|
| Status |
Public on Jan 31, 2018 |
| Title |
genomic DNA from HRAS_3 |
| Sample type |
genomic |
| |
|
| Source name |
Early Transformed BJ Fibroblast Cells Replicate 3
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: BJ
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA was extracted and purified from cell pellets using the Promega Wizard Genomic DNA Purification Kit according to the manufacturer's instructions. Genomic DNA quality was assessed by low concentration agarose gel (0.6%) electrophoresis and spectrometry of OD260/280 and OD 260/230 ratio.
|
| Label |
Cy5 and Cy3
|
| Label protocol |
DNA bisulfite conversion was carried out using EZ DNA Methylation Kit (Zymo Research) by following manufacturer’s manual with modifications for Illumina Infinium Methylation Assay. Briefly, 0.5 – 1.0 ug of genomic DNA was first mixed with 5 ul of M-Dilution Buffer and incubate at 37C for 15 minutes and then mixed with 100 ul of CT Conversion Reagent prepared as instructed in the kit’s manual. Mixtures were incubated in a thermocycler with 16 thermal cycles at 95C for 30 seconds and 50C for one hour. Bisulfite-converted DNA samples were loaded onto 96-column plates provided in the kit for desulphonation and purification. Concentration of eluted DNA was measured using Nanodrop-1000 spectrometer. 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.
|
| Description |
Tranformed BJ fibroblast cells
|
| Data processing |
Data were extracted using Methylation Module of GenomeStudio v1.0 Software. Methylation status of each CpG site was presented as b (beta) value based on following definition: beta value = (signal intensity of methylation-detection probe)/(signal intensity of methylation- detection probe + signal intensity of non-methylation-detection probe + 100).
|
| |
|
| Submission date |
Dec 08, 2016 |
| Last update date |
Jan 31, 2018 |
| Contact name |
Wenbing Xie |
| E-mail(s) |
[email protected]
|
| Organization name |
The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, The Johns Hopkins University School of Medicine
|
| Department |
Department of Oncology
|
| Lab |
Stephen B. Baylin
|
| Street address |
1650 Orleans Street, CRB1, Room 530
|
| City |
Baltimore |
| State/province |
MD |
| ZIP/Postal code |
21205 |
| Country |
USA |
| |
|
| Platform ID |
GPL13534 |
| Series (2) |
| GSE91069 |
DNA methylation patterns separate senescence from malignant transformation potential in human cells [methylation] |
| GSE91071 |
DNA methylation patterns separate senescence from malignant transformation potential in human cells |
|
