title: RPD3-18Myc_WT_vs_NoTag_#2

status: Public on Nov 28 2010

city: Montréal

first: François

last: Robert

state: Québec

address: 110, avenue des Pins Ouest

department: Chromatin and Genomic Expression

institute: Institut de recherches cliniques de Montréal (IRCM)

description: Biological replicate 2 of 2. RPD3-18Myc occupancy measured by the IP/NoTag ratio in WT cells

hyb_protocol: Resuspend colored pellet in 110uL hybridization buffer (100uL DIGEasy Buffer, 5uL 5mg/mL Salmon Sperm DNA, 20uL 8mg/mL yeast tRNA). Put at 95C for 3 minutes. Put at 65C while waiting for hybridization. Follow standard Agilent chamber hybridization protocols. Incubate 16-20h at 40C in hybridization oven at 20RPM rotation speed. Post-Hybridization Washes: Separate coverslip and slide in Wash buffer #1. Wash slides in Wash buffer #1 for 5 minutes at room temperature on an orbital shaker (cover the dish to avoid light on the slides). Wash in 31C Wash buffer #2 for 5 minutes on an orbital shaker. Take slides out of the buffer slowly to dry them, and allow them to air dry for 1 minute. Wash Buffer #1: 6x SSPE, 0.005% N-lauroylsarcosine. Wash Buffer #2: 0.06x SSPE.    Stripping is done in 500 ml of 5 mM Potassium phosphate buffer pH 6.6. 1. Use a 2L beaker, which can fit a slide rack. Pour the stripping buffer, submerge the rack with slides (from 1 to 10) and heat it up slowly while stirring with a stir bar until the liquid boils with big bubbles. It usually takes 15 to 20 minutes. Do not overboil it. 2. Remove the rack and briefly rinse in a container with water. Slowly remove the rack from the liquid. Cy5 channel will be stripped much better than Cy3, but this does not affect the performance of subsequent hybridizations. Stock solution of 1M Potassium phosphate buffer, pH 6.6: Mix 3.81 ml of 1M K2HPO4 + 6.19 ml of 1M KH2PO4. Dilute 2.5 ml of this 1M solution in 500 ml Milli-Q water to obtain 5mM Potassium phosphate buffer, pH 6.6.

scan_protocol: Axon GenePix 4000B scanner and the GenePix Pro extraction software were used. Scan at 100% laser power for both channels. Set the Pixel Size to 5um / pixel. Set the Lines to average to 1. Set the Focus Position to 0um. Adjust PMTs so that approximately 1-2% spots are saturated. This is done to ensure full dynamic range utilization. Adjust PMTs so that the intensity ratio is 0.9 - 1.1 when looking at intensity values greater than or equal to 3000 on the Intensity / Frequence histogram. Images were quantified using Axon GenePix (version 6.1.0.4).

data_processing:
The raw data were corrected (foreground-background) then normalized using the limma's loess function (Yang et al.,2002) in BioConductor (from the ArrayPipe Analysis Pipeline (Hokamp et al., 2004)) and replicates were combined using a weighted average method as described previously (Pokholok et al., 2005). The combined data were next transformed to Z-scores (discarding the mitochondrial values) for the analyses.

Bioconductor packages: Biobase version 1.12.1 and limma version 2.9.1; R version 2.4.0.

label_protocol: Blunting: Do everything on ice! Add 70uL of the blunting solution (11uL NEB Buffer #2, 0.5uL BSA 10mg/mL, 0.5uL 20mM dNTPs, 0.2uL T4 DNA Polymerase 3U/uL, 57.8uL ddH2O) to 40uL of the resuspended DNA obtained at the end of the cleanup step. For WCEs, use 2uL + 38uL ddH2O instead of 40uL. Incubate 20 minutes at 12C. Add 11.5uL 3M NaOAc, pH 5.2, and 0.5uL Glycogen 20mg/mL and vortex briefly. Extract with 120uL Phenol/Chloroform/Isoamyl alcohol (25:24:1). Transfer 110uL to a new tube, and add 230uL -20C 100% EtOH. Put at -80C for 30min, then spin 20 minutes at 4C. Wash the pellet with 500uL -20C 70% EtOH and spin 15 minutes at 4C. Resuspend the pellet in 25uL ddH2O and place on ice.    Ligation: Thaw everything on ice, and do all manipulations on ice. Add 25uL of ligase mix (10uL 5x T4 Ligase Buffer, 6.7uL 15uM unidirectional linkers, 0.5uL T4 DNA Ligase, High Concentration, 8uL ddH2O) to the resuspended pellet from the blunting step. Incubate overnight at 16C. Add 6uL 3M NaOAc, pH 5.2, and vortex. Add 130uL -20C 100% EtOH and centrifuge 20 minutes at 4C. Wash the pellet (should be a smear on the side of the tube) with 500uL -20C 70% EtOH. Spin 15 minutes at 4C. Dry the pellet and resuspend in 25uL ddH2O.     Labeling: Add 15uL of PCR Labeling mix (4uL 10x ThermoPol Buffer, 2uL 5mM aa-dUTP mix, 1.25uL Oligo FR1, 7.75uL ddH2O) to the pellet from the ligation step. Start the PCR program, and during the first step (4 minutes at 55C), add 10uL of the polymerase mix (1uL 10x ThermoPol buffer, 1uL Taq Polymerase 5U/uL, 0.01uL Pfu Polymerase Turbo 2.5U/uL, 8uL ddH2O). PCR cycling conditions: 1) 5:00 at 55C 2) 5:00 at 72C 3) 2:00 at 95C 4) 0:30 at 95C 5) 0:30 at 55C 6) 1:00 at 72C 7) Repeat steps 4-6 31 more times. 8) 4:00 at 72C 9) Forever at 4C Purify PCR reactions with Qiagen PCR Cleanup kits, following their protocol BUT use a phosphate wash buffer and a phosphate elution buffer instead of their buffers PE and EB, respectively. Also, wash the columns only once, and elute twice with 30uL elution buffer. Speed-vac the eluate to dryness. Resuspend the pellets with fresh 4.5uL 0.1M Sodium Carbonate Buffer, pH 9.0. Add 4.5uL of the appropriate NHS-ester Cy-dye. Fresh tubes of NHS-ester Cy-dye should be resuspended in 73uL DMSO. Mix well, and incubate at room temperature in the dark for one hour. Add 35uL 0.1M NaOAc, pH 5.2. Purify using the Qiagen PCR Cleanup protocol using all their buffers, but wash only once and elute twice in 30uL. It is a good idea to elute both samples of a pair (Cy5 and Cy3) in the same tube to avoid DNA loss upon later resuspension. Speed-vac until 10-20uL are left, or to drynes+B33s. Phosphate Wash Buffer (100mL): 0.5mL 1M KPO4, pH 8.5 15.25mL ddH2O 84.25mL 95% EtOH Solution will be cloudy upon mixing. Phosphate Elution Buffer (10mL): 0.04mL 1M KPO4, pH 8.5 9.96mL ddH2O 1M KPO4, pH 8.5 (10mL): 9.5mL 1M K2HPO4 0.5mL 1M KH2PO4 1M Sodium Carbonate Buffer, pH 9.0 (100mL): 10.8g Na2CO3 80mL ddH2O pH to 9.0 with concentrated HCl Adjust volume to 100mL with ddH2O DILUTE 1:10 WHEN USING! Solution will change composition over time. Use only if less than a month old. Reference: Drouin S., Robert F. Methods (in press).

growth_protocol: Cultures were started in 50mL YEP+2% Glucose from one colony of appropriate strain and incubated overnight to OD600 0.8-1.0. Cultures were poured in 50mL Falcon tube containing 1.4mL 37% formaldehyde, and put on rotating wheel for 30min. Crosslinking was stopped by addition of 2.5mL 2.5M Glycine.

extract_protocol: Crosslinked cells are pelleted at 3.7k RPM in Beckman centrifuge (GS 6R Rotor 3.8). Cells are resuspended in 40mL ice cold TBS and pelleted again. This is repeated a second time. Cell pellets are resuspended in the remaining liquid (approx. 1mL) and transfered to 1.5mL screw cap tubes. Cells are pelleted by centrifugation, the supernatant removed by pipetting, and the cells quick-frozen by immersion in liquid nitrogen. Cells are thawed on ice, and resuspended in 700mL lysis buffer with protease inhibitors (solution composition detailed at the end). 500uL of acid-washed glass beads (Sigma) are added, and the cells are lysed using the bead-beater (4x5min cycles, with 5min breaks on ice). The tubes are pierced with a 18-gauge syringe needle to allow elution of lysate, but not of beads. The pierced tubes are put in 2mL collection tubes and spun briefly to 6000 RPM. The lysate is then homogenized by pipetting and sonicated 4x 20sec with a 1 minute break between sonication cycles. The sonicated lysates are centrifuged, and the supernatant is saved and frozen at -80C, or used immediately for ChIP. Lysis buffer: 50mM HEPES-KOH, pH 7.5; 140mM NaCl; 1mM EDTA; 1% Triton X-100; 0.1% Na-deoxycholate; 1mM PMSF; 1mM Benzamidine; 10ug/mL Aprotinin; 1ug/mL Leupeptin; 1ug/mL Pepstatin.    For the immunoprecipitation, beads are washed twice with 10mL PBS + 5% BSA (freshly made), and resuspended in 2mL PBS + 5% BSA. Antibodies (at the concentration indicated below) are added and incubated overnight at 4C in haematological mixer. Beads are washed twice with 10mL PBS + 5% BSA (freshly made), and resuspended in 30uL PBS + 5% BSA / ChIP. 400-500uL of lysate are added to 30uL beads, and incubated overnight at 4C in haematological mixer. Beads are then washed (using the Dynal magnet system) twice with 1mL ice cold Lysis Buffer (no protease inhibitors), twice with 1mL ice cold Lysis Buffer (no protease inhibitors) + additional 360mM NaCl (720uL of 5M NaCl per 10mL lysis buffer), twice with ice cold Wash Buffer, and once with ice cold TE. Remaining liquid is removed by pipetting, and 50uL TE + 1% SDS is added. Beads are incubated overnight at 65C to reverse the crosslinks. Note for Whole Cell Extracts (WCE): WCE are included in the protocol only at the crosslink reversal step. Add 45uL TE + 1% SDS to 5uL WCE, and put overnight at 65C to reverse the crosslinks. Lysis Buffer (no protease inhibitors): 50mM HEPES-KOH, pH 7.5; 140mM NaCl; 1mM EDTA; 1% Triton X-100; 0.1% Na-deoxycholate. Wash Buffer: 10mM Tris-HCl, pH 8.0; 250mM LiCl; 0.5% NP40; 0.5% Na-deoxycholate; 1mM EDTA. Reference: Drouin S., Robert F. Methods (in press). The 9E11 antibody (provided by Alain Verreault, IRIC) was used (10uL / ChIP) with anti-mouse Dynabeads (50uL / ChIP). The 9E10 antibody (from the Young lab) was used (2uL / ChIP) with anti-mouse Dynabeads (50uL / ChIP). The RYH4 (provided by Alain Verreault, IRIC) and 8WG16 anti-Rpb1 CTD (from the Young lab) antibodies were used (2uL / ChIP) with protein G Dynabeads (50uL / ChIP). The H3K36me3 (Abcam ab9050) and H4K5Ac (Upstate 07-327) antibodies were used (4uL / ChIP) with protein G Dynabeads (50uL / ChIP). The H5 antibody (Covance Research, MMS-129R-200) was used (5uL / ChIP) with protein G Dynabeads (50uL / ChIP).    To clean up the DNA, beads are spun down and supernatant is transfered to a new 1.5mL tube. 350uL of a solution of 345uL TE / 3uL RNAse A (10mg/mL) / 2uL Glycogen (20mg/mL) is added to the supernatant and incubated at 37C for 2 hours. 15uL 10% SDS and 7.5uL Proteinase K are then added and incubated for another 2 hours at 37C. The samples are then extracted twice with 450uL phenol/chloroform/isoamyl alcohol (25:24:1). 16uL of 5M NaCl is added to the final extract of about 350uL. 2 volumes of freezing 100% EtOH (-20C) are added, and the samples are put at -80C for 30 minutes. They are then centrifuged at maximum velocity for 20 minutes at 4C. The supernatant is discarded, and the pellet is washed with 500uL 70% EtOH (-20C) and centrifuged 15 minutes at 4C. The supernatant is discarded, the pellet resuspended in 50uL TE and stored at -20C.

