title: delta-ccr4 Replicate3

city: Montreal

first: Andre

last: Nantel

state: QC

address: 6100 Royalmount

department: Biotechnology Research Institute

institute: National Research Council of Canada

hyb_protocol: The microarray slides were pre-hybridized for 2 hours at 42°C with DIGeasy hybridization buffer (Roche) containing 0.5 ug/ul of yeast tRNA (Roche) and 0.5 ug/ul of salmon sperm DNA (Invitrogen) and subsequently washed with 0.1X SSC and air dried. The slides were then hybridized with labeled cDNAs overnight at 42°C in DIGeasy hybridization buffer with yeast tRNA and salmon sperm DNA. Hybridization was done with an Advalytix SlideBooster. The slides were washed twice with SSC-0.2% SDS, once with 0.1X SSC-0.2% SDS and 3 times with 0.1X SSC. A final wash with isopropanol was performed and slides were air-dried.

scan_protocol: Slides were scanned with a ScanArray 5000 scanner (Perkin Elmer) at 10-µm resolution.

data_processing: Signal intensity was quantified with QuantArray software (Perkin Elmer) and final Lowess normalization and inspection of the data was done with the GeneSpring package GX v.7.3 (Agilent Technologies).

source_name: ccr4delta-delta knockout

label_protocol: 20 µg of total RNA was reverse transcribed using oligo(dT)21 in the presence of Cy5-dCTP (Perkin-Elmer-Cetus/NEN) and Superscript III reverse transcriptase (Invitrogen). Thereafter, template RNA was degraded by adding 2.5 units of RNase H (USB) and 1 ug of RNase A (Pharmacia) followed by incubation for 15 min at 37°C. The labeled cDNAs were purified with QIAquick PCR Purification Kit (Qiagen).

extract_protocol: Total RNA was isolated from quadruplicate independent biological samples using the Ambion RiboPure Yeast RNA isolation kit according to manufacturer’s instructions, with the exception that cell lysis was performed by Beadbeater using three cycles of two minutes of lysis with a one minute break between cycles.

treatment_protocol: Overnight cultures of the ccr4delta-delta mutant and the ccr4delta-delta/CCR4 complemented strain (both strains are URA+ ARG+ HIS+) were diluted to OD600=0.1 in 100ml of YPD and grown to OD600=1.0. Cells were recovered by centrifugation and the pellets washed in water. The cell pellets were snap frozen in liquid nitrogen and stored at -80°C.

value: ccr4delta-delta knockout

source_name: CCR4 reintegrant

value: CCR4 reintegrant


title: E0-12h_HDAC11-495_5 extraction5_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 input DNA;

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_HDAC11-495_6 extraction6_array1

description: channel ch1 is input DNA;


title: AdultFemale_H3K9Ac_2 extraction2_array3

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: 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.

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.

value: Adult Female

tag: Sex

value: Female


title: E8-12_CBP_1 extraction1_array2

extract_protocol:
Collection of Drosophila embryos at time-point 12-16h 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: AdultFemale_H3K9Ac_1 extraction1_array3


title: AdultFemale_H3K9Ac_3 extraction3_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: delta-ccr4 Replicate4

type: RNA

molecule: total RNA

organism: Candida albicans


title: FXRSHPko.3wk.rep1

city: Houston

first: Scott

middle: Andrew

last: Ochsner

state: TX

address: One Baylor Plaza

department: Molecular and Cellular Biology

country: USA

institute: Baylor College of Medicine

description: Gene expression in 3wk old FXR-/-/SHP-/- mouse liver

hyb_protocol: Labeled cRNA was hybridized to the Illumina mouse REF8 v1.1 Expression BeadChip using standard protocols (Illumina).

scan_protocol: Standard Illumina scanning protocol

data_processing: The probe profile output from the Illumina BeadStudio toolkit was read into R using the lumi BioC package. Then, the data was VST transformed (Lin, S.M., Du, P., Huber, W., and Kibbe, W.A. 2008. Model-based variance-stabilizing transformation for Illumina microarray data. Nucleic Acids Res 36:e11) followed by quantile normalization. The versions of software and BioC packages were as follows: R_2.8.0, lumi_1.8.2.

organism: Mus musculus

source_name: mouse liver

label_protocol: 500 ng of total RNA was reverse transcribed into cRNA and biotin-UTP labeled using the Illumina TotalPrep RNA Amplification Kit (Ambion). cRNA was quantified using an Agilent Bioanalyzer 2100

growth_protocol: Individual FXR-/- or SHP-/- mice were backcrossed with C57BL/6 mice to generate congenic C57BL/6 FXR-/- or SHP-/- mice. Congenic single knockout animals were bred with each other to generate FXR/SHP double knockout mice. Age-matched, male, DKO congenic and C57BL/6 wild-type (WT) mice were used throughout this study. WT control mice (C57BL/6) were purchased from Harlan Sprague Dawley, Inc. (Indianapolis, IN). Mice were sacrificed at 3 or 5 weeks after birth. Mice were housed on a standard 12-hour light/dark cycle and were fed normal chow and water ad libitum.

extract_protocol: Total RNA was prepared according to manufacturer’s protocol. (Trizol, Invitrogen). The RNA obtained was further purified using qiagen columns.

