title: Candida_parapsilosis_farnesol_treatment_replicate_6

status: Public on Mar 01 2007

city: Dublin

first: Geraldine

last: Butler

address: Belfield

department: School of Biomolecular and Biomedical Science

country: Ireland

institute: University Colege Dublin Conway Institute

description: Data were acquired with the Genepix Pro 5.0 software (Axon Instruments) and low quality spots were automatically flagged. In addition, spots that were saturated, or did not have a background corrected intensity greater than 20 in the Cy3 or the Cy5 channel were flagged.

hyb_protocol: Microarray slides were prehybridized with in 5X SSC, 0.1% SDS, 10 mg ml-1 BSA for 1 h at 42ºC, washed 3 times in water, 1 time in isopropanol and dried. Incorporation of Cy3 and Cy5 dyes into cDNA targets was quantified by measuring the absorbance of each sample at 260 nm, 550 nm or 650 nm. Samples were used if incorporation was greater than 15 pmol. The labeled cDNAs were concentrated by drying under vacuum, resuspended and pooled in 45 ul of hybridization buffer (1X DigEasy (Roche) containing 0.5 mg ml-1 bakers yeast tRNA (Invitrogen) and 0.5 mg ml-1 salmon sperm DNA (Sigma)). The mixture was denatured at 95ºC for 2 min, quickly cooled on ice, applied to the DNA microarray and covered with a 24 mm x 60 mm coverslip. Slides were placed in a hybridization chamber (Corning, Palo Alto, CA) and incubated at 42ºC for approximately 16 h.  The slides were washed in 1 X SSC at 42ºC until the coverslip fell off, and then washed 3 times for 10 min in 1 X SSC, 0.1% SDS at 42ºC, 3 times 5 min in 1 X SSC at 42ºC, and finally rinsed in 0.2 X SSC at room temperature. The slides were dried by centrifugation at 1000 rpm for 5 min.

scan_protocol: The microarrays were scanned with an Axon 4000B scanner (Axon Instruments) at a 10 microns resolution.

data_processing: Normalization of the data was performed using the Lowess method in GeneSpring (Agilent Technologies). Spots present at least in 3 of the 6 slides were included in the subsequent analysis.

organism: Candida parapsilosis

source_name: Candida parapsilosis treated with farnesol

label_protocol: 25 ug of total RNA was first incubated with 200 pmol of anchored oligo dT (Invitrogen) for 10 min at 70ºC. 1X first strand buffer (Invitrogen), 1.5 mM (dATP, dTTP, dGTP), 25 uM dCTP, 10 mM DTT, 2 ul Superscript II reverse transcriptase (Invitrogen) and 37.5 uM Cy3-dCTP or 37.5 uM Cy5-dCTP (Amersham) were added in a total volume of 40 microl. The mixture was incubated 42ºC for 2 h. An additional 1 ul of Superscript II was added to the mixture and the incubation was continued for 1 h at 42ºC.  RNA was degraded by addition of 1 ul of RNAse A (Qiagen) at 50 ug ml-1 and 1 ul of RNAse H (Invitrogen) at 0.05 unit ul-1 and incubated at 37ºC for 30 min. The labeled cDNAs were purified using a QIAquick PCR Purification kit (Qiagen) following the manufacturer’s instructions.

growth_protocol: The strain was grown overnight in YPD medium at 30 degrees C and diluted to an A600 of 0.2 in 50 ml of YPD at 30 degrees C.

extract_protocol: RNA was extracted from 50 ml of culture using the RiboPure yeast kit (Ambion) according to the manufacturer’s instructions after 2 hours of growth. Concentration, quality and integrity of the RNA samples were controlled using an Agilent 2100 Bioanalyzer (Agilent) according to the manufacturer’s instructions. Only samples with a 28S:18S rRNA ratio between 1.6 and 2.2 and showing an absence of degradation were used in subsequent analysis.

treatment_protocol: Fresh trans, trans-Farnesol solution (Sigma) was prepared at a concentration of 40 mM in methanol. Farnesol was added after 4 hours of growth at a final concentration of 50 microM.

tag: Candida parapsilosis strain CLIB214

source_name: Candida parapsilosis untreated

treatment_protocol: Culture was treated with the same volume of methanol as used in channel1 but without farnesol


title: Candida_parapsilosis_farnesol_treatment_replicate_2

type: RNA

description:
Data were acquired with the Genepix Pro 5.0 software (Axon Instruments) and low quality spots were automatically flagged. In addition, spots that were saturated, or did not have a background corrected intensity greater than 20 in the Cy3 or the Cy5 channel were flagged.
This replicate was a dye swap

molecule: total RNA

tag: Culture was treated with the same volume of methanol as used in channel1 but without farnesol


title: Candida_parapsilosis_farnesol_treatment_replicate_3


title: nonsporulating, nonmating A2 sample

status: Public on Mar 30 2007

city: Riverside

first: Howard

middle: S

last: Judelson

state: CA

address: Boyce Hall

department: Plant Pathology

country: USA

institute: University of California

description: Nonmating A2 control

hyb_protocol: standard Affymetrix procedures

scan_protocol: standard Affymetrix procedures

data_processing: MAS5.0, per chip normalization

label: biotin

organism: Phytophthora infestans

source_name: Isolate 618, vegetative culture

label_protocol: Approximately 10 µg of total RNA was processed to produce biotinylated cRNA targets using a standard Affymetrix protocol.

growth_protocol: Grown at 18C in the dark.

extract_protocol: Hyphae were grown to a powder in liquid nitrogen and then total RNA was extracted using the Qiagen Plant Mini RNA kit, using an on-column DNAse treatment protocol.

tag: A2 mating type, grown 3 days on polycarbonate membranes laid upon rye sucrose agar


title: Candida_parapsilosis_farnesol_treatment_replicate_1


title: Candida_parapsilosis_farnesol_treatment_replicate_5


title: Candida_parapsilosis_farnesol_treatment_replicate_4


title: nonsporulating, nonmating A1 sample

description: Nonmating A1 control

source_name: Isolate 88069, vegetative culture

tag: A1 mating type, grown 3 days on polycarbonate membranes laid upon rye sucrose agar


title: HepG2 Heat Inactived Heat Inactived NGC_V_24_repeated 2 (dye swap)

city: Singapore

first: Martin

last: Hibberd

state: Singapore

address: 60, Biopolis Street

department: Population Genetics

country: Singapore

institute: Genome Institute of Singapore

description: HepG2 cells were collected 24 hours after  Heat Inactived Dengue 2 virus (NGC) infection

hyb_protocol: Labeled cDNA were pooled, concentrated, resuspended in DIG EasyHyb (Roche, Basel, Switzerland) buffer and Hybridizated overnight (14-16h) in the MAUI Hybridization chamber( BioMicro, Salt Lake City, UT)

scan_protocol: Microarrays were scanned using a GenePix 4000B scanner in the two channels (Cy3 and Cy5) at the same time. The GenePix Pro 6.0 software was used to grid the microarrays and to calculate the signals for each spot

data_processing: Lowess normalized ratio value (Ch1/Ch2)

organism: Homo sapiens

source_name: HepG2 cell  and dengue virus serotype2 NGC

label_protocol: 20ug of total RNA from experiment samples were reverse transcribed in the presence of Cy3-dUTP  (Amersham Biosciences, Little Chalfont, UK) by using the Superscript?reverse transcription kit (Invitrogen,USA). Resuspend cDNA in the cy3 dye thoroughly, incubate for 1 hour at room temperature in the dark. Mix every 15 minutes

extract_protocol: standard  QIAGEN  Rneasy Mini Protocol for isolation total RNA from Animal Cells

tag: Human hepatoma cell line (HepG2)

source_name: Universal Human Reference (Strategene. USA)

label_protocol: 20ug of total RNA from universal human reference (Strategene. USA)  were reverse transcribed in the presence of Cy5-dUTP  (Amersham Biosciences, Little Chalfont, UK) by using the Superscript?reverse transcription kit (Invitrogen,USA). Resuspend cDNA in the cy5 dye thoroughly, incubate for 1 hour at room temperature in the dark. Mix every 15 minutes

tag: Total RNA prepared from 10 different human cell lines


title: HepG2 Heat Inactived Heat Inactived NGC_V_24_repeated 1 (dye swap)


title: HepG2 Heat Inactived Heat Inactived NGC_V_48_repeated 2 (dye swap)

description: HepG2 cells were collected 48 hours after  Heat Inactived Dengue 2 virus (NGC) infection


title: HepG2 Heat Inactived Heat Inactived NGC_V_72_repeated 2 (dye swap)

description: HepG2 cells were collected 72 hours after  Heat Inactived Dengue 2 virus (NGC) infection


title: HepG2 Heat Inactived Heat Inactived NGC_V_48_repeated 1 (dye swap)


title: HepG2 Heat Inactived Heat Inactived NGC_V_24_repeated 3 (dye swap)


title: HepG2 Heat Inactived Heat Inactived NGC_V_72_repeated 1 (dye swap)


title: HepG2 Heat Inactived Heat Inactived NGC_V_72_repeated 3 (dye swap)


title: HepG2 Heat Inactived Heat Inactived NGC_V_12_repeated 3 (dye swap)

description: HepG2 cells were collected 12 hours after  Heat Inactived Dengue 2 virus (NGC) infection


title: HepG2 NGC_V_72_repeated 3 (dye swap)

description: HepG2 cells were collected 72 hours after Dengue 2 live virus (NGC) infection


title: HepG2 Heat Inactived Heat Inactived NGC_V_12_repeated 2 (dye swap)


title: HepG2 Heat Inactived Heat Inactived NGC_V_6_repeated 3 (dye swap)

description: HepG2 cells were collected 6 hours after  Heat Inactived Dengue 2 virus (NGC) infection


title: HepG2 Heat Inactived Heat Inactived NGC_V_48_repeated 3 (dye swap)


title: HepG2 Heat Inactived Heat Inactived NGC_V_3_repeated 3 (dye swap)

description: HepG2 cells were collected 3 hours after  Heat Inactived Dengue 2 virus (NGC) infection


title: HepG2 Heat Inactived Heat Inactived NGC_V_6_repeated 1 (dye swap)


title: HepG2 Heat Inactived Heat Inactived NGC_V_12_repeated 1 (dye swap)


title: HepG2 Heat Inactived Heat Inactived NGC_V_3_repeated 2 (dye swap)


title: HepG2 Heat Inactived Heat Inactived NGC_V_6_repeated 2 (dye swap)


title: HepG2 NGC_V_72_repeated 2 (dye swap)


title: HepG2 NGC_V_48_repeated 2 (dye swap)

description: HepG2 cells were collected 48 hours after Dengue 2 live virus (NGC) infection


title: HepG2 NGC_V_48_repeated 3 (dye swap)


title: HepG2 NGC_V_24_repeated 3 (dye swap)

description: HepG2 cells were collected 24 hours after Dengue 2 live virus (NGC) infection


title: HepG2 NGC_V_12_repeated 3 (dye swap)

description: HepG2 cells were collected 12 hours after Dengue 2 live virus (NGC) infection


title: HepG2 NGC_V_12_repeated 2 (dye swap)


title: HepG2 NGC_V_24_repeated 1 (dye swap)


title: HepG2 NGC_V_6_repeated 1 (dye swap)

description: HepG2 cells were collected 6 hours after Dengue 2 live virus (NGC) infection


title: HepG2 NGC_V_24_repeated 2 (dye swap)


title: HepG2 NGC_V_48_repeated 1 (dye swap)


title: HepG2 NGC_V_12_repeated 1 (dye swap)


title: HepG2 NGC_V_6_repeated 3 (dye swap)


title: HepG2 NGC_V_3_repeated 2 (dye swap)

description: HepG2 cells were collected 3 hours after Dengue 2 live virus (NGC) infection


title: HepG2 NGC_V_6_repeated 2 (dye swap)


title: HepG2 Heat Inactived Heat Inactived NGC_V_72_repeated 2


title: HepG2 Heat Inactived Heat Inactived NGC_V_48_repeated 1


title: HepG2 Heat Inactived Heat Inactived NGC_V_48_repeated 3


title: HepG2 Heat Inactived Heat Inactived NGC_V_12_repeated 1


title: HepG2 Heat Inactived Heat Inactived NGC_V_72_repeated 1


title: HepG2 NGC_V_3_repeated 3 (dye swap)


