title: Cotyledon-10dag-stm-35SCUC1stm rep2

city: Kumamoto

first: Mitsuhiro

last: Aida

address: 2-39-1 Kurokami, Chuo-ku

institute: Kumamoto University

description: stm 35SCUC1-Cy5/stm-Cy3

hyb_protocol: Hybridized for 17 hours with rotating under 60 degree.

scan_protocol: Scan array (GSI Lumonics) was used for fluorescence detection. Data was collected with 10 μm/pixel resolution. Difference of fluorescence between Cy3 and Cy5 was corected with Human Cot-1DNA (Agilent).

data_processing: Data was processed with Quant Array (GSI Lumonics) . The final output value is the result that the average of four-point background around the spot was subtracted from the fluorescent strength of that spot.

source_name: [reference] Cotyledon from 10-day old seedling, stm

label_protocol: 20 μg of total RNA was used to prepare the Cy3- or Cy5-labelled cDNA probes using the Agilent Arabidopsis1 Oligo Microarray kit.

growth_protocol: Seeds were strerilzed, incubated under dark condition at 4 degree for two days, and grown on growth media under continuous light conditions.

extract_protocol: Total RNA extracted using Rneasy Plant Mini Kit (QIAGEN).

treatment_protocol: Cotyledons were collected and used for the RNA extraction.

tag: ecotype

value: Ler

value: stm-1 mutant

value: Cotyledon

source_name: [test] Cotyledon from 10-day old seedling, 35S:CUC1 stm

value: 35S:CUC1 stm


title: Cotyledon-10dag-stm-35SCUC1stm rep1

status: Public on Mar 31 2011

type: RNA

country: Japan

description: stm 35SCUC1-Cy3/stm-Cy5

molecule: total RNA

organism: Arabidopsis thaliana

tag: genotype/variation

tag: tissue


title: Labellum_4dayspollination_Rep5

city: Lisboa

first: Filipa

middle: Isabel de Almeida

last: Monteiro

address: Ed. ICAT, Faculty of Sciences, Rua Ernesto Vasconcelos, Campo Grande

department: BioFIG

country: Portugal

institute: Plant Systems Biology Lab

description: Biological replicate 5 of 5. Comparison of nonpollinated labella vs. pollinated, 4 days after pollination. Both files coming from Versarray and edited for GEPAS are provided

hyb_protocol: The hybridization mix consisted of 40μL labelled cDNA, 1.7 μL of poly-A DNA (10 mg mL-1) (Sigma), 1.6 μL salmon sperm DNA (5 mg mL-1) (Invitrogen), 1.6 μL of 50X Denhardt´s solution (1% Ficoll (type 400), 1% PVP-40, 1% BSA), 8.45 μL of 20X SSC and 1.69 μL of 10% SDS(w/v). Mixture was denatured at 97ºC for 2 min, cooled to 60ºC and then 1μl of 10X DIG blocking (Roche Diagnostic GmbH, Mannheim, Germany) buffer was added. Hybridization was carried out at 60º C for 17 hours in chambers ArrayIt (Telechem International).

scan_protocol: Slides were scanned using VersArray ChipReader® vs 3.1. build 1.68 (Bio-Rad, USA) and spot and background intensities quantified using VersArray AnalyserTM software vs 4.5.1.46 (Bio-Rad, USA).

data_processing: Data files were imported into GEPAS (http://gepas.bioinfo.cipf.es) and log2-transformed prior to normalization (DNMAD).Print-tip Lowess was used for within-slide normalization considering background subtraction. Impute missing values (median). Patterns with less than 70% removed. VersArray.txt files correspond to raw data files retrieved by VersArray Analyser software (Bio-Rad). GEPAS.txt files correspond to raw data files (with flags indicated) ready to go for GEPAS software for normalization.

organism: Ophrys fusca

source_name: 4days after pollination

label_protocol: 2 µg of each aRNA was used were reverse transcribed and labelled with either Cy3 or Cy5 dye molecules, according the RPN 5660 CyScribeTM cDNA Post Labeling Kit and CyScribeTM GFXTM purification kit (Amersham, GE Healthcare).

growth_protocol: Ophrys fusca plants were sampled from a Portuguese natural occurring population. Hundred plants were selected in the field, accordingly their stage of development with fully opened flowers, with pollinia and with roughly the same stage of development. Five biological replicates was achieved from each condition assessed, each biological replicate represented by 5 labella.Plants were covered with a white and inert net, built specially for preventing pollinator’s visits either in pollinated or unpollinated flowers. Cross- pollination was performed manually with a sterile plastic stick.

extract_protocol: Total RNA from Ophrys fusca labella was isolated following the Hot Borate method (Wan and Wilkins, 1994) with minor modifications. About 1 μg of total RNA extracted from each biological replicate was used for aRNA amplification, by using the MessageAmpTM II aRNA Amplification Kit (Ambion, Applied Biosystems).

treatment_protocol: RNA was extracted from labella from unpollinated and pollinated plants, at both 2 days and 4 days after pollination

tag: condition

value: unpollinated _4DAP

value: labellum

value: pollinated _4DAP


title: 13.5dpc MatDup.dist7 MEF

city: Duarte

first: Xiwei

last: Wu

state: CA

address: 1500 E. Duarte Rd.

department: Computational and Quantitative Medicine

country: USA

institute: City of Hope National Medical Center

description: mouse embryonic fibroblasts form MatDup.dist7

hyb_protocol: The hybridization cocktails containing 2.5 ug of the fragmented, end-labeled cDNA were applied to the GeneChip® Mouse Gene 1.0 ST arrays. Hybridization was performed for 16 hours, and the arrays were washed and stained with the GeneChip Fluidics Station 450 using FS450_0007 script.

scan_protocol: Arrays were scanned using the Affymetrix GCS 3000 7G and GeneChip Operating Software v. 1.4 to produce CEL intensity files.

data_processing: Data were background corrected with GC bin, quantile normalized and summarized by interPLIER algorithm with Affymetrix Expression Console.

label: Biotin

organism: Mus musculus

label_protocol: Standard Affymetrix Protocol, as described in GeneChip Whole Transcript Sense Target Labeling Assay Manual, Version 4 (available at http://www.affymetrix.com/support/technical/product_updates/wt_1_1_assay.affx)

growth_protocol: MEFs were grown in 25 ml of culture medium (Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 10–4 M β-mercaptoethanol, nonessential amino acids, L-glutamine, and antibiotics at standard concentrations).

extract_protocol: RNA was isolated from MatDup.dist7 and PatDup.dist7 MEFs using RNA-Bee according to manufacturer’s instructions (Tel-Test). The pellet was dissolved in DEPC water containing RNasin (Promega) and 10 mM DTT. Contaminating DNA was removed with the DNA-free Kit (Ambion).

tag: cell type

value: embryonic fibroblasts


title: Cotyledon-5dag-Ler-35SCUC1 rep2

source_name: [reference] Cotyledon from 5-day old seedling, Ler

value: wild type

source_name: [test] Cotyledon from 5-day old seedling, 35S:CUC1


title: Cotyledon-5dag-Ler-35SCUC1 rep1


title: LY2_6hr_100nM-4-OHT_2

status: Public on Mar 30 2011

type: other

city: Louisville

first: Carolyn

middle: M

last: Klinge

state: KY

address: 580 S Preston St

department: Biochemistry & Molecular Biology

institute: University of Louisville School of Medicine

description:
CH1 raw data file: 1_Exiqon_13847532_S01_Cropped.txt
CH2 raw data file: 0_Exiqon_13847532_S01_Cropped.txt

hyb_protocol: Samples were sent to Exiqon where they were labeled and hybridized into the miRCURY LNA microarray (miRbase 11.0 human array)

scan_protocol: MiRNA analysis was performed by Exiqon

data_processing: LOESS normalized, background subtracted data obtained from log2 of processed Red signal/processed Green signal. Exiqon performed these calculations/analyses.

molecule: other

organism: Homo sapiens

source_name: Breast Cancer Cells treated with EtOH, E2, or 4-OHT

label_protocol: Cells were serum-starved for 48 h prior to 6 h treatment with Ethanol (EtOH- vehicle control), 10 nM estradiol (E2), or 100 nM 4-hydroxytamoxifen (4-OHT)

growth_protocol: MCF-7 cells were grown in IMEM with 5% FBS + 1% PS and were serum starved (5% dextran-coated charcoal stripped FBS in IMEM phenol red free medium) for 48 h prior to treatment

extract_protocol: RNA was extracted using miRVANA miRNA isolation kit from Ambion

treatment_protocol: Cells were treated with Ethanol (EtOH- vehicle control), 10 nM estradiol (E2), or 100 nM 4-hydroxytamoxifen (4-OHT) for 6 hr

tag: gender

value: female

tag: cell line

value: LY2 breast cancer cell line

tag: tamoxifen sensitivity

value: resistant

tag: agent

source_name: Exiqon Common Reference Pool

tag: reference

value: Exiqon Common Reference Pool


title: PADI4 ChIP-chip (replicate B)

type: genomic

city: Bronx

first: Matthew

middle: Jon

last: Gamble

state: NY

address: 1300 Morris Park Ave / Golding 203

department: Molecular Pharmacology

institute: Albert Einstein College of Medicine

description: no additional information

hyb_protocol: see NimbleGen website

scan_protocol: see NimbleGen website

data_processing: Genomic data analysis was performed using the statistical programming language R (R Development Core Team). All data processing scripts are available upon request. The log2 ratio data from each of the arrays was subjected to lowess normalization.

molecule: genomic DNA

source_name: MCF-7 ChIP input

label_protocol: see NimbleGen website

extract_protocol: MCF-7 breast cancer cells were grown to ~80 to 90% confluence, cross-linked with 1% paraformaldehyde in PBS for 10 min. at 37°C, and quenched in 125 mM glycine in PBS for 5 min at 4°C. The cells were collected by centrifugation and sonicated in lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris•HCl, pH 7.9, 1x protease inhibitor cocktail) to generate chromatin fragments of ~500 bp in length. The material was clarified by centrifugation, diluted 10-fold in dilution buffer (0.5% Triton X-100, 2 mM EDTA, 150 mM NaCl, 20 mM Tris•HCl, pH 7.9, 1x protease inhibitor cocktail), and pre-cleared with protein A-agarose beads. The pre-cleared, chromatin-containing supernatant was used in immunoprecipitation reactions with an antibody against PADI4. The immunoprecipitated genomic DNA and a sample of input material were cleared of protein and residual RNA by digestion with proteinase K and RNase H, respectively. The DNA was then extracted with phenol:chloroform:isoamyl alcohol and precipitated with ethanol. The material was then amplified with LM-PCR.

tag: chip antibody

source_name: MCF-7 PADI4 IP


title: Blood_HighPb_Rec_10

city: Durham

first: Heather

middle: G.

last: LaBreche

state: NC

address: 101 Science Drive

department: Institute for Genome Sciences and Policy

institute: Duke University

hyb_protocol: Following fragmentation, 15ug of labeled cRNA were hybridized on Mouse Genome M430 2.0 GeneChips for 16 hours at 45°C. GeneChips were washed and stained using the Biomex FX liquid handler.

scan_protocol: GeneChips were scanned using the Axon Genepix Scanner (Genepix 4000B).

data_processing: Data were RMA normalized in R/Bioconductor using an R script generated by Jeff Chang available at http://discovery.genome.duke.edu/express/rss/project/resources/2762

label: biotin

source_name: Whole blood from High Pb mice after 2-week recovery

label_protocol: Each sample (150ng) was amplified, labeled and fragmented using the MessageAmp Premier RNA Amplification Kit (Ambion, Austin, TX) according to the manufacturer's instructions.

extract_protocol:
Blood collection:  All blood samples (72 total blood samples) were collected using GoldenRod animal lancets (MEDIpoint, Inc., Mineola, NY) and stabilized using RNAProtect Animal Blood Tubes.
Total RNA was extracted using the RNEasy Protect Animal Blood Kit (Qiagen, Valencia, CA).  Globin transcript was removed using GLOBINclear for mice (Ambion, Austin, TX).