tag: age

value: 3 weeks

tag: gender

value: male

tag: tissue

value: liver

tag: genome/variation

value: FXR-/-,SHP-/- double knock out


title: delta-ccr4 Replicate1


title: FXRSHPko.3wk.rep2


title: delta-ccr4 Replicate2


title: WT.5wk.rep1

description: Gene expression in 5wk old wild type mouse liver

value: 5 weeks

value: wild type


title: FXRko.3wk.rep2

description: Gene expression in 3wk old FXR-/- mouse liver

value: FXR-/-


title: SHPko.3wk.rep2

description: Gene expression in 3wk old SHP-/- mouse liver

value: SHP-/-


title: S. salar_65_dpf_rep1

city: Victoria

first: Ben

middle: F

last: Koop

state: BC

address: PO Box 3020 STN CSC

department: Biology

institute: The University of Victoria

description: Biological replicate 1 of 3

hyb_protocol: cRNA fragmentation mixtures were created following the LIQA kit instructions, using 825 ng of experimental sample and 825 ng of reference pool. These mixtures were incubated at 60 ºC for 30 minutes. After cooling on ice for one minute, hybridization mixtures were prepared by adding 2x GEx Hybridization Buffer HI-RPM and mixing well by pipetting. These reactions were loaded in random arrangements with respect to time point onto 44K oligo salmonid microarrays (Agilent-025055) using Agilent SureHyb Hybridization Chambers. Each of the 4x44K arrays on the microarray slides had 100 uL of hybridization reaction added. The hybridization reactions were allowed to occur for 17 hours at 65 ºC. Slide washes were performed as per the manufacturer’s instructions, including an ozone-protection step using the Agilent Stabilization and Drying Solution.

scan_protocol: Slides were scanned as soon as possible on a ScanArray Express (Perkin Elmer) scanner at 5 µm resolution using a PMT setting of 80 in both channels, a black threshold of 1800, and a full color threshold of 26.8. Slides were stored in a low ozone chamber (typically <5 ppb) until scanned. Images were quantified using Imagene 8.0 (Biodiscovery).

data_processing: Background corrected signal medians converted to threshold of 1.0, log2 transformed, Lowess normalized, baseline transformation to median.

organism: Salmo salar

source_name: Whole tissue, 65 days post fertilization

label_protocol: Cy5 labeled experimental cRNA samples was generated using an Agilent Low Input Quick Amp (LIQA) kit, following the manufacturer’s instructions. For each time point, 40 ng of total RNA from three individuals was used to generate first-strand cDNA. Agilent Spike-In B control RNA was included in each reaction. After the denaturation step (10 min at 65 ºC) and cRNA synthesis step (2 hr at 40 ºC), the reactions were incubated at 70 ºC for 15 minutes to inactivate the AffinityScript enzyme, and subsequently stored at -80 ºC until further use. For the labeling reactions, thawed cRNA samples were each mixed with 16 µL of Transcription Master Mix cocktail containing Cy5 dye, and incubated at 40 ºC for two hours. Purification was performed using Qiagen RNeasy mini spin columns, eluting in 30 µL of RNase-free water. For the generation of the reference pool, equimolar amounts from the three individuals in each time point were pooled to give 120 ng of total RNA used in each first-strand reaction. Spike-In A control RNA was included in each reaction. After labeling with Cy3 and column purification as above, a common reference pool was created by including 2.8 µg of cRNA from each time point, except for D2, for which only 1.3 µg of material was available due to the small size of the animal at this stage.

growth_protocol: Eggs from Atlantic salmon (McConnell (Mowi)) were obtained in November, 2009 from Marine Harvest United Hatchery (Fanny Bay, B.C., Canada). The eggs were fertilized by gently mixing the eggs and milt by hand and then washed with partial exchanges of water. Approximately 2000 fertilized eggs were then transferred and placed in Heath trays (Marisource) at the University of Victoria. The embryos and larvae were raised in fresh water at a temperature of 12°C and a flow rate of 200 liters/h.

extract_protocol: Total RNAs were extracted in TRIzol reagent (Invitrogen) by mixer-mill homogenization (Retsch) and spin-column purified using RNeasy Mini kits (Qiagen).

value: whole organism

value: 65 days post fertilization

source_name: Pooled cRNA from whole tissues in all time points

tag: sample type


title: SHPko.3wk.rep1


title: E0-12h_HDAC11-495_3 extraction3_array1


title: WT.3wk.rep2

description: Gene expression in 3wk old wild type mouse liver


title: AdultFemale_H3K9Me3_3 extraction3_array1

description: channel ch1 is ChIP DNA; Antibody information listed below: official name: H3K9me3;target name: H3K9me3;host: Rabbit;antigen: Synthetic peptide conjugated to KLH derived from within residues 1 - 100 of Human Histone H3 tri methylated at K9.;clonal: Polyclonal;purified: Affinity;company: Abcam;catalog: ab8898;short description: Histone H3 (tri methyl K9) antibody - ChIP Grade (ab8898);


title: FXRko.3wk.rep1


title: E0-12h_HDAC11-495_4 extraction4_array1


title: WT.3wk.rep1


title: HBG3_Day7_r3

city: University Park

first: Shaun

last: Mahony

state: PA

address: 404 South Frear Bldg

department: Biochemistry & Molecular Biology

institute: Penn State University

description: Gene expression data from ES to Motor Neuron differentiation, Day7 (postmitotic motor neuron stage)

hyb_protocol: RNA was hybridized to Affymetrix Mouse Genome 430 2.0 microarrays.

scan_protocol: Arrays were scanned using the GeneChip Scanner 3000.

