title: AdultFemale_H3K9Me3_2 extraction2_array3

extract_protocol:
Collection of Drosophila adult females 2-3 days old 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.


title: E0-12h_HDAC1-500_4 extraction4_array1

status: Public on Dec 10 2010

type: genomic

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 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: 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. In MAT, all Control and IP samples corresponding to the same experiment are analyzed together (and not separately).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.

label: biotin

molecule: genomic DNA

organism: Drosophila melanogaster

label_protocol: Attached as supplementary file

extract_protocol:
1. y; bw cn sp flies are cultivated in cages with apple juice agar plates covered with yeast powder.   2. For a 0-12 hours egg laying, new plates are added after a 2hours pre-egglaying at 9pm and flies are allowed to lay eggs for 12 hours.   3. Embryos are collected at 8.45 am using a filter mesh and a brush. They are rinsed with EWB and cross-linked at 9am 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.

tag: strain

tag: developmental stage

value: Embryo 0-12h

tag: genotype


title: E0-12h_HDAC1-500_1 extraction1_array1

description: channel ch1 is ChIP DNA; Antibody information listed below: official name: HDAC1-500;target name: HDAC1;host: Rabbit;antigen: CRKRPRLDKDTNSNK;clonal: Polyclonal;purified: Crude;company: Other;catalog: Novartis;short description: This is an antibody raised against peptides for different Drosophila HDACs by Novartis in collaboration with Kevin White.;


title: 6475C3_PocR(3)

type: RNA

city: Houston

first: Delphine

last: Saulnier

state: TX

address: Feigin Center, Suite 830, 1102 Bates Avenue

department: Pathology

country: USA

institute: Baylor College of Medicine

description: Biological replicate 3 of 3. Dye swap 1

hyb_protocol: For hybridization, yeast tRNA and salmon testes DNA were added to labeled cDNA along with a formamide hybridization buffer. Samples were applied to slides, and hybridization was carried out at 42 degrees Celsius in Corning microarray hybridization chambers overnight.  Slides were then washed sequentially for four minutes in each of the following buffers: 1X SSC and 0.2% SDS, 0.1X SSC and 0.2% SDS, and 0.1X SSC

scan_protocol: Arrays were scanned on a Axon instruments GenePix 4000B scanner.  Images were processed using GenePix Pro software (version 4.1)  and hybridization was carried out at 42 degrees Celsius in Corning microarray hybridization chambers overnight.  Slides were then washed sequentially for four minutes in each of the following buffers: 1X SSC and 0.2% SDS, 0.1X SSC and 0.2% SDS, and 0.1X SSC

data_processing: Data normalization between and within array using LOWESS

molecule: total RNA

organism: Limosilactobacillus reuteri

source_name: Bacteria

label_protocol: 2 ug of total RNA were primed with 2.5 ug of random hexamers (Qiagen) at 70 degrees Celsius for 10 minutes. Reverse transcription was then carried out using Superscript III (Invitrogen) with 500 uM each dATP, dCTP, and dGTP, 300 uM dTTP, and 200 uM aminoallyl dUTP. After reverse transcription, RNA was removed by hydrolysis and cDNA was cleaned. cDNA was then labeled using CyDye Post-labeling reactive dye packs (Amersham).

growth_protocol: Bacterial cells were grown for 24h in semi-defined medium (LDM III) at 37C in anaerobic conditions

extract_protocol: At the specified times, 5 ml of bacterial were collected and mixed with an equal part of ice-cold methanol. Cell pellets were centrifuged, washed, and resuspended in 0.25 units/ul mutanolysin. After incubation at 37 degrees Celsius for 20 minutes, total RNA was collected according to the manufacturer's instructions with the Qiagen RNeasy Kit.

value: L. reuteri 6475

value: L. reuteri 6475 PocR insertant


title: 6475C3_PocR(2)

description: Biological replicate 2 of 3. Dye swap 1


title: MS121 AR H

city: Madrid

first: Manuel

middle: J

last: Del Rey

address: Avda Córdoba s/n

department: Grupo de Enfermedades Inflamatorias y Autoinmunes

country: Spain

institute: Instituto de Investigación Hospital 12 de Octubre

description:
RASF (rheumatoid arthritis synovial fibroblasts) gene expression under hypoxic conditions
US82003505_251485050865_S01_GE1-v5_10_Apr08_1_2.txt

hyb_protocol: 1.5 ug of Cy3-labelled cRNA (specific activity >10.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 250 ml containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 250 ml of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent Whole Human Genome Oligo Microarrays (G4845A) for 17 hours at 65Â°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37Â°C GE Wash buffer 2 (Agilent), then dried immediately by brief centrifugation.

scan_protocol: Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505B) using one color scan setting for 4x44k array slides (Scan Area 61x21.6 mm, Scan resolution 5um, Dye channel is set to Green and Green PMT is set to 100%).

data_processing: The scanned images were analyzed with Feature Extraction Software 10.7.3.1 (Agilent) using default parameters (protocol GE1-v1_91 and Grid: 014850_D_20070207) to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded.

organism: Homo sapiens

source_name: RASF, hypoxia, 0.5% Ox, 22h

label_protocol: Cyanine-3 (Cy3) labeled cRNA was prepared from 0.5 ug RNA using the One-Color Low RNA Input Linear Amplification PLUS kit (Agilent) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.

extract_protocol: Total RNAs from synovial fibroblasts was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. RNA quality control was performed in 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA). In all cases, RIN (RNA integrity number) was higher than or equal to 9.

treatment_protocol: Synovial tissues were obtained from 6 patients with RA at the time of knee prostetic replacement surgery and 6 sex and age matched adult donors without history of joint disease and macroscopically healthy joints either at necropsy or at elective knee arthroscopic surgery for meniscal tears. Fibroblast cultures were established by explant growth in Dulbecco’s modified Eagle’s medium supplemented with 10% heat inactivated fetal bovine serum (FBS) (Cambrex Bio Science, Verviers, Belgium). Cultures were used after passage 4 in order to avoid contamination by other cell types. Parallel subconfluent cultures were incubated for 22 hours under normoxic or hypoxic conditions in a cell incubator under a controlled (CO2/N2/O2) atmosphere.

tag: cell type

value: Synovial fibroblasts culture

tag: gender

value: Female

tag: age

tag: disease status

value: rheumatoid arthritis patient


title: AdultFemale_H3K9Me3_1 extraction1_array3

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 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: Adult Female

tag: Sex


title: MS96 SANO N

description:
HSF (healthy synovial fibroblasts) gene expression under normoxic conditions
US82003505_251485050865_S01_GE1-v5_10_Apr08_1_3.txt

source_name: HSF, normoxia, 20% Ox, 22h

value: healthy donor


title: 6475C3_PocR(1)

description: Biological replicate 1 of 3. Dye swap 1


title: IgG vs.C20 2 of 2 re-hyb 101978

city: Berlin

first: Liliana

middle: H.

last: Mochmann

state: Berlin

address: Hindenburgdamm 30

department: Hematology and Oncology

country: Germany

institute: Charite

hyb_protocol: The labeled ChIP DNA was precipitated with 0.1 volume 5M NaCl and 1 volume isopropanol, and hybridized in 45 ul of buffer containing 20% formamide, 1.2 M betaine, 0.1 ug/ul herring sperm DNA and 10 ug of human COT1 DNA (Invitrogen). Arrays were hybridized in Maui hybridization stations for 16-18 h at 42C, and then washed in 42C 0.2% SDS/0.2x SSC, room temperature 0.2x SSC, and 0.05x SSC. Hybridization buffers and washes were completed using manufacturer's protocols (http://www.nimblegen.com/products/lit/lit.html)

scan_protocol: Arrays were scanned on an Axon 4000B scanner per manufacturer's protocol (http://www.nimblegen.com/products/lit/lit.html).

data_processing:
Arrays were processed using Nimblegen's standard protocol for Nimblescan 2.4 ChIP data extraction. Roche NimbleGen scales the GFF files by subtracting the bi-weight mean for the log-ratio values from each log-ratio value.
Procedure for Lowess_ratios annalysis
* take pair data from Nimblegen
* normalize them with limma package (script available from Metzler hompage
(http://watson.nci.nih.gov/twiki/bin/view/Main/MeltzerLabSoftware)
* create file with required informations for ACME sofware/Analysis (like ratio (R/G) chromosomal position etc (same as for the first analysis already done)
run program (parameters: window size 2000 bp, threshold 0,90)
calculate p-values (FDR-analysis; false discovery rate) create list with: chromosome, chromosomal position, p-value, accession number

label_protocol: 4 µg ChIP DNA was directly labeled by Klenow (New England Biolabs) random priming with Cy3 nonamers (ChIP DNA isolated from normal rabbit IgG-immunoprecipitated cells) per manufacturer's protocol (http://www.nimblegen.com/products/lit/lit.html).