treatment_protocol: Nine-week old female Balb/c mice were divided into three groups of fifteen mice each.  Each group received drinking water with different concentrations of Pb in the form of lead acetate trihydrate (Sigma Aldrich, St. Louis, MO) for a period of two weeks.  The control group received 0µg/mL; the low exposure group received 5µg/mL; and the high exposure group received 50µg/mL. After two weeks of exposure, peripheral blood was collected by submandibular bleed from half of the control mice (n=7) and all of the exposed mice (n=15 per group).  All mice were then switched to Pb-free drinking water for two additional weeks.  A second blood sample was collected after this recovery period (controls, n=7; low Pb, n=15; high Pb, n=13)

tag: strain

value: Balb/c

tag: age

tag: study phase

value: recovery phase (post-exposure)

tag: time

value: 2 weeks

tag: agent during exposure phase

value: Pb

tag: agent dose during exposure phase

value: peripheral blood


title: Blood_HighPb_Rec_12


title: 13.5dpc PatDup.dist7 MEF

description: mouse embryonic fibroblasts form PatDup.dist7


title: LY2_6hr_100nM-4-OHT_1

description:
CH1 raw data file: 1_Exiqon_13847531_S01_Cropped.txt
CH2 raw data file: 0_Exiqon_13847531_S01_Cropped.txt


title: Blood_HighPb_Rec_9


title: Blood_HighPb_Rec_8


title: Blood_HighPb_Rec_7


title: Blood_HighPb_Rec_11


title: LY2_6hr_EtOH_2

description:
CH1 raw data file: 1_Exiqon_13847530_S01_Cropped.txt
CH2 raw data file: 0_Exiqon_13847530_S01_Cropped.txt

value: EtOH


title: Blood_HighPb_Rec_6


title: Blood_HighPb_Rec_13


title: LY2_6hr_EtOH_1

description:
CH1 raw data file: 1_Exiqon_13847529_S01_Cropped.txt
CH2 raw data file: 0_Exiqon_13847529_S01_Cropped.txt


title: Blood_HighPb_Rec_5


title: Blood_HighPb_Rec_4


title: MCF-7_6hr_EtOH_1

description:
CH1 raw data file: 1_Exiqon_13847527_S01_Cropped.txt
CH2 raw data file: 0_Exiqon_13847527_S01_Cropped.txt

value: MCF-7 breast cancer cell line

value: sensitive


title: E0_4h_rbCBP_3 extraction3_array1

city: Toronto

first: DCC

last: modENCODE

state: Ontario

address: MaRS Centre, South Tower, 101 College Street, Suite 800

country: Canada

institute: Ontario Institute for Cancer Research

description: channel ch1 is ChIP DNA; Antibody information listed below: official name: CBP antibody;target name: nejire;host: Rabbit;antigen: MMADHLDEPPQKRVKMDPTDISYFLEENLPDELVSSNSGWSDQLTGGAGG GNGGGGASGVTTNPTSGPNPGGGPNKPAAQGPGSGTGGVGVGVNVGVGGV VGVGVVPSQMNGAGGGNGSGTGGDDGSGNGSGAGNRISQMQHQQLQHLLQ QQQQGQKGAMVVPGMQQLGSKSPNLQSPNQGGMQQVVGTQMGMVNSMPMS ISNNGNNGMNAIPGMNTIAQGNLGNMVLTNSVGGGMGGMVNHLKQQPGGG GGGMINSVSVPGGPGAGAGGVGAGGGGAVAANQGMHMQNGPMMGRMVGQQ HMLRGPHLMGASGGAGGPGNGPGGGGPRMQNPNMQMTQLNSLPYGVGQYG GPGGGNNPQQQQQQQQQQLLAQQMAQRGGVVPGMPQGNRPVGTVVPMSTL GGDGSGPAGQLVSGNPQQQQMLAQQQTGAMGPRPPQPNQLLGHPGQQQQQ QQQPGTSQQQQQQQGVGIGGAGVVANAGTVAGVPAVAGGGAGGAVQSSGP GGANRDVPDDRKRQIQQQLMLLLHAHKCNRRENLNPNREVCNVNYCKAMK SVLAHMGTCKQSKDCTMQHCASSRQILLHYKTCQNSGCVICYPFRQNHSV FQNANVPPGGGPAGIGGAPPGGGGAGGGAAGAGGNLQQQQQQQQQQQQNQ QPNLTGLVVDGKQGQQVAPGGGQNTAIVLPQQQGAGGAPGAPKTPADMVQ QLTQQQQQQQQQVHQQQVQQQELRRFDGMSQQVVAGGMQQQQQQGLPPVI RIQGAQPAVRVLGPGGPGGPSGPNVLPNDVNSLHQQQQQMLQQQQQQGQN RRRGGLATMVEQQQQHQQQQQQPNPAQLGGNIPAPLSVNVGGFGNTNFGG AAAGGAVGANDKQQLKVAQVHPQSHGVGAGGASAGAGASGGQVAAGSSVL MPADTTGSGNAGNPNQNAGGVAGGAGGGNGGNTGPPGDNEKDWRESVTAD LRNHLVHKLVQAIFPTSDPTTMQDKRMHNLVSYAEKVEKDMYEMAKSRSE YYHLLAEKIYKIQKELEEKRLKRKEQHQQMLMQQQGVANPVAGGAAGGAG SAAGVAGGVVLPQQQQQQQQQQQQQGQQPLQSCIHPSISPMGGVMPPQQL RPQGPPGILGQQTAAGLGVGVGVTNNMVTMRSHSPGGNMLALQQQQRMQF PQQQQQQPPGSGAGKMLVGPPGPSPGGMVVNPALSPYQTTNVLTSPVPGQ QQQQQFINANGGTGANPQLSEIMKQRHIHQQQQQQQQQQQQGMLLPQSPF SNSTPLQQQQQQQQQQQQQQQATSNSFSSPMQQQQQGQQQQQQKPGSVLN NMPPTPTSLEALNAGAGAPGTGGSASNVTVSAPSPSPGFLSNGPSIGTPS NNNNNSSANNNPPSVSSLMQQPLSNRPGTPPYIPASPVPATSASGLAASS TPASAAATCASSGSGSNSSSGATAAGASSTSSSSSAGSGTPLSSVSTPTS ATMATSSGGGGGGGGNAGGGSSTTPASNPLLLMSGGTAGGGTGATTTTST SSSSRMMSSSSSLSSQMAALEAAARDNDDETPSPSGENTNGSGGSGNAGG MASKGKLDSIKQDDDIKKEFMDDSCGGNNDSSQMDCSTGGGKGKNVNNDG TSMIKMEIKTEDGLDGEVKIKTEAMDVDEAGGSTAGEHHGEGGGGSGVGG GKDNINGAHDGGATGGAVDIKPKTETKPLVPEPLAPNAGDKKKKCQFNPE ELRTALLPTLEKLYRQEPESVPFRYPVDPQALGIPDYFEIVKKPMDLGTI RTNIQNGKYSDPWEYVDDVWLMFDNAWLYNRKTSRVYRYCTKLSEVFEAE IDPVMQALGYCCGRKYTFNPQVLCCYGKQLCTIPRDAKYYSYQNSLKEYG VASNRYTYCQKCFNDIQGDTVTLGDDPLQSQTQIKKDQFKEMKNDHLELE PFVNCQECGRKQHQICVLWLDSIWPGGFVCDNCLKKKNSKRKENKFNAKR LPTTKLGVYIETRVNNFLKKKEAGAGEVHIRVVSSSDKCVEVKPGMRRRF VEQGEMMNEFPYRAKALFAFEEVDGIDVCFFGMHVQEYGSECPAPNTRRV YIAYLDSVHFFRPRQYRTAVYHEILLGYMDYVKQLGYTMAHIWACPPSEG DDYIFHCHPTDQKIPKPKRLQEWYKKMLDKGMIERIIQDYKDILKQAMED KLGSAAELPYFEGDFWPNVLEESIKELDQEEEEKRKQAEAAEAAAAANLF SIEENEVSGDGKKKGQKKAKKSNKSKAAQRKNSKKSNEHQSGNDLSTKIY ATMEKHKEVFFVIRLHSAQSAASLAPIQDPDPLLTCDLMDGRDAFLTLAR DKHFEFSSLRRAQFSTLSMLYELHNQGQDKFVYTCNHCKTAVETRYHCTV CDDFDLCIVCKEKVGHQHKMEKLGFDIDDGSALADHKQANPQEARKQSIQ RCIQSLAHACQCRDANCRLPSCQKMKLVVQHTKNCKRKPNGGCPICKQLI ALCCYHAKNCEEQKCPVPFCPNIKHKLKQQQSQQKFQQQQLLRRRVALMS RTAAPAALQGPAAVSGPTVVSGGVPVVGMSGVAVSQQVIPGQAGILPPGA GGMSPSTVAVPSPVSGGAGAGGMGGMTSPHPHQPGIGMKPGGGHSPSPNV LQVVKQVQEEAARQQVSHGGGFGKGVPMAPPVMNRPMGGAGPNQNVVNQL GGMGVGVGGVGGVGVGGVGGVGVNQLNSGGGNTPGAPISGPGMNVNHLMS MDQWGGGGAGGGGANPGGGNPQARYANNTGGMRQPTHVMQTNLIPPQQQQ QMMGGLGGPNQLGGGQMPVGGQHGGMGMGMGAPPMAGTVGGVRPSPGAGG GGGSATGGGLNTQQLALIMQKIKNNPTNESNQHILAILKQNPQIMAAIIK QRQQSQNNAAAGGGAPGPGGALQQQQAGNGPQNPQQQQQQQQQQQVMQQQ QMQHMMNQQQGGGGPQQMNPNQQQQQQQVNLMQQQQQGGPGGPGSGLPTR MPNMPNALGMLQSLPPNMSPGVSTQGGMVPNQNWNKMRYMQMSQYPPPYP QRQRGPHMGGAGPGPGQQQFPGGGGGAGNFNAGGAGGAGGVVGVGGVPGG AGTVPGGDQYSMANAAAASNMLQQQQGQVGVGVGVGVKPGPGQQQQQMGV GMPPGMQQQQQQQQPLQQQQMMQVAMPNANAQNPSAVVGGPNAQVMGPPT PHSLQQQLMQSARSSPPIRSPQPTPSPRSAPSPRAAPSASPRAQPSPHHV MSSHSPAPQGPPHDGMHNHGMHHQSPLPGVPQDVGVGVGVGVGVGVNVNV GNVGVGNAGGALPDASDQLTKFVERL;clonal: Polyclonal;purified: Affinity;company: Lab;catalog: Mattias Mannervik's Lab;short description: Rabbit affinity purified antisera raised against the CBP protein. This is a gift from Mattias Mannervik's Lab.;

hyb_protocol: Affymetrix standard labeling and hybridization reaction of ChIP samples

scan_protocol: Arrays are scanned on Agilent's DNA Microarray Scanner G2505B at a resolution of 5 microns. Data is extracted from the images using Agilent's Feature Extraction Software 9.5.3.1.

data_processing: Data Processing:KW:1 protocol. ChIP-chip data generated using Affymetrix Drosophila tiling array 2.0 are subjected to the analysis using MAT (Johnson et al 2006). Since, the IP and Control samples are hybridized on different arrays. The data are strictly paired.We specified the MAT parameters like Bandwidth, MaxGap and Minprobe parameters to be 200, 100 and 10 respectively. We used non-redundant dm3 BPMAP file and self generated Repeat Library files available for download at http://liulab.dfci.harvard.edu/MAT/Download.htmWe used results at 1% and 5% FDR values. The results are available as GFF3 files.

organism: Drosophila melanogaster

label_protocol: Attached as supplementary file

extract_protocol:
Collection of Drosophila embryos at time-point 0-4h of development for further ChIP experiments
Formaldehyde Cross-linking of Chromatin from any Drosophila tissue (Embryos, Larvae, Pupae, Adult flies).1. Homogenize the material (about 150-200 mg of embryos, pupae, adµlts, larvae, sufficient for four-five independent immuno-precipitations) in 5 ml of buffer A1 + 1.8% formaldehyde (290 µl of 37% solution) at room temperature using first a Potter homogenizer and then a Douncer with type A pestle (3 strokes). Wait 15' (total time starting from beginning of homogenization). Add glycin solution to 225 mM (540 µl of 2.5 M solution for 6 ml of cross-linked mixture), mix and incubate 5'. Put on ice. Keep on ice unless specified.2. Transfer the homogenate to a 15 ml tube. Centrifuge 5 min, at 4000 g at 4°C. Discard supernatant. Add 3 ml of buffer A1, resuspend pellet and spin down the same way. Repeat washing step 3 times.3. Wash once in 3 ml of lysis buffer without SDS. Spin down, 5 min at 4000g.4. Resuspend cross-linked material in 0.5 ml of lysis buffer and add SDS to 0.1% (2.5 µl) and N-lauroylsarcosine to 0.5% (12.5 µl). Incubate 10 min at 4°C in a rotating wheel.5. Sonicate in Polypropylene 15ml Falcon tubes according to .Diagenode instructions. The settings are: High power, 15 minutes, 30 seconds off, 30 seconds on. The water bath shoµld be kept cold either in a cold room or by a cold water pump.6. Rotate 10'. Transfer to eppendorf tubes and centrifuge 5', room temperature at maximum speed. Transfer supernatant to the new tube. Add another 0.5 ml of lysis buffer to the pellet, rotate for 10 min. Repeat centrifugation and combine supernatants. Centrifuge the combined supernatant 2x10 min at max speed. The supernatant from this stage will be referred as chromatin extract. Chromatin can be stored at -80°C for several months at this stage. Add sodium azide to 0.02% for storage.7. [Optional : Purification of chromatin. Put chromatin extract in Centricon YM-100 column (4212), centrifuge 3x40' (or more) at 1000 g while adding lysis buffer. At least 3 vol. of lysis buffer should pass through column. Bring the final volume of chromatin extract to 1 ml by lysis buffer + protease inhibitors.][edit]
Solutions and MaterialsLysis buffer: (see above)5mg/ml RNaseA (DNase free)PAS suspension: 100 mg of CL-4B (Amersham, 17-0780-01) PAS shoµld be resuspended in 1 ml of lysis buffer + protease inhibitors + 0.1 mg/ml BSA for 50 %v suspension. Wash in lysis buffer 2-3 times, overall equilibration time 1h. Store up to one week at 4 C.TE: 10 mM Tris-HCl pH 8.0, 1 mM EDTAElution buffer1: 10 mM EDTA, 1% SDS, 50 mM Tris-Cl pH 8.Elution buffer2: TE+0.67% SDS.LiCL 4M Protocol 1. To an amount of chromatin corresponding to 150 mg of biological material, suspended in a final volume of chromatin extract of 1 ml (in lysis buffer + protease inhibitors), add 100 µl of PAS suspension for preincubation. Incubate several hours or overnight at 4°C, then remove PAS. Crosslinked chromatin at this stage can be stored several days at 4°C or frozen at -70°C.2. [Optional : Check for amount of DNA. From the 1 ml solution above, take a 100 µl aliquot, Add proteinase K up to 100 ug/ml and SDS to 1%, incubate 6h at 60°C, then 20 min at 70°C, add RNAse to 50 ug/ml and incubate additional 2h at 37°C. Phenol-chlorophorm extract, ethanol precipitate. Run on an agarose gel to check amount and size of DNA.]3. Separate chromatin sample into 4x250 µl aliquots (one aliquot is enough for one IP but the amount may be decreased further). Immunoprecipitate chromatin by adding the antibody (Ab) of interest (amount of Ab shoµld be determined empirically, usually the same concentration than for a successfµl IF experiement). Keep a control sample without Ab IP, named "Mock".4. Incubate 4 h at 4°C on a head to head rotating wheel, then add 50 µl of PAS suspension and incubate 4 h or overnight. Spin down PAS and proceed to washes.5. Wash PAS 4x with lysis buffer, followed by 2x with TE (without protease inhibitors). Each wash is for 5 min at 4°C, using 1 ml of solution.6. Elution of precipitated material. Spin down PAS. Add 100 µl of elution buffer, mix and incubate 10 min at 65°C. Spin down PAS and transfer supernatant to new tube. Add 150 µl of elution buffer 2 to PAS, mix, centrifuge at fµll speed and transfer eluate to a tube together with the eluate from the first centrifugation. The combined material is the "chromatin precipitate" (approx. 250 µl).7. Incubate precipitate 6 h (or overnight) at 65 C to reverse cross-links. Add 250 µl of Proteinase K solution, incubate at 50°C 2-3 h.8. Add 55 µl of 4M LiCl and 500 µl of phenol-chlorophorm. Mix and microfuge at fµll speed at RT. Transfer aqueous phase to a new tube and precipitate with 1 ml of 100% ethanol. Wash with 750 µl of 70% ethanol. Spin down and dry precipitate.9. Dissolve in 25 µl of water. This is the chromatin immunoprecipitate or 'Native ChIP' sample.
LM-PCR amplification of the native ChIP sample to obtain a sufficient quantity for further hybridization on microarrays.