data_processing: Data analysis was carried out using the affylmGUI BioConductor package. GCRMA normalization was performed across all arrays, followed by linear model fitting using Limma. Differentially expressed genes after 8 hours of RA treatment were defined by ranking all probesets by the moderated t-statistic-derived p-value (adjusted for multiple hypothesis testing using Benjamini & Hochberg’s method and setting thresholds of p<0.001 and a fold-change of at least 2.

source_name: ES to Motor Neuron differentiation, Day 7

label_protocol: RNA was amplified using NuGen Ovation amplification and labeling kit according to manufacturer’s instructions

growth_protocol: ES cells were grown on ES Medium (DMEM, 15% FBS, Non-essential amino acids, Glutamine, Pen/Strept, 2-mercaptoethanol and LIF) on feeder cells for amplification or on Gelatin for differentiation and experiments.  Motor neuron differentiation was done as described (Wichterle & Peljto, Curr Protoc Stem Cell Biol, 2009). Briefly, ES cells were tripsinized  and allowed to form aggregates in ANDFK medium (Advanced DMEM/F12,  Neurobasal Medium, Knockout-SR, of Pen/Strep, L-Glutamine, and 2-mercaptoethanol. Day 0 is the day of tripsinization. At Day 2, RA and Hh agonist are added.

extract_protocol: RNA was extracted with Qiagen RNAeasy at different time points following manufacturer’s instructions.

tag: cell line


title: HBG3_Day7_r4


title: AdultFemale_H3K9Me3_2 extraction2_array1


title: HBG3_Day7_r5


title: AdultFemale_H3K9Me3_1 extraction1_array1


title: E16-20_H3K9Ac_3 extraction3_array1

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.


title: E16-20_H3K9Ac_2 extraction2_array1


title: E0-12h_HDAC6-497_5 extraction5_array1

description: channel ch1 is input DNA;


title: E0-12h_HDAC11-495_1 extraction1_array1

description: channel ch1 is ChIP DNA; Antibody information listed below: official name: HDAC11-495;target name: HDAC11;host: Rabbit;antigen: SFYEPTELTKDQLRRC;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: E0-12h_HDAC11-495_2 extraction2_array1


title: E0-12h_HDAC6-497_6 extraction6_array1

description: channel ch1 is input DNA;


title: E0-12h_HDAC6-497_4 extraction4_array1


title: HBG3_Day4_r3

description: Gene expression data from ES to Motor Neuron differentiation, Day4 (progenitor motor neuron stage)

source_name: ES to Motor Neuron differentiation, Day 4


title: Input vs. C20 1 of 2 100164

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

organism: Homo sapiens

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.

value: Jurkat

tag: fraction

value: TOTAL, input

tag: antibody

value: ERG C20

tag: antibody manufacturer

value: Santa Cruz Biotechnolgy

value: EXPERIMENTAL


title: L6-HGF sheet

city: Helsinki

first: Antti

last: Siltanen

address: Haartmaninkatu 8

country: Finland

institute: University of Helsinki, Institute of Biomedicine

hyb_protocol: Standard Affymetrix protocol

scan_protocol: Standard Affymetrix protocol

data_processing: RMA normalization performed using Genespring GX 11.

organism: Rattus norvegicus

source_name: Rat L6 myoblast sheet

label_protocol: Standard Affymetrix protocol

extract_protocol:
Trizol
RNeasy, Qiagen

value: Wistar

tag: myoblast culture

value: L6 myoblast sheet

tag: genotype/variation

value: HGF expression


title: Input vs. C17/C20 1 of 2 94520


title: E16-20_H3K9Ac_1 extraction1_array1

value: Unknown


title: HBG3_Day4_r2


title: L6-WT sheet


title: IgG vs.C20 1 of 2 94269

value: IgG

value: NONSPECIFIC-IgG


title: L6-WT monolayer culture

source_name: Rat L6 myoblast cell line

value: L6 rat skeletal myoblast cell line


title: HBG3_Day3_r2

description: Gene expression data from ES to Motor Neuron differentiation, Day3 (neural precursor stage)

source_name: ES to Motor Neuron differentiation, Day 3


title: L6-HGF monolayer culture


title: Ependymoma 15

city: Muenster

first: Björn

last: Koos

address: Domagkstr. 19

institute: Institute of Neuropathology, Muenster

description: Gene expression of ependymoma tumor cells

hyb_protocol: After confirmation of cRNA integrity, fragmented cRNA was hybridized for 16 h under constant rotation to U133A 2.0 gene chips (Affymetrix) representing 18,400 transcripts and variants of 14,500 human genes.

scan_protocol: After washing and staining (Fluidics Station 450; Affymetrix), fluorescence was scanned on a GeneChip Scanner 3000 (Affymetrix).

data_processing: Data files were RMA normalized and evaluated using the GeneSpring Software (Agilent). After global chip quality control and fluorescence gradient correction, differentially expressed probe sets (more than 2 fold) were identified.

source_name: LMPC cells of ependymoma tissue

label_protocol: RNA was amplified using the GeneChip Two-Cycle cDNA Synthesis kit (Affymetrix, Wooburn Green, UK) and subsequently transcribed to complementary RNA using the GeneChip IVT Labeling kit (Affymetrix).

extract_protocol: 10 000 LMPC cells were lysed and RNA was isolated using Qiagen Rneasy Micro Kit according to manufacturer's instructions.

