title: HUES6 hESC, rep2

city: La Jolla

first: Alysson

middle: R

last: Muotri

state: CA

address: 9500 Gilman Drive

department: Pediatrics/CMM

institute: University of California San Diego

description:
Gene expression data.
HUES6-2

hyb_protocol: According to the standard Affymetrix protocol (GeneChip Expression Wash, Stain and Scan User Manual, P/N 702731 Rev. 3).

data_processing: Gene-level signal estimates were derived from the CEL files by RMA-sketch normalization as a method in the apt-probeset-summarize program (see Yeo et al. 2007; PMID 7967047). Hierarchical clustering of the full dataset by probeset values was performed by complete linkage using Euclidean distance as a similarity metric in Matlab.

source_name: HUES6 hESC

label_protocol: GeneChip Whole Transcript (WT) Sense Target Labeling Assay.

growth_protocol: Human ESC and iPSC were grown in feeder-free conditions, on matrigel-coated dishes (BD) using mTeSR1. Fibroblasts were grown on DMEM plus 10% FBS.

extract_protocol: Total cellular RNA was extracted from ~5x10^6 cells using the RNeasy Protect Mini kit (Qiagen, Valencia, CA) according to the manufacturer's instructions.

treatment_protocol: Cells were collected at 50% confluency. For hESC and hiPSC, we first removed any differentiated cell on the plate by manual microdissociation based on morphology.

value: embryonic stem cells (ESC)


title: iPSC-WT clone 2, rep3

status: Public on Nov 11 2010

type: RNA

country: USA

description:
Gene expression data.
WT_ips2-3

scan_protocol: According to the standard Affymetrix protocol (GeneChip Expression Wash, Stain and Scan User Manual, P/N 702731 Rev. 3).

label: biotin

molecule: total RNA

organism: Homo sapiens

source_name: iPSC-WT clone 2

tag: cell type

value: induced pluripotent stem cells (iPSC)

tag: gender

value: female

tag: patient

value: control


title: NT3

city: Hong Kong

first: William

last: Niu

address: #21, Rd. Sassoon

department: Dept. Biochemistry

country: Hong Kong

institute: The University of Hong Kong

description: gene expression data from early passage mouse embryonice fibroblsts

hyb_protocol: According to The GeneChip® Mouse Genome 430 2.0 protocol.  Array

data_processing: GCOS

organism: Mus musculus

source_name: Zmpste24-/- mouse embryonic fibroblasts (P3)

growth_protocol: Three pairs of wild-type and Zmpste24-/- MEFs were generated from E13.5 embryos at passage 0, subcultured into passage 3 and collected for total RNA extraction.

extract_protocol: RNA was isolated by TRIzol (Invitrogen) following the manufacturer protocol.

treatment_protocol: A total of 5 x 105 MEFs were plated on multiple 100-mm dishes for each line in 10% fetal calf serum and Dulbecco's Modified Eagle's Medium (DMEM).

tag: strain

tag: genotype/variation

value: mouse embryonic fibroblasts


title: iPSC-WT clone 1, rep2

description:
Gene expression data.
WT_ips1-2

source_name: iPSC-WT clone 1


title: iPSC-WT clone 1, rep3

description:
Gene expression data.
WT_ips1-3


title: HUES6 hESC, rep3

description:
Gene expression data.
HUES6-3


title: iPSC-WT clone 2, rep2

description:
Gene expression data.
WT_ips2-2


title: WT fibroblast, rep2

description:
Gene expression data.
WT-2

source_name: WT fibroblast

value: fibroblast


title: iPSC-RTT clone 18, rep3

description:
Gene expression data.
Rett_1155del32_ips18-3

source_name: iPSC-RTT clone 18

value: Rett syndrome


title: WT fibroblast, rep3

description:
Gene expression data.
WT-3


title: HUES6 hESC, rep1

description:
Gene expression data.
HUES6-1


title: WT fibroblast, rep1

description:
Gene expression data.
WT-1


title: iPSC-WT clone 1, rep1

description:
Gene expression data.
WT_ips1-1


title: iPSC-WT clone 2, rep1

description:
Gene expression data.
WT_ips2-1


title: iPSC-RTT clone 15, rep3

description:
Gene expression data.
Rett_1155del32_ips15-3

source_name: iPSC-RTT clone 15


title: MG1363 versus NZ9000 in GM17

city: Groningen

first: Anne

middle: de

last: Jong

address: Nijenborgh 7

department: Molecular Genetics

country: Netherlands

institute: university of Groningen

description: Hybridization of strain MG1363 and NZ9000 grown in CDM

hyb_protocol: Hybridization (16 h at 45 °C) of labeled cDNA was performed in Ambion Slidehyb #1 hybridization buffer (Ambion Europe) on superamine glass slides (Array-It; SMMBC), containing technical replicates.

scan_protocol: Slides were scanned using the Genepix 4200AL DNA micro array slide scanner. Quantification of the signal of the spots on slides was done with ArrayPro 4.5.

data_processing: Slide data were processed by using MicroPreP as described previously (10). Prior to the analysis, automatically and manually flagged spots and spots with very low background-subtracted signal intensities (5% of the weakest spots [sum of Cy3 and Cy5 net signals]) were filtered out. Net signal intensities were calculated by using grid-based background subtraction. In postprep, negative and empty values were eliminated, and outliers were removed by the deviation test. Differential expression tests were performed by using a Cyber-T Student's t test for paired data (19).

