title: rpoS_stationary_rep1

status: Public on May 23 2008

type: RNA

city: Sapporo

first: Akinobu

last: Ito

address: North-13, West-8, Kitaku

department: Graduate School of Engineering

country: Japan

institute: Hokkaido University

description: Cells were grown for 12 hours for stationary phase

hyb_protocol: Affymetrix protocols

scan_protocol: Affymetrix protocols

data_processing: Normalization was performed with dchip software using the Invariant Set Normalization method. Sample GSM194057 (wild type_exponential_rep3) was used as the baseline array.

label: biotin

molecule: total RNA

organism: Escherichia coli

source_name: plannktonic stationary phase of rpoS knock-out strain in MOPS minimal media with 0.2% glucose

label_protocol: The isolated RNA was converted to cDNA, and the synthesized cDNA was fragmented and labeled with GeneChip® DNA Labeling Reagent according to Affymetrix protocols.

extract_protocol: Samples were RNA-stabilized with Qiagen RNAProtect Bacterial Reagent, and total RNA was isolated with Qiagen RNeasy mini kit (Qiagen).

tag: 'Strain

value: Escherichia coli MG1655 rpoS knock-out strain'


title: wild type_exponential_rep3

description: Cells were grown for 8 hours for exponential phase.

source_name: plannktonic exponential phase of wild type strain in MOPS minimal media with 0.2% glucose

value: Escherichia coli MG1655 wild type strain'


title: wild type_stationary_rep1

description: Cells were grown for 12 hours for stationary phase.

source_name: plannktonic stationary phase of wild type strain in MOPS minimal media with 0.2% glucose


title: rpoS_stationary_rep3


title: rpoS_stationary_rep2


title: wild type_stationary_rep2


title: wild type_stationary_rep3


title: wild type_biofilm_rep2

description: biofilm were grown for 72 hours.

source_name: biofilm of wild type strain in MOPS minimal media with 0.2% glucose


title: rpoS_exponential_rep3

source_name: plannktonic exponential phase of rpoS knock-out strain in MOPS minimal media with 0.2% glucose


title: rpoS_exponential_rep2


title: wild type_exponential_rep1


title: wild type_exponential_rep2


title: rpoS_biofilm_rep3

description: biofilm were grown for 72 hours.

source_name: biofilm of rpoS knock-out strain in MOPS minimal media with 0.2% glucose


title: wild type_biofilm_rep1

description: biofilm were grown for 72 hours.


title: rpoS_exponential_rep1


title: wild type_biofilm_rep3

description: biofilm were grown for 72 hours.


title: rpoS_biofilm_rep2

description: biofilm were grown for 72 hours.


title: rpoS_biofilm_rep1

description: biofilm were grown for 72 hours.


title: MSC_VUB01_1

status: Public on May 20 2008

city: EVRY

first: Christelle

last: ROCHON-BEAUCOURT

address: 5 rue H. Desbruères

country: France

institute: INSERM/UEVE UMR 861/ISTEM,AFM

description: gene expression data for VUB01 hES cell line-derived MSC

hyb_protocol: Hybridization, washing and staining procedures of the Affymetrix GeneChip® Human Genome U133 Plus 2.0 were carried out as described in the Affymetrix technical manual (see manufacturer's web site)

scan_protocol: Scanning procedures of the Affymetrix GeneChip® Human Genome U133 Plus 2.0 were carried out as described in the Affymetrix technical manual (see manufacturer's web site)

data_processing: The statistical treatment of the expression data was achieved using the Array Assist 4.1 software (Stratagene). Normalization was performed using the GC-RMA algorithm.

label: Biotin

organism: Homo sapiens

source_name: Mesenchymal Stem Cells derived from hES cells line VUB01, biological replicate 1

label_protocol: 100 ng of total RNA was used to generate biotinylated cRNA using the GeneChip® Two-Cycle Target Labeling and Control Reagents from Affymetrix according to the manufactures protocol

growth_protocol:
The hES cell lines were maintained on a feeder layer of mitomycin C-inactivated murine embryonic fibroblast (MEF) cells. The culture media (DMEM/F12 supllemented with 20% KRS and 4ng/ml bFGF) was changed daily and routine passages were performed by mechanical cutting of hES cells on a fresh feeder layer every 4-5 days.
Mesenchymal Stem Cells (MSC) were derived from hES by plating cells on 0,1% Gelatin coated dishes in KO-DMEM medium supplemented with 20% FBS.

extract_protocol: Total RNA was extracted from the cells using the RNeasy Mini kit (Qiagen) according to the manufacturer protocol

tag: Differentiation of hES cells line VUB01 towards MPC-like cells

tag: Mesenchymal stem cells phenotype


title: MSC_VUB01_3

source_name: Mesenchymal Stem Cells derived from hES cells line VUB01, biological replicate 3


title: MSC_SA01_2

description: gene expression data for SA01 hES cell line-derived MSC

source_name: Mesenchymal Stem Cells derived from hES cells line SA01, biological replicate 2

tag: Differentiation of hES cells line SA01 towards MPC-like cells


title: MSC_SA01_1

source_name: Mesenchymal Stem Cells derived from hES cells line SA01, biological replicate 1


title: MSC_SA01_3

source_name: Mesenchymal Stem Cells derived from hES cells line SA01, biological replicate 3


title: MSC_VUB01_2

source_name: Mesenchymal Stem Cells derived from hES cells line VUB01, biological replicate 2


title: Caregiver 3

city: Los Angeles

first: Steve

last: Cole

state: CA

address: 11-934 Factor Bldg, UCLA

department: Medicine

country: USA

institute: UCLA School of Medicine

description: Affymetrix U133A

hyb_protocol: Standard Affymetrix hybridization protocol for gen expression profiling (carried out in UCLA DNA Microarray Core).

scan_protocol: Standard Affymetrixscan protocol for gene expression profiling (carried out in UCLA DNA Microarray Core).

data_processing: RMA (RMAExpress) processing of Affymetrix .CEL files generated by UCLA DNA Microarray Core.

source_name: Peripheral blood CD14+ leukocytes

label_protocol: Standard Affymetrix probe synthesis for gene expression profiling (carried out in UCLA DNA Microarray Core).

extract_protocol: Peripheral blood leukocytes were isolated from fresh whole blood by ficol density gradient, CD14+ cells were isolated by MACS immunomagnetic positive selection, and total RNA was extracted using Qiagen RNEasy.

tag: Caregiver 3


title: Caregiver 11

description: Affymetrix U133A

tag: Caregiver 11


title: 746_Rectum2_Conv

status: Public on May 19 2008

city: Lausanne

first: James

last: Holzwarth

state: Vaud

address: vers chez les blanc

department: Bioanalytical Sciences

country: Switzerland

institute: Nestle Research Center

hyb_protocol: standard Affymetrix procedure

scan_protocol: standard Affymetrix procedure

data_processing: Robust Multiarray Analysis followed by GEA

organism: Mus musculus

label_protocol: 5 ug of cRNA was used to make biotinylated RNA

extract_protocol: TriPure Roche + Column purification

tag: Strain:C3H Tissue

value: Rectum


title: Caregiver 8

description: Affymetrix U133A

tag: Caregiver 8


title: 754_Ileum3_Conv

value: Ileum


title: Caregiver 5

description: Affymetrix U133A

tag: Caregiver 5


title: Caregiver 1

description: Affymetrix U133A

tag: Caregiver 1


title: Caregiver 10

description: Affymetrix U133A

tag: Caregiver 10


title: Control 1

description: Affymetrix U133A

tag: Control 1


title: Control 4

description: Affymetrix U133A

tag: Control 4


title: Control 9

description: Affymetrix U133A

tag: Control 9


title: Caregiver 9

description: Affymetrix U133A

tag: Caregiver 9


title: Control 5

description: Affymetrix U133A

tag: Control 5


title: Caregiver 2

description: Affymetrix U133A

tag: Caregiver 2


title: Control 10

description: Affymetrix U133A

tag: Control 10


title: Caregiver 6

description: Affymetrix U133A

tag: Caregiver 6


title: Control 6

description: Affymetrix U133A

tag: Control 6


title: Control 2

description: Affymetrix U133A

tag: Control 2


title: Control 7

description: Affymetrix U133A

tag: Control 7


title: Control 3

description: Affymetrix U133A

tag: Control 3


title: Control 8

description: Affymetrix U133A

tag: Control 8


title: Caregiver 4

description: Affymetrix U133A

tag: Caregiver 4


title: Woundedge_day15_rep1

status: Public on Apr 01 2008

city: Tromsoe

first: Svetlana

last: Zykova

address: Breivika

department: Medical Faculty

country: Norway

institute: University of Tromsoe

description: 2mm wide skin from wound edge tissues collected at day 15 post-wounding.

hyb_protocol: Fluorescent cRNA was hybridized using Pronto! Universal Hybridisation kit (Corning, catalogue number: 40026) in Hybridization Chambers (Corning, catalogue 2551) according to the manufacturer’s instructions.

scan_protocol: The slides were scanned using a GenePix 4000B microarray scanner (Axon Instruments, Westbury, NY, USA) at laser intensity and photomultiplier tube settings giving the best dynamic range for each chip. Image segmentation and feature extraction were done with the ImaGene 4.1 software (Biodiscovery, Marina Del Rey, USA).

data_processing: Replicated spots (n=8) were averaged to produce a single measurement per gene and chip, and log-transformed mean-values were normalized for signal intensity by dividing with median intensity. Proteoglycan-related genes were tested for differential expression over time and for differential expression between wound bed and wound edge using two-way ANOVA.

label_protocol: 500ng total RNA was amplified and labeled with cyanine 3-CTP (PerkinElmer/NEN Life Sciences, catalogue number NEL580) using Agilent Low RNA Input Fluorescent Linear amplification kit (Agilent Technologies, Inc., CA, USA, catalogue number: 5184-3523) according to the manufacturers instructions. Clean-up reaction was performed with Qiagen Rneasy mini kit (Qiagen Nordic, Sweden).

extract_protocol: RNA-later preserved wound and skin samples were weighed and homogenized with Ultra Turrex T8 homogenizer (IKA/TamroLab). Total RNA was isolated with SV Total RNA Isolation System (Promega GmbH, Germany, catalogue number: Z3100) and treated with DNAse I (Technical Manual No. 048 from Promega).

treatment_protocol: Operations: Wounds were established on the backs of mice under general anesthesia and after chemical depilation and desinfection with chlorhexidine, by excising skin and panniculus carnosus of 1.5x1.5 cm area. A semipermeable transparent polyurethane dressing Opsite FlexigridÒ (Smith & Nephew Medical Ltd., Hull, England) was fixed to the skin with tissue adhesive HistoacrylÒ (B. Braun Melsungen AG, Melsungen, Germany) and eight interrupted 5-0 MonosofÔ sutures (Auto Suture Company, Norwalk, CT, USA).  Post-operative animals received analgesia right after, and following 12 hours after the surgical procedure. To provide sufficient hydration, the animals were given two subcutaneous injections of an isotonic electrolyte solution Ringer Acetate (Fresenius Kabi Norge AS, Halden, Norway) (dose: 0.030 – 0.050 ml/g body wt) immediately and at 2 hours post wounding. After the operation, the mice were put into a heating chamber at initial temperature 27 – 31° C, adjusted to 23 –25° C during awakening. Mice were killed by CO2-suffocation on either day 2, 6 or day 15 post-surgery. Samples were collected into microcentrifuge tubes with RNA-later, kept refrigerated overnight and then frozen at -70°C.

tag: Strain

tag: Gender

value: female

tag: Age

value: 9-13 weeks old


title: 752_Colon1_Conv

tag: Strain:C3H Tissue

value: Colon


title: 747_Rectum3_Conv


title: Woundedge_day2_rep3

description: 2mm wide skin from wound edge tissues collected at day 2 post-wounding.


title: Woundedge_day6_rep1

description: 2mm wide skin from wound edge tissues collected at day 6 post-wounding.


title: 749_Ileum2_GF


title: 750_Ileum3_GF


title: 744_Colon3_Conv


title: Woundedge_day2_rep1

source_name: 2mm wide skin from wound edge tissues collected at day 2 post-wounding.


