|
| Status |
Public on Aug 22, 2017 |
| Title |
CGH_Revertant_1 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
sap1-1 revertant
|
| Organism |
Schizosaccharomyces pombe |
| Characteristics |
strain/background: 972h
genotype/variation: sap1-1
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Grow 20 ml cells to OD595 of 1.0. After cell wall digestion using Zymolyase 100T, cells were resuspended in 0.55 ml TE, 1% SDS, 100ug of proteinase K and incubated at 65°C for 30 min. Add 175 µl 5M potassium acetate. Keep on ice for 5 minutes. Lysate was centrifuged for 15 min at maximum speed for removing the cell debris. Genomic DNA was precipitated with isopropanol. After RNaseA treatment, genomic DNA was purified by phenol/chloroform/isoamyl alcohol (PCI) extraction twice and precipitated with ethanol.
|
| Label |
Cy5
|
| Label protocol |
Genomic DNA from wild type or sap1-1 revertant was digested with AluI and RsaI. Digested DNA was used as template for a genomic DNA labelling reaction using the BioPrime® Array CGH Genomic Labeling kit (Invitrogen) according to the manufacturer's protocol.
|
| |
|
| Channel 2 |
| Source name |
wild-type
|
| Organism |
Schizosaccharomyces pombe |
| Characteristics |
strain/background: 972h
genotype/variation: wild-type
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Grow 20 ml cells to OD595 of 1.0. After cell wall digestion using Zymolyase 100T, cells were resuspended in 0.55 ml TE, 1% SDS, 100ug of proteinase K and incubated at 65°C for 30 min. Add 175 µl 5M potassium acetate. Keep on ice for 5 minutes. Lysate was centrifuged for 15 min at maximum speed for removing the cell debris. Genomic DNA was precipitated with isopropanol. After RNaseA treatment, genomic DNA was purified by phenol/chloroform/isoamyl alcohol (PCI) extraction twice and precipitated with ethanol.
|
| Label |
Cy3
|
| Label protocol |
Genomic DNA from wild type or sap1-1 revertant was digested with AluI and RsaI. Digested DNA was used as template for a genomic DNA labelling reaction using the BioPrime® Array CGH Genomic Labeling kit (Invitrogen) according to the manufacturer's protocol.
|
| |
|
| |
| Hybridization protocol |
Equal amounts of Cy5-labeled DNA and Cy3-labeled DNA were mixed and combined with human Cot1 DNA, Agilent Blocking Agent and Agilent Hybridization buffer, and hybridized to high-density microarrays in Agilent SureHyb hybridization chamber for 24 hours at 65˚C, 10 rpm. After hybridization, slides were washed according to Agilent protocol.
|
| Scan protocol |
Scanned on an Agilent G2600D scanner.
|
| Data processing |
Data were extracted using Agilent Feature Extraction Software. Signal was normalized by combined rank consistency filtering with LOWES intensity normalization.
Relative copy number values were calculated as a log 2 ratio of Cy5 processed signal/ Cy3 processed signal.
|
| |
|
| Submission date |
Mar 21, 2017 |
| Last update date |
Aug 22, 2017 |
| Contact name |
Shiv Grewal |
| Phone |
2407607553
|
| Organization name |
NCI
|
| Department |
LBMB
|
| Lab |
Shiv Grewal
|
| Street address |
NCI bldg 37 Rm 6068 9000 Rockville Pike
|
| City |
Bethesda |
| State/province |
MD |
| ZIP/Postal code |
20892 |
| Country |
USA |
| |
|
| Platform ID |
GPL6503 |
| Series (2) |
| GSE96881 |
Shelterin Mediates Genome Reorganization in Response to Replication Stress (Microarray CGH) |
| GSE96883 |
Shelterin Mediates Genome Reorganization in Response to Replication Stress |
|
