|
| Status |
Public on Mar 04, 2024 |
| Title |
RPE1_WT_passage0 [RPE1] |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
RPE-1 cell line, near-diploid, passage 0
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: hTERT RPE-1
gender: female
tissue: Eye; Pigmented epithelium; Retina
disease: Normal
transferred chromosomes: none
in-vitro evolution: passage 0
|
| Treatment protocol |
no treatment
|
| Growth protocol |
Both HCT116 and RPE-1 cell lines were cultured in DMEM (supplemented with 10% FBS) at 5% CO2, 37°C
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were collected with trypsin and centrifuged. The pellet was washed twice with PBS. DNA isolation was performed using the Qiagen DNA easy Blood and Tissue Kit (Hilden, Germany) following manufacturer’s instructions.
|
| Label |
Cy5
|
| Label protocol |
Equal amounts of genomic DNA for both the experimental and reference samples were sheared by enzymatic digestion. 1.0 µg gDNA of each sample was treated with a combination of Alu I and Rsa I at 37°C for 2 hours. The Genomic DNA Enzymatic Labeling Kit (Agilent Technologies) uses random primers and the exo-Klenow fragment to differentially label the digested gDNA samples with fluorescent_x0002_labeled nucleotides. Cyanine-5 dUTP (dUTP = 2’-deoxyuridine 5’-triphosphate) was used for all experimental samples , whereas Cyanine-3 dUTP was used for the reference samples.
|
| |
|
| Channel 2 |
| Source name |
human genomic DNA, female (#G1521, Promega)
|
| Organism |
Homo sapiens |
| Characteristics |
gender: female
|
| Treatment protocol |
no treatment
|
| Growth protocol |
Both HCT116 and RPE-1 cell lines were cultured in DMEM (supplemented with 10% FBS) at 5% CO2, 37°C
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were collected with trypsin and centrifuged. The pellet was washed twice with PBS. DNA isolation was performed using the Qiagen DNA easy Blood and Tissue Kit (Hilden, Germany) following manufacturer’s instructions.
|
| Label |
Cy3
|
| Label protocol |
Equal amounts of genomic DNA for both the experimental and reference samples were sheared by enzymatic digestion. 1.0 µg gDNA of each sample was treated with a combination of Alu I and Rsa I at 37°C for 2 hours. The Genomic DNA Enzymatic Labeling Kit (Agilent Technologies) uses random primers and the exo-Klenow fragment to differentially label the digested gDNA samples with fluorescent_x0002_labeled nucleotides. Cyanine-5 dUTP (dUTP = 2’-deoxyuridine 5’-triphosphate) was used for all experimental samples , whereas Cyanine-3 dUTP was used for the reference samples.
|
| |
|
| |
| Hybridization protocol |
After purification, equal volumes of the corresponding Cyanine-5- and Cyanine-3-labeled samples were mixed. Human Cot-1 DNA (Invitrogen) was added to block repetitive sequences in the DNA. The combined samples were pre-hybridized before Two-Color based hybridization (Oligo aCGH Hybridization Kit, Agilent Technologies). All combinations were hybridized at 65°C for 24 hrs on Agilent Human Genome CGH Microarrays (4x44K format, AMADID 014950). The hybridized microarrays were washed with increasing stringency using Oligo aCGH Wash Buffers (Agilent Technologies) followed by drying with acetonitrile (SIGMA).
|
| Scan protocol |
Fluorescent signal intensities were detected with Scan Control 8.4.1 Software (Agilent Technologies) on the Agilent DNA Microarray Scanner and extracted from the images using Feature Extraction 10.5.1.1 Software (Agilent Technologies).
|
| Description |
CGH probe signal intensities of hTERT RPE-1 cells compared with female human gDNA (#G1521, Promega)
|
| Data processing |
The Agilent Genomic Workbench 7.0.4.0 was used to normalize and process the extracted singnals, applying probe filtering, as well as GC correction and re-centralization of log2 ratios.
|
| |
|
| Submission date |
Feb 02, 2024 |
| Last update date |
Mar 04, 2024 |
| Contact name |
Jan-Eric Boekenkamp |
| Organization name |
University of Kaiserslautern-Landau
|
| Department |
Molecular Genetics
|
| Street address |
Paul-Ehrlich-Strasse 24
|
| City |
Kaiserslautern |
| ZIP/Postal code |
67663 |
| Country |
Germany |
| |
|
| Platform ID |
GPL5477 |
| Series (1) |
| GSE254936 |
Proteogenomic analysis reveals adaptive strategies to alleviate the consequences of aneuploidy in cancer |
|
