Comment[ArrayExpressAccession] E-GEOD-48456 MAGE-TAB Version 1.1 Public Release Date 2013-07-03 Investigation Title H3K27me3 in mortal and immortal breast cells Comment[Submitted Name] H3K27me3 in mortal and immortal breast cells Experiment Description H3K27me3 changes during immortalization of breast cells. The aim of the study was to determine whether differential epigentic state of TERT gene is involved in immortalization of breast cells. comparison of Pre-Stasis, Post-Selection, and immortal 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 Vrba Vrba Garbe Stampfer Futscher Person First Name Lukas Lukas James Martha Bernard Person Mid Initials C R W Person Email geo@ncbi.nlm.nih.gov Person Affiliation University of Arizona Person Address Arizona Cancer Center, University of Arizona, 1515 N Campbell Ave, Tucson, AZ, USA Person Roles submitter Protocol Name P-GSE48456-1 P-GSE48456-4 P-GSE48456-5 P-GSE48456-8 P-GSE48456-7 P-GSE48456-9 P-GSE48456-2 P-GSE48456-3 P-GSE48456-6 Protocol Description Output from GenePix (*.gpr files) were imported to R using the limma package. Individual channels were first spatially normalized within arrays using ma2D function from the package marray and then loess normalized between arrays using the function normalize.loess from package affy. The RG object was transformed to an MA object and M values were again loess normalized between arrays. ID_REF = VALUE = Normalized log2 ratio (Cy5/Cy3) representing input/immunoprecipitated DNA amplification was based on random prime synthesis using the BioPrime DNA Labeling System (Invitrogen, Carlsbad, CA). The DNA sample or equivalent amount of input in a total volume of 21 ul was mixed with 20 ul of 2.5x Random Primers Solution, denatured by heating for 5 min at 95 C in a thermal cycler, and cooled on ice for 5 min. 5 ul of 10x dUTP Nucleotide Mix, 3 ul of 1 mM dTTP and 1 ul of Exo-Klenow Fragment, were added and incubated at 37 C for 20 h. Amplified DNA was purified using a QIAquick PCR purification kit (Qiagen, Valencia, CA) and quantified on a ND-1000 spectrophotometer (Nanodrop Technologies, Wilmington, DE). The typical yield was 4-5 ug of amplified DNA per 50 ul reaction. Equal amounts (2 ug) of amplified immunoprecipitated and input DNA were labeled using the BioPrime DNA Labeling System (Invitrogen) with Cy3 or Cy5 dyes respectively. DNA in a volume of 42 ul was mixed with 40 ul 2.5x Random Primers Solution, denatured by heating for 5 min at 95 C in a thermal cycler, and cooled on ice for 5 min. 10 ul of 10x dUTP Nucleotide Mix, 4 ul of 1 mM dTTP, 2 ul of Cy3- or Cy5-dUTP (1mM) (GE healthcare, Piscataway, NJ, USA) and 2 ul of Exo-Klenow Fragment were added and the mixture was incubated for 20 h at 37 C. Labeled targets were purified using the QIAquick PCR purification kit (Qiagen). The efficiency of labeling was determined using ND-1000 spectrophotometer (Nanodrop Technologies). The typical yield was 200 - 300 pmol of dye incorporated in 7 - 8 ug of DNA per sample. Combined Cy3 and Cy5 labeled samples were vaccum concentrated to volume of 91.5 µl. 12.5 µl of human Cot-1 DNA (1µg/µl, Invitrogen Cat. No. 15279-011), 26 µl of Agilent blocking agent (10x) and 130 µl of Agilent hybridization buffer (2x) were added. Samples were heated for 3 min at 95 oC, transferred to 37 oC, incubated for 30 min, and then used for microarray hybridization for 28h at 65 oC. After hybridization, slides were washed in Agilent Oligo aCGH/ChIP-on-Chip Wash Buffer 1 for 5 min at room temperature, then in Agilent Oligo aCGH/ChIP-on-Chip Wash Buffer 2 for 5 min at 37 oC, washed in acetronitrile for 10 s at room temperature and finally in stabilization and drying solution for 30 s at room temperature. MCDB170 M87A medium supplemented with oxytocin and cholera toxin at 0.5 ng/ml RPMI 1640 medium supplemented with 5% FBS, 100 units/ml penicillin and 50 μg/ml streptomycin DMEM/F12 with 10% FBS Wash ~2x107 cells in a tissue culture dishes with 10 ml of HBSS (Hanks Balanced Salt Solution) with 2.5mM EDTA. Add 15 ml of HBSS with 2.5mM