organism: Lactococcus cremoris subsp. cremoris MG1363

label_protocol: Synthesis of cDNA and indirect Cy-3/Cy-5-dCTPs labeling of 15–20 µg of total RNA was performed with the CyScribe Post labeling kit (Amersham Biosciences) according to the supplier's instructions.

extract_protocol: RNA was isolated from two replicate cultures of both L. lactis ΔlmrCD and cholate-adapted L. lactis ΔlmrCDR. Cultures were grown at 30 ºC in GM17 medium and cells were harvested at the OD660 of ~ 1.RNA was isolated and purified using the High Pure RNA isolation kit (Roche diagnostics) as described (PMID: 17085554). Contaminating genomic DNA was removed by treatment with RNase-free DNase I (Roche diagnostics). Synthesis, subsequent labeling of cDNA, and microarray hybridization was performed as described (PMID: 17085554 and 15907200). In all cases, dye-swapping was performed.

tag: culture medium

organism: Lactococcus cremoris subsp. cremoris NZ9000


title: MG1363 versus NZ9000 in CDM

value: CDM


title: HRPE-rep1

city: Bethesda

first: R

middle: Krishnan

last: Kutty

state: MD

address: 6 Center Dr., Room 112

department: National Eye Institute

institute: National Institutes of Health

description: Biological replicate 1 of 2

hyb_protocol: hybridization performed using dual-channel microarray μParafloTM microfluidic chip containing miRNA transcripts (LC Sciences)

scan_protocol: Axon GenePix 4000B Microarray Scanner

data_processing: LOWESS normalized, background subtracted

source_name: Control HRPE cells

label_protocol: Control labeled with Cy3. Treated labeled wit Cy5

growth_protocol: Human retinal pigment epithelial (HRPE) cell cultures established from adult donor eyes were grown in a medium containing 10% featal bovine serum and was used between passages 7 and 10

extract_protocol: RNA extracted with Ambion mirVana miRNA Isolation Kit

treatment_protocol: Cells were treated with an inflammatory cytokine mix containing IFN-γ (100 u/ml), TNF-α (10 ng/ml) and IL-1β (10 ng/ml) for 16 hours in a serum-free medium

value: Human retinal pigment epithelial (HRPE) cells

tag: treatment group

source_name: Treated HRPE cells


title: iPSC-RTT clone 15, rep1

description:
Gene expression data.
Rett_1155del32_ips15-1


title: PBMCs from vasculitis patients 13

status: Public on Nov 10 2010

city: Suita

first: Daisuke

last: Okuzaki

state: Osaka

address: Yamadaoka 3-1

department: Immunology Frontier Research Center

country: Japan

institute: Osaka univ.

description: Genes expressed in peripheral blood mononuclear cells from patients with vasculitis.

hyb_protocol: Briefly, 1125 ng labeled cRNA was fragmented according to the manufacturer's protocol (Agilent Technologies), was suspended in a hybridization buffer containing 0.12 M Tris-HCl (pH 7.5), 0.12 M NaCl, and 0.05% Tween 20. The hybridization mixture was heated to 94°C for 2 min, then cooled to 37°C and injected into a hybridization chamber (Mitsubishi Rayon). Hybridization was carried out at 65°C for 16 hr. Microarrays were washed by soaking twice in 0.12 M Tris-HCl (pH 7.5), 0.12 M NaCl, 0.05% and Tween 20 at 65°C for 20 min, then once in 0.12 M Tris-HCl (pH 7.5), 0.12 M NaCl at 65°C for 10 min.

scan_protocol: Microarray was scanned and the image was captured using a cooled charge-coupled device (CCD)-type Microarray Image Analyzer equipped with multibeam excitation technology (Yokogawa Electric Co., Tokyo, Japan).

data_processing: Data analysis with background subtraction and normalization to　alpha-tubulin intensity was conducted using the GeneSpring GX software version 10.0.2, according to the manufacturer’s protocol (Agilent Technologies).

source_name: Peripheral blood cells from vasculitis patient 13

label_protocol: RNA labeling was performed with a Fluorescent Linear Amplification Kit (Agilent Technologies). In brief, cDNA was reverse transcribed from 500ng of total RNA (derived from vasculitis patients 1 with an oligo-dT-T7 promoter primer and MMLV-RT. Second strand synthesis was carried out with random hexamers. Fluorescent antisense cRNA was synthesized with cyanine 5-CTP (Cy5-CTP) and T7 polymerase. The fluorescent-labeled antisense cRNA was purified with RNeasy columns (Qiagen) according to manufacturer's protocols and resuspended in water. The purified products were quantified at A650nm for Cy5-CTP with Agilent 2100 Bioanalyzer (Agilent Technologies).

extract_protocol: Peripheral blood was collected directly into PAXGene tubes (Qiagen, Valencia, CA) and stored at -80℃ until extraction. Total RNA from mononuclear cells was extracted using PAXGene Blood RNA kit with the optimal on-column DNase digestion according to the manufacturer's instructions.