title: HepG2 Heat Inactived Heat Inactived NGC_V_6_repeated 1


title: HepG2 Heat Inactived Heat Inactived NGC_V_24_repeated 3


title: HepG2 Heat Inactived Heat Inactived NGC_V_24_repeated 1


title: HepG2 Heat Inactived Heat Inactived NGC_V_24_repeated 2


title: HepG2 Heat Inactived Heat Inactived NGC_V_12_repeated 2


title: HepG2 Heat Inactived Heat Inactived NGC_V_3_repeated 2


title: HepG2 Heat Inactived Heat Inactived NGC_V_6_repeated 3


title: HepG2 Heat Inactived Heat Inactived NGC_V_6_repeated 2


title: HepG2 Heat Inactived Heat Inactived NGC_V_12_repeated 3


title: HepG2 Heat Inactived Heat Inactived NGC_V_48_repeated 2


title: HepG2 Heat Inactived Heat Inactived NGC_V_3_repeated 1


title: HepG2 NGC_V_72_repeated 2


title: HepG2 NGC_V_72_repeated 1


title: HepG2 Heat Inactived Heat Inactived NGC_V_3_repeated 3


title: HepG2 NGC_V_48_repeated 1


title: HepG2 NGC_V_12_repeated 3


title: HepG2 NGC_V_24_repeated 3


title: HepG2 NGC_V_24_repeated 1


title: HepG2 NGC_V_48_repeated 3


title: HepG2 NGC_V_48_repeated 2


title: HepG2 NGC_V_24_repeated 2


title: HepG2 NGC_V_12_repeated 2


title: HepG2 NGC_V_12_repeated 1


title: HepG2 NGC_V_6_repeated 3


title: HepG2 NGC_V_3_repeated 3


title: HepG2 NGC_V_3_repeated 1


title: HepG2 NGC_V_6_repeated 2


title: CEN.PK113-7D/pYX212-NOX chemostat at Dcrit Rep 2

status: Public on Feb 01 2007

city: Göteborg

first: Goutham

middle: N

last: Vemuri

address: Kemivagen 10

department: Chemical and Biological Engineering

country: Sweden

institute: Chalmers University of Technology

description:
The extraction of RNA, cDNA synthesis, cRNA synthesis and labelling, as well as array hybridisation to Affymetrix Yeast Genome 2.0 arrays (Affymetrix, Santa Clara, CA, USA) were performed as described in the Affymetrix manual.
Affymetrix (2000) Affymetrix GeneChip Expression Analysis Technical Manual. (Affymetrix, Santa Clara, CA).

data_processing: dCHIP (version Jun17, 2005)

organism: Saccharomyces cerevisiae

source_name: Strain: CEN.PK113-7D (Lab reference strain) transformed with pYX212-NOX plasmid.

growth_protocol: Growth on minimal media (Verduyn, (1992) Yeast 8, 501-517) using 10 g/L glucose as the sole carbon source.

tag: Strain

value: CEN.PK113-7D (Lab reference strain) transformed with pYX212-NOX plasmid. Chemostat cultivation at the critical dilution rate.


title: CEN.PK113-7D/pYX212-AOX chemostat at Dcrit Rep 2

hyb_protocol: The extraction of RNA, cDNA synthesis, cRNA synthesis and labelling, as well as array hybridisation to Affymetrix Yeast Genome 2.0 arrays (Affymetrix, Santa Clara, CA, USA) were performed as described in the Affymetrix manual. Affymetrix (2000) Affymetrix GeneChip Expression Analysis Technical Manual. (Affymetrix, Santa Clara, CA).

source_name: Strain: CEN.PK113-7D (Lab reference strain) transformed with pYX212-AOX plasmid.

value: CEN.PK113-7D (Lab reference strain) transformed with pYX212-AOX plasmid. Chemostat cultivation at the critical dilution rate.


title: CEN.PK113-7D/pYX212-AOX chemostat at Dcrit Rep 1

scan_protocol: The extraction of RNA, cDNA synthesis, cRNA synthesis and labelling, as well as array hybridisation to Affymetrix Yeast Genome 2.0 arrays (Affymetrix, Santa Clara, CA, USA) were performed as described in the Affymetrix manual. Affymetrix (2000) Affymetrix GeneChip Expression Analysis Technical Manual. (Affymetrix, Santa Clara, CA).

treatment_protocol: Chemostat cultivation at the critical dilution rate


title: HepG2 NGC_V_6_repeated 1


title: Human control 3

status: Public on Feb 13 2007

city: Kuopio

first: Markus

last: Storvik

address: University of Eastern Finland

department: Pharmacology

country: Finland

institute: University of Eastern Finland

description: Prefrontal cortex of human control 3.

hyb_protocol: According to the manufacturer's instructions.

scan_protocol: At 3-Am resolution using the GeneChip System confocal scanner (Hewlett-Packard, Santa Clara, CA) with a 488-nm emission and detection at 70 nm, controlled by Affymetrix Microarray Suite 5.0 (Affymetrix).

data_processing: Raw

label: Biotin

source_name: Human prefrontal cortex

label_protocol: According to the standard protocol outlined in the Affymetrix GeneChip Expression Analysis Technical Manual

growth_protocol: Human post-mortem samples.

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

treatment_protocol: Human post-mortem samples.

tag: State

value: Normal

tag: Tissue

value: Prefrontal cortex

tag: Treatment

value: Untreated


title: Thatcher_Lr34 (susceptible), mock inoculated distal leaf, replication 3

city: Manhattan

first: Jianfa

last: Bai

state: KS

address: 1800 Denison Ave

department: Diagnostic Medicine and Pathobiology

institute: Kansas State University

description: Wheat fully expanded flag leaf, mock inoculated distal half, biological replication 3.

hyb_protocol: Following fragmentation, 15 microg of cRNA were hybridized for 16 hr at 45C on GeneChip wheat Genome Array. GeneChips were washed and stained in the Affymetrix GeneChip Fluidics Station 450.

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

data_processing: The data were analyzed with Affymetrix GCOS software using default analysis settings and global scaling as normalization method. The trimmed mean target intensity of each array was arbitrarily set to 500.

organism: Triticum aestivum

source_name: Fully expanded wheat flag leaves, distal half, mock inoculated

label_protocol: Biotinylated cRNA were prepared according to the standard Affymetrix protocol from 5 microg total RNA (Expression Analysis Technical Manual, 2001, Affymetrix).

growth_protocol: Plants were grown in 4-liter pots in growth chamber (Conviron PGW-36) under 15C night/20C day (+/- 1C) with a 16 hr photoperiod. Pots were fertilized at planting with 5 g/pot 14-14-14 Osmocote (The Scotts Company, Marysville, OH) and thereafter every two weeks with 0.5 g/pot 15-30-15 Miracle-Gro All Purpose Plant Food  (Scott's Miracle-Gro Products, Inc., Marysville, OH) and 0.1 ml/pot Fertilome Chelated Liquid Iron and Other Micronutrients (Voluntary Purchasing Groups, Inc., Bonham, TX).

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

treatment_protocol: Plants were sprayed with Soltrol oil in the evening and were then incubated in a mist chamber with 100% RH for 12 hr. The plants were returned to the growth chamber until sampling at 48 hr after removal from the mist chamber.

tag: Genotype

tag: Growth stage

value: mid-anthesis


title: HepG2 NGC_V_3_repeated 2


title: CEN.PK113-7D/pYX212 chemostat at Dcrit Rep 1

source_name: Strain: CEN.PK113-7D/pYX212 chemostat at Dcrit

label_protocol:
The extraction of RNA, cDNA synthesis, cRNA synthesis and labelling, as well as array hybridisation to Affymetrix Yeast Genome 2.0 arrays (Affymetrix, Santa Clara, CA, USA) were performed as described in the Affymetrix manual.
Affymetrix (2000) Affymetrix GeneChip Expression Analysis Technical Manual. (Affymetrix, Santa Clara, CA).

value: CEN.PK113-7D (Lab reference strain) transformed with pYX212 plasmid. Chemostat cultivation at the critical dilution rate.


title: Thatcher_Lr34 (susceptible), mock inoculated basal leaf, replication 3

description: Wheat fully expanded flag leaf, mock inoculated basal half, biological replication 3.

source_name: Fully expanded wheat flag leaves, basal half, mock inoculated


title: CEN.PK113-7D/pYX212-NOX chemostat at Dcrit Rep 1

extract_protocol:
The extraction of RNA, cDNA synthesis, cRNA synthesis and labelling, as well as array hybridisation to Affymetrix Yeast Genome 2.0 arrays (Affymetrix, Santa Clara, CA, USA) were performed as described in the Affymetrix manual.
Affymetrix (2000) Affymetrix GeneChip Expression Analysis Technical Manual. (Affymetrix, Santa Clara, CA).


title: Human control 2

description: Prefrontal cortex of human control 2.


title: Thatcher_Lr34 (susceptible), mock inoculated distal leaf, replication 1

description: Wheat fully expanded flag leaf, mock inoculated distal half, biological replication 1.


title: Thatcher_Lr34 (susceptible), rust inoculated basal leaf, replication 2

description: Wheat fully expanded flag leaf, rust inoculated basal half, biological replication 2.

source_name: Fully expanded wheat flag leaves, basal half, rust inoculated

treatment_protocol: Plants were heavily inoculated with the Puccinia triticina race MFBL suspended in Soltrol 170 light oil (Chevron Phillips Chemical Company, The Woodlands, TX), and were then incubated in a mist chamber with 100% RH for 12 hr. The plants were returned to the growth chamber until sampling at 48 hr after removal from the mist chamber.


title: Thatcher_Lr34 (susceptible), rust inoculated distal leaf, replication 2

description: Wheat fully expanded flag leaf, rust inoculated distal half, biological replication 2.

source_name: Fully expanded wheat flag leaves, distal half, rust inoculated


title: Thatcher_Lr34 (susceptible), rust inoculated distal leaf, replication 3

description: Wheat fully expanded flag leaf, rust inoculated distal half, biological replication 3.


title: CEN.PK113-7D/pYX212 chemostat at Dcrit Rep2

source_name: Strain: CEN.PK113-7D (Lab reference strain) transformed with pYX212 plasmid.

value: CEN.PK113-7D (Lab reference strain) transformed with pYX212 plasmid. Replicate 2.


title: Thatcher_Lr34 (susceptible), rust inoculated basal leaf, replication 1

description: Wheat fully expanded flag leaf, rust inoculated basal half, biological replication 1.


title: Thatcher_Lr34 (resistant), mock inoculated distal leaf, replication 3


title: Thatcher_Lr34 (resistant), rust inoculated distal leaf, replication 3


title: Thatcher_Lr34 (susceptible), rust inoculated distal leaf, replication 1

description: Wheat fully expanded flag leaf, rust inoculated distal half, biological replication 1.


title: Thatcher_Lr34 (susceptible), mock inoculated distal leaf, replication 2

description: Wheat fully expanded flag leaf, mock inoculated distal half, biological replication 2.


title: Thatcher_Lr34 (susceptible), mock inoculated basal leaf, replication 2

description: Wheat fully expanded flag leaf, mock inoculated basal half, biological replication 2.


title: Thatcher_Lr34 (susceptible), rust inoculated basal leaf, replication 3

description: Wheat fully expanded flag leaf, rust inoculated basal half, biological replication 3.


title: Thatcher_Lr34 (susceptible), mock inoculated basal leaf, replication 1

description: Wheat fully expanded flag leaf, mock inoculated basal half, biological replication 1.


title: Thatcher_Lr34 (resistant), mock inoculated distal leaf, replication 1


title: Thatcher_Lr34 (resistant), rust inoculated distal leaf, replication 1


title: Thatcher_Lr34 (resistant), mock inoculated basal leaf, replication 1


title: Thatcher_Lr34 (resistant), rust inoculated distal leaf, replication 2


title: Thatcher_Lr34 (resistant), mock inoculated basal leaf, replication 2


title: Thatcher_Lr34 (resistant), mock inoculated distal leaf, replication 2


title: Thatcher_Lr34 (resistant), rust inoculated basal leaf, replication 3


title: Jupateco_Lr34 (susceptible), mock inoculated distal leaf, replication 3


title: Thatcher_Lr34 (resistant), mock inoculated basal leaf, replication 3


title: Thatcher_Lr34 (resistant), rust inoculated basal leaf, replication 2


title: Jupateco_Lr34 (susceptible), mock inoculated basal leaf, replication 3


title: Rat microglia_IFNgamma_1c (technical replicate)

status: Public on Jan 10 2007

city: London

first: Linda

middle: B

last: Moran

address: Fulham Palace Road

department: Neuropathology

country: United Kingdom

institute: Imperial College London

description: Gene expression data from IFNgamma-stimulated primary rat microglial culture.

scan_protocol: Standard Affymetrix Protocols (Affymetrix GeneChip Expression Analysis Technical Manual, 2001). Scanning Hardware – GeneArray Scanner.

data_processing: Scaled to 1 (Microarray Suite version 5.0). Data was normalized using the GC-RMA algorithm.

organism: Rattus norvegicus

source_name: Neonatal rat brain derived microglia cells

growth_protocol: Primary microglia cultures were grown in 75-cm3 culture flasks (37 C, 5% CO2) with tissue from one brain per flask in 10 mL of Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 15% fetal calf serum (FCS). Culture media were changed on days 7 and 11, respectively. On day 15, microglia were plated in Petri dishes (100,000 cells/dish).