title: 745_Rectum1_Conv


title: Caregiver 7

description: Affymetrix U133A

tag: Caregiver 7


title: 739_Ileum1_Conv


title: NPC-076-RR Cy5 vers. NPC-076 Cy3

status: Public on May 01 2008

city: Teipei

first: Yun-Shien

last: Lee

address: No.199, Tunghwa Rd.,

country: Taiwan

institute: Chang Gung Memorial Hospital

description:
Slide : NPC-076 Cy5 vers. NPC076-RR Cy3
Slide : NPC-076-RR Cy5 vers. NPC-076 Cy3
Cy3 and Cy5 dye-swaps

hyb_protocol: Hybridization buffer: Supply by Genisphere 3DNA kits. Slide blocking: 3x SSC, 0.1% SDS, 0.1 mg/mL BSA. Prehybridization for 30 min at 50°C. wash procedure:    1x 10 min with 2x SSC, 0.2% SDS at 60oC;    1x 10 min with 2x SSC, at room temperature;    1x 10 min with 0.2x SSC at room temperature;    spin-dried at 250 x g for 3 min. first step Hybridization: overnight (16 h), 25 µl, at 60 °C; second step Hybridization: 2 h, 25 µl, at 60 °C. hybridization instruments: 10-slide hybridization chamber (Genetix)

scan_protocol: Axon GenePix4000A GenePix Pro 4.1 software (Axon Instrument, Inc.) Expected spot diameter: 140 Micrometer. PixelSize = 10

data_processing:
Normalization strategy : total array
Normalization algorithm : Lowess (local weighted regression), available http://www.stat.berkeley.edu/users/terry/zarray/Html/soft.html

label_protocol:
2 µg of total RNA (Genisphere 3DNA-Random Primer)
Genisphere 3DNA-Random Primer Submicro Expression Array Detection Kit  (category # W300130/W300140);.

extract_protocol:
Cells were grown at 37oC in RPMI1640 medium or DMEM supplemented with 10% FCS and 1% antibiotics.
Cell were trypsinized from culture dish. Cell pellets were harvested and washed with PBS and subjected to RNA extraction.
Total RNA was extracted from the RR cells with TRIzol reagent (Gibco BRL) following the manufacturer’s instructions. The concentration, purity, and amount of total RNA were determined by ultraviolet spectrometry.

tag: NPC-076 radioresistant subclone cells

value: Parental cell line


title: PFK study: Sco8.26 (measurement)

city: Manchester

first: Giles

last: Velarde

address: MIB

country: United Kingdom

institute: University of Manchester

description: Hybridisation of "M145 RNA No. 1 cy5" vs "M145 delta_pfkA2 RNA No. 1 cy3"

hyb_protocol: Pre-hybridisation	For pre-hybridisation of the slides use the Pronto! Universal Microarray	Hybridisation kit (Corning, Cat.# 40026) according to the supplier's instructions.		Purification of probes using Qiagen MinElute columns Alternatively Qiagen QiaQuick PCR purificationcolumns can be used 	 elute in 1 x 20µl	To each labelling reaction:	Add 250µl (5 volumes) buffer PB and spin through column 1min at 13K	Add 500µl buffer PE, spin 1min at 13K	Add 250µl buffer PE, spin2min at 13K		Transfer column to clean tube	Add 10µl MilliQ H2O pH 7-8.5, 1min at RT, spin 1min at 13K	Add another 10µl MilliQ H2O pH 7-8.5, 1min at RT, spin 1min at 13K		For each Cy-cDNA sample measure theconcentration of Cy nucleotides	spectrophotometrically at 550nm (Cy3) or 650nm (Cy5): 	pmol Cy3/ µl \= A550 / 0.15	pmol Cy5/ µl \= A650 / 0.25		Take into account any dilutions of the original eluate prior to spectrophotometry. We	recommend the use of NanoDrop spectrophotometer where Cy-dye incorporation is	provided directly from just 1µl of undiluted sample.		Take an aliquot of Cy-cDNA containing 40pmol of Cy nucleotides. Mix Cy3 and Cy5	samples together and dry the labelled targets completely in a Speed Vac at RT.		Hybridisation	Use 40µl of Pronto! Universal Hybridisation Solution (supplied with the Pronto!	Universal Microarray Hybridisation kit(Corning, Cat.# 40026)) to re-dissolve the	labelled probes (final concentration of each cDNA-incorporated Cy dye 1pmol/ µl).		Denature 99 o C for 2min	Pre-heat pre-hybridised slide and place 30µl 3x SSC in each well ofthe hybridisation	chamber towards end of incubation.	Spin down probe and immediately transfer to arrayed area of slide, avoiding bubbles.	Quickly place 24 x 60mm cover slip over and seal in hybridisation chamber.	Incubate in 42 oC water bath for 16-20 hrs.		Washing	For post-hybridisation washes of the slides use the Pronto! Universal Microarray	Hybridisation kit (Corning, Cat.# 40026) according to the supplier's instructions.		Dry by centrifugation or compressed air 		Required equipment / consumables	Scanner and image analysis software	Coplin jars (Fisher Scientific, Cat. # MNK-820-H) or	staining troughs (Fisher Scientific, Cat. # MNK-836-K) with slide racks (Fisher Scientific,	Cat. # MNK-841-K)	Orbital shaker	Centrifuge with rotor for microtitre plates or 50ml tubes	Microfuge	Water baths / heat block / thermocycler	MinElute PCR purification kit (Qiagen Cat.# 28004)	Pronto! Universal Microarray Hybridisation kit (Corning, Cat.# 40026)	Slide heating block (Scientific Laboratory Supplies Cat. # MIC 4302) or heat block	Hybridisation chambers (CMT -Hybridization Chambers (5), Corning Cat. # 2551)	Air-duster(Dust-Pro Pressurized Duster, Sigma Cat # Z37,952-2)	24 x 60 mm cover slips (HybriSlips, VWR Cat.# 241/ 3025/ 28)

scan_protocol: Microarray slides were scanned for image acquisition at Cy3 (532nm) and Cy5 (635nm) channels sequentially with Affymetrix 428 laser scanner. After a gentle blow with pressurised air to remove any dust, the slides were first scanned at a low resolution preview mode in order to control the photmultiplier tube again to the appropriate level. This level was determined as the maximum possible gain where no gene spots were saturated and the background noise remained minimal relative to the signal intensities. Once the gain level was selected for each channel the array was scanned at a 10-micron resolution and the 16-bit images were stored.

data_processing: The image was analysed using BlueFuse (BlueGnome, UK) according to the supplier's manual. 		Manual flags were applied to spots after quantification when the subgrids were checked for any unreliable spots not flagged by the programme. Such spots included those with background artefacts (dust, hair, locally high background, etc.) or extensively merged neighbouring spots.		Spots flagged E classisfied as 'bad' spots		The program automatically performs background noise substraction.

organism: Streptomyces coelicolor A3(2)

source_name: PFK study: Streptomyces coelicolor M145 wild type

label_protocol: cDNA synthesis and labelling	RNA must be purified and dissolved in H2O	For each sample:	10-15µg RNA (15.2µl max)	1.7µl Random primers	to 16.9µl (Cy3) OR 15.9µl (Cy5) RNase-free H2O		70 o C for 10min, snap cool on ice, spin down.	Then add (make a master mix for multiple labellings):	6µl 5x First StrandBuffer	3µl 100mM DTT	0.6µl dNTPs (25mM each dA/ G/ TTP, 10mM dCTP)	2µl SuperScript III	1.5µl Cy3-dCTP OR 2.5µl Cy5-dCTP		25 o C for 10 min in the dark.	42 o C for 110min in the dark.		Denature RNA	To each RNA labelling reaction:	Add 10µl 1N NaOH	70 o C for 10min	Add 10µl 1N HCl		Store at -20 o C protected from light

growth_protocol: PFK study: inoculum preparation

extract_protocol: PFK study: M145 RNA No. 1

treatment_protocol: PFK study: growth in fermentors and sampling for RNA

tag: S. coelicolor M145 is a prototrophic derivative of S. coelicolor A3(2) strain 1147.  Both plasmids SCP1 and SCP2 are missing. This is the strain used for sequencing of S. coelicolor (Bentley et al (2002) "Complete genome sequence of the model actinomycete Streptomyces coelicolor A3(2)". Nature 417(6885):141-147) and is a spotaneous revertant of a phenylalanine auxotroph. Reference

value: Redenbach, M., Kieser, H.M., Denapaite, D., Eichner, A., Cullum, J., Kinashi, H., and Hopwood, D.A. (1996) "A set of ordered cosmids and a detailed genetic and physical map for the 8Mb Streptomyces coelicolor A3(2) chromosome". Mol Microbiol 21:77-96

source_name: PFK study: Streptomyces coelicolor M145 pfkA2 deletion mutant

extract_protocol: PFK study: M145 delta_pfkA2 RNA No. 1

tag: Inframe replacement of pfkA2 gene (ORF ID

value: SCO5426) with apramycin resistance casette in S. coelicolor M145 supplied by Mathias Redenbach. The replacement was achieved by REDIRECT technology (Gust, B., Challis, G. L., Fowler, K., Kieser, T. and Chater, K. F. (2003) "PCR-targeted Streptomyces gene replacement identifies a protein domain needed for biosynthesis of the sesquiterpene soil odor geosmin", Proc Natl Acad Sci U S A 100, 1541-1546)


title: Woundbed_day15_rep1

description: Excised shaved skin from the operation day, wound bed, collected at day 15 post-wounding.


title: Woundbed_day15_rep3

source_name: Excised shaved skin from the operation day, wound bed, collected at day 15 post-wounding.


title: 743_Colon2_Conv


title: Woundbed_day6_rep1

description: Excised shaved skin from the operation day, wound bed, collected at day 6 post-wounding.


title: 3 BJ-TE, 100% confluent, ns shRNA

city: Wuerzburg

first: Christof

last: Burek

address: Josef-schneider-Str. 2

department: Pathologie

country: Germany

institute: Universitaet Wuerzburg

description: BJ-TE cell line in 100 confluent state treated with shRNA control vector

hyb_protocol: Affymetrix standard protocol

scan_protocol: Affymetrix standard protocol

data_processing: Affymetrix standard protocol

source_name: cell line (BJ-TE)

label_protocol: Affymetrix standard protocol

extract_protocol: trizol

tag: cell line


title: Woundbed_day2_rep3

description: Excised shaved skin from the operation day, wound bed, collected at day 2 post-wounding.


title: 748_Ileum1_GF


title: NPC-BM1 Cy5 vers. NPC-BM1-RR Cy3

description:
Slide, NPC-BM1 Cy5 vers. NPC-BM1-RR Cy3
Slide, NPC-BM1-RR Cy5 vers. NPC-BM1 Cy3
Cy3 and Cy5 dye-swaps


title: Woundbed_day2_rep2

source_name: Excised shaved skin from the operation day, wound bed, collected at day 2 post-wounding.


title: Woundedge_day15_rep3

source_name: 2mm wide skin from wound edge tissues collected at day 15 post-wounding.


title: 4 BJ-TE, 100% confluent, p73 shRNA

description: BJ-TE cells treated with shRNA for p73 in a 100% confluent state

source_name: BJ-TE cell line

extract_protocol: trizol extraction protocol

tag: cell line (BJ-TE)


title: Skin_intact_day0_rep3

description: Excised shaved intact skin

source_name: Excised shaved intact skin


title: 740_Ileum2_Conv


title: Skin_intact_day0_rep2

description: Excised shaved intact skin


title: 2 BJ-TE, 40% confluent, p73 shRNA

description: cell line (BJ-TE) in 40% confluent state treated with shRNA p73

tag: BJ-TE cell line


title: NPC-BM1-RR Cy5 vers. NPC-BM1 Cy3

tag: NPC-BM1-RR radioresistant subclone cells

tag: NPC-BM1 Cell line


title: PFK study: Sco8.31 (measurement)

description: Hybridisation of "M145 RNA No. 2 cy5" vs "M145 delta_pfkA2 RNA No. 2 cy3"

extract_protocol: PFK study: M145 RNA No. 2

extract_protocol: PFK study: M145 delta_pfkA2 RNA No. 2


title: PFK study: Sco8.37 (measurement)

description: Hybridisation of "M145 RNA No. 2 cy3" vs "M145 delta_pfkA2 RNA No. 1 cy5"


title: Woundedge_day15_rep2


title: Woundedge_day6_rep3

source_name: 2mm wide skin from wound edge tissues collected at day 6 post-wounding.


title: Woundedge_day6_rep2


title: NPC-076 Cy5 vers. NPC076-RR Cy3


title: PFK study: Sco8.28 (measurement)

description: Hybridisation of "M145 RNA No. 3 cy3" vs "M145 delta_pfkA2 RNA No. 2 cy5"

extract_protocol: PFK study: M145 RNA No. 3


title: Woundedge_day2_rep2


title: Woundbed_day15_rep2


title: Woundbed_day6_rep3

source_name: Excised shaved skin from the operation day, wound bed, collected at day 6 post-wounding.


title: Woundbed_day6_rep2


title: 50 nM BPDE, biological rep1

status: Public on Jan 01 2008

city: Hangzhou

first: Lu

last: XiangYun

address: 388 Yuhangtang Road

country: China

institute: Zhejiang University School of Medicine

description: Gene expression data from FL cells exposed to 50 nM BPDE

hyb_protocol: The biotinlyated cRNAs were fragmented and hybridized to the microarrays at 45°C for 16 h in the Hybridization Oven 640 rotating at 60 rpm. Following hybridization, the microarrays were washed and stained with streptavidin-phycoerythrin in the Fluidics Station 400,

scan_protocol: Based on the Affymetrix Microarray Suite 5.0 platform, GeneChips were scanned using the GeneChip Scanner 3000, and original Affymetrix .dat proprietary output files were generated.

