|
| Status |
Public on May 31, 2011 |
| Title |
Day14_uninfected_rep5_GenePattern |
| Sample type |
RNA |
| |
|
| Source name |
CD34+-derived Hematopoietic stem cells in vitro differentiated to day 14, uninfected
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: growth factor-mobilized CD34+ hematopoietic stem cells
developmental stage: differentiated to day 14 (orthochromatic stage)
|
| Treatment protocol |
Plasmodium falciparum 3D7 was synchronized by successive rounds of Percoll and sorbitol. Late stage schizonts were used to initiate infection of 2x10^6 erythroblasts at a multiplicity of infection =5. Erythroblasts were infected on day 10 (polychromatic stage) and day 14 (orthochromatic) of culture. Cells were resuspended at 1x10^6/ml in Iscove’s Modified Dulbecco’s Medium supplemented with 30% human serum and 2 units/ml EPO and incubated in 5% CO2-humidified chamber at 37ºC. Cells were harvested after 24 hr for RNA isolation.
|
| Growth protocol |
Human primary erythroblasts were generated by culturing CD34 early hematopoietic progenitors initially isolated from growth factor-mobilized peripheral blood (purchased from ALL Cells, Inc.) using an Isolex 300i cell selection device. The culture contained 15% fetal bovine serum, 15% human serum, Isocove’s modified Dulbecco’s medium (IMDM), 10 ng/ml interleukin-3, 2 units/ml EPO, and 50 ng/ml SCF. During the initial 8 days of culture, cells were fed on days 3 and 6 by adding equal volumes of fresh culture media supplemented with growth factors. However, no new interleukin-3 was added after the initial addition on day 0, and the amount of SCF added to the fresh media was gradually decreased at each feeding (day 3, 25 ng/ml; day 6, 10 ng/ml; day 8, 2 ng/ml). The amount of EPO added was 2 units/ml during each feeding. On day 8 of culture, cells were further purified by flow cytometry sorting for GlyA/CD71 or CD71 cells using a MoFlo high speed flow cytometer. The purity of the population isolated by this method was 98–99%. Sorted cells were cultured in the same media as before with EPO and SCF, except the concentration of SCF was reduced to 2 ng/ml. Cells were fed one more time on day 10 of culture by adding equal volumes of fresh media with only EPO (2 units/ml) during this final feeding.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Trizol extraction of total RNA was performed according to the manufacturer's instructions. Total RNA was purified over Qiagen columns (Rneasy) according to manufacturer's recommendations
|
| Label |
biotin
|
| Label protocol |
Biotinylated cRNA were prepared according to the standard Affymetrix protocol from 200 ng total RNA.
|
| |
|
| Hybridization protocol |
Following fragmentation, 11 ug of cRNA were hybridized for 16 hr at 45C on GeneChip HG-U133 plus 2.0 Array. GeneChips were washed and stained in the GeneChip® Fluidics Station 450.
|
| Scan protocol |
GeneChips were scanned using the GeneChip® Scanner 3000 7G.
|
| Description |
Gene expression data from differentiating hematopoietic stem cells that are preparing to extrude the nucleus
|
| Data processing |
The data were analyzed with 2 programs, GenePattern and Dchip. For GenePattern, Data were normalized with RMA using the “ExpressionFileCreator” module and filtered using the default settings of “PreprocessDataset” module. Dchip data were normalized using model-based expression and default settings.
|
| |
|
| Submission date |
Oct 20, 2010 |
| Last update date |
May 31, 2011 |
| Contact name |
Kasturi Haldar |
| E-mail(s) |
khaldar@nd.edu, ptamez@nd.edu
|
| Organization name |
University of Notre Dame
|
| Department |
Biology
|
| Lab |
Haldar
|
| Street address |
107 Galvin Life Sciences
|
| City |
South Bend |
| State/province |
IN |
| ZIP/Postal code |
46556 |
| Country |
USA |
| |
|
| Platform ID |
GPL570 |
| Series (1) |
| GSE24849 |
Human CD34+-derived erythoblast (polychromatophilic and orthochromatic) response to co-culture with Plasmodium falciparum 3D7 |
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