title: blood_breast cancer, 712

description: Collected in pax tubes.


title: blood_breast cancer, 807

status: Public on Jan 15 2010

type: RNA

city: Oslo

first: Jørgen

middle: Mømb

last: Aarøe

address: Montebello

department: Genetics

country: Norway

institute: The Norwegian Radium Hospital

description: Collected in pax tubes.

hyb_protocol: 10ug labeled and fragmented cRNA was hybridized onto single channel Applied Biosystems Human Genome Survey microarrays v2.0 for 16 hours at 55ºC according to the manufacturer’s protocol.

scan_protocol: After hybridization, slides were manually washed and prepared according to manufacturers’ recommendations before image capturing using the AB1700 reader. Identification and quantification of gene expression signals, signal-to-noise ratios and flagging of failed spots were conducted using the Applied Biosystems Expression System software. Raw data files were exported for further analysis.

data_processing:
Data analysis was performed using R  and tools from the Bioconductor project, adapted to our needs. Data were log2 transformed while individual measurements with signal-to-noise < 3 or flag values >8191 were set as missing. Probes with more than 5% missing values over all 156 arrays were excluded. Preprocessing left 156 samples and 11217 probes for further analyses. Data were standardized (i.e. centered and scaled) and missing values were imputed with k-nearest neighbors imputation using k = 10. Data were Batch-adjusted using the ANOVA-based batch adjustment function available in the pamr package for R. The 26 technical control samples (ControlReplicates) were then excluded. The signal intensities in biological replicates (blood_control_mens.cycle_reps) were averaged for each probe. Thus, 127 arrays, one from each individual remained for analysis. Finally, within-array normalization was conducted by global mean subtraction.

Raw data is available in the 'GSE16443_raw_data.txt' file, which is linked to the GSE16443 record. The Sample 'characteristics: assay name' value is used as the Sample identifier in the raw data file.  The 26 technical control samples in the raw data file were removed after preprocessing and hence are not represented as Sample records (which report only the processed data).

label: Digoxigenin

molecule: total RNA

organism: Homo sapiens

source_name: peripheral blood

label_protocol: For each sample, Digoxigenin-UTP labeled cRNA was generated from 500 ng total RNA according to the NanoAmp RT-IVT Labeling Kit Protocol.

extract_protocol: From each woman, 2.5 ml blood was collected in PAXgene tubes and left overnight at room temperature before storing at -80ºC until use. PAXgene tubes were thawed over night in batches of 12 tubes and total RNA was extracted according to the PAXgeneTM Blood RNA Kit Handbook Protocol and Appendix A (April 2001). Total RNA was stored at -80ºC until use. RNA quality and quantity measures were conducted using the 2100 Bioanalyzer (Agilent Technologies, California, USA) and the NanoDrop ND-1000 spectrophotometer (Thermo Scientific, Delaware, USA), respectively.

treatment_protocol: 121 blood samples were collected in PAXgeneTM tubes (PreAnalytiX, Hombrechtikon, Switzerland) from women called in for a second look after a first suspect mammogram. The PAXgene Blood RNA System is a technology that consolidates and integrates the key steps of whole blood collection, nucleic acid stabilization, and RNA purification. The samples were collected prior to a clinical examination that includes diagnostic mammography and biopsy in the case of a positive mammographic finding. In addition, nine control samples were collected from: three pregnant women, one breast-feeding woman and five samples from two healthy controls at different timepoints in their menstrual cycle.

tag: assay name

tag: status

value: Breast cancer

tag: subject age

tag: passed menopause

tag: gender

value: female

tag: estrogen receptor status

tag: progesterone receptor status

tag: additional clinical information

value: DCIS

tag: tumor grade


title: blood_breast cancer, 825

description: Collected in pax tubes.

value: YES


title: blood_breast cancer, 879

description: Collected in pax tubes.

value: NEG

value: ILC


title: blood_breast cancer, 711

description: Collected in pax tubes.

value: POS

value: IDC


title: blood_breast cancer, 730

description: Collected in pax tubes.


title: blood_breast cancer, 706

description: Collected in pax tubes.

value: NO


title: blood_breast cancer, 690

description: Collected in pax tubes.


title: blood_breast cancer, 689

description: Collected in pax tubes.


title: blood_breast cancer, 651

description: Collected in pax tubes.

value: IDC/DCIS


title: blood_breast cancer, 653

description: Collected in pax tubes.


title: blood_breast cancer, 634

description: Collected in pax tubes.


title: blood_breast cancer, 650

description: Collected in pax tubes.


title: blood_breast cancer, 616

description: Collected in pax tubes.


title: blood_breast cancer, 605

description: Collected in pax tubes.


title: blood_breast cancer, 590

description: Collected in pax tubes.


title: blood_breast cancer, 591

description: Collected in pax tubes.


title: blood_breast cancer, 606

description: Collected in pax tubes.


title: blood_breast cancer, 549

description: Collected in pax tubes.


title: blood_breast cancer, 604

description: Collected in pax tubes.


title: blood_breast cancer, 729

description: Collected in pax tubes.


title: blood_breast cancer, 541

description: Collected in pax tubes.


title: blood_breast cancer, 632

description: Collected in pax tubes.


title: blood_breast cancer, 580

description: Collected in pax tubes.


title: blood_breast cancer, 601

description: Collected in pax tubes.

value: ITC


title: blood_breast cancer, 540

description: Collected in pax tubes.


title: blood_breast cancer, 520

description: Collected in pax tubes.


title: blood_breast cancer, 550

description: Collected in pax tubes.

value: IDC/ITC


title: blood_breast cancer, 542

description: Collected in pax tubes.


title: blood_breast cancer, 581

description: Collected in pax tubes.


title: blood_breast cancer, 569

description: Collected in pax tubes.


title: blood_breast cancer, 519

description: Collected in pax tubes.


title: blood_breast cancer, 582

description: Collected in pax tubes.


title: blood_breast cancer, 560

description: Collected in pax tubes.


title: blood_breast cancer, 534

description: Collected in pax tubes.


title: blood_breast cancer, 518

description: Collected in pax tubes.


title: blood_breast cancer, 379

description: Collected in pax tubes.

value: DCIS (microinvasiv)


title: blood_breast cancer, 371

description: Collected in pax tubes.


title: blood_breast cancer, 366

description: Collected in pax tubes.


title: blood_breast cancer, 372

description: Collected in pax tubes.


title: iPS cell line of HDF cl.8

status: Public on Jun 26 2010

city: Amagasaki

first: Hajime

last: Ohgushi

state: Hyogo

address: 3-11-46 Nakouji

department: Research Institute for Cell Engineering

country: Japan

institute: National Institute of Advanced Industrial Science and Technology

hyb_protocol: Biotinylated cRNA was hybridized with a probe array for 16h at 45C, and the hybridized biotinylated cRNA was stained with streptavidin-PE.

scan_protocol: GeneChips were scanned using the GCS3000 GeneArray Scanner and AGCC was used for image processing

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

label: biotin

source_name: iPS cell line of HDF

label_protocol: cDNA was subjected to in vitro transcription , according to the manufacturer’s protocol (One-Cycle Target Labeling and Control Reagent package).

extract_protocol: Total cellular RNA was isolated with the RNeasy Mini Kit (Qiagen), according to manufacture’s instructions.). Genomic DNA was eliminated by DNase I (TAKARA BIO INC.) treatments.

tag: cell line

value: iPS

tag: cell type

value: HDF


title: blood_breast cancer, 512

description: Collected in pax tubes.


title: blood_breast cancer, 274

description: Collected in pax tubes.


title: blood_breast cancer, 326

description: Collected in pax tubes.


title: blood_breast cancer, 317

description: Collected in pax tubes.


title: blood_breast cancer, 350

description: Collected in pax tubes.

value: Medullary carcinoma


title: blood_breast cancer, 340

description: Collected in pax tubes.


title: blood_breast cancer, 277

description: Collected in pax tubes.


title: blood_control_breast feeding, B59

description: Collected in pax tubes.

value: Healthy

value: Breast-feeding woman


title: blood_breast cancer, 338

description: Collected in pax tubes.

value: Breast cancer

value: female


title: blood_control_mens.cycle, B58-1, rep1

description: Collected in pax tubes.

value: 1.week of mens cycle


title: blood_healthy, 816

description: Collected in pax tubes.

value: benign tumor


title: blood_breast cancer, 275

description: Collected in pax tubes.

value: YES


title: blood_breast cancer, 337

description: Collected in pax tubes.

value: NEG


title: blood_control_mens.cycle, B57-1, rep2

description: Collected in pax tubes.

value: 3.week of mens cycle


title: blood_control_pregnant, B54

description: Collected in pax tubes.

value: Pregnant 4th month


title: blood_control_pregnant, B52

description: Collected in pax tubes.

value: Pregnant 6th month


title: blood_breast cancer, 319

description: Collected in pax tubes.

value: POS

value: IDC


title: blood_control_mens.cycle, B57-1, rep1

description: Collected in pax tubes.


title: blood_control_mens.cycle, B58-1, rep2

description: Collected in pax tubes.

value: 4.week of mens cycle


title: blood_healthy, 812

description: Collected in pax tubes.


title: blood_control_pregnant, B53

description: Collected in pax tubes.


title: blood_healthy, 732

description: Collected in pax tubes.


title: blood_healthy, 797

description: Collected in pax tubes.


title: blood_healthy, 723

description: Collected in pax tubes.


title: blood_healthy, 726

description: Collected in pax tubes.

value: NO

value: cyst


title: blood_breast cancer, 315

description: Collected in pax tubes.

value: ITC


title: blood_healthy, 718

description: Collected in pax tubes.

value: Healthy


title: blood_healthy, 686

description: Collected in pax tubes.


title: blood_healthy, 714

description: Collected in pax tubes.


title: blood_healthy, 676

description: Collected in pax tubes.


title: blood_healthy, 717

description: Collected in pax tubes.


title: blood_healthy, 697

description: Collected in pax tubes.


title: blood_healthy, 660

description: Collected in pax tubes.


title: blood_healthy, 699

description: Collected in pax tubes.


title: blood_healthy, 659

description: Collected in pax tubes.


title: blood_healthy, 681

description: Collected in pax tubes.


title: blood_healthy, 666

description: Collected in pax tubes.


title: blood_healthy, 702

description: Collected in pax tubes.


title: blood_healthy, 671

description: Collected in pax tubes.


title: blood_healthy, 692

description: Collected in pax tubes.


title: blood_healthy, 667

description: Collected in pax tubes.


title: blood_healthy, 645

description: Collected in pax tubes.


title: blood_healthy, 684

description: Collected in pax tubes.


title: blood_healthy, 644

description: Collected in pax tubes.


title: blood_healthy, 643

description: Collected in pax tubes.


title: blood_healthy, 662

description: Collected in pax tubes.


title: blood_healthy, 648

description: Collected in pax tubes.


title: blood_healthy, 629

description: Collected in pax tubes.


title: blood_healthy, 622

description: Collected in pax tubes.


title: blood_healthy, 713

description: Collected in pax tubes.


title: blood_healthy, 642

description: Collected in pax tubes.


title: blood_healthy, 639

description: Collected in pax tubes.


title: blood_healthy, 623

description: Collected in pax tubes.


title: blood_healthy, 627

description: Collected in pax tubes.


title: blood_healthy, 613

description: Collected in pax tubes.


title: blood_healthy, 612

description: Collected in pax tubes.


title: blood_healthy, 611

description: Collected in pax tubes.


title: blood_healthy, 603

description: Collected in pax tubes.


title: blood_healthy, 615

description: Collected in pax tubes.


title: blood_breast cancer, 654

description: Collected in pax tubes.

value: DCIS


title: blood_healthy, 578

description: Collected in pax tubes.


title: blood_healthy, 574

description: Collected in pax tubes.


title: blood_healthy, 610

description: Collected in pax tubes.


title: blood_healthy, 566

description: Collected in pax tubes.


title: blood_healthy, 614

description: Collected in pax tubes.


title: blood_healthy, 570

description: Collected in pax tubes.


title: blood_healthy, 328

description: Collected in pax tubes.

value: hamartoma and fib.ad


title: blood_healthy, 577

description: Collected in pax tubes.

value: female


title: blood_healthy, 314

description: Collected in pax tubes.


title: blood_healthy, 310

description: Collected in pax tubes.


title: blood_healthy, 579

description: Collected in pax tubes.