data_processing: Affymetrix MAS5.0 was used to obtain gene expression signals and normalization of our arrays’ signals by global scaling to the target of 500 for U133A and U133B for each array. Detection call and change call were generated through single array analysis and two-array comparison analysis, respectively. A probe set was delimited as differentially expressed only if it showed at least one “Present” detection call and exhibited the consistent “Increase” or “Decrease” change calls in the orthogonal comparisons of the two BPDE and two control treatment microarrays.

source_name: human amnion epithelial FL cells exposed to 50 nM BPDE for 2 h, and subsequently recovered for additional 4 h

label_protocol: Standard Affymetrix Protocols (Affymetrix GeneChip Expression Analysis Technical Manual, rev.3. 2001)

growth_protocol: Human amnion epithelial FL cells were grown as monolayers in minimal essential medium (MEM, Gibco-Invitrogen, Carlsbad, CA) supplemented with 10% newborn bovine serum (Gibco-Invitrogen), 100 U/mL penicillin, and 100 µg/mL streptomycin in a humidified atmosphere of 5% CO2 at 37°C.

extract_protocol: Total RNA was isolated with Trizol reagents (Invitrogen), and further purified using RNeasy Mini Kit (Qiagen, Valencia, CA) according to the manufacturers’ protocols. Isolated total RNA was analyzed on an Agilent Bioanalyzer 2100 to verify that the starting material was of good quality.

treatment_protocol: At the logarithmic growth phase, the cultures were exposed to vehicle control (0.1%, V/V) and increasing doses (5, 50, 500 nM) of anti-BPDE for 2 h in a culture medium that did not contain 10% newborn bovine serum, followed by additional growth for 4 h in a fresh medium containing 10% newborn bovine serum. The cultures were rinsed twice with Hank’s buffer before each change of the medium.

value: human amnion epithelial FL cells, Gender:female


title: DMSO vehicle control, biological rep2

description: Gene expression data from FL cells exposed to DMSO vehicle control

source_name: human amnion epithelial FL cells exposed to DMSO vehicle control for 2 h, and subsequently recovered for additional 4 h


title: Woundbed_day2_rep1


title: 5 nM BPDE, biological rep2

description: Gene expression data from FL cells exposed to 5 nM BPDE

source_name: human amnion epithelial FL cells exposed to 5 nM BPDE for 2 h, and subsequently recovered for additional 4 h


title: DMSO vehicle control, biological rep1


title: 1 BJ-TE, 40% confluent, ns shRNA

description: cell line (BJ-TE) 40% confluent treated with control shRNA


title: Wild type versus crp gene expression profile 2

status: Public on Dec 31 2007

city: Atlanta

first: Jorge

middle: Antonio

last: Benitez

state: GA

address: 720 Westview Dr., SW

department: Microbiology

institute: Morehouse School of Medicine

description: Biological replicate 1 of 4.

hyb_protocol: Labeled cDNA were purified and the cDNA probes mixed and applied to the array surface for hybridization overnight at 42C for 16 h following protocol M008 available at the pathogen functional genomic resource center (http://pfgrc.tigr.org)

scan_protocol: Scanned on an Agilent G2565AA scanner. Images were quantified using Agilent Feature Extraction Software (version A.7.5).

data_processing: LOWESS normalized, background subtracted data obtained from log2 of processed Red signal/processed Green signal. Agilent software was used. Rank product for differential expression was used.

organism: Vibrio cholerae

source_name: Total RNA from C7258 labeled with Cyanine-5 (red).

label_protocol: Using 15 mg of total RNA per sample, cDNA was synthesized in the presence of 3-aminoallyl-2'-deoxyuridine 5'-triphosphate by reverse transcription with SuperscriptTM II reverse transcriptase (Invitrogen) as per the manufacturer’s instructions. Cy-5 fluorescent dye (Amersham Biosciences) was coupled to the reference (wild type) cDNA; Cy-3 was coupled to the test (mutant) cDNA. Labeled cDNA were purified using the MiniElute PCR purification kit (QIAGEN). The detailed protocol (SOP # M009) can be found at http://pfgrc.tigr.org.

growth_protocol: LB medium to OD600 1.5

extract_protocol: Total RNA extracted using Trizol following manufacturer's instructions followed by RNeasy MiniElute cleanup (Qiagen)

tag: virulent V. cholerae El Tor Ogawa strain C7258 (Peru isolate 1991)

source_name: Total RNA from isogenic crp mutant labeled with Cyanine-3 (green).

tag: isogenic deletion mutant (WL7258) lacking DNA sequences encoding the cAMP receptor protein


title: 50 nM BPDE, biological rep2


title: 500 nM BPDE, biological rep1

description: Gene expression data from FL cells exposed to 500 nM BPDE

source_name: human amnion epithelial FL cells exposed to 500 nM BPDE for 2 h, and subsequently recovered for additional 4 h


title: 500 nM BPDE, biological rep2


title: Wild type versus crp gene expression profile 4

description: Biological replicate 3 of 4.

growth_protocol: LB medium to OD600 1.7


title: Wild type versus crp gene expression profile 3

description: Biological replicate 2 of 4. .

growth_protocol: LB medium to OD600 1.6


title: 5 nM BPDE, biological rep1


title: DMSP_160A_647

status: Public on Dec 09 2007

city: Athens

first: Shulei

last: Sun

address: Department of Marine Science

department: Marine Science

institute: UGA

description: Dye in each single use vial was dissolved in 11 ul DMSO, and added to the aa-aRNA, mixed, and incubated for 1 hour in the dark at room temperature

data_processing: Lowess M log ratio

label: AlexaFluor 555

organism: Ruegeria pomeroyi

source_name: Silicibacter pomeroyi DSS-3, control, 0 min.

label: AlexaFluor 647

source_name: Silicibacter pomeroyi DSS-3, DMSP treatment, 160 min.


title: Skin_intact_day0_rep1

description: Excised shaved intact skin


title: Wild type versus crp gene expression profile 1

description: Biological replicate 4 of 4.

growth_protocol: LB medium to OD600 1.8


title: DMSP_160C_647


title: DMSP_40B_647

source_name: Silicibacter pomeroyi DSS-3, DMSP treatment, 40 min.


title: DMSP_40A_555_swap


title: DMSP_20B_555_swap

description: Dye in each single use vial was dissolved in 11 µl DMSO, and added to the aa-aRNA, mixed, and incubated for 1 hour in the dark at room temperature

data_processing: Lowess

source_name: Silicibacter pomeroyi DSS-3, DSMP treatment, 20 min.

source_name: Silicibacter pomeroyi DSS3, control, 0 min.


title: DMSP_80C_555_swap

source_name: Silicibacter pomeroyi DSS-3, DMSP treatment, 80 min.


title: NF-IL6 KI(LAP) PEC(-)

status: Public on Dec 05 2007

city: Suita

first: Satoshi

last: Uematsu

state: Osaka

address: 3-1 Yamada-oka

department: Department of Host Defense

institute: Research Institute for Microbial Diseases, Osaka University

description: Gene expression data from peritoneal macrophages.

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

scan_protocol: GeneChips were scanned using the Hewlett-Packard GeneArray Scanner G2500A.

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 100.

source_name: Peritoneal macrophages

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

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

tag: Cell

value: PECs


title: DMSP_20C_647

source_name: Silicibacter pomeroyi DSS-3, DMSP treatment, 20 min.


title: ACET_160B_647

data_processing: Lowess M Log ratio

source_name: Silicibacter pomeroyi DSS-3, ACET treatments, 160 min.


title: DMSP_81A_555_swap


title: ACET_160C_555_ori

source_name: Silicibacter pomeroyi DSS-3, ACET treatment, 160 min.


title: ACET_20C_555_swap

data_processing: Lowess normalized log ratio data

source_name: Silicibacter pomeroyi DSS-3, ACET treatments, 20 min.

organism: Ruegeria pomeroyi DSS-3


title: DMSP_40C_555_swap


title: ACET_80A_647

source_name: Silicibacter pomeroyi DSS-3, ACET treatment, 80 min


title: ACET_40A_647

source_name: Silicibacter pomeroyi DSS-3, ACET treatments, 40 min.


title: Cardiomyocyte stretch 1

status: Public on Dec 01 2007

city: Heidelberg

first: Derk

last: Frank

address: Im Neuenheimer Feld 350

department: Dept. of Internal Medicine III

institute: University of Heidelberg

description: stretch condition 1

hyb_protocol: Hybridization was carried out for 16-18 hours in the Affymetrix Oven 640 at 60 rpm and 45°C. Subsequent washing and staining was performed with a fluidics station (Gene Chip® Fluidics station 400, Affymetrix) and controlled by Affymetrix GeneChip Operating System (GCOS).

scan_protocol: The scanning process and primary data analysis was done with the Affymetrix GeneChip Scanner 3000 controlled with GCOS.

data_processing: variance stabilizing normalization procedure (Huber et al., Bioinformatics 2002)

label: biotin + phycoerythrin-streptavidin

organism: Rattus norvegicus

source_name: isolated neonatal cardiomyocytes from rat heart

label_protocol: Affymetrix expression profiling was carried out at the German Ressource Center for Genome Research (RZPD; Berlin, Germany). Briefly, integrity of RNA was checked using an Agilent Bioanalyzer (Eukaryote total RNA assay) followed by cDNA synthesis and in vitro transcription with Ambion’s Message Amp kit according the manufacturer’s instructions. cRNA was subjected to quality control using the Bioanalyzer with the mRNA smear nano assay. Final cRNA concentration was 60 ng/µl.

growth_protocol: Hearts from 1-2 days old Wistar rats (Charles River, Sulzfeld, Germany) were harvested and minced in PBS. Subsequently, up to six digestion steps were carried out with pancreatin (Sigma, Munich, Germany, 0,6 mg/ml) and collagenase type II (Worthington, 0,5 mg/ml) in sterile ADS buffer containing 120 mmol/l NaCl, 20 mmol/l HEPES, 8 mmol/l NaH2PO4, 6 mmol/l glucose, 5 mmol/l KCl, 0.8 mmol/l MgSO4, pH 7.4. Cardiomyocytes were purified from contaminating fibroblasts using a Percoll (Amersham, Germany) gradient centrifugation step. Finally, NRVCMs were resuspended and cultered in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% FCS, penicillin/streptomycin and L-glutamine (all from PAA, Linz, Austria) .

extract_protocol: Total RNA was isolated from NRVCMs using the TRIzolTM reagent (Invitrogen, Germany) according to the manufacturer’s protocol. RNA was resuspended in DEPC (Sigma)–treated H2O. RNA integrity and purity were electrophoretically verified by ethidium bromide staining and measurement of OD260/OD280 absorption ratios. Total RNA was pooled from several preparations and purified from contaminating genomic DNA by a DNase I digestion (Sigma, AMPD1).

treatment_protocol: A transparent silicone membrane (0.25 mm, Specialty Manufacturing, Saginaw, MI, USA) was attached to a 60 mm biaxial stretch device as described previously and coated with a 0,1 mg/ml collagen type I solution (Sigma, Munich, Germany). NRVCMs were seeded out at a density of 1.75 x 10^5 cells per cm2, resulting in a total cell count of 1 x 10^7 cells per stretch device. After 36 h, cells were washed with PBS and serum starved in DMEM medium for another 24 h until biaxial stretch was applied to a total of 112% for the indicated period of time.

tag: induced by biomechanical stretching


title: ACET_20B_647


title: ACET_160C_555_swap


title: DMSP_160B_555_swap

source_name: Silicibacter pomeroyi DSS-3, DSMP treatment, 160 min.


title: ACET_80C_647

source_name: Silicibacter pomeroyi DSS-3, ACET treatment, 80 min.


title: DMSP_80B_647


title: NF-IL6 KO PEC (-)

tag: Strain


title: ACET_80B_555_swap


title: Cardiomyocyte control 2

description: control condition 2

tag: control condition


title: ACET_40C_647

source_name: Silicibacter pomeroyi DSS-3, ACET treatment, 40 min.


title: DMSP_20A_647

source_name: Silicibacter pomeroyi DSS-3, control, 0 min.


title: Cardiomyocyte control 1

description: control condition 1


title: NF-IL6 KI(LAP) PEC LPS+IFN-gamma 12h


title: ACET_40B_555_swap


title: Wild PEC LPS+IFN-gamma

value: PEC


title: NF-IL6 KO PEC LPS+IFN-gamma 12h


title: Cardiomyocyte phenylephrine 2

description: phenylephrine condition 2

treatment_protocol: Pharmacological stimulation was carried out with phenylephrine (PE; Sigma), 50 umol/l, endothelin-1 (ET-1; Sigma), 100 nM, and Angiotensin-II (Ang-II; Calbiochem, Darmstadt, Germany), 100 nM, respectively.

