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Status |
Public on Feb 03, 2011 |
Title |
HUES49 |
Sample type |
RNA |
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Source name |
Human ES cell line HUES49, harvested at passage 17
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Organism |
Homo sapiens |
Characteristics |
cell line: HUES49 Sex: female passage number: 17
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Treatment protocol |
Embryoid body (EB) differentiation was performed as follows: Undifferentiated cells were harvested using dispase or trypsin and plated in suspension in low-adherence plates in the presence of human ES cell culture media without bFGF and plasmanate. Cell aggregates (EBs) were allowed to grow for a total of 16 days, refreshing media every 48h.
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Growth protocol |
The pluripotent cell lines were grown in human ES media consisting of KO-DMEM (Invitrogen), 10% KOSR (Invitrogen), 10% plasmanate (Talecris), 1% glutamax or L-glutamin, non-essential amino acids, penicillin/streptomycin, 0.1% 2-mercaptoethanol and 10-20ng/ml bFGF. All pluripotent cells were grown on a monolayer of irradiated CF1-MEFs (GlobalStem) and passaged using trypsin (0.05%) or dispase (Invitrogen). Before collection of RNA for analysis, ES and iPS cells were either isolated using trypsin (0.05%) or dispase, or plated on matrigel (BD Biosciences) for one passage and fed with human ES cell media conditioned in CF1-MEFs for 24h.
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Extracted molecule |
total RNA |
Extraction protocol |
Total RNA was isolated using RNeasy kit (Qiagen) according to manufacturer’s recommendation.
|
Label |
biotin
|
Label protocol |
Total RNA from the samples was normalized to 50 ng/ul and the GeneChip 3’ IVT Express Protocol was used for amplification in a semi automated process. The total RNA underwent reverse transcription to synthesize first-strand cDNA. This cDNA was then converted into a double-stranded DNA template for transcription. In vitro transcription synthesized aRNA and incorporated a biotin-conjugated nucleotide. The aRNA was then purified to remove unincorporated NTPs, salts, enzymes, and inorganic phosphate. Fragmentation of the biotin-labeled aRNA prepared the samples for hybridization on the microarray.
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Hybridization protocol |
The hybridization and subsequent washing and staining were performed on the Affymetrix GeneChip® Array Station (GCAS) automation platform.
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Scan protocol |
The microarrays were scanned on the GeneChip® HT Array Plate Scanner (Affymetrix 00-0332).
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Description |
Cultured under normal growth conditions
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Data processing |
The data were normalized using GCRMA and Bioconductor 2.4 with default parameters.
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Submission date |
Dec 09, 2010 |
Last update date |
Feb 03, 2011 |
Contact name |
Christoph Bock |
E-mail(s) |
cbock@cemm.oeaw.ac.at
|
Organization name |
CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences
|
Street address |
Lazarettgasse 14
|
City |
Vienna |
ZIP/Postal code |
1090 |
Country |
Austria |
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Platform ID |
GPL3921 |
Series (1) |
GSE25970 |
Reference maps of human ES and iPS cell variation enable high-throughput characterization of pluripotent cell lines |
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