Comment[ArrayExpressAccession]	E-MTAB-465
Investigation Title	Differential expression of sorted Alpha and Beta cells
Comment[Submitted Name]	Differential expression of sorted Alpha and Beta cells
Experiment Description	The goal of this experiment is to identify genes differentially expressed in FACS-sorted Alpha and Beta cells.
Comment[AEExperimentType]	transcription profiling by array
Comment[AEExperimentDisplayName]	Transcription profiling by array of human FACS-sorted alpha and beta cells
Experimental Design	cell_type_comparison_design
Experimental Factor Type	cell_type
Experimental Factor Name	CELLTYPE
Person Last Name	Grompe	Dorrell	Schug
Person First Name	Markus	Craig	Jonathan
Person Email	grompem@ohsu.edu	dorrellc@ohsu.edu	jschug@mail.med.upenn.edu
Person Phone	503-494-6888	503-494-6889	215-898-0773
Person Address	L321, 3181 SW Sam Jackson Park Rd. Portland, OR 97239-3098	L321, 3181 SW Sam Jackson Park Rd. Portland, OR 97239-3098	752A CRB, 415 Curie Blvd, Philadelphia, PA 19104-6145
Person Affiliation	Oregon Health Sciences University Oregon Stem Cell Center	Oregon Health & Science University Molecular and Medical Genetics	University of Pennsylvania Functional Genomics Core
Person Roles	submitter	investigator	investigator
Public Release Date	2011-07-23
Publication Title	Transcriptomes of the major human pancreatic cell types
Protocol Name	P-MTAB-18267	P-MTAB-18268	P-MTAB-18269	P-MTAB-18270	P-MTAB-18271	P-MTAB-18272	P-MTAB-18273	P-MTAB-18274	P-MTAB-18275	P-MTAB-18276
Protocol Type	purify	fractionate	split	nucleic_acid_extraction	labeling	hybridization	image_acquisition	feature_extraction	flag_filter	loess_global_normalization
Protocol Description	Islets were collected after 100-700 min of cold ischemia and cultured in CRML for 6-48h prior to over-night shipment. Cell viability on arrival (assessed by Trypan blue staining) was 95-99%. For preparation of a single cell suspension, islets were washed with DPBS (Hyclone, Logan, UT) before incubation in 3 ml 0.05% HyQ Trypsin (Hyclone). Digestion was continued for 10 min. at 37^^C with gentle dispersal by pipetting with a p1000 micropipettor every 3 min. Undispersed material was removed by passage through a 40 um strainer (BD Falcon, Bedford, MA) and 1 ml fetal bovine serum (FBS) was slowly added to wash cells  through the strainer, inhibit trypsin activity, and gradually increase the calcium concentration. Cells were then washed and resuspended in serum-free CMRL for brief storage prior to antibody labeling or mouse immunization.	Dissociated cells from primary islets were resuspended in 500 ul DPBS + 2% FBS, combined 25 ul of hybridoma supernatant and stored at 4C for 30 minutes. Cells were then washed with cold DPBS and resuspended in 100 ul DPBS + 2% FBS containing a 1:200 dilution of each secondary antibody. Secondary antibodies employed included PE-conjugated anti-mouse IgM (u chain-specific) and APC-conjugated anti-mouse IgG subclasses 1+2a+3 (Jackson ImmunoResearch, West Grove, PA). Dead cells were identified with Propidium iodide (10 ug/ml). FACS sorting was performed using a Cytopeia inFluxV-GS with gates set to exclude cell doublets and dead cells.	The extracts were split.	Cell populations were FACS sorted into Trizol LS and stored at -86C. After chloroform extraction, RNA isolation was completed using an RNeasy Mini kit (Qiagen, Valencia CA).	Cy dye is coupled to the DNA in the dark at room temperature (1 h). The reaction is then purified with a min-elute column (Qiagen).	Agilent two-colour cDNA 4x44k array hybridization according to manufacturer's instruction.	Agilent Technologies Scanner G2505C	Agilent Feature Extraction Software	Filter saturated, non-uniform, and manually flagged spots (Agilent Feature Extraction Software)	The limma's normalizeWithinArrays with method=loess
Protocol Parameters								Software Version		foreground_measurement; numerator_channel; R version; limma version; weights; denominator_channel; background_measurement
Publication Author List	C Dorrell, J Schug, CF Lin, PS Canaday, AJ Fox, O Smirnova, R Bonnah, PR Streeter, CJ Stoeckert Jr., KH Kaestner and M Grompe
SDRF File	E-MTAB-465.sdrf.txt