tag: induced by phenylephrine


title: whole_larvae_rep3

status: Public on Oct 01 2007

city: St. Augustine

first: Marco

middle: V.

last: Neira

state: FL

address: 9505 Oceanshore Blvd.

department: The Whitney Laboratory

institute: University of Florida

description: Sample represents pooled material from 200 individuals

hyb_protocol: The biotin-labeled cRNA was fragmented and hybridized for 16 hours to the Affymetrix GeneChip® Plasmodium/Anopheles Genome Array according to the manufactures’ protocols.  The arrays were then washed and stained with streptavidin-phycoerythrin on an Affymetrix Fluidics Station 450

scan_protocol: Hybridized chips were scanned on a GeneChip Scanner 3000 (Affymetrix, Inc) using default values to generate signal intensities

data_processing: The GeneSifter™ (VizX Labs, Seattle, WA, USA) software package was used to perform all statistical analyses. Array data was background-adjusted, normalized, and log2-transformed by applying the robust multiarray average (RMA) algorithm (Irizarry et al., 2003).

organism: Anopheles gambiae

source_name: Whole 4th. instar An. gambiae larvae

label_protocol: cDNA was synthesized from 5ug of total RNA using the One-Cycle Eukaryotic Target Labeling reagents (Affymetrix, Inc.).  The cDNA was used as a template for in vitro transcription (IVT) in the presence of T7 RNA Polymerase and a biotinytlated nucleotide/ribonucleotide mix for cRNA amplification and biotin labeling using the IVT labeling kit (Affymetrix, Inc).

growth_protocol: Eggs of the G3 strain of An. gambiae (originally collected in the Gambia in 1975) (Romans et al., 1998) were obtained from the Centers for Disease Control and Prevention (CDC; Atlanta, Georgia). Mosquito larvae were reared in distilled water at 28oC, 12h: 12h light: darkness cycle, and provided with a mixture of baker’s yeast and ground Tetramin® flakes as food.

extract_protocol: Total RNA was extracted using the RNAeasy-Mini Kit™ (Qiagen®, Chatsworth, CA, USA) following the manufacturer’s recommendations

treatment_protocol: For each biological repeat, 1,000 fourth-instar larvae were anesthetized on ice and dissected in RNAse-free 70% ETOH. Their guts were extracted and divided into gastric caeca (GC), anterior midgut (AM), posterior midgut (PM) and hindgut / Malpighian tubes (HG/MT). Three biological repeats were performed in order to obtain statistical significance.

tag: Larval instar


title: dDAVP mouse 2 kidney medulla tissue vs. Control mouse 3 kidney medulla tissue

status: Public on Oct 07 2007

city: Tucson

first: Kevin

last: Greer

state: AZ

department: Biomedical Engineering

institute: University of Arizona

scan_protocol: Fluorescent array images were collected for both Alexa Fluor 546 and Alexa Fluor 647 with an arrayWoRxe biochip reader and software.

data_processing: After local background subtraction, data was linlog (base 2) transformed and LOWESS normalized. The VALUE column is the ration of Channel 1/Channel 2.

label: Alexa Fluor 546

source_name: dDAVP mouse 2 kidney medulla tissue

treatment_protocol:
dDAVP (des-amino-D-arginine vasopressin) infusion at 10-10 for 7 days
Saline infusion for 7 days

tag: Kidney medulla tissue

label: Alexa Fluor 647

source_name: Control mouse 3 kidney medulla tissue


title: Cardiomyocyte stretch 2

description: stretch condition 2

tag: induced by biomechanical stretch


title: whole_larvae_rep2


title: Wild Pec(-)

source_name: PEC

value: peritoneal macrophages


title: ACET_20A_555_swap

source_name: Silicibacter pomeroyi DSS-3, ACET treatment, 20 min.


title: dDAVP mouse 1 kidney medulla tissue vs. AVP mouse 2 kidney medulla tissue

source_name: dDAVP mouse 1 kidney medulla tissue

treatment_protocol:
dDAVP (des-amino-D-arginine vasopressin) infusion at 10-10 for 7 days
AVP (arginine vasopressin) infusion at 10-8 for 7 days

source_name: AVP mouse 2 kidney medulla tissue


title: Cardiomyocyte phenylephrine 1

description: phenylephrine condition 1


title: AVP mouse 1 kidney medulla tissue vs. dDAVP mouse 1 kidney medulla tissue

source_name: AVP mouse 1 kidney medulla tissue

treatment_protocol:
AVP (arginine vasopressin) infusion at 10-8 for 7 days
dDAVP (des-amino-D-arginine vasopressin) infusion at 10-10 for 7 days


title: larval_gut_hindgut_rep1

description: Sample represents pooled material from 1,000 dissected individual guts

source_name: Hindgut of 4th. instar An. gambiae larvae


title: whole_larvae_rep1


title: Control mouse 1 kidney medulla tissue vs. AVP mouse 1 kidney medulla tissue

source_name: Control mouse 1 kidney medulla tissue

treatment_protocol:
Saline infusion for 7 days
AVP (arginine vasopressin) infusion at 10-8 for 7 days


title: larval_gut_anterior_midgut_rep3

source_name: Anterior midgut of 4th. instar An. gambiae larvae


title: larval_gut_posterior_midgut_rep3

source_name: Posterior midgut of 4th. instar An. gambiae larvae


title: larval_gut_hindgut_rep2


title: larval_gut_posterior_midgut_rep1


title: larval_gut_hindgut_rep3


title: AVP mouse 2 kidney medulla tissue vs. Control mouse 2 kidney medulla tissue

treatment_protocol:
AVP (arginine vasopressin) infusion at 10-8 for 7 days
Saline infusion for 7 days

source_name: Control mouse 2 kidney medulla tissue


title: larval_gut_anterior_midgut_rep1


title: AVP mouse 3 kidney medulla tissue vs. Control mouse 1 kidney medulla tissue

source_name: AVP mouse 3 kidney medulla tissue


title: larval_gut_anterior_midgut_rep2


title: dDAVP mouse 3 kidney medulla tissue vs. AVP mouse 3 kidney medulla tissue

source_name: dDAVP mouse 3 kidney medulla tissue


title: Control mouse 2 kidney medulla tissue vs. dDAVP mouse 2 kidney medulla tissue

treatment_protocol: Saline infusion for 7 days

treatment_protocol: dDAVP (des-amino-D-arginine vasopressin) infusion at 10-10 for 7 days


title: larval_gut_gastric_caeca_rep2

source_name: Gastric caeca of 4th. instar An. gambiae larvae


title: Control mouse 3 kidney medulla tissue vs. dDAVP mouse 3 kidney medulla tissue

treatment_protocol:
Saline infusion for 7 days
dDAVP (des-amino-D-arginine vasopressin) infusion at 10-10 for 7 days


title: larval_gut_posterior_midgut_rep2


title: larval_gut_gastric_caeca_rep1


title: Breast cancer cell line UACC893

status: Public on Sep 01 2007

city: Lund

first: Johan

last: Staaf

address: Medicon Village

country: Sweden

institute: SCIBLU - Swegene Centre for Integrative Biology at Lund University

description: UACC893 breast cancer cell line.

hyb_protocol: Hybridizations were performed in 4 x SSC, 0.1% SDS, 0,5 NOTE human 0,8-1,0 µg/µl Cot-1 DNA at 42degC overnight. Arrays were washed three times 4xSSC RT, twice 2xSSC / 0.1% SDS at 42degC, one time 0.2xSSC, and  one time 0.1xSSC RT.

scan_protocol: Arrays were scanned in Axon 4000A scanner (Axon Instruments)

data_processing: Image analysis was performed using Genepix v4.0. Result files were uploaded into BASE where they were filtered and normalized.

label_protocol: Direct labeled by random priming (Promega, Pronto)

growth_protocol: Cells were grown according to ATCC recommendations

extract_protocol: Trizol (Invitrogen) followed by Qiaex (Qiagen). RNA integrity was checked on a Bioanalyzer (Agilent).

tag: cancer cell line

source_name: Human Reference RNA (Stratagene)

tag: Reference RNA


title: Cell line MCF10A derived from normal breast epithelium from a women with fibrocystic disease

description: MCF10A breast cancer cell line.


title: Breast cancer cell line CAMA1

description: CAMA1 breast cancer cell line.


title: fnr flhDC

status: Public on Sep 30 2007

type: genomic

city: Birmingham

first: Chrystala

last: Constantinidou

department: School of Biosciences

institute: University of Birmingham

description: We used the CGH method to genetically characterize this regulatory gene deletion mutant we had made in MG1655 using the lamda-Red method of Datsenko and Wanner.  The mutant had been verified by PCR amplification using primers designed to anneal to DNA sequences on either side of the gene that had been replaced by a chloramphenicol antibiotic resistance cassette.  With this method we were able to further characterize the fnr deletion mutant and detect the secondary gross deletion in the flagellar regulon present in some knockout strains.

hyb_protocol: Prior to hybridisation the sildes were treated with the background reducing Pronto! Pre-Soak system (Corning Life Sciences). The slides were incubated for 20 min in pre-warmed Universal Pre-Soak Solution at 42oC and washed twice in 0.1x SSC, 0.1% SDS fro 30s at room temperature.  Slides were immediately transferred into pre-warmed pre-hybridiasation solution )5x SSC, 0.1% SDS and 0.1% bovine serum albumin) and incubated for 2-4 hours at 42oC.  The microarray slides were finally washed at room temperature once in 0.1x SSC, 0.1% SDS fro 1min and twice in 0.1x SSC fro 30s, briefly dipped inwater and then ethanol, and dried by centrifugation for 5 min at 800x g. For each experiment equal quantities (80pmole) of each Cy5- and Cy3- labelled DNA were added to a final volume of 80ul of hybridisation solution containing 25% formamide, 10mg/ml of bovine serum albumin (fraction V), 5x SSC, 0.1% SDS, 8ug of polyA and 1x Denhardts solution.The slides were washed in Advalytix Hybridisation and Wash Stations according to manufacturer’s instructions.

scan_protocol: Slide was scanned at 532 and 600 nm by using a GenePix 4000A scanner (Axon Instruments, Union City, CA)

data_processing: This is the raw data.

molecule: genomic DNA

source_name: fnr 31

label_protocol: 2-3 mirograms of total DNA was labeled in a 50 microlitre reaction with either Fluorolink™ Cy3 or Cy5 d-CTP (GE-Amersham, Little Chalfont, UK) in a reaction containing 60ng/ul random hexamers (Invitrogen, Paisley, UK) 0.1mM dA/dG/dT-NTPs, 0.04mM dCTP (Bioline, London, UK), 50 units of Klenow exo- fragment of DNA polymerase I, and 10x EcoPol buffer (New England Biolabs, Hitchin, UK).  Total DNA was mixed with random hexamers and sterile filtered HPLC-grade water (VWR, Lutterworth, UK) to a volume of 41.5ul, and heated to 95oC for 5 minutes, before rapidly cooling on ice and briefly centrifuging at 13,000g in a microcentrifuge. The remaining components were added, and the reaction incubated for 90 minutes in the dark at 37oC. Cy-dye labeled total DNA was purified using the Qiagen QIAquick PCR purification kit according to the manufacturer’s protocol, and eluted in sterile HPLC grade water, before quantifying the DNA concentration and Cy-dye incorporation using a Nanodrop microspectrophotometer.

growth_protocol: Twenty milliliter cultures of MG1655 wild type and mutant strains were grown in LB broth with chloramphenicol selection at 37oC to an OD600 of 1.7-1.8.

extract_protocol: Ten milliliters of the cells were harvested by centrifugation at 6000g for 10 minutes. Total DNA was prepared using the Qiagen Genomic DNA buffer set (Qiagen, Crawley, UK), and Genomic Tip 500/G columns according to the manufacturer’s protocols, except that the total DNA precipitated by isopropanol was washed three times with 70% (V/V)  of cold ethanol to remove any residual salt, before drying and resuspension in EB buffer. Total DNA was sheared by repeated passage through a 19G sterile needle, and quantified using a Nanodrop™ ND1000 microspectrophotometer (Nanodrop Technologies Inc. USA).

value: MG1655 fnr- flhDC- Chloramphenicol resistant

tag: OD

tag: Media

value: LB

source_name: wt


title: Breast cancer cell line MDA453

description: MDA453 breast cancer cell line.


title: Breast cancer cell line MDA361

description: MDA361 breast cancer cell line.


title: fnr

description: We used the CGH method to genetically characterize this regulatory gene deletion mutant we had made in MG1655 using the lamda-Red method of Datsenko and Wanner.  The mutant had been verified by PCR amplification using primers designed to anneal to DNA sequences on either side of the gene that had been replaced by a chloramphenicol antibiotic resistance cassette.  With this method we were able to further characterize the fnr deletion mutant and confrim that it did not have the secondary gross deletion in the flagellar regulon present in some knockout strains.

