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Sample GSM611257 Query DataSets for GSM611257
Status Public on May 31, 2011
Title Day10_uninfected_rep3 _Dchip
Sample type RNA
 
Source name CD34+-derived Hematopoietic stem cells in vitro differentiated to day 10, uninfected
Organism Homo sapiens
Characteristics cell type: growth factor-mobilized CD34+ hematopoietic stem cells
developmental stage: differentiated to day 10 (polychromatophilic 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 mitotic
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
Relations
Reanalysis of GSM611241

Data table header descriptions
ID_REF
VALUE Dchip normalized signal intensity
CALL

Data table
ID_REF VALUE CALL
AFFX-BioB-5_at 433.32 P
AFFX-BioB-M_at 714.97 P
AFFX-BioB-3_at 467.97 P
AFFX-BioC-5_at 937.06 P
AFFX-BioC-3_at 1292.14 P
AFFX-BioDn-5_at 2473.74 P
AFFX-BioDn-3_at 3776.38 P
AFFX-CreX-5_at 12808.93 P
AFFX-CreX-3_at 12265.7 P
AFFX-DapX-5_at 86.99 P
AFFX-DapX-M_at 688.7 P
AFFX-DapX-3_at 1199.75 P
AFFX-LysX-5_at 40.27 P
AFFX-LysX-M_at 146.08 P
AFFX-LysX-3_at 219.26 P
AFFX-PheX-5_at 62.41 P
AFFX-PheX-M_at 122.09 P
AFFX-PheX-3_at 169.88 P
AFFX-ThrX-5_at 40.41 M
AFFX-ThrX-M_at 130.24 P

Total number of rows: 54675

Table truncated, full table size 1001 Kbytes.




Supplementary file Size Download File type/resource
GSM611257.CEL.gz 4.6 Mb (ftp)(http) CEL
Processed data included within Sample table

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