value: Y cn bw sp

tag: developmental stage

tag: genotype

tag: Sex

value: Unknown


title: E0_4h_rbCBP_2 extraction2_array1


title: E0_4h_rbCBP_4 extraction4_array1

description: channel ch1 is negative control for ChIP; Antibody information listed below: official name: No Antibody Control;target name: Not Applicable;host: None-Control;antigen: NONE;clonal: None-Control;purified: None-Control;company: None-Control;catalog: Not Applicable;reference: Not Applicable;short description: Specific Antibodies were omitted from this immunoprecipitation reaction (as a negative control).;


title: E0_4h_rbCBP_5 extraction5_array1


title: Blood_HighPb_Rec_2

tag: tissue


title: Blood_LowPb_Rec_15

source_name: Whole blood from Low Pb mice after 2-week recovery


title: E0_4h_rbCBP_1 extraction1_array1


title: Blood_HighPb_Rec_3


title: Blood_HighPb_Rec_1


title: MCF-7_6hr_100nM-4-OHT_1

description:
CH1 raw data file: 1_Exiqon_13847528_S01_Cropped.txt
CH2 raw data file: 0_Exiqon_13847528_S01_Cropped.txt


title: Blood_LowPb_Rec_13


title: PADI4 ChIP-chip (replicate A)

description: no additional information


title: E.albidus_OECD St_48h_rep2

city: Aveiro

first: Mónica

last: Amorim

address: Campus Universitário de Santiago, Department of Biology, University of Aveiro

department: Biology

institute: CESAM & University of Aveiro

description: The hybridization performed here was the control sample vs the standard OECD soil sample.

hyb_protocol: Prior to hybridization, arrays were incubated for 30-45 min at 42°C in a prehybridization solution (50% formamide, 5x saline sodium citrate (SSC), 0.1% sodium dodecyl sulphate (SDS), 0.1mg/mL BSA). The vacuum dried targets (both reference and treatment target) were resuspended in a hybridization solution (50% formamide, 5x SSC, 0.1% SDS, 0.1mg/mL BSA and 0.1mg/mL sheared salmon sperm) and denaturated at 95°C. The cooled targets were subsequently applied onto the prehybridized slides and incubated overnight at 42°C. After hybridization, arrays were washed in solutions with increasing stringency (decreasing concentrations of SSC and SDS) and dried with N2.

scan_protocol: Scanning and analysing of the slides was performed using the Genepix personal 4100 Scanner and Genepix pro Software (both Axon instruments, USA). Cy3 and Cy5 fluorescent signals were scanned at respectively 532 and 635nm and the PMT (photomultiplier tube) values were adjusted to reach a ratio (Cy5/Cy3) around 1. Spots were identified and ratio’s quantified by means of the Genepix software 5.0 (Axon Instruments).

data_processing: The BioArray Software Environment Database (BASE 1.2.12,http://www.islab.ua.ac.be/base/) was used for storage, further evaluation and succeeding statistical analysis of the microarray datasets. The Log2 transformed Cy5/Cy3 ratio data were normalized by implementation of a Locally Weighed Scatterplot Smoothing (Lowess) (Yang et al., 2002) step, which is an intensity-based method.

organism: Enchytraeus albidus

source_name: E.albidus exposed to standard OECD soil for 48h

label_protocol: The production of cDNA and subsequent amino-allyl labelling were performed according to the protocol described in detail by Van der Ven et al. (2005) and Soetaert et al. (2006). In brief, 7µg of total RNA was mixed with Lucidea control mRNA spike mix (Amersham, UK) to be reverse-transcribed using the Superscript II, Random hexamer primers (both Invitrogen) in the presence of dNTPs with 2/3 aa-dTTP/dTTP (Sigma-Aldrich, Belgium). After an overnight incubation period (42°C) the amino-allyl-incorporated cDNA was purified using a modified Qiagen PCR spin colomn protocol (Van der Ven et al., 2005). In a next step, Cy3/Cy5 esters (Amersham) were used for covalent coupling to the amino-allyl labelled cDNA. The vacuum dried cDNA samples (dissolved in a 0,1M carbonate buffer (pH 9.0)) were mixed with the Cy3/Cy5 esters (dissolved in 100% DMSO) and incubated for an hour in the dark at room temperature. Hereafter, a second cleanup reaction was performed to establish the removal of the remaining uncoupled dyes (QIAquick PCR purification kit, Qiagen, USA). The quality of eluted and labelled cDNA was analysed using the Nanodrop® spectrophotometer. Samples with an FOI (frequence of incorporated dye) between 20 and 50 were selected for hybridization and an amount of 150 pmol labelled target was vacuum dried.

extract_protocol:
15 worms per replicate were collected. Samples were shock-frozen in liquid nitrogen and stored in RNAlater (Ambion, USA) at -80°C.
All RNA extractions were performed using the TRIzol® method (Invitrogen, Belgium) followed by a DNAse treatment using 1,5U RNAse-free DNAse and 1U RNAse inhibitor (Fermentas, Germany) per 50µl sample and subsequent phenol/chloroform extractions. The purity of the RNA samples was checked using the ND-1000 spectrophotometer (Nanodrop®, USA) through measurement of the 260nm/280nm and 260nm/230nm absorbance. For all used samples these ratios were approximately 2. To verify the intactness of the RNA samples, a denaturating formaldehyde agarose gel electrophoresis was performed.

value: Whole body extracts (15 organisms)

tag: stage of development

value: Adult organisms with well developed clitellum

tag: exposure

value: standard OECD soil

source_name: E.albidus maintained for the same 48h in LUFA 2.2 soil - Control RNA sample


title: E.albidus_OECD St_48h_rep1

extract_protocol: 15 worms per replicate were collected. Samples were shock-frozen in liquid nitrogen and stored in RNAlater (Ambion, USA) at -80°C. All RNA extractions were performed using the TRIzol® method (Invitrogen, Belgium) followed by a DNAse treatment using 1,5U RNAse-free DNAse and 1U RNAse inhibitor (Fermentas, Germany) per 50µl sample and subsequent phenol/chloroform extractions. The purity of the RNA samples was checked using the ND-1000 spectrophotometer (Nanodrop®, USA) through measurement of the 260nm/280nm and 260nm/230nm absorbance. For all used samples these ratios were approximately 2. To verify the intactness of the RNA samples, a denaturating formaldehyde agarose gel electrophoresis was performed.


title: E.albidus_OECD pH_48h_rep2

description: The hybridization performed here was the control sample vs the low pH soil sample.

source_name: E.albidus exposed to OECD soil with lower pH for 48h

value: OECD soil with lower pH


title: Blood_LowPb_Rec_14

tag: gender

value: female


title: Con KDvsTet1 KD ES cells, Ezh2 ChIP-chip (3/4)

city: Boston

first: Hao

state: Massachusetts

address: 149G Warren Alpert Building, 200 Longwood Avenue

department: Genetics

institute: Harvard Medical School/HHMI

description: IP/IP

hyb_protocol: 34 ug of Cy5- and Cy3-labeled samples were combined and hybridized to each mouse whole genome tiling microarray (Roche/NimbleGen 4-array set) for 16-20 h at 42 ˚C using NimbleGen hybridization System 4.

scan_protocol: Microarrays were scanned using the Agilent scanner at 5 micron resolution.

data_processing: Data were extracted and analyzed using NimbleScan v2.5 (Roche/NimbleGen).  For identifying probes associated with a significant decrease in Ezh2 levels or a significant increase in DNA methylation level in Tet1 KD ES cells as compared to Con KD cells, a non-parametric one-sided Kolmogorov-Smirno (KS) test was used (KS score>1.3).

source_name: Tet1 KD ES cells, immunoprecipitated DNA by Ezh2 antibodies

label_protocol: Immunoprecipitated and input DNA were amplified using the Whole Genome Amplification Kit (Sigma), and 5 ug of amplified DNA were labeled (5’ Cy5- or Cy3-random nonamers, TriLink Biotechnologies) using the standard protocol (NimbleGen Arrays User’s Guide for ChIP-chip analysis).

growth_protocol: Mouse ES cells were maintained on gelatin-coated dishes in Glasgow Minimum Essential medium (GMEM; GIBCO), supplemented with 15% heat-inactivated fetal bovine serum, 55 mM b-mercaptoethanol (GIBCO), 2mM L-glutamine, 0.1mM MEM non-essential amino acid, 5,000 units/ml penicillin/streptomycin and 1,000 units/ml of ESGRO (Chemicon) under feeder-free conditions.

extract_protocol: MeDIP-chip was perfromed as previously described with minor modifications (Weber et al., 2005). Briefly, 5 ug sonicated, heat-denatured genomic DNA was immunoprecipitated with 5 ul of monoclonal antibody against 5-methylcytidine (Eurogentec) at 4 C overnight.  Immunoprecipitated methylated DNA was collected with 30 ul Dynal protein-G beads for 2 h at 4 C.  ChIP-chip assay was performed with about 10 million cells following Agilent mammalian ChIP-on-chip protocol (March, 2006). Briefly, cells were cross-linked with formaldehyde for 10 min at room temperature. Sonicated chromatin was immunoprecipitated overnight at 4 C with 10 ug immobilized (on 100 ul Dynal protein-G beads, Invitrogen) antibodies to Ezh2 (Active Motif, 39103).

tag: cell type

value: Mouse ES cells

tag: knockdown

tag: antibody

value: Ezh2 antibody

tag: antibody manufacturer

value: Active Motif

tag: antibody catalog number

tag: antibody catalog #

value: 39103 (already discontinued by the vendor)

source_name: Con KD ES cells, immunoprecipitated DNA by Ezh2 antibodies

value: control

tag: sample type


title: Blood_LowPb_Rec_11

tag: strain

value: Balb/c

tag: age

tag: study phase

value: recovery phase (post-exposure)

tag: time

value: 2 weeks

value: Pb


title: Tet1 KD ES cells, DNA methylation by MeDIP-chip  (4/4)

description: IP/input

source_name: Tet1 KD ES cells, whole cell extract

value: input DNA

source_name: Tet1 KD ES cells, immunoprecipitated DNA by 5-methylcytosine antibody

value: 5-methylcytosine antibody

value: Eurogentec


title: Blood_LowPb_Rec_10

tag: agent during exposure phase

tag: agent dose during exposure phase

value: peripheral blood


title: Blood_LowPb_Rec_5

tag: tissue


title: Con KDvsTet1 KD ES cells, DNA methylation by MeDIP-chip (3/4), exp2

description: IP/IP

source_name: Con KD ES cells, immunoprecipitated DNA by 5-methylcytosine antibody


title: E.albidus_OECD OM_48h_rep1

description: The hybridization performed here was the control sample vs the lower organic matter content soil sample.

source_name: E.albidus exposed to OECD soil with lower organic matter content for 48h

value: Whole body extracts (15 organisms)

tag: stage of development

value: Adult organisms with well developed clitellum


title: E.albidus_OECD pH_48h_rep1

tag: exposure

value: OECD soil with lower pH


title: Con KD ES cells, 5-hydroxymethylcytosine  by hMeDIP-chip, Rb Ab  (4/4)

data_processing: Data were extracted and analyzed using NimbleScan v2.5 (Roche/NimbleGen).  For identifying regions associated with a significant enrichment of 5hmC in Con KD or Tet1 KD ES cells, a non-parametric one-sided Kolmogorov-Smirno (KS) test was used ([-log10 5hmC peak P-value]>2.3).

source_name: Con KD ES cells, whole cell extract

extract_protocol: Genomic DNA was sequentially digested with proteinase K and RNase A, and purified by phenol/chloroform extraction. Purified DNA was sonicated to 200-1000 bp and heat-denatured (95 ˚C, 10 min).  An aliquot of sonicated genomic DNA was saved as input.  5μg fragmented genomic DNA was immunoprecipitated with 5 μg of rat monoclonal 5hmC Ab (Diagenode, cat# MAb-633HMC) or rabbit polyclonal 5hmC Ab (Active Motif, cat#39791) at 4 ˚C overnight in a final volume of 500 μl IP buffer [10 mM sodium phosphate (pH 7.0), 140 mM NaCl, 0.05% Triton X-100].  The DNA-antibody mixture was incubated with 30 μl protein G Dynabeads (Invitrogen) for 2 h at 4 ˚C and washed for three times with 1 ml IP buffer.  The beads were then treated with proteinase K for at least 3 h at 55 ˚C and the methylated DNA was purified by phenol-chloroform extraction followed by ethanol precipitation.

tag: tissue origin of primary cultures

source_name: Con KD ES cells, immunoprecipitated DNA by 5-hydroxymethylcytosine rabbit polyclonal antibody

tag: vendor

tag: cat#


title: E.albidus_OECD OM_48h_rep3

description: The hybridization performed here was the control sample vs the low organic matter content soil sample.