source_name: fnr 315

value: MG1655 fnr Chloramphenicol resistant


title: crp flhDC

description: We used the CGH method to genetically characterize this regulatory gene deletion mutant we had made in MG1655 using the lamda-Red method of Datsenko and Wanner.  The mutant had been verified by PCR amplification using primers designed to anneal to DNA sequences on either side of the gene that had been replaced by a chloramphenicol antibiotic resistance cassette.  With this method we were able to further characterize the crp flhDC deletion mutant and detect the secondary gross deletion in the flagellar regulon present in some knockout strains.

growth_protocol: Twenty milliliter cultures of MG1655 crp flhDC strain was grown in LB broth with chloramphenicol selection at 37oC to an OD600 of 1.7-1.8.

value: MG1655 crp- flhDC- Chloramphenicol resistant

growth_protocol: Twenty milliliter cultures of MG1655 wild type was grown in LB broth with chloramphenicol selection at 37oC to an OD600 of 1.7-1.8.


title: Breast cancer cell line SKBR3

description: SKBR3 breast cancer cell line.


title: Breast cancer cell line ZR7530

description: ZR7530 breast cancer cell line.


title: crp

description: We used the CGH method to genetically characterize this regulatory gene deletion mutant we had made in MG1655 using the lamda-Red method of Datsenko and Wanner.  The mutant had been verified by PCR amplification using primers designed to anneal to DNA sequences on either side of the gene that had been replaced by a chloramphenicol antibiotic resistance cassette.  With this method we were able to further characterize the crp deletion mutant and determine that it lacked the secondary gross deletion in the flagellar regulon present in some knockout strains.

growth_protocol: Twenty milliliter cultures of MG1655 crp strain was grown in LB broth with chloramphenicol selection at 37oC to an OD600 of 1.7-1.8.

value: MG1655 crp- Chloramphenicol resistant


title: creB flhDC

description: We used the CGH method to genetically characterize this regulatory gene deletion mutant we had made in MG1655 using the lamda-Red method of Datsenko and Wanner.  The mutant had been verified by PCR amplification using primers designed to anneal to DNA sequences on either side of the gene that had been replaced by a chloramphenicol antibiotic resistance cassette.  With this method we were able to further characterize the creB deletion mutant and detect the secondary gross deletion in the flagellar regulon present in some knockout strains.

source_name: creB flhDC

growth_protocol: Twenty milliliter cultures of MG1655 creB flhDC strain was grown in LB broth with chloramphenicol selection at 37oC to an OD600 of 1.7-1.8.

value: MG1655 creB- flhDC- Chloramphenicol resistant

source_name: wild type


title: Cisplatin-treated wildtype MEFs-c

city: Chicago

first: Kay

last: Macleod

state: IL

address: 929 E 57th Str, CIS W338

department: Ben May Institute for Cancer Research

institute: The University of Chicago

description: Gene expression data from wildtype MEFs with cisplatin treatment (16microM, 24hrs).

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

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

growth_protocol: MEFs were cultured in DMEM (high glucose, Invitrogen) with 10% FCS (Invitrogen) and passaged upon confluency at the ratio 1:3.  Cells were kept in culture before reaching passage 5.

treatment_protocol: Cells were treated with 16 microM cisplatin for 24 hrs prior to harvest. Upon harvest, cells were washed with PBS and then scraped from plates in the Trizol solution (GibcoBRL).

tag: Genotype

value: wildtype MEFs

tag: Primary mouse embryonic fibroblasts (MEFs) generated from embryos at E13.5


title: Cisplatin-treated wildtype MEFs-b

source_name: wildtype MEFs with cisplatin treatment (16microM, 24hrs)


title: Breast cancer cell line BT474

description: BT474 breast cancer cell line.


title: Cisplatin-treated Rb-/- MEFs-b

description: Gene expression data from Rb null MEFs with cisplatin treatment (16microM, 24hrs).

value: Rb null MEFs


title: larval_gut_gastric_caeca_rep3


title: untreated WT MEFs-1

description: Gene expression data from wildtype MEFs without cisplatin treatment.

treatment_protocol: Upon harvest, cells without cisplatin treatment (controls) were washed with PBS and then scraped from plates in the Trizol solution (GibcoBRL).


title: Cisplatin-treated Rb-/- MEFs-a

source_name: Rb null MEFs with cisplatin treatment (16microM, 24hrs)


title: Breast cancer cell line UACC812

description: UACC812 breast cancer cell line.


title: untreated WT MEFs-3

source_name: wildtype MEFs without cisplatin treatment


title: Cisplatin-treated wildtype MEFs-a


title: untreated WT MEFs-2


title: untreated Rb-/- MEFs-2

description: Gene expression data from Rb null MEFs without cisplatin treatment.


title: untreated Rb-/- MEFs-1

source_name: Rb null MEFs without cisplatin treatment


title: untreated Rb-/- MEFs-3


title: Rep2_CON_Cy3

status: Public on Aug 31 2007

city: Corvallis

first: Susan

middle: C.

last: Tilton

state: OR

address: 1007 ALS Bldg

department: AG Envr & Molec Toxicology

institute: Oregon State University

description:
0.15% DMSO treatment - rep2
Hybridized with reference sample.
Packard ScanArray Model 4000
QuantArray
Genisphere labeling and hybridization kit.
Invitrogen TRIzol extraction followed by cleanup with Qiagen RNeasy according to vendor protocol.

hyb_protocol: Hybridizations were performed with the Genisphere Array 350 kit and instructions (Hatfield, PA) using standard reference design with dye-swapping. Briefly, 7 ug total RNA were reverse-transcribed with Superscript II (Invitrogen) using the Genisphere oligo d(T) primer containing a capture sequence for the Cy3 or Cy5 labelling reagents. Each reaction was spiked with a range of concentrations (0.0049 – 2.5 ng/ul) of the ten SpotReport Alien Oligo controls (Stratagene). Each cDNA sample containing the capture sequence for the Cy3 or Cy5 label was combined with equal amounts of reference cDNA (pooled from sham-initiated controls) containing the capture sequence for the opposite label. cDNA from two of the three biological replicates were dye-swapped and hybridized to two slides as technical replicates. cDNA from the reference sample was also hybridized to dye-swapped slides (against itself) following the same protocol as experimental samples for use as a negative control. Prior to hybridization, microarrays were processed post-printing by washing twice in 0.1% SDS for 5 min, 2X SSC, 0.1% SDS at 47C for 20 min, 0.1X SSC for 5 min, water for 3 min, then dried by centrifugation. The cDNAs (25 ul) were hybridized to arrays in formamide buffer [50% formamide, 8X SSC, 1% SDS, 4X Denhardt’s solution] for 16 h at 47C with 22x25 mm Lifterslips (Erie Scientific, Portsmouth, NH). Arrays were then washed once in 2X SSC, 0.1% SDS at 47C for 10 min, twice in 2X SSC, 0.1% SDS for 5 min, twice in 1X SSC for 5 min, twice in 0.1X SSC for 5 min and dried by centrifugation. Shaded from light, the Cy3 and Cy5 fluorescent molecules (3DNA capture reagent, Genisphere) were hybridized in formamide buffer for 3 h at 49C to the corresponding capture sequences on cDNAs bound to the arrays. Arrays were washed in the dark with SSC containing 0.1 M DTT and dried as described earlier. Slide# Omy005.P94.13171132

scan_protocol: Scanned images (5 um) were acquired with ScanArray Express (PerkinElmer, Boston, MA) at an excitation of 543 nm for Cy3 and 633 nm for Cy5 and at 90% power. The photomultiplier tube (PMT) settings for each fluor were set based on intensity of spiked internal alien controls to normalize among all slides in the experiment. Image files were quantified in QuantArray (PerkinElmer) and raw median signal and background values were exported to BioArray Software Environment (BASE; Saal et al. 2002) for analysis.

data_processing: Raw mean signal and background values are included. For experiment, data were background subtracted and normalized by LOWESS. Stringent criteria were used to filter for genes that were regulated at least 1.8-fold consistently in all features from biological replicates (n=3 per treatment) and had a p-value <0.05 by Welch’s t-test.

organism: Oncorhynchus mykiss

source_name: Control Liver

label_protocol: Labeled using Genisphere Array 350 kit adapted from manufacturer protocol.

treatment_protocol: Juvenile trout, 12-18 months old, were maintained in separate 375-L tanks (n=3 tanks) for each treatment with 5 fish per tank.  Animals were fed a maintenance ration (2.8% w/w) of experimental diets containing 0.15%  DMSO for 14 days.  On day 15, fish were euthanized by deep anesthesia with 250 ppm tricaine methanesulfonate.  Approximately 100 mg liver tissue from individual fish was minced, stored in TRIzol Reagent (Invitrogen, Carlsbad, CA) and quick frozen in liquid nitrogen for gene expression analysis.

value: Mt. Shasta

tag: Age

value: 12-18 months

tag: Tissue

value: liver

tag: Treatment

source_name: Reference


title: Rep3_CON_Cy5

description:
0.15% DMSO treatment - rep3
Hybridized with reference sample.
Packard ScanArray Model 4000
QuantArray
Genisphere labeling and hybridization kit.
Invitrogen TRIzol extraction followed by cleanup with Qiagen RNeasy according to vendor protocol.

hyb_protocol: Hybridizations were performed with the Genisphere Array 350 kit and instructions (Hatfield, PA) using standard reference design with dye-swapping. Briefly, 7 ug total RNA were reverse-transcribed with Superscript II (Invitrogen) using the Genisphere oligo d(T) primer containing a capture sequence for the Cy3 or Cy5 labelling reagents. Each reaction was spiked with a range of concentrations (0.0049 – 2.5 ng/ul) of the ten SpotReport Alien Oligo controls (Stratagene). Each cDNA sample containing the capture sequence for the Cy3 or Cy5 label was combined with equal amounts of reference cDNA (pooled from sham-initiated controls) containing the capture sequence for the opposite label. cDNA from two of the three biological replicates were dye-swapped and hybridized to two slides as technical replicates. cDNA from the reference sample was also hybridized to dye-swapped slides (against itself) following the same protocol as experimental samples for use as a negative control. Prior to hybridization, microarrays were processed post-printing by washing twice in 0.1% SDS for 5 min, 2X SSC, 0.1% SDS at 47C for 20 min, 0.1X SSC for 5 min, water for 3 min, then dried by centrifugation. The cDNAs (25 ul) were hybridized to arrays in formamide buffer [50% formamide, 8X SSC, 1% SDS, 4X Denhardt’s solution] for 16 h at 47C with 22x25 mm Lifterslips (Erie Scientific, Portsmouth, NH). Arrays were then washed once in 2X SSC, 0.1% SDS at 47C for 10 min, twice in 2X SSC, 0.1% SDS for 5 min, twice in 1X SSC for 5 min, twice in 0.1X SSC for 5 min and dried by centrifugation. Shaded from light, the Cy3 and Cy5 fluorescent molecules (3DNA capture reagent, Genisphere) were hybridized in formamide buffer for 3 h at 49C to the corresponding capture sequences on cDNAs bound to the arrays. Arrays were washed in the dark with SSC containing 0.1 M DTT and dried as described earlier. Slide# Omy005.P94.13171137

treatment_protocol: Juvenile trout, 12-18 months old, were maintained in separate 375-L tanks (n=3 tanks) for each treatment with 5 fish per tank.  Animals were fed a maintenance ration (2.8% w/w) of experimental diets containing 0.15% DMSO for 14 days.  On day 15, fish were euthanized by deep anesthesia with 250 ppm tricaine methanesulfonate.  Approximately 100 mg liver tissue from individual fish was minced, stored in TRIzol Reagent (Invitrogen, Carlsbad, CA) and quick frozen in liquid nitrogen for gene expression analysis.


title: Rep1_CON_Cy5

description:
0.15% DMSO treatment - rep1
Hybridized with reference sample.
Packard ScanArray Model 4000
QuantArray
Genisphere labeling and hybridization kit.
Invitrogen TRIzol extraction followed by cleanup with Qiagen RNeasy according to vendor protocol.