tag: antibody

value: Anti-Myc 9E10 (Young Lab)


title: RPD3-18Myc_WT_vs_NoTag_#1

type: genomic

country: Canada

description: Biological replicate 1 of 2. RPD3-18Myc occupancy measured by the IP/NoTag ratio in WT cells

molecule: genomic DNA

organism: Saccharomyces cerevisiae

tag: strain


title: Uremic Diet vrs Control rats replicate 1

status: Public on May 11 2010

type: RNA

city: Jerusalem

first: Mony

last: Shuvy

address: Ein-Karem

department: Cardiology

country: Israel

institute: Hadassah Hospital

description: Technical Replicate 1 of 2

hyb_protocol: Equal amounts of Cy3 or Cy5 labeled cRNA were hybridized to the 4 X 44K Agilent Whole Rat Genome Oligo-Microarray (Agilent Technologies, USA) for 17 hours at 60°c in an Agilent DNA-Microarray Hybridization Oven. The arrays were later washed using Gene Expression Wash Buffer Kit from Agilent.

scan_protocol: The Microarray was scanned using the GenePix4000B Scanner (Axon) and the data was extracted from the resulting images using GenePix Pro 4.1 software (Axon).

data_processing: The resultant files (gpr) produced by GenePix have been analyzed utilizing LIMMA package of the R software. The Cy5 and Cy3 intensities within each array were normalized using the print-tip locally weighted scatter plot smoothing (LOESS) function, while no background correction was applied. To identify differentially expressed genes, a parametric empirical Bayesian approach implemented in LIMMA was used. A moderated t test was performed in parallel, with the use of a false discovery rate correction for multiple testing. LIMMA calculated an emission intensity A value [A = (log2(Cy5) × Cy3)/2] where Cy5 and Cy3 are the normalized emission intensities of each feature. P value < 0.05 confidence level has been used to pinpoint those significantly differentiated genes. Genes had to have an A-value (average expression level for the gene across all arrays and channels) of more than 8.5, thus avoiding faint emissions.

molecule: total RNA

organism: Rattus norvegicus

source_name: Rat Aortic valves_Uremic Diet_9 weeks

label_protocol: One microgram of total RNA was amplified and labeled with a fluorescent dye (either Cy3 or Cy5), using the Low RNA Input Linear Amplification & Labeling kit (Agilent Technologies, Palo Alto, CA, USA). The amount and quality of the resulting labeled cRNA was measured using the ‘micro-array measurement mode’ of the Nanodrop (ND-1000) spectrophotometer (NanoDrop Technologies Wilmintog, Delaware, USA).

growth_protocol: Sixty one Sprague-Dawley female rats, 8 weeks old, each weighing about 250 g, were used for the study. The protocol was approved by the Hebrew University Ethics Committee.

extract_protocol: Sample preparation:1. Homogenize tissue samples in TRI REAGENT (1ml per 50-100 mg of tissue) 2. Add 200ul of chloroform per ml of TRI REAGENT used. Cover the sample tightly, shake vigorously for 15 seconds and allow to stand for 2-15 minutes at room temperature. Centrifuge the resulting mixture at 12,000xg for 15 minutes at 4 C .RNA Isolation:  1. Transfer the aqueous phase to a fresh tube and add 0.5ml of isopropanol per ml of TRI REAGENT used in sample preparation. Allow the sample stand for 5-10 minutes at room temperature. Centrifuge at 12,000 g for 10 min at 4C . The RNA precipitate will form pellet on the side and bottom of the tube. 2. Remove the supernatant and wash the RNA pellet by adding 1 ml (minimum) of 75% ethanol per 1ml of TRI REAGENT used in sample preparation, step 1. Vortex the sample and then centrifuge at 7,500 x g for 5 min at 4C. 3. Briefly dry the RNA pellet for 5-10 min by air drying or under vacuum. Add an appropriate volume of water.To facilitate dissolution, mix by repeated pipetting with a micropipette at 55-60C for 10 min.       Sample preparation:1. Homogenize tissue samples in TRI REAGENT (1ml per 50-100 mg of tissue) 2. Add 200ul of chloroform per ml of TRI REAGENT used. Cover the sample tightly, shake vigorously for 15 seconds and allow to stand for 2-15 minutes at room temperature. Centrifuge the resulting mixture at 12,000xg for 15 minutes at 4 C .RNA Isolation:  1. Transfer the aqueous phase to a fresh tube and add 0.5ml of isopropanol per ml of TRI REAGENT used in sample preparation. Allow the sample stand for 5-10 minutes at room temperature. Centrifuge at 12,000 g for 10 min at 4C . The RNA precipitate will form pellet on the side and bottom of the tube. 2. Remove the supernatant and wash the RNA pellet by adding 1 ml (minimum) of 75% ethanol per 1ml of TRI REAGENT used in sample preparation, step 1. Vortex the sample and then centrifuge at 7,500 x g for 5 min at 4C. 3. Briefly dry the RNA pellet for 5-10 min by air drying or under vacuum. Add an appropriate volume of water.To facilitate dissolution, mix by repeated pipetting with a micropipette at 55-60C for 10 min.

treatment_protocol: Aortic valves samples were obtained from 3 groups: a Uremic-diet group, a reversibility group and a control group (n=7 each). The rats in the Uremic-diet and the reversibility group were fed exclusively with a uraemia induced high-adenine (0.75%), high-phosphate diet (1.5%). The control group received normal rat chow with no treatment. After 9 weeks, rats in the Uremic-diet group and in the control were anaesthetized using ketamine/xylazine and sacrificed; the rats in the reversibility group were kept alive for an additional 10 weeks, on a normal rat chow, and then sacrificed.

value: Sprague-Dawley

tag: sex

value: Female

tag: feed

value: Uremic diet

tag: feeding time

value: 9 weeks

source_name: Rat Aortic valves_Normal Diet_9 weeks

value: Normal Diet


title: SW620_Lysate_rep2

status: Public on Apr 16 2010

city: Nagaizumi-cho

first: Keiichi

last: HATAKEYAMA

state: Sunto-gun, Shizuoka

address: 1007 Shimonagakubo

country: Japan

institute: Shizuoka Cancer Center Research Institute

description: miRNA expression in Cellular lysate of SW620

hyb_protocol: Cy3-labelled miRNA was hybridized to Agilent Human miRNA Microarray Kit (8×15K) Ver3.0 (G4470C) for 20 hours at 55°C in a rotating Agilent hybridization oven.  After hybridization, microarrays were washed 5 minute at room temperature with GE Wash Buffer 1 (Agilent) and 5 minute at 37°C with GE Wash buffer 2 (Agilent), then dried.

scan_protocol: Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2565BA) using one color scan setting for 8x15k array slides (Scan Area 61x21.6 mm, Scan resolution 5um, Dye channel is set to Green and Green PMT is set to 100%, No XDR).

data_processing: The scanned images were analyzed with Feature Extraction Software 10.7.3.1 (Agilent) using default parameters (protocol miRNA_107_Sep09 and Grid: 021827_D_F_20091031) to obtain background subtracted and spatially detrended Processed Signal intensities.

organism: Homo sapiens

source_name: SW620, lysate, replicate 2

label_protocol: Cyanine-3 (Cy3) labeled miRNA was prepared from 100 ng  for cellular RNA analysis and 20 ng for extracellular RNA analysis using the miRNA Complete Labeling Reagent and Hyb Kit (Agilent) according to the manufacturer's instructions (Agilent microRNA microarrays version 2.2).

growth_protocol: Cancer cell lines including AZ-521, AZ-P7a, NIH-H69, SBC-3, DMS53, SW480, and SW620 were grown in RPMI1640 medium (Sigma, St. Louis, MO) supplemented with 10% heat-inactivated fetal bovine serum (Invitrogen, Carlsbad, CA), 100 U/ml penicillin, and 0.1 mg/ml of streptomycin in a humidified 5% CO2 incubator at 37°C.

extract_protocol: Cells, exosomes, and supernatant fractions from culture medium were subjected to isolation of total RNA using miRNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's protocols. The protocols include digestion of genomic DNA with RQ1 RNase-Free DNase (Promega) in the presence of RNasin Plus RNase Inhibitor (Promega, Madison, WI) and the following purification with the RNeasy MinElute Cleanup Kit (Qiagen). RNA was quantified using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Wilmington, DE)  and quality was monitored with the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA).

