title: Foot siScramble Replicate2

description: lincRNA knock downs


title: hfq vs. MS11 (I)

status: Public on Sep 15 2009

type: RNA

city: Berlin

first: Hans-Joachim

last: Mollenkopf

address: Charitéplatz 1

country: Germany

institute: Max-Planck-Institute for Infection Biology

description: Features were extracted with an image analysis tool version A.9.5.3 using the GE2-V5_95_Feb07 protocol (Agilent Technologies).

hyb_protocol: Labeled samples was mixed and hybridized to 8x15K custom-made microarray according to the supplier`s protocol (Agilent Technologies). Washing of the hybridized arrays was done with the SSC protocol according supplier`s protocol (Agilent Technologies).

scan_protocol: Scanning of microarrays was performed with 5 µm resolution using a DNA microarray laser scanner (Agilent Technologies).

data_processing: Data analysis was carried out on the Rosetta Inpharmics platform Resolver Version 7.0. Ratio profiles were combined in an error-weighted fashion with Resolver to create ratio experiments. A 1.75-fold change expression cut-off for ratio experiments was applied together with anti-correlation of ratio profiles rendering the microarray analysis set highly significant (P-value > 0.05), robust and reproducible. The LogRatio (base 10) represents Cy5/Cy3 for each sample pair. Values were generated with FE 9.5.3.1. software (Agilent Technologies) using the GE2-v4_95_Feb07 protocol and default settings. In brief, background, signal biases and errors were calculated applying Signal Correction - Calculate Surface Fiting, Spatial Detrending and the Most Conservative error model. Dye biases were corrected with the Dye Normalization Probe Selection Method using Rank Consistent Probes and the normalization correction methods Linear and Lowess. To determine if a feature is differentially expressed, three measures are important: LogRatio, LogRatioError and the PValueLogRatio. The LogRatio (VALUE) is the log (base 10) of the ratio of the red channel signal to the green channel signal after background adjustment and dye bias correction. LogRatioError is the error about the mean of the log ratio. PValueLogRatio is a measure of confidence that a feature was identified as being differentially expressed. The log ratio error was determined by the error model and therefore was used to help assess the significance of the computed log ratio. The p-value was calculated using the logratio and the log ratio error. Genes with lower p-values have higher confidence of being differentially expressed. In order to combine dye-reversal color-swaps, a negative polarity was added to one of the arrays.

molecule: total RNA

organism: Neisseria gonorrhoeae

source_name: hfq

label_protocol: Equal amounts (10 µg) of total bacterial RNA were reverse transcribed. cDNA was labeled with Cy3 or Cy5 using the CyScribe Postlabelling Kit (GE Healthcare) and labeled cDNA purified using the CyScribe GFX purification kit (GE Healthcare)

extract_protocol: Qiagen RNeasy including on-column DNase-digest

tag: genotype

value: hfq KO mutant (MS11hfq)

tag: biological replicate

value: I

value: wildtype (MS11)


title: MS11 vs. hfq (I)


title: SAT63

status: Public on Sep 14 2009

city: Seattle

first: Christopher

middle: L

last: Plaisier

state: Washington

address: 401 Terry Avenue North

country: USA

institute: Institute for Systems Biology

hyb_protocol: The cRNA probes were then hybridized using standard Affymetrix protocols described in the GeneChip Expression Analysis Technical Manual (Affymetrix P/N 702232).

scan_protocol: The chips were scanned using the GeneArray scanner (Affymetrix) to produce CEL files.

data_processing: A rigorous quality control pipeline was developed to prepare the gene expression microarray data for analysis. First, the CEL files were imported into R 2.8.0 using the justRMA function of the Affymetrix library from Bioconductor, which applies background subtraction and quantile normalization. An alternate CDF file (U133Plus2msk.cdf.gz) was used to exclude mis-targeted and nonspecific probes from the microarrays (Zhang et al. 2005), and probesets with less than 7 remaining probes were also excluded. Probesets with more than 50% absent calls were excluded, as calculated using the panp package from Bioconductor. Finally, the ComBat library in R was used to correct for batch effect (Johnson et al. 2007).

label: biotin

organism: Homo sapiens

source_name: Subcutaneous adipose tissue

label_protocol: Samples were then converted to cRNA probes using standard Affymetrix protocols described in the GeneChip Expression Analysis Technical Manual (Affymetrix P/N 702232).

extract_protocol: Total RNA was extracted by first disrupting the tissue submerged in QIazol (Qiagen Cat. #79306) with a rotor stator homogenizer, layers were separated with Eppendorf Phase Lock gel 2ml tubes (Eppendorf) and the aqueous layer was then cleaned with the RNeasy Lipid Tissue Mini Kit (Qiagen). The integrity of the RNA was assessed using an Agilent 2100 Bioanalyzer (Agilent).

tag: tissue

value: Subcutaneous adipose tissue


title: cv. Plainsman V 5 DAP  heat & drought stressed seed Replicate 2

status: Public on Sep 24 2009

city: Szeged

first: Attila

last: Szűcs

address: Somogyi utca 4

department: Faculty of Medical Research

country: Hungary

institute: University of Szeged

description: 5 DAP stressed and control seed

hyb_protocol: The hybridization and washing was performed as described in Agilent protocol.

scan_protocol: Scanned on Agilent scanner G2505B

data_processing: Image analysis and normalization was done by Agilent Feature Extraction 10.1 software using default GE2-v5_10_Apr08 protocol (Linear and Lowess)

organism: Triticum aestivum

source_name: Total RNA from cv. Plainsman V 5 DAP  heat&drought stressed seed , labelled by Cy3

label_protocol: 1 µg total RNA was used to synthesize Cy3  labeled aRNA using AMBION (Austin, USA) Amino Allyl MessageAmpTM II aRNA Amplification Kit

extract_protocol: RNA extracted using RNAzol (SIGMA)

value: cv. Plainsman V

tag: day after pollination

value: 5 DAP

tag: type of stress

value: heat&drought

source_name: Total RNA from Total RNA from cv. Plainsman V 5 DAP  control seed  labelled by Cy5, labelled by Cy5

label_protocol: 1 µg total RNA was used to synthesize Cy5  labeled aRNA using AMBION (Austin, USA) Amino Allyl MessageAmpTM II aRNA Amplification Kit

value: control


title: SAT67


title: SAT64


title: SAT65


title: SAT62


title: MS11 vs. hfq (II)

value: II


title: SAT59


title: SAT66


title: SAT55


title: SAT50


title: SAT58


title: SAT56


title: SAT54


title: SAT49


title: SAT61


title: SAT57


title: SAT52


title: SAT60


title: SAT51


title: SAT47


title: SAT53


title: SAT45


title: SAT42


title: SAT48


title: SAT43


title: SAT46


title: SAT41


title: SAT44


title: SAT39


title: SAT28


title: SAT37


title: SAT34


title: SAT36


title: SAT40


title: SAT38


title: SAT32


title: SAT31


title: SAT35


title: SAT25


title: SAT24


title: SAT30


title: SAT33


title: SAT27


title: SAT26


title: SAT29


title: SAT17


title: SAT20


title: SAT21


title: SAT22


title: SAT15


title: SAT19


title: SAT16


title: SAT18


title: SAT13


title: SAT23


title: SAT14


title: SAT10


title: SAT12


title: SAT11


title: SAT08


title: SAT09


title: SAT07


title: SAT03


title: SAT01


title: SAT05


title: SAT02


title: SAT04


title: SAT06


title: PNS056

city: Cambridge

first: Jadwiga

last: Bienkowska

state: MA

address: 45 Fayette Str Apt 3

institute: BiogenIdec

description: no additional information

hyb_protocol: 3 ug of fragmented and labeled cDNA was hybridized HGU-133 plus 2 arrays

scan_protocol: Genechip cartridge arrays using the Genechip Array Station (Affymetrix, Santa Clara, CA).

data_processing: Data was normalized with GCRMA and the proccessed using limma Bioconductor package

source_name: whole blood from rheumatoid arthritis patient treated with anti_TNF therapy

label_protocol: RNA was amplified, fragmented and labeled using  FL Ovation cDNA biotin automated module V2 (cat #A4200, Nugen inc., San Carlos CA )  and FL Ovation cDNA biotin automated module V2 (cat #A4200, Nugen inc., San Carlos CA ) using Beckman Arrayplex automated workstation according to manufactures protocol

extract_protocol: RNA was isolated from PaxGene tubes as per the manufacturer’s standard protocol.

treatment_protocol: anti-TNF therapy

value: whole blood

tag: disease state

value: rheumatoid arthritis

tag: protocol

value: anti_TNF therapy

tag: disease activity (das28_v1)

tag: disease activity (das28_v3)

tag: response to anti-tnf therapy

value: RESPONSE


title: PNS058

description: no additional information


title: PNS053

description: no additional information


title: PNS045

description: no additional information

value: MEDIUM


title: PNS061

description: no additional information


title: PNS057

description: no additional information


title: PNS055

description: no additional information


title: PNS042

description: no additional information


title: PNS034

description: no additional information


title: PNS047

description: no additional information


title: PNS044

description: no additional information


title: PNS054

description: no additional information


title: PNS033

description: no additional information

value: NORESPONSE


title: PNS032

description: no additional information


title: PNS030

description: no additional information


title: PNS052

description: no additional information


title: PNS038

description: no additional information


title: PNS059

description: no additional information


title: PNS031

description: no additional information


title: PNS027

description: no additional information


title: PNS026

description: no additional information


title: PNS025

description: no additional information


title: PNS024

description: no additional information


title: PNS020

description: no additional information


title: PNS015

description: no additional information


title: PNS016

description: no additional information


title: PNS023

description: no additional information


title: PNS019

description: no additional information


title: PNS022

description: no additional information


title: PNS010

description: no additional information


title: PAF510

description: no additional information


title: PNS012

description: no additional information


title: PNS017

description: no additional information


title: PAF509

description: no additional information


title: NSG518

description: no additional information


title: NSG519

description: no additional information


title: PNS005

description: no additional information


title: PAF507

description: no additional information


title: PNS001

description: no additional information


title: PNS002

description: no additional information


title: NSG517

description: no additional information


title: PNS004

description: no additional information


title: NSG511

description: no additional information


title: NSG509

description: no additional information


title: NSG514

description: no additional information


title: NSG508

description: no additional information


title: NSG512

description: no additional information


title: NSG510

description: no additional information


title: NSG513

description: no additional information


title: NSG506

description: no additional information


title: NSG505

description: no additional information


title: NSG411

description: no additional information


title: NSG415

description: no additional information


title: NSG413

description: no additional information


title: NSG412

description: no additional information


title: NSG501

description: no additional information


title: NSG404

description: no additional information


title: NSG405

description: no additional information


title: NSG401

description: no additional information


title: NSG027

description: no additional information


title: NSG200

description: no additional information


title: NSG018

description: no additional information


title: NSG300

description: no additional information


title: NSG307

description: no additional information


title: NSG305

description: no additional information


title: NSG201

description: no additional information


title: NSG029

description: no additional information


title: NSG006

description: no additional information


title: NSG004

description: no additional information


title: CD4+CD8+CD5loCD69lo WT - repeat 1 - mAdbID:95119

city: Bethesda

first: Andy

last: Johnson

state: MD

address: 9000 Rockville Pike

institute: NIAID/NIH

description: mAdb experiment ID: 95119

hyb_protocol: Sample Hybridization Protocol Other: For sample hybridization, buffer (30 mM sodium citrate, 300 mM sodium chloride, 50% formamide, 0.4% SDS, pH 7.0) was held at 42°C. Twenty-two ml was added to each 25 ml sample along with 3 ml of 1:1 salmon sperm/tRNA blocker. The mix was heated for 2 min at 98°C, centrifuged for 3 min at 14,000 x g, and 50 ml was then transferred into a microarray hybridization chamber (MAUI Mixer, Biomicro Systems). After overnight hybridization at 45°C, slides were inserted into a jig and submerged in Buffer A, comprised of 1X SSC and 0.05% SDS. The adhesive MAUI Mixer lid was removed and the slide placed in a rack in a jar containing fresh Buffer A. Slides were agitated for 1 min, transferred to fresh Buffer A, agitated for 2 min, transferred to Buffer B (0.1X SSC) for 2 min and then into fresh Buffer B. Slides were centrifuged for 5 min starting at 220 rpm and increasing to 580 in increments of approximately 100 rpm. Once dry, the slides were removed from the centrifuge and scanned on a GenePix4000B Scanner (Axon Instrumets / Molecular Devices).

scan_protocol: Creator: GenePix Pro 6.1.0.4 Scanner: GenePix 4000B [82425] ScanPower: 100;; 33 LaserPower: 3.09;; 3.49 Temperature: 26.85

data_processing:
BRB Data Processing Protocol
Calculation Method: A lowess smoother was used to normalize raw array data. Individual probes were eliminated from analysis if they fit the following criteria: 1. Both spot intensities were >100; 2. The spot flag was not equal to 0; 3. The spot size was >10; 4. The P-value of the log-ratio variation was >0.01; 5. The percent missing/removed via filtering exceeded 35% of the entire dataset.

