Investigation Title Chromatin immunoprecipitation of human cell lines with oestrogen receptor (ER), the E2F transcription factor 4 (E2F4), Comment[Submitted Name] Hs4kMd_pCRM_valid_ER_E2F4 Experimental Design binding_site_identification_design unknown_experiment_design_type ChIP-chip by array Experimental Design Term Source REF The MGED Ontology EFO Comment[ArrayExpressReleaseDate] 2006-05-01 Comment[AEMIAMESCORE] 4 Comment[ArrayExpressAccession] E-MEXP-471 Experimental Factor Name Protein for ChIP Experimental Factor Type compound_treatment_design Experimental Factor Term Source REF Person Last Name Bergeron Chen Robert Deblois LaganiM-^Ore LefM-^Obvre Coulombe GiguM-^Ore Ferretti Bataille Blanchette Poitras Person First Name Dominique Xiaoyu FranM-^Mois GeneviM-^Ove JosM-^Ne CM-^Nline Benoit Vincent Vincent Alain Mathieu Christian Person Mid Initials R Person Email Francois.Robert@ircm.qc.ca Person Phone (514) 984 5737 Person Fax (514) 987 5743 Person Address 110 Ave des Pins Ouest, Montreal, Quebec, H2W 1R7, Canada Person Affiliation Institut de Recherches Cliniques de MontrM-^Nal McGill Center for Bioinformatics Laboratoire de chromatine et expression du gM-^Nnome Molecular Oncology Group McGill Molecular Oncology Group McGill Molecular Oncology Group McGill Institut de Recherches Cliniques de MontrM-^Nal Molecular Oncology Group McGill McGill and GM-^Nnome QuM-^Nbec Innovation Center Institut de Recherches Cliniques de MontrM-^Nal McGill Center for Bioinformatics Institut de Recherches Cliniques de MontrM-^Nal Person Roles submitter Person Roles Term Source REF 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 2006-05-01 PubMed ID 16606704 Publication DOI 16606704 Publication Author List Mathieu Blanchette; Alain Bataille; Xiaoyu Chen; Christian Poitras; JosM-BM-^Ne LaganiM-BM-^Ore; CM-BM-^Nline LefM-BM-^Obvre; GeneviM-BM-^Ove Deblois; Vincent GiguM-BM-^Ore; Vincent Ferretti; Dominique Bergeron; Benoit Coulombe; FranM-BM-^Mois Robert Publication Title Genome-wide computational prediction of transcriptional regulatory modules reveals new insights into human gene expression Publication Status journal_article Publication Status Term Source REF Experiment Description The Goal of the experiment was to validate the predicted regulatory modules identified by an algorithm.For this we took advantage of the the genome-wide location analysis technique (or ?ChIP/chip?). We selected modules predicted to be bound by the estrogen receptor (ER), the E2F transcription factor 4 (E2F4), the signal transducer and activator of transcription 3 (STAT3), and the hypoxia-inducible factor 1 HIF1), to print a DNA microarray. In the current study, the microarray was then probed by ChIP/chip for ER and E2F4. Protocol Name P-MEXP-14736 P-MEXP-14739 P-MEXP-14737 P-MEXP-14738 P-MEXP-14757 P-MEXP-14760 P-MEXP-14740 P-MEXP-14759 P-MEXP-14742 P-MEXP-14743 P-MEXP-9973 P-MEXP-14741 P-MEXP-14744 P-MEXP-9977 Protocol Type grow grow specified_biomaterial_action specified_biomaterial_action nucleic_acid_extraction nucleic_acid_extraction nucleic_acid_extraction nucleic_acid_extraction labeling labeling labeling hybridization feature_extraction bioassay_data_transformation Protocol Description MCF7 cells are grown at 37C 5% CO2 in phenol-red free DMEM supplemented with 10% charcoal-stripped serum at 95% confluence. T98G cells (ATCG)were grown at 37C, 5% CO2 in DMEM containing 10% FBS, 100 U/mL Penicillin, 100 ug/mL Streptomycin. Growth in steroid deprived media is followed by a treatment of 45 minutes 17beta estradiol 100nM. T98G cells were arrested though contact inhibition by allowing cells to reach confluence. Media (DMEM, 10%FBS) was changed after the second day of confluence and cells harvested on the third day MCF7 cells (10, 95% confluent 150mm plates for each IP) were fixed in plate by addition of formaldehyde to a final concentration of 1% (10 min at room temperature). The crosslinking reaction was quenched by addition of glycine to a final concentration of 125mM (5min room temperature) or plates directly rinse twice in ice cold PBS. Cells were centrifuged (1500rpm 10min at 4C) and resuspended in lysis buffer (200ul per 1.5x10E7)(1% SDS, 10mM EDTA, 50mM Tris-HCl pH 8.1 suplemented with complete, mini, EDTA-free protease inhibitor cocktail (Roche)) and incubated 10 min on ice. The lysate are sonicated to obtain DNA fragments of 1000 bp