tag: age

tag: disease status

value: vasculitis


title: PBMCs from 16 healthy controls

source_name: Peripheral mononuclear blood cells from 16 healthy controls

value: healthy controls without any identified diseases

value: 9 females and 7 males


title: iPSC-RTT clone 15, rep2

description:
Gene expression data.
Rett_1155del32_ips15-2


title: iPSC-RTT clone 18, rep1

description:
Gene expression data.
Rett_1155del32_ips18-1


title: PBMCs from vasculitis patients 10

source_name: Peripheral blood cells from vasculitis patient 10


title: PBMCs from vasculitis patients 9

source_name: Peripheral blood cells from vasculitis patient 9

value: male


title: PBMCs from vasculitis patients 8

source_name: Peripheral blood cells from vasculitis patient 8


title: PBMCs from vasculitis patients 7

source_name: Peripheral blood cells from vasculitis patient 7


title: PBMCs from vasculitis patients 11

source_name: Peripheral blood cells from vasculitis patient 11


title: Kidney cortical Thick Ascending Limb of Henle's Loop (CTAL)

type: SAGE

city: Paris

first: Lydie

last: Cheval

address: 15 rue de l'école de Médecine

country: France

institute: Institut des Cordeliers

description: Library was obtained from 1000 microdissected cortical thick ascending limb of Henle's loop from male CD1 mice under control condition. The library was generated using Sau3AI as anchoring enzyme and MmeI as tagging enzyme.The SAGE Adaptation method for Downsized Extracts was used to generate the library. Data were provided after curation as described in the publication.

data_processing: Automatic tag detection and counting was performed with SADELAB (CEA Saclay, France), a web-based integrated platform dedicated to the management of SAGE project.

molecule: polyA RNA

source_name: microdissected cortical thick ascending limb of Henle's loop (CTAL) from kidney

extract_protocol: Dynabeads mRNA direct kit (Dynal) was used to extract mRNA.

value: 10 weeks

tag: tissue

value: kidney cortical thick ascending limb of Henle's loop (CTAL)


title: PBMCs from vasculitis patients 6

source_name: Peripheral blood cells from vasculitis patient 6


title: RTT fibroblast, rep1

description:
Gene expression data.
Rett_1155del32-1

source_name: RTT fibroblast


title: PBMCs from vasculitis patients 4

source_name: Peripheral blood cells from vasculitis patient 4


title: PBMCs from vasculitis patients 3

source_name: Peripheral blood cells from vasculitis patient 3


title: PBMCs from vasculitis patients 2

source_name: Peripheral blood cells from vasculitis patient 2


title: PBMCs from vasculitis patients 1

source_name: Peripheral blood cells from vasculitis patient 1


title: PBMCs from vasculitis patients 5

source_name: Peripheral blood cells from vasculitis patient 5


title: PBMCs from vasculitis patients 12

source_name: Peripheral blood cells from vasculitis patient 12

value: vasculiti


title: iPSC-RTT clone 18, rep2

description:
Gene expression data.
Rett_1155del32_ips18-2

source_name: iPSC-RTT clone 18


title: RTT fibroblast, rep3

description:
Gene expression data.
Rett_1155del32-3


title: RTT fibroblast, rep2

description:
Gene expression data.
Rett_1155del32-2


title: U8724hkyn2

city: Heidelberg

first: Ulrike

middle: Mareike

last: Litzenburger

state: Baden-Württemnberg

address: INF 280

department: neuroimmuology

country: Germany

institute: DKFZ

hyb_protocol: Standard Illumina hybridization protocol

scan_protocol: Standard Illumina scanning protocol

data_processing: After normalisation (global; Bioconductor Lumi package), the genes were ranked according to differential expression. Data were expressed as pair-wise analysis comparing the mean expression of the two groups.

label: Biotin

growth_protocol: Cell were cultured under standard conditions in DMEM at 37°C in a 5 % CO2 atmosphere.

extract_protocol: Total RNA was extracted using the RNAeasy RNA isolation kit (Qiagen). Gene expression differences between untreated and kynurenine treated cells were quantified using a Sentrix HumanRef-8 ExpressionBeadChip.

treatment_protocol: Cells were treated for 8h or 24h with 100µM kynurenine

tag: cell line

tag: tumor source

tag: treatment

value: kynurenine for 24 hours


title: U8724hkyn1

label_protocol: Standard Illumina hybridization protocol


title: U878hkyn1

value: kynurenine for 8 hours


title: U878hut1


title: U878hut2


title: U878hkyn2


title: U8724hut1


title: U8724hut2


title: ahr1 yeast vs WT yeast (replica 1)

city: Montreal

first: Adnane

last: Sellam

state: Quebec

address: 6100 Royalmount

department: Anatomy and Cell Biology

country: Canada

institute: Adnane Sellam

description: Arrays are organized in 48 sub-arrays containing 280 spots each.

hyb_protocol: The microarray slides were pre-hybridized for 2 hours at 42°C with DIGeasy hybridization buffer (Roche) containing 0.5 ug/ul of yeast tRNA (Roche) and 0.5 ug/ul of salmon sperm DNA (Invitrogen) and subsequently washed with 0.1X SSC and air dried. The slides were then hybridized with labeled cDNAs overnight at 42°C in DIGeasy hybridization buffer with yeast tRNA and salmon sperm DNA. Hybridization was done with an Advalytix SlideBooster. The slides were washed twice with 1X SSC-0.2% SDS and 3 times with 0.1X SSC.

scan_protocol: Slides were scanned with a ScanArray 5000 scanner (Perkin Elmer) at 5-µm resolution for tiling arrays and 10-µm for expression arrays.

data_processing: ChIP-Chip data was normalized as described by Lavoie et al. (2010) PLoS Biol 8(3): e1000329. Expression array data were normalized as following : Signal intensity was quantified with QuantArray software (Perkin Elmer) and final Lowess normalization and inspection of the data was done with the GeneSpring package GX v.7.3 (Agilent Technologies).