title: Total_48h-10b

status: Public on Jan 01 2010

city: baltimore

first: chris

last: cheadle

state: MD

address: 5200 Eastern Ave

department: SOM

country: USA

institute: JHU

hyb_protocol: Standard Illumina hybridization protocol

scan_protocol: Standard Illumina scanning protocol

data_processing: The data were normalised using z transformation

source_name: P493-6 B cells

label_protocol: Nuclear run-on RNA was labeled by first adding biotin-modified UTP to isolated nuclei for 30 mins.   Biotinylated nuclear run-on RNA was captured on avidin magnetic beads followed by first strand synthesis (directly on beads) and second strand synthesis (in solution).  Biotinylated cRNA was supsequently prepared using the  Ambion TotalPrep RNA Amplification Kit.  Total RNA was labled with the Ambion TotalPrep RNA Amplification Kit according to manufacturers suggested protocols.

growth_protocol: P493-6 cells were grown in RPMI Medium 1640 supplemented with 10% heat-inactivated fetal bovine serum, MEM non-essential amino acids, and antibiotic-antimycotic, in a humidified incubator (95% air and 5% CO2) at 37⁰C.

extract_protocol: Nuclear run-on RNA (following in vitro labeling) was purified using a modified Qiagen RNeasy RNA Isolation procedure.  Total RNA was extracted with Trizol reagent, followed by clean-up with QIAGEN RNeasy mini kit in accordance with the prescribed protocol provided with the kit. Quality control was performed with Agilent Bioanalyser.

treatment_protocol: P493-6 cells were grown in the presence or absence of tetracycline for the time periods indicated, ectopic Myc gene expression is repressed in the presence of tetracycline and induced following its removal.

tag: time point

value: 48 hours

tag: replicate


title: Total_48h-8b


title: blood_healthy, 572

description: Collected in pax tubes.


title: Total_48h-9b


title: blood_healthy, 568

description: Collected in pax tubes.


title: Total_48h-9a


title: blood_healthy, 276

description: Collected in pax tubes.

value: Fibro adenoma


title: Total_48h-8a


title: Total_48h-10a


title: Total_48h-4


title: Total_6h-8

value: six hours


title: Total_48h-5


title: Total_48h-6


title: Total_6h-10


title: Total_3h-9

value: three hours


title: Total_ 6h-3

description: Total_ 6h-3


title: Total_48h-7


title: Total_ 6h-1

description: Total_ 6h-1


title: Total_ 3h-2

description: Total_ 3h-2


title: Total_6h-9


title: Total_ 3h-1

description: Total_ 3h-1


title: Total_1h-9

value: one hour


title: Total_3h-10


title: Total_ 1h-2

description: Total_ 1h-2


title: Total_ 1h-3

description: Total_ 1h-3


title: Total_ 3h-3

description: Total_ 3h-3


title: Total_30'-9

value: thirty minutes


title: Total_ 1h-1

description: Total_ 1h-1


title: Total_1h-10


title: Total_30'-8


title: Total_ 6h-2

description: Total_ 6h-2


title: Total_ 30'-1

description: Total_ 30'-1


title: Total_ 30'-2

description: Total_ 30'-2


title: Total_30'-10


title: Total_ 15'-3

description: Total_ 15'-3

value: fifteen minutes


title: Total_15'-9


title: Total_ 15'-2

description: Total_ 15'-2


title: Total_ 30'-3

description: Total_ 30'-3


title: Total_0h-9

value: zero time


title: Total_0h-8


title: Total_ 0h-7

description: Total_ 0h-7


title: Total_ 0h-6

description: Total_ 0h-6


title: Total_0h-10


title: blood_control_mens.cycle, B58-1, rep3

description: Collected in pax tubes.

value: 5.week of mens cycle


title: Total_ 0h-3

description: Total_ 0h-3


title: Total_ 0h-2

description: Total_ 0h-2


title: Total_ 0h-5

description: Total_ 0h-5


title: blood_healthy, 576

description: Collected in pax tubes.

value: YES


title: Total_ 0h-4

description: Total_ 0h-4


title: Total_15'-8


title: NRO_ 48h-10a

type: other

description: NRO_ 48h-10a

molecule: other

tag: sample

value: nuclear run-on RNA


title: NRO_ 48h-9b

description: NRO_ 48h-9b


title: Total_ 0h-1

description: Total_ 0h-1


title: Total_ 15'-1

description: Total_ 15'-1


title: NRO_ 48h-9a

description: NRO_ 48h-9a


title: NRO_ 48h-8b

description: NRO_ 48h-8b


title: NRO_ 48h-10b

description: NRO_ 48h-10b


title: NRO_ 48h-7

description: NRO_ 48h-7


title: NRO_ 48h-4

description: NRO_ 48h-4


title: NRO_ 48h-6

description: NRO_ 48h-6


title: NRO_ 6h-8

description: NRO_ 6h-8


title: NRO_ 48h-5

description: NRO_ 48h-5


title: NRO_ 48h-8a

description: NRO_ 48h-8a


title: NRO_ 6h-3

description: NRO_ 6h-3


title: NRO_ 6h-9

description: NRO_ 6h-9


title: NRO_ 3h-3

description: NRO_ 3h-3


title: NRO_ 6h-1

description: NRO_ 6h-1


title: NRO_ 3h-2

description: NRO_ 3h-2


title: NRO_ 3h-8

description: NRO_ 3h-8


title: NRO_ 3h-10

description: NRO_ 3h-10


title: NRO_ 6h-10

description: NRO_ 6h-10


title: NRO_ 1h-10

description: NRO_ 1h-10


title: NRO_ 1h-9

description: NRO_ 1h-9


title: NRO_ 1h-8

description: NRO_ 1h-8


title: NRO_ 3h-1

description: NRO_ 3h-1


title: Total_15'-10


title: NRO_ 1h-2

description: NRO_ 1h-2


title: NRO_ 30'-3

description: NRO_ 30'-3


title: NRO_ 30'-9

description: NRO_ 30'-9


title: NRO_ 30'-2

description: NRO_ 30'-2


title: NRO_ 15'-3

description: NRO_ 15'-3


title: NRO_ 30'-10

description: NRO_ 30'-10


title: NRO_ 1h-3

description: NRO_ 1h-3


title: NRO_ 15'-9

description: NRO_ 15'-9


title: NRO_ 30'-1

description: NRO_ 30'-1


title: Total_3h-8


title: NRO_ 1h-1

description: NRO_ 1h-1


title: NRO_ 15'-2

description: NRO_ 15'-2


title: NRO_ 15'-8

description: NRO_ 15'-8


title: NRO_ 15'-1

description: NRO_ 15'-1


title: NRO_ 15'-10

description: NRO_ 15'-10


title: NRO_ 0h-9

description: NRO_ 0h-9


title: NRO_ 0h-6

description: NRO_ 0h-6


title: NRO_ 0h-5

description: NRO_ 0h-5


title: NRO_ 0h-8

description: NRO_ 0h-8


title: NRO_ 0h-10

description: NRO_ 0h-10


title: NRO_ 30'-8

description: NRO_ 30'-8


title: NRO_ 0h-4

description: NRO_ 0h-4


title: NRO_ 0h-7

description: NRO_ 0h-7


title: NRO_ 0h-3

description: NRO_ 0h-3


title: NRO_ 0h-1

description: NRO_ 0h-1


title: MouseRetina-20mons-1

city: Aberdeen

first: Heping

last: Xu

address: Forresterhill

department: Ophthalmology

country: United Kingdom

institute: Univeristy of Aberdeen

hyb_protocol: 1.65 μg of Cy3-labelled fragmented cRNA  in hybridization buffer was hybridized to Agilent whole Mouse Genome Oligo Microarrays 4X44K for 17 hours at 65°C.  After hybridization, microarrays were washed once at room temperature with 6x SSPE Buffer for 1 minute followed by a second wash with preheated 0.06x SSPE Buffer (37°C) for 1 minute. The last washing step was performed with acetonitrile for 30 second.

scan_protocol: Slides were scanned on the Agilent's Microarry Scanner system (Agilent Technologies).

data_processing: The Agilent Feature Extraction Software (FES) was used to read out and process the microarray image files. The raw data were normalized by dividing the intensity values by their median.

organism: Mus musculus

source_name: Mouse retina, 20months old, pooled sample 1

label_protocol: Cyanine-3 (Cy3) labeled cRNA was prepared from 1μg RNA using the One-Color Low RNA Input Linear Amplification kit (Agilent) according to the manufacturer's instructions.Yields of cRNA and the dye-incorporation rate were checked with the NanoDrop ND-1000 Spectrophotometer(NanoDrop Technologies).

extract_protocol: Mouse retina was dissected immediately after mouse was sacrified. RNA was isloated using standard RNA extraction protocols (Trizol). RNA's was monitored with the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA).

tag: tissue

value: Retina

value: male

tag: age

value: 20 Months


title: MouseRetina-3mons-2

source_name: Mouse retina, 3months old, pooled sample 2

value: 3 Months


title: MouseRetina-20mons-2

source_name: Mouse retina, 20months old, pooled sample 2


title: T5 Total

description:
total RNA
replicate 2
probe group file: HuEx-1_0-st-v2.r2.pgf
meta-probeset file: HuEx-1_0-st-v2.r2.dt1.hg18.core.mps.

hyb_protocol: 5.5 ug of fragmented, labeled sense target DNA generated from either NRO or Total cRNA was hybridized to Affymetrix GeneChip Human Exon 1.0 ST Arrays using the Affymetrix FS450_0001 fluidics station protocol .

scan_protocol: Affymetrix Gene ChIP Scanner 3000 7G

data_processing: Data was processed through Affymetrix GeneChip Operating Software (GCOS) Expression Console and further analyzed (for differential gene expression and alternative splicing) using software from JMP (JMP Genomics 3.0) and Exon and Gene Array Expression Analysis from Biotique-XRAY (Reno, NV).

label_protocol: Nuclear run-on RNA was labeled by first adding biotin-modified UTP to isolated nuclei for 30 mins.   Biotinylated nuclear run-on RNA was captured on avidin magnetic beads followed by first strand synthesis (directly on beads) and second strand synthesis (in solution).  Biotinylated cRNA was supsequently prepared using the  Ambion TotalPrep RNA Amplification Kit.  For Affymetrix Exon arrays, 10 µg nuclear run-on cRNA was converted to cDNA and labeled using the Affymetrix Whole Transcript (WT) Sense Target Labeling Assay (cat. no. 900670) according to the manufacturer’s recommendations.   Total RNA was labeled in a similar fashion using an input of 100 ng RNA without ribosomal RNA reduction.

extract_protocol: Nuclear run-on RNA (following in vitro labeling)was purified using a modified Qiagen RNeasy RNA Isolation procedure.  Total RNA was extracted with Trizol reagent, followed by clean-up with QIAGEN RNeasy mini kit in accordance with the prescribed protocol provided with the kit. Quality control was performed with Agilent Bioanalyser.

tag: treatment

value: Tet maintained


title: T4 NRO

description:
nuclear run-on RNA
replicate 4
probe group file: HuEx-1_0-st-v2.r2.pgf
meta-probeset file: HuEx-1_0-st-v2.r2.dt1.hg18.core.mps.

molecule: nuclear RNA


title: T4 Total

description:
total RNA
replicate 1
probe group file: HuEx-1_0-st-v2.r2.pgf
meta-probeset file: HuEx-1_0-st-v2.r2.dt1.hg18.core.mps.


title: MouseRetina-3mons-1

source_name: Mouse retina, 3months old, pooled sample 1

value: 3 months


title: U6 Total

description:
total RNA
replicate 3
probe group file: HuEx-1_0-st-v2.r2.pgf
meta-probeset file: HuEx-1_0-st-v2.r2.dt1.hg18.core.mps.

value: no tet


title: Bulk (Low grain number) replicate 1

city: New Delhi

first: Rupesh

middle: Kailasrao

last: Deshmukh

state: Delhi

address: Libraryavenue, PUSA

department: Rice Genome Laboratory

country: India

institute: National Research Center on Plant Biotechnology

description: Gene expression profile of bulked RILs bearing low grain number in panicle

hyb_protocol: standard Affymetrix procedures

scan_protocol: standard Affymetrix procedures

data_processing: GCOS 1.2

organism: Oryza sativa Indica Group

source_name: Panicle primordia stage '0'

label_protocol: Biotinylated cRNA were prepared according to the standard Affymetrix protocol from 8 ug total RNA (Expression Analysis Technical Manual, 2001, Affymetrix).

extract_protocol: Total RNA was isolated by using TRIzol Reagent (Sigma) RNA samples were processed as recommended by Affymetrix (Affymetrix geneChip Expression Analysis Technical manual; Affymetrix)

tag: grain number per panicle

value: Low


title: U5 NRO

description:
nuclear run-on RNA
replicate 3
probe group file: HuEx-1_0-st-v2.r2.pgf
meta-probeset file: HuEx-1_0-st-v2.r2.dt1.hg18.core.mps.


title: T7 Total

description:
total RNA
replicate 4
probe group file: HuEx-1_0-st-v2.r2.pgf
meta-probeset file: HuEx-1_0-st-v2.r2.dt1.hg18.core.mps.


