|
Status |
Public on Aug 28, 2010 |
Title |
432_brain_normal |
Sample type |
genomic |
|
|
Channel 1 |
Source name |
Input fraction from normal brain
|
Organism |
Homo sapiens |
Characteristics |
sample type: Input fraction from normal brain tissue: frontal cortex
|
Treatment protocol |
untreated
|
Growth protocol |
genomic DNA was isolated from frozen tissue, i.e. not grown in culture
|
Extracted molecule |
genomic DNA |
Extraction protocol |
Genomic DNA (gDNA) were isolated using the MasterPure genomic DNA isolation kit (EpiCentre) according to the manufacturer’s protocol. For each sample, 5 µg of genomic DNA was randomly sheared into 1.6-3kb DNA size fragments using a Hydroshear device (DigiLab), digested with McrBC, and fractionated on a 1% agarose gel alongside the sheared input (UT) fraction to enrich for the unmethylated (MD) fraction. The UT and MD fractions were purified and whole genome amplified using the WGA2 kit (Sigma) according to the manufacturer's protocol.
|
Label |
Cy3
|
Label protocol |
2 µg of DNA was directly labeled by Klenow (New England Biolabs) random priming with Cy3 nonamers for the input (UT) fraction and Cy5 nonamers for the methyl-depleted (MD) fraction per manufacturer's protocol (http://www.nimblegen.com/products/lit/methylation_userguide_v5p0.pdf).
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|
|
Channel 2 |
Source name |
Methyl-depleted fraction from normal brain
|
Organism |
Homo sapiens |
Characteristics |
tissue: frontal cortex sample type: Methyl-depleted fraction from normal brain
|
Treatment protocol |
untreated
|
Growth protocol |
genomic DNA was isolated from frozen tissue, i.e. not grown in culture
|
Extracted molecule |
genomic DNA |
Extraction protocol |
Genomic DNA (gDNA) were isolated using the MasterPure genomic DNA isolation kit (EpiCentre) according to the manufacturer’s protocol. For each sample, 5 µg of genomic DNA was randomly sheared into 1.6-3kb DNA size fragments using a Hydroshear device (DigiLab), digested with McrBC, and fractionated on a 1% agarose gel alongside the sheared input (UT) fraction to enrich for the unmethylated (MD) fraction. The UT and MD fractions were purified and whole genome amplified using the WGA2 kit (Sigma) according to the manufacturer's protocol.
|
Label |
Cy5
|
Label protocol |
2 µg of DNA was directly labeled by Klenow (New England Biolabs) random priming with Cy3 nonamers for the input (UT) fraction and Cy5 nonamers for the methyl-depleted (MD) fraction per manufacturer's protocol (http://www.nimblegen.com/products/lit/methylation_userguide_v5p0.pdf).
|
|
|
|
Hybridization protocol |
The labeled DNA was precipitated with 0.1 volume 5M NaCl and 1 volume isopropanol, and hybridized in NimbleGen Hybridization solution master mix. Arrays were hybridized in Maui hybridization stations for 16-20 h at 42C, and washes were completed using manufacturer's protocols (http://www.nimblegen.com/products/lit/methylation_userguide_v5p0.pdf).
|
Scan protocol |
Arrays were scanned on a GenePix 4000B scanner per manufacturer's protocol (http://www.nimblegen.com/products/lit/methylation_userguide_v5p0.pdf).
|
Description |
DNA methylation data from human brain
|
Data processing |
Arrays were processed using Nimblegen's standard protocol for Nimblescan 2.4 software and the charm bioconductor package v1.0.1 using a) quantile and b) subset-quantile normalization. Percent methylation calculated.
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|
|
Submission date |
Aug 27, 2010 |
Last update date |
Aug 27, 2010 |
Contact name |
Martin Aryee |
E-mail(s) |
Martin.aryee@ds.dfci.harvard.edu
|
Organization name |
Massachusetts General Hospital
|
Department |
Pathology
|
Street address |
149 13th Street
|
City |
Charlestown |
State/province |
MA |
ZIP/Postal code |
02129 |
Country |
USA |
|
|
Platform ID |
GPL9275 |
Series (1) |
GSE23841 |
DNA methylation data from human liver, frontal cortex, spleen and colon |
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