treatment_protocol: Cells were cultured in complete RPMI-1640 medium (Sigma) for 24 h at 37°C and 5% of CO2, washed twice with phosphate-buffered saline (PBS), and incubated 48 h in phenol red-free RPMI-1640 medium containing 10% heat-inactivated fetal bovine serum which was previously depleted contaminating microvesicles by overnight centrifugation at 100,000×g. Culture medium was collected and centrifuged at 800×g for 5 min and additional 2,000×g for 10 min to remove lifted cells. For isolation of exosomes, the supernatant was subjected to filtration on a 0.1 μm pore polyethersulfone membrane filter (Corning, Corning, NY) to remove cell debris and large vesicles, followed by concentration by CentriPlus-70 with a 100,000 Mw cut-off membrane (Millipore, Billerica, MA). The supernatant was then ultracentrifuged at 100,000×g for 1 h at 4°C using 70Ti rotor (Beckman Coulter, Brea, CA). The resulting pellets were resuspended in PBS and ultracentrifuged at 100,000×g for 1 h at 4°C using 100Ti rotor (Beckman Coulter). For RNA isolation from culture medium, after cells were grown for 48 h as described above, culture medium was collected and centrifuged at 800×g for 5 min and additional 2,000×g for 10 min. The supernatant was subjected to filtration on a 0.22 μm pore polyethersulfone membrane filter (Corning), followed by concentration by CentriPlus-70 with a 5,000 Mw cut-off membrane (Millipore). The supernatant was mixed with equal volume of QIAzol Lysis Reagent (Qiagen) and followed the procedure for RNA isolation described below. For RNA isolation from cells, cells were harvested by treatment of Trypsin-EDTA (Invitrogen) and washed twice with PBS (pH 7.4).

tag: cell line type

value: colon cancer

tag: fraction

value: lysate

tag: cell line


title: SW480_Lysate_rep1

description: miRNA expression in Cellular lysate of SW480

source_name: SW480, lysate, replicate 1


title: SBC-3_exosome_rep2

description: miRNA expression in exosome of SBC-3

source_name: SBC-3, exosome, replicate 2

value: small cell lung carcinoma

value: exosome


title: SW620_exosome_rep2

description: miRNA expression in exosome of SW620

source_name: SW620, exosome, replicate 2


title: SW620_culture_medium_rep1

description: miRNA expression in culture medium of SW620

source_name: SW620, culture medium, replicate 1

value: culture medium


title: SW620_culture_medium_rep2

source_name: SW620, culture medium, replicate 2


title: SW480_exosome_rep2

description: miRNA expression in exosome of SW480

source_name: SW480, exosome, replicate 2


title: SBC-3_Lysate_rep2

description: miRNA expression in Cellular lysate of SBC-3

source_name: SBC-3, lysate, replicate 2


title: SW480_Lysate_rep2

source_name: SW480, lysate, replicate 2


title: SBC-3_culture_medium_rep1

description: miRNA expression in culture medium of SBC-3

source_name: SBC-3, culture medium, replicate 1


title: SBC-3_exosome_rep1

description: miRNA expression in exosome of SBC-3

source_name: SBC-3, exosome, replicate 1


title: SBC-3_culture_medium_rep2

source_name: SBC-3, culture medium, replicate 2


title: SW480_exosome_rep1

description: miRNA expression in exosome of SW480

source_name: SW480, exosome, replicate 1


title: SW620_Lysate_rep1

source_name: SW620, lysate, replicate 1


title: SBC-3_Lysate_rep1

source_name: SBC-3, lysate, replicate 1


title: DMS53_exosome_rep2

description: miRNA expression in exosome of DMS53

source_name: DMS53, exosome, replicate 2


title: SW480_culture_medium_rep2

description: miRNA expression in culture medium of SW480

source_name: SW480, culture medium, replicate 2


title: SW480_culture_medium_rep1

source_name: SW480, culture medium, replicate 1


title: DMS53_exosome_rep1

description: miRNA expression in exosome of DMS53

source_name: DMS53, exosome, replicate 1


title: DMS53_culture_medium_rep2

description: miRNA expression in culture medium of DMS53

source_name: DMS53, culture medium, replicate 2


title: SW620_exosome_rep1

description: miRNA expression in exosome of SW620

source_name: SW620, exosome, replicate 1


title: DMS53_Lysate_rep2

description: miRNA expression in Cellular lysate of DMS53

source_name: DMS53, lysate, replicate 2


title: AZP7a_culture_medium_rep2

description: miRNA expression in culture medium of AZP7a

source_name: AZP7a, culture medium, replicate 2

value: gastric cancer


title: DMS53_culture_medium_rep1

source_name: DMS53, culture medium, replicate 1


title: AZP7a_exosome_rep1

description: miRNA expression in exosome of AZP7a

source_name: AZP7a, exosome, replicate 1


title: DMS53_Lysate_rep1

source_name: DMS53, lysate, replicate 1


title: AZP7a_exosome_rep2

description: miRNA expression in exosome of AZP7a

source_name: AZP7a, exosome, replicate 2


title: AZP7a_culture_medium_rep1

source_name: AZP7a, culture medium, replicate 1


title: AZP7a_Lysate_rep1

description: miRNA expression in Cellular lysate of AZP7a

source_name: AZP7a, lysate, replicate 1


title: AZP7a_Lysate_rep2

source_name: AZP7a, lysate, replicate 2


title: AZ521_culture_medium_rep2

description: miRNA expression in culture medium of AZ521

source_name: AZ521, culture medium, replicate 2


title: AZ521_exosome_rep1

description: miRNA expression in exosome of AZ521

source_name: AZ521, exosome, replicate 1


title: AZ521_Lysate_rep1

description: miRNA expression in Cellular lysate of AZ521

source_name: AZ521, lysate, replicate 1


title: NCI_H69_culture_medium_rep1

description: miRNA expression in culture medium of NCI H69

source_name: NCI H69, culture medium, replicate 1

value: NCI H69


title: AZ521_Lysate_rep2

source_name: AZ521, lysate, replicate 2


title: NCI_H69_Lysate_rep2

description: miRNA expression in Cellular lysate of NCI H69

source_name: NCI H69, lysate, replicate 2


title: AZ521_culture_medium_rep1

source_name: AZ521, culture medium, replicate 1


title: NCI_H69_exosome_rep1

description: miRNA expression in exosome of NCI H69

source_name: NCI H69, exosome, replicate 1


title: NCI_H69_Lysate_rep1

source_name: NCI H69, lysate, replicate 1


title: NCI_H69_culture_medium_rep2

source_name: NCI H69, culture medium, replicate 2


title: Organs_control_rep4

status: Public on Feb 01 2010

city: Madrid

first: Julio

middle: M

last: Coll

state: Madrid

address: crt Coruna km7

department: Biotechnology

country: Spain

institute: INIA

description: VHSV: Viral haemorrhagic septicemia rhabdovirus

hyb_protocol: The slides were pre-treated with 1% BSA fraction V, 5 X SSC, 0.1% SDS for 30 min. at 50 ºC and washed with 2 X SSC (3 min) and 0.2 X SSC (3 min) and hybridized overnight in cocktail containing 1.3 X Denhardt's, 3 X SSC, 0.3% SDS, 2.1 ug/ul polyadenylate and 1 ug/ul yeast tRNA.

scan_protocol: Signal was captured and processed using an Agilent scanner (G2565B, Agilent Technologies). The scanner images were segmented by the Agilent Feature Extraction Software (v9.5) with the protocol GE1-v5_95.

data_processing: Data were processed with Microsoft Excel. Normalized to the mean of four independent RPLO (ribosomal L15) genes.

organism: Danio rerio

source_name: Organs, control, replicate 4

label_protocol: Cyanine-3 (Cy3) labeled cRNA was prepared from 2 ug RNA using SuperScript III reverse transcriptase (Invitrogen).

growth_protocol: Zebrafishes were grown at 25 ºC and then acclimated to 14 ºC for 7 days

extract_protocol: RNA was prepared using the Rneasy Micro Kit (QIAGEN)

treatment_protocol: Zebrafishes were incubated at 14 ºC for 2 days

tag: tissue

value: pooled spleen, head kidney, and liver

tag: infection

value: NONE (CONTROL)


title: Organs_VHSV_rep3

source_name: Organs, 2d after VHSV infection, replicate 3

treatment_protocol: Zebrafishes were infected with VHSV and incubated at 14 ºC for 2 days

value: VHSV


title: Organs_control_rep3

source_name: Organs, control, replicate 3


title: Organs_control_rep1

source_name: Organs, control, replicate 1

value: NONE (CONTROL


title: Organs_VHSV_rep1

source_name: Organs, 2d after VHSV infection, replicate 1


title: Fins_control_rep3

source_name: Fins, control, replicate 3

value: fins


title: Fins_control_rep2

source_name: Fins, control, replicate 2


title: Organs_VHSV_rep2

source_name: Organs, 2d after VHSV infection, replicate 2


title: Fins_control_rep4

source_name: Fins, control, replicate 4


title: Organs_control_rep2

source_name: Organs, control, replicate 2


title: Fins_control_rep1

source_name: Fins, control, replicate 1


title: NCI_H69_exosome_rep2

description: miRNA expression in exosome of NCI H69

source_name: NCI H69, exosome, replicate 2


title: Fins_VHSV_rep3

source_name: Fins, 2d after VHSV infection, replicate 3


title: Fins_VHSV_rep2

source_name: Fins, 2d after VHSV infection, replicate 2


title: Organs_VHSV_rep4

source_name: Organs, 2d after VHSV infection, replicate 4


title: Fins_VHSV_rep4

source_name: Fins, 2d after VHSV infection, replicate 4


title: Fins_VHSV_rep1

source_name: Fins, 2d after VHSV infection, replicate 1

value: Fins


title: 0 Dis V-9.2 (A25041)

status: Public on Feb 25 2010

city: Seattle

first: Maitreya

middle: J.