title: U4 Total


title: T5 NRO


title: Bulk (High grain number) replicate 1

description: Gene expression profile of bulked RILs bearing high grain number in panicle

value: High


title: T6 Total


title: T6 NRO

description:
nuclear run-on RNA
replicate 2
probe group file: HuEx-1_0-st-v2.r2.pgf
meta-probeset file: HuEx-1_0-st-v2.r2.dt1.hg18.core.mps.


title: U4 NRO


title: Total_1h-8


title: U5 Total


title: Bulk (High grain number) replicate 2


title: Bulk (Low grain number) replicate 2


title: U7 Total


title: U6 NRO


title: F verticillioides total RNA_96 hr_rep3

status: Public on Dec 31 2009

city: Peoria

first: Robert

last: Butchko

state: IL

address: 1815 N. University St.

institute: U.S. Dept. of Agriculture

description: This sample is of wild-type Fusarium verticillioides M3125. It is the third of three biological replicates used in this experiment, each from separate cultures.

hyb_protocol: Hybridization was performed by NimbleGen Systems Inc., Madison, WI, USA, following their standard operating protocol. See www.nimblegen.com.

scan_protocol: Scanning was performed by NimbleGen Systems Inc., Madison, WI, USA, following their standard operating protocol. See www.nimblegen.com.

data_processing: The raw data (.pair file) was subjected to RMA (Robust Multi-Array Analysis; Irizarry et al. Biostatistics 4(2):249), quantile normalization (Bolstad et al. Bioinformatics 19(2):185), and background correction as implemented in the NimbleScan software package, version 2.4.27 (Roche NimbleGen, Inc.).

organism: Fusarium verticillioides

source_name: F verticillioides, liquid media, 96 hr

label_protocol: Labeling was performed by NimbleGen Systems Inc., Madison, WI, USA, following their standard operating protocol. See www.nimblegen.com.

growth_protocol: F verticillioides was grown in liquid fumonisin media (GYAM) for 1, 2, 3 and 4 days, shaking at 250 rpm at 27C following Proctor et al, Co-expression of 15 contiguous genes delineates a fumonisin biosynthetic gene cluster in Gibberella moniliformis, Fungal Genetics and Biology, 38:237-249.

extract_protocol: Total RNA was extracted using the RNeasy Mini kit (Qiagen Inc., Valencia, CA, USA).  DNA was removed by on-column DNase digestion with the RNase-Free DNase set (Qiagen).

tag: strain

value: wild type F verticillioides M3125

tag: fumonisin producer

value: yes

tag: time

value: 96 hr


title: U7 NRO

description:
nuclear run-on RNA
replicate 1
probe group file: HuEx-1_0-st-v2.r2.pgf
meta-probeset file: HuEx-1_0-st-v2.r2.dt1.hg18.core.mps.


title: T7 NRO


title: NRO_ 6h-2

description: NRO_ 6h-2

value: six hours


title: F verticillioides total RNA_96 hr_rep1

description: This sample is of wild-type Fusarium verticillioides M3125. It is the first of three biological replicates used in this experiment, each from separate cultures.


title: F verticillioides total RNA_72 hr_rep1

source_name: F verticillioides, liquid media, 48 hr

value: 48 hr


title: F verticillioides total RNA_48 hr_rep3

source_name: F verticillioides, liquid media, 72 hr

value: 72 hr


title: F verticillioides total RNA_48 hr_rep2

description: This sample is of wild-type Fusarium verticillioides M3125. It is the second of three biological replicates used in this experiment, each from separate cultures.


title: F verticillioides total RNA_48 hr_rep1


title: F verticillioides total RNA_72 hr_rep2


title: NRO_ 0h-2

description: NRO_ 0h-2

value: zero time


title: F verticillioides total RNA_24 hr_rep2

source_name: F verticillioides, liquid media, 24 hr

value: 24 hr


title: F verticillioides total RNA_72 hr_rep3


title: F verticillioides total RNA_24 hr_rep3


title: cells at T 40 min, biological rep3

city: Freiburg

first: Claudia

last: Steglich

address: Schaenzlestr. 1

department: Biology

country: Germany

institute: University of Freiburg

description: Gene expression data from Prochlorococcus cells after 40 min rifampicin addition

hyb_protocol: The cDNA was hybridized to the MD4-9313 custom Affymetrix array in aqueous hybridization solution (100 mM MES, 1 M NaCl, 20 mM EDTA, 0.01% Tween-20, 0.1 mg/mL Herring Sperm DNA, 0.5 mg/mL BSA, 7.8 % DMSO and 3 nM prelabeled Affymetrix hybridization B2 oligo control probe mix) during a 16 h incubation at 45 °C in a GeneChip Hybridization Oven 320 rotating at 60 rpm. Washes and stains were carried out on a GeneChip Fluidics Station 450 (Affymetrix) following the ProkGE_WS2v3 Affymetrix protocol. Briefly, following two stringency washes the array was sequentially incubated with 10 µg/mL streptavidin (Pierce Chemical), 5 µg/mL biotinylated anti-streptavidin goat antibody (Vector Laboratories) and 0.1 mg/mL goat IgG (Sigma) and 10 µg/mL streptavidin-phycoerythrin conjugate (Mol. Probes) each for 10 min at 25 °C and given a final wash.

scan_protocol: The arrays were scanned with the GeneChip Scanner (Affymetrix) at a 2.5 µm resolution with excitation set for 570 nm.

data_processing:
The probe set summaries calculated from perfect match probe intensities in Affymetrix CEL files using robust multi-array average (RMA) analysis with quantile normalization (as implemented in the Bioconductor package affy).
We performed different normalization methods and checked by qPCR, which normalization method works best. We tested:  i) scaling to the same medium intensity of all genes; ii) scaling to the same medium intensity of spike controls; iii) scaling to the same medium intensity of RNA genes (assumed to be particularly stable) and iv) no subsequent scaling. Remarkably, the RMA processed microarray data with no subsequent scaling achieved the highest concordance with the quantitative PCR standard (i.e. a mean Spearman correlation coefficient of 0.83).

organism: Prochlorococcus marinus subsp. pastoris str. CCMP1986

label_protocol: Synthesis of complementary DNA (cDNA), labeling, hybridization, staining and scanning was carried out according to Affymetrix protocols for E.coli (http://www.affymetrix.com/support/technical/manual/expression_manual.affx) with minor changes. Total RNA (2 µg) was denatured at 70 °C and annealed to random hexamer primers (25 ng/µl) at 25 °C for 10 min. The RNA was reverse transcribed to produce cDNA with Superscript II (25 U/µl – Invitrogen Life Technologies) and 0.5 mM dNTPs in the presence of 1 U/µl RNase Out RNase Inhibitor (Invitrogen). The mix was incubated at 25 °C for 10 min followed by 60 min incubations at 37 °C and 42 °C respectively. Superscript II was inactivated with a 10 min incubation at 70 °C. Sodium hydroxide (0.25 N) was used to remove RNA during a 30 min incubation at 65 °C, followed by neutralization with HCl. The cDNA was purified with MinElute PCR purification columns (Qiagen). Fragments of cDNA, 50-200 nt long, were produced from a 10 min incubation at 37 °C with DNase I (0.6 U per µg cDNA), followed by heat inactivation of the DNase I enzyme (10 min at 98 °C). The cDNA fragments were end-labeled with biotin using the BioArray Terminal Labeling Kit (Enzo) during a 60 min incubation at 37 °C. The reaction was stopped by freezing at –20 °C. The quality of biotin end-labeling was verified by gel-shift assays with NeutrAvidin (Pierce Chemicals) on 1% TBE agarose gels.

growth_protocol: Prochlorococcus MED4 was grown at 21°C in Sargasso seawater-based Pro99 medium (Moore et al. 2007, Limnol Oceanogr Meth) under 30 µmol quanta m-2 s-1 continuous cool white light with a growth rate of 0.325 day-1.

extract_protocol: Total RNA was extracted from cells on filters using a hot-phenol method described previously (Lindell and Post 2001, Appl Environ Microbiol; Steglich et al. 2006, J Bac).

treatment_protocol: Triplicate cultures were divided into seven 30 ml subcultures each and 1.9 ml rifampicin (2.5 mg/ml dissolved in Pro99 medium) was added to a final concentration of 150 µg/ml. For sampling time point 0 min only 1.9 ml Pro99 medium was added. Cells were harvested after 0 min, 2.5 min, 5 min, 10 min, 20 min, 40 min and 60 min of rifampicin treatment by rapid filtration onto Supor-450 membranes. Filters were immersed in 2 ml RNA resuspension buffer (10 mM sodium acetate pH 5.2, 200 mM sucrose, 5 mM EDTA), snap frozen in liquid nitrogen and subsequently stored at – 80°C.

value: 40 min

tag: agent

value: rifampicin


title: cells at T 40 min, biological rep2

source_name: Prochlorococcus cells after 40 min rifampicin addition


title: cells at T 20 min, biological rep1

description: Gene expression data from Prochlorococcus cells after 20 min rifampicin addition

value: 20 min


title: cells at T 20 min, biological rep3

source_name: Prochlorococcus cells after 20 min rifampicin addition


title: cells at T 60 min, biological rep1

description: Gene expression data from Prochlorococcus cells after 60 min rifampicin addition

value: 60 min


title: F verticillioides total RNA_24 hr_rep1


title: cells at T 20 min, biological rep2


title: cells at T 10 min, biological rep1

description: Gene expression data from Prochlorococcus cells after 10 min rifampicin addition

value: 10 min


title: cells at T 60 min, biological rep3

source_name: Prochlorococcus cells after 60 min rifampicin addition


title: cells at T 40 min, biological rep1


title: cells at T 10 min, biological rep2

source_name: Prochlorococcus cells after 10 min rifampicin addition


title: cells at T 60 min, biological rep2


title: cells at T 10 min, biological rep3


title: cells at T 5 min, biological rep2

description: Gene expression data from Prochlorococcus cellsafter 5 min rifampicin addition

source_name: Prochlorococcus cells after 5 min rifampicin addition

value: 5 min


title: cells at T 2.5 min, biological rep2

description: Gene expression data from Prochlorococcus cells after 2.5 min rifampicin addition

value: 2.5 min


title: cells at T 2.5 min, biological rep3

source_name: Prochlorococcus cells after 2.5 min rifampicin addition


title: F verticillioides total RNA_96 hr_rep2


title: cells at T 5 min, biological rep1

description: Gene expression data from Prochlorococcus cells after 5 min rifampicin addition


title: cells at T 0 min, biological rep3

description: Gene expression data from Prochlorococcus cells without rifampicin


title: cells at T 2.5 min, biological rep1


title: cells at T 0 min, biological rep1

source_name: Prochlorococcus cells without rifampicin


title: cells at T 0 min, biological rep2


title: L24S4F

city: Galway

first: Michael

middle: Taylor

last: Cairns

address: University Road

department: MRI

country: Ireland

institute: NUI Galway

description: The two cDNA samples per slide (e.g. reference cDNA Cy3 labelled and experimental cDNA Cy5 labelled) were mixed and appropriate blockers were added (sheared salmon sperm genomic DNA, poly dA). The cDNA mixture was concentrated by precipitation with 3M NaOAC and absolute ethanol at -20oC for 30 min. followed by centrifugation at 12500 x g for 30 min. Following removal of supernatant, the cDNA pellet was resuspended in 70 microl slide hyb buffer #1 (Ambion) and was then denatured at 95oC for 2 min. Slides were placed in Pre-Hyb solution (3xSSC, 0.1 % BSA, 0.1 % SDS) in 50 ml Sarsted tubes pre-warmed @ 42 oC and pre-hybed @ 42 oC for 30 min. Slides were rinsed 3 times in water and spun dry for 3 min @ 1600rpm. The hybridisation mixture was then added to the pre-hybed microarray slide using a coverslip (Erie Scientific). Hybridisation was performed in a Genetix hybidisation chamber (Genetix, UK) at 42 oC for 16 h. Following hybridisation the slides were washed (2 x 5 min. washes using 0.2XSSC/0.1% SDS and 2 x 5 min. washes using 0.2XSSC) using an Advawash automatic washing station (Advalytix).

hyb_protocol: The two cDNA samples per slide were mixed and appropriate blockers were added (10 micrograms each of sheared salmon sperm genomic DNA (Ambion) and poly dA (Biotez, Ger)). The cDNA mixture was concentrated by precipitation with 3M NaOAc and absolute ethanol at -20 oC for 30 min followed by centrifugation at 12500g for 30 min at 4 oC. Following removal of supernatant, the cDNA pellet was resuspended in 70 microlitres slide hyb buffer 1 (Ambion) and was then denatured at 95 oC for 2 min. Sides were pre-hybridised in 3X SSC/0.1% SDS/0.1% BSA at 60 oC for 1h, washed twice briefly in MQ-water, dipped in 100% ethanol and centrifuged dry. The hybridisation mixture was then added to the pre-hybridised microarray slide using a lifter slip (Erie Scientific). Hybridisation was performed in a Genetix hybridisation chamber (Genetix, UK) at 42 oC for 16 h. Following hybridisation the slides were washed (2 x 5 min washes using 0.2X SSC/0.1 % SDS and 2 x 5 min washes using 0.2X SSC) using an Advawash automatic washing station (Advalytix).

