|
| Status |
Public on Feb 04, 2013 |
| Title |
Prostate cancer metastasis 15979 Enriched |
| Sample type |
genomic |
| |
|
| Source name |
Prostate cancer metastasis
|
| Organism |
Homo sapiens |
| Characteristics |
dna fraction: Enriched
subject type: Patient
subject id: 16
cell type: Tumor
tissue site: Lymph node
description: L Pulm Hilar LN Met
sample id (short form for plotting): 16-5
|
| Treatment protocol |
For MBD-SNP, samples were enriched for methylated DNA using magnetic-bead immobilized methyl-binding domain polypeptides as described previously (Yegnasubramanian et al., Nucl Acids Res, 2006; Yegnasubramanian et al., BMC Genomics, 2011). A unenriched total input sample that was otherwise treated the same, was used as a reference for each sample and was used to derive copy number estimates.
|
| Growth protocol |
DNA was obtained from metastatic prostate cancer tissues from the PELICAN rapid autopsy program at Johns Hopkins University, and from normal prostate tissues from organ donors.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
DNA was extracted using the manufacturer's recommended protocol
|
| Label |
biotin
|
| Label protocol |
In the MBD-SNP assay described here, we use the MBD2-MBD polypeptide to isolate methylated DNA fragments from genomic DNA samples followed by analysis with Affymetrix SNP 6.0 high density oligonucleotide microarrays. Comparison with an unenriched total input fraction then allows genome-scale determination of total and allele-specific methylation and copy number in an integrated fashion for each specimen. Briefly, each genomic DNA specimen was divided into two fractions: i) an enriched methylated fraction (EM) and, ii) a total input fraction (TI). Each of these fractions was digested with the NspI and StyI restriction enzymes in separate reactions. The resulting genomic DNA fragments were then ligated with Affymetrix SNP 6.0 assay adaptors. These restriction digest and adaptor ligation steps were carried out following the Affymetrix SNP 6.0 assay protocols. After adaptor ligation, the EM fraction was subject to enrichment for methylated DNA fragments using MBD2-MBD polypeptides immobilized on magnetic beads as previously described (Yegnasubramanian et al., Nucl Acids Res, 2006; Yegnasubramanian et al., BMC Genomics, 2011) except that the final DNA was eluted in 30 uL of EB1 buffer (0.2X NEBuffer 1 (New England Biolabs, Ipswich, MA), 0.2X BSA (NEB), 0.25X T4 DNA ligase Buffer (NEB)) for the NspI reaction and EB2 buffer (0.2X NEBuffer3, 0.2X BSA (NEB), 0.25X T4 DNA ligase buffer (NEB)) for the StyI reaction. The resulting EM and TI fractions were then subjected to one primer amplification, fragmentation, and labeling according to the Affymetrix SNP 6.0 manufacturer’s protocol.
|
| |
|
| Hybridization protocol |
Samples were hybridized to the Affy6 chip for 18 hrs. We washed and stained the arrays using an Affymetrix fluidics Station 450 and scanned the arrays using a GeneChip Scanner 3000 7G (Affymetrix, Inc., Santa Clara, CA).
|
| Scan protocol |
The Affymetrix GeneChip® Operating Software (GCOS) was used to collect and extract feature data from Affymetrix GeneChip® Scanners.
|
| Data processing |
Data was normalized using the 'asm' R package. Due to the sensitivity profile of the MBD enrichment assay, probes in regions with a CpG density of less than 2.5% were discarded (leaving about 50k probes for analysis).
|
| |
|
| Submission date |
May 21, 2012 |
| Last update date |
Feb 04, 2013 |
| Contact name |
Martin Aryee |
| E-mail(s) |
aryee.martin@mgh.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 |
GPL6801 |
| Series (2) |
| GSE38073 |
DNA methylation profiling of normal prostates and prostate cancer metastases [Affymetrix] |
| GSE38242 |
DNA methylation alterations exhibit striking intra-individual stability and inter-individual heterogeneity across metastatic dissemination |
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