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Status |
Public on Jul 12, 2013 |
Title |
MDA-MB-436_day3_methyl |
Sample type |
genomic |
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Source name |
MDA-MB-436 breast cancer cell line, day 3
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Organism |
Homo sapiens |
Characteristics |
gender: Female disease: Breast cancer er: -
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Growth protocol |
Cells were grown for three days changing media every day
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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.
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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.
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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.
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Scan protocol |
Polymer-coated chips were image-processed using an Illumina’s iScan scanner.
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Description |
MDA-MB-436_day3 MDA-MB-436 d3
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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
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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. |
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