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| Status |
Public on Jan 20, 2017 |
| Title |
Sample4_WA09_Mutant_2 |
| Sample type |
genomic |
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| Source name |
WA09 Human Embryonic Stem Cell
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| Organism |
Homo sapiens |
| Characteristics |
genotype/variation: ch17p13.1 deletion
cell type: WA09 Human Embryonic Stem Cell
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| Treatment protocol |
Single-cell cloning was used to derive pure subpopulations of mutant and wild-type cells from the original mosaic MefEnz culture. MefEnz cells cultured on irradiated mouse embryonic fibroblasts (MEFs) in standard hPSC medium, consisting of Dulbecco’s modified eagle medium DMEM/F12 (Life Technologies) with 20% Knockout Serum Replacement (Life Technologies), 1 mM GlutaMAX (Life Technologies), 0.1 mM nonessential amino acids (NEAA, Life Technologies), 0.1 mM -mercaptoethanol, and 12 ng/ml basic FGF (Life Technologies), were treated for 1 hour with 10 M ROCK inhibitor Y-27632 (Calbiochem) before dissociation. The medium was supplemented with SMC4 (0.58 l SMC4/ 1 ml medium) (BD Biosciences) for one day.
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| Growth protocol |
The four WA09 clones (2 wild-type and 2 mutant) were initially grown on MEF feeder layers in standard hPSC medium. These initial cultures were transitioned to feeder-free conditions in a stepwise fashion by passaging them with Accutase onto Geltrex (Gibco) with MEF-conditioned medium (MEF-CM) supplemented with 12 ng/ml basic FGF. After one passage in MEF-CM, the cultures were either continued in MEF-CM or passaged into E8 medium (Life Technologies). For all cultures, cells were grown at 37°C and 5% CO2 and passaged every three to four days, when they were ~80% confluent. At each passage, 150,000 cells were seeded per well of a 6-well plate (equivalent to a cell plating density of ~15,600 cells/ cm2). The medium was changed daily.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
DNA was extracted from hESC samples using the DNeasy Blood and Tissue Genomic DNA Purification Kit (QIAGEN), and quantified (Qubit dsDNA BR Assay Kits, Life Technologies, Inc.) according to the manufacturer’s protocol.
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| Label |
Cy3 and Cy5
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| Label protocol |
Standard Illumina Protocol
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| Hybridization protocol |
Standard Illumina Protocol
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| Scan protocol |
Arrays were imaged using Illumina's HiScan System using standard recommended Illumina scanner setting
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| Description |
Mutant 2
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| Data processing |
Genotyping calls were made with GenomeStudio (Illumina, Inc.) via the cluster files provided by the manufacturer. The GenCall (v. 6.3.0) threshold was set to 0.15, and the call rates were greater than 0.998. Reproducibility and heritability were calculated in GenomeStudio (Illumina, Inc.). CNVs were identified using the cnvPartition Plug-in v 3.2.0 in GenomeStudio (Illumina, Inc.). The cnvPartition confidence threshold was set at 35, with a minimum number of SNPs per CNV region of 3. All CNVs were visually verified by assessing both the B-allele-frequency and Log R ratios.
Name is the SNP ID, Chr (Chromosome), Position (location on Chr), Genotype: AA,AB,BB,or NC (No Call), Score, Thera, R, B Allele, Log R Ratio
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| Submission date |
Jul 13, 2015 |
| Last update date |
Jan 20, 2017 |
| Contact name |
Robert E Morey |
| E-mail(s) |
robmoreyucsd@gmail.com, remorey@health.ucsd.edu
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| Organization name |
UCSD
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| Department |
Pathology
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| Lab |
Parast
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| Street address |
2880 Torrey Pines Scenic Dr
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| City |
La Jolla |
| State/province |
CA |
| ZIP/Postal code |
92037 |
| Country |
USA |
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| Platform ID |
GPL14157 |
| Series (2) |
| GSE70828 |
Spontaneous single-copy loss of TP53 in human embryonic stem cells markedly increases cell proliferation and survival [BeadChip] |
| GSE70875 |
Spontaneous single-copy loss of TP53 in human embryonic stem cells markedly increases cell proliferation and survival |
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| Supplementary data files not provided |
| Processed data are available on Series record |
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