Investigation Title	Chromatin immunoprecipitation of mouse MIN6 pancreatic beta cells to identify Pdx1 targets	
Comment[Submitted Name]	Identification of direct Pdx1 targets in MIN6 cells	
Experimental Design	binding_site_identification_design	dye_swap_design	transcription profiling by array	
Experimental Design Term Source REF	The MGED Ontology	The MGED Ontology	EFO	
Comment[SecondaryAccession]		
Comment[ArrayExpressReleaseDate]	2009-12-02	
Comment[AEMIAMESCORE]	3	
Comment[ArrayExpressAccession]	E-MTAB-134	
Comment[MAGETAB TimeStamp_Version]	2010-10-16 00:29:56 Last Changed Rev: 14677	
Experimental Factor Name	Antibody	
Experimental Factor Type	antibody	
Experimental Factor Term Source REF	
Person Last Name	Sachdeva	Groff	Schug	White	Stoffers	Yang	Khoo	Claiborn	
Person First Name	Mira	David	Jonathan	Peter	Doris	Juxiang	Cynthia	Kathryn	
Person Mid Initials	
Person Email	miras@mail.med.upenn.edu	dgroff@mail.med.upenn.edu	jschug@mail.med.upenn.edu	pwhite@mail.med.upenn.edu	stoffers@mail.med.upenn.edu	juxiang@mail.med.upenn.edu	cynthiakhoo@gmail.com	kcc2@mail.med.upenn.edu	
Person Phone	(215) 573-6647	(215) 898-0209	(215) 898-0773	(215) 898-0773	(215) 573-5413	(215) 573-6647	(215) 573-6647	(215) 573-6647	
Person Fax	
Person Address	720 Clinical Research Building, 415 Curie Boulevard, Philadelphia, Pennsylvania 19104-6145. USA	720 Clinical Research Building, 415 Curie Boulevard, Philadelphia, Pennsylvania 19104-6145. USA		752B CRB, 415 Curie Blvd, Philadelphia, PA 19104-6145	726 Clinical Research Building, 415 Curie Boulevard, Philadelphia, Pennsylvania 19104-6145. USA	720 Clinical Research Building, 415 Curie Boulevard, Philadelphia, Pennsylvania 19104-6145. USA	720 Clinical Research Building, 415 Curie Boulevard, Philadelphia, Pennsylvania 19104-6145. USA	720 Clinical Research Building, 415 Curie Boulevard, Philadelphia, Pennsylvania 19104-6145. USA	
Person Affiliation	Department of Genetics and Institute for Diabetes, Obesity and Metabolism, University of Pennsylvania	Department of Genetics and Institute for Diabetes, Obesity and Metabolism, University of Pennsylvania	University of Pennsylvania Functional Genomics Core	University of Pennsylvania Functional Genomics Core	Department of Genetics and Institute for Diabetes, Obesity and Metabolism, University of Pennsylvania	Department of Medicine and Institute for Diabetes, Obesity and Metabolism, University of Pennsylvania	Department of Genetics and Institute for Diabetes, Obesity and Metabolism, University of Pennsylvania	Department of Genetics and Institute for Diabetes, Obesity and Metabolism, University of Pennsylvania	
Person Roles	investigator	submitter	investigator	investigator	investigator	investigator	investigator	investigator	
Person Roles Term Source REF	The MGED Ontology	The MGED Ontology	The MGED Ontology	The MGED Ontology	The MGED Ontology	The MGED Ontology	The MGED Ontology	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	2009-12-02	
PubMed ID	19855005	
Publication DOI	19855005	
Publication Author List	Sachdeva MM, Claiborn KC, Khoo C, Yang J, Groff DN, Mirmira RG, Stoffers DA.	
Publication Title	Pdx1 (MODY4) regulates pancreatic beta cell susceptibility to ER stress	
Publication Status	journal_article	
Publication Status Term Source REF	The MGED Ontology	
Experiment Description	The aim of this experiment was to use highthroughput chromatin immunoprecipitation followed by hybridization to promoter microarrays to obtain a comprehensive list of sites in the genome that are physically occupied by Pdx1.  Chromatin was prepared from Min6 insulinoma cells and immunoprecipitated with Pdx1 or control antiserum.  The precipitated chromatin was then purified, amplified and directly sequenced using Illumina technology.	
