|
| Status |
Public on Jan 01, 2014 |
| Title |
Human CD34 replicate 1 |
| Sample type |
RNA |
| |
|
| Source name |
Human CD34 replicate 1
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: CD34
source sample: 1
hybridization batch: 1
|
| Treatment protocol |
No treatment was applied to the cells.
|
| Growth protocol |
Purified adult peripheral CD34 cells were growed in complete StemSpan Serum-Free Expansion Medium for 7 days before collection; iPS and iNS were generated by transfecting CD34+ cells with Sendai virus constructs for SOX-2,Klf-4, OCT3/4 and C-MYC (Invitrogen) at MOI of 20. After 5 days, floating cells were collected and transfered to 6 well plates coated with Cellstart (Invitrogen) in stem cell media for 20 days, with daily media change. iPS colonies were collected and expended in the same culture conditions. For iNS generation, 5 days after transfection, adherent cells were collected by pipetting and transfered to noncoating plates in NPC media for neural stem cell selection. When reaching 60% confluence, the cells were subcultured in neural stem cell media (Invitrogen). To generate NSC from iPS, iPS were collected and replated in non-coating 6 well plates in neural stem cell media for neural stem cell induction. Attached cells were collected after reaching 60% confluence. Primary human NPC were cultured on poly-D-lysine coated 6 well-plates in NPC media.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA extraction was performed using the Total RNA purification kit (Norgen Biotek, Canada) following the manufacturer’s instructions.
|
| Label |
Biotin
|
| Label protocol |
Samples were prepared according to Affymetrix protocols (Affymetrix, Inc). RNA quality and quantity was ensured using the Bioanalyzer (Agilent, Inc) and NanoDrop (Thermo Scientific, Inc) respectively. Per RNA labeling, 200 nanograms of total RNA was used in conjunction with the Affymetrix recommended protocol for the GeneChip 2.0 ST chips.
|
| |
|
| Hybridization protocol |
The hybridization cocktail containing the fragmented and labeled cDNAs for each sample was hybridized to The Affymetrix Human Genome ST 2.0 GeneChip. Hybridization of samples occured over two separate batches. The chips were washed and stained by the Affymetrix Fluidics Station using the standard format and protocols as described by Affymetrix. The probe arrays were stained with streptavidin phycoerythrin solution (Molecular Probes, Carlsbad, CA) and enhanced by using an antibody solution containing 0.5 mg/mL of biotinylated anti-streptavidin(Vector Laboratories, Burlingame, CA).
|
| Scan protocol |
An Affymetrix Gene Chip Scanner 3000 was used to scan the probe arrays. Gene expression intensities were calculated using Affymetrix AGCC software.
|
| Description |
Human CD34 replicate 1
|
| Data processing |
Gene expression intensities were summarized and normalized using the Affymetrix Expression Console with the RMA Sketch option selected (Affymetrix, Inc). Resulting normalized intensities for samples hybridized as part of batch 2 were then baseline subtracted using the mean difference between cell types with those samples hybridized as part of batch 1.
|
| |
|
| Submission date |
Feb 21, 2013 |
| Last update date |
Jan 01, 2014 |
| Contact name |
Kory R Johnson |
| E-mail(s) |
johnsonko@ninds.nih.gov
|
| Phone |
301-402-1956
|
| Organization name |
NINDS/NIH
|
| Department |
DIR IT & Bioinformatics
|
| Lab |
Bioinformatics Section
|
| Street address |
10/3B01, 9000 Rockville Pike
|
| City |
Bethesda |
| State/province |
MD |
| ZIP/Postal code |
20892 |
| Country |
USA |
| |
|
| Platform ID |
GPL16686 |
| Series (1) |
| GSE44532 |
Derivation of Neural Stem Cells from human adult peripheral CD34+ Cells for an Autologous Model of Neuroinflammation |
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