value: OECD soil with lower organic matter content


title: Blood_LowPb_Rec_12

tag: gender

value: female


title: E.albidus_OECD OM_48h_rep2


title: Blood_LowPb_Rec_9


title: Blood_HighPb_Exp_15

source_name: Whole blood from mice after 2-week high-level Pb exposure (50ug/mL drinking water)

value: exposure phase


title: Blood_LowPb_Rec_3


title: Blood_LowPb_Rec_2


title: Blood_HighPb_Exp_14


title: Con KDvsTet1 KD ES cells, Ezh2 ChIP-chip (4/4)

description: IP/IP

tag: cell type

value: Mouse ES cells

tag: knockdown

tag: antibody

value: Ezh2 antibody

tag: antibody manufacturer

value: Active Motif

value: immunoprecipitated DNA by Ezh2 antibodies


title: E.albidus_OECD Clay_48h_rep3

description: The hybridization performed here was the control sample vs the high clay content soil sample.

source_name: E.albidus exposed to OECD soil with higher clay content for 48h

value: OECD soil with higher clay content


title: Blood_LowPb_Rec_8

tag: strain

value: Balb/c

value: recovery phase (post-exposure)

value: 2 weeks


title: Blood_LowPb_Rec_7

tag: age

value: Pb


title: Blood_LowPb_Rec_6

tag: study phase


title: Blood_LowPb_Rec_1

tag: time


title: Con KDvsTet1 KD ES cells, DNA methylation by MeDIP-chip (3/4), exp1

description: IP/IP

value: control

value: 5-methylcytosine antibody

value: Eurogentec

tag: antibody catalog #

tag: sample type

value: immunoprecipitated DNA by 5-methylcytosine antibody


title: Blood_HighPb_Exp_13

tag: agent during exposure phase

tag: agent dose during exposure phase

tag: tissue

value: peripheral blood


title: Blood_LowPb_Rec_4


title: E.albidus_OECD Clay_48h_rep1

value: Whole body extracts (15 organisms)

tag: stage of development

value: Adult organisms with well developed clitellum

tag: exposure


title: Con KDvsTet1 KD ES cells, DNA methylation by MeDIP-chip (4/4), exp1

description: IP/IP


title: Blood_HighPb_Exp_7

value: female

value: exposure phase


title: Blood_HighPb_Exp_6


title: Blood_HighPb_Exp_12


title: E.albidus_Phenmedipham 32 mg/Kg_48h_rep2

description: The hybridization performed here was the control sample vs the 32 mg/Kg Phenmedipham exposure sample.

source_name: E.albidus exposed to 32 mg/Kg of Phenmedipham for 48h

value: 32 mg Phenmedipham/Kg soil


title: E.albidus_Phenmedipham 32 mg/Kg_48h_rep1


title: E.albidus_Copper 320 mg/Kg_48h_rep2

description: The hybridization performed here was the control sample vs the 320 mg/Kg Copper exposure sample.

source_name: E.albidus exposed to 320 mg/Kg of Copper for 48h

value: 320 mg Cu/Kg soil


title: Blood_HighPb_Exp_11


title: Blood_HighPb_Exp_8


title: Con KD ES cells, 5-hydroxymethylcytosine  by hMeDIP-chip, Rb Ab  (3/4)

tag: tissue origin of primary cultures

value: Mouse ES cells

tag: vendor

value: Active Motif

tag: cat#


title: E.albidus_Phenmedipham 10 mg/Kg_48h_rep1

description: The hybridization performed here was the control sample vs the 10 mg/Kg Phenmedipham exposure sample.

source_name: E.albidus exposed to 10 mg/Kg of Phenmedipham for 48h

value: 10 mg Phenmedipham/Kg soil


title: E.albidus_Phenmedipham 10 mg/Kg_48h_rep2


title: Tet1 KD ES cells, DNA methylation by MeDIP-chip  (3/4)

description: IP/input

tag: cell type

tag: knockdown

value: input DNA


title: Tet1 KD ES cells, 5-hydroxymethylcytosine  by hMeDIP-chip, Rb Ab  (4/4)

tag: antibody

source_name: Tet1 KD ES cells, immunoprecipitated DNA by 5-hydroxymethylcytosine rabbit polyclonal antibody


title: Con KD ES cells, DNA methylation by MeDIP-chip  (4/4)

description: IP/input

tag: strain

tag: antibody manufacturer


title: Incomplete X-inactivation initiated by a hypomorphic Xist allele in the mouse(Day6_WT)

city: Tsukuba

first: Rieko

last: Ikeda

state: Ibaraki

address: 3-1-1 Koyadai

department: BioResource Center

institute: RIKEN

hyb_protocol: Hybridizations were performed for 17 hours, according to the manufacturers protocol (Agilent Technologies).

scan_protocol: Hybridized arrays were scanned using the Agilent microarray scanner (Agilent Technologies).

data_processing: Hybridization signals were converted to numerical data with the Feature Extraction software ver. 10.5.1.1 (Agilent Technologies). Agilent Feature Extraction was used for background subtraction and LOWESS normalization. After that, We processed one color of normalization (shift to 50 percentile to 0) each of Agilent Feature Extraction output files(gProcessedSignal), and averaged of biological replicates data.

source_name: Trophoblast stem cell line, DD1

label_protocol: 500 ng of total RNA was labeled with Cy3-CTP using a QuickAmp labeling kit (Agilent Technologies).

extract_protocol: Total RNA was isolated from TS cells using TRIzol (Invitrogen).

tag: time point

value: DBA/2 x C57BL/6

tag: Sex


title: Incomplete X-inactivation initiated by a hypomorphic Xist allele in the mouse(Day6_IVS)

source_name: Trophoblast stem cell line, T5

value: DBA/2 x Xist IVS/Y


title: Incomplete X-inactivation initiated by a hypomorphic Xist allele in the mouse(Day0_WT)


title: Blood_HighPb_Exp_4

value: Balb/c

tag: age

tag: gender

tag: study phase

tag: time

value: 2 weeks

value: Pb


title: Blood_HighPb_Exp_5

tag: agent during exposure phase

tag: agent dose during exposure phase

tag: tissue


title: Tet1 KD ES cells, 5-hydroxymethylcytosine  by hMeDIP-chip, Rb Ab  (3/4)

tag: tissue origin of primary cultures


title: Incomplete X-inactivation initiated by a hypomorphic Xist allele in the mouse(Day0_IVS)


title: Con KD ES cells, DNA methylation by MeDIP-chip  (3/4)

description: IP/input

value: control

tag: sample type

value: immunoprecipitated DNA by 5-methylcytosine antibody

value: 5-methylcytosine antibody

value: Eurogentec

tag: antibody catalog #


title: Con KDvsTet1 KD ES cells, DNA methylation by MeDIP-chip (4/4), exp2

description: IP/IP

tag: cell type


title: E.albidus_OECD Clay_48h_rep2

value: Whole body extracts (15 organisms)

tag: stage of development

value: Adult organisms with well developed clitellum

tag: exposure

value: OECD soil with higher clay content

source_name: E.albidus maintained for the same 48h in LUFA 2.2 soil - Control RNA sample


title: Tet1 KD ES cells, 5-hydroxymethylcytosine  by hMeDIP-chip, Rat Ab  (4/4)

tag: strain

tag: antibody

source_name: Tet1 KD ES cells, immunoprecipitated DNA by 5-hydroxymethylcytosine rat monoclonal antibody

tag: vendor

value: Diagenode

tag: cat#


title: Tu 2

city: New York

first: Richard

middle: Mark

last: White

address: 1275 York Avenue MB 424

department: Cancer Biology & Genetics

institute: Memorial Sloan Kettering Cancer Center

description: This sample is an 80hpf Tu (wild-type embryo)

hyb_protocol: Hybridization was performed by the Genomics Core at Children's Hospital Boston according to standard Affymetrix protocols

scan_protocol: Scanning was performed by the Genomics Core at Children's Hospital Boston according to standard Affymetrix protocols

data_processing: Raw data (*.cel files) was normalized using GC-RMA followed by per gene and per chip normalization

organism: Danio rerio

source_name: tu embryos 2

label_protocol: Labeling was performed by the Genomics Core at Children's Hospital Boston according to standard Affymetrix protocols

growth_protocol: Zebrafish of the indicated genotype were incrossed to produce embryos, which were grown to 80hpf.

extract_protocol: Total RNA was extracted using Trizol (Invitrogen, USA).

tag: genotype/variation

value: Tu

tag: developmental stage

value: embryo


title: Blood_LowPb_Exp_15

source_name: Whole blood from mice after 2-week low-level Pb exposure (5ug/mL drinking water)

tag: age

tag: gender

value: female

tag: study phase

value: exposure phase

value: peripheral blood


title: Blood_HighPb_Exp_1

tag: time

tag: agent during exposure phase

tag: agent dose during exposure phase


title: Blood_HighPb_Exp_3

tag: tissue


title: Blood_LowPb_Exp_13


title: Tu 3

source_name: tu embryos 3


title: Blood_LowPb_Exp_14


title: E.albidus_Copper 320 mg/Kg_48h_rep1

value: 320 mg Cu/Kg soil


title: p53 2

description: This sample is an 80hpf p53-M214K -/- mutant

source_name: p53-/- embryos 1b


title: Blood_LowPb_Exp_11

value: Balb/c


title: Blood_LowPb_Exp_10

value: 2 weeks


title: p53 3

source_name: p53-/- embryos 2


title: Blood_LowPb_Exp_12

value: Pb


title: Blood_HighPb_Exp_10


title: Tu 1

source_name: tu embryos 1


title: BRAF 2

description: This sample is an 80hpf transgenic zebrafish carrying a stable homozygous allele of mitf-BRAFV600E

source_name: tg(mitf-BRAFV600E) embryos 2


title: BRAF;p53 1

description: This sample is an 80hpf transgenic zebrafish carrying a stable homozygous allele of mitf-BRAFV600E in the background of a p53-/- loss of function

source_name: tg(mitf-BRAFV600E);p53-/- embryos 1


title: Blood_HighPb_Exp_2

source_name: Whole blood from mice after 2-week high-level Pb exposure (50ug/mL drinking water)


title: BRAF;p53 3

source_name: tg(mitf-BRAFV600E);p53-/- embryos 3


title: BRAF 3

source_name: tg(mitf-BRAFV600E) embryos 3


title: Blood_HighPb_Exp_9


title: BRAF;p53 2

source_name: tg(mitf-BRAFV600E);p53-/- embryos 2


title: Con KD ES cells, 5-hydroxymethylcytosine  by hMeDIP-chip, Rat Ab  (4/4)

source_name: Con KD ES cells, whole cell extract

tag: tissue origin of primary cultures

value: Mouse ES cells

source_name: Con KD ES cells, immunoprecipitated DNA by 5-hydroxymethylcytosine rat monoclonal antibody


title: Blood_LowPb_Exp_4


title: Blood_LowPb_Exp_9


title: Blood_LowPb_Exp_7


title: p53 1

source_name: p53-/- embryos 1a


title: Blood_LowPb_Exp_6


title: Blood_LowPb_Exp_8


title: BRAF 1

source_name: tg(mitf-BRAFV600E) embryos 1


title: WT skin 2

description: This sample is of WT skin

source_name: WT Skin

growth_protocol: Zebrafish of the indicated genotype were grown to 4-8 months of age, when a visible melanoma could be identified.  The melanoma, and immediately adjacent skin was excised from the fish.  For WT samples, the lateral length of the zebrafish skin was excised.

value: Normal skin


title: Blood_LowPb_Exp_2


title: WT skin 1

description: This sample is of WT skin


title: WT skin 3

description: This sample is of WT skin


title: Blood_LowPb_Exp_3


title: Blood_LowPb_Exp_1


title: Blood_LowPb_Exp_5


title: Blood_Control_Rec_7

source_name: Whole blood from control mice after 2-week recovery period

value: recovery phase (post-exposure)

value: control


title: mitf-BRAFV600E;p53-/- melanoma 5

description: This sample is a BRAF;p53 melanoma

source_name: Melanoma BRAFV600E;p53-/-

value: Melanoma


title: mitf-BRAFV600E;p53-/- melanoma 6

description: This sample is a BRAF;p53 melanoma


title: mitf-BRAFV600E;p53-/- melanoma 4

description: This sample is a BRAF;p53 melanoma


title: mitf-BRAFV600E;p53-/- melanoma 3

description: This sample is a BRAF;p53 melanoma


title: mitf-BRAFV600E;p53-/- melanoma 2

description: This sample is a BRAF;p53 melanoma


title: Blood_Control_Rec_4


title: Blood_Control_Rec_1


title: Blood_Control_Rec_6


title: R13_1-6-GFP_2_1 extraction2_array1

description:
channel ch1 is ChIP DNA; Antibody information listed below: official name: odg-GFP;target name: eGFP;host: Goat;antigen: "MSKGEELFTG VVPILVELDG DVNGHKFSVS GEGEGDATYG KLTLKFICTT GKLPVPWPTL VTTLTYGVQC  FSRYPDHMKR HDFFKSAMPE GYVQERTIFF KDDGNYKTRA EVKFEGDTLV NRIELKGIDF KEDGNILGHK  LEYNYNSHNV YIMADKQKNG IKVNFKIRHN IEDGSVQLAD HYQQNTPIGD GPVLLPDNHY LSTQSALSKD  PNEKRDHMVL LEFVTAAGIT HGMDELYK";clonal: Polyclonal;purified: Affinity;company: Lab;catalog: Francis Stewart Lab Dresden Germany;short description: Antisera raised against a peptide from the GFP protein.  This is a gift from Francis Stewart's lab in Dresden Germany.;
channel ch2 is negative control for ChIP; Antibody information listed below: official name: No Antibody Control;target name: Not Applicable;host: None-Control;antigen: NONE;clonal: None-Control;purified: None-Control;company: None-Control;catalog: Not Applicable;reference: Not Applicable;short description: Specific Antibodies were omitted from this immunoprecipitation reaction (as a negative control).;

hyb_protocol: This protocol is used for hybridization of ChIP samples on 244K Agilent arrays.  The hybridization solution is composed of 5 ug each of Cy3- and Cy5-labeled DNA, 50 ug of salmon sperm DNA, Agilent Blocking Agent and Agilent Hyb Buffer. The mixture is heated to 95*C for 3 min and then 37*C for 30 minutes immediately prior to hybridization. Samples are hybridized for 40 hours at 65*C. After hybridization the slides are washed in Agilent Wash Buffer 1 for 5 minutes at room temperature and then in Agilent Wash Buffer 2 for 5 minutes at 31*C.