hyb_protocol: Hybridizations were performed with the Genisphere Array 350 kit and instructions (Hatfield, PA) using standard reference design with dye-swapping. Briefly, 7 ug total RNA were reverse-transcribed with Superscript II (Invitrogen) using the Genisphere oligo d(T) primer containing a capture sequence for the Cy3 or Cy5 labelling reagents. Each reaction was spiked with a range of concentrations (0.0049 – 2.5 ng/ul) of the ten SpotReport Alien Oligo controls (Stratagene). Each cDNA sample containing the capture sequence for the Cy3 or Cy5 label was combined with equal amounts of reference cDNA (pooled from sham-initiated controls) containing the capture sequence for the opposite label. cDNA from two of the three biological replicates were dye-swapped and hybridized to two slides as technical replicates. cDNA from the reference sample was also hybridized to dye-swapped slides (against itself) following the same protocol as experimental samples for use as a negative control. Prior to hybridization, microarrays were processed post-printing by washing twice in 0.1% SDS for 5 min, 2X SSC, 0.1% SDS at 47C for 20 min, 0.1X SSC for 5 min, water for 3 min, then dried by centrifugation. The cDNAs (25 ul) were hybridized to arrays in formamide buffer [50% formamide, 8X SSC, 1% SDS, 4X Denhardt’s solution] for 16 h at 47C with 22x25 mm Lifterslips (Erie Scientific, Portsmouth, NH). Arrays were then washed once in 2X SSC, 0.1% SDS at 47C for 10 min, twice in 2X SSC, 0.1% SDS for 5 min, twice in 1X SSC for 5 min, twice in 0.1X SSC for 5 min and dried by centrifugation. Shaded from light, the Cy3 and Cy5 fluorescent molecules (3DNA capture reagent, Genisphere) were hybridized in formamide buffer for 3 h at 49C to the corresponding capture sequences on cDNAs bound to the arrays. Arrays were washed in the dark with SSC containing 0.1 M DTT and dried as described earlier. Slide# Omy005.P94.13171504


title: Rep1_CON_Cy3

hyb_protocol: Hybridizations were performed with the Genisphere Array 350 kit and instructions (Hatfield, PA) using standard reference design with dye-swapping. Briefly, 7 ug total RNA were reverse-transcribed with Superscript II (Invitrogen) using the Genisphere oligo d(T) primer containing a capture sequence for the Cy3 or Cy5 labelling reagents. Each reaction was spiked with a range of concentrations (0.0049 – 2.5 ng/ul) of the ten SpotReport Alien Oligo controls (Stratagene). Each cDNA sample containing the capture sequence for the Cy3 or Cy5 label was combined with equal amounts of reference cDNA (pooled from sham-initiated controls) containing the capture sequence for the opposite label. cDNA from two of the three biological replicates were dye-swapped and hybridized to two slides as technical replicates. cDNA from the reference sample was also hybridized to dye-swapped slides (against itself) following the same protocol as experimental samples for use as a negative control. Prior to hybridization, microarrays were processed post-printing by washing twice in 0.1% SDS for 5 min, 2X SSC, 0.1% SDS at 47C for 20 min, 0.1X SSC for 5 min, water for 3 min, then dried by centrifugation. The cDNAs (25 ul) were hybridized to arrays in formamide buffer [50% formamide, 8X SSC, 1% SDS, 4X Denhardt’s solution] for 16 h at 47C with 22x25 mm Lifterslips (Erie Scientific, Portsmouth, NH). Arrays were then washed once in 2X SSC, 0.1% SDS at 47C for 10 min, twice in 2X SSC, 0.1% SDS for 5 min, twice in 1X SSC for 5 min, twice in 0.1X SSC for 5 min and dried by centrifugation. Shaded from light, the Cy3 and Cy5 fluorescent molecules (3DNA capture reagent, Genisphere) were hybridized in formamide buffer for 3 h at 49C to the corresponding capture sequences on cDNAs bound to the arrays. Arrays were washed in the dark with SSC containing 0.1 M DTT and dried as described earlier. Slide# Omy005.P94.13171503

extract_protocol: Invitrogen TRIzol extraction followed by cleanup with Qiagen RNeasy according to vendor protocol.


title: Rep3_DHEA_Cy5

description:
750 ppm DHEA treatment - rep3
Hybridized with reference sample.
Packard ScanArray Model 4000
QuantArray
Genisphere labeling and hybridization kit.
Invitrogen TRIzol extraction followed by cleanup with Qiagen RNeasy according to vendor protocol.

hyb_protocol: Hybridizations were performed with the Genisphere Array 350 kit and instructions (Hatfield, PA) using standard reference design with dye-swapping. Briefly, 7 ug total RNA were reverse-transcribed with Superscript II (Invitrogen) using the Genisphere oligo d(T) primer containing a capture sequence for the Cy3 or Cy5 labelling reagents. Each reaction was spiked with a range of concentrations (0.0049 – 2.5 ng/ul) of the ten SpotReport Alien Oligo controls (Stratagene). Each cDNA sample containing the capture sequence for the Cy3 or Cy5 label was combined with equal amounts of reference cDNA (pooled from sham-initiated controls) containing the capture sequence for the opposite label. cDNA from two of the three biological replicates were dye-swapped and hybridized to two slides as technical replicates. cDNA from the reference sample was also hybridized to dye-swapped slides (against itself) following the same protocol as experimental samples for use as a negative control. Prior to hybridization, microarrays were processed post-printing by washing twice in 0.1% SDS for 5 min, 2X SSC, 0.1% SDS at 47C for 20 min, 0.1X SSC for 5 min, water for 3 min, then dried by centrifugation. The cDNAs (25 ul) were hybridized to arrays in formamide buffer [50% formamide, 8X SSC, 1% SDS, 4X Denhardt’s solution] for 16 h at 47C with 22x25 mm Lifterslips (Erie Scientific, Portsmouth, NH). Arrays were then washed once in 2X SSC, 0.1% SDS at 47C for 10 min, twice in 2X SSC, 0.1% SDS for 5 min, twice in 1X SSC for 5 min, twice in 0.1X SSC for 5 min and dried by centrifugation. Shaded from light, the Cy3 and Cy5 fluorescent molecules (3DNA capture reagent, Genisphere) were hybridized in formamide buffer for 3 h at 49C to the corresponding capture sequences on cDNAs bound to the arrays. Arrays were washed in the dark with SSC containing 0.1 M DTT and dried as described earlier. Slide# Omy005.P94.13171141

source_name: 750 ppm DHEA Liver

treatment_protocol: Juvenile trout, 12-18 months old, were maintained in separate 375-L tanks (n=3 tanks) for each treatment with 5 fish per tank.  Animals were fed a maintenance ration (2.8% w/w) of experimental diets containing 750 ppm dehydroepiandrosterone for 14 days.  On day 15, fish were euthanized by deep anesthesia with 250 ppm tricaine methanesulfonate.  Approximately 100 mg liver tissue from individual fish was minced, stored in TRIzol Reagent (Invitrogen, Carlsbad, CA) and quick frozen in liquid nitrogen for gene expression analysis.

value: 750 ppm DHEA


title: Rep1_DHEA_Cy5

description:
750 ppm DHEA treatment - rep1
Hybridized with reference sample.
Packard ScanArray Model 4000
QuantArray
Genisphere labeling and hybridization kit.
Invitrogen TRIzol extraction followed by cleanup with Qiagen RNeasy according to vendor protocol.

hyb_protocol: Hybridizations were performed with the Genisphere Array 350 kit and instructions (Hatfield, PA) using standard reference design with dye-swapping. Briefly, 7 ug total RNA were reverse-transcribed with Superscript II (Invitrogen) using the Genisphere oligo d(T) primer containing a capture sequence for the Cy3 or Cy5 labelling reagents. Each reaction was spiked with a range of concentrations (0.0049 – 2.5 ng/ul) of the ten SpotReport Alien Oligo controls (Stratagene). Each cDNA sample containing the capture sequence for the Cy3 or Cy5 label was combined with equal amounts of reference cDNA (pooled from sham-initiated controls) containing the capture sequence for the opposite label. cDNA from two of the three biological replicates were dye-swapped and hybridized to two slides as technical replicates. cDNA from the reference sample was also hybridized to dye-swapped slides (against itself) following the same protocol as experimental samples for use as a negative control. Prior to hybridization, microarrays were processed post-printing by washing twice in 0.1% SDS for 5 min, 2X SSC, 0.1% SDS at 47C for 20 min, 0.1X SSC for 5 min, water for 3 min, then dried by centrifugation. The cDNAs (25 ul) were hybridized to arrays in formamide buffer [50% formamide, 8X SSC, 1% SDS, 4X Denhardt’s solution] for 16 h at 47C with 22x25 mm Lifterslips (Erie Scientific, Portsmouth, NH). Arrays were then washed once in 2X SSC, 0.1% SDS at 47C for 10 min, twice in 2X SSC, 0.1% SDS for 5 min, twice in 1X SSC for 5 min, twice in 0.1X SSC for 5 min and dried by centrifugation. Shaded from light, the Cy3 and Cy5 fluorescent molecules (3DNA capture reagent, Genisphere) were hybridized in formamide buffer for 3 h at 49C to the corresponding capture sequences on cDNAs bound to the arrays. Arrays were washed in the dark with SSC containing 0.1 M DTT and dried as described earlier. Slide# Omy005.P94.13171500


title: Rep2_CON_Cy5

hyb_protocol: Hybridizations were performed with the Genisphere Array 350 kit and instructions (Hatfield, PA) using standard reference design with dye-swapping. Briefly, 7 ug total RNA were reverse-transcribed with Superscript II (Invitrogen) using the Genisphere oligo d(T) primer containing a capture sequence for the Cy3 or Cy5 labelling reagents. Each reaction was spiked with a range of concentrations (0.0049 – 2.5 ng/ul) of the ten SpotReport Alien Oligo controls (Stratagene). Each cDNA sample containing the capture sequence for the Cy3 or Cy5 label was combined with equal amounts of reference cDNA (pooled from sham-initiated controls) containing the capture sequence for the opposite label. cDNA from two of the three biological replicates were dye-swapped and hybridized to two slides as technical replicates. cDNA from the reference sample was also hybridized to dye-swapped slides (against itself) following the same protocol as experimental samples for use as a negative control. Prior to hybridization, microarrays were processed post-printing by washing twice in 0.1% SDS for 5 min, 2X SSC, 0.1% SDS at 47C for 20 min, 0.1X SSC for 5 min, water for 3 min, then dried by centrifugation. The cDNAs (25 ul) were hybridized to arrays in formamide buffer [50% formamide, 8X SSC, 1% SDS, 4X Denhardt’s solution] for 16 h at 47C with 22x25 mm Lifterslips (Erie Scientific, Portsmouth, NH). Arrays were then washed once in 2X SSC, 0.1% SDS at 47C for 10 min, twice in 2X SSC, 0.1% SDS for 5 min, twice in 1X SSC for 5 min, twice in 0.1X SSC for 5 min and dried by centrifugation. Shaded from light, the Cy3 and Cy5 fluorescent molecules (3DNA capture reagent, Genisphere) were hybridized in formamide buffer for 3 h at 49C to the corresponding capture sequences on cDNAs bound to the arrays. Arrays were washed in the dark with SSC containing 0.1 M DTT and dried as described earlier. Slide# Omy005.P94.13171134


title: Cisplatin-treated Rb-/- MEFs-c


title: Rep1_DHEA_Cy3

hyb_protocol: Hybridizations were performed with the Genisphere Array 350 kit and instructions (Hatfield, PA) using standard reference design with dye-swapping. Briefly, 7 ug total RNA were reverse-transcribed with Superscript II (Invitrogen) using the Genisphere oligo d(T) primer containing a capture sequence for the Cy3 or Cy5 labelling reagents. Each reaction was spiked with a range of concentrations (0.0049 – 2.5 ng/ul) of the ten SpotReport Alien Oligo controls (Stratagene). Each cDNA sample containing the capture sequence for the Cy3 or Cy5 label was combined with equal amounts of reference cDNA (pooled from sham-initiated controls) containing the capture sequence for the opposite label. cDNA from two of the three biological replicates were dye-swapped and hybridized to two slides as technical replicates. cDNA from the reference sample was also hybridized to dye-swapped slides (against itself) following the same protocol as experimental samples for use as a negative control. Prior to hybridization, microarrays were processed post-printing by washing twice in 0.1% SDS for 5 min, 2X SSC, 0.1% SDS at 47C for 20 min, 0.1X SSC for 5 min, water for 3 min, then dried by centrifugation. The cDNAs (25 ul) were hybridized to arrays in formamide buffer [50% formamide, 8X SSC, 1% SDS, 4X Denhardt’s solution] for 16 h at 47C with 22x25 mm Lifterslips (Erie Scientific, Portsmouth, NH). Arrays were then washed once in 2X SSC, 0.1% SDS at 47C for 10 min, twice in 2X SSC, 0.1% SDS for 5 min, twice in 1X SSC for 5 min, twice in 0.1X SSC for 5 min and dried by centrifugation. Shaded from light, the Cy3 and Cy5 fluorescent molecules (3DNA capture reagent, Genisphere) were hybridized in formamide buffer for 3 h at 49C to the corresponding capture sequences on cDNAs bound to the arrays. Arrays were washed in the dark with SSC containing 0.1 M DTT and dried as described earlier. Slide# Omy005.P94.13171501


title: Rep2_DHEA_Cy3

description:
750 ppm DHEA treatment - rep2
Hybridized with reference sample.
Packard ScanArray Model 4000
QuantArray
Genisphere labeling and hybridization kit.
Invitrogen TRIzol extraction followed by cleanup with Qiagen RNeasy according to vendor protocol.