organism: Candida albicans

source_name: WT under yeast conditions (YPD 30oC)

label_protocol: Indirect labeling with Cy3 and Cy5 was performed as described by Lavoie et al. BMC Genomics 2008, 9:578. The labeled DNA was purified with QIAquick PCR Purification Kit (Qiagen). For expression profiling experiment, 20 µg of total RNA was reverse transcribed using oligo(dT)21 in the presence of either Cy3-dCTP or Cy5-dCTP (Perkin-Elmer-Cetus/NEN) and Superscript III reverse transcriptase (Invitrogen). Thereafter, template RNA was degraded by adding 2.5 units of RNase H (USB) and 1 ug of RNase A (Pharmacia) followed by incubation for 15 min at 37°C. The labeled cDNAs were purified with QIAquick PCR Purification Kit (Qiagen).

extract_protocol: ChIP-chip experiment was performed as described by Lavoie et al. BMC Genomics 2008, 9:578. Chromatin immunoprecipitation was performed by following the protocol available at http://www.ircm.qc.ca/microsites/francoisrobert/en/317.html with the following exceptions: chromatin was sonicated to an average 300 bp, and 700 μl of whole-cell extract (WCE) were incubated with IgG-Sepharose 6 beads Fast Flow (GE Healthcare, catalog number 17-0969-01beads). RNAs were extracted as described by Sellam et al. (2009) Eukaryotic Cell 8 (8), 1174–1183, using RNeasy Mini kit (Qiagen).

treatment_protocol: For ChIP-chip analysis, an overnight cultures in YPD were diluted to OD600nm =0.2 and grown to OD600nm=2 in 40 ml YPD at 30oC. For expression profiling experiment,an overnight cultures grown in YPD media were diluted to OD600nm = 0.1 in YPD and grown until OD600nm = 0.8 at 30oC.

source_name: ahr1 under yeast conditions (YPD 30oC)


title: ahr1 yeast vs WT yeast (replica 2)


title: ahr1 yeast vs WT yeast (replica 3)


title: ahr1 yeast vs WT yeast (replica 4)


title: tTA/TDP-WT_3275_14218_18_MOE430A_v2.0

city: Philadelphia

first: Virginia

last: Lee

address: 3rd Floor Maloney Building; 3600 Spruce St

institute: University of Pennsylvania

description:
Genotype: Presence (+) or absence (-) of transgene, with the first value corresponding the TDP-43 transgene and the second value corresponding to the tTA transgene.  For example, +/- indicates the presence of the TDP-43 transgene and the absence of the tTA transgene
Line: Mouse line according to nomenclature in Igaz et al., JCI, 2012

hyb_protocol: The cRNA products were fragmented to 200 nucleotides or less, heated at 99oC for 5 min and hybridized for 16 h at 45oC to the mouse genome MOE430A.v2 array from Affymetrix.  Hybridization cocktails included 10ug of cRNA sample and the spiked controls provided in the Affymetrix Eukaryotic Control Kit.  The microarrays were then washed at low (6X SSPE) and high (100mM MES, 0.1M NaCl) stringency and stained with streptavidin-phycoerythrin.  Fluorescence was amplified by adding biotinylated anti-streptavidin and an additional aliquot of streptavidin-phycoerythrin stain.

scan_protocol: Hybridization signals were collected using a GeneChip 3000 7G scanner.

data_processing: The Partek Genomics Suite was used to perform microarray data analysis and to generate graphics (Partek GS, St. Louis, MO). Affymetrix array raw fluorescence intensity measures of gene expression were normalized and quantified using robust multi-array analysis. To identify genes differentially expressed between groups, we employed an ANOVA, followed by pairwise contrasts between the groups of interest. Our model corrected for gender. To correct for multiple hypothesis testing, we calculated false discovery rates (FDR) using the Benjamini-Hochberg method. Principal components analyses and hierarchical cluster analyses (Pearson correlation coefficient) were employed to evaluate patterns of global gene expression. Differentially expressed genes were additionally subjected to biological pathway analysis using the Gene Ontology database to find pathways enriched in dysregulated genes; enrichment scores are the negative log10 chi-square p-value.

source_name: Cerebral cortex

label_protocol: All protocols were conducted as described in the Affymetrix GeneChip Expression Analysis Technical Manual.  Briefly, 3ug of total RNA was converted to first-strand cDNA using Superscript II reverse transcriptase primed by a poly(T) oligomer that incorporated the T7 promoter.  Second-strand cDNA synthesis was followed by in vitro transcription for linear amplification of each transcript and incorporation of biotin using the Affymetrix 1-Cycle Target kit.

extract_protocol: Frozen tissue samples were homogenized for 30-60 seconds in TRIzol (Invitrogen) using an Omni Homogenizer with disposable probes (Omni International).  RNA was then isolated from homogenates using TRIzol phase separation as per manufacturer protocol.  Total RNA quality and mass were determined using an Agilent Bioanalyzer 2100 and a Nanodrop spectrophotometer.  All samples showed distinct 28S and 18S ribosomal RNA bands with RINs of 7 or greater.  A260/280 ratios ranged from 2.00 – 2.04, with RNA concentrations ranging from 123 – 221ng/uL.

