Human CD34+-selected stem and progenitor cells were obtained from the Yale Center for Excellence in Molecular Hematology. To obtain primary human erythroid cells, CD34+ cells were cultured and selected as described.(Panzenbock, B., Bartunek, P., Mapara, M. Y., and Zenke, M. (1998) Blood 92, 3658-3668. Migliaccio, A. R., Migliaccio, G., Di Baldassarre, A., and Eddleman, K. (2002) Bone Marrow Transplant 30, 75-80) Briefly, Human CD34-selected stem and progenitor cells were cultured for 9 to 14 days in StemSpan SF expansion medium which contains human transferrin (200 ng/ml) and insulin (10 ng/ml) (StemSpan 09650) and was supplemented with estradiol (1 micromolar), dexamethasone (1 micromolar), Flt3 ligand (100 ng/ml), stem cell factor (100 ng/ml), interleukin-3 (50 ng/ml), interleukin-6 (20 ng/ml), insulin-like growth factor 1 (50 ng/ml), and erythropoietin (3 U/ml). FACS analysis was used to analyze cellular expression of CD71 (transferrin receptor) and CD235a (glycophorin A). These cells represent the R3/R4 cell population of nucleated erythroid cells defined by Zhang et al.(Zhang, J., Socolovsky, M., Gross, A. W., and Lodish, H. F. (2003) Blood 102, 3938-3946)
Extracted molecule
total RNA
Extraction protocol
RNA was prepared using the Qiagen RNeasy kit (Qiagen, Valencia, CA) according to the manufacturer's instructions.
Label
biotin
Label protocol
Double stranded cDNA and biotin-labeledcRNA were synthesized and purified according tothe recommended Illumina protocol using aTotalPrep RNA Amplification kit (Invitrogen).
Hybridization protocol
Double stranded cDNA and biotin-labeled cRNA were synthesized and purified according to the recommended Illumina protocol using a TotalPrep RNA Amplification kit (Applied Biosystems). Samples of 400ng of total RNA were reverse transcribed to cDNA using a T7 oligo(dT) primer. Second-strand cDNA was synthesized, in vitro transcribed, and labeled via incorporation of biotin-16-UTP.
Scan protocol
BeadChips were scanned on the Illumina iScan at the Yale Center for Genome Analysis.
Description
R3R4_6
Data processing
Data were made positive by adding an offset to remove negative values, log2 transformed, and subjected to the VST variance-stabilizing transformation, then quantile normalized using procedures in the R 2.13 bioconductor lumi 2.4.0 package.
