|
| Status |
Public on Sep 20, 2011 |
| Title |
Urothelial cancer cell line EJ replicate 1 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
Bladder cancer cell line, 5-methylcystosine enriched DNA (IP)
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: EJ
cell type: Human bladder carcinoma, fast growing tumour
|
| Growth protocol |
Cancer cell lines (EJ, RT112) were grown in Dulbecco's medium with 10% FCS, and normal human urothelial (NHU) cell line was maintained in keratinocyte serum–free medium containing bovine pituitary extract, epidermal growth factor (Invitrogen), and cholera toxin
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
DNA was extracted with DNeasy Blood and Tissue extraction kit from Qiagen, as per manufacturer's instructions. For MeDIP, genomic DNA is extracted and purified from the cells and sheared by sonication to yield 200-600bp fragments. DNA is incubated with antibody raised against 5-methylcytosine. At the same time, a negative mouse IgG control is used in order to rule out non specific binding of antibodies. The antibody-antigen complex is captured with magnetic beads conjugated to anti-mouse-IgG. In extensive washes, unbound and non specific DNA are removed and methylated DNA is purified. Enrichment of methylated DNA in the immunoprecipitated fraction can be determined by PCR with primers designed to the known methylated gene.
|
| Label |
Cy5
|
| Label protocol |
The samples were fluorescently labelled with Cyanine 3-dUTP (the reference samples) and Cyanine 5-dUTP (immunoprecipitated DNA samples) according to Agilent’s recommendations
|
| |
|
| Channel 2 |
| Source name |
Bladder cancer cell line (non-IP)
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: EJ
cell type: Human bladder carcinoma, fast growing tumour
|
| Growth protocol |
Cancer cell lines (EJ, RT112) were grown in Dulbecco's medium with 10% FCS, and normal human urothelial (NHU) cell line was maintained in keratinocyte serum–free medium containing bovine pituitary extract, epidermal growth factor (Invitrogen), and cholera toxin
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
DNA was extracted with DNeasy Blood and Tissue extraction kit from Qiagen, as per manufacturer's instructions. For MeDIP, genomic DNA is extracted and purified from the cells and sheared by sonication to yield 200-600bp fragments. DNA is incubated with antibody raised against 5-methylcytosine. At the same time, a negative mouse IgG control is used in order to rule out non specific binding of antibodies. The antibody-antigen complex is captured with magnetic beads conjugated to anti-mouse-IgG. In extensive washes, unbound and non specific DNA are removed and methylated DNA is purified. Enrichment of methylated DNA in the immunoprecipitated fraction can be determined by PCR with primers designed to the known methylated gene.
|
| Label |
Cy3
|
| Label protocol |
The samples were fluorescently labelled with Cyanine 3-dUTP (the reference samples) and Cyanine 5-dUTP (immunoprecipitated DNA samples) according to Agilent’s recommendations
|
| |
|
| |
| Hybridization protocol |
Hybridization was performed according to the Agilent protocol. Hybridisation took place in a rotating hybridisation platform at 67°C for 40 hours. Post hybridisation stringency washing was done using the Affymetrix fluidics station again following the protocol outlined in the Agilent instructions
|
| Scan protocol |
Scanned on an Agilent G2565AA scanner.
Images were quantified using Agilent Feature Extraction Software (version v 9.5.3).
|
| Description |
EJ 25.6.10
|
| Data processing |
Agilent Feature Extraction Software (v 9.5.3) was used for background subtraction and LOWESS normalization.
|
| |
|
| Submission date |
Sep 02, 2011 |
| Last update date |
Sep 20, 2011 |
| Contact name |
Ewa Dudziec |
| E-mail(s) |
mdp07ed@sheffield.ac.uk
|
| Organization name |
The University of Sheffield
|
| Department |
ICS
|
| Street address |
Beech Hill Road
|
| City |
Sheffield |
| ZIP/Postal code |
S10 2RX |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL9767 |
| Series (2) |
| GSE31865 |
Global methylation in normal and malignant urothelial cells |
| GSE31866 |
Integrated epigenome profiling of DNA methylation and gene expression in normal and malignant urothelial cells |
|