extract_protocol: Jurkat 1x10E6 cells were fixed with 1% formaldehyde in culture medium for 10 min at room temperature followed by quenching with 0.125 M glycine for 5 min. The cells were washed twice with ice-cold PBS and lysed in 1 ml of lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris pH8.0) for at least 30 min on ice. The crosslinked chromatin was sheared to an average size of 500 bp by ten 30-second pulses using a BiorupterTM sonicator (Diagenode). The lysate was then centrifuged to remove cell debris, and the chromatin was quantified in a UV spectrophotometer. The chromatin (400 µg) was diluted 10-fold in dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA 16.7 mM Tris pH8.10, 167 mM NaCl) and pre-cleared with 25 µl  magnetic beads (in BSA) for 2 h at 4C. The chromatin supernatant was then incubated overnight with 5 µg of purified antibody at 4C. After incubation with protein A magnetic beads (50 µl) for 2h at 4C, the immune complexes were collected by centrifugation and washed with the following buffers each for 10 min at 4C; RIPA buffer (150 mM NaCl, 50 mM Tris pH8.0, 0.1% SDS), high-salt buffer (500 mM NaCl, 50 mM Tris pH8.0, 0.1% SDS, 1% NP40) LiCl buffer (250 mM LiCl, 50 mM Tris pH8.0, 0.5% Na deoxycholate, 1% NP40) and 2x TE (20 mM Tris pH8.0, 2 mM EDTA). The protein-DNA complexes were eluted from the beads in 450 µl elution buffer (1% SDS, 100 mM NaHCO3) at 55C for 2 h followed by the addition of proteinaseK to 500 µg/ml and overnight incubaton at 65C. Genomic DNA was isolated from the precipitated material as well as from the sheared chromatin input (1/100 of the material used for ChIP) by phenol extraction and ethanol precipitation.

tag: antibody

value: IgG

tag: cell line

value: Jurkat

tag: fraction

value: NONSPECIFIC-IgG

value: ERG C20

tag: antibody manufacturer

value: Santa Cruz Biotechnolgy

value: EXPERIMENTAL


title: E0-12h_HDAC11-494_6 extraction6_array1

description: channel ch1 is input DNA;


title: E0-12h_HDAC11-494_5 extraction5_array1

description: channel ch1 is input DNA;


title: E0-12h_HDAC11-494_4 extraction4_array1


title: Input vs. C17/C20 2 of 2 94879

value: TOTAL, input


title: E16-20_H3K9Ac_2 extraction2_array3

extract_protocol:
Collection of Drosophila embryos at time-point 16-20h of development 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.

value: Unknown


title: E0-12h_HDAC1-500_2 extraction2_array1


title: 6475C5_PocR(1)

description: Biological replicate 1 of 3. Dye swap 2


title: AdultMale-K27Me3_3 extraction3_array2

extract_protocol:
Collection of Drosophila adult males 2-3 days old 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.

value: Adult Male

value: Male


title: E0-12h_HDAC11-494_3 extraction3_array1

value: Embryo 0-12h


title: E16-20_H3K9Ac_1 extraction1_array3

description: channel ch1 is ChIP DNA; Antibody information listed below: official name: H3K9Ac;target name: H3K9Ac;host: Rabbit;antigen: Synthetic peptide: ARTKQTARAcKSTG-C conjugated to KLH corresponding to amino acids 1-12 of Human Histone H3.;clonal: Polyclonal;purified: Affinity;company: Abcam;catalog: ab4441;short description: Histone H3 (acetyl K9) antibody - ChIP Grade;


title: E0-12h_HDAC11-494_2 extraction2_array1

description: channel ch1 is ChIP DNA; Antibody information listed below: official name: HDAC11-494;target name: HDAC11;host: Rabbit;antigen: SEEVVWVKRDDARC;clonal: Polyclonal;purified: Crude;company: Other;catalog: Novartis;short description: This is an antibody raised against peptides for different Drosophila HDACs by Novartis in collaboration with Kevin White.;


title: Male-K27Me3_2 extraction2_array2

description: channel ch1 is ChIP DNA; Antibody information listed below: official name: H3K27me3;target name: H3K27me3;host: Rabbit;antigen: KLH-conjugated synthetic 2X-branched peptide containing the sequence ?AR(me3K)SAP? in which me3K corresponds to trimethyl-lysine at residue 27 of human histone H3.;clonal: Polyclonal;purified: Affinity;company: Upstate-Millipore;catalog: 07-449;


title: 09/940 SANO N

description:
HSF (healthy synovial fibroblasts) gene expression under normoxic conditions
US82003505_251485050866_S01_GE1-v5_10_Apr08_1_1.txt


title: 09/940 SANO H

description:
HSF (healthy synovial fibroblasts) gene expression under hypoxic conditions
US82003505_251485050866_S01_GE1-v5_10_Apr08_1_2.txt

source_name: HSF, hypoxia, 0.5% Ox, 22h


title: 07/967 SANO N

description:
HSF (healthy synovial fibroblasts) gene expression under normoxic conditions
US82003505_251485050866_S01_GE1-v5_10_Apr08_1_3.txt


title: E0-12h_HDAC11-494_1 extraction1_array1


title: E16-20_H3K9Ac_3 extraction3_array3


title: Male-K27Me3_1 extraction1_array2


title: MS96 SANO H

description:
HSF (healthy synovial fibroblasts) gene expression under hypoxic conditions
US82003505_251485050865_S01_GE1-v5_10_Apr08_1_4.txt


title: IgG vs. C17/C20 2 of 2 94937


title: 07/967 SANO H

description:
HSF (healthy synovial fibroblasts) gene expression under hypoxic conditions
US82003505_251485050866_S01_GE1-v5_10_Apr08_1_4.txt


title: SAMPLE012 null

city: Austin

first: Robert

last: Cabrera

address: 1400 Barbara Jordan Blvd

institute: DPRI

description:
36227 oligo probes, CodeLink Mouse Whole Genome Bioarray
T00341113

hyb_protocol: Hybridization was according Applied Microarrays Specifications.  Detection of biotin label used Alexa-Fluro

scan_protocol: Agilent DNA Microarray Scanner

data_processing: Initial feature extraction from the images was performed using CodeLink Expression Analysis software v4.0. Global-Median-Normalization.

label: biotin-UTP

organism: Mus musculus

source_name: E9.5 anterior neural tube

label_protocol: RNA that has passed the Agilent Technologies 2100 Bioanalyzer quality control test was used to generate biotin-labeled cRNA via a modified Eberwine RNA amplification protocol

growth_protocol: Embryos were removed from pregnant dams at embryonic day 9.5.  At E 9.5, the average embryo was “C” shaped and had 21.3 pairs of somites and neurul tube closure was complete with the exception of the posterior neuropore.

extract_protocol: The total RNA was extracted from collected posterior parts of E9.5 embryos using PicoPure RNA isolation Kit (Arcturus).

treatment_protocol: The posterior part of the embryo (immediately below the forelimb pit) was cut off and transferred separately to a 600 uL plastic tube containing 15 uL of RNAlater (Ambion).

value: LMBc

tag: genotype/variation

value: Splotch null

tag: tissue

value: posterior neural tube

value: embryo day 9.5


title: SAMPLE011 null

description:
36227 oligo probes, CodeLink Mouse Whole Genome Bioarray
T00341112


title: SAMPLE010 wildtype

description:
36227 oligo probes, CodeLink Mouse Whole Genome Bioarray
T00341110

value: WT


title: WT dicamba treated_biological rep 1

city: Wembley

first: Cynthia

last: Gleason

state: WA

address: Private Bag #5

department: Plant Industry

country: Australia

institute: CSIRO

description: Gene expression data from WT seedlings that contain the GSTF8::LUC reporter treated with dicamba

hyb_protocol: cRNA was hybridized to the GeneChip® Arabidopsis ATH1 Genome Array using a Genechip instrument system with scanner 3000 7G and autoloader for expression array processing and analysis, following manufacturer's protocol.  Labeling, hybing and scanning were

scan_protocol: Genechips were scanned Affymetrix GeneChip scanner 3000 7G

data_processing: The raw data files underwent background correction, normalization, probe specific correction, and summary value computation using the Robust Multichip Average (RMA) method in the Bioconductor package.  To find the differentially expressed genes we utilized the limma package for R to calculate the Log2 fold change, the moderate t-statistic, B value, and the adjusted p value (BenjaminiHochberg FDR) for each array probe.

label: Biotin

organism: Arabidopsis thaliana

source_name: 4 day old seedlings dicamba-treated 10 hr

label_protocol: Biotinylated cRNA were prepared according to the standard Affymetrix protocol from 2 ug total RNA (Expression Analysis Technical Manual, 2001, Affymetrix).

growth_protocol: WT (a Columbia line containing a -800GSTF8::LUC reporter) Arabidopsis seeds were surface sterilzed and sown on MS-media plates.  Plates were vernalized 3 days at 4C in dark before being transferred to controlled growth room set at 21ºCwith a 16 hour ligh

extract_protocol: RNA was extracted using the Qiagen RNeasy Mini Kit, following the manufacturer’s instructions.

treatment_protocol: Plates were flooded with water or 7 mM dicamba (Cadence 0.325 g/mole, Syngenta) for 40 minutes.  The treatments were poured off and the plates incubated for an additional 9 hr 20 min at RT.

tag: ecotype

value: Columbia

value: whole seedling

tag: agent

value: dicamba

tag: biological rep


title: SAMPLE009 null

description:
36227 oligo probes, CodeLink Mouse Whole Genome Bioarray
T00341109

tag: strain


title: Sample 11 null

description:
36227 oligo probes, CodeLink Mouse Whole Genome Bioarray
T00317571

data_processing: Initial feature extraction from the images was performed using CodeLink Expression Analysis software v4.0. Median-Normalization.