hyb_protocol: Hybridizations were performed with the Genisphere Array 350 kit and instructions (Hatfield, PA) using standard reference design with dye-swapping. Briefly, 7 ug total RNA were reverse-transcribed with Superscript II (Invitrogen) using the Genisphere oligo d(T) primer containing a capture sequence for the Cy3 or Cy5 labelling reagents. Each reaction was spiked with a range of concentrations (0.0049 – 2.5 ng/ul) of the ten SpotReport Alien Oligo controls (Stratagene). Each cDNA sample containing the capture sequence for the Cy3 or Cy5 label was combined with equal amounts of reference cDNA (pooled from sham-initiated controls) containing the capture sequence for the opposite label. cDNA from two of the three biological replicates were dye-swapped and hybridized to two slides as technical replicates. cDNA from the reference sample was also hybridized to dye-swapped slides (against itself) following the same protocol as experimental samples for use as a negative control. Prior to hybridization, microarrays were processed post-printing by washing twice in 0.1% SDS for 5 min, 2X SSC, 0.1% SDS at 47C for 20 min, 0.1X SSC for 5 min, water for 3 min, then dried by centrifugation. The cDNAs (25 ul) were hybridized to arrays in formamide buffer [50% formamide, 8X SSC, 1% SDS, 4X Denhardt’s solution] for 16 h at 47C with 22x25 mm Lifterslips (Erie Scientific, Portsmouth, NH). Arrays were then washed once in 2X SSC, 0.1% SDS at 47C for 10 min, twice in 2X SSC, 0.1% SDS for 5 min, twice in 1X SSC for 5 min, twice in 0.1X SSC for 5 min and dried by centrifugation. Shaded from light, the Cy3 and Cy5 fluorescent molecules (3DNA capture reagent, Genisphere) were hybridized in formamide buffer for 3 h at 49C to the corresponding capture sequences on cDNAs bound to the arrays. Arrays were washed in the dark with SSC containing 0.1 M DTT and dried as described earlier. Slide# Omy005.P94.13171128


title: Rep1_CLOF_Cy3

description:
1800 ppm CLOF treatment - rep1
Hybridized with reference sample.
Packard ScanArray Model 4000
QuantArray
Genisphere labeling and hybridization kit.
Invitrogen TRIzol extraction followed by cleanup with Qiagen RNeasy according to vendor protocol.

hyb_protocol: Hybridizations were performed with the Genisphere Array 350 kit and instructions (Hatfield, PA) using standard reference design with dye-swapping. Briefly, 7 ug total RNA were reverse-transcribed with Superscript II (Invitrogen) using the Genisphere oligo d(T) primer containing a capture sequence for the Cy3 or Cy5 labelling reagents. Each reaction was spiked with a range of concentrations (0.0049 – 2.5 ng/ul) of the ten SpotReport Alien Oligo controls (Stratagene). Each cDNA sample containing the capture sequence for the Cy3 or Cy5 label was combined with equal amounts of reference cDNA (pooled from sham-initiated controls) containing the capture sequence for the opposite label. cDNA from two of the three biological replicates were dye-swapped and hybridized to two slides as technical replicates. cDNA from the reference sample was also hybridized to dye-swapped slides (against itself) following the same protocol as experimental samples for use as a negative control. Prior to hybridization, microarrays were processed post-printing by washing twice in 0.1% SDS for 5 min, 2X SSC, 0.1% SDS at 47C for 20 min, 0.1X SSC for 5 min, water for 3 min, then dried by centrifugation. The cDNAs (25 ul) were hybridized to arrays in formamide buffer [50% formamide, 8X SSC, 1% SDS, 4X Denhardt’s solution] for 16 h at 47C with 22x25 mm Lifterslips (Erie Scientific, Portsmouth, NH). Arrays were then washed once in 2X SSC, 0.1% SDS at 47C for 10 min, twice in 2X SSC, 0.1% SDS for 5 min, twice in 1X SSC for 5 min, twice in 0.1X SSC for 5 min and dried by centrifugation. Shaded from light, the Cy3 and Cy5 fluorescent molecules (3DNA capture reagent, Genisphere) were hybridized in formamide buffer for 3 h at 49C to the corresponding capture sequences on cDNAs bound to the arrays. Arrays were washed in the dark with SSC containing 0.1 M DTT and dried as described earlier. Slide# Omy005.P94.13166413

source_name: 1800 ppm CLOF Liver

treatment_protocol: Juvenile trout, 12-18 months old, were maintained in separate 375-L tanks (n=3 tanks) for each treatment with 5 fish per tank.  Animals were fed a maintenance ration (2.8% w/w) of experimental diets containing 1800 ppm clofibrate for 14 days.  On day 15, fish were euthanized by deep anesthesia with 250 ppm tricaine methanesulfonate.  Approximately 100 mg liver tissue from individual fish was minced, stored in TRIzol Reagent (Invitrogen, Carlsbad, CA) and quick frozen in liquid nitrogen for gene expression analysis.

value: 1800 ppm CLOF


title: Rep3_E2_Cy5

description:
5 ppm E2 treatment - rep3
Hybridized with reference sample.
Packard ScanArray Model 4000
QuantArray
Genisphere labeling and hybridization kit.
Invitrogen TRIzol extraction followed by cleanup with Qiagen RNeasy according to vendor protocol.

hyb_protocol: Hybridizations were performed with the Genisphere Array 350 kit and instructions (Hatfield, PA) using standard reference design with dye-swapping. Briefly, 7 ug total RNA were reverse-transcribed with Superscript II (Invitrogen) using the Genisphere oligo d(T) primer containing a capture sequence for the Cy3 or Cy5 labelling reagents. Each reaction was spiked with a range of concentrations (0.0049 – 2.5 ng/ul) of the ten SpotReport Alien Oligo controls (Stratagene). Each cDNA sample containing the capture sequence for the Cy3 or Cy5 label was combined with equal amounts of reference cDNA (pooled from sham-initiated controls) containing the capture sequence for the opposite label. cDNA from two of the three biological replicates were dye-swapped and hybridized to two slides as technical replicates. cDNA from the reference sample was also hybridized to dye-swapped slides (against itself) following the same protocol as experimental samples for use as a negative control. Prior to hybridization, microarrays were processed post-printing by washing twice in 0.1% SDS for 5 min, 2X SSC, 0.1% SDS at 47C for 20 min, 0.1X SSC for 5 min, water for 3 min, then dried by centrifugation. The cDNAs (25 ul) were hybridized to arrays in formamide buffer [50% formamide, 8X SSC, 1% SDS, 4X Denhardt’s solution] for 16 h at 47C with 22x25 mm Lifterslips (Erie Scientific, Portsmouth, NH). Arrays were then washed once in 2X SSC, 0.1% SDS at 47C for 10 min, twice in 2X SSC, 0.1% SDS for 5 min, twice in 1X SSC for 5 min, twice in 0.1X SSC for 5 min and dried by centrifugation. Shaded from light, the Cy3 and Cy5 fluorescent molecules (3DNA capture reagent, Genisphere) were hybridized in formamide buffer for 3 h at 49C to the corresponding capture sequences on cDNAs bound to the arrays. Arrays were washed in the dark with SSC containing 0.1 M DTT and dried as described earlier. Slide# Omy005.P94.13166626

source_name: 5 ppm E2 Liver

treatment_protocol: Juvenile trout, 12-18 months old, were maintained in separate 375-L tanks (n=3 tanks) for each treatment with 5 fish per tank.  Animals were fed a maintenance ration (2.8% w/w) of experimental diets containing 5 ppm E2 for 14 days.  On day 15, fish were euthanized by deep anesthesia with 250 ppm tricaine methanesulfonate.  Approximately 100 mg liver tissue from individual fish was minced, stored in TRIzol Reagent (Invitrogen, Carlsbad, CA) and quick frozen in liquid nitrogen for gene expression analysis.

value: 5 ppm E2


title: Rep2_CLOF_Cy3

description:
1800 ppm CLOF treatment - rep2
Hybridized with reference sample.
Packard ScanArray Model 4000
QuantArray
Genisphere labeling and hybridization kit.
Invitrogen TRIzol extraction followed by cleanup with Qiagen RNeasy according to vendor protocol.

hyb_protocol: Hybridizations were performed with the Genisphere Array 350 kit and instructions (Hatfield, PA) using standard reference design with dye-swapping. Briefly, 7 ug total RNA were reverse-transcribed with Superscript II (Invitrogen) using the Genisphere oligo d(T) primer containing a capture sequence for the Cy3 or Cy5 labelling reagents. Each reaction was spiked with a range of concentrations (0.0049 – 2.5 ng/ul) of the ten SpotReport Alien Oligo controls (Stratagene). Each cDNA sample containing the capture sequence for the Cy3 or Cy5 label was combined with equal amounts of reference cDNA (pooled from sham-initiated controls) containing the capture sequence for the opposite label. cDNA from two of the three biological replicates were dye-swapped and hybridized to two slides as technical replicates. cDNA from the reference sample was also hybridized to dye-swapped slides (against itself) following the same protocol as experimental samples for use as a negative control. Prior to hybridization, microarrays were processed post-printing by washing twice in 0.1% SDS for 5 min, 2X SSC, 0.1% SDS at 47C for 20 min, 0.1X SSC for 5 min, water for 3 min, then dried by centrifugation. The cDNAs (25 ul) were hybridized to arrays in formamide buffer [50% formamide, 8X SSC, 1% SDS, 4X Denhardt’s solution] for 16 h at 47C with 22x25 mm Lifterslips (Erie Scientific, Portsmouth, NH). Arrays were then washed once in 2X SSC, 0.1% SDS at 47C for 10 min, twice in 2X SSC, 0.1% SDS for 5 min, twice in 1X SSC for 5 min, twice in 0.1X SSC for 5 min and dried by centrifugation. Shaded from light, the Cy3 and Cy5 fluorescent molecules (3DNA capture reagent, Genisphere) were hybridized in formamide buffer for 3 h at 49C to the corresponding capture sequences on cDNAs bound to the arrays. Arrays were washed in the dark with SSC containing 0.1 M DTT and dried as described earlier. Slide# Omy005.P94.13171142


title: Rep1_CLOF_Cy5

hyb_protocol: Hybridizations were performed with the Genisphere Array 350 kit and instructions (Hatfield, PA) using standard reference design with dye-swapping. Briefly, 7 ug total RNA were reverse-transcribed with Superscript II (Invitrogen) using the Genisphere oligo d(T) primer containing a capture sequence for the Cy3 or Cy5 labelling reagents. Each reaction was spiked with a range of concentrations (0.0049 – 2.5 ng/ul) of the ten SpotReport Alien Oligo controls (Stratagene). Each cDNA sample containing the capture sequence for the Cy3 or Cy5 label was combined with equal amounts of reference cDNA (pooled from sham-initiated controls) containing the capture sequence for the opposite label. cDNA from two of the three biological replicates were dye-swapped and hybridized to two slides as technical replicates. cDNA from the reference sample was also hybridized to dye-swapped slides (against itself) following the same protocol as experimental samples for use as a negative control. Prior to hybridization, microarrays were processed post-printing by washing twice in 0.1% SDS for 5 min, 2X SSC, 0.1% SDS at 47C for 20 min, 0.1X SSC for 5 min, water for 3 min, then dried by centrifugation. The cDNAs (25 ul) were hybridized to arrays in formamide buffer [50% formamide, 8X SSC, 1% SDS, 4X Denhardt’s solution] for 16 h at 47C with 22x25 mm Lifterslips (Erie Scientific, Portsmouth, NH). Arrays were then washed once in 2X SSC, 0.1% SDS at 47C for 10 min, twice in 2X SSC, 0.1% SDS for 5 min, twice in 1X SSC for 5 min, twice in 0.1X SSC for 5 min and dried by centrifugation. Shaded from light, the Cy3 and Cy5 fluorescent molecules (3DNA capture reagent, Genisphere) were hybridized in formamide buffer for 3 h at 49C to the corresponding capture sequences on cDNAs bound to the arrays. Arrays were washed in the dark with SSC containing 0.1 M DTT and dried as described earlier. Slide# Omy005.P94.13171485


title: Rep2_DHEA_Cy5

hyb_protocol: Hybridizations were performed with the Genisphere Array 350 kit and instructions (Hatfield, PA) using standard reference design with dye-swapping. Briefly, 7 ug total RNA were reverse-transcribed with Superscript II (Invitrogen) using the Genisphere oligo d(T) primer containing a capture sequence for the Cy3 or Cy5 labelling reagents. Each reaction was spiked with a range of concentrations (0.0049 – 2.5 ng/ul) of the ten SpotReport Alien Oligo controls (Stratagene). Each cDNA sample containing the capture sequence for the Cy3 or Cy5 label was combined with equal amounts of reference cDNA (pooled from sham-initiated controls) containing the capture sequence for the opposite label. cDNA from two of the three biological replicates were dye-swapped and hybridized to two slides as technical replicates. cDNA from the reference sample was also hybridized to dye-swapped slides (against itself) following the same protocol as experimental samples for use as a negative control. Prior to hybridization, microarrays were processed post-printing by washing twice in 0.1% SDS for 5 min, 2X SSC, 0.1% SDS at 47C for 20 min, 0.1X SSC for 5 min, water for 3 min, then dried by centrifugation. The cDNAs (25 ul) were hybridized to arrays in formamide buffer [50% formamide, 8X SSC, 1% SDS, 4X Denhardt’s solution] for 16 h at 47C with 22x25 mm Lifterslips (Erie Scientific, Portsmouth, NH). Arrays were then washed once in 2X SSC, 0.1% SDS at 47C for 10 min, twice in 2X SSC, 0.1% SDS for 5 min, twice in 1X SSC for 5 min, twice in 0.1X SSC for 5 min and dried by centrifugation. Shaded from light, the Cy3 and Cy5 fluorescent molecules (3DNA capture reagent, Genisphere) were hybridized in formamide buffer for 3 h at 49C to the corresponding capture sequences on cDNAs bound to the arrays. Arrays were washed in the dark with SSC containing 0.1 M DTT and dried as described earlier. Slide# Omy005.P94.13171129


title: Rep3_CLOF_Cy5

description:
1800 ppm CLOF treatment - rep3
Hybridized with reference sample.
Packard ScanArray Model 4000
QuantArray
Genisphere labeling and hybridization kit.
Invitrogen TRIzol extraction followed by cleanup with Qiagen RNeasy according to vendor protocol.