organism: Mus musculus

source_name: Pooled Reference Sample

label_protocol:
Cy3 Sample Labeling Protocol
Other: 50 ng of RNA was amplified using the Ovation Aminoallyl RNA amplification and labelling system (NuGen) according to the manufacturer’s protocol. 2 ug of amplified cDNA was then labelled using the CyDye Post-Labelling Reactive Dye packs (GE Healthcare).

extract_protocol:
Sample Extraction Protocol
Other: After washing the cells in PBS, RNA was isolated by lysing the cells with TRIZOL reagent (Invitrogen), chloroform extraction and isopropanol precipitation.

tag: strain

value: Thymus

tag: cell type

value: CD4+CD8+CD5loCD69lo; CD4+CD8+CD5+CD69+; and CD4+CD8lo/-H2KloCD24hi thymocytes

source_name: CD4+CD8+CD5loCD69lo WT

label_protocol:
Cy5 Sample Labeling Protocol
Other: 50 ng of RNA was amplified using the Ovation Aminoallyl RNA amplification and labelling system (NuGen) according to the manufacturer’s protocol. 2 ug of amplified cDNA was then labelled using the CyDye Post-Labelling Reactive Dye packs (GE Healthcare).

tag: genotype/variation

value: wildtype

value: CD4+CD8+CD5loCD69lo thymocytes


title: NSG400

description: no additional information


title: NSG301

description: no additional information


title: NSG022

description: no additional information


title: NSG303

description: no additional information


title: NSG025

description: no additional information


title: NSG001

description: no additional information


title: NSG011

description: no additional information


title: NSG021

description: no additional information


title: NSG005

description: no additional information


title: NSG013

description: no additional information


title: NSG202

description: no additional information


title: NSG016

description: no additional information


title: NSG023

description: no additional information


title: NSG003

description: no additional information


title: CD4+CD8lo/-H2K-CD24hi WT - repeat 9 - mAdbID:94957

description: mAdb experiment ID: 94957

scan_protocol: Creator: GenePix Pro 6.1.0.4 Scanner: GenePix 4000B [83871] ScanPower: 100;; 100 LaserPower: 2.52;; 3.79 Temperature: 31.85

source_name: CD4+CD8lo/-H2K-CD24hi WT

value: CD4+CD8lo/-H2K-CD24hi thymocytes


title: NSG010

description: no additional information


title: CD4+CD8lo/-H2K-CD24hi end1-Y489X mutant - repeat 6 - mAdbID:94952

description: mAdb experiment ID: 94952

scan_protocol: Creator: GenePix Pro 6.1.0.4 Scanner: GenePix 4000B [83871] ScanPower: 100;; 100 LaserPower: 2.52;; 3.79 Temperature: 31.15

source_name: CD4+CD8lo/-H2K-CD24hi end1-Y489X mutant

value: induced mutation

value: Themis-Y489X induced stop codon


title: CD4+CD8lo/-H2K-CD24hi end1-Y489X mutant - repeat 8 - mAdbID:94954

description: mAdb experiment ID: 94954

scan_protocol: Creator: GenePix Pro 6.1.0.4 Scanner: GenePix 4000B [83871] ScanPower: 100;; 100 LaserPower: 2.53;; 3.81 Temperature: 31.55


title: CD4+CD8lo/-H2K-CD24hi WT - repeat 7 - mAdbID:94955

description: mAdb experiment ID: 94955

scan_protocol: Creator: GenePix Pro 6.1.0.4 Scanner: GenePix 4000B [83871] ScanPower: 100;; 100 LaserPower: 2.53;; 3.79 Temperature: 31.64


title: CD4+CD8lo/-H2K-CD24hi WT - repeat 8 - mAdbID:94956

description: mAdb experiment ID: 94956

scan_protocol: Creator: GenePix Pro 6.1.0.4 Scanner: GenePix 4000B [83871] ScanPower: 100;; 100 LaserPower: 2.52;; 3.79 Temperature: 31.76


title: CD4+CD8lo/-H2K-CD24hi WT - repeat 6 - mAdbID:94775

description: mAdb experiment ID: 94775

scan_protocol: Creator: GenePix Pro 6.1.0.4 Scanner: GenePix 4000B [82425] ScanPower: 100;; 100 LaserPower: 3.19;; 3.45 Temperature: 32.86


title: CD4+CD8lo/-H2K-CD24hi WT - repeat 4 - mAdbID:94773

description: mAdb experiment ID: 94773

scan_protocol: Creator: GenePix Pro 6.1.0.4 Scanner: GenePix 4000B [82425] ScanPower: 100;; 100 LaserPower: 3.22;; 3.46 Temperature: 32.61


title: NSG012

description: no additional information


title: CD4+CD8lo/-H2K-CD24hi end1-Y489X mutant - repeat 4 - mAdbID:94770

description: mAdb experiment ID: 94770

scan_protocol: Creator: GenePix Pro 6.1.0.4 Scanner: GenePix 4000B [83871] ScanPower: 100;; 100 LaserPower: 2.57;; 3.79 Temperature: 32.31


title: CD4+CD8lo/-H2K-CD24hi end1-Y489X mutant - repeat 7 - mAdbID:94953

description: mAdb experiment ID: 94953

scan_protocol: Creator: GenePix Pro 6.1.0.4 Scanner: GenePix 4000B [83871] ScanPower: 100;; 100 LaserPower: 2.52;; 3.81 Temperature: 31.39


title: CD4+CD8lo/-H2K-CD24hi WT - repeat 5 - mAdbID:94774

description: mAdb experiment ID: 94774

scan_protocol: Creator: GenePix Pro 6.1.0.4 Scanner: GenePix 4000B [83871] ScanPower: 100;; 100 LaserPower: 2.57;; 3.79 Temperature: 32.37


title: CD4+CD8lo/-H2K-CD24hi end1-Y489X mutant - repeat 5 - mAdbID:94771

description: mAdb experiment ID: 94771

scan_protocol: Creator: GenePix Pro 6.1.0.4 Scanner: GenePix 4000B [82425] ScanPower: 100;; 100 LaserPower: 3.16;; 3.46 Temperature: 32.31


title: allogeneic transplanted, anti-ICOSL mAb day 3 treated mice, sample 1

city: Munich

first: Guenther

middle: HS

last: Richter

address: Parzivalstr. 16

department: Pediatrics & Children's Cancer Research Center

institute: Klinikum rechts der Isar, TU-Muenchen

description: allogeneic transplanted C57BL/6 mice, anti-ICOSL mAb day 3 treated

hyb_protocol: standard Affymetrix protocol

scan_protocol: standard Affymetrix protocol

data_processing: Microarray Suite 5.0 was used with the data and for normalization by scaling all probe sets to 500.

label: biotin, streptavidin-phycoerytrin

label_protocol: A total of 10 µg RNA from each sample was used to prepare biotinylated target cRNA as previously described (Staege MS, Hutter C, Neumann I, et al. DNA microarrays reveal relationship of Ewing family tumors to both endothelial and fetal neural crest-derived cells and define novel targets. Cancer Res 2004;64:8213-21). A detailed protocol is available at www.affymetrix.com.

extract_protocol: Total RNA was prepared with acid phenol/chloroform extraction (TRIzol, Invitrogen; Karlsruhe, Germany) followed by purification with RNeasy Mini Kit (Qiagen; Hilden, Germany) according to the manufacturer’s instructions and was quantified spectro-photometrically. The quality of the purified RNA was assessed by agarose gel analysis.

treatment_protocol: B6 recipients were irradiated with 10.0 Gray, administered from a 137 Cs source. Splenocytes from C3H mice were prepared as single cell suspensions in PBS and depleted of red blood cells. 2 – 3 × 10^7 splenocytes in a volume of 200 μl were transplanted into B6 recipients via tail vein injection 4 – 6 h after irradiation. Mice received 500 μg anti-ICOSL mAb i. p starting at day 3, followed by  subsequent injections of 200 μg of mAb every other day. Mouse splenocytes were collected on day 4 after transplantation.

tag: strain recipient

tag: strain donor

tag: age

value: 9-12 weeks old

tag: gender

value: female

value: splenocytes


title: allogeneic transplanted, anti-ICOSL mAb day 3 treated mice, sample 2

source_name: allogeneic transplanted C57BL/6 mice, anti-ICOSL mAb day 3 treated


title: CD4+CD8lo/-H2K-CD24hi end1-Y489X mutant - repeat 3 - mAdbID:94772

description: mAdb experiment ID: 94772

scan_protocol: Creator: GenePix Pro 6.1.0.4 Scanner: GenePix 4000B [83871] ScanPower: 100;; 100 LaserPower: 2.57;; 3.79 Temperature: 32.34


title: allogeneic transplanted, isotype treated mice, sample 1

description: allogeneic transplanted C57BL/6 mice, isotype treated

treatment_protocol: B6 recipients were irradiated with 10.0 Gray, administered from a 137 Cs source. Splenocytes from C3H mice were prepared as single cell suspensions in PBS and depleted of red blood cells. 2 – 3 × 10^7  splenocytes in a volume of 200 μl were transplanted into B6 recipients via tail vein injection 4 – 6 h after irradiation. Mice were treated with isotype control (control rat IgG). Mouse splenocytes were collected on day 4 after transplantation.


title: syngen transplanted C57BL/6 mice

description: syngen transplanted C57BL/6 mice

source_name: syngen transplanted C57BL/6 mice

treatment_protocol: B6 recipients were irradiated with 10.0 Gray, administered from a 137 Cs source. Splenocytes from B6 mice were prepared as single cell suspensions in PBS and depleted of red blood cells. 2 – 3 × 10^7  splenocytes in a volume of 200 μl were transplanted into B6 recipients via tail vein injection 4 – 6 h after irradiation. Mouse splenocytes were collected on day 4 after transplantation.


title: CD4+CD8lo/-H2K-CD24hi end1-Y489X mutant - repeat 2 - mAdbID:94766

description: mAdb experiment ID: 94766

scan_protocol: Creator: GenePix Pro 6.1.0.4 Scanner: GenePix 4000B [83871] ScanPower: 100;; 100 LaserPower: 2.57;; 3.79 Temperature: 32.19


title: CD4+CD8lo/-H2K-CD24hi WT - repeat 1 - mAdbID:94767

description: mAdb experiment ID: 94767

scan_protocol: Creator: GenePix Pro 6.1.0.4 Scanner: GenePix 4000B [82425] ScanPower: 100;; 100 LaserPower: 3.11;; 3.46 Temperature: 30.6


title: allogeneic transplanted, anti-ICOSL mAb day 0 treated mice, sample 1

description: allogeneic transplanted C57BL/6 mice, anti-ICOSL mAb day 0 treated

treatment_protocol: B6 recipients were irradiated with 10.0 Gray, administered from a 137 Cs source. Splenocytes from C3H mice were prepared as single cell suspensions in PBS and depleted of red blood cells. 2 – 3 × 10^7 splenocytes in a volume of 200 μl were transplanted into B6 recipients via tail vein injection 4 – 6 h after irradiation. Mice received 500 μg anti-ICOSL mAb i. p starting at day 0, followed by  subsequent injections of 200 μg of mAb every other day. Mouse splenocytes were collected on day 4 after transplantation.


title: allogeneic transplanted, anti-ICOSL mAb day 0 treated mice, sample 2

source_name: allogeneic transplanted C57BL/6 mice, anti-ICOSL mAb day 0 treated


title: CD4+CD8lo/-H2K-CD24hi WT - repeat 3 - mAdbID:94765

description: mAdb experiment ID: 94765

scan_protocol: Creator: GenePix Pro 6.1.0.4 Scanner: GenePix 4000B [82425] ScanPower: 100;; 100 LaserPower: 3.11;; 3.49 Temperature: 28.43


title: allogeneic transplanted, isotype treated mice, sample 2

source_name: allogeneic transplanted C57BL/6 mice, isotype treated


title: CD4+CD8lo/-H2K-CD24hi end1-Y489X mutant - repeat 1 - mAdbID:94769

description: mAdb experiment ID: 94769

scan_protocol: Creator: GenePix Pro 6.1.0.4 Scanner: GenePix 4000B [82425] ScanPower: 100;; 100 LaserPower: 3.17;; 3.46 Temperature: 31.79


title: CD4+CD8lo/-H2K-CD24hi WT - repeat 2 - mAdbID:94764

description: mAdb experiment ID: 94764

scan_protocol: Creator: GenePix Pro 6.1.0.4 Scanner: GenePix 4000B [83871] ScanPower: 100;; 100 LaserPower: 2.57;; 3.78 Temperature: 31.85


title: 6 week smoke_30236

city: Ottawa

first: Andrew

last: Williams

state: ON

address: 50 Columbine

country: Canada

institute: Health Canada

description: Ratio is the normalized log2 ratio (sample/reference), RawCy3 is the raw median signal intensity for the reference, RawCy5 is the raw median signal intensity for the sample.

hyb_protocol: Agilent Mouse G4121A Microarrays (containing approximately 22,000 probes) were hybridized with 5 µg Cy5-labeled lung RNA and 5 µg Cy3-labeled Universal Mouse Reference RNA (Stratagene, CA, USA), used as a common reference on all arrays.  Arrays were incubated overnight at 60 ºC in Agilent hybridization solution and washed according to manufacturer’s instructions.

