Investigation Title Chromatin immunoprecipitation of adult mouse liver to identify Foxa2 targets
Comment[Submitted Name] Foxa2 ChIP in mouse liver
Experimental Design binding_site_identification_design compound_treatment_design ChIP-chip by array
Experimental Design Term Source REF The MGED Ontology The MGED Ontology EFO
Comment[AEMIAMESCORE] 5
Comment[SecondaryAccession]
Comment[ArrayExpressReleaseDate] 2008-07-04
Comment[ArrayExpressAccession] E-MTAB-32
Comment[MAGETAB TimeStamp_Version] 2010-08-11 18:26:41 Last Changed Rev: 13058
Experimental Factor Name immunoprecipitate
Experimental Factor Type immunoprecipitate
Experimental Factor Term Source REF
Person Last Name Kaestner Tuteja White Brestelli
Person First Name Klaus Geetu Peter John
Person Mid Initials
Person Email kaestner@mail.med.upenn.edu geetu@mail.med.upenn.edu pwhite@mail.med.upenn.edu jbrestel@pcbi.upenn.edu
Person Phone (215)-898-8759 (215)-898-8712 (215) 898-0773 215-746-7019
Person Fax
Person Address 752B Clinical Research Building, 415 Curie Boulevard, Philadelphia, Pennsylvania 19104-6145. USA 752B Clinical Research Building, 415 Curie Boulevard, Philadelphia, Pennsylvania 19104-6145. USA 752B CRB, 415 Curie Blvd, Philadelphia, PA 19104-6145 1403 Blockley Hall, 423 Guardian Dr, Philadelphia PA 10104
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
Person Roles investigator investigator investigator submitter
Person Roles Term Source REF 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 2008-07-04
PubMed ID 18556755
Publication DOI 18556755
Publication Author List Tuteja, Geetu; Jensen, Shane T.; White, Peter; Kaestner, Klaus H.
Publication Title Cis-regulatory modules in the mammalian liver: composition depends on strength of Foxa2 consensus site
Publication Status journal_article
Publication Status Term Source REF The MGED Ontology
Experiment Description We performed genome-wide location analysis for Foxa2 to identify its targets in the adult mouse liver. Chromatin isolated from the liver of five adult mice was cross-linked, sheared and immunoprecipitated with a Foxa2-specific antibody. The resulting material, as well as material that was not immunoprecipitated, was uncross-linked, amplified, labeled and hybridized to the Mouse Promoter Chip BCBC-5A. Statistical analysis resulted in a set of 107 genes that are bound by Foxa2. Using computational analyses, we showed that Foxa2 containing cis-regulatory modules are dependent on the strength of the Foxa2 consensus site present.
Protocol Name P-MTAB-969 P-MTAB-971 P-MTAB-972 P-MTAB-975 P-MTAB-974
Protocol Type nucleic_acid_extraction labeling hybridization loess_group_normalization feature_extraction
Protocol Description Mouse livers were minced finely in cold phosphate-buffered saline (PBS) and cross-linked in 1% formaldehyde for 10 minutes while rotating. Cross-linking was quenched by adding glycine to a final concentration of 0.125M for 5 minutes while rotating. The tissue was rinsed in cold PBS and homogenized with a Dounce homogenizer in cold cell lysis buffer (10mM Tris-Cl, pH 8.0, 10 mM NaCl, 3mM MgCl2, 0.5% NP-40) and protease inhibitors. Cells were incubated at 4 degrees C for 5 minutes to release nuclei. Nuclei were centrifuged at 13,000 x g for 5 minutes to form a pellet. The pellet was resuspended in nuclear lysis buffer (1% sodium dodecyl sulfate [SDS], 5 mM EDTA, 50 mM Tris-Cl, pH 8.1) and protease inhibitors and sonicated using the Diagenode Bioruptor for 10 minutes on High, using 30 second intervals. Debris were removed by centrifugation at 13,000 x g for 10 minutes, and the supernatant was collected and snap frozen in liquid nitrogen. 10ml aliquot was reversed by the addition of NaCl to a final concentration of 192 mM, overnight incubation at 65 degrees C, and purification using a PCR purification kit (QIAGEN). The chromatin concentration was determined by using a NanoDrop 3.1.0 nucleic acid assay (Agilent Technologies). 