growth_protocol: Embryos were removed from pregnant dams at embryonic day 9.5.  At E 9.5, the average embryo was “C” shaped and had 21.6 pairs of somites and neurul tube closure was complete with the exception of the posterior neuropore.

value: Fkbp8 null


title: FADU-Twist1

city: Taipei

first: Muh-Hwa

last: Yang

address: No.155, Sec.2, Li-Nong Street

department: Institute of Clinical Medicine

country: Taiwan

institute: National Yang-Ming University

description: Human hypopharyngeal cancer cell line FADU transfected with the Twist1 expression vector pFLAG-Twist1 were grown to near confluence and collected total RNA for array analysis.

hyb_protocol: Hybridization was performed according to the standard Affymetrix protocol (see complete protocol descriptions: http://www.affymetrix.com/support/technical/manual/expression_manual.affx)

scan_protocol: Arrays were scanned with the Affymetrix GeneChip® Scanner 3000 7G and the data was extracted using Microarray Analysis Suite 5.0 (Affymetrix).

data_processing: We used the Affymetrix GeneChip Suite 5.0 software (MAS 5.0) to calculate raw expression values for each probe sets on the U133 2.0 oligonucleotide arrays.

source_name: human hypopharyngeal squamous cell carcinoma cell line FADU transfected with pFLAG-Twist1

label_protocol: Biotinylated cRNA was prepared according to the standard Affymetrix protocol (see complete protocol descriptions: http://www.affymetrix.com/support/technical/manual/expression_manual.affx)

growth_protocol: cultivated in RPMI + 10% fetal bovine serum

extract_protocol: Cells were lysed by adding 2 ml of trizol reagent (Invitrogen) and homogenized by passing through a syringe with a needle (26G). RNA was picked up in the aqueous phase following phase separation by chloroform. The RNA was then precipitate with isopropanol, washed with ethanol (75%), dissolved in DEPC-treated RNAse/DNAse free water and quantified by spectrophotometry.

tag: cell line source ethnicity

value: Caucasian

tag: cell line source gender

tag: cell line source age

tag: cell line source tissue

value: primary human hypopharyngeal squamous cell carcinoma

tag: transfection

value: FADU


title: WT dicamba treated_biological rep 3


title: SAMPLE005 wildtype

description:
36227 oligo probes, CodeLink Mouse Whole Genome Bioarray
T00341107

tag: age


title: FADU-Bmi1

description: Human hypopharyngeal cancer cell line FADU transfected with the Bmi1 expression vector pcDNA3-Bmi1 were grown to near confluence and collected total RNA for array analysis.

source_name: human hypopharyngeal squamous cell carcinoma cell line FADU transfected with pcDNA3-Bmi1

extract_protocol: Cells were lysed by adding 2 ml of trizol reagent (Invitrogen). RNA was picked up in the aqueous phase following phase separation by chloroform. The RNA was then precipitate with isopropanol, washed with ethanol (75%), dissolved in DEPC-treated RNAse/DNAse free water and quantified by spectrophotometry.

value: primary human hypopharyngeal squamous cell carcinoma p


title: Sample 10 wildtype

description:
36227 oligo probes, CodeLink Mouse Whole Genome Bioarray
T00317570


title: FADU-HIF1a(dODD)

description: Human hypopharyngeal cancer cell line FADU transfected with pHA-HIF1a(dODD) vector which expresses a HIF-1a mutant with an oxygen degradation domain (∆ODD) deletion that functions in a normoxic environment were grown to near confluence and collected total RNA for array analysis.

source_name: human hypopharyngeal squamous cell carcinoma cell line FADU transfected with pHA-HIF1a(dODD)


title: Sample 12 wildtype

description:
36227 oligo probes, CodeLink Mouse Whole Genome Bioarray
T00317572


title: WT dicamba treated_biological rep 2


title: SAMPLE008 wildtype

description:
36227 oligo probes, CodeLink Mouse Whole Genome Bioarray
T00341108


title: PLC8024 CD133- miR-130b

city: Hong Kong

first: Stephanie

last: Ma

address: Rm 47, 1/F, Lab Block, Faculty of Medicine Bldg, 21 Sassoon Rd, Pok Fu Lam

department: School of Biomedical Sciences

country: Hong Kong

institute: The University of Hong Kong

description: gene expression

hyb_protocol: Service by University Health Network (UHN) Microarray Centre (protocol attached)

data_processing: The data were analyzed with RMA using Affymetrix default analysis settings and global scaling as normalization method

source_name: miR-130b transfection

growth_protocol: CD133- cells sorted from PLC8024 HCC cells using flow cytometry cell sorting technique

extract_protocol: Trizol extraction of total RNA was performed according to the manufacturer's instructions

treatment_protocol: PLC8024 CD133- cells lentiviral transduced with miR-130b or empty vector control

tag: cell type

value: HCC

tag: genotype


title: WT mock treated_biological rep 3

description: Gene expression data from WT seedlings that contain the GSTF8::LUC reporter treated with water (mock)

source_name: 4 day old seedlings, mock-treated 10 hr

value: control


title: E0-4_H3K27Ac_2 extraction2_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).;

extract_protocol:
Collection of Drosophila embryos at time-point 0-4h of development 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: developmental stage

tag: Sex

value: Unknown


title: WT mock treated_biological rep 1


title: PLC8024 CD133- EV

description: gene expression

scan_protocol: Service by University Health Network (UHN) Microarray Centre (protocol attached)

source_name: vector control

tag: cell line


title: Sample 9 null

description:
36227 oligo probes, CodeLink Mouse Whole Genome Bioarray
T00317569

tag: genotype/variation


title: FADU-cDNA3

description: Human hypopharyngeal cancer cell line FADU transfected with pcDNA3.1 empty vector were grown to near confluence and collected total RNA for array analysis.

source_name: human hypopharyngeal cancer cell line FADU transfected with pcDNA3.1 empty vector

value: Male

value: pcDNA3.1 empty vector


title: Sample 6 null

description:
36227 oligo probes, CodeLink Mouse Whole Genome Bioarray
T00317567

tag: tissue


title: Depleted HepG2 versus adiponectin treated HepG2

status: Public on Dec 09 2010

city: Pierre Bénite

first: emmanuelle

last: meugnier

address: Cens Eli Bat 2 D- Hopital Lyon Sud

country: France

description: Four human cell lines (Hela, HepG2, Panc-1, Hek293) were grown until confluence.

hyb_protocol: 3µg of labelled nucleic acid was hybridized at 62 degree_C during 17 hours

scan_protocol: Scanned on Axon Genepix 4000B scanner. Images were quantified using Genepix pro 4.0

data_processing: Lowess normalization.

source_name: Depleted HepG2

label_protocol: 500ng of total RNA was amplified with the Amino Allyl MessageAmp II aRNA kit (Ambion) according to the manufacturer’s instructions. Fluorescent probes were synthesized by chemical coupling of 5 µg of aminoallyl aRNA with cyanine (Cy)3 or Cy5 dyes.  Fluorescent probes were purified using Rneasy minikit (Qiagen)

extract_protocol: Total RNA extracted using Rneasy mini kit (Qiagen) following manufacturer's instructions

treatment_protocol: Each cell line were treated during 3 hours with human adiponectin (2,5µg/ml)

tag: HepG2 are human Caucasian hepatocyte carcinoma

source_name: Adiponectin treated HepG2


title: Sample 7 wildtype

description:
36227 oligo probes, CodeLink Mouse Whole Genome Bioarray
T00317568


title: Depleted Panc-1 versus adiponectin treated Panc-1

source_name: Depleted Panc-1

tag: Panc-1 are pancreatic carcinoma of ductal origin from a 56-year-old Caucasian male

source_name: Adiponectin treated Panc-1


title: Depleted Hek293 versus adiponectin treated Hek293

source_name: Depleted Hek293

tag: Hek293 are human embryo kidney cells

source_name: Adiponectin treated Hek293


title: H460v

city: Pamplona

first: Eva

last: Bandres

address: Avda Pio XII, 55

department: Cancer Research Program

institute: Center for Applied Medical Research (CIMA), University of Navarra

hyb_protocol: Following fragmentation, 15 microg of cRNA were hybridized to the Affymetrix HG-U133A 2.0 chip according to the protocols provided by the manufacturer.

scan_protocol: GeneChips were scanned using the GeneChip Scanner 3000 from Affymetrix.

data_processing: RMA processing using R (Bioconductor)

source_name: human non small cell lung cancer H460 wt expressing an empty vector psuperretro

label_protocol: The cDNA was prepared from 2 microg total RNA using the One –Cycle cDNA Synthesis kit from Affymetrix. This cDNA was used for synthezising the cRNA using the IVT labeling kit (Affimetrix). The synthezised cRNA was purified with the GeneChip Sample Cleanup Module (Affimetrix).

growth_protocol: The human non small lung cancer cell line H460 used in this study was from ATCC (American Type Culture Collection). The cell line was maintained in Roswell Park Memorial Institute (RPMI) (Gibco, Invitrogen) supplemented with 10% bovine serum and transfected by Lipofectamine Plus Reagent from Life Technologies as directed by the manufacturer.

extract_protocol: The cells were harvested with TRIzol Reagent (Invitrogen)  and the RNA was extracted according to the manufacturer's instructions. As a last step of the extraction procedure, the RNA was purified with the RNeasy Mini-kit (Qiagen, Hilden, Germany). The RNA quality was confirmed by the ratio of 28S and 18S rRNA after agarose gel electrophoresis.