last: Dunham

state: WA

address: Foege Building, S403B, Box 355065

department: Genome Sciences

country: USA

institute: University of Washington

description:
Detailed protocols are available in 'GSE20464_protocols.zip', which is linked to the Series GSE20464 record as a supplementary file.
0 Dis V-9.2 (A25041)

scan_protocol: Pixel Size : 5 Scan Date : 2007-11-07 Scan Time : 10:52:42 Scanner Make : Agilent Technologies Scanner Scanner Model : G2505B

data_processing:
Extraction Software : Agilent Feature Extractor
Extraction Software Version : 9.1.3.1
Datafile type : Agilent result file
Normalizaton: Default settings of Agilent Feature Extractor

extract_protocol:
Extraction method: Hoffman-Winston prep:
http://dunham.gs.washington.edu/MDyeastDNAprep.pdf

tag: strain name

source_name: wt

value: wt


title: 0 Dis IV-9.1 (A25038)

description:
Detailed protocols are available in 'GSE20464_protocols.zip', which is linked to the Series GSE20464 record as a supplementary file.
0 Dis IV-9.1 (A25038)

scan_protocol: Pixel Size : 5 Scan Date : 2007-11-07 Scan Time : 10:51:41 Scanner Make : Agilent Technologies Scanner Scanner Model : G2505B


title: 0 Dis V-9.1 (A25040)

description:
Detailed protocols are available in 'GSE20464_protocols.zip', which is linked to the Series GSE20464 record as a supplementary file.
0 Dis V-9.1 (A25040)

scan_protocol: Pixel Size : 5 Scan Date : 2007-11-07 Scan Time : 10:52:21 Scanner Make : Agilent Technologies Scanner Scanner Model : G2505B


title: Rpb3-1

status: Public on Jan 01 2010

city: New York

first: Harm

middle: van

last: Bakel

state: New York

address: One Gustave L. Levy Place, Box 1498

department: Genetics and Genomic Sciences

institute: Mount Sinai School of Medicine

description: Rpb3-IP vs. Input genomic DNA in a wild-type background, Replicate 1

hyb_protocol: Samples were hybridized according to standard Agilent protocols.

scan_protocol: Microarrays were scanned using a GenePix 4000B laser scanner (Axon Instrument)

data_processing: Spot quantification was carried out using GenePix (Axon Instruments). Following quantification, the ChIP microarray data was lowess-normalized and a binding ratio (ChIP enrichment) was calculated for each microarray feature by dividing the signal intensity of the immunoprecipitated DNA sample by that of the background control (input DNA).

organism: Schizosaccharomyces pombe

source_name: Rpb3-IP genomic DNA from wild-type S. pombe carrying Rpb3-HA

label_protocol: Fluorophores were resuspended in 9 ul of NaHCO3 0.1 M and incubated with the pelleted DNA for 1 hour. The reaction was stopped by the addition of 4.5 ul of Hydroxylamine 4M and incubation for 15 minutes. The labelled samples were purified using the Qiagen PCR purification kit.

growth_protocol: A culture of 100ml was grown at 32°C in YES medium until mid-log phase. Formaldehyde was added to a final concentration of 3% and cells were crosslinked for 10 mins. The crosslink was stopped by adding 20 ml of glycin 2.5 M and cells were further incubated for 5 min in a shaker.

extract_protocol: After formaldehyde crosslinking, cells were lysed using glass beads and chromain extracts were sonicated on a Branson Sonifier 450 to shear the  chromatin to an average size of ~500 bp. A single pulldown was performed with magnetic beads coated with appropriate antibodies and following decrosslinking and LM-PCR amplification of purified IP DNA, the sample was labeled and hybridized on a Agilent 4x44k array.

value: "536 h+ rpb3-3HA-kanR ade6- ura4-D18 leu1-32"

source_name: Input genomic DNA from wild-type S. pombe carrying Rpb3-HA

extract_protocol: After formaldehyde crosslinking, cells were lysed using glass beads and chromain extracts were sonicated on a Branson Sonifier 450 to shear the  chromatin to an average size of ~500 bp. A small fraction of the chromatin extract was decrosslinked and following LM-PCR amplification of purified input DNA, the sample was labeled and hybridized on an Agilent 4x44k array.


title: Affected ileum subject CD-196 II

city: St Louis

first: Rekha

last: Meyer

state: MO

address: 4320 Forest Park Av

institute: Washington University School of Medicine

hyb_protocol: The resulting probes were hybridized to Agilent Whole Human genome Arrays, using established protocals.

scan_protocol: Arrays are scanned on Axon scanner 4000B (Moleculer Devices, Sunnyvale, CA) to detect Cy3 and Cy5 fluorescence. GenePix Pro 6.1 acquisition and analysis software is used to visualize and analyze the 16-bit TIFF files generated by the scanner

data_processing: A primary statistical analysis of the array data was performed using the significance analysis of microarrays (SAM) procedure.  These analyses were performed on log-transformed mean signal intensities.  SAM assignes a gene-specific t-test (q-value) based on changes in gene expression relative to the standard deviation of repeated measurements for that gene.  the critearia set for differences between groups in the SAM analysis q<0.05, fold change <0.5 or >2.

source_name: macroscopic disease affected ileum, specimen from initial ileocolic resection

label_protocol: The test ileal RNA and a common reference ileal RNA (NOD2s, non IBD) were labled using the Agilent low RNA input linear amplification kit.

growth_protocol: Surgically resected tissue samples were collected from recruited patients.  Diagnosis of Chron's disease was made according to conventional clinical and pathological criteria.  Information about smoking and medication history was also collected from the patients.  Biopsies of macroscopically disease affected and unaffected areas of ileal mucosa were collected from fresh pathologic specimens using Radial Jaw™ 4 large capacity biopsy forceps.  Biopsies of ileal mucosa were also collected from fresh pathological specimens of patients without inflammatory bowel disease undergoing either a right or hemicoletomy or subtotal colectomy for colonic neoplasm or colonic inertia.  A minimum of 4 biopsies were taken within a 2x2 cm2 area from each region.

extract_protocol: RNA samples were amplified using Agilent Linear Ampilfication Kit (Agilent Technologies, CA). 50-250 ng total RNA was mixed with T7 promoter primer in a total volume of 5.8 ul before heating at 65oC for 10 min. 4.4 ul cDNA master mix containing 2 ul 5X first strand buffer, 1 ul 0.1 M DTT, 0.5 ul 10 mM dNTP mix, 0.6 ul MMLV RT and 0.3 ul RNaseOUT were added. The reaction mix was incubated at 40oC for 2 hr and stopped by heating at 65oC for 15 min before cooling and adding 0.5 ul of either 10 mM Cy3-CTP or Cy5-CTP and 14.5 ul transcription master mix containing 3.83 ul nuclearase-free water, 5 ul 4x transcription buffer, 2 ul NTP mix, 1.6 ul 50 % PEG, 0.12 ul RNaseOUT, 0.15 ul inorganic pyrophosphatase and 0.3 ul T7 RNA polymerase. The mix was incubated at 40 oC for 2 hr. The resulting cRNA was purified using RNeasy kit (Qiagen). 1 ug Cy3- and Cy-5 labeled cRNA was mixed and fragmented according to the Agilent Technologies protocol. After overnight hybridization, the arrays were washed and dried according to the commercial protocol.

treatment_protocol: Tissue biopsies were immediately placed in RNA stabilization solution and archived at 80°C.

tag: nod2 genotype

tag: nod2 risk alleles (r = risk allele, nr= not risk allele)

value: NOD2r (2 allele)

tag: smoking history

value: no smoking history prior

tag: tnf infusion

value: no anti TNF medication history prior

tag: disease state

value: Crohn's disease

value: affected ileum

source_name: macroscopic disease unaffected ileum, specimen from initial right colectomy

value: WT/WT

value: NOD2 nr control patient

value: no anti-TNF medication history prior

value: control

value: unaffected ileum


title: Unaffected ileum subject CD-196 NI

source_name: macroscopic disease unaffected ileum, specimen from initial ileocolic resection


title: Rpb3-3

description: Rpb3-IP vs. Input genomic DNA in a wild-type background, Replicate 3


title: 0 Dis IV-9.2 (A25039)

description:
Detailed protocols are available in 'GSE20464_protocols.zip', which is linked to the Series GSE20464 record as a supplementary file.
0 Dis IV-9.2 (A25039)

scan_protocol: Pixel Size : 5 Scan Date : 2007-11-07 Scan Time : 10:52:01 Scanner Make : Agilent Technologies Scanner Scanner Model : G2505B


title: Lsk1-2

description: Lsk1-IP vs. Input genomic DNA in a wild-type background, Replicate 2

source_name: Lsk1-IP genomic DNA from wild-type S. pombe carrying Lsk1-HA

source_name: Input genomic DNA from wild-type S. pombe carrying Lsk1-HA


title: Affected ileum subject CD-124 II

source_name: macroscopic disease affected ileum, resection specimen from initial ileocolic resection

value: single anti-TNF infusion 18 months prior


title: Msc6-4

description: Msc6-IP vs. Input genomic DNA in a wild-type background, Replicate 4

source_name: Msc6-IP genomic DNA from wild-type S. pombe carrying Msc6-HA

source_name: Input genomic DNA from wild-type S. pombe carrying Msc6-HA


title: Unaffected ileum subject CD-124 N1

source_name: macroscopic disease unaffected ileum, resection specimen from initial Ileocolic resection


title: S5A-3

description: S5A-IP vs. Input genomic DNA in a wild-type background, Replicate 3

source_name: S5A-IP genomic DNA from wild-type S. pombe

extract_protocol: After formaldehyde crosslinking, cells were lysed using glass beads and chromain extracts were sonicated on a Branson Sonifier 450 to shear the  chromatin to an average size of ~500 bp. A single pulldown was performed with magnetic beads coated with H5 antibodies (Covance) and following decrosslinking and LM-PCR amplification of purified IP DNA, the sample was labeled and hybridized on a Agilent 4x44k array.