tag: strain/background

value: mixed C57BL/6J x C3HeJ

tag: mouse line

tag: genotype

tag: Sex

value: M


title: tTA/TDP-WT_3275_14220_20_MOE430A_v2.0

value: F


title: Non-transgenic_3275_14214_14_MOE430A_v2.0


title: Non-transgenic_3275_14213_13_MOE430A_v2.0


title: Non-transgenic_3275_14216_16_MOE430A_v2.0


title: tTA/TDP-WT_3275_14219_19_MOE430A_v2.0


title: Non-transgenic_3275_14215_15_MOE430A_v2.0


title: tTA/TDP-WT_3275_14221_21_MOE430A_v2.0


title: Non-transgenic_3275_14217_17_MOE430A_v2.0


title: Non-transgenic_3275_14212_12_MOE430A_v2.0


title: Non-transgenic_3275_14210_10_MOE430A_v2.0


title: Non-transgenic_3275_14211_11_MOE430A_v2.0


title: Genome-wide occupancy of Mcm1p(WT)

type: genomic

description: Starting from sequences from the C. albicans Genome Assembly 21, we extracted a continuous series of 242,860 60-bp oligonucleotides each overlapping by 1 bp. We then eliminated 2062 probes containing stretches of 13 or more A or T nucleotides. The remaining 240,798 sequences were then used to produce an antisense (Crick) whole genome tiling array using the Agilent Technologies eArray service (https://earray.chem.agilent.com/earray/).

molecule: genomic DNA

source_name: Mcm1p-TAP under yeast conditions (YPD 30oC)


title: Genome-wide occupancy of Ahr1p

source_name: Ahr1p-TAP under yeast conditions (YPD 30oC)


title: Monogenic DeltaNLS_3275_14209_9_MOE430A_v2.0


title: Genome-wide occupancy of Mcm1p(ahr1)

source_name: Mcm1p-TAP in ahr1 strain under yeast conditions (YPD 30oC)


title: Monogenic DeltaNLS_3275_14207_7_MOE430A_v2.0


title: Monogenic DeltaNLS_3275_14208_8_MOE430A_v2.0


title: Peripheral mononuclear blood cells in patients with vasculitis 2

hyb_protocol: Before hybridization, 825ng labeled cRNA of each product was fragmented and mixed with control targets and hybridization buffer according to the manufacturer's protocol (Agilent Technologies). Hybridizations were done for approximately 17h at 65°C. The slides were washed according to the manufacturer's manual, and scanning of microarrays was performed with 5µm resolution using a DNA microarray laser scanner (Agilent Technologies).

scan_protocol: The array was scanned using Agilent G2505B DNA microarray scanner.

data_processing: The image files were extracted using Agilent Feature Extraction software version 9.5.1 and LOWESS background subtraction and dye-normalization were used.

source_name: Peripheral mononuclear blood cells from 16 healthy controls

label_protocol: RNA labeling was performed with a Fluorescent Linear Amplification Kit (Agilent Technologies). In brief, cDNA was reverse transcribed from 500ng of total RNA (pooled sample from 16 healthy control patients) with an oligo-dT-T7 promoter primer and MMLV-RT. Second strand synthesis was carried out with random hexamers. Fluorescent antisense cRNA was synthesized with cyanine 3-CTP (Cy3-CTP) and T7 polymerase. The fluorescent-labeled antisense cRNA was purified with RNeasy columns (Qiagen) according to manufacturer's protocols and resuspended in water. The purified products were quantified at A552nm for Cy3-CTP with Agilent 2100 Bioanalyzer (Agilent Technologies).

value: female


title: Monogenic DeltaNLS_3275_14206_6_MOE430A_v2.0


title: Peripheral mononuclear blood cells in patients with vasculitis 13

source_name: Peripheral blood cells from vasculitis patient 13


title: Peripheral mononuclear blood cells in patients with vasculitis 9

source_name: Peripheral blood cells from vasculitis patient 9


title: 221

city: San Francisco

first: Deepak

last: Srivastava

address: 1650 Owens St.

department: Gladstone Institute of Cardiovascular Disease

institute: J. David Gladstone Institutes

description: Mouse_Dicerfl/+,Wnt1Cre,R26R vs. Dicer+/+,Wnt1Cre,R26R_E10.5 embryos_cells from 10 embryos pooled

hyb_protocol: According to manufacturer's (Exiqon) recommendations

data_processing: GPR files were read into R/Bioconductor using the m-array package. GenePix flagged spots were removed from subsequent analysis. Only good (unflagged) miR probes were used for normalization and subsequent analysis. M (log2 ratios) of Cy5 to Cy3 signals were calculated for each array (with NO background subtraction), and normalized by loess normalization. For each miRNA with more than 1 good probes, the median of normalized M of the replicate probes was taken as its summary value for each array.

source_name: S221.2b_Mouse_Dicerfl/+,Wnt1Cre,R26R_YFP+ cells from 10 embryos pooled

label_protocol: Cy5 and Cy3 labeling protocol according to manufacturer's (Exiqon) recommendations

extract_protocol:
Equal number of Dicer heterozygotes and Dicer wildtypes were harvested at E10.5.  After removal from the embryonic sac, the region from just above the eyes to just below pharyngeal arch 6 was dissected out and placed in ice-cold PBS (Ca2+ and Mg2+ free).  The tissue was then sonicated to break up the tissue.  The tissue was then treated with 0.05% Trypsin/EDTA to generate single cells.  The cells were spun, resuspended in DMEM, and passed through a 40um nylon filter.  Cells from ten embryos were sorted into YFP+ populations and pooled for each genotype.
Total RNA was isolated from sorted cells using Trizol following the manufacturer's recommended protocols.

