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