|
| Status |
Public on Jul 12, 2013 |
| Title |
SK-BR-3_day3_methyl |
| Sample type |
genomic |
| |
|
| Source name |
SK-BR-3 breast cancer cell line, day 3
|
| Organism |
Homo sapiens |
| Characteristics |
gender: Female
disease: Breast cancer
er: -
her2: +
|
| Growth protocol |
Cells were grown for three days changing media every day
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA was purified by phenol-chloroform extraction. 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 the EZ DNA Methylation Kit (Zymo Research) following the 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 indicated in the 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 a Nanodrop-1000 spectrometer.
|
| |
|
| Hybridization protocol |
Bisulfite-converted DNA was analyzed using Illumina’s Infinium Human Methylation450 Beadchip Kit (WG-314-1001) by following the 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 0.014 N 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 were loaded onto a 12-sample chip and the chips were assembled into an hybridization chamber as instructed in the manual. After incubation at 48C for 16-20 hours, the 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 using an Illumina’s iScan scanner.
|
| Description |
SK-BR-3_day3
SK-BR-3 d3
|
| Data processing |
Data were extracted using teh Methylation Module of GenomeStudio v1.0 Software. Methylation status of each CpG site was presented as 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).
Unmethylated and methylated signal intensities
|
| |
|
| Submission date |
Mar 04, 2013 |
| Last update date |
Jul 12, 2013 |
| Contact name |
Francescopaolo Di Cello |
| E-mail(s) |
fdicello@hotmail.com
|
| Organization name |
The Johns Hopkin University School of Medicine
|
| Street address |
1650 Orleans Street
|
| City |
Baltimore |
| State/province |
MD |
| ZIP/Postal code |
21287 |
| Country |
USA |
| |
|
| Platform ID |
GPL13534 |
| Series (2) |
| GSE44837 |
Gene methylation profile of breast cancer cells. |
| GSE44838 |
Gene expression and methylation profile of breast cancer cells. |
|
