|
| Status |
Public on Jan 09, 2015 |
| Title |
Prefrontal Cortex 9 input, methyl-depleted |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
Input fraction from PFC9
|
| Organism |
Homo sapiens |
| Characteristics |
sample type: Input fraction
tissue: prefrontal cortex
|
| Treatment protocol |
no treatment
|
| Growth protocol |
genomic DNA was isolated from frozen tissue, i.e not grown in culture
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
DNA was extracted from tissues using the MasterPure DNA Purification kit (Epicentre Biotechnologies) and 10 µg of DNA were sheared using a Hydroshear device (Digilab, Holliston, MA) into 1.6 kb-3 kb fragments. Sheared DNA was then divided into two fractions. One fraction was digested overnight at 37°C with the methyl-sensitive enzyme McrBC (NEB, Ipswich, MA). Following digestion, cut and uncut fractions from the same sample were electrophoresed in adjacent wells of a 1% agarose gel. Areas corresponding to the 1.6 kb-3 kb regions were excised and purified using Qiagen Spin Gel Purification columns (Qiagen, Germantown, MD). The gel-purified DNA was quantified on a NanoDrop 1000 Spectrophotometer (Thermo Scientific, Rockford, IL) and 30 ng of DNA from each fraction was amplified using a GenomePlex Whole Genome Amplification Kit (Sigma-Aldrich, St. Louis, MO). The amplified DNA was then isolated with a Qiagen PCR Purification column, then quantified on NanoDrop.
|
| 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).
|
| |
|
| Channel 2 |
| Source name |
Methyl-depleted fraction from PFC9
|
| Organism |
Homo sapiens |
| Characteristics |
sample type: Methyl-depleted fraction
tissue: prefrontal cortex
|
| Treatment protocol |
no treatment
|
| Growth protocol |
genomic DNA was isolated from frozen tissue, i.e not grown in culture
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
DNA was extracted from tissues using the MasterPure DNA Purification kit (Epicentre Biotechnologies) and 10 µg of DNA were sheared using a Hydroshear device (Digilab, Holliston, MA) into 1.6 kb-3 kb fragments. Sheared DNA was then divided into two fractions. One fraction was digested overnight at 37°C with the methyl-sensitive enzyme McrBC (NEB, Ipswich, MA). Following digestion, cut and uncut fractions from the same sample were electrophoresed in adjacent wells of a 1% agarose gel. Areas corresponding to the 1.6 kb-3 kb regions were excised and purified using Qiagen Spin Gel Purification columns (Qiagen, Germantown, MD). The gel-purified DNA was quantified on a NanoDrop 1000 Spectrophotometer (Thermo Scientific, Rockford, IL) and 30 ng of DNA from each fraction was amplified using a GenomePlex Whole Genome Amplification Kit (Sigma-Aldrich, St. Louis, MO). The amplified DNA was then isolated with a Qiagen PCR Purification column, then quantified on NanoDrop.
|
| 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.
|
| |
|
| Submission date |
Sep 30, 2011 |
| Last update date |
Jan 09, 2015 |
| Contact name |
Jerry Guintivano |
| E-mail(s) |
[email protected]
|
| Organization name |
Johns Hopkins University
|
| Street address |
720 Rutland Avenue, Ross 1068
|
| City |
Baltimore |
| State/province |
MD |
| ZIP/Postal code |
21205 |
| Country |
USA |
| |
|
| Platform ID |
GPL9275 |
| Series (1) |
| GSE32528 |
DNA methylation profiles of human prefrontal cortex |
|