value: ependymoma tissue

tag: malignancy

value: neoplastic


title: Ependymoma 13


title: AdultMale-K27Me3_3 extraction3_array3

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_HDAC6-497_3 extraction3_array1

value: Embryo 0-12h


title: HBG3_Day4_r1


title: HBG3_Day3_r3


title: Ependymoma 14


title: IgG vs. C17/C20 1 of 2 100126

value: Jurkat


title: E0-12h_HDAC6-497_2 extraction2_array1

description: channel ch1 is ChIP DNA; Antibody information listed below: official name: HDAC6-497;target name: HDAC6;host: Rabbit;antigen: EHFERLKETSGIRDDERC;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_array3

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: E0-12h_HDAC6-497_1 extraction1_array1


title: HBG3_Day3_r1


title: Male-K27Me3_1 extraction1_array3


title: Ependymoma 12


title: E0-12h_HDAC6-496_5 extraction5_array1

description: channel ch1 is input DNA;


title: HBG3_Day2_RA_r3

description: Gene expression data from ES to Motor Neuron differentiation, Day2 + 8hrs exposure to retinoic acid

source_name: ES to Motor Neuron differentiation, Day 2 + 8hrs exposure to retinoic acid


title: E0-12h_HDAC6-496_6 extraction6_array1

description: channel ch1 is input DNA;


title: E0-4_H3K9Me3_3 extraction3_array3

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.


title: E0-4_H3K9Me3_2 extraction2_array3


title: HBG3_Day2_RA_r1


title: E0-4_H3K27Ac_2 extraction2_array3


title: E0-12h_HDAC6-496_3 extraction3_array1


title: E0-12h_HDAC6-496_2 extraction2_array1

description: channel ch1 is ChIP DNA; Antibody information listed below: official name: HDAC6-496;target name: HDAC6;host: Rabbit;antigen: CGDERPSGSKPKVKVK;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: E0-4_H3K27Ac_1 extraction1_array3

description: channel ch1 is ChIP DNA; Antibody information listed below: official name: H3K27Ac;target name: H3K27Ac;host: Rabbit;antigen: Synthetic peptide conjugated to KLH derived from within residues 1 - 100 of Human Histone H3 acetylated at K27.;clonal: Polyclonal;purified: Affinity;company: Abcam;catalog: ab4729;short description: Histone H3 (acetyl K27) antibody - ChIP Grade;


title: E0-12h_HDAC6-496_1 extraction1_array1


title: Ependymoma 11


title: Ependymoma 09


title: Ependymoma 10


title: Ependymoma 08


title: E0-4_H3K27Ac_3 extraction3_array3

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


title: E0-4_H3K9Me3_1 extraction1_array3


title: E12-16_PolII_3 extraction3_array1

description: channel ch1 is ChIP DNA; Antibody information listed below: official name: PolII;target name: RNA Polymerase II;host: Mouse;antigen: The antibody 8WG16 was developed using RNA polymerase (RNA pol II) purified from wheat germ extract. It recognizes the C-terminal heptapeptide repeat present on the largest subunit of pol II. Clone 8WG16 has been used to investigate the role of the C-terminal domain of the 220kD RNA pol II subunit in the formation of the transcription complex.;clonal: Polyclonal;purified: Affinity;company: Covance;catalog: 8wg16;short description: Monoclonal Antibody against RNA Polymerase II;

extract_protocol:
Collection of Drosophila embryos at time-point 12-16h 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.


title: E0-12h_HDAC4a-493_6 extraction6_array1

description: channel ch1 is input DNA;


title: HBG3_Day2_r3

description: Gene expression data from ES to Motor Neuron differentiation, Day2 (embryoid body stage)

source_name: ES to Motor Neuron differentiation, Day 2


title: E12-16_PolII_2 extraction2_array1


title: E0-12h_HDAC4a-493_5 extraction5_array1

description: channel ch1 is input DNA;


title: HBG3_Day2_r2


title: HBG3_Day2_r4


title: HBG3_Day2_r1


title: E12-16_PolII_1 extraction1_array1


title: HBG3_Day0_r3

description: Gene expression data from ES to Motor Neuron differentiation, Day0 (ES stage)

source_name: ES to Motor Neuron differentiation, Day 0


title: E0-12h_HDAC6-496_4 extraction4_array1


title: Ependymoma 04


title: E0-12h_HDAC4a-493_4 extraction4_array1


title: Ependymoma 03


title: Ependymoma 06


title: Ependymoma 05


title: E0-12h_HDAC4a-493_3 extraction3_array1


title: HBG3_Day0_r2


title: Ependymoma 01


title: MS89 SANO N1

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:
HSF (healthy synovial fibroblasts) gene expression under normoxic conditions
US82003505_251485044354_S01_GE1-v5_10_Apr08_1_1.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.