data_processing: Agilent ChIP data processing:KW:1 protocol. We preprocessed microarray data to normalize it and reduce experimental noise as described in Qi et al. (Qi et al., Nature biotechnology 2006). In short, the raw intensities from each channel was divided by the median intensity from that channel before computing a ratio to arrive at a median adjusted ratio. We further subtracted average value of negative control probes on the array and then added one as a regularization parameter. For factors that mark regions (e.g. H3K27me3), the data was quantile normalized, replicates were merged and HMM based segmentation was carried out.

extract_protocol:
1. y; bw cn sp flies are cultivated in cages with apple juice agar plates covered with yeast powder.   2. For a 1-6 hours egg laying, new plates are added after a 2hours pre-egglaying at 9am and flies are allowed to lay eggs for 5 hours.   3. Embryos are collected at 2.45 pm using a filter mesh and a brush. They are rinsed with EWB and cross-linked at 3pm in 1.8% formaldehyde at room temperature for 15 min.
Formaldehyde Cross-linking of Chromatin from any Drosophila tissue (Embryos, Larvae, Pupae, Adult flies).1. Homogenize the material (about 150-200 mg of embryos, pupae, adµlts, larvae, sufficient for four-five independent immuno-precipitations) in 5 ml of buffer A1 + 1.8% formaldehyde (290 µl of 37% solution) at room temperature using first a Potter homogenizer and then a Douncer with type A pestle (3 strokes). Wait 15' (total time starting from beginning of homogenization). Add glycin solution to 225 mM (540 µl of 2.5 M solution for 6 ml of cross-linked mixture), mix and incubate 5'. Put on ice. Keep on ice unless specified.2. Transfer the homogenate to a 15 ml tube. Centrifuge 5 min, at 4000 g at 4°C. Discard supernatant. Add 3 ml of buffer A1, resuspend pellet and spin down the same way. Repeat washing step 3 times.3. Wash once in 3 ml of lysis buffer without SDS. Spin down, 5 min at 4000g.4. Resuspend cross-linked material in 0.5 ml of lysis buffer and add SDS to 0.1% (2.5 µl) and N-lauroylsarcosine to 0.5% (12.5 µl). Incubate 10 min at 4°C in a rotating wheel.5. Sonicate in Polypropylene 15ml Falcon tubes according to .Diagenode instructions. The settings are: High power, 15 minutes, 30 seconds off, 30 seconds on. The water bath shoµld be kept cold either in a cold room or by a cold water pump.6. Rotate 10'. Transfer to eppendorf tubes and centrifuge 5', room temperature at maximum speed. Transfer supernatant to the new tube. Add another 0.5 ml of lysis buffer to the pellet, rotate for 10 min. Repeat centrifugation and combine supernatants. Centrifuge the combined supernatant 2x10 min at max speed. The supernatant from this stage will be referred as chromatin extract. Chromatin can be stored at -80°C for several months at this stage. Add sodium azide to 0.02% for storage.7. [Optional : Purification of chromatin. Put chromatin extract in Centricon YM-100 column (4212), centrifuge 3x40' (or more) at 1000 g while adding lysis buffer. At least 3 vol. of lysis buffer should pass through column. Bring the final volume of chromatin extract to 1 ml by lysis buffer + protease inhibitors.][edit]
Solutions and MaterialsLysis buffer: (see above)5mg/ml RNaseA (DNase free)PAS suspension: 100 mg of CL-4B (Amersham, 17-0780-01) PAS shoµld be resuspended in 1 ml of lysis buffer + protease inhibitors + 0.1 mg/ml BSA for 50 %v suspension. Wash in lysis buffer 2-3 times, overall equilibration time 1h. Store up to one week at 4 C.TE: 10 mM Tris-HCl pH 8.0, 1 mM EDTAElution buffer1: 10 mM EDTA, 1% SDS, 50 mM Tris-Cl pH 8.Elution buffer2: TE+0.67% SDS.LiCL 4M Protocol 1. To an amount of chromatin corresponding to 150 mg of biological material, suspended in a final volume of chromatin extract of 1 ml (in lysis buffer + protease inhibitors), add 100 µl of PAS suspension for preincubation. Incubate several hours or overnight at 4°C, then remove PAS. Crosslinked chromatin at this stage can be stored several days at 4°C or frozen at -70°C.2. [Optional : Check for amount of DNA. From the 1 ml solution above, take a 100 µl aliquot, Add proteinase K up to 100 ug/ml and SDS to 1%, incubate 6h at 60°C, then 20 min at 70°C, add RNAse to 50 ug/ml and incubate additional 2h at 37°C. Phenol-chlorophorm extract, ethanol precipitate. Run on an agarose gel to check amount and size of DNA.]3. Separate chromatin sample into 4x250 µl aliquots (one aliquot is enough for one IP but the amount may be decreased further). Immunoprecipitate chromatin by adding the antibody (Ab) of interest (amount of Ab shoµld be determined empirically, usually the same concentration than for a successfµl IF experiement). Keep a control sample without Ab IP, named "Mock".4. Incubate 4 h at 4°C on a head to head rotating wheel, then add 50 µl of PAS suspension and incubate 4 h or overnight. Spin down PAS and proceed to washes.5. Wash PAS 4x with lysis buffer, followed by 2x with TE (without protease inhibitors). Each wash is for 5 min at 4°C, using 1 ml of solution.6. Elution of precipitated material. Spin down PAS. Add 100 µl of elution buffer, mix and incubate 10 min at 65°C. Spin down PAS and transfer supernatant to new tube. Add 150 µl of elution buffer 2 to PAS, mix, centrifuge at fµll speed and transfer eluate to a tube together with the eluate from the first centrifugation. The combined material is the "chromatin precipitate" (approx. 250 µl).7. Incubate precipitate 6 h (or overnight) at 65 C to reverse cross-links. Add 250 µl of Proteinase K solution, incubate at 50°C 2-3 h.8. Add 55 µl of 4M LiCl and 500 µl of phenol-chlorophorm. Mix and microfuge at fµll speed at RT. Transfer aqueous phase to a new tube and precipitate with 1 ml of 100% ethanol. Wash with 750 µl of 70% ethanol. Spin down and dry precipitate.9. Dissolve in 25 µl of water. This is the chromatin immunoprecipitate or 'Native ChIP' sample.
LM-PCR amplification of the native ChIP sample to obtain a sufficient quantity for further hybridization on microarrays.

value: Embryo 1-6h

tag: genotype

value: eveR13; [BACRO1LO1 eve YFP]attP2

tag: transgene


title: Blood_Control_Rec_3


title: mitf-BRAFV600E;p53-/- melanoma 1

description: This sample is a BRAF;p53 melanoma


title: R13_1-6-GFP_1_1 extraction1_array1


title: mitf-BRAFV600E;p53-/- skin 4

description: This sample is BRAF;p53 skin adjacent to a melanoma

source_name: Skin BRAFV600E;p53-/-

value: Abnormal skin


title: Blood_Control_Exp_7

source_name: Whole blood from control mice after 2-week exposure period


title: mitf-BRAFV600E;p53-/- skin 5


title: Blood_Control_Rec_2


title: Blood_Control_Exp_6


title: Blood_Control_Exp_5


title: ISO1_8-16_mod_mdg4_3_1 extraction3_array1

description:
channel ch1 is ChIP DNA; Antibody information listed below: official name: KW4-mod(mdg4)-D2;target name: mod(mdg4);host: Rabbit;antigen: MPRASARYKI ETVDDGLGDE KQSTTQIVIQ TTAAPQATIV QQQQPQQAAQ QIQSQQLQTG TTTTATLVST NKRSAQRSSL TPASSSAGVK RSKTSTSANV MDPLDSTTET GC;clonal: Polyclonal;purified: Protein A/G;company: Other;catalog: Aviva-KW4-mod(mdg4)-D2;short description: Antisera raised against a peptide from the MOD(MDG4) protein. Produced commercially for the modENCODE Project by K. White's group.;
channel ch2 is negative control for ChIP; Antibody information listed below: official name: No Antibody Control;target name: Not Applicable;host: None-Control;antigen: NONE;clonal: None-Control;purified: None-Control;company: None-Control;catalog: Not Applicable;reference: Not Applicable;short description: Specific Antibodies were omitted from this immunoprecipitation reaction (as a negative control).;

extract_protocol:
1. y; bw cn sp flies are cultivated in cages with apple juice agar plates covered with yeast powder.   2. For a 8-16 hours egg laying, new plates are added after a 2 hours pre-egglaying at 12 pm and flies are allowed to lay eggs for 8 hours.   3. Embryos are collected at 7.45 am using a filter mesh and a brush. They are rinsed with EWB and cross-linked at 8am in 1.8% formaldehyde at room temperature for 15 min.
Formaldehyde Cross-linking of Chromatin from any Drosophila tissue (Embryos, Larvae, Pupae, Adult flies).1. Homogenize the material (about 150-200 mg of embryos, pupae, adµlts, larvae, sufficient for four-five independent immuno-precipitations) in 5 ml of buffer A1 + 1.8% formaldehyde (290 µl of 37% solution) at room temperature using first a Potter homogenizer and then a Douncer with type A pestle (3 strokes). Wait 15' (total time starting from beginning of homogenization). Add glycin solution to 225 mM (540 µl of 2.5 M solution for 6 ml of cross-linked mixture), mix and incubate 5'. Put on ice. Keep on ice unless specified.2. Transfer the homogenate to a 15 ml tube. Centrifuge 5 min, at 4000 g at 4°C. Discard supernatant. Add 3 ml of buffer A1, resuspend pellet and spin down the same way. Repeat washing step 3 times.3. Wash once in 3 ml of lysis buffer without SDS. Spin down, 5 min at 4000g.4. Resuspend cross-linked material in 0.5 ml of lysis buffer and add SDS to 0.1% (2.5 µl) and N-lauroylsarcosine to 0.5% (12.5 µl). Incubate 10 min at 4°C in a rotating wheel.5. Sonicate in Polypropylene 15ml Falcon tubes according to .Diagenode instructions. The settings are: High power, 15 minutes, 30 seconds off, 30 seconds on. The water bath shoµld be kept cold either in a cold room or by a cold water pump.6. Rotate 10'. Transfer to eppendorf tubes and centrifuge 5', room temperature at maximum speed. Transfer supernatant to the new tube. Add another 0.5 ml of lysis buffer to the pellet, rotate for 10 min. Repeat centrifugation and combine supernatants. Centrifuge the combined supernatant 2x10 min at max speed. The supernatant from this stage will be referred as chromatin extract. Chromatin can be stored at -80°C for several months at this stage. Add sodium azide to 0.02% for storage.7. [Optional : Purification of chromatin. Put chromatin extract in Centricon YM-100 column (4212), centrifuge 3x40' (or more) at 1000 g while adding lysis buffer. At least 3 vol. of lysis buffer should pass through column. Bring the final volume of chromatin extract to 1 ml by lysis buffer + protease inhibitors.][edit]
Solutions and MaterialsLysis buffer: (see above)5mg/ml RNaseA (DNase free)PAS suspension: 100 mg of CL-4B (Amersham, 17-0780-01) PAS shoµld be resuspended in 1 ml of lysis buffer + protease inhibitors + 0.1 mg/ml BSA for 50 %v suspension. Wash in lysis buffer 2-3 times, overall equilibration time 1h. Store up to one week at 4 C.TE: 10 mM Tris-HCl pH 8.0, 1 mM EDTAElution buffer1: 10 mM EDTA, 1% SDS, 50 mM Tris-Cl pH 8.Elution buffer2: TE+0.67% SDS.LiCL 4M Protocol 1. To an amount of chromatin corresponding to 150 mg of biological material, suspended in a final volume of chromatin extract of 1 ml (in lysis buffer + protease inhibitors), add 100 µl of PAS suspension for preincubation. Incubate several hours or overnight at 4°C, then remove PAS. Crosslinked chromatin at this stage can be stored several days at 4°C or frozen at -70°C.2. [Optional : Check for amount of DNA. From the 1 ml solution above, take a 100 µl aliquot, Add proteinase K up to 100 ug/ml and SDS to 1%, incubate 6h at 60°C, then 20 min at 70°C, add RNAse to 50 ug/ml and incubate additional 2h at 37°C. Phenol-chlorophorm extract, ethanol precipitate. Run on an agarose gel to check amount and size of DNA.]3. Separate chromatin sample into 4x250 µl aliquots (one aliquot is enough for one IP but the amount may be decreased further). Immunoprecipitate chromatin by adding the antibody (Ab) of interest (amount of Ab shoµld be determined empirically, usually the same concentration than for a successfµl IF experiement). Keep a control sample without Ab IP, named "Mock".4. Incubate 4 h at 4°C on a head to head rotating wheel, then add 50 µl of PAS suspension and incubate 4 h or overnight. Spin down PAS and proceed to washes.5. Wash PAS 4x with lysis buffer, followed by 2x with TE (without protease inhibitors). Each wash is for 5 min at 4°C, using 1 ml of solution.6. Elution of precipitated material. Spin down PAS. Add 100 µl of elution buffer, mix and incubate 10 min at 65°C. Spin down PAS and transfer supernatant to new tube. Add 150 µl of elution buffer 2 to PAS, mix, centrifuge at fµll speed and transfer eluate to a tube together with the eluate from the first centrifugation. The combined material is the "chromatin precipitate" (approx. 250 µl).7. Incubate precipitate 6 h (or overnight) at 65 C to reverse cross-links. Add 250 µl of Proteinase K solution, incubate at 50°C 2-3 h.8. Add 55 µl of 4M LiCl and 500 µl of phenol-chlorophorm. Mix and microfuge at fµll speed at RT. Transfer aqueous phase to a new tube and precipitate with 1 ml of 100% ethanol. Wash with 750 µl of 70% ethanol. Spin down and dry precipitate.9. Dissolve in 25 µl of water. This is the chromatin immunoprecipitate or 'Native ChIP' sample.
LM-PCR amplification of the native ChIP sample to obtain a sufficient quantity for further hybridization on microarrays.

