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| Status |
Public on May 01, 2009 |
| Title |
U2OS IP2 |
| Sample type |
genomic |
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| Source name |
OS cell line 1
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| Organism |
Homo sapiens |
| Characteristics |
U2OS cell line (ATCC # HTB-96)
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| Treatment protocol |
Cells were harvested with Trypsin (Invitrogen) as per manifacturer's instructions
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| Growth protocol |
The human OS cell lines U2OS (ATCC # HTB-96) and MG63 (ATCC # CRL-1427) were purchased from American Type Culture Collection ATCC (Rockville, MD) and maintained in alpha-Minimum Essential Medium (alpha-MEM) supplemented with 10% heat inactivated Fetal Bovine Serum and 2 mM L-Glutamine. 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 |
Methyl-Cytosine Immunoprecipitated fraction - Replicate 2
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| Data processing |
The Me-DIP-chip .cel files for HOB, U2OS and MG63 (2 IP and 2 IN) were log2 transformed, normalized and imported into Partek Genomic Suite Software as previously described [ Sadikovic B, Andrews J, Carter D, Robinson J, Rodenhiser DI (2008) Genome-wide H3K9 histone acetylation profiles are altered in benzopyrene-treated MCF7 breast cancer cells. J Biol Chem 283: 4051-4060]. 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 U2OS or MG63 relative to HOB, we used the PGS 1-way ANOVA tool and calculated the fold change using the geometric mean (for log-transformed data). Thus generated signals represented baseline-normalized, HOB-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 PGA by applying it to the fold change data for both U2OS and MG63. In order to capture significant differences in enrichment in each cancer cell line vs. HOB 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 cutoffs 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.transcript.csv file. Normalized data provided as Series supplementary file.
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| Submission date |
May 12, 2008 |
| Last update date |
May 12, 2008 |
| 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) |
| GSE11413 |
Me-DIP-chip data from osteosarcoma cell lines |
| GSE11416 |
Comparison of osteosarcoma cell lines and normal human osteoblasts |
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| Supplementary file |
Size |
Download |
File type/resource |
| GSM287994.CEL.gz |
20.3 Mb |
(ftp)(http) |
CEL |
| Processed data are available on Series record |
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