in average (on Virtis Virsonic 60, 5times 7 to 8 sec at setting 10 for 200ul). Chromatin fragment solution was centrifuged 10 min, the supernatants collected and diluted 10X in ChIP dilution buffer (0.5% Triton X-100, 2mM EDTA, 150mM NaCl, 20mM Hepes pH8) to achieve a final SDS concentration of 0.1%. At this step an aliquot corresponding to 10% of the total amount for one IP was set aside for the non-IP DNA and the rest used for the IP. The preclearing of the diluted chromatin was achieved for 1 hour at 4 degrees with salmon sperm DNA/protein A agarose (Upstate). IP was performed overnight at 4C using 20ug antibody ERa (HC20 sc-543). Following IP 400ul of salmon sperm DNA/protein A agarose was added and the incubation pursued for two more hours. The precipitate was washed sequencially for 10 min each with low salt wash buffer (0.1% SDS, 1% Triton X-100, 2mM EDTA, 20mM Tris-HCl pH 8.1, 150mM NaCl), high salt wash buffer (0.1% SDS, 1% Triton X-100, 2mM EDTA, 20mM Tris-HCl pH 8.1, 500mM NaCl), LiCl wash buffer (0.25M LiCl, 1% NP-40, 1% deoxycholate, 1mM EDTA, 10mM Tris-HCl pH 8.1), 3 times with TE buffer and eluted twice with 75ul of elution buffer (1% SDS, 0.1M NaHCO3) for 15 to 30 min each time on vortex. The reverse formaldehyde crosslink of the pool eluate and the non-IP DNA was carried at 65C for at least 6 hours and the isolated DNA fragments purified according to the QIAquick spin Kit protocol. MCF7 cells (10, 95% confluent 150mm plates for each IP) were fixed in plate by addition of formaldehyde to a final concentration of 1% (10 min at room temperature). The crosslinking reaction was quenched by addition of glycine to a final concentration of 125mM (5min room temperature) or plates directly rinse twice in ice cold PBS. Cells were centrifuged (1500rpm 10min at 4C) and resuspended in lysis buffer (200ul per 1.5x10E7)(1% SDS, 10mM EDTA, 50mM Tris-HCl pH 8.1 suplemented with complete, mini, EDTA-free protease inhibitor cocktail (Roche)) and incubated 10 min on ice. The lysate are sonicated to obtain DNA fragments of 1000 bp in average (on Virtis Virsonic 60, 5times 7 to 8 sec at setting 10 for 200ul). Chromatin fragment solution was centrifuged 10 min, the supernatants collected and diluted 10X in ChIP dilution buffer (0.5% Triton X-100, 2mM EDTA, 150mM NaCl, 20mM Hepes pH8) to achieve a final SDS concentration of 0.1%. At this step an aliquot corresponding to 10% of the total amount for one IP was set aside for the non-IP DNA and the rest used for the IP. The reverse formaldehyde crosslink of the pool eluate and the non-IP DNA was carried at 65C for at least 6 hours and the isolated DNA fragments purified according to the QIAquick spin Kit protocol. Confluent T98G cells were fixed in plate by addition of formaldehyde to a final concentration of 1% (10 min at room temperature)(10 150mm plates per IP). The crosslinking reaction was quenched by addition of glycine to a final concentration of 125mM (5 min at room temperature). The cell monolayer was rinsed twice with cold PBS. Cells were harvested and rinse one more time in cold PBS prior to be flash frozen in liquid nitrogen. Cells were lyse in cell lysis buffer (10 min at 4C on rotating platform)(5mM Pipes pH8.0, 85mM KCl, 0.5% NP40, 1mM Phenylmethylsulfonyl fluoride, 1mM benzamidine, 1ug/mL aprotinin, 1ug/mL of leupeptin, and 1ug/mL pepstatin), spin at low speed and peleted nucleis lysed in nuclei lysis buffer (10 min at 4C)(50mM Tris pH 8.0, 10mM EDTA, 1% SDS, 1mM phenylmethylsulfonyl fluoride, 1mM benzamidine, 1ug/mL aprotinin, 1ug/mL of leupeptin, and 1ug/mL pepstatin). The solution was sonicated to obtain DNA fragments of 600 bp in average (on Brandson 250 sonifier, 20 times 30 sec 1 min on ice between pulse). Chromatin fragment solution was centrifuged (14000rpm 10 min at 4C) to get rid of the cell debris. The Solution was then preclear by addition of 50ul preblock dynabeads (Dyna) for 1hour to 2 hours at 4C on a rotating platform. At this step 100ul of the supernatant solution is set aside for the non-IP DNA and the rest used for the IP.The remaining solution is diluted with 2 volume of ChIP dillution buffer (0.01% SDS, 1.1% Triton X-100, 1.2mM EDTA, 16.7mM Tris pH 8.0, 167mM NaCl). IP was carried overnight at 4C with 10ug of anti-E2F4 antibody (sc-1082, Santa-Cruz), and the immunocomplex collected using 100ul of preblock Dynabeads (Dyna). Peleted beads