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

data_processing: A blocked factorial design, using the date of hybridization and the date of exposure, was used to analyse lung microarray data. The data were normalized using a LOWESS curve using the R MAANOVA library. Ratio intensity plots and heat maps for the raw and normalized data were also constructed using R.  Differentially expressed genes were determined using the MAANOVA library in R. The main effects in the model included treatment, duration of exposure and break period, as well all two-way and the three-way interaction. This model was applied to the log2 of the relative intensities. The Fs statistic, a shrinkage estimator for the gene-specific variance components, was used to test main effects, interactions and pair-wise comparisons. The p-values for all statistical tests were estimated by the permutation method using residual shuffling, followed by adjustment for multiple comparisons by using the false discovery rate (FDR) approach.

source_name: Total RNA from mouse lungs labeled with Cyanine-5 (red)

label_protocol: Individual 2.5 µg aliquots of RNA from each sample were amplified and labelled using the Low RNA Input Fluorescent Linear Amplification Kit (Agilent, (5184-3523)).

extract_protocol: RNA was isolated using the TRIzol reagent (Invitrogen Canada Inc., Burlington, ON, Canada), and further purified using RNeasy Mini Kits (Qiagen Inc., Mississauga, ON, Canada).  RNA was quantified using UV spectrophotometer, and quality was confirmed using the Agilent 2100 Bioanalyzer and RNA 6000 NanoLab Chip Kit (Agilent Technologies Canada Inc., Mississauga, ON, Canada).

tag: Tissue

value: part of lung

tag: Gender

value: Male

tag: Age

value: 8-10 week

tag: C57BL/6 x CBA F1 hybrid mice

tag: Date of exposure

tag: Exposure

value: Two cigarettes daily at a rate of 0.08 litres per minute, 1 (20ml) puff per 52 seconds, 5 days/week for a total of 6 or 12 weeks, including the 2 week lead-up period

tag: Time post exposure

value: 3 h

tag: micro array ID:30236

tag: Hybridization Block 2

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

extract_protocol: Unknown: Material was purchased as total RNA (universal mouse reference total RNA (Stratagene, CA, USA))

tag: Total RNA derived from 11 mouse cell lines


title: 12 week sham_30235

value: restrainers only

tag: micro array ID:30235

tag: Hybridization Block 3


title: 12 week sham_30232

tag: micro array ID:30232

tag: Hybridization Block 5


title: 12 week smoke-6 week break_30233

tag: micro array ID:30233


title: 12 week smoke-break sham_30234

tag: micro array ID:30234


title: CD4+CD8+CD5loCD69lo WT - repeat 9 - mAdbID:94756

description: mAdb experiment ID: 94756

scan_protocol: Creator: GenePix Pro 6.1.0.4 Scanner: GenePix 4000B [83871] ScanPower: 100;; 100 LaserPower: 2.51;; 3.81 Temperature: 31.58

tag: strain

tag: tissue

tag: cell type

tag: genotype/variation


title: NSG410

description: no additional information

scan_protocol: Genechip cartridge arrays using the Genechip Array Station (Affymetrix, Santa Clara, CA).

value: whole blood

tag: disease state

value: rheumatoid arthritis

tag: protocol

value: anti_TNF therapy

tag: disease activity (das28_v1)

tag: disease activity (das28_v3)

tag: response to anti-tnf therapy

value: MEDIUM


title: CD4+CD8+CD5loCD69lo WT - repeat 8 - mAdbID:94755

description: mAdb experiment ID: 94755

scan_protocol: Creator: GenePix Pro 6.1.0.4 Scanner: GenePix 4000B [83871] ScanPower: 100;; 100 LaserPower: 2.52;; 3.81 Temperature: 31.42

value: Thymus

value: CD4+CD8+CD5loCD69lo; CD4+CD8+CD5+CD69+; and CD4+CD8lo/-H2KloCD24hi thymocytes

value: wildtype

value: CD4+CD8+CD5loCD69lo thymocytes


title: CD4+CD8+CD5loCD69lo WT - repeat 7 - mAdbID:94754

description: mAdb experiment ID: 94754

scan_protocol: Creator: GenePix Pro 6.1.0.4 Scanner: GenePix 4000B [83871] ScanPower: 100;; 100 LaserPower: 2.52;; 3.79 Temperature: 31.21


title: CD4+CD8+CD5loCD69lo end1-Y489X mutant - repeat 9 - mAdbID:94753

description: mAdb experiment ID: 94753

scan_protocol: Creator: GenePix Pro 6.1.0.4 Scanner: GenePix 4000B [83871] ScanPower: 100;; 100 LaserPower: 2.52;; 3.79 Temperature: 30.97

source_name: CD4+CD8+CD5loCD69lo end1-Y489X mutant

value: induced mutation

value: Themis-Y489X induced stop codon


title: CD4+CD8+CD5loCD69lo end1-Y489X mutant - repeat 8 - mAdbID:94747

description: mAdb experiment ID: 94747

scan_protocol: Creator: GenePix Pro 6.1.0.4 Scanner: GenePix 4000B [82425] ScanPower: 100;; 100 LaserPower: 3.11;; 3.52 Temperature: 30.32


title: 6 week smoke and 6 week break_30189

tag: Tissue

tag: Gender

tag: Age

tag: C57BL/6 x CBA F1 hybrid mice

tag: Date of exposure

tag: Exposure

tag: Time post exposure

tag: micro array ID:30189


title: 12 week smoke-break sham_30025

tag: micro array ID:30025

tag: Hybridization Block 2

tag: Total RNA derived from 11 mouse cell lines


title: 6 week sham_30188

tag: micro array ID:30188

tag: Hybridization Block 5


title: 6 week smoke-break sham_30190

tag: micro array ID:30190


title: 6 week smoke_30187

tag: micro array ID:30187


title: CD4+CD8+CD5+CD69+ WT - repeat 6 - mAdbID:93450

description: mAdb experiment ID: 93450

scan_protocol: Creator: GenePix Pro 6.0.1.27 Scanner: GenePix 4000B [83871] ScanPower: 100;; 100 LaserPower: 2.56;; 3.81 Temperature: 30.66

tag: strain

tag: tissue

tag: cell type

source_name: CD4+CD8+CD5+CD69+ WT

tag: genotype/variation

value: CD4+CD8+CD5+CD69+ thymocytes


title: CD4+CD8+CD5+CD69+ end1-Y489X mutant - repeat 9 - mAdbID:93449

description: mAdb experiment ID: 93449

scan_protocol: Creator: GenePix Pro 6.0.1.27 Scanner: GenePix 4000B [82425] ScanPower: 100;; 100 LaserPower: 3.14;; 3.49 Temperature: 29.87

source_name: CD4+CD8+CD5+CD69+ end1-Y489X mutant


title: CD4+CD8+CD5loCD69lo end1-Y489X mutant - repeat 7 - mAdbID:94752

description: mAdb experiment ID: 94752

scan_protocol: Creator: GenePix Pro 6.1.0.4 Scanner: GenePix 4000B [82425] ScanPower: 100;; 100 LaserPower: 3.11;; 3.52 Temperature: 29.84


title: CD4+CD8+CD5+CD69+ WT - repeat 7 - mAdbID:93451

description: mAdb experiment ID: 93451

scan_protocol: Creator: GenePix Pro 6.0.1.27 Scanner: GenePix 4000B [82425] ScanPower: 100;; 100 LaserPower: 3.16;; 3.5 Temperature: 31.06


title: CD4+CD8+CD5+CD69+ WT - repeat 8 - mAdbID:93452

description: mAdb experiment ID: 93452

scan_protocol: Creator: GenePix Pro 6.0.1.27 Scanner: GenePix 4000B [83871] ScanPower: 100;; 100 LaserPower: 2.56;; 3.82 Temperature: 30.87


title: Wild-type vs HP0244-KO-pH2.5 replicate 3

city: Los Angeles

first: Yi

last: Wen

state: CA

address: 11301 Wilshire Blvd., Bldg.113, Rm. 324

department: Physiology Department

institute: UCLA/VAGLAHS

description: Biological replicate 3 of 3. pH2.5

hyb_protocol: Helicobacter pylori microarrays containing 1,534 predicted ORFs of Helicobacter pylori strain 26695 were hybridized in a final volume of 20 µl containing 3 × SSC, 0.8 mg/ml salmon sperm DNA and 0.2% SDS.  Prior to hybridization, combined Cy3 and Cy5 labeled probes (representing mutant and wild type strains) were heated to 95˚C for 2 min and pipetted directly onto microarray slides.  A glass cover slip (22 mm × 22 mm) was applied and the arrays hybridized overnight at 65˚C in a humidified hybridization chamber.  Following hybridization, slides were washed for 5 min in 0.2 × SSC with 0.1% SDS, followed by two washes of 3 min each in 0.2 × SSC and rinsed in 0.05 × SSC.  To remove residual salts, slides were spun at 500 rpm for 5 min prior to scanning.

scan_protocol: Scanned on an Agilent G2565AA scanner with dual-image output.

data_processing: Signal intensities for each spot were determined using Phoretix Array software (Nonlinear Dynamic).  Following background subtraction, signal intensities were calculated as the percent of total signal from all spots as a means of normalization.  These values were used to determine the ratio of wild type to HP0244-defecient mutant signals. A threshold of a minimum acceptable signal (two standard deviations above background intensity) was used to eliminate expression ratios that were extremely high or low due to too low signals in wild type or mutant samples.

organism: Helicobacter pylori 26695

source_name: Wild type Helicobacter pylori-pH2.5

label_protocol: For each comparative array hybridization, labeled cDNA was synthesized by reverse transcription from RNAs of wild-type strain after treatment at pH 7.4, pH 4.5 without urea, or pH 2.5 (30 mM urea) with Cy5-dCTP, and from RNAs of HP0244-deficent mutant strain (after corresponding treatment at different pH conditions) with Cy3-dCTP, using the Superscript II reverse-transcription kit (Invitrogen).  For each reverse transcription reaction, 1 µg of random primers (Invitrogen) and 40 µg of Helicobacter pylori RNA were mixed in a final volume of 12.5 µl, heated to 70oC for 10 min and chilled on ice.  Fluorescent cDNA probe synthesis was performed at 42˚C for 3 h in a 25 µl reaction volume containing 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2 10 mM DTT, 0.5 mM dATP, 0.5 mM dGTP, 0.5 mM dTTP, 0.2 mM dCTP, 0.1 mM either Cy3 or Cy5 conjugated dCTP (Amersham), 40 U RNAsin (Promega), and 200 U Superscript II reverse transcriptase (Invitrogen.).  Following reverse transcription, the RNA template was degraded by incubating for 40 min at 65 oC in 0.27 M NaOH, followed by neutralization with Tris-HCl buffer.  Labeled cDNA was purified and concentrated prior to hybridization using Microcon 30 concentrators (Amicon).

growth_protocol: In preparation for an experiment, bacteria were scraped from the plates, suspended in 1 mM phosphate HP buffer (138 mM NaCl, 5 mM KCl, 1 mM CaCl2, 0.5 mM MgCl2, 10 mM glucose, 1 mM glutamine), pH 7.0, and transferred to fresh plates for 24 hours.

extract_protocol: Total RNA was isolated from Helicobacter pylori strains using TRIzol reagent (Invitrogen, CA) combined with RNeasy columns (Qiagen, CA).  The bacterial pellet was resuspended in 500 µl of TRIzol reagent (Invitrogen) and lysed at room temperature for 5 min before 100 µl of chloroform was added. After spinning at 12000 g for 10 minutes at 4oC, the supernatant was mixed with 250 µl ethanol and applied to an RNeasy spin column (Qiagen), and RNA purification was processed following manufacturer’s instructions (beginning with the application to the column). RNA concentration was quantified by absorbance at 260 nm and the quality was evaluated by capillary electrophoresis using an Agilent 2100 Bioanalyzer with the RNA 6000 Nano Assay kit (Agilent Technologies).

treatment_protocol: For exposure to experimental low pH conditions, the overnight culture of Helicobacter pylori on TSA (trypticase soy agar) plates supplemented with 5% sheep blood were suspended in Brain Heart Infusion (BHI) medium (Difco) to an OD600 of 0.20 ~ 0.25.  The pH of BHI was adjusted to pH 7.4, pH 4.5, or pH 2.5 using concentrated HCl followed by filtration to remove any precipitate.  Helicobacter pylori were then incubated in the presence of urea (at an indicated concentration) with shaking (120 rpm) under microaerobic conditions at 37˚C for 30 minutes.