Protocol Name	P-MTAB-5132	P-MTAB-5133	P-MTAB-5134	P-MTAB-5135	P-MTAB-5138	P-MTAB-5137	
Protocol Type	nucleic_acid_extraction	PCR_amplification	labeling	hybridization	transformation_protocol_series	feature_extraction	
Protocol Description	PreBind  beads to IP antibody overnight. For each IP sample in 1ml cold PBS bind 35ul of (50:50) bead slurry with 10ug of IP Ab on rotary shaker in cold. Wash once with cold PBS. Wash 10cm Plate with 1 1xPBS, aspirate, add 10 ml 1xPBS, crosslink by adding formaldehyde to final concentration of 1% (270ul of 37% per plate) for 10 minutes at Room Temp. Quench crosslinking by adding 1ml of 1.25M glycine (fresh) to a finla concnetration of 0.125M. Swirl to mix at Room Temp. Aspirate off supernatant, wash twice with COLD 1xPBS, keep plates on ice. Harvest cells with cold 1xPBS +Protease Inhibitors (500ul/plate) scrape into 1.5ml tube (can place two plates per tube), spin down cells at 2000rpm for 5min @4C. Lyse Cells by resuspending in 350ul of cell lysis buffer with 1:200 protease inhibitors, 1:100 Pefablock. Resuspend by pipetting. Combine same samples use 2ml tube ,incubate on ice for 10minutes. Spin at 2500rpm at 4C to recover nuclei, remove supernatant. Lyse nuclei with 1ml culei lysis buffer, add 0.6 ml of IP dilution. Transfer to 15ml tube, sonicate on low for max 4 minutes, 30sec on 30 sec off. Transfer to 2ml tube, spin at max speed 4C for 5 min transfer to 15ml, add 3.4ml IP dilution buffer,. Preclear by adding 100ul of each bead and 125ug of each control Ab. Incubate O/N rotary shake at 4C. Spin down 2 min at 3000rom. transfer lysate to new tube avoid beads, divide among prewashed Ab-beads. Set aside 750ul for IP. Incbate with rotation 4C for 6 hours, was once with 1ml IP wash 1, spin at 7500 rpm for 2 min, let sit for 1 min before removing supernatant. Wash twice with High Salt buffer, wash once with IP wash 2, wash twice with 1ml TE, elute by adding 110ul elution buffer made fresh and at room temp using specials SDS for CHIP. vortex, spin and transfer 100ul to new tube. Add 2 ul of Rnase A and 12ul fo 5M NaCl. Final conc 0.2M to IP samples. Add 2ul of Rnase A and 12ul of 5 M NaCl to input samples +20ul TE. Incubate O/N at 67C. Digest Protein by addin 3ul of Proteinase K (20mg/ml) incubate at 42C for 2 hours. Dilute samples with TE to 400ul. Phenol extract samples, vortex, spin at RT 5minutes at max speed, Once for IP twice for input. Cholorform extrac with eaqual volume spin ma speed 5 min. Add 15ug of glycogen, ETOH PP by adding 2.5 volumes -20C ETOH, incubate on ice 2 minutes, spin max speed 40minutes 4C. Wash pellets with cold 70% ETOH 750ul, Spin 5 min, dry on 65C heat block with caps open, cover in saran wrap for 2 hours, resuspend IP in 60ul PCR water, inputs in 133ul.	Start with 5ng, Blunt end w/T4 DNA pol (Invitrogen), rxn volume 50 ul, 5 ul of 10X T4 Buffer, 10mM dNTP 2ul, 5U/ul T4 DNA Pol, water and sample to 50ul. Inucbate 12C for 15 mintues, Min Elute Cleanup, Add 1/10 volume 3M Na Acetate, ph 5.2, 300ul ERC buffer, Apply to column, Spin 1 min full speed, discard flow through, spin 1 min full speed, Place into new eppendorf, Apply 20ul EB or water (used 1/5 EB). Spin 1 min full speed, keep eluate. Ligate: Make Annealed Oligo Linker (6.7uM), 100uM OJW102 linker, 6.7ul, 100uM OJW103 linker 6.7ul, add water to 100ul. Heat to 95 allow to slowly cool by removing blcok from incubator	LM-PCR amplified ChIP DNA was labeled using the BioPrime¨ Array CGH Genomic Labeling System (Invitrogen Life Technologies, CA) as per manufacturerÕs instructions. Briefly, 1 _g of DNA was mixed with Random Primers and denatured at 95¡C for 5 min, then cooled briefly on ice. Next, the appropriate Cyanine dUTP fluorescent nucleotides (PerkinElmer Life And Analytical Sciences, Inc, MA) were added, along with the nucleotide mix and Exo Klenow fragment. This was gently mixed and incubated at 37¡C for 2 hours. The Cy3 and Cy5 labeled samples were purified using the MinElute PCR Purification Kit (Qiagen, CA), and the efficiency of dye incorporation and yield was determined using the NanoDrop¨ ND-1000 UV-Vis Spectrophotometer.	The Cy5 and Cy3 labeled samples were combined and 1 _g of Mouse Cot1 DNA (Invitrogen Life Technologies, CA) was added to each sample and denatured at 95¡C for 5 min. The samples were then cooled to 42¡C and an equal volume of 2 x hybridization buffer (50% formamide, 10 x SSC, and 0.2% SDS) was added, mixed, and applied to the array.  Microarray slides were hybridized overnight.	Median foreground intensities were obtained for each spot and imported into the mathematical software package ÒRÓ, which is used for all data input, diagnostic plots, normalization and quality checking steps of the analysis process using scripts developed in-house by Peter White. The ratio of expression for each element on the array was calculated in terms of M(log2(Red/Green)) and A ((log2(Red) + log2(Green))/2)). The dataset was filtered to remove positive control elements (Cy3 anchors and SpotReport elements) and any elements that had been manually flagged as bad. The M values were then normalized by the print tip loess method using the ÒmarrayÓ microarray processing package in ÒRÓ. Statistical analysis was performed in ÒRÓ using both the LIMMA and SAM packages	Images were analyzed with GenePix 5.0 software (Axon Instruments).	
Protocol Parameters	Antibody;				spots used for loess curve;marray version;signal column;limma version;Script version;R version;	software version;	
Protocol Hardware	
Protocol Software	
Protocol Contact	
Protocol Term Source REF		mo				The MGED Ontology	
SDRF File	E-MTAB-134.sdrf.txt	
Term Source Name	EFO	nci_thesaurus	The MGED Ontology	ArrayExpress	The MGED Ontology	EFO	mo	
Term Source File	http://www.ebi.ac.uk/efo/	http://nciterms.nci.nih.gov/NCIBrowser/Dictionary.do	http://mged.sourceforge.net/ontologies/MGEDontology.php	http://www.ebi.ac.uk/arrayexpress	http://mged.sourceforge.net/ontologies/MGEDontology.php	http://www.ebi.ac.uk/efo/	http://mged.sourceforge.net/ontologies/MGEDontology.php	
Term Source Version