treatment_protocol: The DUSP1 pSuperRetro-derived vectors were constructed as described (Chattoppadhyay et al., 2006). Clones expressing siRNA for DUSP1 were selected for their ability to grow in the presence of puromycin and kept for selection. Stable transfection was confirmed by qRT-PCR and Western Blot. RNA was extracted and quality control was checked before hybridization on Affymetrix microarrays.

value: non small cell lung cancer


title: ATM-/-8h

first: Guohui

last: Wan

address: 7777 Knight Road

department: Cancer Biology

institute: The University of Texas M.D. Anderson Cancer Center

description: Gene expression data from MCFs after DNA damage

hyb_protocol: Labelled miRNAs were hybridized by miRNA OneArray™ Hybridization (OneArray) according to the manufacturer's instruction.

scan_protocol: MiRNAs-Chips were scanned using the Molecular Devixes™ GenePix™ Scanner.

data_processing: The data were analyzed with Array Studio Analysis Software by Rosetta Resolver.

label: Haptens Fluorophores

label_protocol: MicroRNA were prepared and labelled according to Kreatech ULS™ miRNA Labeling Kit (Standard, Phalanxbiotech) from 4 ug total RNA.

growth_protocol: The MEFs were plated on multiple 100-mm dishes for each line in 10% fetal calf serum and Dulbecco's modified Eagle's medium (DMEM) in 37℃ with 5% CO2.

extract_protocol: Trizol extraction of total RNA was performed according to the manufacturer's instructions (Invitrogen).

treatment_protocol: MEFs were treated with a radiomimetic drug, neocarzinostatin (NCS) that generates DSBs. Cells were harvested at varying time points (0-24 h).

value: Mouse Embryonic Fibroblast

value: ATM-/-


title: ATM-/-4h


title: WT mock treated_biological rep 2

tag: ecotype

tag: agent

tag: biological rep


title: ATM+/+8h

value: ATM+/+


title: ATM+/+24h


title: ATM+/+0h


title: ATM+/+4h


title: ATM-/-24h


title: phr1 -Pi-DEX-CHX biological rep3

city: Campus de Cantoblanco

first: Regla

last: Bustos

state: Madrid

address: Darwin 3

institute: CNB-CSIC

description: Gene expression data from phr1 plants grown in Pi-lacking Johnson liquid media treated with cycloheximide.

hyb_protocol: 10µg of fragmented cRNA were hybridized to the Arabidopsis ATH1 Genome array (Affymetrix, SantaClara, CA), containing 22500 transcript variants from 24000 well -characterized Arabidopsis thaliana genes. Each sample was added to a hybridization solution containing 100mM2-(N-morpholino) ethanesulfonic acid, 1M Na+,and 20mM of EDTA in the presence of 0.01% of Tween-20 to a final cRNA concentration of 0.05µg/ml. Hybridization was performed for 16 h at 45ºC . Each microarray was washed and stained with streptavidin-phycoerythrin in a Fluidics station 450 (Affymetrix)

scan_protocol: Each microarray was scanned at 2.5 µm resolution in a GeneChip® Scanner 3000 7G System (Affymetrix)

data_processing: Analysis was performed using the affylmaGUI R package (Wettenhall et al, 2006). Robust Muti-array Analysis (RMA) algorithm was used for background correction, normalization and expression levels summarization (Irizarry et al., 2003).

source_name: phr1 plants grown in -Pi treated with cycloheximide

label_protocol: Each RNA preparation was tested for degradation using the Agilent 2100 Bioanalyzer (Agilent technologies, Palo Alto, CA). cDNA was synthesized from 4 µg of total RNA using One-cycle target labeling and control reagents(Affymetrix, Santa Clara, CA) to produce biotin-labeled cRNA. The cRNA preparations (15 µg) were fragmented at 94ºC for 35 min into 35-200 bases in length.

growth_protocol: Plants were grown for 7 days in liquid Johnson medium containing 2% sucrose and 2mM phosphate (KH2PO4), 2 days in phosphate-lacking medium (-Pi) and then supplemented with 5 µM dexamethasone (DEX) and 10 µM cycloheximide (CHX) for 6 hours before harvesting.

extract_protocol: The RNA from complete plants was isolated using RNeasy plant mini kit from Quiagen

treatment_protocol: 5 µM dexamethasone (DEX) and 10 µM cycloheximide (CHX) for 6 hours in phosphate-lacking Johnson liquid medium.

tag: strain

value: phr1 mutant


title: phr1 -Pi-DEX-CHX biological rep1


title: phr1 -Pi-DEX-CHX biological rep2


title: OxPHR1 -Pi-DEX-CHX biological rep3

description: Gene expression data from OxPHR1 phr1 plants grown in Pi-lacking Johnson liquid media treated with cycloheximide.

source_name: OXGR:PHR1 phr1 plants grown in -Pi treated with cycloheximide


title: Depleted Hela versus adiponectin treated Hela

source_name: Depleted Hela

tag: Hela are cervix carcinoma from a 31 year old Negro

source_name: Adiponectin treated Hela


title: OxPHR1 -Pi-DEX-CHX biological rep2


title: colon_fishoil_10wk_rep2 [236.2]

city: College Station

first: Jennifer

last: Goldsby

address: 112 Cater-Mattil

department: Nutrition and Food Sciences

institute: Texas A&M University

hyb_protocol: Codelink Gene Expression System: Single-Assay Bioarray Hybridization and Detection according to manufacturer's instructions http://www.appliedmicroarrays.com/pdf/codelink_bioarray_processing_protocol.pdf

scan_protocol: Codelink Gene Expression System: Single-Assay Bioarray Hybridization and Detection according to manufacturer's instructions http://www.appliedmicroarrays.com/application_notes/63005079.pdf

data_processing: Median normalized values within each array as calculated in Microsoft Excel version 2003

label: biotin, Alexa-647 Streptavidin

organism: Rattus norvegicus

source_name: colonic mucosa, fed fish oil, terminated 10 wk after carcinogen injection

label_protocol: Codelink Gene Expression System: Manual Labelled cRNA Target Preparation according to manufacturer's instructions http://www.appliedmicroarrays.com/pdf/iexpress2006_web.pdf

growth_protocol: Rats were fed diets that differed in type of fat (corn oil or fish oil or olive oil).

extract_protocol: Ambion Totally RNA kit followed by DNAFree treatment.

treatment_protocol: Rats were treated with the carcinogen azoxymethane at 15 mg/kg body weight and terminated 12 h later or were given a second injection and terminated 9 wk later.

tag: diet

value: fish oil

tag: time point

value: Sprague Dawley

tag: gender

value: Weanling male

value: colon


title: colon_fishoil_10wk_rep1 [236.1]

value: terminated 10 wk after carcinogen injection


title: OxPHR1 -Pi-DEX-CHX biological rep1

tag: genotype


title: colon_oliveoil_10wk [334]

source_name: colonic mucosa, fed olive oil, terminated 10 wk after carcinogen injection

value: olive oil


title: colon_oliveoil_10wk_rep1 [336.1]


title: colon_fishoil_10wk_rep3 [236.3]


title: colon_oliveoil_10wk_rep1 [334.1]


title: colon_oliveoil_10wk [332]


title: colon_oliveoil_10wk [335]


title: colon_oliveoil_10wk [333]


title: colon_oliveoil_10wk [336]


title: colon_fishoil_10wk_rep1 [234.1]


title: colon_fishoil_10wk [232]


title: colon_fishoil_10wk [233]


title: colon_fishoil_10wk [234]


title: colon_fishoil_10wk [236]


title: colon_cornoil_10wk_rep3 [136.3]

source_name: colonic mucosa, fed corn oil, terminated 10 wk after carcinogen injection

value: corn oil


title: colon_cornoil_10wk [136]


title: colon_cornoil_10wk [135]


title: colon_cornoil_10wk_rep1 [136.1]


title: H460cri

source_name: human non small cell lung cancer H460 expressing DUSP1 siRNA

tag: cell type


title: colon_cornoil_10wk_rep2 [136.2]


title: colon_cornoil_10wk [134]


title: colon_cornoil_10wk [131]


title: MedB1_unstim_12h

city: Munich

first: Andreas

last: Roller

address: Max-Lebsche-Platz 31

institute: Munich Leukemia Laboratory

description: GeneChip Human Gene 1.0 ST Arrays (Affymetrix)

hyb_protocol: Following cDNA-Cleanup, 5.5ug ssDNA were fragmented, labeled and hybridized for 17h at 45C. GeneChips were washed and stained in the Affymetrix Fluidics Station 450.

scan_protocol: Fluorescence intensities of the hybridized arrays were acquired with the GeneChip Scanner 3000 and the operating software AGCC (Affymetrix).

data_processing: Data processing was performed using the Limma toolbox (Smyth et al., 2005) provided by Bioconductor (Gentleman et al., 2004). The RMA approach was used for normalization and background correction. In addition, probe sets were filtered out by the genefilter package.

label_protocol: Biotinylated cRNA were prepared according to the One-Cycle Target Labeling Assay from 2.5ug total RNA (Expression Analysis Technical Manual, 2004, Affymetrix).

growth_protocol: Cells were cultivated in RPMI medium supplemented with 10% FCS.

