Investigation Title Transcription profiling of human hematopoietic stem/progenitor cells treated with TGF-beta1 to idetntify its target genes Comment[Submitted Name] TGF-beta1 target genes in human hematopoietic stem/progenitor cells. Experimental Design unknown_experiment_design_type transcription profiling by array Experimental Design Term Source REF EFO Comment[AEMIAMESCORE] 4 Comment[ArrayExpressReleaseDate] 2008-06-13 Comment[SecondaryAccession] GSE5150 Comment[ArrayExpressAccession] E-GEOD-5150 Comment[MAGETAB TimeStamp_Version] 2010-08-05 19:04:38 Last Changed Rev: 13058 Experimental Factor Name Experimental Factor Type Experimental Factor Term Source REF Person Last Name Zenke Person First Name Martin Person Mid Initials Person Email Martin.Zenke@rwth-aachen.de Person Phone Person Fax Person Address Cell Biology, Institute for Biomedical Engineering, Universitatsklinikum Aachen, RWTH, Aachen, 52057, Germany Person Affiliation Institute for Biomedical Engineering Person Roles submitter Person Roles Term Source REF The MGED Ontology Quality Control Type Quality Control Term Source REF Replicate Type Replicate Term Source REF Normalization Type Normalization Term Source REF Date of Experiment Public Release Date 2008-06-13 PubMed ID Publication DOI Publication Author List Publication Title Publication Status Publication Status Term Source REF Experiment Description CD34+ hematopoietic stem/progenitor cells were isolated from human cord blood and amplified in vitro for 10-14 days in serum-free medium with specific cytokines (Ju et al., Eur. J. Cell Biol. 82, 75-86, 2003; Hacker et al., Nat. Immunol. 4, 380-386, 2003). Cells were then treated with TGF-beta1 for various periods of time (2, 4, 16 hours) and RNA was prepared and subjected to microarray analysis. Experiment Overall Design: CD34+ hematopoietic stem/progenitor cells (HPC) were amplified in vitro and treated with TGF-beta1 (10 ng/ml) for 2, 4 and 16 hours. Experiment Overall Design: HPC untreated Experiment Overall Design: HPC + TGF-beta1 for 2 hours Experiment Overall Design: HPC + TGF-beta1 for 4 hours Experiment Overall Design: HPC + TGF-beta1 for 16 hours Protocol Name P-G5150-5 P-G5150-1 P-G5150-9 P-G5150-6 P-G5150-2 P-G5150-10 P-G5150-8 P-G5150-4 P-G5150-7 P-G5150-3 Affymetrix:Protocol:Hybridization-Unknown P-AFFY-6 Protocol Type grow grow specified_biomaterial_action specified_biomaterial_action specified_biomaterial_action specified_biomaterial_action nucleic_acid_extraction nucleic_acid_extraction labeling labeling hybridization feature_extraction Protocol Description CD34+ hematopoietic stem/progenitor cells (HPC) were isolated using the MACS system (Miltenyi Biotec, Bergisch-Gladbach, Germany). Purity was 83-90%. CD34+ cells (0.3-0.5x106 cells/ml) were cultured in StemSpan serum free medium (StemCell Technologies Inc., Vancouver, Canada) with 100 ng/ml SCF, 50 ng/ml Flt3 ligand (FL), 20 ng/ml thrombopoietin (Tpo) and 10 ng/ml hyper-IL-6 (Ju et al., Eur. J. Cell Biol. 82, 75-86, 2003; Hacker et al., Nat. Immunol. 4, 380-386, 2003.). Growth factors were added every 2 days and cells were maintained at 1x10E6 cells/ml cell density. Cells were amplified in vitro for 10-14 days. CD34+ hematopoietic stem/progenitor cells (HPC) were isolated using the MACS system (Miltenyi Biotec, Bergisch-Gladbach, Germany). Purity was 83-90%. CD34+ cells (0.3-0.5x106 cells/ml) were cultured in StemSpan serum free medium (StemCell Technologies Inc., Vancouver, Canada) with 100 ng/ml SCF, 50 ng/ml Flt3 ligand (FL), 20 ng/ml thrombopoietin (Tpo) and 10 ng/ml hyper-IL-6 (Ju et al., Eur. J. Cell Biol. 82, 75-86, 2003; Hacker et al., Nat. Immunol. 4, 380-386, 2003.). Growth factors were added every 2 days and cells were maintained at 1x106 cells/ml cell density. Cells were amplified in vitro for 10-14 days. Hematopoietic stem/progenitor cells treated for 4 hours with TGF-beta1. Hematopoietic stem/progenitor cells treated for 2 hours with TGF-beta1. Hematopoietic stem/progenitor cells untreated (control) Hematopoietic stem/progenitor cells treated for 16 hours with TGF-beta1. Total RNA was extracted using RNeasy Midi kit (Qiagen, Hilden, Germany) using the manufacturer buffers and protocols. An additional DNAse digestion with RNAse free DNAse (Qiagen) was performed. First and second strand cDNA synthesis were done with 7 mg total RNA with the SuperScript Choice system (Gibco BRL) and a T7-(dT)24 Primer (Biotez, Berlin, Germany). Total RNA was extracted using RNeasy Midi kit (Qiagen, Hilden, Germany) using the manufacturer’s buffers and protocols. An additional DNAse digestion with RNAse free DNAse (Qiagen) was performed. First and second strand cDNA synthesis were done with 7 mg total RNA with the SuperScript Choice system (Gibco BRL) and a T7-(dT)24 Primer (Biotez, Berlin, Germany). In vitro transcription employing double stranded cDNA as template and biotinylated UTP and CTP (Perkin Elmer/ENZO) as label was performed with the MEGAscript kit from Ambion according to the manufacturer's instructions. Labeled RNA was fragmented by heating to 95 C for 35 minutes in 200 mM TRIS (pH 8.1), 500 mM KOAc, 150 mM MgOAc buffer. In vitro transcription employing double stranded cDNA as template and biotinylated UTP and CTP (Perkin Elmer/ENZO) as label was performed with the MEGAscript kit from Ambion according to the manufacturer's instructions. Labeled RNA was fragmented by heating to 95°C for 35 minutes in 200 mM TRIS (pH 8.1), 500 mM KOAc, 150 mM MgOAc buffer. Title: Affymetrix Generic Hybridization. Description: Title: Affymetrix CEL analysis. Description: Protocol Parameters Protocol Hardware Protocol Software MicroArraySuite 5.0 MicroArraySuite 5.0 Protocol Contact Protocol Term Source REF mo SDRF File E-GEOD-5150.sdrf.txt Term Source Name mo The MGED Ontology ArrayExpress EFO The MGED Ontology mo Term Source File http://mged.sourceforge.net/ontologies/MGEDontology.php http://mged.sourceforge.net/ontologies/MGEDontology.php http://www.ebi.ac.uk/arrayexpress http://www.ebi.ac.uk/efo/ http://mged.sourceforge.net/ontologies/MGEDontology.php http://mged.sourceforge.net/ontologies/MGEDontology.php Term Source Version