source_name: HSF, normoxia, 20% Ox, 22h, experiment 1

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: disease status

value: healthy donor


title: MS89 SANO H1

description:
HSF (healthy synovial fibroblasts) gene expression under hypoxic conditions
US82003505_251485044354_S01_GE1-v5_10_Apr08_1_2.txt

source_name: HSF, hypoxia, 0.5% Ox, 22h, experiment 1


title: Ependymoma 02


title: E8-12_CBP_2 extraction2_array1

description: channel ch1 is ChIP DNA; Antibody information listed below: official name: CBP antibody;target name: nejire;host: Mouse;

tag: strain

tag: developmental stage


title: Ependymoma 07


title: E0-12h_HDAC4a-493_2 extraction2_array1

description: channel ch1 is ChIP DNA; Antibody information listed below: official name: HDAC4a-493;target name: HDAC4a;host: Rabbit;antigen: CSALETLKVEHDES;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.;

tag: genotype


title: MS89 SANO H2

description:
HSF (healthy synovial fibroblasts) gene expression under hypoxic conditions
US82003505_251485044354_S01_GE1-v5_10_Apr08_1_3.txt

source_name: HSF, hypoxia, 0.5% Ox, 22h, experiment 2

tag: age


title: E0-12h_HDAC4a-493_1 extraction1_array1


title: HBG3_Day0_r1


title: E8-12_CBP_1 extraction1_array1

tag: Sex


title: Ependymal tissue 07

description: Gene expression of non neoplastic ependymal cells

source_name: LMPC cells of ependymal tissue

tag: tissue

value: ependymal tissue

value: non neoplastic


title: AdultFemale_H3K9Ac_3 extraction3_array1

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;

value: Adult Female

value: Female


title: E0-12h_HDAC4a-492_5 extraction5_array1

description: channel ch1 is input DNA;


title: AdultFemale_H3K9Ac_2 extraction2_array1


title: E0-12h_HDAC4a-492_6 extraction6_array1

description: channel ch1 is input DNA;


title: AdultFemale_H3K9Ac_1 extraction1_array1


title: E0-12h_HDAC4a-492_4 extraction4_array1


title: AdultFemale_H3K9Me3_3 extraction3_array2

description: channel ch1 is ChIP DNA; Antibody information listed below: official name: H3K9me3;target name: H3K9me3;host: Rabbit;antigen: Synthetic peptide conjugated to KLH derived from within residues 1 - 100 of Human Histone H3 tri methylated at K9.;clonal: Polyclonal;purified: Affinity;company: Abcam;catalog: ab8898;short description: Histone H3 (tri methyl K9) antibody - ChIP Grade (ab8898);


title: E0-12h_HDAC4a-492_2 extraction2_array1

description: channel ch1 is ChIP DNA; Antibody information listed below: official name: HDAC4a-492;target name: HDAC4a;host: Rabbit;antigen: PIHDLPSEAGSDERC;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: AdultFemale_H3K9Me3_2 extraction2_array2


title: Ependymal tissue 05


title: MS89 SANO N2

description:
HSF (healthy synovial fibroblasts) gene expression under normoxic conditions
US82003505_251485044354_S01_GE1-v5_10_Apr08_1_4.txt

source_name: HSF, normoxia, 20% Ox, 22h, experiment 2

tag: gender


title: E0-12h_HDAC4a-492_1 extraction1_array1


title: Ependymal tissue 02


title: MS80 AR H1

description:
RASF (rheumatoid arthritis synovial fibroblasts) gene expression under hypoxic conditions
US82003505_251485032535_S01_GE1-v5_10_Apr08_1_1.txt

source_name: RASF, hypoxia, 0.5% Ox, 22h, experiment 1

value: rheumatoid arthritis patient


title: AdultFemale_H3K9Me3_1 extraction1_array2


title: Ependymal tissue 04


title: Ependymal tissue 03


title: MS80 AR N2

description:
RASF (rheumatoid arthritis synovial fibroblasts) gene expression under normoxic conditions
US82003505_251485032535_S01_GE1-v5_10_Apr08_1_4.txt

source_name: RASF, normoxia, 20% Ox, 22h, experiment 2


title: MS80 AR N1

description:
RASF (rheumatoid arthritis synovial fibroblasts) gene expression under normoxic conditions
US82003505_251485032535_S01_GE1-v5_10_Apr08_1_2.txt

source_name: RASF, normoxia, 20% Ox, 22h, experiment 1


title: MS80 AR H2

description:
RASF (rheumatoid arthritis synovial fibroblasts) gene expression under hypoxic conditions
US82003505_251485032535_S01_GE1-v5_10_Apr08_1_3.txt

source_name: RASF, hypoxia, 0.5% Ox, 22h, experiment 2


title: E16-20_H3K9Ac_3 extraction3_array2

value: Unknown


title: E0-12h_HDAC3-499_5 extraction5_array1

description: channel ch1 is input DNA;


title: E0-12h_HDAC4a-492_3 extraction3_array1

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


title: Ependymal tissue 01


title: MS83 AR H1

description:
RASF (rheumatoid arthritis synovial fibroblasts) gene expression under hypoxic conditions
US82003505_251485043584_S01_GE1-v5_10_Apr08_1_1.txt


title: E0-12h_HDAC3-499_4 extraction4_array1


title: E16-20_H3K9Ac_2 extraction2_array2


title: MS83 AR H2

description:
RASF (rheumatoid arthritis synovial fibroblasts) gene expression under hypoxic conditions
US82003505_251485043584_S01_GE1-v5_10_Apr08_1_3.txt


title: Ependymal tissue 06


title: E0-12h_HDAC3-499_3 extraction3_array1


title: AdultMale-K27Me3_3 extraction3_array1


title: E0-12h_HDAC3-499_6 extraction6_array1

description: channel ch1 is input DNA;


title: MS83 AR N2

description:
RASF (rheumatoid arthritis synovial fibroblasts) gene expression under normoxic conditions
US82003505_251485043584_S01_GE1-v5_10_Apr08_1_4.txt


title: MS83 AR N1

description:
RASF (rheumatoid arthritis synovial fibroblasts) gene expression under normoxic conditions
US82003505_251485043584_S01_GE1-v5_10_Apr08_1_2.txt


title: CD4Tcells_C57BL/6_PbA-infected_rep4

city: Herston

first: Ashraful

last: Haque

state: Brisbane

address: 300 Herston Road

department: Immunology

country: Australia

institute: Queensland Institute of Medical Research

hyb_protocol: Labelled cRNA was hybridized to Sentrix BeadChip Arrays, MouseWG-6 v2 (Illumina Inc, San Diego, CA, USA) according to standard Illumina protocols.

