Investigation Title	Primary human erythroid progenitor cells NK57 treatment samples	
Comment[Submitted Name]	Primary human erythroid progenitor cells NK57 treatment samples	
Experimental Design	transcription profiling by array	
Experimental Design Term Source REF	EFO	
Comment[SecondaryAccession]		
Comment[SecondaryAccession]	GSE22368	
Comment[ArrayExpressReleaseDate]	2010-07-29	
Comment[AEMIAMESCORE]	4	
Comment[ArrayExpressAccession]	E-GEOD-22368	
Comment[MAGETAB TimeStamp_Version]	2010-10-14 13:35:02 Last Changed Rev: 14677	
Experimental Factor Name	TREATMENT	
Experimental Factor Type	treatment	
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Person Last Name	Ebert	Bradner	Ross	
Person First Name	Benjamin	James	Kenneth	
Person Mid Initials	L	E	N	
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Public Release Date	2010-07-29	
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Experiment Description	Gene expression profiling was performed on primary human erythroid progenitor cells left untreated or treated with 2uM NK57 for 3 days. The worldwide burden of sickle cell disease is enormous, with over 200,000 infants born with the disease each year in Africa alone. Induction of fetal hemoglobin is a validated strategy to improve symptoms and complications of this disease. The development of targeted therapies has been limited by the absence of discrete druggable targets. We developed a novel bead-based strategy for the identification of inducers of fetal hemoglobin transcripts in primary human erythroid cells. A small-molecule screen of bioactive compounds identified remarkable class-associated activity among histone deacetylase (HDAC) inhibitors. Using a chemical genetic strategy combining focused libraries of biased chemical probes and reverse genetics by RNA interference, we have identified HDAC1 and HDAC2 as molecular targets mediating fetal hemoglobin induction. Our findings suggest the potential of isoform-selective inhibitors of HDAC1 and HDAC2 for the treatment of sickle cell disease. Gene expression profiling was performed on primary human erythroid progenitor cells left untreated (n=7) or treated with 2uM NK57 for 3 days (n=2).	
Protocol Name	P-GSE22368-2	P-GSE22368-4	P-GSE22368-3	P-GSE22368-5	P-GSE22368-6	P-GSE22368-7	P-GSE22368-8	P-GSE22368-1	
Protocol Type	specified_biomaterial_action	nucleic_acid_extraction	grow	labeling	hybridization	image_aquisition	feature_extraction	bioassay_data_transformation	
Protocol Description	Primary erythroid cells were left untreated or treated with 2uM NK57 for 3 days.	RNA was purified from mononuclear cells using Trizol (Invitrogen). Linear amplification of 20 ng of total RNA was performed using the Ovation Biotin RNA Amplification and Labeling System (Nugen).	Cryopreserved human bone marrow CD34+ cells were obtained from Cambrex (Poietics, Cambrex).  Umbilical cord blood was harvested at Brigham and Women’s Hospital under an IRB approved protocol.  Mononuclear cells were purified by Ficoll Hypaque sedimentation, and CD34+ cells were selected using magnetic beads coupled to a CD34 monoclonal antibody (Miltenyi Biotec).  Erythroid differentiation was induced in vitro in two steps.(1)  For the first seven days, cells were cultured in Serum Free Expansion Medium (SFEM, Stem Cell Technologies) supplemented with 100 U/mL penicillin/streptomycin, 2 mM glutamine, 100 ng/mL stem cell factor (SCF), 10 ng/mL interleukin-3 (IL-3),  40 ug/mL lipids, and 0.5 IU/mL erythropoietin (Epo).  After 7 days, cells were cultured in the same medium supplemented with 3 IU/mL Epo.	First strand cDNA synthesis was generated using a T7-linked oligo-dT primer, followed by second strand synthesis. An in vitro transcription reaction was performed to generate cRNA containing biotinylated UTP and CTP, which was subsequently chemically fragmented at 95°C for 35 minutes.	Ten micrograms of the fragmented, biotinylated cRNA was hybridized in MES buffer (2-[N-Morpholino]ethansulfonic acid) containing 0.5 mg/ml acetylated bovine serum albumin (Sigma) to Affymetrix U133A arrays at 45°C for 16 hours.	Scans were performed on Affymetrix scanners.	Raw expression values were normalized using Robust Multiarray Averaging (RMA).	ID_REF = <br>VALUE = RMA normalized signal	
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Protocol Term Source REF	The MGED Ontology						The MGED Ontology	The MGED Ontology	
SDRF File	E-GEOD-22368.sdrf.txt	
Term Source Name	The MGED Ontology		ArrayExpress	EFO	The MGED Ontology	
Term Source File	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	
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