scan_protocol: Microarray slides were scanned at two wavelengths (543nm and 633nm) at 5 micrometer resolution using a confocal laser scanner (ScanArray Express HT, Perkin Elmer). The 543nm channel is referred to as 570 and 633nm is referred to as 670.

data_processing: The 2 channel TIFF image files were imported into Genepix Pro (version 5.0.1.24) (Axon instruments) for feature finding and alignment using a batch alignment process. Features were flagged as present, absent or bad by this software program and pixel intensities for feature and background were quantified. Output Genepix results (GPR) files were imported into the Genespring GX 7.3 software program (Agilent technologies). To account for dye swap, the signal channel and control channel measurements for appropriate samples were reversed. Values below 0.01 were set to 0.01. A Lowess curve was fit to the log-intensity versus log-ratio plot using 20.0% of the data to calculate the Lowess fit at each point. This curve was used to adjust the control value for each measurement. If the control channel was lower than 10 then 10 was used instead.

organism: Sparus aurata

source_name: Sparus aurata 24 H stress fish 4 111

label_protocol: Ten micrograms of total RNA per fish and ten micrograms of reference RNA per slide were indirectly labelled following an in house protocol with the red fluorescent dye ester Cyanine 5 (Cy5) and green fluorescent dye ester Cyanine 3 (Cy3) both from GE Healthcare (UK). In brief, cDNA was synthesised using Superscript III MMLV reverse transcriptase (Invitrogen) incorporating aminoallyl dUTP (Sigma Aldrich). Synthesis took place at 42 oC for 2 h. RNA was hydrolysed with 10M NaOH/0.5M EDTA for 15 min and the reaction was then neutralised with 1M HEPES. Reverse transcription reactions were purified using Microcon 30 columns (Millipore, UK) according to manufacturers instructions. Purified cDNAs were dried using a vacuum concentrator (Eppendorf) and were then resuspended in 0.1M sodium bicarbonate buffer. Cy dyes were resuspended in the same buffer and coupling to the Cy dye ester took place in the dark for 2 h at room temperature. The removal of unincorporated dye was performed using the Illustra CyScribe GFX purification kit (GE Healthcare). Labelled cDNA samples were tested for successful dye incorporation using a Nanodrop spectrophotometer (Nanodrop technologies).

extract_protocol: Qiagen midi kit and Qiazol reagent

tag: gender

value: male

tag: confinement

value: stress

tag: timepoint

source_name: Confinement stress reference pool of RNA from all stress and control fish except L6S1 L6S4 L6C3 L6C4 L120C1

tag: reference

value: confinement stress reference pool of RNA from all stress and control fish except L6S1 L6S4 L6C3 L6C4 L120C1


title: L24S3R

source_name: Sparus aurata 24 H stress fish 3 123 G


title: L24S2R

source_name: Sparus aurata 24 H stress fish 2 126 G


title: L24C1R

source_name: Sparus aurata 24 H Control fish 1 127 G

value: control


title: L24S3F


title: L24C5R

source_name: Sparus aurata 24 H Control fish 5 120 G


title: L24C2R

source_name: Sparus aurata 24 H Control fish 2 110 G


title: L120S3R

source_name: Sparus aurata, 120H Stress fish 3  129 G


title: L24C3R

source_name: Sparus aurata 24 H Control fish 3 138 G


title: L120S3F


title: L24S1F

source_name: Sparus aurata 24 H stress fish 1 105 G


title: L24C4F

source_name: Sparus aurata 24 H Control fish 4 121 G


title: L24C2F


title: L24C1F


title: L24C5F


title: L24S1R


title: L24S2F


title: NRO_ 3h-9

description: NRO_ 3h-9

source_name: P493-6 B cells

value: three hours

value: nuclear run-on RNA


title: L120S5F

source_name: Sparus aurata, 120H Stress fish 5  123 G


title: L120C3R

source_name: Sparus aurata, 120H CONTROL fish 3  128 G


title: L120C5F

source_name: Sparus aurata, 120H CONTROL fish 5  145 G


title: L120S1R

source_name: Sparus aurata, 120 H Stress fish 1  149 G


title: L120C4R

source_name: Sparus aurata, 120H CONTROL fish 4  112 G


title: L120S1F


title: L24C3F


title: L120C4F


title: L120C5R


title: L120C3F


title: L120C1R

source_name: Sparus aurata, 120H CONTROL fish 1  108 G


title: L120S4F

source_name: Confinement stress reference pool of RNA from all stress and control fish except L6S1 L6S4 L6C3 L6C4 L120C1

source_name: Sparus aurata, 120H Stress fish 4  137 G


title: L120S5R


title: L120C1F


title: L72S4F

source_name: Sparus aurata, Stress fish 4  118 G


title: L72S2R

source_name: Sparus aurata, Stress fish 2  118 G


title: L72S3R

source_name: Sparus aurata, Stress fish 3  150 G


title: L72S5R

source_name: Sparus aurata, Stress fish 2  121 G


title: L72C1F

source_name: Sparus aurata 72 H Control fish 1 129G


title: L72S4R


title: L6S5R

source_name: 6 H stress fish 5 118 G Tank 6-5S


title: L72S2F


title: L6S3F

source_name: 6 H stress fish 3 123g Tank 6-3S


title: L72C2R

source_name: Sparus aurata 72 H Control fish 2 129 g


title: L120C2F

source_name: Sparus aurata, 120H CONTROL fish 2  137 G


title: L72C3F

source_name: Sparus aurata 72 H Control fish 3 113 G


title: L6S4R

source_name: 6 H stress fish 4 139G Tank 6-4S


title: L72S1F

source_name: Sparus aurata, Stress fish 1  119 G


title: L72S1R


title: L72C5R

source_name: Sparus aurata, Control fish 5  130 G


title: L72C3R


title: L72S3F


title: L6S5F


title: L6S3R


title: L72C1R


title: L72C2F


title: L6S4F


title: @52055800697862121408403718160344

city: Valbonne - Sophia-Antipolis

first: Kevin

last: Lebrigand

address: 660 route des lucioles

country: France

institute: IPMC/CNRS

description:
2825 WT
Date:2008/02/08

hyb_protocol: Affy-169 : 300.0 ng at 45 degree_C during 17 hours

scan_protocol: Arrays were scanned using the GeneChip Scanner 3000 7G (Affymetrix).

data_processing: Interslide norm. : none, Intraslide norm. : none, Background norm. : rma, Software used : expr_console --> VALUE data obtained from log2 normalized expression signal.

label: Biotin-Affy

extract_protocol: Frozen tissues were homogenized in Trizol (Invitrogen Life Technologies, Gaithersburg, MD, USA). After extraction with isopropanol, total RNAs were purified on RNeasy Qiagen columns (Qiagen Inc., Valencia, CA, USA).

tag: subject

tag: type

value: wild type

value: Mouse skeletal muscle


title: @52055800697862121408403718160371

description:
2827 TG
Date:2008/02/08

value: transgenic


title: L6S2F

source_name: Sparus aurata, 120H Stress fish 2  142 G


title: L6S1R

source_name: 6 H stress fish 1 117 G Tank 6-1S


title: L6C5R

source_name: Control fish 5 124 G Tank 6-5C

source_name: Confinement stress reference pool of RNA from all stress and control fish except L6S1 L6S4 L6C3 L6C4 L120C1


title: cwc21-bud13-null expression profile

city: New York

first: Harm

middle: van

last: Bakel

state: New York

address: One Gustave L. Levy Place, Box 1498

department: Genetics and Genomic Sciences

institute: Mount Sinai School of Medicine

description: cwc21-bud13-null expression profile

hyb_protocol: 6 μg  of biotinylated cDNA was hybridized at 45C for 16 hours, and then washed and stained according to Affymetrix protocol EukGE-WS2v4_450 in an Affymetrix Fluidics Station 450.

scan_protocol: Arrays were scanned in an Affymetrix 7G scanner.

data_processing: Raw data files were normalized with Affymetrix Tiling Analysis Software (TAS) v1.1. Treatment and control samples were quantile normalized in pairs using only perfect match probes with a bandwidth of 20.

organism: Saccharomyces cerevisiae

source_name: Total RNA from a cwc21 and bud13 null mutant

label_protocol: cDNA was fragmented by nuclease digestion in a solution containing 1X One-Phor-All buffer (GE Healthcare) and DNase I (GE Healthcare) at 37ｰC until the average fragment size was approximately 70 bp. The fragmentation reaction was stopped by incubating at 95C for 10 min. Fragmented samples were biotinylated with Biotin-N6-ddATP (Enzo Life Sciences) using terminal deoxynucleotidyl transferase at 37C for 2 hours.

growth_protocol: Cell cultures were grown to an early mid-log phase (OD600 0.8) in rich medium (YPD) at 30C while shaking.

extract_protocol: Total RNA was extracted from yeast cells by vortexing with glass beads in the presence of hot acid phenol and cleaned by phenol:chlorophorm extraction followed by ethanol precipitation. After DNaseI (GE Healthcare) treatment to remove residual genomic DNA, 25 ug of total RNA was incubated with random hexamers at 70C for 10 min, incubated at 15C for 30 min and reverse transcribed to single-stranded cDNA for 2 hours at 42C with Superscript II (Invitrogen) in the presence of 6 μg/ml Actinomycin D to prevent antisense artifacts. RNA was subsequently removed by hydrolysis and cDNA was cleaned up and concentrated on MinElute PCR purification columns (Qiagen).

label: Biotin

source_name: Total RNA from wild-type yeast


title: L6S2R

source_name: 6 H stress fish 2 121 G Tank 6-2S


title: L6C4R

source_name: Control fish 4 123G Tank 6-5C


title: L6C5F


title: @52055800697862121408403718160373

description:
2824 TG
Date:2008/02/08


title: L6S1F


title: @52055800697862121408403718160409

description:
2828 WT
Date:2008/02/08


title: L72S5F

source_name: Sparus aurata, Stress fish 2  121 G


title: isy1-null expression profile

description: isy1-null expression profile

source_name: Total RNA from an isy1 null mutant


title: L24S4R

source_name: Sparus aurata 24 H stress fish 4 111


title: cwc21-isy1-null expression profile

description: cwc21-isy1-null expression profile

source_name: Total RNA from a cwc21 and isy1 null mutant


title: bud13-null expression profile

description: bud13-null expression profile

source_name: Total RNA from a bud13 null mutant


title: cwc21-null expression profile

description: cwc21-null expression profile

source_name: Total RNA from a cwc21 null mutant


title: HUVEC

type: SAGE

anchor: NlaIII

city: Hsinchu

first: Yung-Jen

last: Chuang

state: Taiwan

address: No. 101, Sec. 2, Kuang Fu Road

department: Department of Medical Science

country: Taiwan

institute: National Tsing Hua University

description:
Human umbilical cords were obtained from the Lucille Packard Children Hospital. In this study, we devised a rapid isolation scheme to preserve the in vivo phenotype of each endothelial subtype. ECs were first isolated from umbilical cords by collagenase perfusion through the vein or artery as described. Cells were further purified using a Percoll density gradient (Amersham Biosciences, Piscataway, NJ) to remove residual erythrocytes and platelets. 1-5 x 106 ECs were then cultured overnight on gelatin-coated T12.5 flasks in Clonetics EGM-2 media (Cambrex Bio Science, Walkersville, MD) in which the ECs generally reached confluence the next morning. The confluent EC were trypsinized and subjected to two rounds of immuno-magnetic beads purification, adapted from a published purification protocol. There was a negative selection step using CD14, CD45 and CD64 to remove residual contaminating leukocytes, followed by positive selection using a mouse anti-endothelial cell monoclonal antibody (anti-CD146/clone P1H12 purchased from Chemicon, Temecula, CA). Total processing time was limited to 20 to 24 hours. The homogeneous, viable, primary ECs were used immediately to construct the library. The construction of SAGE libraries was performed with the I-SAGE kit (Invitrogen, Carlsbad, CA) per manufacturer?s instructions.
Keywords = endothelial cell
Keywords = vein

source_name: Primary human umbilical vein endothelial cell

extract_protocol: Trizol


title: @52055800697862121408403718160406

description:
2826 TG
Date:2008/02/08


title: L6C1F

source_name: Control fish 1 136g Tank 6-1C


title: L24S5R

source_name: Sparus aurata 24 H stress fish 5 151 G


title: L6C1R


title: L24S5F


title: No28LCMHpre

city: Kanazawa

first: Masao

last: Honda

address: Takara-Machi 13-1

department: Gastroenterology

institute: Kanazawa University

description: Before therapy

hyb_protocol: The biotin-labeled cDNA was suspended in hybridization cocktail (NuGen), and used for the hybridization. Affymetrix Human 133 Plus 2.0 microarray chip (Affymetrix, Santa Clara, CA, USA) containing 54,675 gene transcripts was used for the analysis. Hybridization (45°C for 16hrs at 60 rpm), washing and staining (using Fluidics Station) were performed according to the standard protocol (Affymetrix).