tag: pH2.5, 30 mM urea, 30min

organism: Helicobacter pylori

source_name: HP0244-deficient muant-pH2.5


title: Wild-type vs HP0244-KO-pH2.5 replicate 2

description: Biological replicate 2 of 3. pH2.5


title: CD4+CD8+CD5+CD69+ end1-Y489X mutant - repeat 7 - mAdbID:93447

description: mAdb experiment ID: 93447

scan_protocol: Creator: GenePix Pro 6.0.1.27 Scanner: GenePix 4000B [83871] ScanPower: 100;; 100 LaserPower: 2.55;; 3.81 Temperature: 30.39


title: CD4+CD8+CD5+CD69+ WT - repeat 5 - mAdbID:93277

description: mAdb experiment ID: 93277

scan_protocol: Creator: GenePix Pro 6.1.0.2 Scanner: GenePix 4000B [83871] ScanPower: 100;; 100 LaserPower: 2.55;; 3.81 Temperature: 30.36


title: CD4+CD8+CD5+CD69+ end1-Y489X mutant - repeat 8 - mAdbID:93448

description: mAdb experiment ID: 93448

scan_protocol: Creator: GenePix Pro 6.0.1.27 Scanner: GenePix 4000B [82425] ScanPower: 100;; 100 LaserPower: 3.12;; 3.49 Temperature: 29.5


title: CD4+CD8+CD5+CD69+ WT - repeat 4 - mAdbID:93276

description: mAdb experiment ID: 93276

scan_protocol: Creator: GenePix Pro 6.1.0.2 Scanner: GenePix 4000B [83871] ScanPower: 100;; 100 LaserPower: 2.56;; 3.84 Temperature: 30.02


title: 12 week smoke-6 week break_30022

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

tag: Tissue

value: part of lung

tag: Gender

tag: Age

tag: C57BL/6 x CBA F1 hybrid mice

tag: micro array ID:30022

tag: Hybridization Block 3


title: 6 week sham_29900

value: Male

tag: Date of exposure

tag: Exposure

tag: Time post exposure

tag: micro array ID:29900


title: 6 week smoke-break sham_29902

value: 8-10 week

tag: micro array ID:29902

tag: Total RNA derived from 11 mouse cell lines


title: 12 week sham_29876

tag: micro array ID:29876

tag: Hybridization Block 2


title: 12 week smoke-6 week break_29877

value: Two cigarettes daily at a rate of 0.08 litres per minute, 1 (20ml) puff per 52 seconds, 5 days/week for a total of 6 or 12 weeks, including the 2 week lead-up period

tag: micro array ID:29877


title: 6 week smoke_29899

value: 3 h

tag: micro array ID:29899


title: 12 week smoke-break sham_30024

value: restrainers only

tag: micro array ID:30024


title: 12 week smoke_29903

tag: Tissue

tag: micro array ID:29903


title: 6 week smoke and 6 week break_29901

tag: Gender

tag: Age

tag: micro array ID:29901


title: Wild-type vs HP0244-KO-pH4.5 replicate 3

description: Biological replicate 3 of 3. pH4.5

source_name: Wild type Helicobacter pylori-pH4.5

tag: pH4.5 without urea, 30min

source_name: HP0244-deficient muant-pH4.5


title: Wild-type vs HP0244-KO-pH2.5 replicate 1

description: Biological replicate 1 of 3. pH2.5

tag: pH2.5, 30 mM urea, 30min


title: Wild-type vs HP0244-KO-pH4.5 replicate 2

description: Biological replicate 2 of 3. pH4.5


title: Wild-type vs HP0244-KO-pH4.5 replicate 1

description: Biological replicate 1 of 3. pH4.5


title: Wild-type vs HP0244-KO-pH7.4 replicate 3

description: Biological replicate 3 of 3. pH7.4

source_name: Wild type Helicobacter pylori-pH7.4

source_name: HP0244-deficient muant-pH7.4


title: 12 week smoke_29870

tag: C57BL/6 x CBA F1 hybrid mice

tag: Date of exposure

tag: Exposure

tag: micro array ID:29870


title: Wild-type vs HP0244-KO-pH7.4 replicate 1

description: Biological replicate 1 of 3. pH7.4


title: CD4+CD8+CD5+CD69+ WT - repeat 3 - mAdbID:93275

description: mAdb experiment ID: 93275

scan_protocol: Creator: GenePix Pro 6.1.0.2 Scanner: GenePix 4000B [82425] ScanPower: 100;; 100 LaserPower: 3.11;; 3.5 Temperature: 28.1

source_name: Pooled Reference Sample

tag: strain

tag: tissue

tag: cell type

tag: genotype/variation


title: CD4+CD8+CD5+CD69+ end1-Y489X mutant - repeat 5 - mAdbID:93273

description: mAdb experiment ID: 93273

scan_protocol: Creator: GenePix Pro 6.1.0.2 Scanner: GenePix 4000B [82425] ScanPower: 100;; 100 LaserPower: 3.09;; 3.51 Temperature: 26.51


title: Wild-type vs HP0244-KO-pH7.4 replicate 2

description: Biological replicate 2 of 3. pH7.4


title: CD4+CD8+CD5+CD69+ end1-Y489X mutant - repeat 6 - mAdbID:93274

description: mAdb experiment ID: 93274

scan_protocol: Creator: GenePix Pro 6.1.0.2 Scanner: GenePix 4000B [83871] ScanPower: 100;; 100 LaserPower: 2.56;; 3.83 Temperature: 29.74


title: CD4+CD8+CD5+CD69+ end1-Y489X mutant - repeat 4 - mAdbID:93272

description: mAdb experiment ID: 93272

scan_protocol: Creator: GenePix Pro 6.1.0.2 Scanner: GenePix 4000B [83871] ScanPower: 100;; 100 LaserPower: 2.56;; 3.83 Temperature: 29.59


title: CD4+CD8+CD5+CD69+ WT - repeat 2 - mAdbID:93216

description: mAdb experiment ID: 93216

scan_protocol: Creator: GenePix Pro 6.1.0.2 Scanner: GenePix 4000B [82425] ScanPower: 100;; 100 LaserPower: 3.24;; 3.44 Temperature: 31.64


title: 6 week smoke-break sham_29869

tag: Time post exposure

tag: micro array ID:29869

tag: Hybridization Block 2

tag: Total RNA derived from 11 mouse cell lines


title: 6 week sham_29860

tag: Tissue

tag: Gender

tag: micro array ID:29860

tag: Hybridization Block 4


title: CD4+CD8+CD5+CD69+ WT - repeat 1 - mAdbID:93215

description: mAdb experiment ID: 93215

scan_protocol: Creator: GenePix Pro 6.1.0.2 Scanner: GenePix 4000B [82425] ScanPower: 100;; 100 LaserPower: 3.15;; 3.43 Temperature: 31


title: LONG_OSA32

city: Utrecht

first: Gayathri

middle: Thevi

last: Selvarajah

state: Utrecht

address: Yalelaan 104

department: Clinical Sciences of Companion Animals

country: Netherlands

institute: University Utrecht

description: Long survival group 16 out of 16

hyb_protocol: Microarray slides were washed for 1 min in 2x SSC containing 0.05% Sodium dodecyl sulphate (SDS) at room temperature (RT), incubated in 0.25% sodium borohydride and 0.25% SDS in 2x SSC for 30 min at 42C  and then rinsed 5 times in double distilled water at RT.  Slides were incubated for 45 minutes at 42C in pre-warmed pre-hybridization buffer (2X) consisting of 10xSSC, 25% formamide, 0.1% SDS and 1% BSA. Slides were washed 5 times in water followed by 5 times in isopropanol at RT and air dried. To 40 µl containing 2.5 µg of each Cy3 and Cy5 labeled RNA 40 µl hybridization buffer was added consisting of 50% formamide, 0.2% SDS and 200 µg/ml sheared herring sperm DNA. The mixture was heated at 95°C for 5 min, centrifuged for 2 min at 12,000xg and hybridized to the slides at 42C for 16 hours using hybridization chambers (Corning Life Sciences) in a water bath. After hybridization, slides were washed three times: first a low-stringency wash for 4 min at RT in 1xSSC and 0.2% SDS, next a high-stringency wash in 0.1% SDS and 0.2% SDS for 4 min and finally 2 washes in 0.1xSSC at room temperature. Slides were blown dry using compressed nitrogen.

scan_protocol: Slides were scanned using an Agilent G2565AA DNA microarray scanner (100% laser power, 30% photomultiplier tube)

data_processing: Image analysis was carried out using Imagene 5.0 software (Biodiscovery Inc). Data were normalized based on Lowess print-tip normalization by using the gene spots to calculate the Lowess line for each sub grid and then applying the these lines to all spots.

organism: Canis lupus familiaris

source_name: Osteosarcoma 32, long survival time

label_protocol: Total RNA isolated from bone powder was used for cRNA synthesis. About 2 µg total RNA was reverse transcribed using Superscript II reverse transcriptase and T7 promoter coupled poly T primers (5' GGCCAGTGAATTGTAATACGACTCACCTATAGGGAGG CGGT). After second strand synthesis, cDNA products were purified using QIAquick PCR Purification Kit and transcribed using the T7 Megascript kit (Ambion) using a UTP to 5-(3-aminoallyl)-UTP ratio of 1/3.5. The RNeasy mini kit (Qiagen) was used to clean the resulting cRNA. Three µg cRNA  was coupled to Cy3 or Cy5 fluorophores. Labeled cRNA was cleaned using ChromaSpin-30 columns and cRNA samples with specific activity of 2-5% dye-labeled nucleosides were used for microarray hybridization

tag: Long_312 Days

source_name: Reference pool

extract_protocol: Snap frozen bone tumour samples were pulverized for 45 seconds at a speed of 2000 rounds per minute in the Mikro Dismembrator®. Total RNA was isolated from bone powder using Qiagen RNA isolation (mini) kit and DNase treatment kit.

tag: All 32 OSA samples.


title: 12 week smoke-6 week break_9920

tag: Age

tag: C57BL/6 x CBA F1 hybrid mice

tag: Date of exposure

tag: micro array ID:9920


title: 6 week sham_29867

tag: Exposure

tag: micro array ID:29867


title: 6 week smoke and 6 week break_29868

tag: micro array ID:29868


title: CD4+CD8+CD5+CD69+ end1-Y489X mutant - repeat 3 - mAdbID:93213

description: mAdb experiment ID: 93213

scan_protocol: Creator: GenePix Pro 6.1.0.2 Scanner: GenePix 4000B [82425] ScanPower: 100;; 100 LaserPower: 3.14;; 3.44 Temperature: 29.9

tag: strain

tag: tissue

tag: cell type

tag: genotype/variation


title: LONG_OSA31

description: Long survival group 15 out of 16

source_name: Osteosarcoma 31, long survival time

extract_protocol: Snap frozen bone tumour samples were pulverized for 45 seconds at a speed of 2000 rounds per minute in the Mikro Dismembrator®. Total RNA was isolated from bone powder using Qiagen RNA isolation (mini) kit and DNase treatment kit

tag: Long_920 Days


title: CD4+CD8+CD5+CD69+ end1-Y489X mutant - repeat 2 - mAdbID:93212

description: mAdb experiment ID: 93212

scan_protocol: Creator: GenePix Pro 6.1.0.2 Scanner: GenePix 4000B [82425] ScanPower: 100;; 100 LaserPower: 3.14;; 3.45 Temperature: 27.85


title: 12 week smoke-break sham_9921

tag: Tissue

tag: Gender

tag: Time post exposure

tag: micro array ID:9921

tag: Total RNA derived from 11 mouse cell lines


title: 12 week sham_9898

tag: Age

tag: micro array ID:9898

tag: Hybridization Block 1


title: 12 week sham_9919

tag: C57BL/6 x CBA F1 hybrid mice

tag: Date of exposure

tag: micro array ID:9919

tag: Hybridization Block 4


title: LONG_OSA30

description: Long survival group 14 out of 16

source_name: Osteosarcoma 30, long survival time

tag: Long_312 Days

tag: All 32 OSA samples.


title: LONG_OSA29

description: Long survival group 13 out of 16

source_name: Osteosarcoma 29, long survival time

tag: Long_348 Days


title: LONG_OSA28

description: Long survival group 12 out of 16

source_name: Osteosarcoma 28, long survival time

tag: Long_705 Days


title: LONG_OSA27

description: Long survival group 11 out of 16

source_name: Osteosarcoma 27, long survival time

tag: Long_239 Days


title: CD4+CD8+CD5+CD69+ end1-Y489X mutant - repeat 1 - mAdbID:93211

description: mAdb experiment ID: 93211

scan_protocol: Creator: GenePix Pro 6.1.0.2 Scanner: GenePix 4000B [83871] ScanPower: 100;; 100 LaserPower: 2.61;; 3.81 Temperature: 34.17

tag: strain

tag: tissue

tag: cell type

tag: genotype/variation


title: LONG_OSA26

description: Long survival group 10 out of 16

source_name: Osteosarcoma 26, long survival time

tag: Long_284 Days


title: 12 week smoke-6 week break_9897

source_name: Total RNA from mouse lungs labeled with Cyanine-5 (red)

tag: Tissue

tag: Gender

tag: Age

tag: Exposure

tag: Time post exposure

tag: micro array ID:9897

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

tag: Total RNA derived from 11 mouse cell lines


title: LONG_OSA25

description: Long survival group 9 out of 16

source_name: Osteosarcoma 25, long survival time

tag: Long_445 Days


title: CD4+CD8+CD5loCD69lo WT - repeat 5 - mAdbID:93209

description: mAdb experiment ID: 93209

scan_protocol: Creator: GenePix Pro 6.1.0.2 Scanner: GenePix 4000B [82425] ScanPower: 100;; 100 LaserPower: 3.21;; 3.44 Temperature: 32.03

source_name: CD4+CD8+CD5loCD69lo WT


title: CD4+CD8+CD5loCD69lo WT - repeat 4 - mAdbID:93208

description: mAdb experiment ID: 93208

scan_protocol: Creator: GenePix Pro 6.1.0.2 Scanner: GenePix 4000B [82425] ScanPower: 100;; 100 LaserPower: 3.17;; 3.45 Temperature: 30.9


title: CD4+CD8+CD5loCD69lo end1-Y489X mutant - repeat 6 - mAdbID:93207

description: mAdb experiment ID: 93207

scan_protocol: Creator: GenePix Pro 6.1.0.2 Scanner: GenePix 4000B [82425] ScanPower: 100;; 100 LaserPower: 3.18;; 3.45 Temperature: 29.74

source_name: CD4+CD8+CD5loCD69lo end1-Y489X mutant


title: LONG_OSA22

description: Long survival group 6 out of 16

tag: All 32 OSA samples.