extract_protocol: Total RNA was extracted using Qiagen RNAeasy Mini Kit (performed according to the manufacturer's instructions).

treatment_protocol: For activation studies, fresh media containing IFN-gamma (100 U/ml) were added and left overnight (16 hours).

tag: Lewis rat (newborn)

tag: Primary microglia cultures (15 day old)


title: Jupateco_Lr34 (resistant), mock inoculated basal leaf, replication 2


title: Rat microglia_CON6

description: Gene expression data from unstimulated (Control) primary rat microglial culture.

hyb_protocol: Standard Affymetrix Protocols (Affymetrix GeneChip Expression Analysis Technical Manual, 2001).

treatment_protocol: For control studies, culture plates received only fresh culture media.


title: Rat microglia_CON3

label_protocol: Standard Affymetrix Protocols (Affymetrix GeneChip Expression Analysis Technical Manual, 2001).


title: Rat microglia_CON5


title: Rat microglia_IFNgamma_2


title: Thatcher_Lr34 (resistant), rust inoculated basal leaf, replication 1


title: Rat microglia_IFNgamma_1a (technical replicate)


title: Rat microglia_IFNgamma_1b (technical replicate)


title: Jupateco_Lr34 (susceptible), mock inoculated basal leaf, replication 2


title: Jupateco_Lr34 (susceptible), mock inoculated distal leaf, replication 2


title: Rat microglia_IFNgamma_3


title: Jupateco_Lr34 (resistant), mock inoculated distal leaf, replication 3


title: Rat microglia_CON2


title: Jupateco_Lr34 (susceptible), mock inoculated basal leaf, replication 1


title: Jupateco_Lr34 (resistant), mock inoculated basal leaf, replication 1


title: Rat microglia_CON1


title: Jupateco_Lr34 (susceptible), mock inoculated distal leaf, replication 1


title: Jupateco_Lr34 (resistant), mock inoculated basal leaf, replication 3


title: Jupateco_Lr34 (resistant), mock inoculated distal leaf, replication 2


title: TUV93-0 B

status: Public on Jan 30 2007

city: Birmingham

first: Chrystala

last: Constantinidou

department: School of Biosciences

institute: University of Birmingham

description: E. coli O157 strain TUV93-0 has been shown to secrete high levels of EspD and Tir effector proteins when cultured in MEM-HEPES, typically 5-10 fold higher than when the same strains are cultured in DMEM. Expression conditions were used that are highly selective for up-regulation of the LEE. The E. coli O157:H7 transcriptome was compared from bacteria cultured in MEM-HEPES with DMEM.

hyb_protocol: Prior to hybridization, the slides were treated with the background-reducing Pronto! Pre-Soak System (Corning Life Sciences). The slides were incubated for 20 min in prewarmed Universal Pre-Soak solution at 42 °C and washed twice in 0.1_SSC, 0.1% SDS for 30 s at room temperature. Slides were immediately transferred into prewarmed prehybridization solution (5_ SSC, 0.1% SDS, and 0.1% bovine serum albumin) and incubated for 2–4 h at 42 °C. The microarray slides were finally washed at room temperature once in 0.1_ SSC, 0.1% SDS for 1 min and twice in 0.1_ SSC for 30 s, briefly dipped in water and then ethanol, and dried by centrifugation for 5 min at 800_g. For each experiment, equal quantities (80 pmol) of each Cy5- and Cy3-labeled cDNA were added to a final volume of 80 _l of hybridization solution containing 25% formamide, 10 mg of bovine serum albumin (fraction V) per ml, 5_ SSC (1_ SSC is 0.15 M NaCl, 0.015 M sodium citrate), 0.1% SDS, 8 _g of poly(A), and 1_Denhardt’s solution. The cDNA probes were denatured at 95 °C for 3 min and hybridized for 16 h at 42 °C. After hybridization was complete, the slides were washed in 2_SSC, 0.1% SDS at 42 °C for 2 min in 0.1_SSC, 0.1% SDS for 2 min at room temperature, and finally twice in 0.1_ SSC for 2 min at room temperature. The microarray slides were dried by centrifugation for 5 min at 800g.

scan_protocol: Slide was scanned at 532 and 630 nm by using a Genepix 4000A scanner (Axon Instruments, Union City, CA).

data_processing: Data was analysed using GeneSpring

organism: Escherichia coli

source_name: TUV93-0 DMEM

label_protocol:
20 ug total RNA was converted to Cy5-labelled cDNA
using the CyScribe Post-Labelling Kit (Amersham Biosciences/GE
Healthcare) according to manufacturer’s instructions. Amino-allyllabelled
nucleotides were incorporated into the cDNA by reverse
transcription of the total RNA, followed by a direct chemical coupling
of Cy5 NHS-dye esters to the amino-allyl-labelled
cDNAs.

growth_protocol: Sample was grown shaking at 37oC in DMEM to OD600 = 0.6.

extract_protocol: An RNeasy minikit was used to prepare total RNA according to the manufacturer’s instructions (QIAGEN Ltd.). Any contaminating DNA was removed using a DNAase column kit (QIAGEN Ltd).

treatment_protocol: 15 ml of culture was mixed with 30 ml of RNAprotect bacterial reagent (QIAGEN Ltd).

value: O157:H7 EHEC TUV93-0

tag: OD

tag: Media

value: DMEM

source_name: TUV93-0 MEM-HEPES

label_protocol:
20 ug total RNA was converted to Cy3-labelled cDNA
using the CyScribe Post-Labelling Kit (Amersham Biosciences/GE
Healthcare) according to manufacturer’s instructions. Amino-allyllabelled
nucleotides were incorporated into the cDNA by reverse
transcription of the total RNA, followed by a direct chemical coupling
of Cy3 NHS-dye esters to the amino-allyl-labelled
cDNAs.

value: MEM, 25mM HEPES


title: TUV93-0 C


title: FNR- mutant vs. WT (Dye swap-3)

status: Public on Jan 05 2007

city: St. Cloud

first: Ryan

middle: Cristiano

last: Fink

state: MN

address: 720 4th Ave S

department: Biology

institute: St. Cloud State University

description: The labelling of the total RNA was performed using indirect labelling with AA-dUTP and subsequent coupling.

data_processing: Scanarray 4.0 has been used. Lowess normalization.

organism: Salmonella enterica subsp. enterica serovar Typhimurium

source_name: WT

tag: strain

value: ATCC14028s, OD600=0.3, anaerobic, LB medium, 20 mM Xylose

source_name: FNR null mutant

value: NC983, OD600=0.3, anaerobic, LB medium, 20 mM Xylose


title: Jupateco_Lr34 (resistant), mock inoculated distal leaf, replication 1


title: FNR- mutant vs. WT (Dye swap-1)


title: FNR- mutant vs. WT (2)


title: FNR- mutant vs. WT (3)


title: FNR- mutant vs. WT


title: FNR- mutant vs. WT (Dye swap-2)


title: TUV93-0 A


title: STAT1 IFN-gamma ENCODE NimbleGen hg16 tiling array replicate 2/3

status: Public on Jan 01 2007

type: genomic

city: La Jolla

first: Bing

last: Ren

address: 9500 Gilman Drive

department: Department of Cellular and Molecular Medicine

institute: Ludwig Institute for Cancer Research

description: HeLa S3 cells (ATCC CCL-2.2) were cultured under adherent conditions in DMEM + 10% FBS. At 80% confluency, half of the cells were treated with 10 ng/mL interferon gamma (IFNγ, Sigma I-3265) and incubated for 30 min under normal culture conditions (37ºC, 5% CO2).

hyb_protocol: NimbleGen

scan_protocol: NimbleGen

data_processing: raw data

molecule: genomic DNA

source_name: Chromatin immunoprecipitated DNA from HeLa cells using STAT1 antibody

label_protocol: Cy3 input Cy5 sample NimbleGen

extract_protocol: Three biological replicates of treated and untreated cells were crosslinked and harvested as previously described with the following modifications. Cells were crosslinked for 20 minutes at 37ºC in normal culture media plus 1/10 volume formaldehyde crosslinking solution in large culture plates, followed by glycine quenching and PBS wash at room temperature. Crosslinked cells were collected by scraping and centrifugation. Chromatin was isolated and fragmented as previously described, though generating fragments of 1.5 Kbp in length required 12 x 30 sec cycles of sonication.

tag: cells

value: HeLa

source_name: input chromatin DNA control


title: H3K4me1 IFN-gamma stimulated ENCODE NimbleGen hg17 tiling array replicate 3.

source_name: Chromatin immunoprecipitated DNA from IFN-gamma stimulated HeLa cells using H3K4me1 antibody

extract_protocol: Treated and untreated cells were crosslinked and harvested as previously described with the following modifications. Cells were crosslinked for 20 minutes at 37ºC in normal culture media plus 1/10 volume formaldehyde crosslinking solution in large culture plates, followed by glycine quenching and PBS wash at room temperature. Crosslinked cells were collected by scraping and centrifugation. Chromatin was isolated and fragmented as previously described, though generating fragments of 1.5 Kbp in length required 12 x 30 sec cycles of sonication. ChIP-chip, DNA purification, and LM-PCR were performed as previously described using commercially available antibodies (α-histone H3, Abcam ab1791; α-H4ac, Upstate 06-866; α-H3ac, Upstate 06-599; α-H3K4me1, Abcam ab8895; α-H3K4me2, Upstate 07-030; α-H3K4me3, Upstate 07-473; α-RNAPII, Covance MMS-126R; α-TAF1, Santa Cruz sc-735; α-p300, Santa Cruz sc-585; α-TRAP220, Santa Cruz sc-5334).


title: STAT1 IFN-gamma ENCODE NimbleGen hg16 tiling array replicate 1/3


title: STAT1 IFN-gamma ENCODE NimbleGen hg16 tiling array replicate 3/3


title: p300 IFN-gamma stimulated ENCODE NimbleGen hg17 tiling array replicate 4.

source_name: Chromatin immunoprecipitated DNA from IFN-gamma stimulated HeLa cells using p300 antibody


title: RNAP unstimulated ENCODE NimbleGen hg17 tiling array replicate 3.

source_name: Chromatin immunoprecipitated DNA from unstimulated HeLa cells using RNAP antibody


title: H4ac IFN-gamma stimulated ENCODE NimbleGen hg17 tiling array replicate 3.

source_name: Chromatin immunoprecipitated DNA from IFN-gamma stimulated HeLa cells using H4ac antibody


title: H3 IFN-gamma stimulated ENCODE NimbleGen hg17 tiling array replicate 1.

source_name: Chromatin immunoprecipitated DNA from IFN-gamma stimulated HeLa cells using H3 antibody


title: TAF1 IFN-gamma stimulated ENCODE NimbleGen hg17 tiling array replicate 1.

source_name: Chromatin immunoprecipitated DNA from IFN-gamma stimulated HeLa cells using TAF1 antibody


title: H3 unstimulated ENCODE NimbleGen hg17 tiling array replicate 2.

source_name: Chromatin immunoprecipitated DNA from unstimulated HeLa cells using H3 antibody


title: TAF1 unstimulated ENCODE NimbleGen hg17 tiling array replicate 2.

source_name: Chromatin immunoprecipitated DNA from unstimulated HeLa cells using TAF1 antibody


title: TRAP220 unstimulated ENCODE NimbleGen hg17 tiling array replicate 3.

source_name: Chromatin immunoprecipitated DNA from unstimulated HeLa cells using TRAP220 antibody


title: p300 IFN-gamma stimulated ENCODE NimbleGen hg17 tiling array replicate 2.


title: TRAP220 unstimulated ENCODE NimbleGen hg17 tiling array replicate 1.


title: TAF1 IFN-gamma stimulated ENCODE NimbleGen hg17 tiling array replicate 2.


title: H3K4me1 IFN-gamma stimulated ENCODE NimbleGen hg17 tiling array replicate 2.


title: H3 IFN-gamma stimulated ENCODE NimbleGen hg17 tiling array replicate 2.


title: H3K4me1 unstimulated ENCODE NimbleGen hg17 tiling array replicate 3.

source_name: Chromatin immunoprecipitated DNA from unstimulated HeLa cells using H3K4me1 antibody


title: TAF1 unstimulated ENCODE NimbleGen hg17 tiling array replicate 3.


title: p300 unstimulated ENCODE NimbleGen hg17 tiling array replicate 3.

source_name: Chromatin immunoprecipitated DNA from unstimulated HeLa cells using p300 antibody


title: p300 unstimulated ENCODE NimbleGen hg17 tiling array replicate 2.


title: p300 IFN-gamma stimulated ENCODE NimbleGen hg17 tiling array replicate 3.


