Comment[ArrayExpressAccession]	E-GEOD-50910
MAGE-TAB Version	1.1					
Public Release Date	2013-09-17
Investigation Title	Gene expression of thymic settling progenitors from embryos at E13 compared to E18					
Comment[Submitted Name]	Gene expression of thymic settling progenitors from embryos at E13 compared to E18
Experiment Description	We used whole genome transcriptome as gene discovery to further understand the differential behaviour of the first (embryonic) and second waves of thymic settling progenitors in the murine embryo. Mouse embryos at days 13 and 18 of gestation were isolated and the thymic lobes were dissected. Single cell suspension was prepared and treated with biotinilated antibodies to CD3, CD4, CD8, TER119, Gr1, CD25, CD19, NK1.1 and CD11c. Cell suspensions were then incubated with streptavidin Miltenyi Macs beads. Lineage negative thymocytes were recovered in Macs columns. Cell suspensions were then incubated with antibodies against CD135, CD117, CD44, CD24 and Streptavidin bound to PECy7. Lin-, CD117+, CD44+, CD24low, CD135+ cells were then isolated by cell sorting. 3 biological replicates of each sample were isolated with 104-3x104 cells					
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	Cumano	Cumano				
Person First Name	Ana	Ana				
Person Email	ana.cumano@pasteur.fr					
Person Affiliation	Pasteur Institute					
Person Phone	+33145688255					
Person Address	Immunology, Pasteur Institute, 25 Rue du Dr. Roux, Paris, France					
Person Roles	submitter					
Protocol Name	P-GSE50910-1	P-GSE50910-4	P-GSE50910-5	P-GSE50910-2	P-GSE50910-3	P-GSE50910-6
Protocol Description	The Agilent Feature Extraction Software (FES) was used to read out and process the  microarray image files. The software determines feature intensities (including background  subtraction), rejects outliers and calculates statistical confidences. For determination of  differential gene expression FES derived output data files were further analyzed using the  Rosetta ResolverM-oM-^CM-" gene expression data analysis system (Rosetta Biosoftware). This  software offers M-bM-^@M-^S among other features M-bM-^@M-^S the possibility to compare two single intensity  profiles in a ratio experiment (table 4). All samples were labeled with Cy3, here, the ratio  experiments are designated as control versus (vs.) sample experiments (automated data  output of the ResolverM-oM-^CM-" system).  Please note, that the ratios are always calculated by dividing sample signal intensity through  control signal intensity. ID_REF =  VALUE = processed Cy3 signal intensity (Agilent gProcessedSignal)	The complete amount of each total RNA sample (except 1 M-NM-<l backup) was used for the T7 based amplification step. To produce Cy3-labeled cRNA, the RNA samples were amplified and labeled using the Agilent Low Input Quick Amp Labeling Kit (Agilent Technologies) following the manufacturerM-bM-^@M-^Ys protocol. Yields of cRNA and the dye-incorporation rate were measured with the ND-1000 Spectrophotometer (NanoDrop Technologies).	The hybridization procedure was performed according to the Agilent 60-mer oligo microarray processing protocol using the Agilent Gene Expression Hybridization Kit (Agilent Technologies). Briefly, 600 ng Cy3-labeled fragmented cRNA in hybridization buffer was hybridized overnight (17 hours, 65 M-0C) to Agilent Whole Mouse Genome Oligo Microarrays 8x60K using AgilentM-bM-^@M-^Ys recommended hybridization chamber and oven.	Freshly isolated thymocytes were depleted of lineage negative cells and thymic settling progenitors were sorted on a MoFlo cell sorted directly to RLT buffer.	RNA was isoalted using the MicroRNAeasy kit from Quiagen France that included a DNAse treatment according to the manufacture's instructions. RNA quality was checked in an Agilent 2100 Bioanalyzer platform (Agilent Technologies).	Fluorescence signals of the hybridized Agilent Microarrays were detected using AgilentM-bM-^@M-^Ys Microarray Scanner System (Agilent Technologies).
Protocol Type	normalization data transformation protocol	labelling protocol	hybridization protocol	growth protocol	nucleic acid extraction protocol	array scanning protocol
Experimental Factor Name	AGE					
Experimental Factor Type	age					
Comment[SecondaryAccession]	GSE50910					
Comment[GEOReleaseDate]	2013-09-17					
Comment[ArrayExpressSubmissionDate]	2013-09-16					
Comment[GEOLastUpdateDate]	2013-09-19					
Comment[AEExperimentType]	transcription profiling by array					
Comment[AdditionalFile:Data1]	GSE50910_E13_1_vs_E18_1.txt					
Comment[AdditionalFile:Data2]	GSE50910_E13_2_vs_E18_2.txt					
Comment[AdditionalFile:Data3]	GSE50910_E13_3_vs_E18_3.txt					
Comment[AdditionalFile:Data4]	GSE50910_E13_Pool1-3_vs_E18_Pool_1-3.txt					
SDRF File	E-GEOD-50910.sdrf.txt