|
| Status |
Public on Dec 03, 2010 |
| Title |
Day 7 differentiated H1 |
| Sample type |
mixed |
| |
|
| Channel 1 |
| Source name |
differentiated H1, day 7
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: H1
time of differentiation: 7 days
differentiation state: differentiated
|
| Treatment protocol |
The media for H1 hESC maintenance, colony-forming culture and MSC propagation contained 10-20 ng/ml basic fibroblast growth factor (FGF2). The differentiation medium for H1 hESC/OP9 cocultures contained 10% fetal bovine serum (FBS).
|
| Growth protocol |
H1 were maintained on irradiated MEFs (Amit et al. Dev. Biol. 2000; 271:278). Differentiation was induced by transferring H1 cells on OP9 stromal cells (Vodyanik et al: Blood 2005; 105:617-626). Mouse cells (MEFs, OP9) were depleted by magnetic sorting (Vodyanik, Slukvin: Curr Ptotoc Cell Biol 2007; 36:23.6.1-23.6.28); the purity of H1 and H1-derived cells used for analysis was >99%. H1-derived APLNR+ cells were isolated by magnetic sorting and cultured in the semisolid colony-forming culture to generate mesenchymal colonies, from which MSC lines were derived.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using the RiboPure kit (Ambion, Austin, TX) and treated with DNAse (Ambion).
|
| Label |
Cy5
|
| Label protocol |
Amino Allyl labeling method.
|
| |
|
| Channel 2 |
| Source name |
undifferentiated H1
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: embryonic stem cells
cell line: H1
differentiation state: undifferentiated
|
| Treatment protocol |
The media for H1 hESC maintenance, colony-forming culture and MSC propagation contained 10-20 ng/ml basic fibroblast growth factor (FGF2). The differentiation medium for H1 hESC/OP9 cocultures contained 10% fetal bovine serum (FBS).
|
| Growth protocol |
H1 were maintained on irradiated MEFs (Amit et al. Dev. Biol. 2000; 271:278). Differentiation was induced by transferring H1 cells on OP9 stromal cells (Vodyanik et al: Blood 2005; 105:617-626). Mouse cells (MEFs, OP9) were depleted by magnetic sorting (Vodyanik, Slukvin: Curr Ptotoc Cell Biol 2007; 36:23.6.1-23.6.28); the purity of H1 and H1-derived cells used for analysis was >99%. H1-derived APLNR+ cells were isolated by magnetic sorting and cultured in the semisolid colony-forming culture to generate mesenchymal colonies, from which MSC lines were derived.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA was extracted using the phenol/chloroform extraction method.
|
| Label |
Cy3
|
| Label protocol |
Amino Allyl labeling method.
|
| |
|
| |
| Hybridization protocol |
Array hybridization and washes were performed according to NimbleGen’s recommended protocol.
|
| Scan protocol |
Arrays were scanned using a GenePix 4000B scanner and the PMT settings were followed by NimbleGen array manual.
|
| Description |
The second channel (genomic DNA) served as common reference across the experiments.
DAY7_74308
|
| Data processing |
1. Image extraction: NimbleScan provided by NimbleGen is used to extract signal data from image files (*.TFF). Signal meansurements were saved in .pair files. 2. Normalization: Signal intensities from RNA and gDNA samples are normalized with Robust Multiple-chip Analysis (RMA) algorithm (Irizarry et al., 2003) separately. A median-adjusted ratio is calculated for each gene by dividing its signal intensity from the RNA sample by the sum of the corresponding signal intensity from the gDNA sample and median signal intensity of all genes from the gDNA channel.
|
| |
|
| Submission date |
Feb 01, 2010 |
| Last update date |
Sep 21, 2010 |
| Contact name |
Igor Slukvin |
| E-mail(s) |
islukvin@wisc.edu
|
| Phone |
608-263-0058
|
| Organization name |
University of Wisconsin
|
| Department |
Department of Pathology and Laboratory Medicine
|
| Street address |
1220 Capitol Court
|
| City |
Madison |
| State/province |
WI |
| ZIP/Postal code |
53706 |
| Country |
USA |
| |
|
| Platform ID |
GPL6602 |
| Series (2) |
| GSE20045 |
A mesoderm-derived mesenchymal stem/stromal cells (MSC) precursor: time course experiment |
| GSE20047 |
A mesoderm-derived mesenchymal stem/stromal cells (MSC) precursor |
|