EDTA, with 400 ul of 37% formaldehyde added (final conc 1%). Incubate at 37 C for 10 minutes. Aspirate off formaldehyde solution and add 5 ml of ice cold HBSS, 2.5mM EDTA with protease inhibitors (PIs) at a final concentration of 1 mM PMSF, 1 ug/ml Pepstatin A, 1 ug/ml Aprotinin. Gently detach cells with a cell scraper and transfer the cells to a conical tube. Wash remaining cells out of flask with 5 ml of HBSS with PIs. Spin cells down ~700 g for 5 min at 4 C. After spin, aspirate HBSS then add PIPES buffer (5 mM PIPES buffer pH 8.0, 85mM KCl, 0.5% NP40) with protease inhibitors 400 ul/sample, transfer sample to microfuge tube, then incubate on ice for 10 min. After incubation, pellet cells at 700 g for 5 min at 4 C. Remove PIPES buffer and add SDS lysis buffer (1% SDS, 10mM EDTA, 50mM Tris-HCl, pH 8.1) with protease inhibitors. Again 10 min on ice. Store at -80 C. Thaw samples on ice. Sonicate samples 6 x for 10 seconds on setting 7-8 watts until the majority of chromatin is between 500-1000 bp in length when checked on 1% agarose gel. After sonication spin samples at max speed at 4 C for 10 min to remove any cellular debris. Dilute supernatant fraction 10 fold with ChIPs dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2mM EDTA, 16.7mM Tris-HCl, pH 8.1, 167mM NaCl) with PIs. Set aside 10 % of sample into a fresh microfuge tube - Input sample. [immunoprecipitation protocol] Pre-clear the diluted sample by adding 120 ul of protein-A sepharose slurry (Pharmacia). Rotate for 1hr at 4 C. Spin down samples at 400 g at 4 C for 2 min to pellet beads. Divide samples equally into microfuge tubes for each Ab sample and add Abs. Rotate tubes overnight at 4 C. Next day add 90 ul of Pro-A sepharose slurry to each Ab tube. Rotate at 4 C for 1hr. Spin samples in picofuge for 10 s, discard all but 400 ul of supernatant, resuspend sepahrose beads in remaining volume and transfer to Handee spin cup columns (Pierce), spin in picofuge for 10 s, discard flow through. Wash beads for 5 min at 4 C with 400 ul each of the following buffers: low salt buffer (0.1% SDS, 1% Triton X100, 2mM EDTA, 20mM Tris-HCl, pH 8.1, 150mM NaCl), high salt buffer (0.1% SDS, 1% Triton X100, 2mM EDTA, 20mM Tris-HCl, pH 8.1, 500mM NaCl), LiCl buffer (0.25M LiCl, 1% NP40, 1% deoxycholate, 1mM EDTA, 10mM Tris-HCl, pH 8.1), wash twice with TE (10mM Tris-HCl, 1mM EDTA, pH 8.0). After each wash, spin columns in picofuge for 10 s. After the final TE wash, elute the DNA with 250 ul of elution buffer (1% SDS, 0.1M NaHCO3). Mix well and rotate for 15 min at room temp. Spin down, save flow through to a new microfuge tube. Repeat elution and combine eluates. Total volume ~ 500 ul. Bring the Input sample volume up to 500 ul using the elution buffer. To reverse the cross-links add 40 ul of 5M NaCl to eluate or diluted input and incubate at 65 C for 4 hr. After incubation add 100 ul of 0.1M EDTA, 40 ul of 1M Tris-Cl pH 7.5, and 2 ul of 20 mg/ml proteinase K to each sample and incubate at 45 C for 1 hr. Purify samples using QIAquick PCR purification kit (Qiagen, Valencia, CA): Transfer samples to 10 ml tubes. Add 5 volumes of PB buffer, mix well, apply to Qiagen columns under vacuum, wash 2x with 750 ul PE buffer. Elute twice with 30 ul of PCR H2O. Vacuum concentrate. Slides were scanned using an Axon GenePix 4000B microarray scanner (Axon Instruments, Inc., Foster City, CA, USA) and GenePix 6.0 software at 5 µm resolution and PMT settings 750 (635nm) and 600 (532nm) Protocol Type normalization data transformation protocol labelling protocol hybridization protocol growth protocol growth protocol growth protocol growth protocol nucleic acid extraction protocol array scanning protocol Experimental Factor Name CHIP ANTIBODY CELL TYPE SPECIMEN Experimental Factor Type chip antibody cell type specimen Comment[SecondaryAccession] GSE48456 Comment[GEOReleaseDate] 2013-07-03 Comment[ArrayExpressSubmissionDate] 2013-07-01 Comment[GEOLastUpdateDate] 2013-07-03 Comment[AEExperimentType] ChIP-chip by tiling array SDRF File E-GEOD-48456.sdrf.txt