source_name: Osteosarcoma 22, long survival time

tag: Long_1185 Days


title: LONG_OSA19

description: Long survival group 3 out of 16

source_name: Osteosarcoma 19, long survival time

tag: Long_1752 Days


title: LONG_OSA24

description: Long survival group 8 out of 16

source_name: Osteosarcoma 24, long survival time

tag: Long_257 Days


title: CD4+CD8+CD5loCD69lo WT - repeat 6 - mAdbID:93210

description: mAdb experiment ID: 93210

scan_protocol: Creator: GenePix Pro 6.1.0.2 Scanner: GenePix 4000B [82425] ScanPower: 100;; 100 LaserPower: 3.26;; 3.44 Temperature: 32.58

source_name: Pooled Reference Sample

tag: strain


title: LONG_OSA20

description: Long survival group 4 out of 16

source_name: Osteosarcoma 20, long survival time

tag: Long_307 Days


title: 12 week smoke-break sham_9895

tag: C57BL/6 x CBA F1 hybrid mice

tag: Date of exposure

tag: micro array ID:9895

tag: Hybridization Block 1


title: LONG_OSA23

description: Long survival group 7 out of 16

source_name: Reference pool

source_name: Osteosarcoma 23, long survival time

tag: Long_1289 Days


title: 12 week smoke_9892

tag: Tissue

tag: Gender

tag: Age

tag: micro array ID:9892

tag: Hybridization Block 4


title: LONG_OSA18

description: Long survival group 2 out of 16

source_name: Osteosarcoma 18, long survival time

tag: Long_274 Days


title: 6 week smoke-break sham_9891

tag: Exposure

tag: Time post exposure

tag: micro array ID:9891

tag: Total RNA derived from 11 mouse cell lines


title: CD4+CD8+CD5loCD69lo end1-Y489X mutant - repeat 4 - mAdbID:93205

description: mAdb experiment ID: 93205

scan_protocol: Creator: GenePix Pro 6.1.0.2 Scanner: GenePix 4000B [82425] ScanPower: 100;; 100 LaserPower: 3.15;; 3.46 Temperature: 28.28

tag: strain

tag: tissue

tag: cell type

tag: genotype/variation


title: SHORT_OSA13

description: Short survival group 13 out of 16

tag: All 32 OSA samples.

source_name: Osteosarcoma 13, short survival time

tag: Short_50 Days


title: LONG_OSA17

description: Long survival group 1 out of 16

source_name: Osteosarcoma 17, long survival time

tag: Long_352 Days


title: 6 week smoke_9888

tag: Tissue

tag: C57BL/6 x CBA F1 hybrid mice

tag: Date of exposure

tag: micro array ID:9888


title: LONG_OSA21

description: Long survival group 5 out of 16

source_name: Osteosarcoma 21, long survival time

tag: Long_1619 Days


title: SHORT_OSA11

description: Short survival group 11 out of 16

source_name: Osteosarcoma 11, short survival time

tag: Short_140 Days


title: SHORT_OSA12

description: Short survival group 12 out of 16

source_name: Osteosarcoma 12, short survival time

tag: Short_61 Days


title: SHORT_OSA15

description: Short survival group 15 out of 16

source_name: Osteosarcoma 15, short survival time

tag: Short_65 Days


title: SHORT_OSA14

description: Short survival group 14 out of 16

source_name: Osteosarcoma 14, short survival time

tag: Short_47 Days


title: CD4+CD8+CD5loCD69lo WT - repeat 3 - mAdbID:93204

description: mAdb experiment ID: 93204

scan_protocol: Creator: GenePix Pro 6.1.0.2 Scanner: GenePix 4000B [83871] ScanPower: 100;; 100 LaserPower: 2.6;; 3.82 Temperature: 33.96

source_name: CD4+CD8+CD5loCD69lo WT


title: SHORT_OSA16

description: Short survival group 16 out of 16

source_name: Osteosarcoma 16, short survival time

tag: Short_6 Days


title: 6 week smoke and 6 week break_9890

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

source_name: Total RNA from mouse lungs labeled with Cyanine-5 (red)

tag: Gender

tag: Age

tag: Exposure

tag: Time post exposure

tag: micro array ID:9890

tag: Hybridization Block 4

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

tag: Total RNA derived from 11 mouse cell lines


title: CD4+CD8+CD5loCD69lo end1-Y489X mutant - repeat 5 - mAdbID:93206

description: mAdb experiment ID: 93206

scan_protocol: Creator: GenePix Pro 6.1.0.2 Scanner: GenePix 4000B [82425] ScanPower: 100;; 100 LaserPower: 3.13;; 3.45 Temperature: 29.23

tag: strain

tag: tissue

tag: cell type

source_name: CD4+CD8+CD5loCD69lo end1-Y489X mutant

tag: genotype/variation


title: SHORT_OSA9

description: Short survival group 9 out of 16

tag: All 32 OSA samples.

source_name: Osteosarcoma 9, short survival time

tag: Short_77 Days


title: CD4+CD8+CD5loCD69lo WT - repeat 2 - mAdbID:93203

description: mAdb experiment ID: 93203

scan_protocol: Creator: GenePix Pro 6.1.0.2 Scanner: GenePix 4000B [83871] ScanPower: 100;; 100 LaserPower: 2.59;; 3.81 Temperature: 33.77

source_name: Pooled Reference Sample


title: 6 week smoke and 6 week break_135

tag: Tissue

tag: C57BL/6 x CBA F1 hybrid mice

tag: Date of exposure

tag: micro array ID:135

tag: Hybridization Block 1


title: SHORT_OSA8

description: Short survival group 8 out of 16

source_name: Osteosarcoma 8, short survival time

tag: Short_66 Days

source_name: Reference pool


title: SHORT_OSA10

description: Short survival group 10 out of 16

source_name: Osteosarcoma 10, short survival time

tag: Short_164 Days


title: SHORT_OSA7

description: Short survival group 7 out of 16

source_name: Osteosarcoma 7, short survival time

tag: Short_119 Days


title: CD4+CD8+CD5loCD69lo end1-Y489X mutant - repeat 3 - mAdbID:93201

description: mAdb experiment ID: 93201

scan_protocol: Creator: GenePix Pro 6.1.0.2 Scanner: GenePix 4000B [82425] ScanPower: 100;; 100 LaserPower: 3.13;; 3.49 Temperature: 28.43


title: 6 week smoke_133

tag: Gender

tag: Age

tag: Exposure

tag: Time post exposure

tag: micro array ID


title: 12 week smoke_111

tag: micro array ID:111

tag: Total RNA derived from 11 mouse cell lines


title: FIA T cells - 5

status: Public on Sep 12 2009

type: protein

city: Stanford

first: William

last: Robinson

address: 269 Campus Dr

institute: Stanford University

description:
1:200 dilution
Plasma derived from SJL mice injected with FIA T cells.

hyb_protocol: Synovial antigen arrays were produced using a robotic microarrayer to print peptides and proteins on ArrayIt SuperEpoxy microscope slides (TeleChem International, Sunnyvale, CA). On each slide, antigens are printed at 0.2 mg/ml in four replicates. Slides were then blocked overnight (phosphate-buffered saline (PBS), 3% fetal bovine serum, 0.1% Tween 20) and incubated for 1h at 4°C with individual mouse plasma diluted at 1:200 in PBS/3% fetal bovine serum.  Slides were washed with PBS, water-rinsed, and spun dry.

scan_protocol: Slides were then scanned to determine pixel intensities for each antigen feature (Genepix 4000B; Molecular Devices, Sunnyvale, CA, USA). Genepix Pro 5.0 software (Axon Instruments) at 532 nm was used to detect IgG and IgM reactivity. Images were saved as tiff files.

data_processing: GenePix Pro 5.0 software (Axon Instruments) was used to determine the net median pixel intensities for each antigen feature (each value is background-corrected by removing the background around each spotted protein). The median intensities for each protein were centered around 300 and log-scaled. A level of 10 digital fluorescence intensity units was set as a threshold for significant reactivity. The formula for values below 10 is log(10/300,2), and for all other values it is log(median pixel intensity/300,2).  Data analysis was performed using Significance Analysis for Microarrays (SAM) software (http://www-stat-class. stanford.edu /SAM/servlet/ SAMServlet) to identify antigen features with statistically significant differences in reactivities between the experimental groups. Cluster software was then used to hierarchically group the samples and antigen features on the basis of a pairwise similarity function, and TreeView software was used to display the data as a heat map (http://rana.lbl.gov/EisenSoftware.htm).

molecule: protein

source_name: FIA T cells - 5

label_protocol: After washing with phosphate-buffered saline (PBS)/3% fetal bovine serum/0.1% Tween 20, reactive antibodies were detected using Cy3-conjugated goat-anti-human or goat-anti-mouse IgG/IgM secondary antibody (diluted 1:4,000 in PBS/3% fetal bovine serum; Jackson Immunoresearch). After incubating for 45 minutes with the secondary antibody, the slides were washed with washing solution (PBS/3 % fetal bovine serum/0.1% Tween 20).

growth_protocol: Female SJL/J mice were obtained from Jackson Laboratories (Bar Harbor, Maine) and were between 7 and 9 weeks of age when experiments were initiated. Adoptive transfer recipients were between 5 and 7 weeks of age.

extract_protocol: At the end of the injection courses, the mice were bled for their plasma.

treatment_protocol: Whole splenocytes and lymphocytes from mice with FIA were cultured in vitro for 72 h with 0.01 mg/ml fibrinogen. Cells were harvested and washed with PBS. CD3+ T cells were isolated using CD3+ T-cell enrichment columns (R&D systems, Minneapolis, MN). 50 million T cells were injected intravenously into naïve recipients.

tag: sample

value: Plasma

tag: gender

value: female

tag: genotype

value: SJL/J


title: CD4+CD8+CD5loCD69lo end1-Y489X mutant - repeat 2 - mAdbID:93200

description: mAdb experiment ID: 93200

scan_protocol: Creator: GenePix Pro 6.1.0.2 Scanner: GenePix 4000B [82425] ScanPower: 100;; 100 LaserPower: 3.17;; 3.48 Temperature: 28.95

tag: strain

tag: tissue

tag: cell type

tag: genotype/variation


title: SHORT_OSA5

description: Short survival group 5 out of 16

source_name: Osteosarcoma 5, short survival time

tag: Short_121 Days

tag: All 32 OSA samples.


title: CD4+CD8+CD5loCD69lo end1-Y489X mutant - repeat 1 - mAdbID:93199

description: mAdb experiment ID: 93199

scan_protocol: Creator: GenePix Pro 6.1.0.2 Scanner: GenePix 4000B [82425] ScanPower: 100;; 100 LaserPower: 3.14;; 3.47 Temperature: 29.47


title: SHORT_OSA3

description: Short survival group 3 out of 16

scan_protocol: Slides were scanned using an Agilent G2565AA DNA microarray scanner (100% laser power, 30% photomultiplier tube)

source_name: Osteosarcoma 3, short survival time

tag: Short_13 Days


title: FIA plasma - 4

description:
1:200 dilution,
Plasma derived from SJL mice injected with FIA plasma

source_name: FIA plasma - 4

treatment_protocol: Plasma collected from mice with FIA were pooled, and 0.3 ml was injected intravenously into naïve SJL mice on days 0 and 2.


title: FIA T cells - 3

source_name: FIA T cells - 3


title: FIA T cells - 1

source_name: FIA T cells - 1


title: SHORT_OSA4

description: Short survival group 4 out of 16

source_name: Osteosarcoma 4, short survival time

tag: Short_169 Days


title: FIA plasma - 3

source_name: FIA plasma - 3


title: FIA plasma - 2

source_name: FIA plasma - 2


title: SHORT_OSA2

description: Short survival group 2 out of 16

source_name: Osteosarcoma 2, short survival time

tag: Short_79 Days


title: Naive - 4

description:
1:200 dilution
Plasma

source_name: Naive - 4


title: SHORT_OSA1

description: Short survival group 1 out of 16

source_name: Osteosarcoma 1, short survival time

tag: Short_36 Days


title: FIA plasma - 1

source_name: FIA plasma - 1


title: SHORT_OSA6

description: Short survival group 6 out of 16

source_name: Osteosarcoma 6, short survival time

tag: Short_66 Days


title: FIA T cells - 4

source_name: FIA T cells - 4


title: Naive - 1

description:
1:200 dilution
Plasma

source_name: Naive - 1


title: FIA T cells - 2

source_name: FIA T cells - 2


title: Naive - 3

description:
1:200 dilution
Plasma

source_name: Naive - 3


title: FIA preboost -6

description:
1:150 dilution
Plasma derived from SJL mice immunized with fibrinogen emulsified in CFA or with CFA alone.

source_name: FIA preboost -6

growth_protocol: Female SJL/J mice were obtained from Jackson Laboratories (Bar Harbor, Maine) and were between 7 and 9 weeks of age when experiments were initiated.