scan_protocol: Standard Illumina scanning protocol.

data_processing: The data were extracted in GenomeStudio (Illumina) using default analysis settings and no normalization method. The resulting data were imported into GeneSpring GX v11.0 (Agilent Technologies). The expression values were normalized using Percentile Shift Normalization with default settings.  Briefly, Percentile Shift Normalization is a global normalization, where the location of all the spot intensities in an array are adjusted. This normalization takes each column in an experiment independently, and computes the nth percentile of the expression values for this array, across all spots (where n has a range from 0-100 and n=50 is the median; n = 75 for this experiment).  It then subtracts this value from the expression value of each entity.  In GeneSpring GX, log transformation is done on the dataset before the normalization and hence the percentile is subtracted from the expression value.

source_name: Spleen, CD4 T cells, C57BL/6, Plasmodium berghei ANKA

label_protocol: Biotinylated cRNA was prepared with the Illumina TotalPrep RNA Amplification Kit (Ambion, Austin, TX).

extract_protocol: Total RNA was extracted using the RNeasy mini kit, according to manufacturer’s instructions, and contaminants were removed by passing samples over RNeasy mini-columns with on-column DNase treatment (Qiagen). RNA integrity was assessed by denaturing agarose gel electrophoresis, samples were included based on 28S:18S ratios ≥ 1.7. RNA concentrations were assessed using the Nanodrop ND-1000 (NanoDrop Technologies Inc, Wilmington, DE).

treatment_protocol: Mice were infected intravenously (i.v.) with 100,000 PbA-parasitized red blood cells (pRBCs). At day 4 post-infection, animals were sacrificed by CO2 asphyxiation, in accordance with animal ethics guidelines. Following sacrifice, spleens were removed. CD4+ T cells were isolated by fluorescence activated cell sorting. Cells were homogenised using QIAshredder spin columns and stored in RLT buffer from the RNeasy mini kit (Qiagen, Doncaster, Australia).

value: 8-10 weeks

tag: treatment

value: Plasmodium berghei ANKA

tag: time point

value: Day 4 post-infection

value: Spleen

value: CD4 T cells


title: Male-K27Me3_2 extraction2_array1

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;

value: Adult Male


title: CD4Tcells_C57BL/6_uninfected_rep4

source_name: Spleen, CD4 T cells, C57BL/6, uninfected

value: Uninfected


title: CD4Tcells_GzmB-/-_PbA-infected_rep4

source_name: Spleen, CD4 T cells, B6.GzmB-/-, Plasmodium berghei ANKA


title: Male-K27Me3_1 extraction1_array1

value: Male


title: E0-12h_HDAC3-499_1 extraction1_array1

description: channel ch1 is ChIP DNA; Antibody information listed below: official name: HDAC3-499;target name: HDAC3;host: Rabbit;antigen: CRQDNANSKQYLELIVK;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.;

value: Embryo 0-12h


title: E16-20_H3K9Ac_1 extraction1_array2


title: CD4Tcells_C57BL/6_uninfected_rep3


title: E0-12h_HDAC3-498_6 extraction6_array1

description: channel ch1 is input DNA;


title: E0-12h_HDAC3-499_2 extraction2_array1


title: MS95 AR N2

description:
RASF (rheumatoid arthritis synovial fibroblasts) gene expression under normoxic conditions
US82003505_251485043585_S01_GE1-v5_10_Apr08_1_3.txt

value: Synovial fibroblasts culture


title: MS95 AR H1

description:
RASF (rheumatoid arthritis synovial fibroblasts) gene expression under hypoxic conditions
US82003505_251485043585_S01_GE1-v5_10_Apr08_1_2.txt


title: CD4Tcells_GzmB-/-_PbA-infected_rep3


title: E0-12h_HDAC3-498_5 extraction5_array1

description: channel ch1 is input DNA;


title: E0-4_H3K9Me3_2 extraction2_array2


title: MS95 AR H2

description:
RASF (rheumatoid arthritis synovial fibroblasts) gene expression under hypoxic conditions
US82003505_251485043585_S01_GE1-v5_10_Apr08_1_4.txt


title: MS95 AR N1

description:
RASF (rheumatoid arthritis synovial fibroblasts) gene expression under normoxic conditions
US82003505_251485043585_S01_GE1-v5_10_Apr08_1_1.txt


title: E0-12h_HDAC3-498_4 extraction4_array1


title: MS86 SANO H1

description:
HSF (healthy synovial fibroblasts) gene expression under hypoxic conditions
US82003505_251485044320_S01_GE1-v5_10_Apr08_1_2.txt