source_name: Input genomic DNA from wild-type S. pombe


title: Rpb3-4

description: Rpb3-IP vs. Input genomic DNA in a wild-type background, Replicate 4


title: S2A-4

description: S2A-IP vs. Input genomic DNA in a wild-type background, Replicate 4

source_name: S2A-IP genomic DNA from wild-type S. pombe


title: Lsk1-3

description: Lsk1-IP vs. Input genomic DNA in a wild-type background, Replicate 3


title: Msc6-1

description: Msc6-IP vs. Input genomic DNA in a wild-type background, Replicate 1


title: S5A-2

description: S5A-IP vs. Input genomic DNA in a wild-type background, Replicate 2


title: Rpb3-2

description: Rpb3-IP vs. Input genomic DNA in a wild-type background, Replicate 2


title: Msc6-2

description: Msc6-IP vs. Input genomic DNA in a wild-type background, Replicate 2


title: Cdk9-3

description: Cdk9-IP vs. Input genomic DNA in a wild-type background, Replicate 3

source_name: Cdk9-IP genomic DNA from wild-type S. pombe carrying Cdk9-HA

source_name: Input genomic DNA from wild-type S. pombe carrying Cdk9-HA


title: S5A-1

description: S5A-IP vs. Input genomic DNA in a wild-type background, Replicate 1


title: S2A-2

description: S2A-IP vs. Input genomic DNA in a wild-type background, Replicate 2


title: Cdk9-4

description: Cdk9-IP vs. Input genomic DNA in a wild-type background, Replicate 4


title: Arrb1+Arrb2 MO 12hpf zebrafish

status: Public on Oct 30 2009

city: Shanghai

first: Rui

last: Yue

address: 320 Yueyang Road

country: China

institute: Shanghai Institute of Biochemistry and Cell Biology

data_processing: Agilent GeneSpring version 7.3

source_name: 12hpf zebrafish embryos

growth_protocol: 28.5 degree centigrade

extract_protocol:
Sigma Trizol reagent extraction;
Refer to Agilent "One-Color Microarray-Based Gene Expression Analysis Protocol, version 5.5"

treatment_protocol: 4ng Arrb1+ 4ng Arrb2 morpholino injection per embryo

value: AB/WT

tag: time point


title: Cdk9-2

description: Cdk9-IP vs. Input genomic DNA in a wild-type background, Replicate 2


title: Ctrl MO 12hpf zebrafish

hyb_protocol: Refer to Agilent "One-Color Microarray-Based Gene Expression Analysis Protocol, version 5.5"

source_name: 12 hpf zebrafish embryos

treatment_protocol: 4ng control morpholino injection per embryo


title: Arrb1 MO 12hpf zebrafish

scan_protocol: Refer to Agilent "One-Color Microarray-Based Gene Expression Analysis Protocol, version 5.5"

treatment_protocol: 4ng Arrb1 morpholino injection per embryo


title: WT vs Hypomethylated Forebrain Gene Expression

status: Public on Dec 12 2009

city: Los Angeles

first: Masakazu

last: Namihira

state: CA

address: 695 Charles E. Young Dr.

department: Human Genetics

institute: UCLA

description:
Using the cre-loxP binary gene deletion strategy, we crossed female mice homozygous for the Dnmt1 conditional allele (Dnmt12lox (Fan et al., 2001; Jackson-Grusby et al., 2001)) with male mice carrying the Emx1-cre insertion (Emx1-cre, kindly provided by Dr. S. Itohara at RIKEN Brain Research Institute, Japan. See Iwasato et al., 2000) to generate both control, heterozygous, and mutant offspring in expected Mendelian ratios.
RNA samples were extracted from the dorsal cortex of WT and mutant mice at the postnatal day (P) 5.

hyb_protocol: 1ug of RNA for each channel was labelled using Agilent Low Input Linear Amplification protocol. Labelled cRNA mixed with Agilent 10x Control targets and Agilent 25x Fragmentation Buffer. This was incubated at 37C for 30 min and the reaction was stopped by the addition of 2x Agilent hybridization buffer. This mix was hybridized to arrays for 17 hours at 65C at 4rpms. Arrays were washed as per Agilent protocol and scanned immediately to prevent ozone degradation.

scan_protocol: The arrays were scanned with Agilent DNA microarray scanner (G2565BA), and probe features were extracted using Feature Extraction Software (version 8.5)

data_processing: Arrays were processed using Agilent Feature Extraction (Version 8.5). Log 10 ratio were calculated from processed Cy5/ processed Cy3 signals. Replicates 1 - 3 were labeled - mutant mutant with Cy5 and WT with Cy3.

organism: Mus musculus

source_name: dorsal cortex of P5 Emx1-cre Dnmt1 mutant mice

label_protocol: We converted the RNA into cDNA and then the cDNA into cRNA using the Agilent Low RNA Input Linear Amplification Kit (Agilent). We using a Nanodrop (Nanodrop) to quantify the labeled cRNA. Cy5 Labelled with Agilent Low Input Linear Amplification Kit

extract_protocol: Trizol Extracted and cleaned with Qiagen RNeasy minElute column and tested the quality of the RNA on a NanoChip (Agilent).

tag: dorsal cortex of P5 Emx cre Dnmt1 mutant mice

tag: (Back born strain; C57BL/6)

source_name: dorsal cortex of P5 WT mice

label_protocol: We converted the RNA into cDNA and then the cDNA into cRNA using the Agilent Low RNA Input Linear Amplification Kit (Agilent). We using a Nanodrop (Nanodrop) to quantify the labeled cRNA. Cy3 Labelled with Agilent Low Input Linear Amplification Kit.

tag: dorsal cortex of P5 WT mice


title: S2A-3

description: S2A-IP vs. Input genomic DNA in a wild-type background, Replicate 3

tag: strain


title: S2A-1

description: S2A-IP vs. Input genomic DNA in a wild-type background, Replicate 1


title: OS177 replicate 2

status: Public on Sep 08 2009

city: Toronto

first: Bekim

last: Sadikovic

state: Ontario

address: 555 University Avenue

institute: The SickKids Hospital and Princess Margaret Hospital

description: The Agilent Human Genome CGH microarray 244A (Agilent Technologies, Inc., Palo Alto, USA) were used for the array-CGH experiments. Three μg of Human Genomic DNA from multiple anonymous male donors (Promega Corporation, Madison, USA) and 3 μg of test genomic DNA sample were digested with AluI (5 units) and RsaI (5 units) (Promega) for a minimum of 2 hours at 37C. Digestion quality was assessed by the DNA 1000 LabChip Kit (Agilent 2100 Bioanalyzer, Agilent Technologies). Labelling reactions were performed using the Agilent Genomic DNA Labeling Kit PLUS (Agilent Technologies, Inc., Palo Alto, USA) according to the manufacturer’s directions. Briefly, the reference and sample DNA were labelled with 1.5 - 3 mM Cy3-dUTP or Cy5-dUTP (Agilent Technologies, Inc., Palo Alto, USA), and purified using a Centricon YM-30 filter (Millipore, Billerica, USA). Probe mixture of Cy3-labelled sample DNA, Cy5-labelled reference DNA, 50 μl of 1.0mg/ ml of human Cot-1 DNA (Invitrogen, USA), 52 μl of Agilent 10X Blocking Agent and 260 μl of Agilent 2X Hybridization Buffer (Agilent Technologies, Inc.) was denatured at 100C for 1 minute 30 seconds and incubated at 37C for 30 minutes. The probe was applied to the array using an Agilent microarray hybridization chamber, and hybridized for 40 hours at 65C in a rotating oven (Robbins Scientific, Sunnyvale, USA) at 20 rpm. Arrays were washed according to the manufacturer’s recommendations; air dried, and scanned using an Agilent 2565AA DNA microarray scanner (Agilent Technologies, Inc), and processed using Agilent’s Feature Extraction software. Dye-swapped duplicate experiments were carried out.

hyb_protocol: Probe mixture of Cy3-labelled sample DNA, Cy5-labelled reference DNA, 50 μl of 1.0mg/ ml of human Cot-1 DNA (Invitrogen, USA), 52 μl of Agilent 10X Blocking Agent and 260 μl of Agilent 2X Hybridization Buffer (Agilent Technologies, Inc.) was denatured at 100C for 1 minute 30 seconds and incubated at 37C for 30 minutes. The probe was applied to the array using an Agilent microarray hybridization chamber, and hybridized for 40 hours at 65C in a rotating oven (Robbins Scientific, Sunnyvale, USA) at 20 rpm. Arrays were washed according to the manufacturer’s recommendation and air dried.

scan_protocol: Arrays were scanned using an Agilent 2565AA DNA microarray scanner (Agilent Technologies, Inc).

data_processing: Data were processed using Agilent’s Feature Extraction software protocol for array-CGH data. To analyze genomic imbalance tumour samples (2 arrays each) the  processed R signal and processed G signal columns from the Agilent  Feature Extraction-generated a-CGH .txt files were imported into PGS.  The tumour-specific signal across all probes was normalized as a ratio  to baseline using Normalise to Baseline Tool in PGS, where baseline  data corresponded to the normal human DNA. The data was then log2  transformed using the PGS Normalization and Scaling Tool. In order to  detect regions of genomic gain and loss we applied the Genomic  Segmentation tool with segmentation parameters set at: min. probes:  10, p-value threshold: 0.01, and signal to noise: 0.1. Region report  was set at values bellow -.5/+.5 (log2) and p-value threshold of 0.05  for 2/2 (replicate) arrays in individual analysis or at 40% cut-off in  cumulative analysis (i.e. 4/10 tumour samples). Regions of significant  gain or loss were annotated to the corresponding genes present on the  Affymetrix Gene 1.0 Array using the  HuGene-1_0-st-v1.na24.hg18.transcript.csv file. Visualizations and  VENN analysis were performed in PGS.

source_name: Normal Human Genomic DNA

label_protocol: Labelling reactions were performed using the Agilent Genomic DNA Labeling Kit PLUS (Agilent Technologies, Inc., Palo Alto, USA) according to the manufacturer’s directions. Briefly, DNA was labelled with 1.5 - 3 mM Cy5-dUTP (Agilent Technologies, Inc., Palo Alto, USA), and purified using a Centricon YM-30 filter (Millipore, Billerica, USA).

extract_protocol: DNA was extracted after harvesting the cells by Trypsin (Invitrogen) followed by phenol-chloroform extraction and subsequent precipitation in 100% ethanol. DNA precipitate was washed with 70% ethanol then eluted in DNAse free water.

source_name: Osteosarcoma tumor sample 177

label_protocol: Labelling reactions were performed using the Agilent Genomic DNA Labeling Kit PLUS (Agilent Technologies, Inc., Palo Alto, USA) according to the manufacturer’s directions. Briefly, DNA was labelled with 1.5 - 3 mM Cy3-dUTP (Agilent Technologies, Inc., Palo Alto, USA), and purified using a Centricon YM-30 filter (Millipore, Billerica, USA).

extract_protocol: Comercial DNA extracted as per manifacturers instructions - Human Genomic DNA from multiple anonymous male donors (Promega Corporation, Madison, USA)

tag: Paediatric Human Osteosarcoma Tissue Sample


title: OS177 replicate 1

tag: Human Genomic DNA from multiple anonymous male donors (Promega Corporation, Madison, USA)


title: OS138 replicate 1

source_name: Osteosarcoma tumor sample 138


title: OS178 replicate 1

source_name: Osteosarcoma tumor sample 178


title: OS138 replicate 2


title: 67LR positive population (jhu-251485011211-4.txt_Cy5)

status: Public on May 28 2009

city: Baltimore

first: Luigi

last: Marchionni

state: MD

address: 1550 Orleans St., CRB2, Room 1M52

department: Oncology

institute: Johns Hopkins University

description: 67LR- Positive

hyb_protocol: 1.5 µg of each sample labeled with Cy3 or Cy5 was mixed with control targets (Agilent Technologies). Fragmentation was carried out by incubating for 30 minutes at 60 degrees Celsius and stopped by adding an equal volume of 2x hybridization bu?er (Agilent Technologies). Fragmented targets were added to the microarrays, assembled into a hybridization chamber (Agilent Technologies) and hybridized at 65 degrees Celsius for 17 hours in a hybridization oven with rotation. Hybridized microarrays were washed and dried according to the Agilent microarray processing protocol.