treatment_protocol: Mice were immunized subcutaneously with 0.2 mg human fibrinogen (which contains citrulline modifications) (Sigma, St. Louis, MO or Calbiochem, Gibbstown, NJ) in PBS (without calcium or magnesium) emulsified with an equal volume of complete Freund’s adjuvant (CFA) consisting of incomplete Freund’s adjuvant (IFA, Sigma, St. Louis, MO) and 0.5 mg heat-inactivated Mycobacterium tuberculosis (strain H37 RA; Difco Laboratories, Detroit, MI).  Twenty-one days later, the mice were boosted subcutaneously with a second injection of human fibrinogen in IFA.


title: FIA preboost -3

source_name: FIA preboost -3


title: CFA preboost -3

source_name: CFA preboost -3


title: FIA preboost -1

source_name: FIA preboost -1


title: FIA preboost -4

source_name: FIA preboost -4


title: FIA preboost -2

source_name: FIA preboost -2


title: 6 week sham_134

source_name: Total RNA from mouse lungs labeled with Cyanine-5 (red)

tag: Tissue

tag: Gender

tag: Age

tag: C57BL/6 x CBA F1 hybrid mice

tag: Date of exposure

tag: Exposure

tag: Time post exposure

tag: micro array ID:134

tag: Hybridization Block 1

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


title: CFA preboost -1

source_name: CFA preboost -1

tag: sample

tag: gender

tag: genotype


title: 6 week smoke-break sham_109

tag: micro array ID:109

tag: Total RNA derived from 11 mouse cell lines


title: CFA preboost -4

source_name: CFA preboost -4


title: BXD28a Progenitor batch1

city: Haren

first: Bruno

last: Tesson

address: Kerklaan 30

department: Groningen Bioinformatics Center

institute: University of Groningen

hyb_protocol: Standard Illumina hybridization protocol

scan_protocol: Standard Illumina scanning protocol

data_processing: Quantile normalization was applied on log2 transformed data.

source_name: Progenitor

label_protocol: Biotinylated cRNA was prepared using the Illumina TotalPrep RNA Amplification Kit (Ambion) according to the manufacturer’s specifications starting with 100 ng total RNA

extract_protocol: Total RNA was isolated using the RNeasy Mini kit (Qiagen) in accordance with the manufacturer’s protocol.

tag: mouse id

tag: bxd strain

tag: cell type

value: Sca-Kit+

tag: array batch


title: BXD2 Myeloid batch1

source_name: Myeloid


title: BXD40 Stem batch1

source_name: Stem

value: Sca+Kit+


title: BXD15 Stem batch1


title: BXD12 Erythroid batch1

source_name: Erythroid


title: BXD2 Progenitor batch1


title: BXD2 Erythroid batch1


title: BXD31 Stem batch1


title: BXD1 Myeloid batch1


title: BXD23 Progenitor batch1


title: BXD33a Myeloid batch1


title: BXD28a Stem batch1


title: FIA preboost -5

source_name: FIA preboost -5


title: BXD40 Erythroid batch1


title: BXD19 Stem batch1


title: BXD14 Progenitor batch3


title: BXD12 Stem batch1


title: BXD21B Progenitor TechnicalRep2 batch3

description: This sample is a technical replicate of sample GdH127 and they were averaged after normalization


title: BXD1 Erythroid batch1


title: BXD23 Erythroid batch1


title: BXD8 Erythroid batch3


title: BXD14 Erythroid batch3


title: BXD6 Stem batch1


title: BXD33b Progenitor batch3


title: BXD36 Myeloid batch1


title: BXD13 Myeloid batch1


title: BXD8 Stem batch3


title: BXD29 Myeloid batch3


title: BXD29 Progenitor batch3


title: BXD21B Stem batch3


title: BXD32B Myeloid batch3


title: CFA preboost -2

source_name: CFA preboost -2


title: BXD21B Erythroid batch3


title: BXD32B Erythroid batch3


title: BXD21B Progenitor TechnicalRep1 batch3

description: This sample is a technical replicate of sample GdH127_2 and they were averaged after normalization


title: BXD34B Erythroid batch3


title: BXD34 Progenitor batch3


title: BXD28b Progenitor TechnicalRep2 batch2


title: BXD31 Myeloid batch3


title: BXD18 Erythroid batch2


title: BXD8 Myeloid batch3


title: Naive - 2

description:
1:200 dilution
Plasma

source_name: Naive - 2


title: BXD29 Erythroid batch3


title: BXD33b Stem TechnicalRep2 batch2

description: This sample is a technical replicate of sample GdH111 and they were averaged after normalization


title: BXD29 Stem batch3


title: BXD6 Myeloid batch1


title: BXD8 Progenitor batch3


title: BXD31 Erythroid batch2


title: BXD12 Myeloid batch2


title: BXD14 Myeloid batch3


title: BXD18 Progenitor batch2


title: BXD27 Stem batch2


title: BXD18 Myeloid batch2


title: BXD2 Stem batch1


title: BXD33b Myeloid batch3


title: BXD1 Progenitor batch2


title: BXD15 Progenitor batch1


title: BXD23 Myeloid batch1


title: BXD28a Myeloid batch1


title: BXD31 Progenitor batch1


title: BXD42 Myeloid batch2


title: BXD33a Progenitor batch2


title: BXD14 Stem batch3


title: BXD12 Progenitor batch1


title: BXD40 Progenitor batch1


title: BXD27 Myeloid batch1


title: BXD16 Progenitor batch2


title: BXD13 Progenitor batch2


title: BXD11 Progenitor batch1


title: BXD18 Stem batch1


title: BXD16 Stem batch1

description: This sample was later excluded from analysis


title: BXD28a Erythroid batch1


title: BXD27 Erythroid batch1


title: BXD21B Myeloid batch3


title: BXD32 Progenitor batch1


title: BXD6 Progenitor batch1


title: BXD33b Stem TechnicalRep1 batch1

description: This sample is a technical replicate of sample GdH111_2 and they were averaged after normalization


title: BXD11 Myeloid batch1


title: BXD19 Myeloid batch1


title: BXD9 Myeloid batch1


title: BXD40 Myeloid batch1


title: BXD23 Stem batch1


title: BXD32 Stem batch1


title: BXD42 Progenitor batch1


title: BXD15 Erythroid batch1


title: BXD27 Progenitor batch1


title: BXD34 Stem batch1


title: BXD19 Erythroid batch1


title: BXD1 Stem batch1


title: BXD16 Erythroid batch1


title: BXD16 Myeloid batch1


title: BXD33a Stem batch1


title: BXD11 Stem batch1


title: BXD28b Stem batch1


title: BXD6 Erythroid batch1


title: BXD13 Stem batch1


title: BXD13 Erythroid batch1


title: Expression Profiling of wt mice brain samples after placebo treatment (in comparison with CCI-779) sample III

city: Tuebingen

first: Michael

last: Bonin

state: BW

address: CAlwerstr. 7

department: Medical Genetics

institute: University Tuebingen

description:
Mice (male) were administered CCI-779 or placebo from 5 weeks of age for a period of 6 weeks. Mice were then sacrificed and brains dissected and snap frozen on dry ice. CCI-779 was administered by intraperitoneal injections three times a week at a dose of 20mg/kg. Control mice were given a placebo injection with the same frequency.
All our processing steps follows affymetrix protocols.

hyb_protocol: We used standard Affymetrix hybridisation condtions.

scan_protocol: We used standard Affymetrix scanning parameters.

data_processing: All samples were normalized by GC-RMA algorithm and analyzed by t-tests.

source_name: brain

label_protocol: We used standard Affymetrix labeling protocols.

extract_protocol: We used standard Qiagen RNAeasy Midi protocols for the brain tissue.

value: male

tag: tissue

value: brain

tag: agent

value: placebo


title: Expression Profiling of wt mice brain samples after placebo treatment (in comparison with CCI-779) sample IV

value: wildtype


title: BXD28b Progenitor TechnicalRep1 batch1


title: Expression Profiling of wt mice brain samples after placebo treatment (in comparison with CCI-779) sample I


title: BXD36 Progenitor batch1


title: Expression Profiling of SCA3 tg mice brain samples after placebo treatment (in comparison with CCI-779) sample V


title: BXD9 Stem batch1


title: Expression Profiling of SCA3 transgenic mice brain samples after treatment with CCI-779 sample V


title: Expression Profiling of SCA3 tg mice brain samples after placebo treatment (in comparison with CCI-779) sample IV


title: Expression Profiling of SCA3 tg mice brain samples after placebo treatment (in comparison with CCI-779) sample I


title: Expression Profiling of SCA3 tg mice brain samples after placebo treatment (in comparison with CCI-779) sample II


title: dental follicle cells differentiated 2 A364_4_Diff_437

city: Regensburg

first: Christian

last: Morsczeck

address: Franz-Josef Strauss Allee 11

institute: University of Regensburg

description: Gene expression of dental follicle cells after osteogenic differentiation

hyb_protocol: Hybridization to Affymetrix oligonucleotide microarray Human Gene 1.0 ST array was carried out overnight with biotin-labeled cRNA in a hybridization oven 640

scan_protocol: GeneChip Scanner 3000 (Affymetrix)

data_processing: Affymetrix GeneChip Operating Software (GCOS) 1.4 and the Expression Console 1.1. Software

source_name: dental follicle cells after differentiation

label_protocol: Double-stranded cDNA was prepared from 5 μg of total cellular RNA (cDNA synthesis kit, Affymetrix Santa Clara, CA, USA), in vitro transcribed using an IVT labeling kit, and the cRNA product purified using a GeneChip Sample Cleanup Module (Affymetrix). All procedures according to the Affymetrix protocol.

growth_protocol: Cell were grown in in Mesenchym Stem Medium (PAA, Pasching, Austria) at 37°C in 5% CO2

extract_protocol: Total RNAs were extracted using the kit NucleoSpin® RNA II kit (Macherey Nagel, Düren, Germany) and quality-controlled using the RNA 6000 Nano LabChip (Agilent Technologies, Santa Clara, CA, USA)

treatment_protocol: Osteogenic differentiation was induced after cultivation in alpha-MEM (PAA) supplemented with 10% fetal bovine serum (PAA), 100 µmol/L ascorbic acid 2-phosphate, 2.8 mmol/l KH2PO4, 1 × 10−7 mol/l dexamethasone sodium phosphate (Sigma-Aldrich) and HEPES (20 mmol/L).

value: dental progenitor/stem cells


title: dental follicle cells differentiated 3 A364_5_Diff_158


title: dental follicle cells differentiated 4 A364_6_Diff_159


title: Expression Profiling of SCA3 transgenic mice brain samples after treatment with CCI-779 sample I


title: dental follicle cells differentiated 1 A364_3_Diff_436


title: BXD36 Erythroid batch1


title: Expression Profiling of SCA3 transgenic mice brain samples after treatment with CCI-779 sample II


title: Expression Profiling of wt mice brain samples after treatment with CCI-779 sample II


title: Expression Profiling of SCA3 transgenic mice brain samples after treatment with CCI-779 sample III


title: dental follicle cells undifferentiated 1 A364_1_co_447

description: Gene expression of dental follicle cells before differentiation

source_name: dental follicle cells before differentiation control


title: Expression Profiling of wt mice brain samples after treatment with CCI-779 sample IV


title: Expression Profiling of wt mice brain samples after treatment with CCI-779 sample III


title: BXD9 Erythroid batch1


title: dental follicle cells undifferentiated 2 A364_2_co_448


title: Expression Profiling of wt mice brain samples after treatment with CCI-779 sample V


title: Expression Profiling of wt mice brain samples after treatment with CCI-779

data_processing: All samples were normalized by GC-RMA algorithm and analyszed by  t-tests.