scan_protocol: The probe array was scanned by GeneChip® Scanner 3000(Affymetrix).

data_processing: Hybridized data files (CEL) were obtained with the GeneChip® Operating Software 1.4 (GCOS) (Affymetrix).The intensity of each gene chip was normalized by one patient using GeneChip® Operating Software 1.4 (GCOS) and further processed by BRB-ArrayTools (http://linus.nci.nih.gov/BRB-ArrayTools.htm)

source_name: Liver biopsy LCM

label_protocol: Total RNA isolated from the LCM specimens was amplified twice with the TargetAmp™ 2-Round Aminoallyl-aRNA Amplification Kit 1.0 (EPICENTRE, Madison, WI, USA). 20 μg aRNA was used for further biotin labeling using Biotin-X-X-NHS (provided by EPICENTRE) according to the manufacturer’s protocol.

extract_protocol: Total RNA in LCM was isolated with a carrier nucleic acid (20ng poly C) using RNAqueousTM-Micro (Ambion).

value: Liver biopsy LCM

tag: genotype

value: 2 RVR No28

tag: cell type

value: hepatocytes

tag: time point

value: before therapy


title: No28LCMHinf

description: Under therapy

tag: tissue

value: under therapy


title: No28LCMPpre

description: Before therapy

value: lymphocytes


title: No27LCMHinf

description: Under therapy

value: 2 RVR No27


title: HUAEC

description:
Human umbilical cords were obtained from the Lucille Packard Children Hospital. In this study, we devised a rapid isolation scheme to preserve the in vivo phenotype of each endothelial subtype. ECs were first isolated from umbilical cords by collagenase perfusion through the vein or artery as described. Cells were further purified using a Percoll density gradient (Amersham Biosciences, Piscataway, NJ) to remove residual erythrocytes and platelets. 1-5 x 106 ECs were then cultured overnight on gelatin-coated T12.5 flasks in Clonetics EGM-2 media (Cambrex Bio Science, Walkersville, MD) in which the ECs generally reached confluence the next morning. The confluent EC were trypsinized and subjected to two rounds of immuno-magnetic beads purification, adapted from a published purification protocol. There was a negative selection step using CD14, CD45 and CD64 to remove residual contaminating leukocytes, followed by positive selection using a mouse anti-endothelial cell monoclonal antibody (anti-CD146/clone P1H12 purchased from Chemicon, Temecula, CA). Total processing time was limited to 20 to 24 hours. The homogeneous, viable, primary ECs were used immediately to construct the library. The construction of SAGE libraries was performed with the I-SAGE kit (Invitrogen, Carlsbad, CA) per manufacturer?s instructions.
Keywords = endothelial cell
Keywords = artery

source_name: Primary human umbilical arterial endothelial cell


title: No27LCMPinf

description: Under therapy


title: L6C2F

source_name: Control fish 2 110 G Tank 6-2C


title: No29LCMPpre

description: Before therapy

value: 2 RVR No29


title: L6C3F

source_name: Control fish 3 117 G Tank T6-3C

value: male

source_name: Confinement stress reference pool of RNA from all stress and control fish except L6S1 L6S4 L6C3 L6C4 L120C1


title: No29LCMPinf

description: Under therapy


title: No29LCMHpre

description: Before therapy


title: L6C3R


title: @52055800697862121408403718160364

description:
2830 WT
Date:2008/02/08


title: No20LCMPpre

description: Before therapy

value: 1 nonRVR No20


title: No29LCMHinf

description: Under therapy


title: No27LCMPpre

description: Before therapy


title: No20LCMHpre

description: Before therapy


title: No28LCMPinf

description: Under therapy


title: L6C4F

value: confinement stress reference pool of RNA from all stress and control fish except L6S1 L6S4 L6C3 L6C4 L120C1


title: L6C2R

value: control


title: No20LCMPinf

description: Under therapy


title: No18LCMHinf

description: Under therapy

value: 1 nonRVR No18


title: No27LCMHpre

description: Before therapy


title: No18LCMHpre

description: Before therapy


title: No18LCMPpre

description: Before therapy


title: No14LCMPinf

description: Under therapy

value: 1 nonRVR No14


title: No15LCMPpre

description: Before therapy

value: 1 nonRVR No15


title: No17LCMHpre

description: Before therapy

value: 1 RVR No17


title: No14LCMHpre

description: Before therapy


title: No14LCMPpre

description: Before therapy


title: No17LCMPpre

description: Before therapy


title: No17LCMPinf

description: Under therapy


title: No15LCMHinf

description: Under therapy


title: No13LCMHinf

description: Under therapy

value: 1 nonRVR No13


title: No7LCMPinf

description: Under therapy

value: Liver biopsy LCM

value: 1 RVR No7


title: No13LCMPpre

description: Before therapy


title: No13LCMHpre

description: Before therapy

value: hepatocytes


title: No17LCMHinf

description: Under therapy


title: No7LCMHpre

description: Before therapy

value: before therapy


title: No7LCMPpre

description: Before therapy


title: No14LCMHinf

description: Under therapy

value: under therapy


title: No18LCMPinf

description: Under therapy

value: lymphocytes


title: No4LCMPinf

description: Under therapy

value: 1 nonRVR No4


title: No7LCMHinf

description: Under therapy


title: No1LCMPinf

description: Under therapy

value: 1 RVR No1


title: No4LCMHinf

description: Under therapy


title: No1LCMPpre

description: Before therapy


title: No4LCMHpre

description: Before therapy


title: No28inf

description: Under therapy

source_name: Liver biopsy

label_protocol: Aliquots of total RNA (50 ng) isolated from the liver biopsy specimens were subjected to amplification with the WT-OvationTM Pico RNA Amplification System (NuGen, San Carlos, CA, USA) as recommended by the manufacturer (http://www.nugeninc.com/).5 microgram of resultant cDNA was used for fragmentation and biotin labeling using the FL-OvationTM cDNA Biotin Module V2 (NuGen) as recommended by the manufacturer.

extract_protocol: Total RNA in each liver biopsy specimen was isolated using the RNAqueous® kit (Ambion, Austin, TX, USA)

value: Liver biopsy

value: 2 No28RVR


title: No27pre

description: Before therapy

value: 2 No27RVR


title: No29inf

description: Under therapy

value: 2 No29RVR


title: No13LCMPinf

description: Under therapy


title: No1LCMHinf

description: Under therapy


title: No30pre

description: Before therapy

value: 2 No30RVR


title: No28pre

description: Before therapy


title: No25inf

description: Under therapy

value: 2 No25RVR


title: No30inf

description: Under therapy


title: No1LCMHpre

description: Before therapy


title: No25pre

description: Before therapy


title: No29pre

description: Before therapy


title: No26pre

description: Before therapy

value: 2 No26RVR


title: No15LCMPinf

description: Under therapy

value: 1 nonRVR No15


title: No26inf

description: Under therapy


title: No24pre

description: Before therapy

value: 2 No24RVR


title: No15LCMHpre

description: Before therapy

value: Liver biopsy LCM


title: No27inf

description: Under therapy


title: No24inf

description: Under therapy


title: No21inf

description: Under therapy

value: 1 No21 nonRVR


title: No22pre

description: Before therapy

value: 1 No22RVR


title: No20inf

description: Under therapy

value: 1 No20 nonRVR


title: No19pre

description: Before therapy

value: 1 No19RVR

value: before therapy


title: No22inf

description: Under therapy

value: under therapy


title: No17inf

description: Under therapy

value: 1 No17RVR


title: No18inf

description: Under therapy

value: Liver biopsy

value: 1 No18 nonRVR


title: No21pre

description: Before therapy


title: No20pre

description: Before therapy


title: No15inf

description: Under therapy

value: 1 No15 nonRVR


title: No17pre

description: Before therapy


title: No23inf

description: Under therapy

value: 1 No23 nonRVR


title: No16inf

description: Under therapy

value: 1 No16RVR


title: No19inf

description: Under therapy


title: No18pre

description: Before therapy


title: No14inf

description: Under therapy

value: 1 No14 nonRVR


title: No13inf

description: Under therapy

value: 1 No13 nonRVR


title: No15pre

description: Before therapy


title: No12inf

description: Under therapy

value: 1 No12 nonRVR


title: No14pre

description: Before therapy


title: No16pre

description: Before therapy


title: No13pre

description: Before therapy


title: No11pre

description: Before therapy

value: 1 No11RVR


title: No9inf

description: Under therapy

value: 1 No9 nonRVR


title: No23pre

description: Before therapy


title: No11inf

description: Under therapy


title: No12pre

description: Before therapy


title: No7inf

description: Under therapy

value: 1 No7RVR


title: No8pre

description: Before therapy

value: 1 No8 nonRVR


title: No9pre

description: Before therapy


title: No10pre

description: Before therapy

value: 1 No10RVR


title: No10inf

description: Under therapy


title: No6pre

description: Before therapy

value: 1 No6 nonRVR


title: No7pre

description: Before therapy

value: before therapy


title: No8inf

description: Under therapy

value: under therapy


title: No5inf

description: Under therapy

value: 1 No5 nonRVR


title: ID0211

city: Muenster

first: Jochen

last: Seggewiss

address: Roentgenstr. 21

department: Integrated Functional Genomics

institute: University of Muenster

hyb_protocol: As recommended by the manufacturer (SCIENION AG, Berlin, Germany).

scan_protocol: The hybridized microarrays were scanned with a GenePix 4000B microarray scanner (MDS Analytical Technologies GmbH, Ismaning, Germany). A geometric raster was laid over the resulting microarray picture to distinguish the signals from the background.

data_processing: The hybridization patterns and intensities were quantitatively analyzed using the Imagene 6 software (BioDiscovery, El Segundo, CA). After localization of single spots, intensities and global background were calculated automatically. The replicates were averaged, and the spots identified by Imagene 6 (BioDiscovery) as flawed were omitted. The data set was normalized by application of the LOWESS algorithm. In a next step, the intensity values of all arrays for each time point as well as for all time points combined were used for t tests. Genes with a change of <0.5- or ≥2.0-fold were characterized as having significantly differing amounts of transcripts based on t tests with a P value cut-off of at least 0.05. Gene functions were assigned to the respective accession numbers and annotations as compiled on DOGAN, a web page for S. aureus N315 (http://www.bio.nite.go.jp/dogan/MicroTop?GENOME_ID=n315G1).

organism: Staphylococcus aureus

source_name: S. aureus wild-type strain, timepoint 2

label_protocol: cDNA was synthesized from total RNA with the LabelStar Array Kit from QIAGEN using the QIAGEN protocol with slight modifications: Random hexamer primer were used (Invitrogen, Karlsruhe, Germany) and Cy3- and Cy5-dCTP were purchased from Perkin-Elmer (Rodgau - Juegesheim, Germany).

extract_protocol: For RNA isolation, cultures (100 ml TSB in 500 ml shaking flasks as used in previous studies) were incubated in TSB at 37°C and 160 rpm starting at an OD578nm of 0.05 by inoculation from overnight cultures. Samples were collected and processed in triplicate to analyze at least three RNA samples for each strain and time point. Cells of the wild-type strain and the mutant strain were harvested after 150, 270, 375, 480, and 600 min as performed previously. Ten ml of both, the wild-type strain and the mutant strain cultures, were immediately mixed with 20 ml of RNAprotect (QIAGEN, Hilden, Germany), vortexed for 5 s, incubated for 5 min at room temperature, and pelleted by centrifugation for 10 min at 4,000 x g. The bacterial pellets were resuspended in 1 ml RNApro solution (Qbiogene, Heidelberg, Germany) and purified on a Matrix E column (Qbiogene). Cells were separated by mechanical lysis using a FastPrep Instrument (Qbiogene): once at 30 s, attitude of disruption 6.5, 30 s on ice, and once at 30 s, attitude 6.5. Further, RNA purification was performed using the RNeasy Mini Kit (QIAGEN) according to the manufacturer´s recommendations. Contaminating DNA in the RNA preparations was removed using DNase treatment as described by the manufacturer (QIAGEN). The RNA quantity and quality were determined by measurement of the concentration and absorbance at 260 and 280 nm (Eppendorf BioPhotometer; Hamburg, Germany) and by the Agilent 2100 Bioanalyzer using the RNA 6000 Nano Kit and the 2100 Expert Software (v. B.02.06) (all Agilent Technologies Deutschland GmbH, Boeblingen, Germany). Purified RNA was stored at –70°C.