scan_protocol: Hybridized slides were scanned using a G2565BA Agilent scanner, with 100% PMT and 10 micrometer resolution according to the manufacturer`s directions. The scanner was operated by the Agilent Scan Control 7.0 software. Data were extracted with the Agilent Feature Extraction 9.5.3.1 software. Images were subjected to quality checks recommended by the manufacturer. Images were visually inspected for artifacts and distributions of red and green channels, both for foreground and background, to identify anomalous arrays. No abnormalities were found and all arrays were retained for subsequent analyses.

data_processing: Within-array dye e?ects were corrected by the ?loess? normalization method. The ?scale? normalization method was applied to standardize the M- and the A-values distributions across different arrays, by using the median-absolute-value as scale estimator. No background subtraction was performed prior to normalization. Positive and negative microarray control features were not used to compute the ?loess? smoothing and were further excluded from the subsequent analysis. After scaling the normalized A and M values were deconvoluted into single channel intensities

source_name: BRIGHT_2; Cy5; 67LR Positive population of sw 780 tumor

label_protocol: Sample amplification and labeling procedures for microarray analysis were carried out by using the Low RNA Input Fluorescent Linear Amplification Kit (Agilent Technologies). 400 ng of total RNA was reverse transcribed into first strand and second strand cDNA by MMLV-RT using an oligo dT primer (System Biosciences) that incorporates a T7 promoter sequence. The cDNA was then used as a template for in vitro transcription in the presence of T7 RNA polymerase and Cyanine labeled CTPs. The labeled cRNA was purified using RNeasy mini kit (Qiagen), followed by quantification based on both concentrations of cRNA and dye. RNA spike-in controls (Agilent Technologies) were added to RNA samples before amplification and labeling according to manufacturer`s protocol.

extract_protocol: Total RNA was purified using RNAzol (Invitrogen), followed by a secondary purification using the RNEasy system (Qiagen). RNA integrity was determined the Agilent 2100 Bioanalyzer. RNA concentration for individual samples was > 250 ng/µl, with 28S/18S ratio > 1.4 and  RIN score > 9.4.

tag: cell type

value: 67LR Positive

value: urothelial cancer


title: Whole population (jhu-251485011212-1.txt_Cy3)

source_name: BULK_2; Cy3; whole population of sw780 tumor

value: 67 Positive and Negative


title: OS87B replicate 2

source_name: Osteosarcoma tumor sample 87B


title: Whole population (jhu-251485011211-3.txt_Cy3)

source_name: BULK_1; Cy3; whole population of sw780 tumor


title: DNA packaging of human spermatozoal chromatin revealed by salt extraction and restriction digestion (SRD)

status: Public on Jun 09 2009

city: Leeds

first: David

last: Miller

address: Clarendon Way

department: Leeds Institute for Genetics and Health Therapeutics

country: United Kingdom

institute: Leeds University

description: Spermatozoal DNA from a healthy human donor was partitioned through a salt extraction to profile histone and protamine binding of the chromatin.

hyb_protocol: For hybridisation, equal quantities of labelled DNA (500 - 1000 ng) were mixed with Cot-1 DNA (1 mg/ml) and incubated at 95°C for 3 minutes and then re-incubated at 37°C for 30 minutes. The DNAs were applied to either Agilent's 44K (100 μl) or 244K (490 μl) arrays, sealed and then incubated for 40 hours (244K arrays) or 24 hours (44K arrays) at 65°C with constant rotation (20 rpm).

scan_protocol: Scanning was conducted an accordance with the Agilent protocol.

data_processing:
Data was processed for feature extraction, background subtraction and normalized in the Agilent CGH analytics software package.
The default settings for the Agilent Analytics CGH v9.1 protocols were used (selection of a subset of rank invariant probes and using these for a linear median normalization between channels).

source_name: Spermatozoal DNA partitioned with a salt extraction (SRDI; Insoluble Fraction)

label_protocol:
Labelling with cy3 or cy5 (GE Healthcare, Amersham UK) was accomplished using a
random priming method in the presence of Klenow DNA polymerase (Invitrogen,
Paisley, UK) according to the suggested Agilent protocol. Unincorporated nucleotides
were removed by passing the reaction mixture through a sephadex G50 column.

extract_protocol:
CGH profiling was carried out according to the manufacturer's (Agilent) instructions.
Prior to processing, all DNA samples (including those pre-digested with EcoR1 and
BamH1 or MNase were double re-digested with 10 units each of Alu1 and Rsa1
(New England Biolabs, Hitchin, UK) that generates fragments of 500 bp or less.

treatment_protocol:
In brief, semen
was washed free of seminal plasma by diluting the semen 1:1 with HEPES buffered
HSOF and centrifuging for 10 minutes at 500 x g. Pellets were resuspended in HSOF
and pelleted as before. This was repeated until the supernatants were clear. Pellets
were resuspended in STE buffer (100mM NaCl, 10 mM Tris-Cl, pH 8.0, 1 mM EDTA)
14
containing 0.2 mM phenylmethylsulphonylfluoride (PMSF) and re-pelleted to remove
HSOF. Pellets were subsequently processed as described previously with sequential
extraction in 10% (w/v) mixed CTAB (alkly-trimethylammonium bromide), which
removes the flagella, including principal pieces, thus ensuring no contamination of
the samples with non-genomic DNA followed by sequential incubation in 1% w/v
CTAB in 10 mM Tris-Cl pH 8.0 containing 0.05% w/v digitonin; Tris buffered saline
(TBS) containing 0.05% w/v digitonin, 10 mM Tris-Cl pH 8.0 containing 1mM EDTA,
0.05% w/v digitonin, EDTA-free protease inhibitor cocktail (Roche) and finally
extraction in 0.65M NaCl to release soluble histones.The supernatant following salt
extraction was retained.
Nuclear pellets were re-suspended in BamH1 buffer containing 0.05% digitonin
and digested with ~150 units each of BamH1 and EcoR1 (Invitrogen, Paisley, UK:
2,000 U per 5 x 108 nuclei) for 90 minutes at 37°C. Suspensions were centrifuged at
3,000 x g for 5 minutes and the supernatants removed. STOP buffer comprising
proteinase K (Bioline: - 200 μg/ml) in TBS, 0.5% w/v sodium dodecyl sulphate (SDS)
was added to this suspension and incubated for 10 hours at 55°C. Pellets were also
resuspended in STOP buffer and concurrently digested under identical conditions.
DNA was isolated from both soluble and insoluble fractions by incubation in STOP
buffer containing RNAse A at 10mg/ml for 1 hour at 37°C and extraction in phenol
chloroform (1:1) followed by ethanol precipitation. Precipitated DNA was dissolved in
TE buffer and stored at 4°C.

value: Spermatozoa

tag: donor

value: healthy human donor

source_name: Spermatozoal DNA partitioned with a salt extraction (SRDS; soluble Fraction)


title: L-CD31+_sample #7

status: Public on Dec 22 2009

city: Biella

first: giovanna

last: chiorino

address: via malta 3

department: Cancer Genomics

country: Italy

institute: Fondo Edo Tempia

description:
Technical replicates (normal and dye-swap) of L-CD31+_sample #7.
Normal: 7_L-CD31+_NORM
Dye-swap: 7_L-CD31+_DS

hyb_protocol: Equal amounts (0.75 ug) of labeled specimens from sample and reference were put together, fragmented and hybridized to oligonucleotide glass arrays representing 41K human unique genes and transcripts (Human Whole Genome Oligo Microarray Array, Agilent Technologies). All steps were performed using the In Situ Hybridization kit-plus (Agilent Technologies) and following the 60-mer oligo microarray processing protocol (Agilent Technologies). Then, slides were washed with the SSPE wash procedure. For each sample, a dye-swap replicate was performed.

scan_protocol: Slides were scanned with the dual-laser microarray scanner Agilent G2505B.

data_processing:
Images were analyzed using Feature Extraction software (Agilent Technologies) version 7.6. Output files containing feature and background intensities and the related statistical parameters for red and green signals were then loaded into the Resolver SE System (Rosetta Biosoftware, Seattle, WA) together with the scan images and the Agilent Human Whole Genome pattern file. Data processing and normalization were performed using the Agilent Human Whole Genome platform-specific error model. Replicated expression profiles were combined to form ratio experiments where each gene is associated to an expression fold-change and a p-value to assess the statistical significance of its modulation in the sample compared to the reference. New ratio experiments were also built by applying the “ratio split with common reference” option and calculating ratios over the average of non tumor samples (CMN). A “weight by error” divisive clustering algorithm available within the Rosetta Resolver system (with cosine correlation as similarity metrics) was applied to such new ratios to compare their expression profiles. At the same time, data treatment with the LIMMA (LInear Models for MicroArray data) package available within Bioconductor (http://www.bioconductor.org/) was also performed applying normexp background calculation, lowess within-array and A-quantile between-array normalization methods.