value: Mixed genders

value: Mixed background

tag: developmental state

tag: tissue extraction

value: The entire region from just above the eye to below pharyngeal arch 6 was dissected out and subjected to FACS and YFP+ cells were pooled from 10 embryos

source_name: S221.2a_Mouse_Dicer+/+,Wnt1Cre,R26R_YFP+ cells from 10 embryos pooled

value: The entire region from just above the eye to below pharyngeal arch 6 was dissected out and subjected to FACS and YFP- cells were pooled from 10 embryos


title: Peripheral mononuclear blood cells in patients with vasculitis 12

tag: gender

value: vasculitis


title: tTA/TDP-DeltaNLS_3275_14205_5_MOE430A_v2.0


title: Peripheral mononuclear blood cells in patients with vasculitis 11

value: healthy controls without any identified diseases

source_name: Peripheral blood cells from vasculitis patient 11


title: Peripheral mononuclear blood cells in patients with vasculitis 3

value: 9 females and 7 males

source_name: Peripheral blood cells from vasculitis patient 3


title: tTA/TDP-DeltaNLS_3275_14201_1_MOE430A_v2.0


title: Peripheral mononuclear blood cells in patients with vasculitis 8

source_name: Peripheral blood cells from vasculitis patient 8


title: Peripheral mononuclear blood cells in patients with vasculitis 10

source_name: Peripheral blood cells from vasculitis patient 10


title: 220

description: Mouse_Wnt1Cre-R26R_YFP+ vs. YFP-_E10.5 embryos_cells from 10 embryos pooled

scan_protocol: According to manufacturer's (Exiqon) recommendations

source_name: S220.1b_Mouse_Wnt1Cre-R26R_YFP+ cells from 10 embryos pooled

extract_protocol:
Equal number of Wnt1Cre,R26R YFP+ and Wnt1Cre,R26R YFP- were harvested at E10.5.  After removal from the embryonic sac, the region from just above the eyes to just below pharyngeal arch 6 was dissected out and placed in ice-cold PBS (Ca2+ and Mg2+ free).  The tissue was then sonicated to break up the tissue.  The tissue was then treated with 0.05% Trypsin/EDTA to generate single cells.  The cells were spun, resuspended in DMEM, and passed through a 40um nylon filter.  Cells from ten E10.5 embryos were sorted into YFP+ and YFP- populations and pooled.
Total RNA was isolated from sorted cells using Trizol following the manufacturer's recommended protocols.

tag: strain

source_name: S220.1a_Mouse_Wnt1Cre-R26R_YFP- cells from 10 embryos pooled


title: tTA/TDP-DeltaNLS_3275_14202_2_MOE430A_v2.0


title: Peripheral mononuclear blood cells in patients with vasculitis 4

source_name: Peripheral blood cells from vasculitis patient 4


title: Peripheral mononuclear blood cells in patients with vasculitis 1

source_name: Peripheral blood cells from vasculitis patient 1


title: Peripheral mononuclear blood cells in patients with vasculitis 5

source_name: Peripheral blood cells from vasculitis patient 5


title: RNA extraction 70h, WT rep 1

city: Delft

first: Wilbert

last: Heijne

address: A.Fleminglaan 1

institute: DSM Biotechnology Center

description: Gene expression data from Streptomyces clavuligerus ATCC 27064 after 70h, at the end of exponential phase

hyb_protocol: For hybridization, 5 μg and 7 μg biotinylated cDNA were used per Affymetrix GeneChip

scan_protocol: GeneChips were scanned using an Affymetrix GeneChip scanner

data_processing: The data were analyzed with Affymetrix GCOS software using default analysis settings and global scaling as normalization method. The mean target intensity of each array was arbitrarily set to 100.

organism: Streptomyces clavuligerus

source_name: Streptomyces clavuligerus WT during antibiotics production phase

label_protocol: Biotinylated cDNA was prepared after fragmentation according to the standard Affymetrix protocol using GC rich (average 72%) primers from 10 μg total RNA.

growth_protocol: Cultivations were performed in shake flasks directly inoculated with spore suspensions at 28ºC and 280 rpm. The semi-synthetic growth medium used consisted of 30 g/ l glycerol, 5 g/l wheat gluten, 3.5 g/l asparagine monohydrate, 1.5 g/l L-lysine, 0.7 g/l KH2PO4, 0.3 g/l MgSO4•7H2O, 0.2 g/l CaCl2•2H2O, 0.2 g/l FeSO4•7H2O, 10 g/l MOPS, 0.1 ml/l Basilodon, and 1 ml/l trace elements solution at pH7.0. The trace element solution consists of 20.4 g/l H2SO4, 50 g/l citric acid H2O , 16.75 g/l ZnSO4•7H2O, 1.6 g/l CuSO4. 5 H2O, 1.5 g/l MnCl2•4H2O, 2 g/l H3BO3, 2 g/l Na2MoO4•2H2O.

extract_protocol: After 70 h of cultivation, the cells were harvested by centrifugation, treated with RNAprotect (Qiagen) and directly frozen with liquid nitrogen and stored at −80°C. To isolate total RNA, the frozen mycelium was ground in a mortar, re-suspended in TE buffer with 5 mg/l lysozyme and incubated for 5 min at room temperature. RNA isolation and purification were performed using phenol extraction (TRIzol reagent, Invitrogen) and RNeasy Kit (Qiagen). The RNA was quantified by measuring the absorbance at 260 nm.

