|
| Status |
Public on Mar 13, 2015 |
| Title |
A353V1 |
| Sample type |
RNA |
| |
|
| Source name |
IPS cells with A353V mutation of DKC1 gene, biological duplication 1
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: human IPS cells
genotype: A353V mutation
|
| Treatment protocol |
N/A
|
| Growth protocol |
IPS cells were cultured in 10-cm dish coated with mouse embryonic fibroblasts (MEFs) and grown in iPS cell medium (ES medium, DMEM/F12, supplemented with 20% knockout serum replacement, 0.1 mM nonessential amino acids, 1 mM l-glutamine [Invitrogen, Carlsbad, CA USA], 10 ng/ml recombinant human fibroblast like growth factor–basic (Peprotech, NJ, USA.), and 0.1 mM 2-mercaptoethanol (Sigma, St Louis, MO USA).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted by using RNeasy Mini Kit (Qiagen)
|
| Label |
Biotin
|
| Label protocol |
250ng of total RNA was converted to first-strand cDNA using reverse transcriptase primed by poly(T) and random oligomers that incorporated the T7 promoter sequence. Second-strand cDNA synthesis was followed by in vitro transcription with T7 RNA polymerase for linear amplification of each transcript, and the resulting cRNA was converted to cDNA, fragmented, assessed by Bioanalyzer, and biotinylated by terminal transferase end labeling
|
| |
|
| Hybridization protocol |
Five and a half micrograms of labeled cDNA were added to Affymetrix hybridization cocktails, heated at 99ºC for 5 min and hybridized for 16 h at 45ºC to Human Exon 1.0 ST GeneChips (Affymetrix Inc., Santa Clara CA) using the GeneChip Hybridization oven 645. The microarrays were then washed at low (6X SSPE) and high (100mM MES, 0.1M NaCl) stringency and stained with streptavidin-phycoerythrin. Fluorescence was amplified by adding biotinylated anti-streptavidin and an additional aliquot of streptavidin-phycoerythrin stain.
|
| Scan protocol |
A GeneChip 3000 7G scanner was used to collect fluorescence signal.
|
| Data processing |
Affymetrix Command Console and Expression Console were used to quantitate expression levels for targeted genes; default values provided by Affymetrix were applied to all analysis parameters. Probes for each targeted gene were averaged and inter-array normalization performed using the RMA algorithm
|
| |
|
| Submission date |
Mar 12, 2015 |
| Last update date |
Mar 13, 2015 |
| Contact name |
BAIWEI GU |
| E-mail(s) |
baiweigu@gmail.com
|
| Organization name |
The Children's Hospital of Philadelphia
|
| Street address |
3615 Civic center BLVD
|
| City |
Philadelphia |
| State/province |
PA |
| ZIP/Postal code |
19104 |
| Country |
USA |
| |
|
| Platform ID |
GPL5188 |
| Series (1) |
| GSE66849 |
Study the transcriptional level changes of induced pluripotent stem (iPS) cells from X-linked Dyskeratosis Congenita (DC) Patients |
|