The raw and processed data output are from Feature Extraction Software version 7.6 (Agilent Technologies). Data from technical replicates (normal and dye-swap labelling) were combined and then the data was normalized. Log10 ratios representing test/reference are reported in the VALUE column of the Sample data tables.

label: Cy3, Cy5

source_name: CD31+ transfected CLL cells from patient #7

label_protocol: RNA isolated from each cell culture and from the human universal reference (BD™ Human Universal Reference Total RNA, Clontech, Palo Alto, CA) was amplified by means of the Amino Allyl MessageAmp II aRNA Kit (Ambion) to obtain amino allyl antisense RNA (aaRNA) following the method developed by Eberwine and coworkers. Two rounds of amplification were performed to obtain the necessary quantity of aaRNA for labeling. Briefly: mRNA was reverse transcribed in cDNA single strand; after the second strand synthesis (in the second round of amplification), cDNA was in vitro transcribed in aaRNA including amino allyl modified nucleotides (aaUTP). Both dsDNA and aaRNA underwent a purification step using columns provided with the kit. Labeling was performed using NHS ester Cy3 or Cy5 dyes (Amersham Biosciences, Buckinghamshire UK) able to react with the modified RNA. mRNA quality was checked by means of RNA 6000 nano chip assays (Agilent Technologies). At least 5 ug of mRNA for each sample were labeled and purified with columns.

extract_protocol: All the samples were homogenized (Ultra – TurraxT8, Ika – Werke, Staufen, Germany) and total RNA was isolated by using TriReagent (Sigma Aldrich). TotRNA quality was checked by means of RNA 6000 pico chip assays (Agilent Technologies, Palo Alto, CA) run on the Agilent 2100 bioanalyzer.

tag: Gender

value: F

tag: Age

source_name: commercial human universal reference

tag: Clontech human universal reference


title: Cdx2+/-Cdx4-/- dyeswap

status: Public on Oct 19 2009

city: Utrecht

first: Jacqueline

last: Deschamps

address: Uppsalalaan 8

country: Netherlands

institute: Hubrecht Institute

hyb_protocol: According to Agilent guidelines

scan_protocol: According to Agilent guidelines

data_processing: Microarray signal and background information were retrieved using Feature Extraction (V9.5.3, Agilent technologies).

source_name: Posterior parts of 13 somite embryos (n=7)

label_protocol: According to Agilent guidelines

extract_protocol: Posterior tissues from pools of 7 embryos of each genotype were pooled and stored in RNAlater (Ambion, cat. AM1924). RNAs were purified using a kit from Qiagen (Cat 74104).

tag: genotype

value: wildtype


title: Cdk9-1

description: Cdk9-IP vs. Input genomic DNA in a wild-type background, Replicate 1

source_name: Cdk9-IP genomic DNA from wild-type S. pombe carrying Cdk9-HA

source_name: Input genomic DNA from wild-type S. pombe carrying Cdk9-HA


title: 67LR negative population (jhu-251485011211-1.txt_Cy5)

description: 67LR- Negative

source_name: DIM_1; Cy5; 67LR Negative population of sw 780 tumor

value: 67LR Negative


title: 67LR negative population (jhu-251485011211-2.txt_Cy3)

description: 67LR -Negative

source_name: DIM_1; Cy3; 67LR Negative population of sw 780 tumor


title: Whole population (jhu-251485011212-4.txt_Cy5)

source_name: BULK_2; Cy5; whole population of sw780 tumor


title: 67LR positive population (jhu-251485011212-3.txt_Cy5)


title: 67LR positive population (jhu-251485011212-4.txt_Cy3)

source_name: BRIGHT_2; Cy3; 67LR Positive population of sw 780 tumor


title: Whole population (jhu-251485011212-2.txt_Cy3)


title: 67LR positive population (jhu-251485011211-1.txt_Cy3)

description: 67LR -Positive

source_name: BRIGHT_1; Cy3; 67LR Positive population of sw 780 tumor


title: 67LR positive population (jhu-251485011212-1.txt_Cy5)

description: 67LR- Positive

source_name: BRIGHT_1; Cy5; 67LR Positive population of sw 780 tumor


title: Cdx2+/-Cdx4-/-


title: 67LR negative population (jhu-251485011212-3.txt_Cy3)

source_name: DIM_2; Cy3; 67LR Negative population of sw 780 tumor


title: 67LR positive population (jhu-251485011211-3.txt_Cy5)

description: 67LR- Positive


title: Whole population (jhu-251485011211-4.txt_Cy3)


title: Whole population (jhu-251485011211-2.txt_Cy5)

source_name: BULK_1; Cy5; whole population of sw780 tumor


title: E_78

status: Public on Apr 06 2009

first: Hegang

last: Chen

address: 660 West Redwood Street

institute: University of Maryland School of Medicine

hyb_protocol: Hybridization was carried out using the conditions specified in the Agilent Human Whole Genome Expression system.

scan_protocol: Scanned images were processed for quality assessment and preprocessing of image data using the Agilent Feature Extraction software (Agilent Technologies)

data_processing: The software quantifies feature signals and their background, performs dye normalization and calculates feature log ratios and error estimates. The error estimates, based on an extensive error model and pixel level statistics calculated from the feature and background for each spot, are used to generate a p-value for each log ratio.

source_name: LnCap cells treated with shCon-DHT

label_protocol: see manufacturer's web site at http://www.agilent.com/

tag: cell line

value: LnCap

tag: treatment

value: shCon-DHT

source_name: LNCap cells treated with no DHT

value: LNCap

value: no DHT, serving as a common reference


title: E_81

source_name: LNCap cells treated with shCon+DHT

extract_protocol: see manufacturer's web site at http://www.agilent.com/

value: shCon+DHT


title: E_79

source_name: LNCap cells treated with shRNF6-DHT


title: E_80

source_name: LNCap cells treated with 1 nM DHT

value: 1 nM DHT


title: Zebrafish von Hippel-Lindau (vhl) tumor suppressor mutants compared to siblings at 7 dpf_1

status: Public on Mar 01 2009

first: Ellen

last: van Rooijen

institute: Hubrecht Institute- KNAW and UMC Utrecht

description: Duplicate samples were run in dye-swap. This experiment is described in "Zebrafish von Hippel-Lindau (vhl) tumor suppressor mutants compared to siblings at 7 dpf_2".

hyb_protocol: Labeled samples were hybridized for 17 hours at 60 degrees Celcius to a 4x44K zebrafish expression array (G2519F, Agilent Technologies) using the Gene Expression Hybridization Kit (5188-5242, Agilent Technologies). Arrays were washed according to the manufacturers protocol.

scan_protocol: The microarray was scanned using the G2505C Agilent DNA microarray scanner using default parameters.

data_processing: Images were processed using the Feature Extraction software (Agilent Technologies), and analyzed (P-value ≤ 0.01) using Limma in R software (Smyth GK. Limma: linear models for microarray data. In: Gentleman RC, V.; Huber, W.; Irizarry, R.; Dudoit, S., ed. Bioinformatics and Computational Biology Solutions using R and Bioconductor: Springer, New York; 2005:397-420).

source_name: vhl hu2081/hu2117 mutants

label_protocol: cDNA synthesis and labeling were performed using the two color Quick-Amp Labeling Kit (5190-0444, Agilent Technologies), and samples were purified using the RNeasy Mini kit (Qiagen). Concentration and Cy5 incorporation were determined using a Nanodrop spectrophotometer.

growth_protocol: Embryos from transheterozygous crosses vhl hu2081/+ x vhl hu2117/+ were raised in standard E3 embryo medium at 28 degrees Celcius. At 7 dpf vhl mutant embryos were isolated based on morphological phenotype.

extract_protocol: Total RNA from 40 vhl mutants (equally pooled from four different clutches) at 7 dpf was isolated using TRIzol (Invitrogen) according to the manufacturers protocol.

treatment_protocol: no treatment

value: vhl hu2081/hu2117 transheterozygous mutants

tag: age

value: 7 days post-fertilization (dpf)

source_name: siblings of vhl hu2081/hu2117 mutants

label_protocol: cDNA synthesis and labeling were performed using the two color Quick-Amp Labeling Kit (5190-0444, Agilent Technologies), and samples were purified using the RNeasy Mini kit (Qiagen). Concentration and Cy3 incorporation were determined using a Nanodrop spectrophotometer.

growth_protocol: Embryos from transheterozygous crosses vhl hu2081/+ x vhl hu2117/+ were raised in standard E3 embryo medium at 28 degrees Celcius. At 7 dpf sibling embryos were isolated based on morphological phenotype.

extract_protocol: Total RNA from 40 siblings (equally pooled from four different clutches) at 7 dpf was isolated using TRIzol (Invitrogen) according to the manufacturers protocol.

value: normal


title: 67LR negative population (jhu-251485011212-2.txt_Cy5)

source_name: DIM_2; Cy5; 67LR Negative population of sw 780 tumor

tag: disease state


title: E_82

source_name: LNCap cells treated with shRNF6+DHT


title: Zebrafish von Hippel-Lindau (vhl) tumor suppressor mutants compared to siblings at 7 dpf_2

description: Duplicate samples were run in dye-swap. This experiment is described in "Zebrafish von Hippel-Lindau (vhl) tumor suppressor mutants compared to siblings at 7 dpf_1".


title: COPxACI F2 #37

status: Public on Mar 31 2006

city: Madison

first: James

last: Shull

state: WI

address: 1400 University Avenue

department: McArdle Laboratory for Cancer Research

institute: University of Wisconsin-Madison

description: Copenhagen x ACI F2 rats were derived by crossing female COP rats with male ACI rats to generate the F1 generation, which was then intercrossed to generate F2 animals. At 9 weeks of age, a silastic tubing implant containing 5mg of the synthetic estrogen diethylstilbestrol (DES) was surgically implanted subcutaneously in the interscapular region of male rats. Each animal received an intraperitoneal injection of BrdU (50mg/kg) 4 hours prior to sacrifice. Animals were sacrificed at 21 weeks of age by decapitation and pituitary glands were immediately harvested, weighed and flash-frozen in liquid nitrogen. Tissues were homogenized through a 22-gauge needle and total RNA was extracted using the Absolutely RNA Kit (Stratagene). A total of 43 DES-treated F2 males were analyzed by microarray.

hyb_protocol: standard Affymetrix procedures

scan_protocol: standard Affymetrix procedures

data_processing: Affymetrix GeneChip Operating Software version 1.2

label: biotin

source_name: anterior pituitary

label_protocol: Approximately 2 µg of total RNA was processed to produce biotinylated cRNA targets.

extract_protocol: Anterior pituitary gland was harvested at the time of sacrifice, weighed and flash-frozen in liquid nitrogen. Tissue was homogenized in lysis buffer by passage through an 18- and then 22-guage needle. Total RNA was isolated using the Absolutely RNA Kit from Stratagene.

