|
Status |
Public on May 08, 2014 |
Title |
genomic dna from colon_HCT116_AZA_D14 |
Sample type |
genomic |
|
|
Source name |
colon_HCT116_AZA
|
Organism |
Homo sapiens |
Characteristics |
cell line: HCT116 cell type: colon cancer cell line sample group: AZA_D14
|
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 |
|