To induce lineage-specific differentiation, growth factors (R&D Systems, Wiesbaden-Nor-denstadt, Germany, were added as follows: for erythropoietic differentiation, SCF (50 ng/ml), Flt3-ligand (50 ng/ml), IL-3 (10 ng/ml), EPO (10 U/ml); for granulopoietic differentiation, SCF (50 ng/ml), Flt3-ligand (50 ng/ml), IL-3 (10 ng/ml), G-CSF, and GM-CSF (each, 10 ng/ml); for megakaryopoietic differentiation, SCF (50 ng/ml), Flt3-ligand (50 ng/ml), TPO (20 ng/ml)
Growth protocol
CD34+ PBCs were cultured in X-VIVO10 supplemented with 1% human serum albumin (HSA). At least 1 x 106 CD34+ cells were assayed in 6-well plates and incubated at 37°C and 5% CO2 in a fully humidified atmosphere in air.
Extracted molecule
total RNA
Extraction protocol
RNA extraction was performed using the RNEasy plus kit following qigen's protocol
Label
biotin
Label protocol
Target labeling of RNA for Human exon arrays was done using the Affymetrix/Ambion WT cDNA synthsis and labeling protocol
Hybridization protocol
Labeled and Fragmented ST cDNA was hybridized to Human Exon 1.0St arrays for 16 hours at 45C in an hybridization oven. Hybridized probes were detected by Streptavidin-Phycoerythrin stain
Scan protocol
Stained chips were scanned in Affymetrix GCS 3000G scanner
Description
differentiated CD34+ cells lineage-specific differentiation, was induced using growth factors for erythropoietic differentiation, SCF (50 ng/ml), Flt3-ligand (50 ng/ml), IL-3 (10 ng/ml), EPO (10 U/ml); for granulopoietic differentiation, SCF (50 ng/ml), Flt3-ligand (50 ng/ml), IL-3 (10 ng/ml), G-CSF, and GM-CSF (each, 10 ng/ml); for megakaryopoietic differentiation, SCF (50 ng/ml), Flt3-ligand (50 ng/ml), TPO (20 ng/ml).
Transcriptome Profiling and Sequencing of differentiated Human Hematopoietic Stem cells Reveal Lineage Specific Expression and Alternative Splicing of Genes