value: Y cn bw sp

value: Embryo 8-16h


title: Blood_Control_Rec_5

value: female


title: ISO1_8-16_mod_mdg4_1_1 extraction1_array1


title: ISO1_8-16_Ez_3_1 extraction3_array1

description:
channel ch1 is ChIP DNA; Antibody information listed below: official name: KW4-E(z)-D2;target name: Enhancer of zeste;host: Rabbit;antigen: MCSFLFNLNN DFVVDATRKG NKIRFANHSI NPNCYAKVMM VTGDHRIGIF AKRAIQPGEE LFFDYRYGPT EQLKFVGIER EMEIVC;clonal: Polyclonal;purified: Protein A/G;company: Other;catalog: Aviva-KW4-E(z)-D2;short description: Antisera raised against a peptide from the E(Z) protein. Produced commercially for the modENCODE Project by K. White's group.;
channel ch2 is negative control for ChIP; Antibody information listed below: official name: No Antibody Control;target name: Not Applicable;host: None-Control;antigen: NONE;clonal: None-Control;purified: None-Control;company: None-Control;catalog: Not Applicable;reference: Not Applicable;short description: Specific Antibodies were omitted from this immunoprecipitation reaction (as a negative control).;


title: mitf-BRAFV600E;p53-/- skin 3


title: mitf-BRAFV600E;p53-/- skin 1


title: ISO1_8-16_Ez_2_1 extraction2_array1


title: ISO1_8-16_mod_mdg4_2_1 extraction2_array1


title: mitf-BRAFV600E;p53-/- skin 2


title: LEFLUNOMIDE 3

description: This is a experimental embryo treated with Leflunomide 6.5uM from 50% epiboly until 24hpf

source_name: 24hpf embryos treated with Leflunomide 6.5uM

growth_protocol: Zebrafish of the Tu strain were treated with either DMSO or leflunomide 6.5uM from 50% epiboly until 24hpf

value: Tu

value: embryo

tag: treatment group

value: treated with Leflunomide 6.5uM


title: LEFLUNOMIDE 1


title: ISO1_8-16_Ez_1_1 extraction1_array1

tag: strain


title: LEFLUNOMIDE 2


title: Blood_Control_Exp_4

value: Balb/c

value: exposure phase

value: peripheral blood


title: Blood_Control_Exp_1

value: 2 weeks


title: L2_PolII_1_3 extraction3_array1

description:
channel ch1 is ChIP DNA; Antibody information listed below: official name: PolII;target name: RNA Polymerase II;host: Mouse;antigen: The antibody 8WG16 was developed using RNA polymerase (RNA pol II) purified from wheat germ extract. It recognizes the C-terminal heptapeptide repeat present on the largest subunit of pol II. Clone 8WG16 has been used to investigate the role of the C-terminal domain of the 220kD RNA pol II subunit in the formation of the transcription complex.;clonal: Polyclonal;purified: Affinity;company: Covance;catalog: 8wg16;short description: Monoclonal Antibody against RNA Polymerase II;
channel ch2 is negative control for ChIP; Antibody information listed below: official name: No Antibody Control;target name: Not Applicable;host: None-Control;antigen: NONE;clonal: None-Control;purified: None-Control;company: None-Control;catalog: Not Applicable;reference: Not Applicable;short description: Specific Antibodies were omitted from this immunoprecipitation reaction (as a negative control).;

extract_protocol:
Collection of Drosophila second instar larvae for further ChIP experiments or RNA extraction.
Formaldehyde Cross-linking of Chromatin from any Drosophila tissue (Embryos, Larvae, Pupae, Adult flies).1. Homogenize the material (about 150-200 mg of embryos, pupae, adµlts, larvae, sufficient for four-five independent immuno-precipitations) in 5 ml of buffer A1 + 1.8% formaldehyde (290 µl of 37% solution) at room temperature using first a Potter homogenizer and then a Douncer with type A pestle (3 strokes). Wait 15' (total time starting from beginning of homogenization). Add glycin solution to 225 mM (540 µl of 2.5 M solution for 6 ml of cross-linked mixture), mix and incubate 5'. Put on ice. Keep on ice unless specified.2. Transfer the homogenate to a 15 ml tube. Centrifuge 5 min, at 4000 g at 4°C. Discard supernatant. Add 3 ml of buffer A1, resuspend pellet and spin down the same way. Repeat washing step 3 times.3. Wash once in 3 ml of lysis buffer without SDS. Spin down, 5 min at 4000g.4. Resuspend cross-linked material in 0.5 ml of lysis buffer and add SDS to 0.1% (2.5 µl) and N-lauroylsarcosine to 0.5% (12.5 µl). Incubate 10 min at 4°C in a rotating wheel.5. Sonicate in Polypropylene 15ml Falcon tubes according to .Diagenode instructions. The settings are: High power, 15 minutes, 30 seconds off, 30 seconds on. The water bath shoµld be kept cold either in a cold room or by a cold water pump.6. Rotate 10'. Transfer to eppendorf tubes and centrifuge 5', room temperature at maximum speed. Transfer supernatant to the new tube. Add another 0.5 ml of lysis buffer to the pellet, rotate for 10 min. Repeat centrifugation and combine supernatants. Centrifuge the combined supernatant 2x10 min at max speed. The supernatant from this stage will be referred as chromatin extract. Chromatin can be stored at -80°C for several months at this stage. Add sodium azide to 0.02% for storage.7. [Optional : Purification of chromatin. Put chromatin extract in Centricon YM-100 column (4212), centrifuge 3x40' (or more) at 1000 g while adding lysis buffer. At least 3 vol. of lysis buffer should pass through column. Bring the final volume of chromatin extract to 1 ml by lysis buffer + protease inhibitors.][edit]
Solutions and MaterialsLysis buffer: (see above)5mg/ml RNaseA (DNase free)PAS suspension: 100 mg of CL-4B (Amersham, 17-0780-01) PAS shoµld be resuspended in 1 ml of lysis buffer + protease inhibitors + 0.1 mg/ml BSA for 50 %v suspension. Wash in lysis buffer 2-3 times, overall equilibration time 1h. Store up to one week at 4 C.TE: 10 mM Tris-HCl pH 8.0, 1 mM EDTAElution buffer1: 10 mM EDTA, 1% SDS, 50 mM Tris-Cl pH 8.Elution buffer2: TE+0.67% SDS.LiCL 4M Protocol 1. To an amount of chromatin corresponding to 150 mg of biological material, suspended in a final volume of chromatin extract of 1 ml (in lysis buffer + protease inhibitors), add 100 µl of PAS suspension for preincubation. Incubate several hours or overnight at 4°C, then remove PAS. Crosslinked chromatin at this stage can be stored several days at 4°C or frozen at -70°C.2. [Optional : Check for amount of DNA. From the 1 ml solution above, take a 100 µl aliquot, Add proteinase K up to 100 ug/ml and SDS to 1%, incubate 6h at 60°C, then 20 min at 70°C, add RNAse to 50 ug/ml and incubate additional 2h at 37°C. Phenol-chlorophorm extract, ethanol precipitate. Run on an agarose gel to check amount and size of DNA.]3. Separate chromatin sample into 4x250 µl aliquots (one aliquot is enough for one IP but the amount may be decreased further). Immunoprecipitate chromatin by adding the antibody (Ab) of interest (amount of Ab shoµld be determined empirically, usually the same concentration than for a successfµl IF experiement). Keep a control sample without Ab IP, named "Mock".4. Incubate 4 h at 4°C on a head to head rotating wheel, then add 50 µl of PAS suspension and incubate 4 h or overnight. Spin down PAS and proceed to washes.5. Wash PAS 4x with lysis buffer, followed by 2x with TE (without protease inhibitors). Each wash is for 5 min at 4°C, using 1 ml of solution.6. Elution of precipitated material. Spin down PAS. Add 100 µl of elution buffer, mix and incubate 10 min at 65°C. Spin down PAS and transfer supernatant to new tube. Add 150 µl of elution buffer 2 to PAS, mix, centrifuge at fµll speed and transfer eluate to a tube together with the eluate from the first centrifugation. The combined material is the "chromatin precipitate" (approx. 250 µl).7. Incubate precipitate 6 h (or overnight) at 65 C to reverse cross-links. Add 250 µl of Proteinase K solution, incubate at 50°C 2-3 h.8. Add 55 µl of 4M LiCl and 500 µl of phenol-chlorophorm. Mix and microfuge at fµll speed at RT. Transfer aqueous phase to a new tube and precipitate with 1 ml of 100% ethanol. Wash with 750 µl of 70% ethanol. Spin down and dry precipitate.9. Dissolve in 25 µl of water. This is the chromatin immunoprecipitate or 'Native ChIP' sample.
LM-PCR amplification of the native ChIP sample to obtain a sufficient quantity for further hybridization on microarrays.

tag: Sex

value: Unknown


title: Blood_Control_Exp_2


title: Tet1 KD + Nanog overexpression (OE), rep3

hyb_protocol: 15 ug of fragmented cRNA was then added to a hybridization cocktail (0.05 mg/ml fragmented cRNA, 50 pM control oligonucleotide B2, BioB, BioC, BioD, and cre hybridization controls, 0.1 mg/ml herring sperm DNA, 0.5 mg/ml acetylated BSA, 100 mM MES, 1 M [Na+], 20 mM EDTA, 0.01% Tween 20). 10 ug of cRNA were used for hybridization to Affymetrix GeneChip Mouse Genome 430A 2.0 Array. Hybridization was carried out at 45°C for 16 hours.

scan_protocol: The array was then washed and stained with R-phycoerythrin streptavidin before scanning. Washing, scanning and basic analysis was carried out using Affymetrix GeneChip Microarray Suite 5.0 software.

data_processing: Raw data (.cel files) were normalized using the RMA method in affy (R/Bioconductor).

source_name: Tet1 KD + Nanog OE mouse ES cells

label_protocol: Biotinylated cRNA was then generated from the cDNA reaction using the BioArray High Yield RNA Transcript Kit. The cRNA was then fragmented in fragmentation buffer (40 mM Tris-acetate, pH8.1, 100 mM KOAc and 150 mM MgOAc) at 94°C for 35 minutes before microarray hybridization.

growth_protocol: Mouse E14Tg2A ES cells were maintained on gelatin-coated dishes in Glasgow Minimum Essential medium (GMEM; GIBCO), supplemented with 15% heat-inactivated fetal bovine serum, 55 mM b-mercaptoethanol (GIBCO),        2mM L-glutamine, 0.1mM MEM non-essential amino acid, 5,000 units/ml penicillin/streptomycin and 1,000 units/ml of ESGRO (Chemicon) under feeder-free conditions.

extract_protocol: For expression microarray analysis comparing control KD, Tet1 KD and Nanog rescued Tet1 KD mouse ES cells, 2 µg of total RNA purified from GFP sorted cells were reverse-transcribed into cDNA with a T7-(dT)24 primer from a custom kit (Life Technologies).

treatment_protocol: For Tet1 knockdown (KD), mouse ES cells were infected with lentiviruses expressing both the GFP reporter and short-hairpin RNA (shRNA) specific for Tet1. All analyses were performed using  Tet1-depleted ES cells that were purified on the basis of GFP fluorescence by FACS 8 days after lentiviral transduction. Lentiviruses expressing GFP alone was used as a control. Short-hairpin RNA (shRNA) sequences were cloned into pTY vector under the control of the U6 promoter. To rescue Tet1 knockdown with Nanog, complementary DNA of Nanog was placed downstream of the puromycin-resistant gene and foot-and-mouth disease virus 2A segment.

tag: cell type

value: mouse ES cells

tag: knockdown

tag: overexpression

value: Nanog


title: Tet1 KD + Nanog overexpression (OE), rep4


title: DMSO 3

description: This is a control embryo treated with DMSO from 50% epiboly until 24hpf

source_name: 24hpf embryos treated with DMSO

tag: genotype/variation

value: treated with DMSO


title: L2_PolII_1_1 extraction1_array1

tag: developmental stage


title: Blood_Control_Exp_3

tag: age

tag: gender

value: control


title: DMSO 2


title: Tet1 KD + Nanog overexpression (OE), rep1


title: A375 LEF25 2

description: A375 cells treated with Leflunomide 25uM for 3 days

hyb_protocol: Standard Illumina hybridization protocol

scan_protocol: Standard Illumina scanning protocol

data_processing: The data were normalised using average normalisation with Illumina Genome Studio

source_name: Cancer cells in DMEM/10% FBS

label_protocol: Biotinylated cRNA were prepared with the Ambion MessageAmp kit for Illumina arrays

growth_protocol: A375 cells were grown in DMEM/10% FBS until confluent.  Cells were then split to 100,000 cells/T75 flask and grown for 3 days in the presence of DMSO or leflunomide 25uM

extract_protocol: RNA was extracted with Trizol reagent, followed by clean-up and DNase I treatment with QIAGEN RNeasy mini kit in accordance with the prescribed protocol provided with the kit. Quality control was performed with Agilent Bioanalyser.