title: TAF1 IFN-gamma stimulated ENCODE NimbleGen hg17 tiling array replicate 3.


title: p300 IFN-gamma stimulated ENCODE NimbleGen hg17 tiling array replicate 1.


title: H3K4me1 unstimulated ENCODE NimbleGen hg17 tiling array replicate 2.


title: H3 unstimulated ENCODE NimbleGen hg17 tiling array replicate 1.


title: TRAP220 unstimulated ENCODE NimbleGen hg17 tiling array replicate 2.


title: H3K4me1 IFN-gamma stimulated ENCODE NimbleGen hg17 tiling array replicate 1.


title: p300 unstimulated ENCODE NimbleGen hg17 tiling array replicate 1.


title: H3ac IFN-gamma stimulated ENCODE NimbleGen hg17 tiling array replicate 3.

source_name: Chromatin immunoprecipitated DNA from IFN-gamma stimulated HeLa cells using H3ac antibody


title: H3K4me1 unstimulated ENCODE NimbleGen hg17 tiling array replicate 1.


title: H3K4me3 IFN-gamma stimulated ENCODE NimbleGen hg17 tiling array replicate 3.

source_name: Chromatin immunoprecipitated DNA from IFN-gamma stimulated HeLa cells using H3K4me3 antibody


title: E2: Time:12h

city: Quebec

first: Ezequiel

middle: L

last: Calvo

state: Quebec

address: 2705 Boul. Laurier

department: Molecular Endocrinilogy

country: Canada

institute: CRCHUL

description: Analysis of uteri of 10-11 weeks old C57Bl/6 females sacrified 12h after a single injection of E2.

hyb_protocol: Following fragmentation, 15 ug of cRNA were hybridized for 16 hr at 45C on GeneChip MOE 430 2.0 Mouse Genome Array. GeneChips were washed and stained in the Affymetrix Fluidics Station 450.

scan_protocol: GeneChips were scanned using the Affymetrix Scanner 3000 G7.

data_processing: The data were analyzed with Microarray Suite version 5.0 (MAS 5.0) using Affymetrix default analysis settings and global scaling as normalization method. The trimmed mean target intensity of each array was arbitrarily set to 500.

organism: Mus musculus

source_name: Uteri of 10-11 weeks old C57Bl/6 females sacrified 12h after a single injection of 0.05ug/muose of Estradiol (E2).

label_protocol: Biotinylated cRNA were prepared according to the standard Affymetrix protocol from 20 ug total RNA (Expression Analysis Technical Manual, 2001, Affymetrix).

growth_protocol: Ten week-old female C57BL6 mice were received from Charles River (St Constant, Canada) and were allowed to acclimate for 3 weeks. The animals were housed individually in an environmentally-controlled room (temperature: 22 ± 3°C; humidity: 50 ± 20%; 12-h light-12-h dark cycles, lights on at 07:15h). The mice had free access to tap water and a certified rodent feed (Lab Diet 5002 (pellet), Ralston Purina, St Louis, MO).

extract_protocol: Mice were anesthetized by inhalation of isoflurane and exsanguinated via cardiac puncture followed by cervical dislocation. Them, total uterus was collected and any adhering tissue was removed. Tissue was then rapidly frozen in liquid nitrogen and kept at -80C until RNA extraction. Trizol extraction of total RNA was performed from pooled tissues, according to the manufacturer's instructions (Invitrogen).

treatment_protocol: Ten 10-11 weeks old C57Bl/6 mice were gonadectomized under isoflurane-induced anesthesia and sacrified seven days after GDX.  Mice  were injected s.c with E2 (0.05ug/mice) or vehicle, at 0.5, 1, 3, 6, 12 or 24h before sacrifice. Uteri was removed and pooled before total RNA extraction.

tag: Genotype C57BL6

tag: Age

value: 10-11 weeks old


title: RNAP IFN-gamma stimulated ENCODE NimbleGen hg17 tiling array replicate 3.

source_name: Chromatin immunoprecipitated DNA from IFN-gamma stimulated HeLa cells using RNAP antibody


title: RNAP IFN-gamma stimulated ENCODE NimbleGen hg17 tiling array replicate 2.


title: E2: Time:24h

description: Analysis of uteri of 10-11 weeks old C57Bl/6 females sacrified 24h after a single injection of E2.

source_name: Uteri of 10-11 weeks old C57Bl/6 females sacrified 24h after a single injection of 0.05ug/mouse of Estradiol (E2).


title: TAF1 unstimulated ENCODE NimbleGen hg17 tiling array replicate 1.

source_name: input chromatin DNA control


title: H3ac unstimulated ENCODE NimbleGen hg17 tiling array replicate 3.

source_name: Chromatin immunoprecipitated DNA from unstimulated HeLa cells using H3ac antibody


title: H4ac IFN-gamma stimulated ENCODE NimbleGen hg17 tiling array replicate 2.


title: H4ac unstimulated ENCODE NimbleGen hg17 tiling array replicate 3.

source_name: Chromatin immunoprecipitated DNA from unstimulated HeLa cells using H4ac antibody


title: H4ac IFN-gamma stimulated ENCODE NimbleGen hg17 tiling array replicate 1.


title: E2: Time:3h

description: Analysis of uteri of 10-11 weeks old C57Bl/6 females sacrified 3h after a single injection of E2.

source_name: Uteri of 10-11 weeks old C57Bl/6 females sacrified 3h after a single injection of 0.05ug/muose of Estradiol (E2).

label_protocol: Biotinylated cRNA was prepared according to the standard Affymetrix protocol from 20 ug total RNA (Expression Analysis Technical Manual, 2001, Affymetrix).

extract_protocol: Mice were anesthetized by inhalation of isoflurane and exsanguinated via cardiac puncture followed by cervical dislocation. Then, total uterus was collected and any adhering tissue was removed. Tissue was then rapidly frozen in liquid nitrogen and kept at -80C until RNA extraction. Trizol extraction of total RNA was performed from pooled tissues, according to the manufacturer's instructions (Invitrogen).


title: E2: Time:1h

description: Analysis of uteri of 10-11 weeks old C57Bl/6 females sacrified 1h after a single injection of E2.

source_name: Uteri of 10-11 weeks old C57Bl/6 females sacrified 1h after a single injection of 0.05ug/muose of Estradiol (E2).


title: H3K4me2 unstimulated ENCODE NimbleGen hg17 tiling array replicate 2.

source_name: Chromatin immunoprecipitated DNA from unstimulated HeLa cells using H3K4me2 antibody


title: H3K4me3 IFN-gamma stimulated ENCODE NimbleGen hg17 tiling array replicate 2.


title: H4ac unstimulated ENCODE NimbleGen hg17 tiling array replicate 2.


title: H3ac unstimulated ENCODE NimbleGen hg17 tiling array replicate 2.


title: H3ac IFN-gamma stimulated ENCODE NimbleGen hg17 tiling array replicate 1.


title: H3K4me2 unstimulated ENCODE NimbleGen hg17 tiling array replicate 3.


title: H3K4me3 unstimulated ENCODE NimbleGen hg17 tiling array replicate 1.

source_name: Chromatin immunoprecipitated DNA from unstimulated HeLa cells using H3K4me3 antibody


title: H3ac IFN-gamma stimulated ENCODE NimbleGen hg17 tiling array replicate 2.


title: H3K4me3 unstimulated ENCODE NimbleGen hg17 tiling array replicate 3.


title: H3K4me2 IFN-gamma stimulated ENCODE NimbleGen hg17 tiling array replicate 2.

source_name: Chromatin immunoprecipitated DNA from IFN-gamma stimulated HeLa cells using H3K4me2 antibody


title: E2: Time:6h

description: Analysis of uteri of 10-11 weeks old C57Bl/6 females sacrified 6h after a single injection of  0.05ug/muose of Estradiol (E2).

source_name: Uteri of 10-11 weeks old C57Bl/6 females sacrified 6h after a single injection of 0.05ug/muose of Estradiol (E2).


title: STAT1 HeLa RenLab ENCODE PCR tiling array replicate 4

description: HeLa S3 cells (ATCC CCL-2.2) were cultured under adherent conditions in DMEM + 10% FBS. At 80% confluency, half of the cells were treated with 10 ng/mL interferon gamma (IFNγ, Sigma I-3265) and incubated for 30 min under normal cultur (37ºC, 5% CO2).

hyb_protocol: Hybridization procedure and parameters: Equal amounts (12 ug) of Cy5 and Cy3 labeled DNA samples were mixed, and 4 uL 2.94 nM Xenohybe control oligos (an equimolar mixture of 5'TTGCCGATGCTAACGACGCATCAGACTGCGTACGCCTAAGCAACGCTA3' and 5'CATTGCTGTGCGTACGCAGTCAAGTCGATCACGCTAACTCGTTGCGAC3') was added to the mixture. The sample was vacuum dried under low heat until the volume of sample was less than 14.4 uL. The final volume of DNA was adjusted to 14.4 uL with dH2O. To this sample, 11.25 uL 20X SSC, 18 uL 100% formamide, 0.45 uL 10% SDS, 0.45 uL 10X TE (100mM Tris, 10mM EDTA), and 0.45 uL equimolar mixture of Cy3 and Cy5 labeled CPK6 oligonucleotides (5'TTCCTCTCGCTGTAATGACCTCTATGAATAATCCTATCAAACAACTCA3' and 5'TTCCTCTCGCTGTAATGACCTCTATGAATAATCCTATCAAACAACTCA3', respectively) were added to prepare the hybridization mixture. The hybridization sample was heated to 95 °C and was applied to the slide and incubated in the MAUIÂ® Hybridization Station (BioMicro Systems, Inc.) at 42 °C for 16-20 hours.  The hybridized slides from the MAUI® Hybridization Station were washed once in Wash 1 (0.2X SSC, 0.2% SDS, 0.1 mM DTT) for 10-15 seconds and followed by another wash in Wash 1 (0.2X SSC, 0.2% SDS, 0.1 mM DTT) for 2 minutes with gentle agitation. The slides were then washed in Wash 2 (0.2X SSC and 0.1mM DTT) for 1 minute and followed by a wash in Wash 3 (0.05X SSC and 0.1 mM DTT) for 15 seconds. The slides were dried by centrifugation

scan_protocol: Measurement data and specifications: The hybridized arrays were scanned on an Axon GenePix 4000B scanner (Axon Instruments Inc.) at wavelengths of 532nm for control (Cy3), and 635nm (Cy5) for experimental sample. PCR arrays were processed using GenePix 4.0 software while NimbleGen data were extracted from the scanned images using the NimbleScan 2.0 program (NimbleGen Systems, Inc.). The arrays were gridded using the automated gridding algorithm, and extracted in two channels using a mean intensity calculation of the interior of the gridded rectangular features upon extraction, and each pair of N probe signals were converted into a scaled log ratio using the function:  R(i) = Log (Experimental(i) / Control(i))

source_name: Chromatin immunoprecipitated DNA from HeLa cells using STAT1 antibody

label_protocol: One microgram (ug) of LM-PCR products were used for labeling and hybridization to each array. One microgram of immunoprecipitated or total genomic LM-PCR DNA was mixed with 40 uL of 1 uM Cy5 or Cy3 end labeled random prime nonamer oligonucleotides (TriLink Biotechnologies) respectively with the bacterial label control DNA in a total volume of 88 uL. The DNA and random primers were annealed by heating the sample to 98°C for 5 minutes and chilled quickly in ice water for 2-3 minutes. Two microliter of (100 units) of E. coli DNA polymerase Klenow fragment and 10 uL of 10 mM equimolar mixture of dATP, dTTP, dCTP, and dGTP were added to the annealed DNA sample and incubated at 37°C for 2 hours. The reaction was stopped by addition of 10 uL of 0.5 M EDTA. The labeled sample was ethanol precipitated by addition of 11uL 5 M NaCl and 110 uL isopropanol. The precipitate was collected by centrifugation and the resulting labeled DNA pellet was washed with 80% ethanol (V/V). The pellet was dried under vacuum for 5-15 minutes to remove any remaining liquid, and the resulting dry labeled DNA pellet was resuspended in 10 uL dH2O.

extract_protocol: Three biological replicates of treated and untreated cells were crosslinked and harvested as previously described [Kim, T. H. et al. A high-resolution map of active promoters in the human genome. Nature 436, 876-80 (2005)] with the following modifications. Cells were crosslinked for 20 minutes at 37ºC in normal culture media plus 1/10 volume formaldehyde crosslinking solution in large culture plates, followed by glycine quenching and PBS wash at room temperature. Crosslinked cells were collected by scraping and centrifugation. Chromatin was isolated and fragmented as previously described, though generating fragments of 1.5 Kbp in length required 12 x 30 sec cycles of sonication.

