|
| Status |
Public on Dec 26, 2018 |
| Title |
HSR-GBM1+5azacitidine replicate 3 |
| Sample type |
genomic |
| |
|
| Source name |
Glioblastoma
|
| Organism |
Homo sapiens |
| Characteristics |
gender: Male
tissue: brain tumor
disease state: Glioblastoma
cell line: HSR-GBM1
|
| Treatment protocol |
HSR-GBM1 cells were cultured in the presence of 0.5 μM of 5-azacytidine (Sigma-Aldrich) for 48 hours or 100 μg/mL of Vitamin C (L-ascorbic acid, Sigma-Aldrich) for 72 hours. Culture media was changed every 24 hours.
|
| Growth protocol |
HSR-GBM1 cells were cultured as neurospheres on ultra-low attachment plates (Sigma) in Dulbecco's Modified Eagle Medium /Nutrient Mixture (DMEM/F-12) Media (Gibco) containing 2% StemPro neural supplement (Invitrogen Life Technologies), 20 ng/ml basic fibroblast growth factor (bFGF; Millipore) and 20 ng/ml epidermal growth factor (EGF; Millipore) at 37 °C, 5% CO2 and 95% humidity.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Total genomic DNA were extracted from cultured cell pellets using the ISOLATE II Genomic DNA Kit (Bioline) according to manufacturer’s protocols . The DNA samples were treated with 3 μL of RNase solution (Qiagen) and Proteinase K Solution (20 μg/μL; Qiagen) at 65 °C for 10 minutes.
|
| Label |
C-Bio and A-DNP
|
| Label protocol |
Approximately 200ng of genomic DNA was whole-genome amplified using neutralisation mix (MA2) and amplification master mix (MSM) and incubated for 22 hours at 37°C. After incubation, the amplified DNA was fragmented with fragmentation solution (FMS), precipitated with precipitation solution (PM1) and 2-propanol and resuspended in resuspension/hybridisation solution (RA1).
|
| |
|
| Hybridization protocol |
The fragmented, resuspended DNA was loaded onto a BeadChip and incubated for 18 hours at 48°C. After incubation, single-base extension and allele-specific staining was performed on a Te-Flow Chamber Rack (Tecan, Maennedorf, Switzerland).
|
| Scan protocol |
After allele-specific staining, the BeadChip was coated with XC4/EtOH, dried in a vacuum desiccator for approximately 1 hour and scanned on an iScan System (Illumina) using the Infinium LCG scan setting.
|
| Description |
Genomic DNA extracted from HSR-GBM1cells and the cells treated with vitamin C/5-azacitidine were genotyped using Infinium Global Screening Array-24 (Illumina).
|
| Data processing |
DNA samples were genotyped using Infinium Global Screening Array-24 (Illumina). Data were analyzed usign GenomeStudio 2.0 Genotyping Module (Illumina).
|
| |
|
| Submission date |
May 01, 2017 |
| Last update date |
Dec 26, 2018 |
| Contact name |
Justin C St. John |
| E-mail(s) |
justin.stjohn@hudson.org.au
|
| Phone |
+61399024749
|
| Organization name |
Hudson Institute of Medical Research
|
| Department |
Centre for Genetic Diseases
|
| Lab |
Mitochondrial Genetics
|
| Street address |
27-31 Wright St
|
| City |
Clayton |
| State/province |
Vic |
| ZIP/Postal code |
3168 |
| Country |
Australia |
| |
|
| Platform ID |
GPL23391 |
| Series (2) |
| GSE98377 |
Impact of DNA demethylation agents (5-azacitidine or vitamin C) on DNA copy number in gliobalstoma HSR-GBM1 cells |
| GSE98693 |
Induced DNA demethylation, genome instability and transcription |
|