treatment_protocol: DMSO or leflunomide treatment for 3 days

value: Melanoma cell line

tag: cell line

value: leflunomide treatment for 3 days


title: Tet1 KD + Nanog overexpression (OE), rep2


title: Tet1 KD, rep2

source_name: Tet1 KD mouse ES cells


title: Con KDvsTet1 KD ES cells, Ezh2 ChIP-chip (1/4)

description: IP/IP

source_name: Tet1 KD ES cells, immunoprecipitated DNA by Ezh2 antibodies

value: Mouse ES cells

tag: antibody

value: Ezh2 antibody

tag: antibody manufacturer

value: Active Motif

tag: antibody catalog number

tag: antibody catalog #

value: 39103 (already discontinued by the vendor)

source_name: Con KD ES cells, immunoprecipitated DNA by Ezh2 antibodies

tag: sample type


title: DMSO 1

value: Tu

tag: tissue

value: embryo


title: Tet1 KD, rep3


title: A375 LEF25 1

tag: treatment group


title: L2_PolII_1_2 extraction2_array1

tag: strain

tag: genotype


title: mitf-BRAFV600E;p53-/- skin 6

value: Abnormal skin


title: Tet1 KD, rep4

tag: cell type


title: A375 LEF25 3


title: Tet1 KD, rep1

tag: knockdown


title: Tet1 KD ES cells, 5-hydroxymethylcytosine  by hMeDIP-chip, Rb Ab  (1/4)

source_name: Tet1 KD ES cells, whole cell extract

tag: tissue origin of primary cultures

source_name: Tet1 KD ES cells, immunoprecipitated DNA by 5-hydroxymethylcytosine rabbit polyclonal antibody

tag: vendor

tag: cat#


title: A375 DMSO 2

description: A375 cells treated with DMSO for 3 days

value: DMSO treatment for 3 days


title: Con KDvsTet1 KD ES cells, DNA methylation by MeDIP-chip (1/4), exp2

description: IP/IP

source_name: Con KD ES cells, immunoprecipitated DNA by 5-methylcytosine antibody

value: 5-methylcytosine antibody

value: Eurogentec

source_name: Tet1 KD ES cells, immunoprecipitated DNA by 5-methylcytosine antibody


title: 13852255 - WT_c vs gollum_c

city: BEAUCOUZE

first: Sandra

last: Pelletier

address: 42, rue Georges Morel - BP 60057

country: France

institute: INRA

description: Elucidate the role of Gollum gene in the plant development by a RNAi approach

hyb_protocol: gollum_c Cy5 / WT_c Cy3 : 30pmol. (Hybridization_Protocol.txt) Hybridization Protocol: CATMA slides (Corning Microarray Technology, CORNING) are pretreated in the prehybridisation solution (1 % BSA, 0.1 SDS, 5X SSC ) at 42°C for 60 min. They are dipped a couple of times in distilled water at room temperature, then in isopropanol and dried immediately by compressed nitrogen stream. Slides were placed in Corning hybridization chambers with a 25x60 lifterslip and 10ul of distilled water for each groove. The target was diluted to a final volume of 60 µL as follows 15µl of purified, labeled cDNA, 15 µl of 4X Hybridization Buffer ( 20X SDS, 0.4 % SDS), 30 µL formamide. The target mixture is heated for 3 min at 95°C, put on ice for 30 sec and centrifuged to remove dust for 1 min. The target mixture was put on the chip as quickly as possible. The microarray is sealed in a chamber and submerged in a 42°C water bath for approximately 16 h. The microarray is washed for 4 min in 1xSSC, 0.2% SDS (42°C); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC (RT); dipped a few times in distilled water and dried immediately by compressed nitrogen stream.

scan_protocol: GenePix Pro 3.0, Cy3:pmt voltage 532nm,480V,laser power 35%, Cy5:635nm,pmt voltage 480V,laser power 30%

data_processing: The raw data comprised the logarithm of median feature pixel intensity at wavelength 635 nm (red) and 532 nm (green). No background was subtracted. An array-by-array normalization was performed to remove systematic biases. First, we excluded spots that were considered badly formed features by the experimenter (Flags=-100). Then we performed a global intensity-dependent normalization using the loess procedure (see Yang et al., 2002) to correct the dye bias. Finally, on each block, the log-ratio median is subtracted from each value of the log-ratio of the block to correct a print-tip effect on each metablock.

label_protocol: labelling Cy3 and Cy5 direct, amplification=yes, aRNA 5 ug. Labelling protocol: 5 µg of aRNA (8µl) mixed with 2 µl of random nonamers 1µg/µl and 0.5 µl of RNAse Out 40 U/µl, denatured 10 min at 70 °C and chilled on ice. The followig components were added to the sample 4 µl of first strand buffer 5 X, 1 µl of 10 mM dNTP/2mM dCTP, 2 µl of 0.1 M DTT, 1.5 µl of Cy3-dCTP or Cy5-dCTP (Amersham Pharmacia Biotech, 25nmol tube, PA5502), 1 µl of SuperScript II RT 200U/µl. Then it was incubated at 42°C for 2.5 hours and chilled on ice. The sample was denaturated by adding 2 µl of NaOH 2.5M and incubated at 37 °C for exactly 15 min, then was added 10 µl of 2M MOPS and put on ice. The dyes were purified with the QIAquick PCR Purification Kit (QIAGEN).

growth_protocol: caulin leaf - media: soil hygrometry: 0.6 temperature: 22°C light: 16h/24h at 250µmol.m2.s-1

extract_protocol: gollum_c:10ug. (Qiagen_RNeasy.pdf)

treatment_protocol: no treatment

tag: arabidopsis thaliana (columbia)  mutant (gollum) - harvest date

extract_protocol: WT_c:10ug. (Qiagen_RNeasy.pdf)

tag: arabidopsis thaliana (columbia)  - harvest date


title: MCF-7_6hr_100nM-4-OHT_4

description:
CH1 raw data file: 1_Exiqon_13973534_S01_Cropped.txt
CH2 raw data file: 0_Exiqon_13973534_S01_Cropped.txt

source_name: Breast Cancer Cells treated with EtOH, E2, or 4-OHT

extract_protocol: RNA was extracted using miRVANA miRNA isolation kit from Ambion

tag: gender

value: female

tag: cell line

value: MCF-7 breast cancer cell line

tag: tamoxifen sensitivity

value: sensitive

tag: agent

source_name: Exiqon Common Reference Pool

tag: reference

value: Exiqon Common Reference Pool


title: MCF-7_6hr_10nM-E2_4

description:
CH1 raw data file: 1_Exiqon_13973531_S01_Cropped.txt
CH2 raw data file: 0_Exiqon_13973531_S01_Cropped.txt


title: E0-4_H3K4Me3_1_3 extraction3_array1

description:
channel ch1 is ChIP DNA; Antibody information listed below: official name: H3K4me3;target name: H3K4me3;host: Rabbit;antigen: Synthetic peptide conjugated to KLH derived from within residues 1 - 100 of Human Histone H3 tri methylated at K4.;clonal: Polyclonal;purified: Affinity;company: Abcam;catalog: ab8580;short description: Histone H3 (tri methyl K4) antibody - ChIP Grade;
channel ch2 is negative control for ChIP; Antibody information listed below: official name: No Antibody Control;target name: Not Applicable;host: None-Control;antigen: NONE;clonal: None-Control;purified: None-Control;company: None-Control;catalog: Not Applicable;reference: Not Applicable;short description: Specific Antibodies were omitted from this immunoprecipitation reaction (as a negative control).;

data_processing: Agilent ChIP data processing:KW:1 protocol. We utilized Agilent 244 K array sets for carrying out ChIP-chip experiments. The Drosophila melanogaster genome is tiled using 3 array sets with mean probe resolution of ~160 base pairs. We preprocessed microarray data to normalize it and reduce experimental noise as described in Qi et al. (Qi et al., Nature biotechnology 2006). In short, the raw intensities from each channel was divided by the median intensity from that channel before computing a ratio to arrive at a median adjusted ratio. We further subtracted average value of negative control probes on the array and then added one as a regularization parameter. For factors that mark regions (e.g. H3K27me3), the data was quantile normalized, replicates were merged and HMM based segmentation was carried out.

value: Y cn bw sp

tag: developmental stage

tag: Sex

value: Unknown


title: 13852254 - gollum_c vs WT_c

hyb_protocol: WT_c Cy5 / gollum_c Cy3 : 30pmol. (Hybridization_Protocol.txt) Hybridization Protocol: CATMA slides (Corning Microarray Technology, CORNING) are pretreated in the prehybridisation solution (1 % BSA, 0.1 SDS, 5X SSC ) at 42°C for 60 min. They are dipped a couple of times in distilled water at room temperature, then in isopropanol and dried immediately by compressed nitrogen stream. Slides were placed in Corning hybridization chambers with a 25x60 lifterslip and 10ul of distilled water for each groove. The target was diluted to a final volume of 60 µL as follows 15µl of purified, labeled cDNA, 15 µl of 4X Hybridization Buffer ( 20X SDS, 0.4 % SDS), 30 µL formamide. The target mixture is heated for 3 min at 95°C, put on ice for 30 sec and centrifuged to remove dust for 1 min. The target mixture was put on the chip as quickly as possible. The microarray is sealed in a chamber and submerged in a 42°C water bath for approximately 16 h. The microarray is washed for 4 min in 1xSSC, 0.2% SDS (42°C); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC (RT); dipped a few times in distilled water and dried immediately by compressed nitrogen stream.


title: A375 DMSO 3

description: A375 cells treated with DMSO for 3 days

tag: tissue

value: Melanoma cell line

tag: treatment group


title: MsmPS48h_HL2

city: St Louis

first: Federico

middle: E

last: Rey

state: MO

address: 4444 Forest Park Blvd

department: Pathology

institute: Washington University

description: Gene expression data from cultures in log phase growth with 2.8mM formate

hyb_protocol: Following fragmentation, 5 ug of cRNA were hybridized for 16 hr at 45C on GeneChip BacEubMsa custom Genome Array. GeneChips were washed and stained in the Affymetrix Fluidics Station 400.

scan_protocol: GeneChips were scanned using the Hewlett-Packard GeneArray Scanner G2500A.

data_processing: The data were analyzed with Microarray Suite version 5.0 (MAS 5.0) using Affymetrix default analysis settings and global scaling as normalization method. The trimmed mean target intensity of each array was arbitrarily set to 100.

organism: Methanobrevibacter smithii ATCC 35061

source_name: Methanobrevibacter smithii PS in vitro culture

label_protocol: Enzo Biochemicals Bioarray terminal labeling kit

growth_protocol: 48 hour cultures were grown in modified MBC media under 30psi of 80% hydrogen and 20% CO2, replenished every 6 hours

extract_protocol: acid-phenol chloroform; mega clear kit

treatment_protocol: Aliquots were combined with an equal volume of RNA-Protect (QIAGEN), incubated for 5 min at room temperature, and then centrifuged to pellet.

value: 2.8mM formate

tag: time

value: 48 h post innoculation


title: MCF-7_6hr_10nM-E2_3

description:
CH1 raw data file: 1_Exiqon_13973530_S01_Cropped.txt
CH2 raw data file: 0_Exiqon_13973530_S01_Cropped.txt


title: Control KD, rep4

source_name: control KD mouse ES cells

tag: strain

tag: cell type

value: mouse ES cells

value: control

tag: overexpression


title: E0-4_H3K4Me3_1_2 extraction2_array1

tag: genotype


title: Control KD, rep3

tag: knockdown


title: MCF-7_6hr_10nM-E2_2

description:
CH1 raw data file: 1_Exiqon_13973529_S01_Cropped.txt
CH2 raw data file: 0_Exiqon_13973529_S01_Cropped.txt


title: A375 DMSO 1

description: A375 cells treated with DMSO for 3 days


title: Control KD, rep1


title: Control KD, rep2


title: E0-4_H3K4Me3_1_3 extraction3_array3


title: MCF-7_6hr_10nM-E2_1

description:
CH1 raw data file: 1_Exiqon_13973528_S01_Cropped.txt
CH2 raw data file: 0_Exiqon_13973528_S01_Cropped.txt


title: MsmPS48h_HL1


title: MsmPS48h_HH1

description: Gene expression data from cultures in log phase growth with 44.1mM formate

value: 44.1mM formate


title: MsmPS48h_HH2


title: UMS84_WT_MMnit_ara_12h_1

city: Karlsruhe

first: Jörg

middle: Thomas

last: Kämper

address: Hertzstrasse 16, Geb. 06.40

department: Department for Applied Biosciences

country: Germany

institute: Karlsruhe Institute of Technology

description: Effect of ectopic clp1-expression on b-dependent gene expression 12h after nar1P- and crg1P- induction

hyb_protocol: EukGE-WS2 protocol on a GeneChip Fluidics Station 400

scan_protocol: Affymetrix GCS2500

label: biotin/phycoerythrin

organism: Mycosarcoma maydis

source_name: U. maydis strain UMS84 grown for 12h in array medium/nitrate/arabinose

label_protocol: Target preparation was performed according to the standard Affymetrix protocol using the Enzo BioArray RNA transcript labelling kit, except for a reaction temperature of 50°C for first-strand cDNA synthesis.

growth_protocol: Strain UMS84 was grown on a rotary shaker (200 rpm) to an OD600 of 0.4 to 0.6 at 28°C in liquid array medium: 6.25% (w/v) salt solution, 30 mM L-glutamine, 1% (w/v) glucose, pH 7.0 (filter sterilized). bE1 and bW2 gene expression was induced for 12 hours in liquid array medium containing 3 g/l nitrate instead of glutamine as a nitrogen source and 1% arabinose instead of glucose as a carbon source.

extract_protocol: Total RNA was extracted using the TRIZOL-reagent (Invitrogen) according to the manufacturers instructions. RNA samples were further column purified (Quiagen RNeasy) and the quality checked using a Bioanalyzer with a RNA 6000 Nano LabChip kit (Agilent).

value: 12h array medium/nitrate/arabinose

value: UMS84 is a FB2 (a2 b2) derivative in which the induction of the bE1 and bW2 genes is under the control of the nitrate-inducible nar1 promoter and the expression of the clp1 gene is under the control of the arabinose-inducible crg1 promoter.