title: Female Infected vs Control replicate 4

status: Public on Sep 11 2009

city: Planegg-Martinsried

first: Stephan

last: Hutter

state: Bavaria

address: Grosshaderner Strasse 2

department: Department of Biology II

institute: University of Munich

description: Competitive hybridization between Infected and Control

hyb_protocol: Hybridizations were performed following DGRC protocols (https://dgrc.cgb.indiana.edu/microarrays/) using Corning microarray hybridization chambers (Corning Incorporated, Corning, NY, USA)

scan_protocol: Scanned on an aQuire microarray scanner (Genetix, New Milton, UK), images were quantified using Genetix QScan software (Version 1.1.8.10)

data_processing: Normalized 647/555 ratios were obtained using CARMAweb (https://carmaweb.genome.tugraz.at/). Dye-swap replicates were normalized as pairs. Local background effects, within-array variation, and between-array variation was corrected using the "minimum", "printtiploess", and "quantile" methods respectively

label: Alexa Fluor 647

organism: Drosophila melanogaster

source_name: Infected

label_protocol: 30 µg of total RNA were reverse transcribed and labeled following DGRC protocols (https://dgrc.cgb.indiana.edu/microarrays/) using SuperScript Plus Indirect cDNA Labeling System and Alexa Flour dyes (Invitrogen, Carlsbad, California, USA)

growth_protocol: Flies were reared at 25ºC on a 12 hour light-dark cycle

extract_protocol: TriZol procedure

tag: strain

tag: sex

tag: developmental stage

value: adult

value: whole fly

tag: infection

value: Sigma virus isolate AP30

label: Alexa Fluor 555

source_name: Control

label_protocol: 30 µg of total RNA were reverse transcribed and labeled following DGRC protocols (https://dgrc.cgb.indiana.edu/microarrays/) using SuperScript Plus Indirect cDNA Labeling System and Alexa Flour dyes (Invitrogen, Carlsbad, CA, USA)


title: Expression Profiling of SCA3 tg mice brain samples after placebo treatment (in comparison with CCI-779) sample III


title: Female Infected vs Control replicate 1 dye-swap

description: Competitive hybridization between Control and Infected


title: Female Infected vs Control replicate 4 dye-swap


title: Expression Profiling of wt mice brain samples after placebo treatment (in comparison with CCI-779) sample II


title: Female Infected vs Control replicate 2


title: Female Infected vs Control replicate 2 dye-swap


title: Female Infected vs Control replicate 3


title: Male Infected vs Control replicate 5 dye-swap


title: Female Infected vs Control replicate 1


title: Female Infected vs Control replicate 3 dye-swap


title: Expression Profiling of SCA3 transgenic mice brain samples after treatment with CCI-779 sample IV


title: Male Infected vs Control replicate 4


title: Male Infected vs Control replicate 3 dye-swap


title: Male Infected vs Control replicate 4 dye-swap


title: Male Infected vs Control replicate 1 dye-swap


title: Male Infected vs Control replicate 2 dye-swap


title: Male Infected vs Control replicate 1


title: Potato_leaves_control_Vs_salt-Treated_Day3_Rep3

city: Belvaux

first: Danièle

last: Evers

address: 41, rue du Brill

department: EVA

country: Luxembourg

institute: CRP-Gabriel Lippmann

hyb_protocol: Probe hybridization was performed according to the TIGR protocol. Probes were precipitated during 1 h at  80°C. After centrifugation at 16000×g at 4°C during 20 min, the probes were washed with cold 75% EtOH, dried, resuspended in 60 µL hybridisation buffer (5X SSC, 50% formamide, 0.1% SDS) and finally denatured at 95°C for 3 min. Hybridisation in individual chambers was done at 42°C during 16 h. Cy5-labelled cDNAs of salt-treated plants were hybridized versus Cy3-labelled cDNAs of control plants. Two slides were performed with a dye-swap (inverted labelling). Slides were washed (see supplemental data) and dried by centrifugation at 30×g during 5 min. The TIGR 10K potato microarrays containing 15,264 cDNAs repeated twice (http://www.jcvi.org/potato/sol_ma_microarrays.shtml) were used for the experiments.

scan_protocol: Slides were scanned using a Professional 4200A microarray scanner (Molecular Devices Corporation, Union City, CA, USA). Two laser channels were used (532 nm and 635 nm) at different settings for the photomultiplier tube (PMT gain). The two PMT gains were automatically adjusted with the Genepix® Pro 6.0 software (Molecular Devices Corporation, Union City, CA, USA) to reach a global intensity ratio close to 1 with pixel saturation equal to 0.001%. Spots with a diameter smaller than 60 µm, spots with a signal intensity below background intensity plus two standard deviations and finally clones annotated “Do not use” were excluded from further analysis. MA plots (log ratio (stress/control) versus mean (log(stress),log(control))) were performed to check the quality of the arrays.

data_processing: To analyse gpr files generated by Genepix® Pro 6.0 software, data were imported into Acuity 4.0 software (Molecular Devices Corporation, Union City, CA, USA) and normalised with the print-tip Lowess method. A “one sample t-test” was first performed for the three slides of each time-point.In order to retain spots with repeatable results, only probes with a p-value below 0.05 were selected. As a second step, probes with a log ratio below -0.5 and above 0.5 were selected to generate the final list.

organism: Solanum tuberosum

label_protocol: Labelling of 15 µg RNA per sample was performed following the TIGR protocol. Reverse transcription was performed using Superscript II kit (Invitrogen) during 2 hours (see supplemental data). The reaction was stopped by addition of 10 µL 0.5 M EDTA. Untranscribed RNA was degraded by addition of 10 µL 1 M NaOH and incubation at 65°C during 15 min. Sixty microliters of 1 M Tris-HCl were added for neutralisation. aa-cDNA purification was performed using the QIAquick PCR purification kit (Qiagen, Leusden, The Netherlands). Washing steps were performed using 500 µL of 5 mM KPO4 pH=8.5, 80% EtOH solution and elution was done using two times 30 µL of 4 mM KPO4 pH=8.5 (final volume 60 µL). Precipitation of aa-cDNA was performed (see supplemental data for details) during 1 hour at  80°C. After centrifugation at 16000×g during 20 min, the pellet was washed using cold 75% EtOH and vacuum-dried for 10 min. The pellet was solubilised in 4.5 µL daily-prepared 0.1 M Na2CO3. A volume of 4.5 µL of dye (previously resuspended in DMSO following manufacturer instructions) was then added and the labelling reaction took place during 1 h at room temperature. To remove uncoupled dye, the QIAquick PCR purification kit was used and the elution was performed twice with supplied elution buffer EB (final volume of 65 µL). Cyanine incorporation was controlled by measuring the absorbance at 260 nm (cDNA), 550 nm (Cy3) and 650 nm (Cy5), followed by an adjustment of the Cy3- and Cy5-coupled cDNA quantities.

growth_protocol: After in vitro multiplication, plantlets of a Solanum tuberosum Cv. PS3 were transferred for acclimatization in a 13 cm diameter pot with 0.25 dm3 of 3:1 soil:sand mixture during 2 months before salt exposure. The culture room parameters were set at 21°C/18°C (day/night), 12 hours daylight and a light intensity of 40 μmol.m-2.s-1.

extract_protocol: The first and second leaves were sampled after 1 and 3 days on two different sets of plants. Leaves were immediately frozen in liquid nitrogen before being stored at -80°C. For each treatment and time-point, three biological replicates were sampled. Total RNA was extracted from 100 mg of frozen leaves with the RNeasy Plant Mini Kit (Qiagen, Leusden, The Netherlands) including DNase treatment, following the manufacturer’s instructions. Quality control was performed with the RNA Nano assay using a 2100 Bioanalyzer (Agilent Technologies, Diegem, Belgium). RNA samples with a RIN (RNA integrity number adapted to plant rRNA profiles) lower than 7 were excluded from the experiment. RNA purity and concentration were measured by the absorbance at 260 nm and 280 nm using a DU800 spectrophotometer (Beckman Coulter, Villepinte, France).

treatment_protocol: Salt treatment was started at day 0 by watering the plants with 75 mL of 150 mM NaCl solution while the control plants were watered with the same amount of tap water.

tag: reference

value: control

tag: time point

tag: replicate

tag: cultivar

value: Cv. PS3

value: Leaves

value: Salt


title: Potato_leaves_control_Vs_salt-Treated_Day3_Rep2


title: Male Infected vs Control replicate 2


title: Potato_leaves_control_Vs_salt-Treated_Day3_Rep1


title: A4p84_1(cy3) vs. A4p15_1 (cy5) - Replicate 3

city: Pune

first: Sharmila

last: Bapat

address: NCCS Complex, Ganeshkhind

country: India

institute: National Centre for Cell Science

description: Biological replicate 3 of 3

hyb_protocol: The fragmented cRNA were mixed with 25ul of 2x GE Hybridization Buffer (Agilent). About 45ul of the resulting mixture was applied to the Microarray and hybridized at 65degC for 17 hours in an Agilent Microarray Hybridization Chamber (SureHyb: G2534A) with Hybridization Oven. After hybridization, slides were washed with Agilent Gene expression Wash Buffer I for 1 minute at room temperature followed by a 1 min wash with Agilent Gene expression Wash Buffer II for 37C. Slides were finally rinsed with Acetonitrile for cleaning up and drying.

scan_protocol: Laser detection of Cyanine 3 and Cyanine 5 fluorescence is performed using a confocal scanning instrument containing two tuned lasers, which excite Cyanine dyes at the appropriate wavelengths.Microarrays were scanned on an Agilent scanner (G2565AA) at 100% laser power, 30% PMT.Data extraction was carried out with Agilent Feature Extraction software (version 9.1), and normalization was done using linear per array algorithm according to the manufacturer’s protocol.

data_processing:
Feature extracted data was analyzed using GeneSpring GX v 7.3.1 software from Agilent. Normalization of the data was done in GeneSpring GX using the recommended one color Per Chip and Per Gene Data Transformation: Set measurements less than 0.01 to 0.01, Per Chip: Normalize to 50th percentile, Per Gene: Normalize to Specific Samples. Further quality control of normalized data was done using correlation based condition tree to eliminate bad experiments. Differentially regulated Genes were filtered with cutoff of > 1.5 for Up regulation and < 0.55 for Down Regulation were obtained. Differentially regulated genes were clustered using gene tree to identify significant gene expression patterns.
The normalization was done using GeneSpring GX using the recommended Per Spot and Per Chip:    Intensity dependent (Lowess) normalization.

source_name: Transformed A4 cells at passage 84 - replicate 3

label_protocol: T7 promoter based-linear amplification to generate labeled complementary RNA

growth_protocol: A4 cells at early passages (less than ~20) were non-tumorigenic since they fail to exhibit anchorage independence growth in soft agar and form tumors in nude mice. After further culture (more than 20 passages in vitro), the cells expressed all features suggestive of transformation including capabilities of anchorage independence growth, clonogenecity and reproducibility of the human disease in mice models as assessed through tumor and ascites formation in nude mice, and serial transplantion of tumor-derived cells over 3-4 mouse generations. The tumors generated in nude mice were also identified to be histologically similar to the primary tumor of the patient.

extract_protocol: The whole of tissue was used for RNA isolation by using Qiagen Rneasy Minikit (Cat No: 74106) as per the manufacturer’s instructions . The RNA was eluted in 30 µl of RNase free water (Ambion, AM9939). RNA concentration and purity was determined at an optical density ratio of 260/280 using the Nanodrop® ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE) and the integrity of total RNA was verified on an Agilent 2100 Bioanalyzer using the RNA 6000 Nano LabChip (Agilent Technologies). RNA was stored at −80 °C until use.

treatment_protocol: A4 cells at passage 15 were considered as representative of untransformed cells; A4 cells at passage 84 were considered representative of tumorigenic (transformed) cells

tag: sample type

value: Transformed A4 cells at passage 84

tag: passage

tag: cell line

tag: phenotype

value: transformed

source_name: Non-tumorigenic cells at passage 15 - replicate 3

value: Non-tumorigenic cells at passage 15

value: non-tumorigenic


title: A4p84_1(cy5) vs. A4p15_1 (cy3) - Replicate 2

description: Biological replicate 2 of 3

source_name: Transformed A4 cells at passage 84 - replicate 2

source_name: Non-tumorigenic cells at passage 15 - replicate 2


title: Potato_leaves_control_Vs_salt-Treated_Day1_Rep1


title: Male Infected vs Control replicate 5


title: Potato_leaves_control_Vs_salt-Treated_Day1_Rep2


title: Potato_leaves_control_Vs_salt-Treated_Day1_Rep3


title: A4p84_1(cy5) vs. A4p15_1 (cy3) - Replicate 1

description: Biological replicate 1 of 3

source_name: Transformed A4 cells at passage 84 - replicate 1

source_name: Non-tumorigenic cells at passage 15 - replicate 1


title: KO vs KO introduced with BBF2H7 N-terminal

city: Kiyotake, Miyazaki

first: Atsushi

last: Saito

address: Kihara, 5200

department: Anatomy

country: Japan

institute: Miyazaki university

description: CIBEX: CAR257

hyb_protocol: TORAY standard procedure

scan_protocol: GenePix 4000B (Molecular Devices)

data_processing: Data analysis were performed with an Excel program (Microsoft)

label_protocol: Amino Allyl MessageAmpTM II aRNA Amplification kit (Ambion)

growth_protocol: Primary cultured chondrocytes were prepared from E18.5 rib cartilage of WT and Bbf2h7-/- mice. Chondrocytes were isolated using 0.2% collagenase D (Roche) after adherent connective tissue was removed by 0.2% trypsin (Sigma) and collagenase pretreatment. Isolated chondrocytes were maintained in α-MEM (Gibco) supplemented with 10% FCS and 50 µg/mL ascorbic acid. Adenovirus vectors expressing the mouse p60 BBF2H7 (1-377 aa, BBF-N) was constructed with the AdenoX Expression system (Clontech), according to the manufacturer’s protocol. The cells were infected with adenoviruses 30 h before analysis.

extract_protocol: Experimental sample RNAs were isolated using RNeasy (QIAGEN).

treatment_protocol: Bbf2h7-/- mice were generated using a targeting vector containing a neomycin resistant gene. The Bbf2h7 targeting vector was electroporated into embryonic stem (ES) cells derived from 129/Sv (D3). Homologous recombination was identified by genomic PCR and Southern blotting. Germ-line transmission of the mutant allele was achieved using C57BL/6 mice.

value: primary cultured chondrocytes

source_name: BBF2H7 KO-2


title: HCV+cirrhosis without HCC 46

city: Columbus

first: Kellie

middle: J.