source_name: Input DNA from HeLa cells

label_protocol: same as for Channel 1

extract_protocol: same as for Channel 1

tag: Input DNA from HeLa cells


title: RNAP unstimulated ENCODE NimbleGen hg17 tiling array replicate 1.

source_name: Chromatin immunoprecipitated DNA from unstimulated HeLa cells using RNAP antibody


title: RNAP unstimulated ENCODE NimbleGen hg17 tiling array replicate 2.


title: H3ac unstimulated ENCODE NimbleGen hg17 tiling array.


title: H3K4me3 IFN-gamma stimulated ENCODE NimbleGen hg17 tiling array replicate 1.


title: E2: Time:0h

description: Analysis of uteri of 10-11 weeks old C57Bl/6 females sacrified 24h after a single injection of vehicle (Control animal).

source_name: Uteri of 10-11 weeks old C57Bl/6 females sacrified 24h after a single injection of vehicle.

treatment_protocol: Ten 10-11 weeks old C57BL6 mice were ovariectomized under isoflurane-induced anesthesia and sacrified seven days after GDX.  24h before sacrifice, mice  were injected s.c vehicle solution (Control group).


title: STAT1 HeLa RenLab ENCODE PCR tiling array replicate 2

tag: Chromatin immunoprecipitated DNA from HeLa cells using STAT1 antibody


title: CTCF HeLa replicate 3

description: ChIP-chip analyses were performed for various transcription factors and chromatin modifications in various cell types. Details to be described in publications.

source_name: Chromatin immunoprecipitated DNA from HeLa cells using CTCF antibody

extract_protocol: NimbleGen


title: H3K4me2 unstimulated ENCODE NimbleGen hg17 tiling array replicate 1.


title: H4ac unstimulated ENCODE NimbleGen hg17 tiling array.


title: H3K4me3 unstimulated ENCODE NimbleGen hg17 tiling array replicate 2.


title: STAT1 HeLa RenLab ENCODE PCR tiling array replicate 1


title: H3K4me2 IFN-gamma stimulated ENCODE NimbleGen hg17 tiling array.


title: SOX13 regulated genes in CD4+CD8+ double positive thymocytes-control B

status: Public on Dec 20 2006

city: Worcester

first: joonsoo

last: kang

state: MA

address: 55 lake ave n.

department: Pathology

institute: University of Massachusetts Medical School

description: control, baseline, wild type immature alphabeta T cell lineage cells not expressing enodgenous Sox13 gene to determine if ectopic Sox13 expression can impose alternate T cell lineage molecular program

hyb_protocol: Per Affymetrix Protocol

scan_protocol: Per Affymetrix Protocol

source_name: Ex vivo wild type CD4+CD8+ double positive DP thymocytes; wild type

label_protocol: Per Affymetrix Protocol

extract_protocol: Trizol

treatment_protocol: Ex vivo flow cytometer sorted

value: C57BL/6 Age:6 weeks old Gender:Female Tissue:Thymus Sample:sorted CD4+CD8+ double positive thymocytes


title: CTCF HeLa replicate 2

tag: Chromatin immunoprecipitated DNA from HeLa cells using CTCF antibody


title: SOX13 regulated genes in CD4+CD8+ double positive thymocytes-U74A-Control

description: Sorted, control, baseline, wild type immature alphabeta T cell lineage cells not expressing enodgenous Sox13 gene to determine if ectopic Sox13 expression can impose alternate T cell lineage molecular program in vivo

source_name: Ex vivo CD4+CD8+ double positive DP thymocytes; wild type

treatment_protocol: Ex vivo flow cytometer sorted, using  monoclonal Antibodies to CD4 and CD8 antigens.

tag: Strain

value: 6 weeks old

tag: Gender

value: Female

value: Thymus

tag: Sample

value: sorted CD4+CD8+ double positive thymocytes


title: CTCF HeLa replicate 1


title: H3K4me3 IMR90 replicate 3

source_name: Chromatin immunoprecipitated DNA from IMR90 cells using H3K4me3 antibody

source_name: Input DNA from IMR90 cells

tag: Input DNA from IMR90 cells


title: SOX13 regulated genes in CD4+CD8+ double positive thymocytes-U74A

description: Ex vivo immature alphabeta T cell lineage cells ectopically expressing Sox13 gene to determine if Sox13 can impose alternate T cell lineage molecular program in vivo.

source_name: Ex vivo CD4+CD8+ double positive DP thymocytes; sox13 transgene positive

value: C57BL/6, Sox13 transgenic mice

value: 6 weeks old Gender:Female Tissue:Thymus Sample:sorted CD4+CD8+ double positive thymocytes


title: H3K4me3 HeLa replicate 3

source_name: Chromatin immunoprecipitated DNA from HeLa cells using H3K4me3 antibody


title: SOX13 regulated genes in CD4+CD8+ double positive thymocytes-control A


title: H3K4me2 IFN-gamma stimulated ENCODE NimbleGen hg17 tiling array replicate 3.

source_name: input chromatin DNA control


title: RNAP IFN-gamma stimulated ENCODE NimbleGen hg17 tiling array replicate 1.

source_name: Chromatin immunoprecipitated DNA from IFN-gamma stimulated HeLa cells using RNAP antibody


title: CTCF U937 replicate 1

source_name: Chromatin immunoprecipitated DNA from U937 cells using CTCF antibody

source_name: Input DNA from U937 cells

tag: Input DNA from U937 cells


title: 11K-B SOX13 regulated genes in CD4+CD8+ double positive thymocytes

description: immature alphabeta T cell lineage cells ectopically expressing Sox13 gene to determine if Sox13 can impose alternate T cell lineage molecular program

extract_protocol: Per Affymetrix Protocol

treatment_protocol: Ex vivo flow cytometer sorted sample

tag: Tissue


title: CTCF U937 replicate 3

tag: Chromatin immunoprecipitated DNA from U937 cells using CTCF antibody


title: H3K4me3 IMR90 replicate 2

tag: Chromatin immunoprecipitated DNA from IMR90 cells using H3K4me3 antibody


title: STAT1 HeLa RenLab ENCODE PCR tiling array replicate 5


title: GW3965 + LPS

status: Public on Dec 15 2006

first: Christopher

last: Glass

address: 9500 Gilman Dr.

department: CMM

institute: University of California, San Diego

hyb_protocol: In 25 ul total volume, add 10 ug of cRNA to 5 ul of 5x fragmentation buffer and incubate at 94 C for 20 min. Bring 10 ug of fragmented cRNA, 78 ul of hybridization buffer component A, and 130 ul of hybridization buffer component B to a final volume of 260 ul with water. Incubate at 90C for 5 min and immediately chill on ice for 5 min. Slowly inject 250 ul of hybridization reaction mixture into array input port and seal ports with sealing strips. Set the shaker speed to 300 rpm and incubate slides for 18-24 h at 37C. Remove the Flex Chamber using the hybridization removal tool. Then, place the bioarrays into the bioarray rack while it sits inside the medium reagent reservoir containing 0.75xTNT. Transfer rhe bioarray rack to the large reagent reservoir containing preheated 0.75xTNT, and incubate at 46 C for 1 hour. Fill each slot of the small reagent reservoir with 3.4 ml of Cy5-Streptavidin working solution. Transfer the bioarray rack from the large reagent reservoir in to the small reagent reservoir and incubate bioarrays at RT for 30 min. Wash the bioarrays four times with 1xTNT . Rinse the bioarrays in o.1xSSC/0.05%Tween for 30 sec. Immediately follow the rinse with centrifugation to dry bioarrays, and store dried bioarrays in the dark

scan_protocol: We scaned bioarrays with Agilent Scanner G2505B and analyzed with CodeLink Expression Analysis software.

data_processing: Data were normalized using a multi-loess technique described in Corbeil et al (Journal of Molecular Endocrinology (2004) 33:1–9).

source_name: primary macrophages

label_protocol:
Assess the concentration and quality of the total RNA sample by Agilent Bioanalyzer.
Incubate total RNA (0.5 ug), diluited bacterial mRNA controls and T7 oligo (dT) primer for 10 min at 70 C. Add first-strand reaction components and incubate 2 hours at 42C.
Add the first-strand cDNA product to the second-strand reaction components and incubate for 2 hours at 16 C. Purify and elute twice with 30 ul EB buffer each time. Concentrate the purified cDNA to 9.5 ul per tube. Prepare IVT mix of biotinylated UTP, ribonucleotides, and the 10x T7 enzyme mix and add to the resuspended cDNA. Incubate at 37C for 14 hours. Purify cRNA and elute the cRNA twice, each time with 50 ul nuclease-free water. Measure the absorbance at 260 nm and 280 nm to determine the ratio (A260:A280=2).

extract_protocol: Standard protocol from Qiagen RNeasy Mini Kit (Cat no. 74106).

value: female

value: Thioglycollate-elicited macrophage


title: SOX13 regulated genes in CD4+CD8+ double positive thymocytes

value: thymus

tag: Subset

value: CD4+CD8+ DP thymocyte


title: No treatment


title: TSA + LPS


title: H3K4me3 IMR90 replicate 1


title: TSA + GW3965 + LPS


title: H3K4me3 HeLa replicate 2

tag: Chromatin immunoprecipitated DNA from HeLa cells using H3K4me3 antibody


title: CTCF U937 replicate 2


title: H3K4me3 HeLa replicate 1


title: LPS


title: GW7845 + PolyIC 2

data_processing: Data Processing: Data were normalized using a multi-loess technique described in Corbeil et al (Journal of Molecular Endocrinology (2004) 33:1–9).


title: GW7845 + PolyIC 1


title: TSA


title: GW396 5+ PolyIC 1


title: No treatment 2


title: Polyl C 1


title: GW3965 + PolyIC 2


title: GW7845 + LPS 1

source_name: Primary macrophages


title: PolyI C 2


title: No treatment 1


title: GW3965 + LPS 1

scan_protocol: We scaned bioarrays with Agilent Scanner G2505B and analyzed with CodeLink Expression Analysis software

extract_protocol: Standard protocol from Qiagen RNeasy Mini Kit (Cat no. 74106)


title: GW3965 + LPS 2


title: LPS 1

hyb_protocol: In 25 ul total volume, add 10 ug of cRNA to 5 ul of 5x fragmentation buffer and incubate at 94 C for 20 min. Bring 10 ug of fragmented cRNA, 78 ul of hybridization buffer component A, and 130 ul of hybridization buffer component B to a final volume of 260 ul with water. Incubate at 90C for 5 min and immediately chill on ice for 5 min. Slowly inject 250 ul of hybridization reaction mixture into array input port and seal ports with sealing strips. Set the shaker speed to 300 rpm and incubate slides for 18-24 h at 37C. Remove the Flex Chamber using the hybridization removal tool. Then, place the bioarrays into the bioarray rack while it sits inside the medium reagent reservoir containing 0.75xTNT. Transfer rhe bioarray rack to the large reagent reservoir containing preheated 0.75xTNT, and incubate at 46 C for 1 hour. Fill each slot of the small reagent reservoir with 3.4 ml of Cy5-Streptavidin working solution. Transfer the bioarray rack from the large reagent reservoir in to the small reagent reservoir and incubate bioarrays at RT for 30 min. Wash the bioarrays four times with 1xTNT . Rinse the bioarrays in o.1xSSC/0.05%Tween for 30 sec. Immediately follow the rinse with centrifugation to dry bioarrays, and store dried bioarrays in the dark.

source_name: Primary Macrophage


title: STAT1 HeLa RenLab ENCODE PCR tiling array replicate 3

source_name: Chromatin immunoprecipitated DNA from HeLa cells using STAT1 antibody

source_name: Input DNA from HeLa cells


title: Gastrocnemius tissue at time 0 from aged calorie restricted mouse, biological rep7

status: Public on Dec 01 2006

city: Aurora

first: Michael

last: Edwards

state: CO

address: 12700 East 19th Avenue, Box C272

department: Pulmonary

institute: UC Denver

description:
Gene expression data from gastrocnemius tissue from 25 month-old mice on calorie restricted diet
gastrocnemius tissue at time 0 from aged calorie restricted mouse, biological rep7

hyb_protocol: The hybridization cocktail (200 µl) containing 10 µg of fragmented cRNA was injected into the MOE430A array (Affymetrix, Santa Clara, CA). The GeneChip was placed in a 45°C oven at 60 rpm for 16 h. After hybridization, the GeneChips were washed and stained in a fluidic station (model 800101, Affymetrix) with signal amplification protocol using antibody.

scan_protocol: GeneChips were scanned at a resolution of 3 µm twice using a Hewlett-Packard GeneArray Scanner (model 900154, Affymetrix), and the averaged images were used for further analysis.

data_processing: Robust Multi-Array Analysis (RMA) was used to pre-process and normalize the raw Affymetrix GeneChip data.  Microarray Suite version 5.0 (MAS5.0) was used to compute absent/present calls.