tag: growth phase

value: growing cells at the end of exponential phase


title: tTA/TDP-DeltaNLS_3275_14204_4_MOE430A_v2.0

source_name: Cerebral cortex


title: RNA extraction 70h, WT rep 2

description: Gene expression data from Streptomyces clavuligerus ATCC 27064 a\fter 70h, at the end of exponential phase


title: Peripheral mononuclear blood cells in patients with vasculitis 6

source_name: Peripheral blood cells from vasculitis patient 6


title: tTA/TDP-DeltaNLS_3275_14203_3_MOE430A_v2.0


title: RNA extraction 70h, DS48802 rep 2

description: Gene expression data from Streptomyces clavuligerus DS48802 after 70h, at the end of exponential phase

source_name: Streptomyces clavuligerus DS48802 during antibiotics production phase


title: RNA extraction 70h, DS48802 rep 1


title: BEAS-2B cells_TNF&IFN-treated 18h_HuR IP-rep3

city: Baltimore

first: Jinshui

last: Fan

address: 5200 Eastern Avenue

department: Division of Allergy & Clinical Immunology

institute: Johns Hopkins University

description:
HuR antibody
TNF-IFN18h.HuR-set3

hyb_protocol: Labeled probes were hybridized following the manufacturer’s instructions against a GEArrayTM S series Human Autoimmune and Inflammatory Response Gene Array (SuperArray Bioscience, Frederick, MD), which contains full-length cDNAs for 364 known human genes.

scan_protocol: The results were collected by exposure to a phosphoimager screen and then scanning by a Typhoon 8600 Variable Mode Image (GE Healthcare).

data_processing:
Analysis was performed with the P-SCAN analysis software using a custom array option available.
Arrays were normalized within each treatment/control set using a subset of genes which were reproducibly precipitated by IgG control only. Fold changes were independently calculated between HuR
antibody and IgG matched replicates within each set and tested for significance using the paired t test. Genome Ontology analysis was performed using the DAVID gene functional classification tool (NIAID, NIH, 2008).
Data were analyzed using repeated measures ANOVA test with post-hoc analysis or paired T test when appropriate.

source_name: TNF and IFN, HuR IP was done using anti-HuR antibody, replicate 3

label_protocol:
The IP RNA was reverse-transcribed according to an established labeling protocol to generate 33P-labeled probes (for detail, please refer to Fan et al. Global analysis of stress-regulated mRNA turnover by using cDNA arrays.
Proceedings Natl Acad Sci USA  2002; 99:10611-16).

growth_protocol:
The BEAS-2B cells were cultured in F12/DMEM (Gibco/Invitrogen, Frederick, MD) containing 5% heat-inactivated fetal calf serum, 2 mM L-glutamine, penicillin (100 U/ml) and streptomycin (100 mg/ml).
Primary bronchial epithelial cells (PBECs) were isolated by pronase digestion from bronchi of cadaveric lungs.  The purity of these preparations was confirmed by immunohistochemical staining for cytokeratin.
PBECs were cultured on collagen-coated flasks and maintained in serum-free LHC-9 medium (Biofluids, Rockville, MD).  Cultures of PBEC were used during the first passage only, while BEAS-2B cells were
used from passages 36 to 45.  All cells were cultured at 37°C in humidified air containing 5% CO2.

extract_protocol:
(1) IP and RNA extraction: BEAS-2B cell monolayers cultured in T-175 flasks were stimulated at ~70% confluence in the absence or presence of TNFalpha (50 ng/ml) in combination with IFNgamma at 50 ng/ml (R&D Systems,
Minneapolis, MN).  Cytokine stimulation did not affect cell viability, measured by trypan blue exclusion. Cells were then harvested by trypsinization and their viability  was assessed by trypan blue exclusion (consistently >95%).
An equal number of cells per condition (30 x 106) was pelleted and resuspended in approximately two cell-pellet volumes of polysome lysis buffer (PLB) containing 100 mM KCl, 5 mM MgCl2, 10 mM Hepes,
pH 7.0, 0.5% Nonidet P-40 with 1 mM DTT, 100 Units/ml RNaseOUT (Gibco BRL), 0.2% vanadyl-ribonucleoside complex (Gibco BRL), 0.2 mM PMSF, 1 mg/ml pepstatin A, 5 mg/ml bestatin, and 20
mg/ml leupeptin added fresh immediately before use.  Cell lysates were then frozen at -80°C for storage.  At time of use, cell lysates were thawed and centrifuged at 16,000 x g for 10 min at 4°C.  The cell lysate
contained approximately 30 mg/ml total protein and was immunoprecipitated with anti-HuR or the irrelevant antibody, according to adaptation of an established protocol.   Briefly, Protein-A Sepharose
beads (Sigma-Aldrich, St. Louis, MO) were swollen 1:1 v/v in NT2 buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1 mM MgCl2/0.05% Nonidet P40) supplemented with 5% BSA.  A 100 ul aliquot of the preswollen
protein-A bead slurry was used for IP reaction and incubated 4 h at RT with excess immunoprecipitating antibody (30 ug), using either a mouse monoclonal antibody specific for HuR, 3A2 (Santa Cruz
Biotechnology, Santa Cruz, CA) (55) or an IgG1 isotype control antibody (B&D Life Sciences, Franklin Lanes, NJ).  The antibody-coated beads were washed with ice-cold NT2 buffer and resuspended in 900
ul of NT2 buffer supplemented with 100 Units/ml RNaseOUT, 0.2% vanadyl-ribonucleoside complex, 1 mM DTT, and 20 mM EDTA.  The beads were vvortexed briefly, and 100 ul of the mRNP cell lysate was
added and immediately centrifuged.  A 100-ul aliquot was removed to represent total cellular RNA.  The IP reactions were tumbled at room temperature for 2 h and then washed six times with ice-cold NT2
buffer.  Washed beads were resuspended in 100 ul NT2 buffer supplemented with 0.1% SDS and 30 ug proteinase K, incubated for 30 min at 55°C, then cytoplasmic RNA was extracted using phenol-
chlorophorm-isoamylalcohol and precipitation in ethanol.
(2) RNA Isolation and Analysis: Total RNA was extracted using TRIzol (Invitrogen/ Life Technologies).  Cytoplasmic RNA was isolated and DNase-treated using the RNAeasy kit (Qiagen).  RNA was reverse-transcribed
using the Gene Amp Kit (Perkin-Elmer, Waltham, MA) and PCR amplified using the SYBR Green reagent Kit (Perkin-Elmer).  The PCR primers (forward/reverse) for detection of CCL2, CCL8, CCL13, CXCL1, CXCL2
and GAPDH, used for normalization, are listed in Table 1.  The conditions for amplification by real-time PCR for all targets were as follows: 50 °C, 2 minutes for UNG activation, then 95 °C, 10 min (1 cycle each, hot-start
to activate the Taq polymerase), then 95 °C, 15 sec followed by 63 °C, 1 min, for 40 cycles.  All samples were run in triplicate (SD <0.1) for real-time fluorimetric determination in an ABI 7300 sequence detector
(Applied Biosystems/LifeTechnologies) and quantified using the comparative cycle threshold (CT) method.