last: Archer

state: OH

address: 1841 Neil Ave

department: Division of Biostatistics

institute: Ohio State University

description: Gene expression using RMA method

hyb_protocol: Following fragmentation, 10 ug of cRNA were hybridized for 16 hr at 45C on the Affymetrix GeneChips. GeneChips were washed and stained in the Affymetrix Fluidics Station 400.

scan_protocol: GeneChips were scanned using GeneChip® Scanner 3000 by Affymetrix.

data_processing: The RMA method in the R affy package was used to obtain probe set expression summaries.

source_name: liver tissue

label_protocol: Biotinylated cRNA were prepared according to the standard Affymetrix protocol from 6 ug total RNA (Expression Analysis Technical Manual, 2001, Affymetrix).

growth_protocol: Fresh tissue specimens were collected from the consented patients in the operating room and processed immediately after extraction to minimize RNA degradation. Liver tissue samples were collected in liquid nitrogen or RNA later solution (Ambion, Inc, Austin, TX), and stored at -80°C until use.

extract_protocol: Trizol extraction of total RNA was performed according to the manufacturer's instructions.The precipitated product was further purified using RNeasy Mini Kit (Qiagen, CA).

treatment_protocol: Five mm frozen section slides were made and stained with H&E for Pathologist evaluation to determine tumor content. After Pathologist evaluation, normal/necrotic tissue was macrodissected. Twenty 20 mm sections were performed in a 15 ml tube with 10 ml of Trizol.

tag: disease state

value: HCV + cirrhosis without HCC

value: liver


title: Male Infected vs Control replicate 3

value: male


title: HCV+cirrhosis without HCC 45

description: Gene expression using RMA method


title: HCV+cirrhosis without HCC 47

description: Gene expression using RMA method


title: HCV+cirrhosis without HCC 44

description: Gene expression using RMA method


title: HCV+cirrhosis with HCC 16

description: Gene expression using RMA method

value: HCV + cirrhosis with HCC


title: HCV+cirrhosis without HCC 41

description: Gene expression using RMA method


title: HCV+cirrhosis without HCC 40

description: Gene expression using RMA method


title: HCV+cirrhosis without HCC 39

description: Gene expression using RMA method


title: HCV+cirrhosis without HCC 38

description: Gene expression using RMA method


title: HCV+cirrhosis without HCC 34

description: Gene expression using RMA method


title: HCV+cirrhosis without HCC 42

description: Gene expression using RMA method


title: HCV+cirrhosis without HCC 37

description: Gene expression using RMA method


title: HCV+cirrhosis with HCC 15

description: Gene expression using RMA method


title: HCV+cirrhosis without HCC 36

description: Gene expression using RMA method


title: HCV+cirrhosis with HCC 13

description: Gene expression using RMA method


title: HEK293-AIRE1_PolII

type: genomic

city: Tartu

first: Tõnis

last: Org

address: Riia 23

department: Institute of Molecular and Cell Biology

country: Estonia

institute: University of Tartu

hyb_protocol: Hybridization to custom made tiling arrays was performed by Nimblegen (Roche) using standard protocols (see http://www.nimblegen.com/).

scan_protocol: Scanning was performed by Nimblegen (Roche) using standard protocols (see http://www.nimblegen.com/).

data_processing: Signal intensity data was extracted from the scanned images of each array using NimbleScan TM, Nimblegen's data extraction software. For each probe, a scaled log2-ratio was calculated. This is the ratio of the input signals for the experimental and test samples that where co-hybridized to the array. Scaling was performed by subtracting the bi-weight mean for the log2-ratio values for all probes on the array from each log2-ratio value.

molecule: genomic DNA

source_name: Chromatin immunoprecipiated DNA using PolII antibody, from HEK293 cell-line expressing AIRE protein

label_protocol: The immunoprecipitated material was amplified using WGA2 and WGA1 GenomePlex Whole Genome Amplification Kits (Sigma) according to manufacturers protocol. Sample labeling was performed by Nimblegen (Roche) using standard protocols (see http://www.nimblegen.com/).

extract_protocol:
DAY I
Crosslinking:
1.	Add 1/10th volume of crosslinking mix directly to the plate containing growth medium giving a final concentraion of 1% of FA and rotate gently for 10 minutes at room temperature.
2.	Quench the crosslinking reaction by the addition of 1/10th volume of 1.25M glycine giving 125 mM final concentration. Remove the medium from the plate. Aspirate medium. Wash cells twice with 10 ml ice cold PBS.
3.	Scrape cells from one dish with about 2-3 X 1 ml of ice cold PBS and move the cells into 15 ml conical Falcon tube. I usually combine cells from 4 plates into one Falcon tube.
4.	Pellet cells for 10 minutes at 1000 rpm at 4ºC.
Preparation of chromatin: (you can skip if you use Histone antibodies).
1.	Resuspend cell pellet (about 4x107 HEK293 cells from 4x10 cm dishes) in 1 ml of (Nuclear/Chromatin preparation) NCP Buffer I + protease inhibitors. Spin 2000 g 5 min at 4°C.
2.	Discard supernatant. Wash nuclei (the pellet) with resuspending (use pipet again) with 1 ml of NCP II + protease inhibitors. Spin 2000 g 5 min at 4°C.
Lysis and sonication:
1.	Resuspend in 600 µl of Lysis Buffer + protease inhibitors.
2.	Incubate for 10 min at RT, put back on ice.
3.	Sonicate lysate to shear DNA to lengths between 200 and 1000 base pairs. Be sure to keep the samples ice cold. I use 8-10 x 10 sec at cycle 7 using power 70 % and at least 50 sec pauses
4.	Dilute the sonicated suspension 2 x with IP buffer. Centrifuge samples for 2x10 minutes at 11,000 g at 4°C.
5.	Optional: Take 50 µl of supernatant, dilute 3 x with IP buffer and incubate on 65ºC over night. Next day you can use this sample to control efficiency of sonication.
6.	Move the rest of the sup (about 1100  µl) into a 15 ml Falcon tube and dilute 5 x with IP buffer (the final SDS conts should be 0.1 %, the total vol will be about 6 ml)
Preclearing and IP:
7.	To reduce nonspecific background, pre-clear the sonicated and diluted cell lysate with adding 100-150 µl (20ul 50% slurry per IP) of protein G beads slurry pre-absorbed with 100µg/ml BSA and 500µg/ml sheared salmon sperm DNA (see suppl. protocol) Incubate at least two hours at 4°C.
8.	Pellet agarose by brief centrifugation (1min @ 1000rpm) and collect the supernatant fraction.
9.	Add the immunoprecipitating antibody (the amount will vary per antibody, I use 4 µg) to the 1 - 1.5 ml supernatant fraction (you can do 4-6 IP-s from 6 ml of lysate) and incubate overnight at 4ºC with rotation. For a control, use the same amount of IgG or non-specific antibody for immunoprecipitation.
10.	For a negative control, you can include a sample where you have instead of cell lysate 1 - 1.5 ml of IP buffer:Lysis 9:1
DAY II
Collection of the antibody/protein complexes:
1.	Carry the lysates over to the tubes containing 40 µl of 50%slurry and pre-absorbed Protein G Agarose Slurry and incubate additional two hours at 4ºC with rotation.  Be sure you have the same amount of beads in each tube.
2.	Pellet agarose by gentle centrifugation (5000 rpm for 1min). Carefully remove the supernatant that contains unbound, non-specific DNA.
3.	Store 250 µl of supernatants for your input fractions
Washings:
Wash the protein G sepharose/ antibody/ protein complex for 3-5 minutes on a rotating platform with each of the buffers listed in the order as given below:
1.	Low Salt Immune Complex Wash Buffer, WB I (3 X 1 ml)
2.	High Salt Immune Complex Wash Buffer, WB II (1 to 2 X 1 ml)
3.	LiCl Immune Complex Wash Buffer, WB III (1 X 1 ml)
4.	TE (2 X 1 ml)
Note: Washing conditions may differ slightly depending upon antibody affinity for antigen. More washes will give a lower background
Elution:
1.	Elute the histone complex from the antibody by adding 100 µl of elution buffer to the pelleted protein G sepharose/antibody/protein complex. I place the tubes on vortexer and mix at medium setting for 15 min.
2.	Spin down agarose, and carefully transfer the supernatant fraction (eluate) to another tube and repeat elution 2 X 75 µl of elution buffer and combine eluates (total volume=approximately 250 µl).
Reversing the cross-links:
4.	Add 10 µl 5M NaCl to the combined eluates (250 µl) and reverse protein/histone-DNA cross-links by heating at 65ºC for 4 hours to overnight. Reverse the cross-links of your input fractions as well.
DAY III
DNA purification:
A) Phenol/Chlorophorm protocol
1. Add 5  µl of 5M NaCl to each sample and to inputs to bring the final NaCl conts 0.25-0.3M and precipitate the DNA with 2.5 vol EtOH (375 µl)
2. Dissolve the pellets in 100ul TE and add
- 25 µl 5X Prot K buffer
- 1.5 µl Prot K (stock 10mg/ml)
3. incubate for 2 hours at 45ºC.
4. Add 75 µl of TE and extract with 200 µl of phenol or phenol/chlorophorm and 150 µl of chlorophorm
5. Precipitate with adding 1/10 vol of NaOAc, 2.5 vol of EtOH. To IP fractions add glycogen.
6. Resuspend in 50 µl TE

B) Recover DNA using Qiagen PCR purification Kit. Final elution is in 50ul EB.
DAY IV
Q-PCR
Buffers and Reagents:
Crosslinking mix: 	11% Formaldehyde (FA)
			100mM NaCl
			0.5mM EGTA
			50mM HEPES, pH 8.0
			Prepeare freshly
NCP I: 10 mM EDTA
	0.5 mM EGTA
	10 mM Hepes pH 6.5
0.25% TritonX-100
	Protease Inhibitors – add freshly
NCP II: 1 mM EDTA
	 0.5 mM EGTA
	 10 mM Hepes pH 6.5
	 200 mM NaCl
	Protease Inhibitors – add freshly
Lysis Buffer:     10mM EDTA
 20 mM Tris-HCl, pH 8.1.
0.5 % NP-40
1% SDS
Protease Inhibitors – add freshly
IP Buffer:         2 mM EDTA
150 mM NaCl
20 mM Tris-HCl, pH 8.1,
1 % Triton X-100
Protease Inhibitors – add freshly
Low Salt Immune Complex Wash Buffer (WB I):  	2 mM EDTA
20 mM Tris-HCl, pH 8.1
150 mM NaCl
0.1% SDS
1%Triton X-100

High Salt Immune Complex Wash Buffer (WB II):     2 mM EDTA
20 mM Tris-HCl, pH 8.1
500 mM NaCl
0.1% SDS
1%Triton X-100
LiCl Immune Complex Wash Buffer: 1 mM EDTA,
10 mM Tris-HCl, pH 8.1
0.25 M LiCl,1% NP40,
1% deoxycholate
			0.5 % NP-40
TE Buffer: 	10mM Tris-HCl
		1mM EDTA,pH 8.0.
Elution buffer: 1%SDS,
		0.1M NaHCO3
		Prepeare freshly 10 ml (84mg NaHCO3/1ml 10%SDS/9 ml dH20)
5x Proteinase K buffer:	50mM TrisHCl pH 7.5
				25mM EDTA
				1.25% SDS
Supplementary Protocol:
To make pre-absorbed protein G sepharose beads:
1.	Take 60 µl 50% slurry per IP (20 for preclearing, 40 for IP) of protein G sepharose beads slurry per 4x107 cells from 4x10 cm dishes.
2.	Wash the protein G beads in a final volume of 5 ml of 9:1 IP Buffer : Lysis Buffer
3.	Pre-absorb the beads with 100µg/ml BSA and 500µg/ml salmon sperm DNA and rotate overnight at 4°C or 2 hours at RT.
      Note: salmon sperm DNA should be shared by heating 30 min at 99.5 °C).
4.	Before using the beads in pre-clearing or IP, remove the pre-absorption mix and wash the pre-absorbed beads twice in 9:1 IP Buffer : Lysis Buffer

tag: antibody

value: PolII

value: HEK293 stably expressing AIRE protein

source_name: Input genomic DNA from HEK293 cell-line expressing AIRE protein

label_protocol: Input material was amplified using WGA2 and WGA1 GenomePlex Whole Genome Amplification Kits (Sigma) according to manufacturers protocol. Sample labeling was performed by Nimblegen (Roche) using standard protocols (see http://www.nimblegen.com/).

value: none (input genomic DNA)


title: HCV+cirrhosis without HCC 35

description: Gene expression using RMA method


title: HCV+cirrhosis without HCC 43

description: Gene expression using RMA method


title: HCV+cirrhosis with HCC 14

description: Gene expression using RMA method


title: HCV+cirrhosis without HCC 33

description: Gene expression using RMA method


title: BBF2H7 KO

hyb_protocol: Affymetrix standard procedure

scan_protocol: GeneChips were scanned using GeneChip Scanner 3000

data_processing: The data were analyzed with Expression Console version 1.1 using Affymetrix RMA algorithm and quantile as normalization method.

source_name: primary cultured chondrocytes OASIS knock out

label_protocol: Biotinylated cRNA were prepared according to the standard Affymetrix protocol using Two cycle target labeling kit (Affymetrix)

tag: cell type


title: HCV+cirrhosis with HCC 11

description: Gene expression using RMA method

tag: tissue