10 micrograms of chromatin per sample was precleared by adding 90ml of protein G-agarose in 1 ml of ChIP dilution buffer (0.01% SDS, 1.1% Triton X-100, 167 mM NaCl, 16.7 mM Tris-Cl, pH 8.1) and rotating the sample for 1 h at 4 degreesC. Protein G-agarose was sedimented by centrifugation at 3,000xg for 30 seconds. The sample was split and two micrograms of rabbit anti-Foxa2 serum (provided by J.A. Whitsett), was added to the supernatant of one (the other being the control sample). These were incubated overnight at 4 degrees C. Protein G-agarose was blocked overnight at 4 degrees C with 1 mg/ml bovine serum albumin and 0.1 mg/ml herring sperm DNA in ChIP dilution buffer, added to the chromatin, and rotated for 1 hour at 4 degrees C. Following three consecutive washes of 5 min each with TSE I (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-Cl, pH 8.1, 150 mM NaCl), TSE II (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-Cl, pH 8.1, 500 mM NaCl), and ChIP buffer III (0.25 M LiCl, 1% NP-40, 1% deoxycholate, 1 mM EDTA, 10 mM Tris-Cl, pH 8.1), chromatin was eluted by adding 100ml of freshly made ChIP elution buffer (1% SDS, 0.1 M NHCO3) to the pellet and rotating the sample for 10 min. Elution was repeated with an additional 100ml of ChIP elution buffer, and the eluates were combined. Cross-linking was reversed by the addition of NaCl to a final concentration of 192 mM and overnight incubation at 65 degreesC.
DNA blunting, linker ligation and amplification were carried out following the Agilent Mammalian ChIP-on-chip Protocol. (http://www.chem.agilent.com/scripts/LiteraturePDF.asp?iWHID=42637) DNA was labeled using the BioPrime Array CGH Genomic Labeling System (Invitrogen Life Technologies, CA) as per manufacturers instructions. Briefly, 1mg of DNA was mixed with Random Primers and denatured at 95 degrees 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 degrees 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 1mg of Mouse Cot1 DNA (Invitrogen Life Technologies, CA) was added to each sample and denatured at 95 degrees C for 5 min. The samples were then cooled to 42 degrees 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. The BCBC-5A chip contains over 18,000 proximal and distal promoter regions. Promoter regions were determined from full length cDNA libraries and Reference Sequences (RefSeqs). Over 12,000 well characterized genes were chosen and are represented by either a 1 kb or 2 kb tile, PCR amplified from genomic material. Microarray slides were hybridized overnight. Median foreground intensities were obtained for each spot and imported into the mathematical software package R (http://www.r-project.org/), which is used for all data input, diagnostic plots, normalization and quality checking steps of the analysis process using scripts developed in-house. 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 poor quality. The M values were then normalized by the print tip loess method using the marray microarray processing package in R. Images were analyzed with GenePix software (Axon Instruments).
Protocol Parameters amount;antibody; marray version;foreground_measurement;denominator_channel;spots used for loess curve;numerator_channel; version;
Protocol Hardware
Protocol Software
Protocol Contact
Protocol Term Source REF The MGED Ontology The MGED Ontology The MGED Ontology
SDRF File E-MTAB-32.sdrf.txt
Term Source Name The MGED Ontology NCI_thesaurus ArrayExpress The MGED Ontology EFO
Term Source File http://mged.sourceforge.net/ontologies/MGEDontology.php ncithesaurus.obo.alt http://www.ebi.ac.uk/arrayexpress http://mged.sourceforge.net/ontologies/MGEDontology.php http://www.ebi.ac.uk/efo/
Term Source Version