extract_protocol: The quality of total RNA samples extracted from the two cell types was assessed with the Bioanalyzer 2100 (Agilent) to ensure that 28S/18S rRNA ratios were in the range of 1.5 to 2.0 and quality were comparable between samples. For each time point, 200ng of total RNA were used for the hybridization procedure. Labeled cDNA were prepared according Whole Transcript (WT) Sense Target Labeling Assay (Affymetrix).

treatment_protocol: Cells were starved in RPMI medium supplemented with 1 mg/ml BSA and then stimulated with 20 ng/ml IL13 (MedB-1 cells) or 40 ng/ml IL13 (L1236 cells) for 0, 0.5, 1, 1.5, 2, 3, 4, 6, 8 and 12 h or left untreated.

tag: condition

value: unstimulated

value: primary mediastinal B-cell lymphoma cell line (MedB-1)


title: colon_oliveoil_12h [306]

source_name: colonic mucosa, fed olive oil, terminated 12 h after carcinogen injection


title: colon_oliveoil_12h [305]

value: terminated 12 h after carcinogen injection


title: colon_cornoil_10wk [132]


title: MedB1_stim_12h

value: stimulated with IL13

tag: cell line


title: MedB1_unstim_8h


title: colon_oliveoil_12h [304]


title: colon_oliveoil_12h [302]


title: ATM-/-0h

tag: genotype/variation


title: MedB1_stim_6h


title: 4 mg/ml of coal tar diluted in DMSO 5D2R8 incubation 8 hours

city: Ottawa

first: Andrew

last: Williams

state: ON

address: 50 Columbine

institute: Health Canada

hyb_protocol: Agilent Mouse G4121B Microarrays (containing approximately 22,000 probes) were hybridized with 5 µg Cy5-labelled FE1 cells (lung) RNA and 5 µg Cy3-labelled Universal Mouse Reference RNA (Stratagene, CA, USA), used as a common reference on all arrays.  Arrays were incubated overnight at 60 ºC in Agilent hybridization solution and washed according to manufacturer’s instructions.

scan_protocol: Arrays were scanned using a ScanArray Express (Perkin-Elmer Life Sciences, Woodbridge, ON, Canada), and data were acquired with ImaGene 5.5 (BioDiscovery, CA, USA).

data_processing: The median signal intensity of each microarray sample was used. Technical replicates were not averaged. Data for each probe was flagged (flag=1) if the median signal intensity for that probe was less than the mean of the negative controls ((-)3xSLv1) plus three standard deviations. RawCy5 and RawCy3 are the median signal intensities for the Cy5 and Cy3 channels on each microarray. NormCy5 and NormCy3 are the loess normalized signal intensities for the Cy5 and Cy3 channels within each array; this is a within-array normalization performed separately for each array. Flag indicates whether the median signal intensity falls in the background, i.e below threshold calculated from the negative controls (mean plus three standard deviations of the negative controls.) Ratio is calculated as log2(NormCy5/NormCy3).

source_name: Total RNA from cultured FE1 cells dreived from lung labeled with Cyanine-5 (red)

label_protocol: Individual 2.5 µg aliquots of RNA from each sample were amplified and labelled using the Low RNA Input Fluorescent Linear Amplification Kit (Agilent).

extract_protocol:
RNA was isolated using the TRIzol reagent (Invitrogen Canada Inc., Burlington, ON, Canada), and further purified using RNeasy Mini Kits (Qiagen Inc., Mississauga, ON, Canada).  RNA was quantified using UV spectrophotometer and quality was confirmed using the Agilent 2100 Bioanalyzer and RNA 6000 NanoLab Chip Kit (Agilent Technologies Canada Inc., Mississauga, ON, Canada).
Material was purchased as total RNA (universal mouse reference total RNA (Stratagene, CA, USA)

value: FE1 cells from lung

tag: time of growth

value: Cells were grown for 72 hrs

tag: growth conditions

value: 24hrs prior to exposure, the cells were washed and incubated with serum free media

tag: passage

value: Cells of passage 14 at 70% confluence

tag: date of exposure

tag: exposure

value: 6 hours exposure to 4 mg/ml of coal tar diluted in DMSO

tag: incubation

value: 8 hours

tag: id

value: Microarray ID Cy5 60

source_name: universal mouse reference total RNA (Catalog #740100; Stratagene, La Jolla, CA, USA)

tag: sample type

value: Total RNA derived from 11 mouse cell lines

tag: source

value: Stratagene, La Jolla, CA, USA


title: 4 mg/ml of coal tar diluted in DMSO 5D2R4 incubation 4 hours

value: 4 hours

value: Microarray ID Cy5 57


title: colon_oliveoil_12h [301]

tag: diet

value: olive oil

tag: strain

value: Sprague Dawley

value: Weanling male

tag: tissue

value: colon


title: MedB1_stim_4h

tag: time point

value: primary mediastinal B-cell lymphoma cell line (MedB-1)


title: MedB1_unstim_4h

value: unstimulated


title: MedB1_unstim_3h


title: 1 mg/ml of coal tar diluted in DMSO 5D1R4 incubation 4 hours

tag: cell type

value: FE1 cells from lung

value: 6 hours exposure to 1 mg/ml of coal tar diluted in DMSO

value: Microarray ID Cy5 56


title: 1 mg/ml of coal tar diluted in DMSO 5D1R8 incubation 8 hours

value: Cells were grown for 72 hrs

value: 24hrs prior to exposure, the cells were washed and incubated with serum free media

value: Cells of passage 14 at 70% confluence

value: Microarray ID Cy5 59


title: MedB1_stim_3h

tag: condition

value: stimulated with IL13


title: colon_fishoil_12h_rep1 [206.1]

source_name: colonic mucosa, fed fish oil, terminated 12 h after carcinogen injection

value: fish oil

tag: gender


title: MedB1_unstim_6h

tag: cell line


title: MedB1_stim_2h


title: colon_oliveoil_12h_rep1 [301.1]

value: terminated 12 h after carcinogen injection


title: DMSO only 5C1R4 incubation 4 hours

tag: time of growth

value: 6 hours exposure to DMSO only

value: 4 hours

value: Microarray ID Cy5 55

value: Total RNA derived from 11 mouse cell lines

value: Stratagene, La Jolla, CA, USA


title: DMSO only 5C1R8 incubation 8 hours

tag: growth conditions

value: 8 hours

value: Microarray ID Cy5 58


title: colon_fishoil_12h [205]

value: Sprague Dawley

value: Weanling male

value: colon


title: colon_fishoil_12h [206]


title: colon_fishoil_12h [203]


title: colon_fishoil_12h_rep1 [202.1]


title: MedB1_stim_1_5h

value: primary mediastinal B-cell lymphoma cell line (MedB-1)


title: MedB1_unstim_2h

value: unstimulated


title: 4 mg/ml of coal tar diluted in DMSO 4D2R4 incubation 4 hours

value: FE1 cells from lung

value: Cells were grown for 72 hrs

value: 24hrs prior to exposure, the cells were washed and incubated with serum free media

tag: passage

value: Cells of passage 14 at 70% confluence

value: 6 hours exposure to 4 mg/ml of coal tar diluted in DMSO

value: Microarray ID Cy5 45


title: 4 mg/ml of coal tar diluted in DMSO 4D2R8 incubation 8 hours

tag: date of exposure

value: Microarray ID Cy5 48


title: colon_fishoil_12h [202]

value: fish oil

value: terminated 12 h after carcinogen injection


title: 1 mg/ml of coal tar diluted in DMSO 4D1R8 incubation 8 hours

tag: exposure

value: 6 hours exposure to 1 mg/ml of coal tar diluted in DMSO

value: Microarray ID Cy5 47

value: Total RNA derived from 11 mouse cell lines

value: Stratagene, La Jolla, CA, USA


title: colon_fishoil_12h [201]

value: Sprague Dawley

value: Weanling male


title: colon_cornoil_12h [106]

source_name: colonic mucosa, fed corn oil, terminated 12 h after carcinogen injection

value: corn oil

value: colon


title: 1 mg/ml of coal tar diluted in DMSO 3D1R8 incubation 8 hours

value: FE1 cells from lung

tag: incubation

value: 8 hours

value: Microarray ID Cy5 35


title: MedB1_unstim_1_5h

value: unstimulated

value: primary mediastinal B-cell lymphoma cell line (MedB-1)


title: MedB1_stim_1h

value: stimulated with IL13


title: MedB1_unstim_1h


title: 1 mg/ml of coal tar diluted in DMSO 4D1R4 incubation 4 hours

value: Cells were grown for 72 hrs

value: 24hrs prior to exposure, the cells were washed and incubated with serum free media

value: Cells of passage 14 at 70% confluence

value: 4 hours

tag: id

value: Microarray ID Cy5 44


title: 4 mg/ml of coal tar diluted in DMSO 3D2R8 incubation 8 hours

value: 6 hours exposure to 4 mg/ml of coal tar diluted in DMSO

value: Microarray ID Cy5 36

tag: sample type

value: Total RNA derived from 11 mouse cell lines


title: DMSO only 3C1R8 incubation 8 hours

value: 6 hours exposure to DMSO only

value: Microarray ID Cy5 34

tag: source

value: Stratagene, La Jolla, CA, USA


title: MedB1_stim_0_5h


title: 4 mg/ml of coal tar diluted in DMSO 3D2R4 incubation 4 hours

value: FE1 cells from lung

value: Microarray ID Cy5 33


title: MedB1_stim_8h


title: colon_cornoil_12h_rep1 [104.1]

