|
| Status |
Public on Jul 30, 2010 |
| Title |
Cyt49_feeder-free_d2-B [miRNA] |
| Sample type |
RNA |
| |
|
| Source name |
Cyt49 cells, day 2 of differentiation
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: Cyt49 human embryonic stem cell line
growth protocol: feeder-free conditions
time: 2 days
|
| Treatment protocol |
For differentiation, growth medium was replaced with RPMI with 0% serum, 100ng/mL activin A and 25 ng/mL Wnt3A for 1 day, followed by RPMI with 0.2% serum and 100ng/mL Activin A for 2 days, followed by RPMI with 2% serum and 100ng/mL Activin A for 1 days.
|
| Growth protocol |
Cyt49 were plated on BD matrigel and cultured MEF conditioned medium, containing DMEM/F12 supplemented with 20% knockout serum replacement, 4 ng/mL basic FGF, and 10ng/mL activin A
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Trizol extraction of total RNA was performed according to the manufacturer's instructions. The miRNA-enriched fraction of small RNAs was purified from total RNA by polyacrylamide gel electrophoresis using Ambion’s flashPAGE™ kits
|
| Label |
biotin
|
| Label protocol |
The 3' ends of the RNA molecules were tailed and biotin-labeled using the mirVana miRNA Labeling Kit (Ambion Inc., Austin, TX). The kit's dNTP mixture in the tailing reaction was replaced with a proprietary nucleotide mixture containing biotin-modified nucleotides (PerkinElmer, Waltham, MA).
|
| |
|
| Hybridization protocol |
Hybridization, washing, staining, imaging and signal extraction were performed according to Affymetrix-recommended procedures, excet that the 20X GeneChip Eikaryotic Hybridization Control cocktail was omitted from the hybridization.
|
| Scan protocol |
Affymetrix GeneChip® 3000 7G scanner
|
| Description |
miRNA expression data from hESCs differentiated 2 days
|
| Data processing |
The signal processing implemented for the Ambion miRCHIP is a multi-step process involving probe specific signal detection calls, background estimate and correction, constant variance stabilization and either array scaling or global normalization. For each probe, an estimated background value is subtracted that is derived from the median signal of a set of G-C matched anti-genomic controls. Arrays within a specific analysis experiment were normalized together according to the variance stabilization methods described by Huber et al. (Huber et al., 2002). Detection calls were based on a Wilcoxon rank-sum test of the miRNA probe signal compared to the distribution of signals from GC-content matched anti-genomic probes. For statistical hypothesis testing, a two-sample t-Test, with assumption of equal variance, was applied. One-way ANOVA was used for experimental designs with more than two experimental groupings or levels of the same factor. These tests define which probes are considered to be significantly differentially expressed, or significant, based on a default p-value of 0.001 and log2 difference > 1.
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| |
|
| Submission date |
Jun 17, 2009 |
| Last update date |
Jul 30, 2010 |
| Contact name |
Andrew Hinton |
| E-mail(s) |
ahinton@ucsd.edu
|
| Organization name |
UC, San Diego
|
| Department |
Pediatrics
|
| Lab |
Hayek
|
| Street address |
3525 John Hopkins Ct.
|
| City |
San Diego |
| State/province |
CA |
| ZIP/Postal code |
92121 |
| Country |
USA |
| |
|
| Platform ID |
GPL8733 |
| Series (2) |
| GSE16678 |
MicroRNA expression data from differentiation of human Cyt49 ESCs into definitive endoderm in feeder-free conditions |
| GSE16690 |
A distinct microRNA signature for definitive endoderm derived from human embryonic stem cells |
|