title: UMS84_WT_MMnit_ara_12h_2


title: E0-4_H3K4Me3_1_2 extraction2_array3

extract_protocol:
Collection of Drosophila embryos at time-point 0-4h of development for further ChIP experiments
Formaldehyde Cross-linking of Chromatin from any Drosophila tissue (Embryos, Larvae, Pupae, Adult flies).1. Homogenize the material (about 150-200 mg of embryos, pupae, adµlts, larvae, sufficient for four-five independent immuno-precipitations) in 5 ml of buffer A1 + 1.8% formaldehyde (290 µl of 37% solution) at room temperature using first a Potter homogenizer and then a Douncer with type A pestle (3 strokes). Wait 15' (total time starting from beginning of homogenization). Add glycin solution to 225 mM (540 µl of 2.5 M solution for 6 ml of cross-linked mixture), mix and incubate 5'. Put on ice. Keep on ice unless specified.2. Transfer the homogenate to a 15 ml tube. Centrifuge 5 min, at 4000 g at 4°C. Discard supernatant. Add 3 ml of buffer A1, resuspend pellet and spin down the same way. Repeat washing step 3 times.3. Wash once in 3 ml of lysis buffer without SDS. Spin down, 5 min at 4000g.4. Resuspend cross-linked material in 0.5 ml of lysis buffer and add SDS to 0.1% (2.5 µl) and N-lauroylsarcosine to 0.5% (12.5 µl). Incubate 10 min at 4°C in a rotating wheel.5. Sonicate in Polypropylene 15ml Falcon tubes according to .Diagenode instructions. The settings are: High power, 15 minutes, 30 seconds off, 30 seconds on. The water bath shoµld be kept cold either in a cold room or by a cold water pump.6. Rotate 10'. Transfer to eppendorf tubes and centrifuge 5', room temperature at maximum speed. Transfer supernatant to the new tube. Add another 0.5 ml of lysis buffer to the pellet, rotate for 10 min. Repeat centrifugation and combine supernatants. Centrifuge the combined supernatant 2x10 min at max speed. The supernatant from this stage will be referred as chromatin extract. Chromatin can be stored at -80°C for several months at this stage. Add sodium azide to 0.02% for storage.7. [Optional : Purification of chromatin. Put chromatin extract in Centricon YM-100 column (4212), centrifuge 3x40' (or more) at 1000 g while adding lysis buffer. At least 3 vol. of lysis buffer should pass through column. Bring the final volume of chromatin extract to 1 ml by lysis buffer + protease inhibitors.][edit]
Solutions and MaterialsLysis buffer: (see above)5mg/ml RNaseA (DNase free)PAS suspension: 100 mg of CL-4B (Amersham, 17-0780-01) PAS shoµld be resuspended in 1 ml of lysis buffer + protease inhibitors + 0.1 mg/ml BSA for 50 %v suspension. Wash in lysis buffer 2-3 times, overall equilibration time 1h. Store up to one week at 4 C.TE: 10 mM Tris-HCl pH 8.0, 1 mM EDTAElution buffer1: 10 mM EDTA, 1% SDS, 50 mM Tris-Cl pH 8.Elution buffer2: TE+0.67% SDS.LiCL 4M Protocol 1. To an amount of chromatin corresponding to 150 mg of biological material, suspended in a final volume of chromatin extract of 1 ml (in lysis buffer + protease inhibitors), add 100 µl of PAS suspension for preincubation. Incubate several hours or overnight at 4°C, then remove PAS. Crosslinked chromatin at this stage can be stored several days at 4°C or frozen at -70°C.2. [Optional : Check for amount of DNA. From the 1 ml solution above, take a 100 µl aliquot, Add proteinase K up to 100 ug/ml and SDS to 1%, incubate 6h at 60°C, then 20 min at 70°C, add RNAse to 50 ug/ml and incubate additional 2h at 37°C. Phenol-chlorophorm extract, ethanol precipitate. Run on an agarose gel to check amount and size of DNA.]3. Separate chromatin sample into 4x250 µl aliquots (one aliquot is enough for one IP but the amount may be decreased further). Immunoprecipitate chromatin by adding the antibody (Ab) of interest (amount of Ab shoµld be determined empirically, usually the same concentration than for a successfµl IF experiement). Keep a control sample without Ab IP, named "Mock".4. Incubate 4 h at 4°C on a head to head rotating wheel, then add 50 µl of PAS suspension and incubate 4 h or overnight. Spin down PAS and proceed to washes.5. Wash PAS 4x with lysis buffer, followed by 2x with TE (without protease inhibitors). Each wash is for 5 min at 4°C, using 1 ml of solution.6. Elution of precipitated material. Spin down PAS. Add 100 µl of elution buffer, mix and incubate 10 min at 65°C. Spin down PAS and transfer supernatant to new tube. Add 150 µl of elution buffer 2 to PAS, mix, centrifuge at fµll speed and transfer eluate to a tube together with the eluate from the first centrifugation. The combined material is the "chromatin precipitate" (approx. 250 µl).7. Incubate precipitate 6 h (or overnight) at 65 C to reverse cross-links. Add 250 µl of Proteinase K solution, incubate at 50°C 2-3 h.8. Add 55 µl of 4M LiCl and 500 µl of phenol-chlorophorm. Mix and microfuge at fµll speed at RT. Transfer aqueous phase to a new tube and precipitate with 1 ml of 100% ethanol. Wash with 750 µl of 70% ethanol. Spin down and dry precipitate.9. Dissolve in 25 µl of water. This is the chromatin immunoprecipitate or 'Native ChIP' sample.
LM-PCR amplification of the native ChIP sample to obtain a sufficient quantity for further hybridization on microarrays.


title: Tet1 KD ES cells, DNA methylation by MeDIP-chip  (1/4)

description: IP/input

value: Mouse ES cells

tag: sample type

value: input DNA

value: immunoprecipitated DNA by 5-methylcytosine antibody

tag: antibody

tag: antibody manufacturer

tag: antibody catalog #


title: MCF-7_6hr_EtOH_4

description:
CH1 raw data file: 1_Exiqon_13973532_S01_Cropped.txt
CH2 raw data file: 0_Exiqon_13973532_S01_Cropped.txt

tag: gender

value: female

tag: cell line

tag: tamoxifen sensitivity

value: EtOH


title: E0-4_H3K4Me3_1_1 extraction1_array1

tag: developmental stage


title: Con KD ES cells, 5-hydroxymethylcytosine  by hMeDIP-chip, Rb Ab  (1/4)

source_name: Con KD ES cells, whole cell extract

tag: tissue origin of primary cultures

source_name: Con KD ES cells, immunoprecipitated DNA by 5-hydroxymethylcytosine rabbit polyclonal antibody

tag: vendor

value: Active Motif

tag: cat#


title: E0-4_H3K4Me3_1_1 extraction1_array3

tag: Sex


title: UMS84_WT_MMnit_ara_5h_2

description: Effect of ectopic clp1-expression on b-dependent gene expression 5h after nar1P- and crg1P- induction

source_name: U. maydis strain UMS84 grown for 5h in array medium/nitrate/arabinose

growth_protocol: Strain UMS84 was grown on a rotary shaker (200 rpm) to an OD600 of 0.4 to 0.6 at 28°C in liquid array medium: 6.25% (w/v) salt solution, 30 mM L-glutamine, 1% (w/v) glucose, pH 7.0 (filter sterilized). bE1 and bW2 gene expression was induced for 5 hours in liquid array medium containing 3 g/l nitrate instead of glutamine as a nitrogen source and 1% arabinose instead of glucose as a carbon source.

tag: time

value: 5h array medium/nitrate/arabinose

tag: strain


title: MCF-7_6hr_EtOH_3

description:
CH1 raw data file: 1_Exiqon_13973526_S01_Cropped.txt
CH2 raw data file: 0_Exiqon_13973526_S01_Cropped.txt

value: MCF-7 breast cancer cell line

value: sensitive

tag: agent

tag: reference

value: Exiqon Common Reference Pool


title: E0-4_H3K4Me3_1_3 extraction3_array2

value: Y cn bw sp

tag: genotype

value: Unknown


title: AB33_WT_MMnit_ara_12h_2

description: b-dependent gene expression 12h after nar1P-induction

source_name: U. maydis strain AB33 grown for 12h in array medium/nitrate/arabinose

growth_protocol: Strain AB33 was grown on a rotary shaker (200 rpm) to an OD600 of 0.4 to 0.6 at 28°C in liquid array medium: 6.25% (w/v) salt solution, 30 mM L-glutamine, 1% (w/v) glucose, pH 7.0 (filter sterilized). bE1 and bW2 gene expression was induced for 12 hours in liquid array medium containing 3 g/l nitrate instead of glutamine as a nitrogen source and 1% arabinose instead of glucose as a carbon source.

value: AB33 is a FB2 (a2 b2) derivative in which the induction of the bE1 and bW2 genes is under the control of the nitrate-inducible nar1 promoter.


title: E0-4_H3K4Me3_1_2 extraction2_array2


title: Con KD ES cells, DNA methylation by MeDIP-chip  (1/4)

description: IP/input

tag: cell type

tag: knockdown

value: control

tag: sample type

value: 5-methylcytosine antibody

value: Eurogentec


title: LY2_6hr_100nM-4-OHT_3

description:
CH1 raw data file: 1_Exiqon_13973523_S01_Cropped.txt
CH2 raw data file: 0_Exiqon_13973523_S01_Cropped.txt

value: LY2 breast cancer cell line

value: resistant


title: LY2_6hr_100nM-4-OHT_4

description:
CH1 raw data file: 1_Exiqon_13973524_S01_Cropped.txt
CH2 raw data file: 0_Exiqon_13973524_S01_Cropped.txt


title: UMS84_WT_MMnit_ara_5h_1

value: UMS84 is a FB2 (a2 b2) derivative in which the induction of the bE1 and bW2 genes is under the control of the nitrate-inducible nar1 promoter and the expression of the clp1 gene is under the control of the arabinose-inducible crg1 promoter.


title: E0-4_H3K4Me3_1_1 extraction1_array2


title: Con KDvsTet1 KD ES cells, DNA methylation by MeDIP-chip (1/4), exp1

description: IP/IP

value: Mouse ES cells

tag: antibody

tag: antibody manufacturer

tag: antibody catalog #

value: immunoprecipitated DNA by 5-methylcytosine antibody


title: MCF-7_6hr_EtOH_2

description:
CH1 raw data file: 1_Exiqon_13973525_S01_Cropped.txt
CH2 raw data file: 0_Exiqon_13973525_S01_Cropped.txt

tag: gender

value: female

tag: cell line

tag: tamoxifen sensitivity

value: EtOH


title: LY2_6hr_EtOH_4

description:
CH1 raw data file: 1_Exiqon_13973522_S01_Cropped.txt
CH2 raw data file: 0_Exiqon_13973522_S01_Cropped.txt


title: AB33_WT_MMnit_ara_12h_1

value: 12h array medium/nitrate/arabinose


title: LY2_6hr_EtOH_3

description:
CH1 raw data file: 1_Exiqon_13973521_S01_Cropped.txt
CH2 raw data file: 0_Exiqon_13973521_S01_Cropped.txt


title: AB33_WT_MMnit_ara_5h_1

description: b-dependent gene expression 5h after nar1P-induction

source_name: U. maydis strain AB33 grown for 5h in array medium/nitrate/arabinose

growth_protocol: Strain AB33 was grown on a rotary shaker (200 rpm) to an OD600 of 0.4 to 0.6 at 28°C in liquid array medium: 6.25% (w/v) salt solution, 30 mM L-glutamine, 1% (w/v) glucose, pH 7.0 (filter sterilized). bE1 and bW2 gene expression was induced for 5 hours in liquid array medium containing 3 g/l nitrate instead of glutamine as a nitrogen source and 1% arabinose instead of glucose as a carbon source.

value: 5h array medium/nitrate/arabinose


title: AB33_WT_MMnit_ara_5h_2


title: agr mutant strain 6911 rep 2

status: Public on Mar 29 2011

city: Hamilton

first: Dan

last: Sturdevant

state: MT

address: 903 S 4th street

department: NIAID

institute: NIH

description: Gene expression data from_agr mutant strain 6911 rep 2

hyb_protocol: Following fragmentation, 10 ug of cRNA were hybridized for 16 hr at 40C on our custom Array. GeneChips were washed and stained in the Affymetrix Fluidics Station 450.

scan_protocol: GeneChips were scanned using the Affymetrix GeneChip Scanner 7GPlus.

data_processing: The data were analyzed with GCOS 1.4 using Affymetrix default analysis with a scale filter for Ft genes setting and global scaling as normalization method. The trimmed mean target intensity of each array was arbitrarily set to 500.

organism: Staphylococcus aureus

source_name: agr mutant strain 6911 rep 2

label_protocol: Biotinylated cRNA were prepared according to the standard Affymetrix protocol from 10 ug total RNA (Expression Analysis Technical Manual, 2001, Affymetrix).

growth_protocol: standard broth cultures to log phase

extract_protocol: Total RNA was isolated by use of an RNeasy Mini Kit (Qiagen) recommended by a standard protocol.

tag: genotype/variation

value: agr mutant


title: MCF-7_6hr_100nM-4-OHT_2

description:
CH1 raw data file: 1_Exiqon_13973527_S01_Cropped.txt
CH2 raw data file: 0_Exiqon_13973527_S01_Cropped.txt

value: MCF-7 breast cancer cell line

value: sensitive

value: Exiqon Common Reference Pool


title: agr mutant strain LAC rep 3

description: Gene expression data from_agr mutant strain LAC rep 3

source_name: agr mutant strain LAC rep 3

value: LACagr


title: wild type strain 252 rep 3

description: Gene expression data from_wild type strain 252 rep 3

source_name: wild type strain 252 rep 3

value: wild type


title: agr mutant strain LAC rep 2

description: Gene expression data from_agr mutant strain LAC rep 2

source_name: agr mutant strain LAC rep 2


title: agr mutant strain LAC rep 1

description: Gene expression data from_agr mutant strain LAC rep 1

source_name: agr mutant strain LAC rep 1


title: agr mutant strain 6911 rep 3

description: Gene expression data from_agr mutant strain 6911 rep 3

source_name: agr mutant strain 6911 rep 3


title: agr mutant strain 252 rep 2

description: Gene expression data from_agr mutant strain 252 rep 2

source_name: agr mutant strain 252 rep 2


title: wild type strain LAC rep 2

description: Gene expression data from_wild type strain LAC rep 2

source_name: wild type strain LAC rep 2

value: LAC


title: agr mutant strain 6911 rep 1

description: Gene expression data from_agr mutant strain 6911 rep 1

source_name: agr mutant strain 6911 rep 1


title: agr mutant strain 252 rep 1

description: Gene expression data from_agr mutant strain 252 rep 1

source_name: agr mutant strain 252 rep 1