tag: diet

value: corn oil

value: terminated 12 h after carcinogen injection

value: Sprague Dawley

value: Weanling male

value: colon


title: colon_cornoil_12h [105]

tag: strain


title: DMSO only 3C1R4 incubation 4 hours

value: Cells were grown for 72 hrs

value: 24hrs prior to exposure, the cells were washed and incubated with serum free media

value: Microarray ID Cy5 31


title: 4 mg/ml of coal tar diluted in DMSO 2D2R8 incubation 8 hours

value: Cells of passage 14 at 70% confluence

value: 8 hours

value: Microarray ID Cy5 24


title: MedB1_stim_0h

value: stimulated with IL13

value: primary mediastinal B-cell lymphoma cell line (MedB-1)


title: MedB1_unstim_0_5h

value: unstimulated


title: 1 mg/ml of coal tar diluted in DMSO 3D1R4 incubation 4 hours

value: 6 hours exposure to 1 mg/ml of coal tar diluted in DMSO

value: 4 hours

value: Microarray ID Cy5 32

value: Total RNA derived from 11 mouse cell lines

value: Stratagene, La Jolla, CA, USA


title: colon_cornoil_12h [103]

value: corn oil

value: terminated 12 h after carcinogen injection

value: Sprague Dawley

tag: tissue


title: 1 mg/ml of coal tar diluted in DMSO 2D1R8 incubation 8 hours

value: FE1 cells from lung

value: Cells were grown for 72 hrs

value: Microarray ID Cy5 23


title: MedB1_unstim_0h

tag: time point


title: colon_cornoil_12h [102]

value: Weanling male

value: colon


title: colon_cornoil_12h_rep1 [103.1]


title: L1236_stim_8h

value: Hodgkin lymphoma cell line (L1236)


title: DMSO only 2C1R8 incubation 8 hours

tag: cell type

value: 24hrs prior to exposure, the cells were washed and incubated with serum free media

value: Cells of passage 14 at 70% confluence

value: 6 hours exposure to DMSO only

value: 8 hours

value: Microarray ID Cy5 22


title: L1236_stim_6h

tag: condition

value: stimulated with IL13


title: 4 mg/ml of coal tar diluted in DMSO 2D2R4 incubation 4 hours

value: 6 hours exposure to 4 mg/ml of coal tar diluted in DMSO

value: 4 hours

value: Microarray ID Cy5 21

value: Total RNA derived from 11 mouse cell lines


title: DMSO only 2C1R4 incubation 4 hours

value: Microarray ID Cy5 19

value: Stratagene, La Jolla, CA, USA


title: 1 mg/ml of coal tar diluted in DMSO 1D1R8 incubation 8 hours

value: FE1 cells from lung

value: Cells were grown for 72 hrs

value: 6 hours exposure to 1 mg/ml of coal tar diluted in DMSO

value: Microarray ID Cy5 11


title: 4 mg/ml of coal tar diluted in DMSO 1D2R8 incubation 8 hours

value: Microarray ID Cy5 12


title: cells_treated_rep3

city: Richmond

first: Mikhail

last: Dozmorov

state: VA

address: 830 E Main St

department: Biostatistics

institute: Virginia Commonwealth University

description: Replicate 3

hyb_protocol: Briefly, cDNA was reverse transcribed from the total RNA after priming withT7-oligo-dT, and cRNA was synthesized in vitro from the T7 promoter with the incorporation of biotinylated UTP. Biotinylated cRNA was hybridized overnight to The Illumina WG-6_v3 BeadChips containing probes for >48,000 transcripts. Microarray chips were washed at high stringency and labeled with streptavidin -Cy3 (Amersham Biosciences,Piscataway, NJ) prior to scanning on an Illumina BeadArray Reader.

scan_protocol: Images were obtained on an Illumina Beadchip scanner.

data_processing:
The images and raw data from each chip were transferred automatically to the microarray database using one of the specified microarray core servers
Non-normalized - Raw signali intensity; Normalized - log10 values processed using two-step normalization (Dozmorov I, Lefkovits I., Nucleic Acids Res. 2009 Oct;37(19):6323-39.)

source_name: H441 lung adenocarcinoma cells

label_protocol: Illumina HumanWG-6 v3.0 arrays containing >48,000 probes were handled according to standard protocols established by the manufacturer. Briefly, 200ng of total RNA from each sample were used to generate biotin-labeled cRNA probes using an Illumina MessageAmp cRNA labeling kit protocol (Ambion).

growth_protocol: Human lung adenocarcinoma cell line NCI-H441 (ATCC Number: HTB-174) was obtained from the American Type Culture Collection (Manassas, VA).  H441 cells were maintained at 37 °C with 5% CO2 in McCoy's 5A Medium (Invitrogen, Carlsbad, California) supplemented with 5% heat-inactivated fetal bovine serum (FBS).  All media contained gentamicin at 50 µg/ml (GIBCO Laboratories, Grand Island, NY).

extract_protocol: After 12 h of treatment with CLEFMA (1 µM), the cells were washed twice with Dulbecco’s PBS. The total RNA was extracted using RNAeasy kit (Qiagen, CA) as per the manufacturer’s instructions. The quality of RNA was ascertained by Nanodrop 2000. A total of 250 ng of total RNA from three biological replicates of untreated H441 cells and cells treated with CLEFMA were labeled using the Illumina Total Prep RNA Amplification Kit following manufacturer's directions (Ambion, Austin. TX).

treatment_protocol: H441 cells were seeded in 6-well tissue culture plates at a density of 1x106 cells per well. The cells were allowed to attach and grow overnight. CLEFMA was solubilized in dimethyl sulfoxide (DMSO), filtered through 0.2 µm nylon filter and added to the treated wells (n=4) at 1 µM concentration in culture medium supplemented with 5% FBS. The DMSO concentration was maintained at 0.1% per well. Control wells (n=4) received equivalent volume of DMSO without any test compound. The cells were allowed to remain in the treatment medium for 12 hours.

value: lung adenocarcinoma cells

value: NCI-H441 (ATCC Number:HTB-174)

tag: treatment

value: Treated


title: L1236_unstim_6h

value: unstimulated

tag: cell line

value: Hodgkin lymphoma cell line (L1236)


title: 1 mg/ml of coal tar diluted in DMSO 1D1R4 incubation 4 hours

tag: time of growth

value: 24hrs prior to exposure, the cells were washed and incubated with serum free media

value: Cells of passage 14 at 70% confluence

value: Microarray ID Cy5 8


title: cells_treated_rep2

description: Replicate 2


title: L1236_unstim_4h


title: L1236_stim_4h

value: stimulated with IL13


title: cells_treated_rep4

description: Replicate 4


title: cells_treated_rep5

description: Replicate 5


title: L1236_unstim_8h


title: DMSO only 1C1R8 incubation 8 hours

tag: growth conditions

value: 6 hours exposure to DMSO only

value: 8 hours

value: Microarray ID Cy5 10

value: Total RNA derived from 11 mouse cell lines


title: L1236_stim_3h


title: L1236_unstim_3h


title: cells_control_rep4

description: Replicate 4

value: Untreated


title: cells_control_rep5

description: Replicate 5


title: DMSO only 1C1R4 incubation 4 hours

value: FE1 cells from lung

value: Cells were grown for 72 hrs

tag: passage

value: 4 hours

value: Microarray ID Cy5 7

value: Stratagene, La Jolla, CA, USA


title: cells_treated_rep1

description: Replicate 1

value: lung adenocarcinoma cells

value: NCI-H441 (ATCC Number:HTB-174)

value: Treated


title: Nuocytes cultured for 9 days rep3

city: CAMBRIDGE

last: MCKENZIE

address: HILLS ROAD

country: United Kingdom

institute: MRC-LMB

description: Gene expression data from nuocytes isolated from the spleen following 3 daily administrations of IL-25 and cultured with IL-33 and IL-7 (both at 10 nanograms /ml) for 9 days.

hyb_protocol: Hybridization were done on Affymetrix GeneChips (Mouse Genome 430 2.0 cartridge arrays) for 16 hours, 48C and 55rpm, then washed on an Affymetrix FS450 using the EukGE-WS2v4_450 protocol instrument

scan_protocol: Arrays were scanned using an Affymetrix GCS3000 7G system

data_processing: RMA normalization of the data followed by two-way ANOVAs using the default analysis settings in Array Studio

source_name: IL-25-induced nuocytes cultured with IL-33 and IL-7 for 9 days

label_protocol: Labeling was done using NuGEN Encore Biotin Module according to manufacturer’s protocol

growth_protocol: Nuocytes were purified directly from spleen and prepared for microarray or cultured with IL-33 and IL-7 (both at 10 nanograms/ml) for 9 days before being prepared for microarray gene expression analysis

extract_protocol: RNA was extracted with the Qiagen RNAeasy Mini Kit according to the manufacturer's protocol.

treatment_protocol: Nuocytes were purified by flow cytometry from the spleens of mice treated with 3 daily doses of IL-25

value: Splenic nuocytes

tag: time

value: 9 days in culture

tag: trreatment


title: Nuocytes cultured for 9 days rep2

value: 3 daily administrations of IL-25 and cultured with IL-33 and IL-7 (both at 10 nanograms /ml) for 9 days.