molecule: polyA RNA

source_name: gastrocnemius tissue in calorie restricted-fed 25 month-old C57BL/6 mice

label_protocol: One microgram of poly(A)+ RNA was converted into double-stranded cDNA (ds-cDNA) by using the SuperScript Choice System (Life Technologies) with an oligo-dT primer containing a T7 RNA polymerase promoter (Genset, La Jolla, CA). The ds-cDNA was extracted with phenol-chloroform-isoamyl alcohol and precipitated with pellet paint coprecipitant (Novagen, Madison, WI). Biotin-labeled RNA was synthesized in vitro using a high-yield RNA transcript labeling kit (BioArray; Enzo, Farmingdale, NY). The biotin-labeled antisense cRNA was purified using the RNeasy affinity column (Qiagen) and fragmented randomly.

growth_protocol: Male C57BL/6NHsd mice were purchased from Harlan Sprague-Dawley at 6-7 weeks of age and individually housed for the remainder of their lifespan.

extract_protocol: Trizol extraction of total RNA was performed according to the manufacturer's instructions and polyadenylate [poly(A)+] RNA was purified from the total RNA with oligo-dT-linked Oligotex resin (Qiagen, Valencia, CA).

treatment_protocol: Mice were fed one of two life-long dietary regimens: normal diet (ND) and caloric restriction (CR).  Mice on ND were provided acidic water ad libitum and fed 84 kcal/wk which is ~15% less than the average ad libitum diet. C57BL/6NHsd mice on this dietary regiment typically have an average lifespan of 30 mo. in our colony. Mice on CR were fed a diet that resulted in a 26% difference in caloric intake to the ND animals. The diet fed to CR mice was enriched in protein, vitamins and minerals such that CR and ND mice were fed nearly identical amounts of these components.

value: 25 month-old

tag: Diet

value: calorie restricted


title: Gastrocnemius tissue at time 0 from aged calorie restricted mouse, biological rep3

description:
Gene expression data from gastrocnemius tissue from 25 month-old mice on calorie restricted diet
gastrocnemius tissue at time 0 from aged calorie restricted mouse, biological rep3


title: Gastrocnemius tissue at time 0 from aged calorie restricted mouse, biological rep6

description:
Gene expression data from gastrocnemius tissue from 25 month-old mice on calorie restricted diet
gastrocnemius tissue at time 0 from aged calorie restricted mouse, biological rep6


title: Gastrocnemius tissue at time 0 from aged calorie restricted mouse, biological rep2

description:
Gene expression data from gastrocnemius tissue from 25 month-old mice on calorie restricted diet
gastrocnemius tissue at time 0 from aged calorie restricted mouse, biological rep2


title: Gastrocnemius tissue at time 0 from aged mouse, biological rep5

description:
Gene expression data from gastrocnemius tissue from 25 month-old mice on normal diet
gastrocnemius tissue at time 0 from aged mouse, biological rep5

source_name: gastrocnemius tissue in control-fed 25 month-old C57BL/6 mice

value: normal


title: Gastrocnemius tissue at time 0 from aged calorie restricted mouse, biological rep5

description:
Gene expression data from gastrocnemius tissue from 25 month-old mice on calorie restricted diet
gastrocnemius tissue at time 0 from aged calorie restricted mouse, biological rep5


title: Gastrocnemius tissue at time 0 from aged mouse, biological rep4

description:
Gene expression data from gastrocnemius tissue from 25 month-old mice on normal diet
gastrocnemius tissue at time 0 from aged mouse, biological rep4


title: GW7845 + LPS 2


title: Gastrocnemius tissue at time 0 from aged mouse, biological rep2

description:
Gene expression data from gastrocnemius tissue from 25 month-old mice on normal diet
gastrocnemius tissue at time 0 from aged mouse, biological rep2


title: Gastrocnemius tissue at time 0 from young mouse, biological rep4

description:
Gene expression data from gastrocnemius tissue from 5 month-old mice on normal diet
gastrocnemius tissue at time 0 from young mouse, biological rep4

source_name: gastrocnemius tissue in control-fed 5 month-old C57BL/6 mice

value: 5 month-old


title: Gastrocnemius tissue at time 0 from young mouse, biological rep5

description:
Gene expression data from gastrocnemius tissue from 5 month-old mice on normal diet
gastrocnemius tissue at time 0 from young mouse, biological rep5


title: Gastrocnemius tissue at time 0 from young mouse, biological rep3

description:
Gene expression data from gastrocnemius tissue from 5 month-old mice on normal diet
gastrocnemius tissue at time 0 from young mouse, biological rep3


title: Gastrocnemius tissue at time 0 from aged mouse, biological rep1

description:
Gene expression data from gastrocnemius tissue from 25 month-old mice on normal diet
gastrocnemius tissue at time 0 from aged mouse, biological rep1


title: Gastrocnemius tissue at time 0 from aged mouse, biological rep3

description:
Gene expression data from gastrocnemius tissue from 25 month-old mice on normal diet
gastrocnemius tissue at time 0 from aged mouse, biological rep3


title: LPS 2

data_processing: Data were normalized using a multi-loess technique described in Corbeil et al (Journal of Molecular Endocrinology (2004) 33:1–9)


title: Gastrocnemius tissue at time 0 from young mouse, biological rep1

description:
Gene expression data from gastrocnemius tissue from 5 month-old mice on normal diet
gastrocnemius tissue at time 0 from young mouse, biological rep1


title: Gastrocnemius tissue at time 0 from young mouse, biological rep2

description:
Gene expression data from gastrocnemius tissue from 5 month-old mice on normal diet
gastrocnemius tissue at time 0 from young mouse, biological rep2


title: cpst12 deletion mutant (sample B, dye-swap)

status: Public on Nov 30 2006

city: Rockville

first: Donald

middle: Lee

last: Nuss

state: MD

address: 9600 Gudelsky Drive

department: Center for Biosystems Research

institute: University of Maryland Biotechnology Institute

hyb_protocol: Printed slides were prepared for hybridization by the addition of 30 μl of prehybridization solution, which contained 50% formamide, 6X SSPE (1X SSPE is 0.15 M NaCl, 0.001 M NaH2PO4 and 0.001 M EDTA), 0.5% SDS, 5X Denhardt’s solution, and 100 μg of salmon sperm DNA/ml, to the arrayed surface of a glass slike (covered with a coverslip to evenly distribute the prehybridization solution). The slide was incubated for 30 min at 42 C in a hybridization chamber. Fluorescence-labeled probes were dried and resuspended in 20 μl of hybridization solution, which contained 50% formamide, 6x SSPE, 0.5% SDS, 5x Denhardt's solution, blocking solution [2 µg of poly(dA), 4 µg of yeast tRNA, 10 µg of salmon sperm DNA)], 14 µl of master mix solution (70% formamide, 3x Denhardt's solution, 0.7% SDS), and 6 µl of 20x SSPE. The resuspended probes were heated to 100°C for 2 min, vortexed, and collected by a brief spin in a microcentrifuge. The probes were applied to the arrayed surface, covered with a coverslip, and placed in a hybridization chamber overnight at 42°C.

scan_protocol: Hybridized slides were washed in each of three solutions (solution 1 is 1x SSC- 0.1% SDS, solution 2 is 2x SSC- 0.1% SDS, and solution 3 is 2x SSC) for 10 min, spun dry, scanned in both Cy3 and Cy5 channels with an Affymetrix 418 Scanner at a 10-µm resolution and a 70% photomultiplier tube value, and exported as 16-bit TIFF images for analysis.

data_processing: Integrated pixel intensity values for each spot were calculated using TIGR Spotfinder software (TIGR, Rockville, MD; http://www.tigr.org/software) and saved in tab-delimited format for analysis in Mathematica 5.1 (Wolfram Research Inc., Champaign, IL; http://www.wolfram.com). All hybridization data among two sets of dye-swap experiments (representing a total of 4 datasets) were processed according to the following pipeline: (Step 1) Each hybridization dataset was individually processed using a locally weighted linear regression (Lowess) algorithm (smoothing factor = 0.15) to remove systemic dye bias present in each group of spots within a single metarow, metacolumn block of the microarray chip. (Step 2) Following the intra-slide normalization routine described above, all lowess-normalized datasets were loaded simultaneously to rescale each spot through the use of a Z-transformation. Specifically, the global log2 (cy3/cy5) mean and standard deviations were calculated across all four datasets simultaneously and then used to rescale each spot in all four normalized datasets using Equation 1: the rescaled log2 (cy3/cy5) value of an individual spot = [the spot’s own log2 (cy3/cy5) value – global mean of all log2 (cy3/cy5) values] / by the global standard deviation of all log2 (cy3/cy5) values.

organism: Cryphonectria parasitica

source_name: cpst12 deletion mutant

label_protocol: Fluorescence-labeled cDNA probes were prepared from total RNA (25 μg per probe) by the direct incorporation of Cy3-UTP using a CyScribe first-strand cDNA labeling kit (Amersham pharmacia) primed with oligo (dT) according to the manufacturer’s instructions. Unincorporated nucleotides were removed with a Microcon-30 spin column.

growth_protocol: Grow at 22-24 C with a 12-h light-dark cycle at 1300-1600 lx.

extract_protocol: Cultures used for RNA isolation were grown on PDA-cellophane for 6 days and harvested by freezing the mycelia in liquid nitrogen and then immediately grinding the mycelia into a fine powder by using a mortar and pestle. The powder was resuspended in RNA extraction buffer (200 mM NaCl, 100 mM Tris-Cl [pH 8.0], 4 mM EDTA [pH 8.0], 2% sodium dodecyl sulfate [SDS], 2 mM dithiothreitol) at a ratio of 4 ml of buffer per g of mycelia. This step was followed sequentially by extraction with equal volumes of water-saturated phenol, acid phenol-chloroform-isoamyl alcohol (25:24:1) (pH 4.5), and chloroform. Single-stranded RNA was precipitated on ice for 2 h by the addition of LiCl to a final concentration of 2 M. The single-stranded RNA precipitate was collected by centrifugation, resuspended in 2 ml of double-distilled H2O, reprecipitated by the addition of 2.5 volumes of ice-cold ethanol and 0.1 volume of 3 M sodium acetate (pH 5.2), and incubated for 30 min at -20°C. The RNA was collected by centrifugation, washed with 2 ml of ice-cold 75% ethanol, dried, and treated with 2 U of RQ1 DNase (Promega) in 0.5 ml of 20 mM Tris (pH 8.0)-20 mM MgCl2 in the presence of 40 U of RNasin (Promega) for 1 h at 37°C. Following phenol-chloroform and chloroform extractions, the RNA was precipitated with ethanol and resuspended in 100 µl of double-distilled H2O.

tag: cpst12 deletion mutant strain cpst-E1 grown on PDA with cellophane overlay (sample B).

source_name: wild type EP155 strain

label_protocol: Fluorescence-labeled cDNA probes were prepared from total RNA (25 μg per probe) by the direct incorporation of Cy5-UTP using a CyScribe first-strand cDNA labeling kit (Amersham pharmacia) primed with oligo (dT) according to the manufacturer’s instructions. Unincorporated nucleotides were removed with a Microcon-30 spin column.

tag: Strain EP155 grown on PDA with cellophane overlay (sample B).


title: cpst12 deletion mutant (sample A, dye-swap)

tag: cpst12 deletion mutant strain cpst-E1 grown on PDA with cellophane overlay (sample A).

tag: Strain EP155 grown on PDA with cellophane overlay (sample A).


title: cpst12 deletion mutant (sample B)

tag: cpst12 deletion mutant strain cpst-E1 grown on PDA with cellophane overlay(sample B).


title: Regulatory Network of Inflammation Downstream Proteinase-Activated Receptors 17

status: Public on Nov 29 2006

city: Oklahoma City

first: Ricardo

last: Saban

state: OK

institute: University of Oklahoma Health Sciences Center

hyb_protocol: The radioactively labeled complex cDNA probes were hybridized overnight to AtlasTM mouse plastic 5K arrays (Clontech, Palo Alto, CA) using ExpressHybTM hybridization solution with continuous agitation at 68 °C.  After two high- stringency washes, the hybridized membranes were exposed (at room temperature) to a ST Cyclone phosphor screen overnight.

scan_protocol: Spots on the arrays were quantified by BD AtlasImage™ 2.7 software (Clontech, Palo Alto, CA).

data_processing: Data was normalized by a robust linear regression analysis using only genes expressed above background and the ratio of gene-expression between LPS- and SP-, and saline-challenge was obtained. PAR1-/- dependent genes were selected according to the following criterion:  a. In tissues isolated from WT mice, the expression of particular gene should be up-regulated (ratio between LPS- or SP- and saline-treated >3.0) in at least one of the time points (4 and 24 hours post-challenge); b. in tissues isolated from PAR1-/- mice, the expression of same gene should not be altered in response to LPS or SP in any of the time points.