source_name: S. aureus mutant strain, timepoint 2


title: ID0105

source_name: S. aureus wild-type strain, timepoint 5

source_name: S. aureus mutant strain, timepoint 5


title: ID0107

source_name: S. aureus wild-type strain, timepoint 1

source_name: S. aureus mutant strain, timepoint 1


title: ID085

source_name: S. aureus wild-type strain, timepoint 4

source_name: S. aureus mutant strain, timepoint 4


title: No5pre

description: Before therapy

value: Liver biopsy


title: No4inf

description: Under therapy

value: 1 No4 nonRVR


title: No4LCMPpre

description: Before therapy

value: Liver biopsy LCM

value: 1 nonRVR No4

value: lymphocytes


title: No4pre

description: Before therapy


title: No6inf

description: Under therapy


title: No3pre

description: Before therapy

value: 1 No3RVR


title: No2inf

description: Under therapy

value: 1 No2RVR


title: No2pre

description: Before therapy


title: ID0207

source_name: S. aureus wild-type strain, timepoint 3

source_name: S. aureus mutant strain, timepoint 3


title: ID0206


title: No1inf

description: Under therapy

value: 1 No1RVR


title: No3inf

description: Under therapy


title: ID0209


title: ID0103


title: ID084

hyb_protocol: As recommended by the manufacturer (SCIENION AG, Berlin, Germany)

scan_protocol: The hybridized microarrays were scanned with a GenePix 4000B microarray scanner (MDS Analytical Technologies GmbH, Ismaning, Germany). A geometric raster was laid over the resulting microarray picture to distinguish the signals from the background. After localization of single spots, intensities and global background were calculated automatically.


title: No20LCMHinf

description: Under therapy

value: 1 nonRVR No20

value: hepatocytes


title: C0D4R3_vs_C5D4R2

city: Antwerpen

first: Dries

last: Knapen

address: Groenenborgerlaan 171

country: Belgium

institute: University of Antwerp

description: Microarray is part of a loop design.

hyb_protocol: Agilent's two-colour microarray-based gene expression analysis protocol version 5.7.

data_processing: Data analysis was performed using the R package limma. Background correction: normexp. Normalization: loess.

organism: Danio rerio

source_name: cadmium 0 degrees C day 4 replicate 3

extract_protocol: Trizol RNA extraction according to Invitrogen's instructions

value: liver

source_name: cadmium 5 degrees C day 4 replicate 2


title: ID0208


title: No1pre

description: Before therapy


title: T26D4R1_vs_T34D28R2

description: Microarray is part of a loop design.

scan_protocol: Agilent's two-colour microarray-based gene expression analysis protocol version 5.7.

source_name: temperature 26 degrees C day 4 replicate 1

source_name: temperature 34 degrees C day 28 replicate 2


title: T26D4R3_vs_T34D4R2

description: Microarray is part of a loop design.

source_name: temperature 26 degrees C day 4 replicate 3

label_protocol: Agilent's two-colour microarray-based gene expression analysis protocol version 5.7.

source_name: temperature 34 degrees C day 4 replicate 2


title: T34D4R1_vs_T26D4R3

description: Microarray is part of a loop design.

source_name: temperature 34 degrees C day 4 replicate 1


title: T26D4R2_vs_T34D4R1

description: Microarray is part of a loop design.

source_name: temperature 26 degrees C day 4 replicate 2


title: T18D28R2_vs_T26D28R1

description: Microarray is part of a loop design.

source_name: temperature 18 degrees C day 28 replicate 2

source_name: temperature 26 degrees C day 28 replicate 1


title: T34D28R1_vs_T18D28R1

description: Microarray is part of a loop design.

source_name: temperature 34 degrees C day 28 replicate 1

source_name: temperature 18 degrees C day 28 replicate 1


title: C5D4R1_vs_C0D4R3

description: Microarray is part of a loop design.

source_name: cadmium 5 degrees C day 4 replicate 1


title: C5D4R2_vs_C0D4R2

description: Microarray is part of a loop design.

source_name: cadmium 0 degrees C day 4 replicate 2


title: C0D28R2_vs_C5D28R1

description: Microarray is part of a loop design.

source_name: cadmium 0 uM day 28 replicate 2

source_name: cadmium 5 uM day 28 replicate 1


title: T34D4R2_vs_T26D4R2

description: Microarray is part of a loop design.


title: C0D4R2_vs_C5D4R1

description: Microarray is part of a loop design.


title: T26D28R1_vs_T34D28R1

description: Microarray is part of a loop design.


title: T26D28R2_vs_T34D28R1

description: Microarray is part of a loop design.

source_name: temperature 26 degrees C day 28 replicate 2


title: T34D28R2_vs_T26D28R2

description: Microarray is part of a loop design.


title: T26D28R2_vs_T18D28R2

description: Microarray is part of a loop design.


title: T18D28R1_vs_T26D4R1

description: Microarray is part of a loop design.


title: C1D28R2_vs_C0D28R1

description: Microarray is part of a loop design.

source_name: cadmium 1 uM day 28 replicate 2

source_name: cadmium 0 uM day 28 replicate 1


title: Csac grown on xylan vs xyloglucan

status: Public on Dec 29 2009

city: Raleigh

first: Amy

last: VanFossen

state: NC

address: 840 Main Campus Dr.  Rm3305

department: Chemical and Biomolecular Engineering

institute: NC State Unversity

description: Scatterplots were used to verify overall quality of the data making sure both channels are equal.

hyb_protocol: Sample preparation steps for microarray hybridization: Prepare Prehyb buffer: 225mL water, 75mL 20X SSC, 0.3g SDS, 0.3g BSA. Preheat Prehyb buffer 45 min 42 degree C. Prehybidize slides at 42 degree C for 45 min. Wash slides by dipping 5 time in sterile dH2O at RT. Dip twice in isopropanol at RT. Dry slide in slide spinner. Add 1 ul COT1-DNA (20mg/ml) to samples. Heat probe mixture to 95oC in heat block for 2 min to denature DNA, do a quick spin. Add 30 ul of 2x hyb. buffer that has been preheated to 42oC to each tube. Hybridization buffer:  27.2uL water, 20uL 20X SSC, 8uL 50X Denhardts solution, 24uL formamide, 0.8uL 10% SDS. Apply hyb. mix to slide and cover with a 20x60 mm polyethylene hydrophobic coverslip. Place slide in water proof container, cover well with foil and place in 42 degree C bath for 18-20 hours. The following is a standard hybridization method that seems to work well for this procedure: 1 wash cycle at 42 degree C with 2X SSC w/ 0.2% SDS. 1 wash cycle at RT with 0.5X SSC w/ 0.2% SDS. 3 wash cycles at RT with 0.5X SSC.

scan_protocol: Images were aquired with a scanarray 5000 (PE) scanner with 2 lasers. Channels were balanced using the line scan feature and by balancing the overall signals without the use of external standards.

data_processing: Signals for each channel were obtained using ScanArray express and were background subtracted using the median local intensity around the spot.  Once all the slides were quantitated, data from the loop was analyzed with JMP Genomics 3.2 (SAS, NC), using a mixed effects ANOVA model [Wolfinger et al. J Comput Biol 8 625-637 (2001)].

label: Cy™3 dye (GE Healthcare)

organism: Caldicellulosiruptor saccharolyticus

label_protocol: Producing cDNA: In a sterile PCR tube add 20 ug RNA with 6 ug random primers. Then add nuclease free water so the volume is 10 uL. Incubate at 70 degree C for 10 min. Place tube on ice for 1 min. Add 8 uL of RT Master Mix (recipe below) to each tube and swirl to mix.  Quick spin before incubation. RT Master Mix: 4uL 5X strand buffer, 1uL 0.1M DTT, 1uL dNTP mix (see below), 2uL superscript III  [Invitrogen]. dNTP mix:  3uL dATP (100mM), 3uL dGTP (100mM), 3uL dCTP(100mM), 1.8uL dTTP (100mM), 2.4uL aa-dUTP(50mM) [Ambion], 6.8uL nuclease free water. Incubate at 46 degree C for at least 3 hours. Hydrolyze RNA by adding 10uL 1N NaOH to each tube. To stop reaction add 10 uL 0.5M EDTA to each tube. Mix and incubate at 65 degree C for 15 min. Add 10uL 1N HCl to each tube neutralize reaction mix. Transfer samples into labeled tubes of appropriate sizes. Add five volumes PB Buffer (Qiagen) to each tube and mix well. Add 300 ul of RT product to a labeled Qiagen PCR cleanup column. Spin at 5,000 rpm for 15 sec., empty collection tube, and repeat until all of the solution has passed through the column. Complete wash One, add 750 ul of phosphate wash buffer (recipe below) to column and spin at 13,000 rpm for 1 min., empty tube. Complete wash Two, add 750 ul of phosphate wash buffer to column and spin at 13,000 rpm for 1 min., empty tube. Phosphate wash buffer: 0.1mL 1M KPO4( pH 8.5), 3.05mL autoclaved dH2O and 16.85mL 95% EtOH. Spin an additional 1 min. with the same collection tube and discard the flow through. Transfer to new 1.5 mL tube, add 60uL phosphate elution buffer to the membrane and incubate at room temp. for 1 min.  Spin at 13,000 rpm. Phosphate elution buffer: 4mM KPO4. Repeat phosphate elution into same tube using 60uL. Fluorescent Labeling: Dry all RT samples completely in speed vac. Resuspend each dried sample cDNA in 4.5 uL of 0.1M sodium carbonate buffer, pH 9.0.  Mix up and down with pipette for good mixing. Dissolve and mix thoroughly the Cy™ dye to be used with 20uL of DMSO. Typically Cy dyes contain four applications in each vial. Mix 4.5 uL of dye with prepared cDNA sample. Incubate in the dark for 2 hour @ R.T. Mix RT-PCR products with 35uL 100mM NaOAc PH 5.2 and mix by vortexing. Mix with 250uL Qiagen Buffer PB and filter through spin columns (13,000 rpm). Wash spin column  with 750 uL of Buffer PE (Qiagen). Spin an additional 1 min. with the same collection tube and discard the flow through. Elute labeled cDNA 2 times with 30 uL of EB Buffer (Qiagen). Combine different labeled probes Speed vacuum sample until dry (not too dry, i.e. don’t leave in speed vacuum for over 1 hour). Combine different labeled probes and speed vacuum sample until dry. Resuspend samples in 29uL sterilized dH2O.

growth_protocol: C. saccharolyticus was subcultured (overnight) 7 times on the substrate of interest in modified DSMZ 640 medium before inoculating 0.4-liter culture containing 0.5 gram substrate per liter. Cells were grown at 70 °C until mid-logarithmic phase (cell density 3-5*10^-7)

extract_protocol: RNA Isolation Protocol: Collect all 400mL of  cells into precooled centrifuge tubes and cool down in a dry ice and ethanol back. Spin down 10 min at 6500 rpm at 4 ºC. Store pellets at -80 ºC (at least overnight). Resuspend in 1 ml Trizol and transfer to new eppindoff tube.  Then sonicate for 10 seconds and incubate for 5 min at room temperature. Add 0.2 ml chloroform, shake for 15 second and incubate for 5 min at room temperature. Spin down for 15 min at 10.000 rpm at 4 ºC. Transfer aqueous phase to new tube. Add an equal amount of 70% EtOH. Transfer the sample to an RNeasy spin column. Close lid and centrifuge for 15 sec at 10.000 RPM. After centrifugation, carefully remove the spin column form the collection tube so that the column does not contact the flow-through. Pipet the DNase I incubation mix (10 ul DNase I stock and 70 ul RDD buffer) directly onto the RNeasy membrane and incubate at room temperature for 15 min. Pipet 350 ul RW1 into the RNeasy column, incubate 5 min. and centrifuge 15s at 10,000 RPM. Wash with 700 ul RW1 and throw away the collection tube. Take a collection new tube and wash column twice with 500 ul RPE. Centrifuge the column 1 min at 10.000 RPM. Place the column onto a new tube and elute twice with 30 ul RNase free water, incubate for 5 min at room temperature. Store at -80°C.

value: whole cells

label: Cy™5 dye (GE Healthcare)


title: C0D28R2_vs_C1D28R2

description: Microarray is part of a loop design.


title: R1881, biological rep1

status: Public on Dec 30 2009

city: Ann Arbor

first: Adaikkalam

last: Vellaichamy

state: MI

address: 1400 E. Med. Center Dr.