title: L1236_unstim_2h

value: unstimulated

value: Hodgkin lymphoma cell line (L1236)


title: L1236_stim_2h

value: stimulated with IL13


title: Nuocytes cultured for 9 days rep1


title: cells_control_rep1

description: Replicate 1


title: Ex vivo nuocytes day 0 rep1

description: Gene expression data from nuocytes directly isolated from the spleen following 3 daily administrations of IL-25.

source_name: IL-25-induced nuocytes

value: 0 days in culture

value: 3 daily administrations of IL-25.


title: cells_control_rep2

description: Replicate 2

value: Untreated


title: colon_cornoil_12h [104]

value: corn oil

value: terminated 12 h after carcinogen injection

value: Sprague Dawley

tag: gender

value: Weanling male

value: colon


title: L1236_stim_1_5h


title: L1236_stim_12h


title: cells_control_rep3

description: Replicate 3

value: lung adenocarcinoma cells


title: L1236_unstim_1_5h


title: Ex vivo nuocytes day 0 rep2


title: 8 ng/ml camptothecin-treated Jurkat cells

first: Qun

last: Pan

address: 160 College St.

institute: University of Toronto

description: This Sample represents two hybridizations.

hyb_protocol: Hybridization of Cy3- and Cy5-labelled cDNA synthesized from polyA+ RNA from DRB, camptothecin or DMSO-treated Jurkat cells was performed using a HS4800 Pro Hybridization Station (Tecan) at 42°C for 22 hours.

scan_protocol: Microarray slides were scanned with an Agilent microarray scanner at 5µm resolution and TIFF images were processed using Feature Extraction Software (Agilent).

data_processing: Microarray slides were scanned with an Agilent microarray scanner at 5µm resolution and TIFF images were processed using Agilent Feature Extraction Software (version 9.5). Processed data were then analyzed using the GenASAP algorithm (Pan et al. 2004; Shai et al., 2006).

label: Cy3, Cy5

label_protocol: The mRNA (1–2 μg) was reverse-transcribed with random nonamer primers (1 μg per reaction) and T18VN (0.25 μg per reaction) to synthesize cDNA. The reaction contained a 1:1 mixture of 5-(3-aminoallyl) thymidine 5'-triphosphate (Sigma, St. Louis, USA) and thymidine triphosphate (TTP) in place of TTP alone. The cDNA products were bound to QIAquick PCR Purification columns (Qiagen, Hilden, Germany) following the manufacturer's instructions, washed three times with 80% ethanol, and eluted with water. Purified cDNA was reacted with N-hydroxy succinimide esters of Cy3 or Cy5 (Amersham Pharmacia Biotech, Piscataway, USA) following the manufacturer's instructions.

extract_protocol: RNA was extracted using homogenization and Trizol reagent (Invitrogen, Carlsbad, USA) following the instructions from the manufacturer. Prior to cDNA synthesis, polyA+ RNA was purified from total RNA using the Nucleotrap mRNA Midi kit (Clontech) as recommended by the manufacturer with two rounds of purification.

value: Jurkat

value: 8 ng/ml camptothecin


title: 16 ng/ml camptothecin-treated Jurkat cells

value: 16 ng/ml camptothecin


title: L1236_unstim_1h


title: L1236_unstim_12h


title: L1236_stim_1h


title: mpkCCD clone 11 exposed to dDAVP for 5 days, biological replicate 2

first: Ming-Jiun

last: Yu

address: Rm 816, No. 1 Ren-Ai Road Section 1

department: Institute of Biochemistry and Molecular Biology

institute: National Taiwan University College of Medicine

description:
vasopressin-mediated endogenous aquaporin 2 expression
Gene expression data from cells in response to 0.1nM dDAVP exposure for 5 days

hyb_protocol: Labeled cRNA were hybridized to Mouse Genome 430 2.0 Arrays in Affymetrix Hybridization Oven 640 and washed in GeneChip® Fluidics Station 450 according to the manufacturer's recomended protocols.

scan_protocol: Affymetrix GeneChip® Scanner 3000 according to the manufacturer's recomended protocols

data_processing: Transcript intensity values were obtained with Affymetrix GeneChip Operating System software version 1.4.  Detected signals were normalized based on MAS5 and RMA algorithms using Affymetrix Expression Console software version 1.1.  Probe sets that had absent calls in all 6 arrays were eliminated from further analysis.  Statistical analysis was based on RMA normalized signal intensity values.

source_name: mpkCCD clone 11 cells

label_protocol: Biotinylated cRNA were prepared according to the standard Affymetrix protocols.

growth_protocol: The mpkCCD cells were grown on membrane supports to permit polarization.

extract_protocol: Trizol extraction of total RNA was performed according to the manufacturer's instructions.

treatment_protocol: Once transepithelial resistance reached 5kohm per centimeter square and higher, the cells were exposed to the vasopressin V2 receptor analog, dDAVP, at a physiological concentration, 0.1nM, for 5 days.  Control experiments were done with cells exposed to vehicle alone.

value: mouse kidney cortical collecting duct cells

value: mpkCCD clone 11


title: mpkCCD clone 11 exposed to dDAVP for 5 days, biological replicate 3


title: Cells_Treated_rep3

description: Replicate 3

hyb_protocol: Labeled cRNA probes were hybridized to Illumina arrays according to manufacturer's protocol

data_processing: The images and raw data from each chip were transferred automatically to the microarray database using one of the specified microarray core servers. Normalized - log10 values processed using two-step normalization (Dozmorov I, Lefkovits I., Nucleic Acids Res. 2009 Oct;37(19):6323-39.)

source_name: SKBR-3 cells

growth_protocol: The SKBR-3 cells were cultured in DMEM F-12 medium supplemented with fetal bovine serum (10%) and 100μg/ml streptomycin

extract_protocol: The total RNA was isolated using RNeasy Kit (Qiagen, USA) according to the manufacturer’s instructions.

treatment_protocol: Normally cultured cells were compared with cells treated with our designed phage mimetic nanorods

value: Treated


title: BAPHK_R16 transcriptome profile

city: Cambridge

first: Tsute

last: Chen

state: MA

address: 245 First Street

department: Microbiology

institute: The Forsyth Institute

hyb_protocol: RNA-DNA hybridization was performed on microarray slides covered with a HybriWell chamber (Grace Bio-Labs, Bend, OR, USA).  Prehybridization was carried out for 90 min at 42°C in a 400 μl solution containing 343 μl Long Oligo hybridization buffer [94 mM Tris/HCl pH 7.0, 9.4 mM EDTA, 29.15% formamide, 5.83X SSC, 0.12% SDS], 0.5 mg/ml BSA, and 0.1 mg/ml salmon sperm DNA (Applied Biosystems/Ambion) . The HybriWell chamber was then removed and the slide washed with nuclease-free water. The slide was spin-dried at low-speed for 2 min, and a new HybriWell chamber was sealed onto the slide. For each microarray 3 μg of labeled RNA was denatured at 65°C for 5 min in a final volume of 300 μl hybridization solution containing 257 μl Long Oligo hybridization buffer, 3 μl Alignment Oligo (NimbleGen), and 0.7 mg/ml salmon sperm DNA. Denatured RNA was then loaded in the chamber and hybridization was carried out at 42°C for 16 hours with 10 rpm rotation in an oven (Labnet, Edison, NJ, USA). The slide was washed according to NimbleGen’s protocol (NimbleGen Arrays User’s Guide Gene Expression Analysis).

scan_protocol: After washing and drying, the microarray slides were immediately scanned in a GenePix 4000B Scanner (Axon Instruments, Union City, CA, USA) using the provided GenePix Pro 6.1 software. NimbleScan v2.5 software was then used for spot features extraction from the scanned images.

data_processing: Two types of normalizations were performed – normalization by DNA reference array and between-array normalization. For DNA reference array normalization, intensities of RNA signals were normalized with signals from DNA reference arrays (raw array data provided additionally) that were hybridized with fragmented genomic DNA chemically labeled with The Label IT Cy3 Reagent (Mirus Bio, Madison, WI, USA) . Both normalizations were done using the Bioconductor R package “tilingArray”.

organism: Porphyromonas gingivalis

source_name: Laboratory culture on BAPHK blood plate

label_protocol: The Label IT Cy3 Reagent (Mirus Bio, Madison, WI, USA) was used to directly label total RNA. To optimize the RNA labeling efficiency, 1 μg RNA was mixed with 4 μl Label IT Reagent and incubated in a 100-μl final volume for 4 hours at 37°C. To improve hybridization efficiency, the labeled RNA was fragmented to an average size of 80-100 nucleotides with 0.25 volume of 5X Fragmentation Buffer (Mirus Bio) and incubated at 94°C for 15 min. The fragmented and fluorescently labeled RNA was purified with the mirVana miRNA Isolation Kit before hybridization.

growth_protocol: P. gingivalis strain W83 was cultured anaerobically on trypticase soy agar (TSA) containing sheep blood, hemin and vitamin K (BAPHK) for 48 hours at 37°C. Upon harvest, a solution containing 2:1 (v:v) ratio of RNAprotect Bacteria Reagent (Qiagen, Valencia, CA, USA) and 1X PBS was poured onto the plates with colonies and incubated anaerobically for 5 min. The cells were mixed and suspended in this solution, centrifuged down in a microcentrifuge tube at 5,000×g, 4°C for 10 min, and then subjected to either RNA or DNA extraction.