source_name: urinary bladder

label_protocol: cDNA probes were prepared from DNase-treated RNAs obtained from each of the experimental groups.  Five microg of DNase-treated RNA was reverse-transcribed to cDNA and labeled with [alpha -33P]dATP, according to the manufacturer's protocol (Clontech, Palo Alto, CA).

extract_protocol: Briefly, eight bladders from each group were homogenized together in Ultraspec RNA solution (Biotecx Laboratories Inc. Houston, TX) for isolation and purification of total RNA.  Mouse bladders were pooled to ensure enough RNA for gene array analysis.  The justification for this approach is that there is not enough RNA in a single mouse bladder for performing cDNA array experiments and the step of purification reduces the amount of total RNA.  RNA was DNase-treated according to manufacturer’s instructions (Clontech Laboratories, Palo Alto, CA), and the quality of 10 microg was evaluated by denaturing formaldehyde/agarose gel electrophoresis.

treatment_protocol: Anesthetized mice were challenged into the bladder with   WITH SUBSTANCE P 10 MICROm and euthanized 24 hours later.

tag: female wild type (C57BL/6J)


title: Regulatory Network of Inflammation Downstream Proteinase-Activated Receptors 11

treatment_protocol: Anesthetized mice were challenged into the bladder with  WITH Escherichia coli LPS strain 055:B5 (Sigma, St. Louis, MO; 100 µg/ml) and euthanized 24 hours later.

tag: FEMALE PAR1 KO


title: Actin-binding proteins in Human

type: protein

city: cambridge

first: Nazila

last: Salamat-Miller

address: 700 main street

institute: Shire

description: Interactions were considered statistically significant when they displayed a mean signal greater than the median signal of all protein spots on the array plus one times the standard deviation (+1SD).  This +1SD threshold has a Z-score which is greater than 1.  For such threshold values, polyanion binding proteins with low, medium, and high confidence levels are included in the dataset.  Proteins which bound polyanions at the +2 and +3 SD level demonstrate a higher confidence level.  We decided, however, to generally include the greatest number of proteins involved in any type of binding to polyanions (specific or non-specific) and, therefore, chose to study PABPs at the level of +1SD, unless otherwise indicated.

scan_protocol: Experiments were conducted according to the manufacturer’s protocol.  Briefly, protein arrays were probed with 120 ul of 50 ug/ml of each polyanion in the presence and absence of 0.15 M NaCl in the probing solution.  After subsequent washings, the arrays were exposed to strepavidin-Alexa Fluor® 647.  The arrays were then washed, dried, and scanned with the GenePix®4000B at 635 nm to acquire a fluorescence image.  The resultant files were produced using GenePix® Pro 5.0 software.  Each image was analyzed using the appropriate grid downloaded from the Invitrogen Inc. website.  For all of the scanned images, a program available through Invitrogen Inc. (ProtoArray™ Prospector, v. 3.1.1) was used to obtain the identity of the proteins which demonstrated significant interaction with the probes.

molecule: protein

organism: Bos taurus

source_name: Lyophilized G-actin from bovine muscle (MW 43 kDa) was purchased from Sigma (St. Louis, MO).

label_protocol: Actin (~2.0 mg/ml) was biotinylated according to the manufacturer’s instructions using the biotin-XX-sulfosuccinimidyl ester (5 nmole/ul) included in the Invitrogen protein array kit.  Excess biotin was removed using a gel filtration resin provided in the kit.  The biotinylation efficiency was assessed by performing Tris-Glycine SDS-PAGE and a western blot of the biotinylated polyanions and the provided reference proteins according to standard protocols.  Detection and visualization of samples were performed using a Strepavidin- alkaline phosphatase conjugate and a chemiluminescent substrate, respectively.

extract_protocol:
(From Sigma website http://www.sigmaaldrich.com/catalog/search/ProductDetail/SIGMA/A3653) A3653 was prepared from the acetone powder of bovine skeletal muscle2 by the method of Spudich and Watt. Muscle acetone power is extracted with buffer A (2 mM Tris buffer, pH 8.0 containing 0.2 mM ATP,
0.5 mM ß-mercaptanol and 0.2 mM CaCl2). The filtrate is clarified by centrifugation, then the actin is polymerized by addition of 50 mM KCl and 2 mM MgCl2 (final concentration). The KCl concentration is then
increased to 0.6 M and the solution is stirred for 1.5 hour (critical for obtaining high purity actin). The F-actin is then collected by centrifugation at 80,000 x g, resuspended in Buffer A and extensively dialyzed to convert it actin to G-actin.  Protein content (Biuret) is approximately 90%, (the remainder being buffer salts).

treatment_protocol: Actin was dialyzed against 10 mM phosphate buffered saline (PBS) overnight to avoid a cross-reaction between the Tris salt contained in its lyophilized form and the biotinylation reagent.


title: Regulatory Network of Inflammation Downstream Proteinase-Activated Receptors 16

treatment_protocol: Anesthetized mice were challenged into the bladder with   WITH SUBSTANCE P 10 MICROm and euthanized 4 hours later.


title: Heparin-binding proteins in human

data_processing: The resultant files were produced using GenePix® Pro 5.0 software.  Each image was analyzed using the appropriate grid downloaded from the Invitrogen Inc. website.

organism: Sus scrofa

source_name: Heparin from porcine intestinal mucosa with mean molecular weight of 18 kDa was purchased from Sigma (St. Louis, MO).

label_protocol: Compounds were dissolved in 0.1 M MES buffer to a final concentration of approximately 4.0 mg/ml.  The biotinylation reaction was conducted by addition of 25 ul of biotin hydrazide in dry DMSO and 12.5 ul of freshly-prepared EDC, with final concentrations of 50 and 5.0 mM, respectively, to the MES/heparin or MES/HS solution.  Solutions were stirred overnight at room temperature.  The carbodiimide chemistry coupled biotin-LC-hydrazide to heparin and HS through uronic acids (carboxylate groups).  Excess biotin was removed using either dialysis cassettes (MWCO 3,500) or polyacrylamide desalting columns (Pierce, Rockford, IL).  Biotinylation efficiency was assessed by performing a dot blot on nitrocellulose membranes (Schleicher & Schuell BioScience, Inc., Keene, NH).  Approximately 2 ul of each biotinylated sample was placed on the membrane and the blot was air dried.  Biotinylated tubulin or BSA (both at a molar ratio of 9/1), and non-biotinylated polysaccharide or buffer were used as positive and negative controls, respectively.  This procedure was repeated three times, followed by blocking and washing of the membrane according to the western blot protocols.  Detection and visualization of samples were performed using a Strepavidin- alkaline phosphatase conjugate and a chemiluminescent substrate, respectively.

treatment_protocol: No treatment prior to biotinylation procedure


title: Heparan sulfate (HS)-binding proteins in human

description: For concentration determination of carbohydrates, azure A, a metachromatic dye, was utilized.  A stock solution of Azure A was prepared in 0.2% (v/v) aqueous formic acid, pH 3.5, at a final concentration of 5.04 × 10-5 M.  One ml of the dye stock solution was titrated with 1-10 ul of 1.0 mg/ml of carbohydrate solution prepared in PBS and vortexed.  The absorbance of each sample was measured at 620 nm, where the extent of disappearance of Azure A was followed as a function of carbohydrate concentration.  In each case, the non-biotinylated heparin and HS were used as standards for the biotinylated samples.  The absorbance spectroscopy confirmed that Azure A was bound to both biotinylated and non-biotinylated heparin and HS.

data_processing: The arrays were then washed, dried, and scanned with the GenePix®4000B at 635 nm to acquire a fluorescence image.  Interactions were considered statistically significant when they displayed a mean signal greater than the median signal of all protein spots on the array plus one times the standard deviation (+1SD).  This +1SD threshold has a Z-score which is greater than 1.  For such threshold values, polyanion binding proteins with low, medium, and high confidence levels are included in the dataset.  Proteins which bound polyanions at the +2 and +3 SD level demonstrate a higher confidence level.  We decided, however, to generally include the greatest number of proteins involved in any type of binding to polyanions (specific or non-specific) and, therefore, chose to study PABPs at the level of +1SD, unless otherwise indicated.

source_name: HS from porcine intestinal mucosa with mean molecular weight of 14 kDa was purchased from Sigma (St. Louis, MO).

label_protocol: Compounds were dissolved in 0.1 M MES buffer to a final concentration of approximately 4.0 mg/ml.  The biotinylation reaction was conducted by addition of 25 ul of biotin hydrazide in dry DMSO and 12.5 ul of freshly-prepared EDC, with final concentrations of 50 and 5.0 mM, respectively, to the MES/heparin or MES/HS solution.  Solutions were stirred overnight at room temperature.  The carbodiimide chemistry coupled biotin-LC-hydrazide to heparin and HS through uronic acids (carboxylate groups) (25).  Excess biotin was removed using either dialysis cassettes (MWCO 3,500) or polyacrylamide desalting columns (Pierce, Rockford, IL).  Biotinylation efficiency was assessed by performing a dot blot on nitrocellulose membranes (Schleicher & Schuell BioScience, Inc., Keene, NH).  Approximately 2 l of each biotinylated sample was placed on the membrane and the blot was air dried.  Biotinylated tubulin or BSA (both at a molar ratio of 9/1), and non-biotinylated polysaccharide or buffer were used as positive and negative controls, respectively.  This procedure was repeated three times, followed by blocking and washing of the membrane according to the western blot protocols.  Detection and visualization of samples were performed using a Strepavidin- alkaline phosphatase conjugate and a chemiluminescent substrate, respectively.


title: Regulatory Network of Inflammation Downstream Proteinase-Activated Receptors 12

treatment_protocol: Anesthetized mice were challenged into the bladder with  WITH substance P 20 microM and euthanized 4 hours later.


title: Regulatory Network of Inflammation Downstream Proteinase-Activated Receptors 14

treatment_protocol: Anesthetized mice were challenged into the bladder with   WITH Escherichia coli LPS strain 055:B5 (Sigma, St. Louis, MO; 100 µg/ml) and euthanized 4 hours later.


title: Regulatory Network of Inflammation Downstream Proteinase-Activated Receptors 10

treatment_protocol: Anesthetized mice were challenged into the bladder with  WITH Escherichia coli LPS strain 055:B5 (Sigma, St. Louis, MO; 100 µg/ml) and euthanized 4 hours later.

tag: FEMALE PAR1KO


title: cpst12 deletion mutant (sample A)

data_processing: Integrated pixel intensity values for each spot were calculated using TIGR Spotfinder software (TIGR, Rockville, MD; http://www.tigr.org/software) and saved in tab-delimited format for analysis in Mathematica 5.1 (Wolfram Research Inc., Champaign, IL; http://www.wolfram.com). All hybridization data among two sets of dye-swap experiments (representing a total of 4 datasets) were processed according to the following pipeline: (Step 1) Each hybridization dataset was individually processed using a locally weighted linear regression (Lowess) algorithm (smoothing factor = 0.15) to remove systemic dye bias present in each group of spots within a single metarow, metacolumn block of the microarray chip.  (Step 2) Following the intra-slide normalization routine described above, all lowess-normalized datasets were loaded simultaneously to rescale each spot through the use of a Z-transformation.  Specifically, the global log2 (cy3/cy5) mean and standard deviations were calculated across all four datasets simultaneously and then used to rescale each spot in all four normalized datasets using Equation 1: the rescaled log2 (cy3/cy5) value of an individual spot = [the spot’s own log2 (cy3/cy5) value – global mean of all log2 (cy3/cy5) values] / by the global standard deviation of all log2 (cy3/cy5) values.

tag: cpst12 deletion mutant strain cpst-E1 grown on PDA with cellophane overlay(sample A).


title: Regulatory Network of Inflammation Downstream Proteinase-Activated Receptors 2

treatment_protocol: Anesthetized mice were challenged into the bladder with  WITH saline and euthanized 24 hours later.

tag: PAR1 KO


title: Regulatory Network of Inflammation Downstream Proteinase-Activated Receptors 13

treatment_protocol: Anesthetized mice were challenged into the bladder with  WITH substance P 20 microM and euthanized 24 hours later.


title: Regulatory Network of Inflammation Downstream Proteinase-Activated Receptors 15

treatment_protocol: Anesthetized mice were challenged into the bladder with   WITH Escherichia coli LPS strain 055:B5 (Sigma, St. Louis, MO; 100 µg/ml) and euthanized 24 hours later.


title: DNA-binding protein in human

data_processing: The arrays were then washed, dried, and scanned with the GenePix®4000B at 635 nm to acquire a fluorescence image.

source_name: Calf thymus double-stranded DNA was purchased from Sigma (St. Louis, MO).