treatment_protocol: diethylstilbestrol (DES) treatment for 12 weeks starting at 9 weeks of age

tag: Copenhagen x ACI F2 rat, male, age 21 weeks, treated with the synthetic estrogen diethylstilbestrol (DES) for 12 weeks


title: Lsk1-4

description: Lsk1-IP vs. Input genomic DNA in a wild-type background, Replicate 4

source_name: Lsk1-IP genomic DNA from wild-type S. pombe carrying Lsk1-HA

source_name: Input genomic DNA from wild-type S. pombe carrying Lsk1-HA


title: COPxACI F2 #34


title: COPxACI F2 #39


title: COPxACI F2 #40


title: COPxACI F2 #33


title: COPxACI F2 #43


title: COPxACI F2 #36


title: COPxACI F2 #32


title: COPxACI F2 #28


title: COPxACI F2 #41


title: COPxACI F2 #25


title: COPxACI F2 #42


title: COPxACI F2 #24


title: COPxACI F2 #31


title: COPxACI F2 #30


title: COPxACI F2 #21


title: COPxACI F2 #20


title: COPxACI F2 #26


title: COPxACI F2 #22


title: COPxACI F2 #38


title: COPxACI F2 #35


title: COPxACI F2 #19


title: COPxACI F2 #23


title: COPxACI F2 #29


title: COPxACI F2 #18


title: COPxACI F2 #17


title: COPxACI F2 #16


title: COPxACI F2 #27


title: COPxACI F2 #15


title: COPxACI F2 #14


title: COPxACI F2 #13


title: COPxACI F2 #12


title: COPxACI F2 #7


title: COPxACI F2 #5


title: COPxACI F2 #11


title: Ept6 DES-treated replicate 2

description: Ept6 congenic rats were generated by introgressing COP alleles onto an ACI background within the Ept6 locus on rat chromosome 3. At 9 weeks of age, a silastic tubing implant containing 5mg of the synthetic estrogen diethylstilbestrol (DES) was surgically implanted subcutaneously in the interscapular region of male rats. Each animal received an intraperitoneal injection of BrdU (50mg/kg) 4 hours prior to sacrifice. Animals were sacrificed at 21 weeks of age by decapitation and pituitary glands were immediately harvested, weighed and flash-frozen in liquid nitrogen. Tissues were homogenized through a 22-gauge needle and total RNA was extracted using the Absolutely RNA Kit (Stratagene). A total of 3 DES-treated males were used, resulting in two independent biological replicates.

tag: Ept6 congenic rat, male, age 21 weeks, treated with the synthetic estrogen diethylstilbestrol (DES) for 12 weeks


title: COPxACI F2 #9


title: COPxACI F2 #6


title: COPxACI F2 #3


title: COPxACI F2 #4


title: COPxACI F2 #10


title: Ept9 DES-treated replicate 3

description: Ept9 congenic rats were generated by introgressing COP alleles onto an ACI background within the Ept9 locus on rat chromosome 10. At 9 weeks of age, a silastic tubing implant containing 5mg of the synthetic estrogen diethylstilbestrol (DES) was surgically implanted subcutaneously in the interscapular region of male rats. Each animal received an intraperitoneal injection of BrdU (50mg/kg) 4 hours prior to sacrifice. Animals were sacrificed at 21 weeks of age by decapitation and pituitary glands were immediately harvested, weighed and flash-frozen in liquid nitrogen. Tissues were homogenized through a 22-gauge needle and total RNA was extracted using the Absolutely RNA Kit (Stratagene). A total of 3 DES-treated males were used, resulting in two independent biological replicates.

tag: Ept9 congenic rat, male, age 21 weeks, treated with the synthetic estrogen diethylstilbestrol (DES) for 12 weeks


title: Ept1 DES-treated replicate 3

description: Ept1 congenic rats were generated by introgressing COP alleles onto an ACI background within the Ept1 locus on rat chromosome 6. At 9 weeks of age, a silastic tubing implant containing 5mg of the synthetic estrogen diethylstilbestrol (DES) was surgically implanted subcutaneously in the interscapular region of male rats. Each animal received an intraperitoneal injection of BrdU (50mg/kg) 4 hours prior to sacrifice. Animals were sacrificed at 21 weeks of age by decapitation and pituitary glands were immediately harvested, weighed and flash-frozen in liquid nitrogen. Tissues were homogenized through a 22-gauge needle and total RNA was extracted using the Absolutely RNA Kit (Stratagene). A total of 3 DES-treated males were used, resulting in two independent biological replicates.

tag: Ept1 congenic rat, male, age 21 weeks, treated with the synthetic estrogen diethylstilbestrol (DES) for 12 weeks


title: Ept2 DES-treated replicate 3

description: Ept2 congenic rats were generated by introgressing COP alleles onto an ACI background within the Ept2 locus on rat chromosome 3. At 9 weeks of age, a silastic tubing implant containing 5mg of the synthetic estrogen diethylstilbestrol (DES) was surgically implanted subcutaneously in the interscapular region of male rats. Each animal received an intraperitoneal injection of BrdU (50mg/kg) 4 hours prior to sacrifice. Animals were sacrificed at 21 weeks of age by decapitation and pituitary glands were immediately harvested, weighed and flash-frozen in liquid nitrogen. Tissues were homogenized through a 22-gauge needle and total RNA was extracted using the Absolutely RNA Kit (Stratagene). A total of 3 DES-treated males were used, resulting in two independent biological replicates.

tag: Ept2 congenic rat, male, age 21 weeks, treated with the synthetic estrogen diethylstilbestrol (DES) for 12 weeks


title: Ept6 DES-treated replicate 1


title: COPxACI F2 #1


title: Arrb2 MO 12hpf zebrafish

source_name: 12hpf zebrafish embryos

label_protocol: Refer to Agilent "One-Color Microarray-Based Gene Expression Analysis Protocol, version 5.5"

treatment_protocol: 4ng Arrb2 morpholino injection per embryo

tag: strain

value: AB/WT

tag: time point


title: Ept2 DES-treated replicate 2


title: Ept6 DES-treated replicate 3


title: COPxACI F2 #8


title: Ept1 DES-treated replicate 2


title: Ept9 DES-treated replicate 1


title: Ept1 DES-treated replicate 1


title: Ept2 DES-treated replicate 1


title: Ept9 DES-treated replicate 2


title: BN DES-treated replicate 1

description: BN rats were obtained from Harlan (Indianapolis, IN). At 9 weeks of age, a silastic tubing implant containing 5mg of the synthetic estrogen diethylstilbestrol (DES) was surgically implanted subcutaneously in the interscapular region of male rats. Each animal received an intraperitoneal injection of BrdU (50mg/kg) 4 hours prior to sacrifice. Animals were sacrificed at 21 weeks of age by decapitation and pituitary glands were immediately harvested, weighed and flash-frozen in liquid nitrogen. Tissues were homogenized through a 22-gauge needle and total RNA was extracted using the Absolutely RNA Kit (Stratagene). A total of 4 DES-treated males were used, resulting in three independent biological replicates.

tag: BN strain, male, age 21 weeks, treated with the synthetic estrogen diethylstilbestrol (DES) for 12 weeks


title: BN control replicate 3

description: BN rats were obtained from Harlan (Indianapolis, IN). Empty silastic tubing implants were surgically inserted into control animals’ interscapular regions, as sham treatment, at 9 weeks of age. Each animal received an intraperitoneal injection of BrdU (50mg/kg) 4 hours prior to sacrifice. Animals were sacrificed at 21 weeks of age by decapitation and pituitary glands were immediately harvested, weighed and flash-frozen in liquid nitrogen. Tissues were homogenized through a 22-gauge needle and total RNA was extracted using the Absolutely RNA Kit (Stratagene). A total of 3 control males were used, resulting in two independent biological replicates.

tag: BN strain, male, age 21 weeks


title: COPxACI F2 #2


title: BN DES-treated replicate 4


title: BN DES-treated replicate 3


title: COP DES-treated replicate 4

description: COP rats were obtained from Charles River Laboratories (Wilmington, MA). At 9 weeks of age, a silastic tubing implant containing 5mg of the synthetic estrogen diethylstilbestrol (DES) was surgically implanted subcutaneously in the interscapular region of male rats. Each animal received an intraperitoneal injection of BrdU (50mg/kg) 4 hours prior to sacrifice. Animals were sacrificed at 21 weeks of age by decapitation and pituitary glands were immediately harvested, weighed and flash-frozen in liquid nitrogen. Tissues were homogenized through a 22-gauge needle and total RNA was extracted using the Absolutely RNA Kit (Stratagene). A total of 4 DES-treated males were used, resulting in three independent biological replicates.

tag: COP strain, male, age 21 weeks, treated with the synthetic estrogen diethylstilbestrol (DES) for 12 weeks


title: BN DES-treated replicate 2


title: BN control replicate 2


title: COP DES-treated replicate 2


title: ACI DES-treated replicate 2

description: ACI rats were obtained from Harlan (Indianapolis, IN). At 9 weeks of age, a silastic tubing implant containing 5mg of the synthetic estrogen diethylstilbestrol (DES) was surgically implanted subcutaneously in the interscapular region of male rats. Each animal received an intraperitoneal injection of BrdU (50mg/kg) 4 hours prior to sacrifice. Animals were sacrificed at 21 weeks of age by decapitation and pituitary glands were immediately harvested, weighed and flash-frozen in liquid nitrogen. Tissues were homogenized through a 22-gauge needle and total RNA was extracted using the Absolutely RNA Kit (Stratagene). A total of 4 DES-treated males were used, resulting in three independent biological replicates.

tag: ACI strain, male, age 21 weeks, treated with the synthetic estrogen diethylstilbestrol (DES) for 12 weeks


title: ACI DES-treated replicate 4


title: ACI DES-treated replicate 3


title: COP control replicate 2

description: COP rats were obtained from Charles River Laboratories (Wilmington, MA). Empty silastic tubing implants were surgically inserted into control animals’ interscapular regions, as sham treatment, at 9 weeks of age. Each animal received an intraperitoneal injection of BrdU (50mg/kg) 4 hours prior to sacrifice. Animals were sacrificed at 21 weeks of age by decapitation and pituitary glands were immediately harvested, weighed and flash-frozen in liquid nitrogen. Tissues were homogenized through a 22-gauge needle and total RNA was extracted using the Absolutely RNA Kit (Stratagene). A total of 3 control males were used, resulting in two independent biological replicates.

tag: COP strain, male, age 21 weeks


title: ACI control replicate 3

description: ACI rats were obtained from Harlan (Indianapolis, IN). Empty silastic tubing implants were surgically inserted into control animals’ interscapular regions, as sham treatment, at 9 weeks of age. Each animal received an intraperitoneal injection of BrdU (50mg/kg) 4 hours prior to sacrifice. Animals were sacrificed at 21 weeks of age by decapitation and pituitary glands were immediately harvested, weighed and flash-frozen in liquid nitrogen. Tissues were homogenized through a 22-gauge needle and total RNA was extracted using the Absolutely RNA Kit (Stratagene). A total of 3 control males were used, resulting in two independent biological replicates.

tag: ACI strain, male, age 21 weeks


title: COP DES-treated replicate 1


title: COP control replicate 3


title: BN control replicate 1


title: COP control replicate 1


title: ACI control replicate 2


title: ACI control replicate 1


title: ACI DES-treated replicate 1


title: COP DES-treated replicate 3