extract_protocol: Lysis of bacterial cells was performed according to the protocol provided with the MasterPure RNA Purification Kit (Epicentre, Madison, WI, USA). The lysate was treated with Proteinase K at 65°C for 15 min and then placed on ice. To increase the recovery rate of small RNA 0.1 volume of 5 M NaCl was added to the lysate. Acid-phenol: chloroform (5:1) (v:v) extraction was then done using Phase-Lock Gel Heavy tubes (Eppendorf, Hauppauge, NY, USA). Total RNA in the upper aqueous phase was purified with a solid-phase extraction filter supplied in the mirVana miRNA Isolation Kit (Applied Biosystems/Ambion, Austin, TX, USA) according to the recommended protocol. The filter-trapped RNA was then washed and eluted with the provided wash solutions and elution buffer. To completely remove genomic DNA, the RNA extract was treated twice with Turbo DNase (Applied Biosystems/Ambion) at 37°C for 30 min, and purified again with the mirVana miRNA Isolation Kit.

tag: media

value: BAPHK

tag: labeled molecule

value: RNA


title: BAPHK_cDNA3 transcriptome profile

hyb_protocol: Microarray hybridization was carried out with the customer service provided by NimbleGen.

data_processing: Two types of normalizations were performed – normalization by DNA reference array and between-array normalization. For DNA reference array normalization, intensities of cDNA signals were normalized with signals from DNA reference arrays (raw array data provided additionally) that were hybridized with fragmented genomic DNA labeled with biotin. Both normalizations were done using the Bioconductor R package “tilingArray”.

label_protocol: Synthesis of cDNA and biotin end-labeling were performed according to NimbleGen’s protocol (Prokaryotic Biotin-Label Procedure). First-strand cDNA was synthesized from 10 μg total RNA. The RNA together with 3 μg random hexamer primers (Invitrogen, Carlsbad, CA, USA) in a 12-μl volume was denatured at 70°C for 10 min. The solution was then cooled to 25°C and mixed to a final concentration of 0.5 mM dNTP, 20 mM DTT, 1X first strand buffer and 0.75 U/μl RNaseOUT (Invitrogen) followed by heating to 42°C. A total of 1200 U SuperScript II reverse transcriptase (Invitrogen) was added to a final volume of 54 μl and the reaction incubated overnight at 42°C. The cDNA product was treated with 0.02 U/μl RNase H (Invitrogen) and 0.01 μg/μl RNase A (Epicentre) in a 100-μl reaction to eliminate RNA contaminants. The cDNA was then purified with standard procedures for phenol: chloroform extraction using Phase-Lock Gel Light tubes (Eppendorf) followed by ethanol precipitation. Purified cDNA was fragmented to 50-200 nucleotides in size with DNase I (Applied Biosystems/Ambion), and labeled at the 3’end in a 100-μl reaction containing 0.5 U/μl Terminal Deoxynucleotidyl Transferase (Promega, Madison, WI, USA) and 0.025 mM Biotin-N6-ddATP (Enzo Life Sciences Inc., Farmingdale, NY, USA). The labeling reaction was incubated for 2 hours at 37°C. The biotin-labeled cDNA was then concentrated using Microcon YM-10 filters (Millipore, Billerica, MA, USA).

value: cDNA


title: BAPHK_cDNA2 transcriptome profile


title: 50 ug/ml DRB-treated Jurkat cells

city: Toronto

source_name: 50 ug/ml DRB-treated Jurkat cells

value: 50 ug/ml 5,6-dichloro-1-beta-D-ribofuranosyl-benzimidazole (DRB)


title: L1236_stim_0_5h

value: stimulated with IL13

value: Hodgkin lymphoma cell line (L1236)


title: 4 ng/ml camptothecin-treated Jurkat cells

value: 4 ng/ml camptothecin


title: Cells_Treated_rep1

description: Replicate 1


title: mpkCCD clone 11 exposed to dDAVP for 5 days, biological replicate 1


title: Cells_Treated_rep2

description: Replicate 2


title: 10 ug/ml DRB-treated Jurkat cells

source_name: 10 ug/ml DRB-treated Jurkat cells

value: 10 ug/ml 5,6-dichloro-1-beta-D-ribofuranosyl-benzimidazole (DRB)


title: BAPHK_R15 transcriptome profile


title: L1236_stim_0h


title: L1236_unstim_0_5h

value: unstimulated


title: 25 ug/ml DRB-treated Jurkat cells

source_name: 25 ug/ml DRB-treated Jurkat cells

value: 25 ug/ml 5,6-dichloro-1-beta-D-ribofuranosyl-benzimidazole (DRB)


title: Cells_Control_rep2

description: Replicate 2

value: Untreated


title: Cells_Control_rep3

description: Replicate 3


title: L1236_unstim_0h


title: mpkCCD clone 11 exposed to vehicle for 5 days, biological replicate 3

description:
vasopressin-mediated endogenous aquaporin 2 expression
Gene expression data from cells in response to vehicle exposure for 5 days


title: mpkCCD clone 11 exposed to vehicle for 5 days, biological replicate 2


title: Cells_Control_rep1

description: Replicate 1


title: mpkCCD clone 11 exposed to vehicle for 5 days, biological replicate 1


title: DMSO-treated Jurkat cells

source_name: DMSO-treated Jurkat cells

value: Jurkat

value: DMSO


title: MAS98.12_33VSd10con_Ref2

status: Public on Dec 08 2010

city: Oslo

first: Eldrid

last: Borgan

address: Ullernchausseen 70

department: Genetics

country: Norway

institute: Oslo University Hospital Radiumhospitalet

description:
MAS98.12_33VSd10con
raw data file: 200509_251485044331_S01_GE1-v5_10_Apr08_1_3.txt

hyb_protocol: 1.65 μg cRNA was hybridized to 4 × 44 k Agilent Whole Human Genome Oligo Microarrays at 60°C and 10 rpm for 17 hours, according to Agilent's protocol (February 2007).

scan_protocol: The arrays were scanned using an Agilent G2565A DNA microarray scanner

data_processing:
The data were extracted using Feature Extraction (v 10.1.1.1, Agilent), The microarray data was normalized and analysed using R (v 2.9.0) and the LIMMA Bioconductor package. The microarray data was normalized seperately for the reference 1 and 2, because slightly different samples were included (See SAMPLES overview). The raw signals were corrected for multiplicative detrending effects and the arrays were quantile normalized and log2 transformed. Control probes, probes which were flagged as outliers by the Feature Extraction software, and probes that were present in less than 30% of the samples were removed. The signal intensities were averaged between duplicate probes, and the data matrices were imputed using k nearest neighbour imputing (impute package, Bioconductor).
The two sets of normalized data (Ref1 and Ref2) are included as separate SAMPLES. To avoid duplication, the raw data (Agilent Feature Extraction) files are provided in the RAW_AFE.tar archive on the Series record. Please see 'description' above for raw data file name. The 105 transcripts representing choline metabolism (used in reference 1) and 38 transcripts representing glucose metabolism (used in reference 2) are also provided as supplementary files on the Series record.

source_name: Breast tumor tissue from establlished orthotopic xenograft mouse model MAS98.12

label_protocol: Total RNA (700 ng) was amplified and labelled with Cy3 with Low RNA input linear amplification kit PLUS and RNA spike-in kit, according to Agilent's protocol (February 2007).

growth_protocol: The animals were fed RM1 diet (Scanbur BK, Norway) and distilled tap water ad libitum. The drinking water was supplemented with 17-β-estradiol at a concentration of 4 μg/ml in order to ensure stimulation of the estrogen receptors and promote tumor growth in the MAS98.06 xenografts. To provide equal experimental conditions, the MAS98.12 xenografts also received estradiol supplement.

extract_protocol: Trizol reagent (Invitrogen) was used to extract total RNA from fresh frozen tumor material according to the manufacturers protocol. Nanodrop and 2100 Bioanalyzer (Agilent) was used to assess the quantity and quality of RNA.

treatment_protocol: Established orthotopic xenograft models were sacrificed by cervical dislocation, tumor tissue was harvested from for 6 animals from each model for gene expression microarray analyses, at tumor diameters of approximately 13-15 mm. Samples were put in cryogenic vials and immersed in liquid nitrogen immediately after dissection and stored under cryogenic conditions until RNA extraction.

value: Breast tumor tissue

tag: xenograft model

tag: intrinsic subtype

value: BasalLike

tag: biological replicate

tag: used in reference


title: MAS98.12_53HSd3con_Ref2

description:
MAS98.12_53HSd3con
raw data file: 130509_251485044309_S02_GE1-v5_10_Apr08_1_4.txt

tag: tissue


title: MAS98.12_36HSd10con_Ref2

description:
MAS98.12_36HSd10con
raw data file: 130509_251485044310_S01_GE1-v5_10_Apr08_1_4.txt


title: MAS98.12_56HSd3con_Ref2

description:
MAS98.12_56HSd3con
raw data file: 050509_251485044290_S01_GE1-v5_10_Apr08_1_3.txt


title: MAS98.12_60HSd3con_Ref2

description:
MAS98.12_60HSd3con
raw data file: 120509_251485044294_S01_GE1-v5_10_Apr08_1_1.txt


title: MAS98.06_23HSd10con_Ref2

description:
MAS98.06_23HSd10con
raw data file: 040609_251485044341_S01_GE1-v5_10_Apr08_1_2.txt

source_name: Breast tumor tissue from establlished orthotopic xenograft mouse model MAS98.06

value: LuminalLike