value: healthy donor


title: CD4Tcells_C57BL/6_PbA-infected_rep3


title: CD4Tcells_C57BL/6_uninfected_rep1


title: E0-4_H3K9Me3_1 extraction1_array2

description: channel ch1 is ChIP DNA; Antibody information listed below: official name: H3K9me3;target name: H3K9me3;host: Rabbit;antigen: Synthetic peptide conjugated to KLH derived from within residues 1 - 100 of Human Histone H3 tri methylated at K9.;clonal: Polyclonal;purified: Affinity;company: Abcam;catalog: ab8898;short description: Histone H3 (tri methyl K9) antibody - ChIP Grade (ab8898);


title: E0-4_H3K9Me3_3 extraction3_array2


title: E0-12h_HDAC3-498_3 extraction3_array1


title: E0-12h_HDAC3-498_2 extraction2_array1

description: channel ch1 is ChIP DNA; Antibody information listed below: official name: HDAC3-498;target name: HDAC3;host: Rabbit;antigen: CSDPDVRISVADEDK;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: CD4Tcells_C57BL/6_PbA-infected_rep2


title: E0-4_H3K27Ac_1 extraction1_array1

description: channel ch1 is ChIP DNA; Antibody information listed below: official name: H3K27Ac;target name: H3K27Ac;host: Rabbit;antigen: Synthetic peptide conjugated to KLH derived from within residues 1 - 100 of Human Histone H3 acetylated at K27.;clonal: Polyclonal;purified: Affinity;company: Abcam;catalog: ab4729;short description: Histone H3 (acetyl K27) antibody - ChIP Grade;


title: E0-12h_HDAC3-498_1 extraction1_array1


title: E12-16_PolII_3 extraction3_array2

description: channel ch1 is ChIP DNA; Antibody information listed below: official name: PolII;target name: RNA Polymerase II;host: Mouse;antigen: The antibody 8WG16 was developed using RNA polymerase (RNA pol II) purified from wheat germ extract. It recognizes the C-terminal heptapeptide repeat present on the largest subunit of pol II. Clone 8WG16 has been used to investigate the role of the C-terminal domain of the 220kD RNA pol II subunit in the formation of the transcription complex.;clonal: Polyclonal;purified: Affinity;company: Covance;catalog: 8wg16;short description: Monoclonal Antibody against RNA Polymerase II;


title: E0-12h_HDAC1-501_6 extraction6_array1

description: channel ch1 is input DNA;


title: CD4Tcells_C57BL/6_uninfected_rep2


title: CD4Tcells_GzmB-/-_PbA-infected_rep2


title: E0-12h_HDAC1-501_5 extraction5_array1

description: channel ch1 is input DNA;


title: E12-16_PolII_2 extraction2_array2


title: MS86 SANO N1

description:
HSF (healthy synovial fibroblasts) gene expression under normoxic conditions
US82003505_251485044320_S01_GE1-v5_10_Apr08_1_1.txt


title: MS86 SANO N2

description:
HSF (healthy synovial fibroblasts) gene expression under normoxic conditions
US82003505_251485044320_S01_GE1-v5_10_Apr08_1_3.txt


title: E0-12h_HDAC1-501_4 extraction4_array1

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


title: CD4Tcells_C57BL/6_PbA-infected_rep1


title: E12-16_PolII_1 extraction1_array2


title: CD4Tcells_GzmB-/-_PbA-infected_rep1


title: MS86 SANO H2

description:
HSF (healthy synovial fibroblasts) gene expression under hypoxic conditions
US82003505_251485044320_S01_GE1-v5_10_Apr08_1_4.txt


title: MS90 SANO N1

description:
HSF (healthy synovial fibroblasts) gene expression under normoxic conditions
US82003505_251485044353_S01_GE1-v5_10_Apr08_1_2.txt


title: MS90 SANO H2

description:
HSF (healthy synovial fibroblasts) gene expression under hypoxic conditions
US82003505_251485044353_S01_GE1-v5_10_Apr08_1_3.txt


title: E0-12h_HDAC1-501_3 extraction3_array1


title: MS82 AR N

description:
RASF (rheumatoid arthritis synovial fibroblasts) gene expression under normoxic conditions
US82003505_251485044356_S01_GE1-v5_10_Apr08_1_1.txt

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


title: E8-12_CBP_3 extraction3_array3

description: channel ch1 is ChIP DNA; Antibody information listed below: official name: CBP antibody;target name: nejire;host: Mouse;


title: E8-12_CBP_2 extraction2_array3


title: MS82 AR H

description:
RASF (rheumatoid arthritis synovial fibroblasts) gene expression under hypoxic conditions
US82003505_251485044356_S01_GE1-v5_10_Apr08_1_2.txt

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


title: E0-12h_HDAC1-501_2 extraction2_array1

description: channel ch1 is ChIP DNA; Antibody information listed below: official name: HDAC1-501;target name: HDAC1;host: Rabbit;antigen: SGSGSGAGAKGAKENNI;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: NFAT5/TonEBP KO MEF cells, isotonic conditions

city: Philadelphia

first: Todd

last: Lamitina

address: 3700 Hamilton Walk, A702

department: Physiology

institute: University of Pennsylvania

hyb_protocol: The microarrays were washed at low (6X SSPE) and high (100mM MES, 0.1M NaCl) stringency and stained with streptavidin-phycoerythrin. Fluorescence was amplified by adding biotinylated anti-streptavidin and an additional aliquot of streptavidin-phycoerythrin stain

scan_protocol: A confocal scanner was used to collect fluorescence signal at 3um resolution after excitation at 570 nm. The average signal from two sequential scans was calculated for each microarray feature.