were rinse at 4C twice with 1.5mL bufferI (2mM EDTA, 50mM Tris pH 8.0, 0.2% sarkosyl) four times with 1.5mL bufferII (100mM Tris pH 9.0, 500mM LiCl, 1% NP40, 1% deoxycholate) and elute with 2x 150uL of ChIP elution buffer (20 min vortex)(1% SDS, 50mM NaHCO3). Non-IP DNA is thaw and diluted to 300ul with ChIP elution buffer. NaCl is added to the eluted sample and non-IP DNA to a final concentration of 0.2M. The reverse formaldehyde crosslink was carried overnight at 65C. Samples were ethanol precipitate, resuspended in 100ul TE and treated with 0.04mg proteinase K after addition of 5x Proteinase K buffer (1 o 2 hours at 45C)(50mM Tris-HCl pH 7.5, 25mM EDTA, 1.25% SDS). Finally chromatin was purified using Qiaquick PCR purification kit (elution in 2x 30ul Quiagen elution buffer). Confluent T98G cells were fixed in plate by addition of formaldehyde to a final concentration of 1% (10 min at room temperature)(10 150mm plates per IP). The crosslinking reaction was quenched by addition of glycine to a final concentration of 125mM (5 min at room temperature). The cell monolayer was rinsed twice with cold PBS. Cells were harvested and rinse one more time in cold PBS prior to be flash frozen in liquid nitrogen. Cells were lyse in cell lysis buffer (10 min at 4C on rotating platform)(5mM Pipes pH8.0, 85mM KCl, 0.5% NP40, 1mM Phenylmethylsulfonyl fluoride, 1mM benzamidine, 1ug/mL aprotinin, 1ug/mL of leupeptin, and 1ug/mL pepstatin), spin at low speed and peleted nucleis lysed in nuclei lysis buffer (10 min at 4C)(50mM Tris pH 8.0, 10mM EDTA, 1% SDS, 1mM phenylmethylsulfonyl fluoride, 1mM benzamidine, 1ug/mL aprotinin, 1ug/mL of leupeptin, and 1ug/mL pepstatin). The solution was sonicated to obtain DNA fragments of 600 bp in average (on Brandson 250 sonifier, 20 times 30 sec 1 min on ice between pulse). Chromatin fragment solution was centrifuged (14000rpm 10 min at 4C) to get rid of the cell debris. The Solution was then preclear by addition of 50ul preblock dynabeads (Dyna) for 1hour to 2 hours at 4C on a rotating platform. At this step 100ul of the supernatant solution is set aside for the non-IP DNA and the rest used for the IP. When IPs are eluted non-IP DNA is thaw and diluted to 300ul with ChIP elution buffer. NaCl is added to the eluted sample and non-IP DNA to a final concentration of 0.2M. The reverse formaldehyde crosslink was carried overnight at 65C. Samples were ethanol precipitate, resuspended in 100ul TE and treated with 0.04mg proteinase K after addition of 5x Proteinase K buffer (1 o 2 hours at 45C)(50mM Tris-HCl pH 7.5, 25mM EDTA, 1.25% SDS). Finally chromatin was purified using Qiaquick PCR purification kit (elution in 2x 30ul Quiagen elution buffer). The immunoprecipitated DNA fragments are blunted with T4 DNA polymerase and ligated to unidirectional linkers to be amplified and by ligation-mediated PCR (LM-PCR). The labeling is done post PCR, using 2x1ug from the PCR reaction, by random priming (Bioprime DNA Labeling system, invitrogen). In this reaction klenow enzyme incorporate Cy5-dUTP in the product. The resulting label sample is purified Using QIAquick PCR purification kit and dry in speed vac prior hybridization. The immunoprecipitated DNA fragments are blunted with T4 DNA polymerase and ligated to unidirectional linkers to be amplified and by ligation-mediated PCR (LM-PCR). The labeling is done post PCR, using 2x1ug from the PCR reaction, by random priming (Bioprime DNA Labeling system, invitrogen). In this reaction klenow enzyme incorporate Cy3-dUTP in the product. The resulting label sample is purified Using QIAquick PCR purification kit and dry in speed vac prior hybridization. The immunoprecipitated DNA fragments are blunted with T4 DNA polymerase and ligated to unidirectional linkers to be amplified and labeled by ligation-mediated PCR (LM-PCR) where aminoallyl-modified dUTP is incorporated in the product. The labeling is done post-PCR using monoreactive Cy-dye NHS esters (Pharmacia) that will react specifically with the aminoallylmodified dUTP. Cy5 labeled IP are resuspended in 5ul of water and combine with corresponding Cy3 labeled sample. 20ul of human Cot-1 DNA (1mg/ml) and 5ul of Yeast tRNA (8mg/ml) are added, the solution is then dry in speed vac before resuspenssion in 50ul of hybe buffer per sample (25% formamide, 5X SSC, 0.1% SDS, 0.2% BSA). Sample (hyb solution) are denature at 95M-