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| Status |
Public on Sep 08, 2009 |
| Title |
177 IN1 |
| Sample type |
genomic |
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| Source name |
osteosarcoma tissue sample 177
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| Organism |
Homo sapiens |
| Characteristics |
tissue: pediatric osteosarcoma
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| Treatment protocol |
Cells were harvested with Tripsin (Invitrogen) as per manifacturers instructions
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| Growth protocol |
Normal human osteoblasts (HOB) are primary osteoblasts from the hip bone of a normal male donor that were purchased from PromoCell (Heidelberg, Germany, Catalogue # C-12760) and maintained in medium provided by the manufacturer and used at culture passage 3. Three days after plating (~80% confluent), cells where harvested for DNA or RNA extractions.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
DNA was extracted by phenol-chloroform extraction and subsequent precipitation in 100% ethanol. DNA precipitate was washed with 70% ethanol then eluted in DNAse free water
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| Label |
streptavidin/phycoerythrin
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| Label protocol |
The uracil glycosilase treatment, streptavidin/phycoerythrin labeling, hybridization and microarray scanning were performed as per Affymetrix Chromatin Immunoprecipitation Assay Protocol (http://www.affymetrix.com/products/arrays/specific/human_promoter.affx) at The Centre for Applied Genomics (The Hospital for Sick Children, Toronto, ON, Canada)
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| Hybridization protocol |
The uracil glycosilase treatment, streptavidin/phycoerythrin labeling, hybridization and microarray scanning were performed as per Affymetrix Chromatin Immunoprecipitation Assay Protocol (http://www.affymetrix.com/products/arrays/specific/human_promoter.affx) at The Centre for Applied Genomics (The Hospital for Sick Children, Toronto, ON, Canada)
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| Scan protocol |
The uracil glycosilase treatment, streptavidin/phycoerythrin labeling, hybridization and microarray scanning were performed as per Affymetrix Chromatin Immunoprecipitation Assay Protocol (http://www.affymetrix.com/products/arrays/specific/human_promoter.affx) at The Centre for Applied Genomics (The Hospital for Sick Children, Toronto, ON, Canada)
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| Description |
Input DNA - Replicate 1
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| Data processing |
The Me-DIP-chip .cel files for osteoblasts and tumour samples (2 IP and 2 IN) were log2 transformed, normalized and imported into PGS. We baseline normalized the signal using the matched-pair normalization tool in PGS, by subtracting log2 IN signal intensity at each of 4.2 million from cell type-matched IP signals resulting in 6 corrected datasets (IP1 cor. and IP2 cor. for each cell line). In order to determine the relative enrichment of IP cor. signals in tumours relative to osteoblasts, we used the PGS 1-way ANOVA tool and calculated the fold change using the geometric mean (for log-transformed data) for individual tumours, or alternatively for all tumours vs. osteoblasts in cumulative analysis. Thus generated signals represented baseline-normalized, osteoblast-relative, and cancer cell-specific enrichment levels for each of 4.2 million probes. Significant region detection (both enrichment/hypermethylation and depletion/hypomethylation) was performed using Hidden Markov Model (HMM) tool in PGS by applying it to the fold change data for individual tumour or cumulative analysis. In order to capture significant differences in enrichment in tumours vs. normal osteoblast across shorter genomic regions (min. ~350 nucleotides) with robust enrichment differences as well as intermediate (min. ~500 nucleotides) and large regions (min. 1400 nucleotides), three HMM algorithms were applied (s-HMM, m-HMM, and l-HMM). Following cut-offs were used: s-HMM (min. probes: 10, detection states: -5,5, ignore state: 0, max. probability: 0.99, genomic decay: 10,000, sigma: 2), m-HMM (min. probes: 15, detection states: -3,3, ignore state: 0, max. probability: 0.99, genomic decay: 10,000, sigma: 1), and l-HMM (min. probes: 40, detection states: -1.5,1.5, ignore state: 0, max. probability: 0.99, genomic decay: 10,000, sigma: 1). Significantly enriched/hypermethylated and depleted/hypomethylated HMM regions were annotated to the corresponding genes present on the Affymetrix Gene 1.0 Array using the HuGene-1_0-st-v1.na24.hg18.transcriptmod.csv file which was annotated to detect regions associated with specific genes 10 Kb 5’ and 3 Kb 3’ from transcriptional start sites to correspond to the coverage on the Affy Promoter 1.0R Array.
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| Submission date |
Sep 19, 2008 |
| Last update date |
Aug 10, 2012 |
| Contact name |
Bekim Sadikovic |
| E-mail(s) |
bsadikov@gmail.com
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| Organization name |
The SickKids Hospital and Princess Margaret Hospital
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| Street address |
555 University Avenue
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| City |
Toronto |
| State/province |
Ontario |
| ZIP/Postal code |
M5G 1X8 |
| Country |
Canada |
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| Platform ID |
GPL5082 |
| Series (2) |
| GSE12864 |
Me-DIP-chip data from osteosarcoma tumours |
| GSE12885 |
Genome-wide changes in DNA methylation and copy number play a role in deregulation of gene expression in osteosarcoma |
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| Supplementary file |
Size |
Download |
File type/resource |
| GSM322673.CEL.gz |
18.1 Mb |
(ftp)(http) |
CEL |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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