|
| Status |
Public on May 08, 2014 |
| Title |
genomic dna from ovarian_OVCAR5_d3_Aza |
| Sample type |
genomic |
| |
|
| Source name |
ovarian_OVCAR5_Aza
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: OVCAR5
cell type: ovarian cancer cell line
sample group: D3_Aza
|
| Treatment protocol |
Cell lines were treated with 500 nM 5-azacitidine for 72 hours while in log-growth phase, changing the media and drug every 24 hours for AZA treatment. Cells were harvested at 1, 3, 7, 10, 14, or 21 days following initial application of drug.
|
| Growth protocol |
Breast, colon, and ovarian cancer cell lines were maintained under ATCC recommended growth conditions.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA was extracted and purified from cell pellets using the Qiagen DNeasy Kit according to the manufacturer's instructions.
|
| Label |
Cy5 and Cy3
|
| Label protocol |
Bisulfite-converted DNA was analyzed using Illumina’s Infinium Human Methylation450 Beadchip Kit (WG-314-1001) by following manufacturer’s manual. Beadchip contains 485,577 CpG loci in human genome. Briefly, 4 ul of bisulfite-converted DNA was added to a 0.8 ml 96-well storage plate (Thermo Scientific), denatured in 0.014N sodium hydroxide, neutralized and amplified with kit-provided reagents and buffer at 37C for 20-24 hours. Samples were fragmented using kit-provided reagents and buffer at 37C for one hour and precipitated by adding 2-propanol. Re-suspended samples were denatured in a 96-well plate heat block at 95C for 20 minutes. 12 ul of each sample was loaded onto a 12-sample chip and the chips were assembled into hybridization chamber as instructed in the manual. After incubation at 48C for 16-20 hours, chips were briefly washed and then assembled and placed in a fluid flow-through station for primer-extension and staining procedures.
|
| |
|
| Hybridization protocol |
Bisulfite-converted DNA was analyzed using Illumina’s Infinium Human Methylation450 Beadchip Kit (WG-314-1001) by following manufacturer’s manual. Beadchip contains 485,577 CpG loci in human genome. Briefly, 4 ul of bisulfite-converted DNA was added to a 0.8 ml 96-well storage plate (Thermo Scientific), denatured in 0.014N sodium hydroxide, neutralized and amplified with kit-provided reagents and buffer at 37C for 20-24 hours. Samples were fragmented using kit-provided reagents and buffer at 37C for one hour and precipitated by adding 2-propanol. Re-suspended samples were denatured in a 96-well plate heat block at 95C for 20 minutes. 12 ul of each sample was loaded onto a 12-sample chip and the chips were assembled into hybridization chamber as instructed in the manual. After incubation at 48C for 16-20 hours, chips were briefly washed and then assembled and placed in a fluid flow-through station for primer-extension and staining procedures.
|
| Scan protocol |
Polymer-coated chips were image-processed in Illumina’s iScan scanner.
|
| Data processing |
Data were extracted using Methylation Module of GenomeStudio v1.0 Software. The 'signal_intensities.txt' contains unmethylated and methylated signal intensities. Methylation status of each CpG site was presented as b (beta) value based on following definition:
b value = (signal intensity of methylation-detection probe)/(signal intensity of methylation- detection probe + signal intensity of non-methylation-detection probe + 100).
|
| |
|
| Submission date |
May 06, 2014 |
| Last update date |
May 08, 2014 |
| Contact name |
Stephen Baylin |
| E-mail(s) |
sbaylin@jhmi.edu
|
| Phone |
4109558506
|
| Organization name |
Johns Hopkins UniversityJohns Hopkins Medical Institutions
|
| Department |
Oncology
|
| Street address |
1650 Orleans St CRB I RM 541
|
| City |
Baltimore |
| State/province |
Maryland |
| ZIP/Postal code |
21287 |
| Country |
USA |
| |
|
| Platform ID |
GPL13534 |
| Series (2) |
| GSE57342 |
Immune regulation by low doses of the DNA methyltransferase inhibitor 5-azacitidine in common human epithelial cancers [methylation] |
| GSE57343 |
Immune regulation by low doses of the DNA methyltransferase inhibitor 5-azacitidine in common human epithelial cancers |
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