|
| Status |
Public on Apr 29, 2010 |
| Title |
MCF-7_1 [MeDIP-chip] |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
methylated ChIP DNA from MCF-7 cells
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: MCF-7 breast cancer cell line
antibody: anti-5-methyl cytidine (MAb-335MEC-500; Monoclonal Methyl DNA IP-grade; 1 μg/μl; Diagenode)
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Methylated DNA from MCF7 cells was prepared according to the protocol published by Schubeler and coworkers (Weber et al. 2005). Briefly, high-quality genomic DNA was sheared to 300 bp (fragment size ranging from 200-600 bp) using Bioruptor. Fragmented DNA was heat denatured to produce single-stranded DNA to enhance the immunoselection step. Antibodies against 5-methyl cytidine (MAb-335MEC-500; Monoclonal Methyl DNA IP-grade; 1 μg/μl; Diagenode) were complexed to the magnetic beads (pan-mouse IgG Dynal Beads, Invitrogen). The immunoprecipitated methylated DNA fragments were processed by the NimbleGen Methylation Microarray Service (NimbleGen Systems, Inc. Madison, WI). Methylation analysis was performed on the NimbleGen Two-Array HG18 Promoter Set.
|
| Label |
Cy5
|
| Label protocol |
Every sample was hybridized with MeDIP sample (Cyanine-5 dye) and sonicated genomic DNA (Cyanine-3 dye).
|
| |
|
| Channel 2 |
| Source name |
input DNA from MCF-7 cells
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: MCF-7 breast cancer cell line
antibody: none
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Methylated DNA from MCF7 cells was prepared according to the protocol published by Schubeler and coworkers (Weber et al. 2005). Briefly, high-quality genomic DNA was sheared to 300 bp (fragment size ranging from 200-600 bp) using Bioruptor. Fragmented DNA was heat denatured to produce single-stranded DNA to enhance the immunoselection step. Antibodies against 5-methyl cytidine (MAb-335MEC-500; Monoclonal Methyl DNA IP-grade; 1 μg/μl; Diagenode) were complexed to the magnetic beads (pan-mouse IgG Dynal Beads, Invitrogen). The immunoprecipitated methylated DNA fragments were processed by the NimbleGen Methylation Microarray Service (NimbleGen Systems, Inc. Madison, WI). Methylation analysis was performed on the NimbleGen Two-Array HG18 Promoter Set.
|
| Label |
Cy3
|
| Label protocol |
Every sample was hybridized with MeDIP sample (Cyanine-5 dye) and sonicated genomic DNA (Cyanine-3 dye).
|
| |
|
| |
| Hybridization protocol |
The labeled ChIP DNA was precipitated with 0.1 volume 5M NaCl and 1 volume isopropanol, and hybridized in 45 ul of buffer containing 20% formamide, 1.2 M betaine, 0.1 ug/ul herring sperm DNA and 10 ug of human COT1 DNA (Invitrogen). Arrays were hybridized in Maui hybridization stations for 16-18 h at 42C, and then washed in 42C 0.2% SDS/0.2x SSC, room temperature 0.2x SSC, and 0.05x SSC. Hybridization buffers and washes were completed using manufacturer's protocols (http://www.nimblegen.com/products/lit/lit.html)
|
| Scan protocol |
Arrays were scanned on an Axon 4000B scanner per manufacturer's protocol (http://www.nimblegen.com/products/lit/lit.html).
|
| Description |
meDIP-chip MCF-7 cells with 5-methyl cytidine antibodies
|
| Data processing |
Arrays were processed using Nimblegen's standard protocol for Nimblescan 2.4 ChIP data extraction. The intensity ratio of the mDIP-chip sample to total DNA was plotted genomic position to identify regions of enrichment. P-values files were generated from the scaled log2-ratio data to test positive enrichement of DNA methylation for each probe against all probes on the array. Peak files identifying regions of DNA methylation were generated from the P-value files and the enriched peaks were then mapped to the transcription start site of each gene.
|
| |
|
| Submission date |
Apr 01, 2010 |
| Last update date |
Apr 29, 2010 |
| Contact name |
Pei-Yin Hsu |
| Organization name |
OSU
|
| Street address |
BRT836, 460W 12th Ave
|
| City |
Columbus |
| State/province |
OH |
| ZIP/Postal code |
43220 |
| Country |
USA |
| |
|
| Platform ID |
GPL6325 |
| Series (1) |
| GSE21068 |
Estrogen-mediated Epigenetic Repression of Large Chromosomal Regions through DNA Looping |
|