data_processing: Affymetrix Microarray Suite 5.0 was used to quantitate expression levels for targeted genes; default values provided by Affymetrix were applied to all analysis parameters. Border pixels were removed, and the average intensity of pixels within the 75th percentile was computed for each probe. The average of the lowest 2% of probe intensities occurring in each of 16 microarray sectors was set as background and subtracted from all features in that sector. Probe pairs were scored positive or negative for detection of the targeted sequence by comparing signals from the perfect match and mismatch probe features. The number of probe pairs meeting the default discrimination threshold (tau = 0.015) was used to assign a call of absent, present or marginal for each assayed gene, and a p-value was calculated to reflect confidence in the detection call. A weighted mean of probe fluorescence (corrected for nonspecific signal by subtracting the mismatch probe value) was calculated using the One-step Tukey's Biweight Estimate. This Signal value, a relative measure of the expression level, was computed for each assayed gene. Global scaling was applied to allow comparison of gene Signals across multiple microarrays: after exclusion of the highest and lowest 2%, the average total chip Signal was calculated and used to determine what scaling factor was required to adjust the chip average to an arbitrary target of 150. All Signal values from one microarray were then multiplied by the appropriate scaling factor.

label: Biotin

source_name: NFAT5/TonEBP knockout MEF cells, isotonic conditions

label_protocol: Total RNA was converted to first-strand cDNA using Superscript II reverse transcriptase primed by a poly(T) oligomer that incorporated the T7 promoter. Second-strand cDNA synthesis was followed by in vitro transcription for linear amplification of each transcript and incorporation of biotinylated CTP and UTP. The cRNA products were fragmented to 200 nucleotides or less, heated at 99degC for 5 min and hybridized for 16 h at 45degC to Affymetrix Mouse Gene-1_0-st-v1 microarrays

extract_protocol: Total RNA was isolated using a combined Trizol / Qiagen RNAeasy method

treatment_protocol: Non-synchronized cells were cultured for 20 hours in isotonic culture media.

value: mouse embryonic fibroblast (MEF) cells

value: NFAT5/TonEBP knockout

tag: condition

value: isotonic


title: AdultFemale_H3K9Ac_3 extraction3_array2

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;

value: Adult Female

value: Female


title: E0-12h_HDAC1-501_1 extraction1_array1


title: E8-12_CBP_1 extraction1_array3

value: Unknown


title: NFAT5/TonEBP KO MEF cells, hypertonic conditions

source_name: NFAT5/TonEBP knockout MEF cells, hypertonic conditions

treatment_protocol: Non-synchronized cells were cultured for 20 hours in hypertonic culture media (450 mosml / kg by addition of NaCl).

value: hypertonic


title: MS90 SANO N2

description:
HSF (healthy synovial fibroblasts) gene expression under normoxic conditions
US82003505_251485044353_S01_GE1-v5_10_Apr08_1_4.txt


title: Wild type MEF cells, isotonic conditions

value: wild type


title: Wild type MEF cells, hypertonic conditions


title: MS114 AR N

description:
RASF (rheumatoid arthritis synovial fibroblasts) gene expression under normoxic conditions
US82003505_251485044356_S01_GE1-v5_10_Apr08_1_3.txt

value: rheumatoid arthritis patient


title: E0-12h_HDAC1-500_6 extraction6_array1

description: channel ch1 is input DNA;


title: MS121 AR N

description:
RASF (rheumatoid arthritis synovial fibroblasts) gene expression under normoxic conditions
US82003505_251485050865_S01_GE1-v5_10_Apr08_1_1.txt


title: E0-12h_HDAC1-500_5 extraction5_array1

description: channel ch1 is input DNA;


title: AdultFemale_H3K9Ac_2 extraction2_array2


title: MS114 AR H

description:
RASF (rheumatoid arthritis synovial fibroblasts) gene expression under hypoxic conditions
US82003505_251485044356_S01_GE1-v5_10_Apr08_1_4.txt


title: MS90 SANO H1

description:
HSF (healthy synovial fibroblasts) gene expression under hypoxic conditions
US82003505_251485044353_S01_GE1-v5_10_Apr08_1_1.txt


title: AdultFemale_H3K9Ac_1 extraction1_array2


title: E0-4_H3K27Ac_3 extraction3_array1


title: E8-12_CBP_3 extraction3_array1


title: 6475C5_PocR(3)

first: Delphine

last: Saulnier

address: Feigin Center, Suite 830, 1102 Bates Avenue

department: Pathology

description: Biological replicate 3 of 3. Dye swap 2

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

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: 6475C5_PocR(2)

description: Biological replicate 2 of 3. Dye swap 2


title: AdultFemale_H3K9Me3_3 extraction3_array3

description: channel ch1 is ChIP DNA; Antibody information listed below: official name: H3K9me3;target name: H3K9me3;host: Rabbit;antigen: Synthetic peptide conjugated to KLH derived from within residues 1 - 100 of Human Histone H3 tri methylated at K9.;clonal: Polyclonal;purified: Affinity;company: Abcam;catalog: ab8898;short description: Histone H3 (tri methyl K9) antibody - ChIP Grade (ab8898);


title: E0-12h_HDAC1-500_3 extraction3_array1

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.


title: Input vs. C20 2 of 2 94878

tag: cell line

value: Jurkat

tag: fraction

value: TOTAL, input

tag: antibody

value: ERG C20

value: Santa Cruz Biotechnolgy

value: EXPERIMENTAL