institute: University of Michigan

description: Gene expression data from R1881 treated cells

hyb_protocol: Following fragmentation, 20 ug of cRNA were hybridized for 16 hr at 45C on GeneChip Human U133 Plus 2.0 Array. GeneChips were washed and stained in the Affymetrix Fluidics Station 450.

scan_protocol: GeneChips were scanned using the Affymetrix 3000 7G GeneChip Scanner with Autoloader.

data_processing: Probe intensity values were converted to expression values using the Robust Multi-Array Average (RMA) method following Irizarry et al (Irizarry, RA, Hobbs, B, Collin, F, Beazer-Barclay, YD, Antonellis, KJ, Scherf, U, Speed, TP (2003) Exploration, Normalization, and Summaries of High Density Oligonucleotide Array Probe Level Data. Biostatistics. Vol. 4, Number 2: 249-264). Log2 transformed expression values were obtained. This was implemented in the R statistical language using the affy package of Bioconductor.

source_name: LNCaP cells treated with R1881

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

growth_protocol: LNCaP cells were grown in RPMI 1640 medium at 37C, under 5% CO2 and 90% humidity

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

treatment_protocol: Cells were deprived of androgen in RPMI medium containing charcoal stripped serum, and treated with 1nM of R1881 for 48h

value: metastistic prostate cancer cell, androgen receptor positive

tag: cell line

value: LNCaP


title: Csac grown on xyloglucan vs xyloglucan oligosaccharides


title: Csac grown on xyloglucan oligosaccharides vs xylose


title: C0D4R1_vs_C5D28R2

description: Microarray is part of a loop design.

source_name: cadmium 0 uM day 4 replicate 1

source_name: cadmium 5 uM day 28 replicate 2


title: Csac grown on glucose vs xylan


title: C1D28R1_vs_C0D4R1

description: Microarray is part of a loop design.

source_name: cadmium 1 uM day 28 replicate 1


title: C5D28R1_vs_C1D28R1

description: Microarray is part of a loop design.


title: C0D28R1_vs_C5D28R1

description: Microarray is part of a loop design.


title: R1881, biological rep2


title: Csac grown on xylose vs sugar mixture

scan_protocol: Images were aquired with a Packard BioChip Scanarray 4000 scanner with 2 lasers. Channels were balanced using the line scan feature and by balancing the overall signals without the use of external standards.

label_protocol: Producing cDNA: In a sterile PCR tube add 10 ug RNA from two different RNA isolation samples with 6 ug random primers. Then add nuclease free water so the volume is 10 uL. Incubate at 70 degree C for 10 min. Place tube on ice for 1 min. Add 8 uL of RT Master Mix (recipe below) to each tube and swirl to mix.  Quick spin before incubation. RT Master Mix: 4uL 5X strand buffer, 1uL 0.1M DTT, 1uL dNTP mix (see below), 2uL superscript III  [Invitrogen]. dNTP mix:  3uL dATP (100mM), 3uL dGTP (100mM), 3uL dCTP(100mM), 1.8uL dTTP (100mM), 2.4uL aa-dUTP(50mM) [Ambion], 6.8uL nuclease free water. Incubate at 46 degree C for at least 3 hours. Hydrolyze RNA by adding 10uL 1N NaOH to each tube. To stop reaction add 10 uL 0.5M EDTA to each tube. Mix and incubate at 65 degree C for 15 min. Add 10uL 1N HCl to each tube neutralize reaction mix. Transfer samples into labeled tubes of appropriate sizes. Add five volumes PB Buffer (Qiagen) to each tube and mix well. Add 300 ul of RT product to a labeled Qiagen PCR cleanup column. Spin at 5,000 rpm for 15 sec., empty collection tube, and repeat until all of the solution has passed through the column. Complete wash One, add 750 ul of phosphate wash buffer (recipe below) to column and spin at 13,000 rpm for 1 min., empty tube. Complete wash Two, add 750 ul of phosphate wash buffer to column and spin at 13,000 rpm for 1 min., empty tube. Phosphate wash buffer: 0.1mL 1M KPO4( pH 8.5), 3.05mL autoclaved dH2O and 16.85mL 95% EtOH. Spin an additional 1 min. with the same collection tube and discard the flow through. Transfer to new 1.5 mL tube, add 60uL phosphate elution buffer to the membrane and incubate at room temp. for 1 min.  Spin at 13,000 rpm. Phosphate elution buffer: 4mM KPO4. Repeat phosphate elution into same tube using 60uL. Fluorescent Labeling: Dry all RT samples completely in speed vac. Resuspend each dried sample cDNA in 4.5 uL of 0.1M sodium carbonate buffer, pH 9.0.  Mix up and down with pipette for good mixing. Dissolve and mix thoroughly the Cy™ dye to be used with 20uL of DMSO. Typically Cy dyes contain four applications in each vial. Mix 4.5 uL of dye with prepared cDNA sample. Incubate in the dark for 2 hour @ R.T. Mix RT-PCR products with 35uL 100mM NaOAc PH 5.2 and mix by vortexing. Mix with 250uL Qiagen Buffer PB and filter through spin columns (13,000 rpm). Wash spin column  with 750 uL of Buffer PE (Qiagen). Spin an additional 1 min. with the same collection tube and discard the flow through. Elute labeled cDNA 2 times with 30 uL of EB Buffer (Qiagen). Combine different labeled probes Speed vacuum sample until dry (not too dry, i.e. don’t leave in speed vacuum for over 1 hour). Combine different labeled probes and speed vacuum sample until dry. Resuspend samples in 29uL sterilized dH2O.


title: Control, biological rep1

description: Gene expression data from vehicle treated cells

source_name: LNCaP cells treated with ethanol as vehicle


title: Csac grown on sugar mixture vs xylose


title: Control, biological rep3


title: Control, biological rep2


title: s73_1.4.006

city: Cologne

first: Fernando

last: Andres

address: Carl von Linne 10

department: Plant Developmental Biology

institute: Max Planck Institute for Plant Breeding Research

description: Cy3-wt, Cy5-mutant

hyb_protocol: From http://ag.arizona.edu/microarray/Microarraymethod1.doc The hybridization buffer was prepared mixing both targets (1 ug of each one) with 6 ul of 20X SSC, 3.6 ul of Liquid Block (Amersham; Cat # RPN3601) in a final volume of 60 ul. The labeled target was denatured by incubating tube at 65o C for 5 min and transfered tube to ice immediately or apply on to the slides directly. The pre-heated sample was applied to the lifterslip and the chamber was sealed and placed into a hybridization oven at 55oC (12 h).

scan_protocol: The hybridized microarray slides were washed and scanned using a Gene Pix Autoloader (Axon/Molecular Devices, 4200A01, Sunnyvale, CA). Spot finding and data extraction was done using GenePix Pro 6 software (Axon/Molecular Devices, Sunnyvale, CA).

data_processing:
Quality control, normalization and the determination of differentially expressed genes were performed in R using the Limma package (Smyth, 2005) of Bioconductor (http://www.bioconductor.org), with local background subtraction and Lowess normalization performed for each microarray slide.

The final statistical analysis of this study is available in the 'GSE16796_stat_analysis.txt' file, which is linked to the Series GSE16796 record as a supplementary file.

organism: Oryza sativa

source_name: Bahia Wild Type

label_protocol: For the dye coupling reaction, 10 μg of aRNA of each sample were used following the “Dye Coupling and Labeled aRNA Cleanup” protocol (Ambion).

growth_protocol: The plants were cultured in controlled growth rooms during 3 weeks under photoperiodic conditions of 12 h light and 12 h dark and then were transfered to long-day conditions (14 h light and 10 h dark) for an additional week. The samples were harvested 1 hour after the beginning of the period of light.

extract_protocol: Total RNAs were isolated with the RNeasy plant mini kit (QIAgen, Hilden, Germany; Ref. 74904), following the manufacturer’s instructions. The aRNA amplification and labeling were performed with the AminoAllyl MessageAmpTM II aRNA Amplification kit (Ambion) according to the manufacturer’s instructions. One microgram of total RNA of each sample was used for the amplification. The final concentration of obtained aRNA was determined with NanoDrop ND1000 Spectrophotometer.

tag: cultivar

value: Bahia

value: wild type

tag: age

value: 4 weeks

source_name: S73 mutant

value: s73 mutant

tag: generation

value: gamma rays 250 Gy


title: s73_1.4.007

description: Cy5-wt, Cy3-mutant


title: s73_1.4.009

description: Cy5-wt, Cy3-mutant


title: s73_1.4.008

description: Cy3-wt, Cy5-mutant


title: Csac grown on mannose vs glucose


title: Csac grown on xylose vs fructose


title: Csac grown on xylose vs glucose


title: Csac grown on galactose vs arabinose


title: Csac grown on glucose vs sugar mixture


title: Csac grown on glucose vs galactose


title: Csac grown on sugar mixture vs glucose


title: cow_7743_PMN_NEB_cy5

status: Public on Dec 25 2009

city: Tjele

first: Kasey

middle: M

last: Moyes

address: Blichers Allé 20, P.O. BOX 50

department: Animal Health, Welfare, and Disease

country: Denmark

institute: University of Aarhus

description: 7743 24-5

hyb_protocol: A 13,257 oligonucleotide microarray was used for gene expression profiling (Loor et al., 2007). Prior to hybridization, slides were denatured in boiling water, rinsed in ethanol, and blocked in prehybridization buffer. Probes, consisting of the experimental sample and reference (control), were denatured by boiling and hybridized to the array under cover slips for 2 d at 42 oC. Slides were then washed for 4 min each in 1 x SSC 0.03% SDS, 0.2 x SSC, 0.1 x SSC, and 0.05 x SSC prior to scanning. All samples were hybridized to duplicate slides and repeated with reverse labeling for a total of 2 slides, or 4 spots per gene per experiment.

scan_protocol: Slides were scanned for both dye channels (Cy3 and Cy5) with a Scanarray 4000 (GSI-Lumonics, Billerica,MA) dual-laser confocal scanner and images were edited and processed using GenePix 6.0 (Axon Instruments, Inc., CA).

data_processing: Oligonucleotides that were flagged with -100 by GenePix were removed from the analysis, and the remaining data were normalized to control for dye effects using the median of control elements on the microarray.  In a subsequent normalization step, the log2 normalized ratio of PMN versus reference (i.e., RNA mixture of  different bovine tissues) signal intensities were adjusted for global dye and PMN effects and normalized by Lowess.  A mixed-effects model was then fitted to the adjusted ratios (PMN/reference) using Proc MIXED of SAS (2003).  The model consisted of treatment (TRT; NEB and PEB).  The fixed effect was dye with cow and microarray as random effects.  Statistical significance probability values for TRT effects were adjusted for the number of comparisons using Benjamini and Hochberg’s FDR.  Differentially expressed genes were based on FDR P-value ≤ 0.05, which corresponded to an unadjusted P ≤ 0.01.

organism: Bos taurus

source_name: neutrophils, negative energy balance

label_protocol: Labelled with Cy3 and Cy5 during cDNA synthesis from total RNA using Superscript III Kit (Invitrogen) according to manufacturer's instructions. Samples were purified using the QIAGEN PCR purification kit before hybridization.

extract_protocol: Total RNA was extracted using TRIZol (Invitrogen) and cleaned using the RNeasy mini kit Column (QIAGEN).