hyb_protocol: Hybridizations were performed with the Genisphere Array 350 kit and instructions (Hatfield, PA) using standard reference design with dye-swapping. Briefly, 7 ug total RNA were reverse-transcribed with Superscript II (Invitrogen) using the Genisphere oligo d(T) primer containing a capture sequence for the Cy3 or Cy5 labelling reagents. Each reaction was spiked with a range of concentrations (0.0049 – 2.5 ng/ul) of the ten SpotReport Alien Oligo controls (Stratagene). Each cDNA sample containing the capture sequence for the Cy3 or Cy5 label was combined with equal amounts of reference cDNA (pooled from sham-initiated controls) containing the capture sequence for the opposite label. cDNA from two of the three biological replicates were dye-swapped and hybridized to two slides as technical replicates. cDNA from the reference sample was also hybridized to dye-swapped slides (against itself) following the same protocol as experimental samples for use as a negative control. Prior to hybridization, microarrays were processed post-printing by washing twice in 0.1% SDS for 5 min, 2X SSC, 0.1% SDS at 47C for 20 min, 0.1X SSC for 5 min, water for 3 min, then dried by centrifugation. The cDNAs (25 ul) were hybridized to arrays in formamide buffer [50% formamide, 8X SSC, 1% SDS, 4X Denhardt’s solution] for 16 h at 47C with 22x25 mm Lifterslips (Erie Scientific, Portsmouth, NH). Arrays were then washed once in 2X SSC, 0.1% SDS at 47C for 10 min, twice in 2X SSC, 0.1% SDS for 5 min, twice in 1X SSC for 5 min, twice in 0.1X SSC for 5 min and dried by centrifugation. Shaded from light, the Cy3 and Cy5 fluorescent molecules (3DNA capture reagent, Genisphere) were hybridized in formamide buffer for 3 h at 49C to the corresponding capture sequences on cDNAs bound to the arrays. Arrays were washed in the dark with SSC containing 0.1 M DTT and dried as described earlier. Slide# Omy005.P94.13171126


title: Rep2_CLOF_Cy5

hyb_protocol: Hybridizations were performed with the Genisphere Array 350 kit and instructions (Hatfield, PA) using standard reference design with dye-swapping. Briefly, 7 ug total RNA were reverse-transcribed with Superscript II (Invitrogen) using the Genisphere oligo d(T) primer containing a capture sequence for the Cy3 or Cy5 labelling reagents. Each reaction was spiked with a range of concentrations (0.0049 – 2.5 ng/ul) of the ten SpotReport Alien Oligo controls (Stratagene). Each cDNA sample containing the capture sequence for the Cy3 or Cy5 label was combined with equal amounts of reference cDNA (pooled from sham-initiated controls) containing the capture sequence for the opposite label. cDNA from two of the three biological replicates were dye-swapped and hybridized to two slides as technical replicates. cDNA from the reference sample was also hybridized to dye-swapped slides (against itself) following the same protocol as experimental samples for use as a negative control. Prior to hybridization, microarrays were processed post-printing by washing twice in 0.1% SDS for 5 min, 2X SSC, 0.1% SDS at 47C for 20 min, 0.1X SSC for 5 min, water for 3 min, then dried by centrifugation. The cDNAs (25 ul) were hybridized to arrays in formamide buffer [50% formamide, 8X SSC, 1% SDS, 4X Denhardt’s solution] for 16 h at 47C with 22x25 mm Lifterslips (Erie Scientific, Portsmouth, NH). Arrays were then washed once in 2X SSC, 0.1% SDS at 47C for 10 min, twice in 2X SSC, 0.1% SDS for 5 min, twice in 1X SSC for 5 min, twice in 0.1X SSC for 5 min and dried by centrifugation. Shaded from light, the Cy3 and Cy5 fluorescent molecules (3DNA capture reagent, Genisphere) were hybridized in formamide buffer for 3 h at 49C to the corresponding capture sequences on cDNAs bound to the arrays. Arrays were washed in the dark with SSC containing 0.1 M DTT and dried as described earlier. Slide# Omy005.P94.13171136


title: Rep2_Hi-PFOA_Cy5

description:
1800 ppm PFOA treatment - rep2
Hybridized with reference sample.
Packard ScanArray Model 4000
QuantArray
Genisphere labeling and hybridization kit.
Invitrogen TRIzol extraction followed by cleanup with Qiagen RNeasy according to vendor protocol.

hyb_protocol: Hybridizations were performed with the Genisphere Array 350 kit and instructions (Hatfield, PA) using standard reference design with dye-swapping. Briefly, 7 ug total RNA were reverse-transcribed with Superscript II (Invitrogen) using the Genisphere oligo d(T) primer containing a capture sequence for the Cy3 or Cy5 labelling reagents. Each reaction was spiked with a range of concentrations (0.0049 – 2.5 ng/ul) of the ten SpotReport Alien Oligo controls (Stratagene). Each cDNA sample containing the capture sequence for the Cy3 or Cy5 label was combined with equal amounts of reference cDNA (pooled from sham-initiated controls) containing the capture sequence for the opposite label. cDNA from two of the three biological replicates were dye-swapped and hybridized to two slides as technical replicates. cDNA from the reference sample was also hybridized to dye-swapped slides (against itself) following the same protocol as experimental samples for use as a negative control. Prior to hybridization, microarrays were processed post-printing by washing twice in 0.1% SDS for 5 min, 2X SSC, 0.1% SDS at 47C for 20 min, 0.1X SSC for 5 min, water for 3 min, then dried by centrifugation. The cDNAs (25 ul) were hybridized to arrays in formamide buffer [50% formamide, 8X SSC, 1% SDS, 4X Denhardt’s solution] for 16 h at 47C with 22x25 mm Lifterslips (Erie Scientific, Portsmouth, NH). Arrays were then washed once in 2X SSC, 0.1% SDS at 47C for 10 min, twice in 2X SSC, 0.1% SDS for 5 min, twice in 1X SSC for 5 min, twice in 0.1X SSC for 5 min and dried by centrifugation. Shaded from light, the Cy3 and Cy5 fluorescent molecules (3DNA capture reagent, Genisphere) were hybridized in formamide buffer for 3 h at 49C to the corresponding capture sequences on cDNAs bound to the arrays. Arrays were washed in the dark with SSC containing 0.1 M DTT and dried as described earlier. Slide# Omy005.P94.13171498

source_name: 1800 ppm PFOA Liver

treatment_protocol: Juvenile trout, 12-18 months old, were maintained in separate 375-L tanks (n=3 tanks) for each treatment with 5 fish per tank.  Animals were fed a maintenance ration (2.8% w/w) of experimental diets containing 1800 ppm PFOA for 14 days.  On day 15, fish were euthanized by deep anesthesia with 250 ppm tricaine methanesulfonate.  Approximately 100 mg liver tissue from individual fish was minced, stored in TRIzol Reagent (Invitrogen, Carlsbad, CA) and quick frozen in liquid nitrogen for gene expression analysis.

value: 1800 ppm PFOA


title: Rep2_E2_Cy3

description:
5 ppm E2 treatment - rep2
Hybridized with reference sample.
Packard ScanArray Model 4000
QuantArray
Genisphere labeling and hybridization kit.
Invitrogen TRIzol extraction followed by cleanup with Qiagen RNeasy according to vendor protocol.

hyb_protocol: Hybridizations were performed with the Genisphere Array 350 kit and instructions (Hatfield, PA) using standard reference design with dye-swapping. Briefly, 7 ug total RNA were reverse-transcribed with Superscript II (Invitrogen) using the Genisphere oligo d(T) primer containing a capture sequence for the Cy3 or Cy5 labelling reagents. Each reaction was spiked with a range of concentrations (0.0049 – 2.5 ng/ul) of the ten SpotReport Alien Oligo controls (Stratagene). Each cDNA sample containing the capture sequence for the Cy3 or Cy5 label was combined with equal amounts of reference cDNA (pooled from sham-initiated controls) containing the capture sequence for the opposite label. cDNA from two of the three biological replicates were dye-swapped and hybridized to two slides as technical replicates. cDNA from the reference sample was also hybridized to dye-swapped slides (against itself) following the same protocol as experimental samples for use as a negative control. Prior to hybridization, microarrays were processed post-printing by washing twice in 0.1% SDS for 5 min, 2X SSC, 0.1% SDS at 47C for 20 min, 0.1X SSC for 5 min, water for 3 min, then dried by centrifugation. The cDNAs (25 ul) were hybridized to arrays in formamide buffer [50% formamide, 8X SSC, 1% SDS, 4X Denhardt’s solution] for 16 h at 47C with 22x25 mm Lifterslips (Erie Scientific, Portsmouth, NH). Arrays were then washed once in 2X SSC, 0.1% SDS at 47C for 10 min, twice in 2X SSC, 0.1% SDS for 5 min, twice in 1X SSC for 5 min, twice in 0.1X SSC for 5 min and dried by centrifugation. Shaded from light, the Cy3 and Cy5 fluorescent molecules (3DNA capture reagent, Genisphere) were hybridized in formamide buffer for 3 h at 49C to the corresponding capture sequences on cDNAs bound to the arrays. Arrays were washed in the dark with SSC containing 0.1 M DTT and dried as described earlier. Slide# Omy005.P94.13171487


title: Rep1_Hi-PFOA_Cy5

description:
1800 ppm PFOA treatment - rep1
Hybridized with reference sample.
Packard ScanArray Model 4000
QuantArray
Genisphere labeling and hybridization kit.
Invitrogen TRIzol extraction followed by cleanup with Qiagen RNeasy according to vendor protocol.

hyb_protocol: Hybridizations were performed with the Genisphere Array 350 kit and instructions (Hatfield, PA) using standard reference design with dye-swapping. Briefly, 7 ug total RNA were reverse-transcribed with Superscript II (Invitrogen) using the Genisphere oligo d(T) primer containing a capture sequence for the Cy3 or Cy5 labelling reagents. Each reaction was spiked with a range of concentrations (0.0049 – 2.5 ng/ul) of the ten SpotReport Alien Oligo controls (Stratagene). Each cDNA sample containing the capture sequence for the Cy3 or Cy5 label was combined with equal amounts of reference cDNA (pooled from sham-initiated controls) containing the capture sequence for the opposite label. cDNA from two of the three biological replicates were dye-swapped and hybridized to two slides as technical replicates. cDNA from the reference sample was also hybridized to dye-swapped slides (against itself) following the same protocol as experimental samples for use as a negative control. Prior to hybridization, microarrays were processed post-printing by washing twice in 0.1% SDS for 5 min, 2X SSC, 0.1% SDS at 47C for 20 min, 0.1X SSC for 5 min, water for 3 min, then dried by centrifugation. The cDNAs (25 ul) were hybridized to arrays in formamide buffer [50% formamide, 8X SSC, 1% SDS, 4X Denhardt’s solution] for 16 h at 47C with 22x25 mm Lifterslips (Erie Scientific, Portsmouth, NH). Arrays were then washed once in 2X SSC, 0.1% SDS at 47C for 10 min, twice in 2X SSC, 0.1% SDS for 5 min, twice in 1X SSC for 5 min, twice in 0.1X SSC for 5 min and dried by centrifugation. Shaded from light, the Cy3 and Cy5 fluorescent molecules (3DNA capture reagent, Genisphere) were hybridized in formamide buffer for 3 h at 49C to the corresponding capture sequences on cDNAs bound to the arrays. Arrays were washed in the dark with SSC containing 0.1 M DTT and dried as described earlier. Slide# Omy005.P94.13171486