|
| Status |
Public on Sep 15, 2010 |
| Title |
IHA_69_2 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
Input fraction from IHA_69_2
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: lymphocyte
bmi: 33.8105180096942
Sex: male
subject_id: 69
timepoint: 1991
hybe_date: 20090729
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA (gDNA) were isolated using DNeasy kit (Qiagen) according to the manufacturer’s protocol. For each sample, 1~5 µg of genomic DNA was sheared into 1.6-3kb DNA size fragments, digested with the endonuclease McrBC, and fractionated on a 1% agarose gel alongside the sheared input (UT) fraction to enrich for the methyl-depleted (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.
|
| |
|
| Channel 2 |
| Source name |
Methyl-depleted fraction from IHA_69_2
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: lymphocyte
bmi: 33.8105180096942
Sex: male
subject_id: 69
timepoint: 1991
hybe_date: 20090729
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA (gDNA) were isolated using DNeasy kit (Qiagen) according to the manufacturer’s protocol. For each sample, 1~5 µg of genomic DNA was sheared into 1.6-3kb DNA size fragments, digested with the endonuclease McrBC, and fractionated on a 1% agarose gel alongside the sheared input (UT) fraction to enrich for the methyl-depleted (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.
|
| |
|
| |
| 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.
|
| Scan protocol |
Arrays were scanned on a GenePix 4000B scanner per manufacturer's protocol.
|
| Description |
IHA_69_2 input
IHA_69_2 methyl-depleted
|
| Data processing |
Arrays were processed using Nimblescan 2.4 software. The data processing methods are described in Ayree, Z. Wu, C. Ladd-Acosta, B. Herb, A. Feinberg, S. Yegnasubramanian, R. Irizarry. Accurate genome-scale percentage DNA methylation estimates from microarray data. Biostatistics, (2010).
Briefly, processing involved background correction using a modified RMA convolution model, a modified control-probe loess procedure for within-array normalization, between-array normalization using quantile normalization, and estimation of percentages.
|
| |
|
| Submission date |
Sep 09, 2010 |
| Last update date |
Sep 10, 2010 |
| Contact name |
Peter Murakami |
| E-mail(s) |
[email protected]
|
| Organization name |
Johns Hopkins University
|
| Department |
Center for Epigenetics
|
| Street address |
855 N Wolfe St, Rangos 580
|
| City |
Baltimore |
| State/province |
MD |
| ZIP/Postal code |
21205 |
| Country |
USA |
| |
|
| Platform ID |
GPL9275 |
| Series (1) |
| GSE23858 |
DNA methylation data from non-immortalized lymphocyte samples from participants of the AGES Reykjavik Study |
|