tag: breed

value: Holstein

value: blood

value: neutrophil

tag: diet

value: restricted

tag: s. uberis infection

value: infected

source_name: Pooled RNA from bovine tissues

value: mixed

tag: sample type

value: reference standard


title: Csac grown on arabinose vs xylose


title: cow_7703_PMN_PEB_cy3

description: 7703 24-3

source_name: neutrophils, positive energy balance

value: ad libitum


title: cow_7697_PMN_NEB_cy3

description: 7697 24-3


title: cow_7750_PMN_PEB_cy3

description: 7750 24-3


title: cow_7743_PMN_NEB_cy3

description: 7743 24-3


title: cow_7703_PMN_PEB_cy5

description: 7703 24-5


title: cow_7698_PMN_PEB_cy3

description: 7698 24-3


title: cow_7783_PMN_PEB_cy3

description: 7783 24-3


title: cow_7750_PMN_PEB_cy5

description: 7750 24-5


title: cow_7697_PMN_NEB_cy5

description: 7697 24-5


title: cow_7783_PMN_PEB_cy5

description: 7783 24-5


title: cow_7585_PMN_NEB_cy3

description: 7585 24-3


title: cow_7698_PMN_PEB_cy5

description: 7698 24-5


title: Csac grown on fructose vs mannose


title: cow_7585_PMN_NEB_cy5

description: 7585 24-5


title: cow_7384_PMN_NEB_cy5

description: 7384 24-5


title: cow_7390_PMN_NEB_cy5

description: 7390 24-5


title: cow_7390_PMN_NEB_cy3

description: 7390 24-3


title: cow_7198_PMN_PEB_cy5

description: 7198 24-5


title: cow_7198_PMN_PEB_cy3

description: 7198 24-3


title: Eyl liver_rep3

status: Public on Dec 01 2009

city: Neuherberg

first: Martin

last: Irmler

state: Bayern

address: Ingolstaedter Landstrasse 1

department: Institute of Experimental Genetics

institute: Helmholtz Zentrum München GmbH

description: RNA isolation, Labelling, Hybridisation and Scanning procedure are described in the fields above

hyb_protocol: This hybridization mixture was placed on a prehybridised microarray, under a cover slip, placed into a hybridization chamber (Genetix) and immersed in a thermostatic bath at 42°C for at least 16 hours. After hybridization slides were washed in 40ml of 3x , 1x, 0.25x and 0.1x SSC at room temperature. For drying slides were placed in an empty 50 ml Falcon tube (Becton Dickinson, USA) and centrifuged at 4000m/s2. Beckers, J., Herrmann, F., Rieger, S., Drobyshev, A., Horsch, M., Hrabé de Angelis, M. and Seliger, B. (2005): Identification and validation of novel ERBB2 (Her2, NEU) targets including genes involved in angiogenesis. Int. J. Cancer 114: 590-597. Greenwood AD, Horsch M, Stengel A, Vorberg I, Lutzny G, Maas E, Schädler S, Erfle V, Beckers J, Schätzl H and Leib-Mösch C (2005): Cell Line Dependent RNA Expression Profiles of Prion-infected Mouse Neuronal Cells. JMB 349: 487-500 Seltmann M, Horsch M, Drobyshev A, Chen Y, Hrabé de Angelis M, Beckers J (2005): Assessment of a Systematic Expression Profiling. Approach in ENU-Induced Mouse Mutant Lines. Mam Genome 16 (1): 1-10

scan_protocol: Dried slides were scanned with a GenePix 4000A microarray scanner and the images were analyzed using the GenePix Pro3.0 image processing software (Axon Instruments, USA) Greenwood AD, Horsch M, Stengel A, Vorberg I, Lutzny G, Maas E, Schädler S, Erfle V, Beckers J, Schätzl H and Leib-Mösch C (2005): Cell Line Dependent RNA Expression Profiles of Prion-infected Mouse Neuronal Cells. JMB 349: 487-500 Seltmann M, Horsch M, Drobyshev A, Chen Y, Hrabé de Angelis M, Beckers J (2005): Assessment of a Systematic Expression Profiling. Approach in ENU-Induced Mouse Mutant Lines. Mam Genome 16 (1): 1-10

data_processing: Normalisation with GenePix Pro 6.1: Ratio based normalized data in each image so that the mean of ratio of means of all of the features is equal to 1. The VALUE column contains the log2 ratio of the result file after normalisation

label_protocol:
For labeling 20 µg of total RNA were used for reverse transcription and indirectly labeled with Cy3 or Cy5 fluorescent dye according the TIGR protocol (Hedge et al., 2000). Labeled cDNA was dissolved in 30 µl hybridization buffer (6x SSC, 0.5% SDS, 5x Denhardt’s solution and 50% formamide) and mixed with 30 µl of reference cDNA solution (pool from six control animals) labeled with the second dye.
HegdeP, Qi R, Abernathy R, Gay C, Dharap S, et al (2000): A concise guide to cDNA microarray analysis-II. Biotechniques 29: 548-562

extract_protocol: Total RNA was isolated just before processing for expression profiling. For preparation of total RNA individual organs were thawed in buffer containing chaotropic salt (RLT buffer, Qiagen) and homogenised using a Polytron homogeniser. Total RNA from individual samples was obtained according to manufacturer’s protocols using RNeasy Midi kits (Qiagen). The concentration was calculated from OD260/280 measurement and 2 µg RNA aliquots were run on a formaldehyde agarose gel to check for RNA integrity. The RNA was stored at -80°C in RNase free water (Qiagen).

tag: gender

value: male

value: 17 weeks

tag: strain

value: liver

source_name: wt pool

value: pool of 4 wildtype mice


title: Eyl liver_rep4

tag: reference


title: Eyl liver_rep1

source_name: Wt RNA pool


title: Eyl liver_rep5


title: Eyl liver_rep2


title: ECT 1

status: Public on Nov 01 2009

city: Ljubljana

first: Vid

last: Mlakar

address: Korytkova 2

department: Department for molecular genetics

country: Slovenia

institute: Faculty of Medicine

description: no additional information

hyb_protocol: All hybridizations were performed on HS400 (Tecan) according to the manufacturer’s instructions (Schott Nexterion). 2 micro g of both (Cy3 and Cy5) labeled aaRNA were disolved in 60 micro l hybridization buffer and used in hybridization. Wash 2, Wash 3, Wash 4 and 10 mg/ml BSA were provided in Nexterion 70mer Oligo Microarraying Kit (Schott Nexterion) Step   Temp   Wash (s)   Soak (s)   Agitation   Drying (s) Liquid 1         25          30          30                                     0,1% SDS 2         25          30           0                                      water 3         42          10          30 min                                10 mg/ml BSA 4         40          20           0                                      10 mg/ml BSA 5         40                                                                injection sample 6         40                       60 min     Medium                  hybridization 7         25          60          30                                      Wash 2 8         25          60          30                                      Wash 3 9         25          60          30                                      Wash 4 10

scan_protocol: LS200 laser scanner (Tecan) at 6 micro m resolution was used for scanning the microarrays at 543 nm (Cy3) and 633 nm (Cy5) excitation vawelenght. Scanner was operated using GenePro software.

data_processing:
We used Array-Pro Analyzer 4.5 (Media Cybernetics, Bethesda, MD, USA) for feature extraction after imaging of microarrays.
Out of each feature we extracted:
Median raw intensity for chanel 1 and 2
Mean raw intensity for chanel 1 and 2
Standard deviation of raw intensity for chanel 1 and 2
Median background intensity for chanel 1 and 2
Mean background intensity for chanel 1 and 2
Standard deviation of background intensity for chanel 1 and 2
Acuity (Molecular devices, USA) was used for filtration of bad signals, Lowess normalization and microarray data analysis. Features showing signal intensity of more than 65000 were flagged as bad. Features with signal less than 2 times the intensity of background or coefficient of variation (CV, ratio between standard deviation of the background and the median feature intensity) greater than 0.3 were considered not significantly expressed and were filtered out. Log2 ratios were normalized using LOWESS fit and median from four replicates was used to calculate average gene expression for single sample.

source_name: Malignant melanoma

label_protocol: Preparation of labeled aaRNA was performed with an Amino Allyl MessageAmpTM II aRNA Amplification Kit (Ambion) according to the manufacturer’s recommendations. For each hybridization, we labelled 5 micro g of tumour mRNA. After removing the excess dye, the RNAs were dissolved in Nexterion Hybridization solution (Schott Nexterion).

growth_protocol: Human malignant melanoma cells (SK-MEL28) were grown as monolayer in MEM with Glutamax (Gibco), supplemented with 10% FBS (Gibco) and gentamicin (30 μg/mL) (Gibco). Cells were routinely subcultured twice a week and incubated in an atmosphere with 5% CO2 at 37°C.

extract_protocol: RNA from cells was isolated using TRI REAGENTTM (Sigma Aldrich) and a PureLinkTM Micro-to-Midi Total RNA Purification System (Invitrogen), according to the manufacturer's instructions. Briefly, cells were trypsinized, washed in MEM with FBS for trypsin inactivation and resuspended in PBS. After centrifugation at 1500 x g for 5 min, all excess liquid was removed and 1 mL of TRI REAGENTTM was added to each sample. Samples were mixed by hand for 15 s and allowed to stand for 2 - 15 min at room temperature. The resulting mixture was centrifuged at 12000 x g for 15 min at 4 ºC. The aqueous phase was transferred to a fresh microcentrifuge tube and an equal amount of 70% ethanol was added. Samples were transferred to a PureLinkTM Micro-to-Midi Total RNA Purification System column (Invitrogen) and processed according to the manufacturer's protocol. All samples were washed from the column with 75 µl of RNAse free water.

value: Malignant melanoma cell line SK-MEL28

tag: protocol

value: Not treated

source_name: Malignant melanoma ECT

treatment_protocol: Confluent cell cultures were trypsinized, washed in MEM with FBS for trypsin inactivation and once in electroporation buffer (125 mM saccharose; 10 mM K2HPO4; 2,5 mM KH2PO4; 2 mM MgCl2·6H20) at 4°C. Final cell suspension was prepared in electroporation buffer at 4°C at a concentration of 22 x 106 cells/mL. Aliquots of final cell suspension (3 x 106 cells) were placed between two parallel electrodes and subjected to eight electric pulses for electrochemotherapy (electric field intensity 1300 V/cm, pulse duration 100 μs and frequency 1 Hz) or eight electric pulses for electrogenetherapy (electric field intensity 600 V/cm, pulse duration 5 ms and frequency 1 Hz). One aliquot of cell suspension was not subjected to any electric pulses and served as control treatment. After electroporation, cells were incubated at room temperature for 5 minutes, diluted in normal growth media and then plated in culture flasks for 16 hours.

value: Exposed to electrochemotherapeutic pulses


title: Cisplatin ECT 1

description: no additional information

treatment_protocol: no treatment

source_name: Malignant melanoma cisplatin ECT

treatment_protocol: Confluent cell cultures were trypsinized, washed in MEM with FBS for trypsin inactivation and once in electroporation buffer (125 mM saccharose; 10 mM K2HPO4; 2,5 mM KH2PO4; 2 mM MgCl2·6H20) at 4°C. Final cell suspension was prepared in electroporation buffer at 4°C at a concentration of 22 x 106 cells/mL. For clonogenic assay, 90 μL of final cell suspension was mixed with 10 μL of CDDP solution in concentration range from 2 to 80 μg/mL. 50 μL of the mixture (1 x 106 cells) was placed between two parallel electrodes and subjected to eight electric pulses with electric field intensity 1300 V/cm, pulse duration 100 μs and frequency 1 Hz. Other 50 μL of mixture (1 x 106 cells)  served as a control of CDDP treatment alone. For adhesion, migration and invasion assays, the volume of cell suspension subjected to electric pulses was 100 μL (2 x 106 cells). After electroporation cells were incubated at room temperature for 5 minutes, dilluted in 2 mL of growth media and then plated for clonogenic assay. For adhesion, migration and invasion assays, cells were plated in petri dishes for 48 h in MEM with serum.

value: Treated with cisplatin and electroporation


title: PHBE mock vs. PHBE RSV 24h

status: Public on Nov 16 2009

city: RTP

first: NIEHS

last: Microarray Core

address: 111 T.W. Alexander Drive

department: DIR

institute: NIEHS

description: Primary human bronchial epithelial cells were left uninfected or were infected with RSV at multiplicity of infection (MOI) of 2.5 plaque forming unit (pfu)/cell. After 24 hr total cellular RNA was isolated using the TRIzol reagent (Invitrogen, Carlsbad, CA) according to the manufacturers’ instructions.

hyb_protocol: For each two color comparison, 750ng of each Cy3 and Cy5 labeled cRNAs were mixed and fragmented using the Agilent In Situ Hybridization Kit protocol.  Hybridizations were performed for 17 hours in a rotating hybridization oven using the Agilent 60-mer oligo microarray processing protocol.

scan_protocol: Slides were washed as indicated in the Agilent 60-mer oligo microarray processing protocol and then scanned with an Agilent Scanner. Data was obtained using the Agilent Feature Extraction software (v9.1), using defaults for all parameters.

data_processing: Data was obtained using the Agilent Feature Extraction software (v9.1), using defaults for all parameters and loaded into GeneSpring (v7.3).

source_name: PHBE mock

label_protocol: Total RNA was amplified using the Agilent Low RNA Input Fluorescent Linear Amplification Kit protocol.  Starting with 500ng of total RNA, Cy3 labeled cRNA was produced according to manufacturer’s protocol.

growth_protocol: The primary human bronchial epithelial (PHBE) cells were grown as a monolayer at 37oC in a 5% CO2 humidified chamber in a serum-free epithelial cell growth medium .

extract_protocol: Total cellular RNA was isolated using the TRIzol reagent (Invitrogen, Carlsbad, CA).

tag: Uninfected Normal Human Bronchial Epithelial Cells

source_name: PHBE RSV 24h

label_protocol: Total RNA was amplified using the Agilent Low RNA Input Fluorescent Linear Amplification Kit protocol.  Starting with 500ng of total RNA, Cy5 labeled cRNA was produced according to manufacturer’s protocol.

tag: Normal Human Bronchial Epithelial Cells infected with 2.5 plaque forming units (pfu)/cell for 24 hours.