Comment[ArrayExpressAccession]	E-GEOD-45374
MAGE-TAB Version	1.1										
Public Release Date	2013-03-22
Investigation Title	Cell-autonomous function of Runx1 transcriptionally regulates megakaryocytic maturation in mice										
Comment[Submitted Name]	Cell-autonomous function of Runx1 transcriptionally regulates megakaryocytic maturation in mice
Experiment Description	This SuperSeries is composed of the SubSeries listed below. Refer to individual Series										
Term Source Name	ArrayExpress	EFO
Term Source File	http://www.ebi.ac.uk/arrayexpress/	http://www.ebi.ac.uk/efo/efo.owl									
Person Last Name	Jaschek										
Person First Name	Ram										
Person Email	ramijaschek@gmail.com										
Person Affiliation	Weizmann Institute of Science										
Person Address	Weizmann Institute of Science, P.O Box 26, Rechovot, Israel										
Person Roles	submitter										
Protocol Name	P-GSE45374-1	P-GSE45374-9	P-GSE45374-6	P-GSE45374-3	P-GSE45374-11	P-GSE45374-5	P-GSE45374-10	P-GSE45374-2	P-GSE45374-7	P-GSE45374-4	P-GSE45374-8
Protocol Description	ID_REF =  VALUE = dChip signal intensity	Samples were hybridized using Affymetrix hybridization kit materials.	FL cells were derived from E14.5 embryos and cultured in DMEM medium supplemented with 10% FBS (Gibco, US), 2mM L-glutamine and penicillin/streptomycin and TPO (50ng ml-1) at 37ºC and 10% CO2. After 7 days in culture, mature MKs were isolated by two sequential 2%-4% BSA gradients.	Culture in DMEM.	Data was normalized using dChip.	Base-calling: CASAVA_1.6 Alignment: Bowtie. Genome build mm9. Peak-calling: Genomic stretches at a resolution of 50bp showing coverage in the top 0.1% of the genome for P300 or RUNX1 and not showing NIS in the top 1% of the genome. Genome_build: MGSCv37	Microarrays were scanned using GeneChip scanner 3000 7G.	Murine FL-derived mature MKs were produced essentially as previously described by Shivdasani RA, Schulze H (2005) Culture, expansion, and differentiation of murine megakaryocytes. Curr Protoc Immunol Chapter 22: Unit 22F 26. Briefly, FL cells were derived from E14.5 embryos and cultured in DMEM medium supplemented with 10% FBS (Gibco, US), 2mM L-glutamine and penicillin/streptomycin and TPO (50ng ml-1) at 37ºC and 10% CO2. After 7 days in culture, mature MKs were isolated by two sequential 2%-4% BSA gradients.	RNA was isolated using EZ-RNA (Biological Industries, Beit Haemek, Israel) according to the manufacturer's instructions.	ChIP samples were prepared using 5*10^7 cells per sample. Chromatin was prepared as described in Ainbinder et. Al Mol Cell Biol. 2002 (PMID 12192035). Antibodies used: NIS - non-immune serum was taken from our rabbits; RUNX1- our home-made antibody, described in Aziz-Aloya RB, Levanon D, Karn H, et al. Expression of AML1-d, a short human AML1 isoform, in embryonic stem cells suppresses in vivo tumor growth and differentiation. Cell Death Differ. 1998;5(9):765-773 (PMID 10200536); and P300 - C-20:rabbit polyclonal IgG, Santa Cruz Biotechnology sc-585, lot #E2010. ChIP libraries were created following the Illumina sample preparation protocol from 10ng of DNA. Following end-repair and A-base addition, primers were ligated and samples were size selected on a 2% agarose gel. Fragments of 200bp were excised and purified using the Qiagen gel-extraction kit with gel melting at room temperature. Enrichment was carried out using 18 cycles of amplification. Library yield was quantified using Qubit (Invitrogen), while final fragment sizes were determined using Agilent BioAnalyzer. The ChIP-seq libraries were run on an Illumina GAIIx at the Department of Biological Services at WIS, using Illumina cluster generation kits and sequencing reagents.	Purified RNA was reverse-transcribed, amplified and labeled with the Affymetrix GeneChip Whole Transcript Sense Target Labeling kit.
Protocol Type	bioassay_data_transformation	hybridization	grow	grow	feature_extraction	feature_extraction	image_aquisition	specified_biomaterial_action	nucleic acid library construction protocol	nucleic acid library construction protocol	labeling
Experimental Factor Name	CHIP ANTIBODY	CELL TYPE	GENOTYPE								
Experimental Factor Type	chip antibody	cell type	genotype								
Comment[SecondaryAccession]	GSE45374										
Comment[GEOReleaseDate]	2013-03-22										
Comment[ArrayExpressSubmissionDate]	2013-03-21										
Comment[GEOLastUpdateDate]	2013-03-25										
Comment[AEExperimentType]	ChIP-seq										
Comment[AEExperimentType]	transcription profiling by array										
SDRF File	E-GEOD-45374.hyb.sdrf.txt	E-GEOD-45374.seq.sdrf.txt