label_protocol: A final concentration of 200 M EZ-link Psoralen-PEO3-biotin in doubly deionized H2O was added to a 1 mg/ml solution of DNA in TE buffer (10 mM Tris, 1mM EDTA, pH 7.4) after boiling and immediate cooling of the DNA solution.  The reaction centrifuge tube was placed on ice and irradiated with a long wavelength UV source (Mini Ralight lamp, UVP Inc., San Gabrieal, CA) for 40 min.  The biotinylated solutions of DNA were stored at -20 °C.  Detection and visualization of samples were performed using a Strepavidin- alkaline phosphatase conjugate and a chemiluminescent substrate, respectively.

extract_protocol:
(From Sigma website) This product is an activated or "nicked" DNA from calf thymus. It is prepared by a modification of the known procedure using DNase I (Aposhian, H.V., and Kornberg, A., J. Biol. Chem.,
237, 519 (1962).  It is suitable as a substrate for DNA polymerase. This product is primarily double stranded. It is prepared from male and female calf thymus tissue. DNA from calf thymus is 41.9 mole % G-C and 58.1 mole % A-T.1 An absorbance of 1.0 at 260 nm corresponds to approximately 50 mg of double-stranded DNA.

tag: Calf thymus double-stranded DNA


title: Tubulin binding proteins in human

source_name: Bovine brain tubulin (Mw 100 kDa) was a kind donation from Dr. Richard Himes of the Division of Biological Sciences at the University of Kansas

label_protocol: Tubulin (~2.0 mg/ml) was biotinylated according to the manufacturer’s instructions using the biotin-XX-sulfosuccinimidyl ester (5 nmole/ul) included in the Invitrogen protein array kit.  Excess biotin was removed using a gel filtration resin provided in the kit.  The biotinylation efficiency was assessed by performing Tris-Glycine SDS-PAGE and a western blot of the biotinylated polyanions and the provided reference proteins according to standard protocols.  Detection and visualization of samples were performed using a Strepavidin- alkaline phosphatase conjugate and a chemiluminescent substrate, respectively.

growth_protocol: not available

extract_protocol: not available

treatment_protocol: Tubulin was dialyzed against two exchanges of 10 mM PBS for three hours at 4°C.

tag: Bovine brain tubulin (Mw 100 kDa)


title: Regulatory Network of Inflammation Downstream Proteinase-Activated Receptors 1


title: 1412082088D_Affected7_1

city: Los Angeles

first: Yelena

last: Bykhovskaya

address: 8700 Beverly Blvd

institute: Cedars-Sinai Medical Center

description: Comparison of genome-wide expression in EBV-transformed lymphoblastoid cell lines of patients and controls

hyb_protocol: Refer to the Illumina Gene Expression System Manual

data_processing: Raw data for each sample was output using the Illumina BeadStudio

label: Biotin-Labeled cRNA

source_name: EBV-transformed cell line of lymphoblasts extrated from peripheral blood

label_protocol: Illlumina® TotalPrep RNA amplification kit, manufactured by Ambion, Inc (Austin, TX) according to the manufacturer’s protocol

growth_protocol: RPMI-1640 medium (Invitrogen, Inc., Carlsbad, CA), containing 2 mmol/L L-glutamine, 100 ug/ml streptomycin, and 10% fetal calf serum

extract_protocol: Trizol reagent (Invitrogen, Inc.)

treatment_protocol: No treatment

tag: Immortalized lymphoblastoid cell line


title: 1412082072B_Control10

scan_protocol: Refer to the Illumina Gene Expression System Manual


title: 1412082072C_Control11


title: 1412082088E_Control8_1


title: 1412082072E_Affected7_2


title: 1412082023E_Affected4_1


title: 1412082088F_Affected9


title: 1412082088B_Parent5


title: 1412082023C_Affected3


title: 1412082023E_Affected4_2


title: 1412082023C_Parent2


title: 12h bufalin vs. 12h DMSO in HL60

city: Beijing

first: Chen

middle: Ai

last: Liang

address: Haidian District

department: Medical  School

country: China

institute: Tsinghua University

description: the genomic influence of bufalin on HL60 after treatment 12h

hyb_protocol: Labeled control and test samples were quantitatively adjusted based on the efficiency of Cy5-dCTP or Cy3-dCTP incorporation and mixed into 30 μl hybridization solution (3×SSC, 0.2% SDS, 25% formamide and 5×Denhart’s solution). DNA in hybridization solution was denatured at 95℃ for 3 min before loading onto a microarray. The array was hybridized at 42℃ overnight and washed with two consecutive washing solutions (0.2% SDS, 2×SSC at 42°C for 5 min, and 0.2% SSC for 5 min at room temperature).

scan_protocol: Fluorescent array images were collected for both Cy3 and Cy5 with a ScanArray Express fluorescent scanner and image intensity data were extracted and analyzed with GenePix Pro 4.0 analysis software.

data_processing:
A space and intensity-dependent normalization based on a LOWESS program was employed. For each test and control sample, two hybridizations were performed by using a reversal fluorescent strategy. Only genes whose alteration tendency kept consistency (both above 1.5-fold) in both microarray were selected as differentially expressed genes
Raw data files for the dye-swap hybridizations are linked below as supplementary files:
GSM146699.txt [bufalin_Cy3 vs. 12h_DMSO_Cy5]
GSM146699_dyeswap.txt [bufalin_Cy5 vs. 12h_DMSO_Cy3]

extract_protocol: RNA isolation was accomplished with Qiagen RNeasy Mini Kit reagents.

treatment_protocol: treatment with bufalin for 12h

label_protocol: Sample_label_protocol_ch1 = Fluorescent dye (Cy5 and Cy3-dCTP, Amersham Pharmacia Biotech, Piscataway, NJ) labeled DNA was produced through Eberwine's linear RNA amplification method.and subsequent enzymatic reaction. the amplified RNA (aRNA) was purified with RNeasy Mini Kit (Qiagen). Because sense oligonucleotide arrays were used here, we took a cDNA labeling approach with Klenow enzyme after reverse transcription.

treatment_protocol: treatment with DMSO for 12h


title: 1412082088C_Parent6


title: 24h bufalin vs. 24h DMSO in HL60

description: the genomic influence of bufalin on HL60 after treatment 24h

data_processing:
A space and intensity-dependent normalization based on a LOWESS program was employed. For each test and control sample, two hybridizations were performed by using a reversal fluorescent strategy. Only genes whose alteration tendency kept consistency (both above 1.5-fold) in both microarray were selected as differentially expressed genes
Raw data files for the dye-swap hybridizations are linked below as supplementary files:
GSM146700.txt [bufalin_Cy3 vs. 24h_DMSO_Cy5]
GSM146700_dyeswap.txt [bufalin_Cy5 vs. 24h_DMSO_Cy3]

label_protocol: Fluorescent dye (Cy5 and Cy3-dCTP, Amersham Pharmacia Biotech, Piscataway, NJ) labeled DNA was produced through Eberwine's linear RNA amplification method.and subsequent enzymatic reaction. the amplified RNA (aRNA) was purified with RNeasy Mini Kit (Qiagen). Because sense oligonucleotide arrays were used here, we took a cDNA labeling approach with Klenow enzyme after reverse transcription.

treatment_protocol: treatment with bufalin for 24h

treatment_protocol: treatment with DMSO for 24h


title: 1412082072D_Control12


title: RNA Polymerase II ChIP-chip on GM06990 cells using polyclonal antibody (N-20) Sample 2

city: New Haven

first: Guoneng

last: Zhong

state: CT

address: 300 George St

institute: Yale Med

description: RNA polymerase II ChIP-chip in ENCODE region in GM06990 cells using anti-Pol II (N-20) rabbit polyclonal antibody

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

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

data_processing: value = log_2(Signal for Cy5 / Signal for Cy3)

source_name: RNA Polymerase II ChIP in GM06990 Cells

label_protocol: For NimbleGen arrays, ChIP DNA prepared from 1 x 10^8 was directly labeled by Klenow (New England Biolabs) random priming with Cy5 nonamers (ChIP DNA isolated from Pol II-immunoprecipitated cells) per manufacturer's protocol (http://www.nimblegen.com/products/lit/lit.html).

extract_protocol: GM06990 cells were grown in RPMI medium supplemented with 15% FBS and antibiotics (kanamycin) at 37C in 5% CO2, to a density of 5 x10^5 cells/ml. Cells were fixed with 1% formaldehyde at room temperature for 10 min. Fixation was terminated with 125 mM glycine, and cells were washed twice in cold 1x PBS, and then swelled on ice in hypotonic lysis buffer (20 mM Hepes, pH 7.9, 10 mM KCl, 1 mM EDTA, pH 8, 10% glycerol, 1 mM DTT, 0.5 mM PMSF, and protease inhibitors). Cell lysates were homogenized with 30 strokes in a Dounce homogenizer. Nuclear pellets were collected and lysed in 1x RIPA buffer (10 mM Tris-Cl, pH 8.0, 140 mM NaCl, 1% Triton X-100, 0.1% SDS, 1% deoxycholic acid, 0.5 mM PMSF, 1 mM DTT, and protease inhibitors). Chromatin was sheared with a Branson 250 Sonifier (output setting 2, 100% duty cycle, five 30-second pulses) to an average of 700 bp as verified on a 1.5% agarose gel. Clarified lysates were agitated overnight at 4C with anti-Pol II (N-20) rabbit polyclonal antibody (Santa Cruz). Following this incubation Protein A agarose (Upstate) was added and allowed to bind for 60 min at 4C. Immunoprecipitates were washed three times in 1x RIPA, once in 1x PBS, and then eluted in 1xTE/1%SDS. Crosslinks were reversed overnight at 65C. ChIP DNA was purified by incubation with RNase A for 1 h at 37C, with 200 ug/ml Proteinase K for 2 h at 45C, phenol:chloroform:isoamyl alcohol extraction, and precipitation with 0.1 volumes of 3M sodium acetate, and 2 volumes of 100% ethanol.

tag: Polyclonal antibody for Pol II (N-20)

source_name: IgG control ChIP in GM06990 Cells

label_protocol: For NimbleGen arrays, ChIP DNA prepared from 1 x 10^8 was directly labeled by Klenow (New England Biolabs) random priming with Cy3 nonamers (ChIP DNA isolated from normal rabbit IgG-immunoprecipitated cells) per manufacturer's protocol (http://www.nimblegen.com/products/lit/lit.html).

extract_protocol: GM06990 cells were grown in RPMI medium supplemented with 15% FBS and antibiotics (kanamycin) at 37C in 5% CO2, to a density of 5 x10^5 cells/ml. Cells were fixed with 1% formaldehyde at room temperature for 10 min. Fixation was terminated with 125 mM glycine, and cells were washed twice in cold 1x PBS, and then swelled on ice in hypotonic lysis buffer (20 mM Hepes, pH 7.9, 10 mM KCl, 1 mM EDTA, pH 8, 10% glycerol, 1 mM DTT, 0.5 mM PMSF, and protease inhibitors). Cell lysates were homogenized with 30 strokes in a Dounce homogenizer. Nuclear pellets were collected and lysed in 1x RIPA buffer (10 mM Tris-Cl, pH 8.0, 140 mM NaCl, 1% Triton X-100, 0.1% SDS, 1% deoxycholic acid, 0.5 mM PMSF, 1 mM DTT, and protease inhibitors). Chromatin was sheared with a Branson 250 Sonifier (output setting 2, 100% duty cycle, five 30-second pulses) to an average of 700 bp as verified on a 1.5% agarose gel. Clarified lysates were agitated overnight at 4C with normal rabbit IgG (Santa Cruz). Following this incubation Protein A agarose (Upstate) was added and allowed to bind for 60 min at 4C. Immunoprecipitates were washed three times in 1x RIPA, once in 1x PBS, and then eluted in 1xTE/1%SDS. Crosslinks were reversed overnight at 65C. ChIP DNA was purified by incubation with RNase A for 1 h at 37C, with 200 ug/ml Proteinase K for 2 h at 45C, phenol:chloroform:isoamyl alcohol extraction, and precipitation with 0.1 volumes of 3M sodium acetate, and 2 volumes of 100% ethanol.

tag: IgG reference


title: 6h bufalin vs. 6h DMSO in HL60

description: the genomic influence of bufalin on HL60 after treatment 6h

data_processing:
A space and intensity-dependent normalization based on a LOWESS program was employed. For each test and control sample, two hybridizations were performed by using a reversal fluorescent strategy. Only genes whose alteration tendency kept consistency (both above 1.5-fold) in both microarray were selected as differentially expressed genes
Raw data files for the dye-swap hybridizations are linked below as supplementary files:
GSM146698.txt [bufalin_Cy3 vs. 6h_DMSO_Cy5]
GSM146698_dyeswap.txt [bufalin_Cy5 vs. 6h_DMSO_Cy3]

treatment_protocol: treatment with bufalin for 6h

treatment_protocol: treatment with DMSO for 6h