treatment_protocol:
(1) Vector transfection: HuR was overexpressed in PBEC and BEAS-2B cells by infection with an adenoviral vector (AdCMV-HuR) in parallel with the empty vector (Ad-EV) (ViraQuest, Inc, North Liberty, IA).
Cells were cultured in six-well plates in serum-free DMEM, and infections were carried out at 80% cell confluency.  Titration experiments showed that significant upregulation of HuR levels was achieved with
6.25 PFU/cell.  The same protocol was used for transduction of Ad (CA)AMPK, expressing a cDNA encoding residues 1 to 312 of the  1 subunit of AMPK, which encodes for a constitutively active
form of the enzyme.
(2) siRNA transfection: For HuR silencing, PBEC and BEAS-2B cells were transiently transfected with a previously described siRNA (5’-aaGAGUGAAGGAGUUGAAACU-3’) or a scrambled control
(5’-GCCAAUUCAUCAGCAAUGG) (Qiagen, Valencia, CA). Cells were seeded in 6-well plates at a density of ~250,000/well and transfected 24 h later, when cells reached ~50 % confluence, using the non-liposomal
cationic vehicle FuGene (Roche Applied Science, Indianapolis, IN) according to the manufacturer's specifications.  Briefly, 3 µl of FuGene per sample were resuspended (5 min at RT) in serum-free Opti-MEM
(Life Technologies, Carlsbad, CA) and allowed to complex with 1 µg of plasmid DNA for 15 min at RT.  The plasmid/FuGene mixture was then overlayed on the cells in a final volume of 2 ml of complete medium.
After incubation for 48 h at 37°C, cells were treated according to the experimental protocols.  The efficiency of the transient transfection of cells, assessed with a CMV promoter-driven expression vector coding for
green fluorescence protein (Promega, Madison, WI), was ~ 22% of total cell count performed by nuclear staining with Hoechst dye.  Changes in HuR levels were determined by Western blot 48 h after transfection.
(3) Western blot: Western blot was performed using primary antibodies 2 µg/ml of mouse monoclonal anti-HuR 3A2 or 0.3 µg/ml of anti-alpha-tubulin (both from Santa Cruz), or a 1:1000 dilution of the AMPK or pAMPK abs (Cell
Signaling, Danvers, MA) in 1x PBS containing 1% Tween for 1 h at RT with continuous shaking.  After washes with 1x PBS containing 0.1% Tween (10 min, RT), membranes were incubated for 1 h at RT with
a horseradish peroxidase-conjugated goat anti-mouse secondary Ab.  After a final washing step (1x PBS containing 0.1% Tween, 10 min, RT), immunoreactive bands were visualized by enhanced
chemiluminescence (Amersham Biosciences, Piscataway, NJ).
(4) In vitro biotin pull-down assay: To generate the labeled RNA probes, PCR products encompassing the coding regions or 3’UTR of the selected HuR targets were generated by RT-PCR of BEAS-2B RNA using
corresponding primers and purified from agarose gels. The resulting cDNAs were used as templates for the synthesis of biotinylated RNAs using T7 RNA polymerase and biotin-conjugated CTP (MaxiScript T7 In Vitro
Transcription Kit, Ambion, Austin, TX).  Biotinylated transcripts were amplified by RT-PCR using specific primers. Cytoplasmic fractions of unstimulated BEAS-2B cells (40 µg) were incubated for 1 h at RT with
1 µg of biotinylated transcripts, then ribonucleoprotein complexes were isolated with streptavidin-conjugated Dynabeads (Invitrogen/Life Technologies).  The presence of HuR in the pull-down pellet was verified by
standard Western blot analysis using the mouse monoclonal anti-HuR antibody 3A2 (Santa Cruz Biotechnology) and the enhanced chemiluminescence protocol (GE Healthcare).
(5) Immunofluorescence: BEAS-2B cells and PBEC were seeded at 40-50% confluency on sterile coverslips and incubated in complete medium overnight.  Following treatment, cells were fixed for 15 min in phosphate-buffered
saline (PBS) containing 4% paraformaldehyde for a period of 15 min, followed by permeabilization in PBS containing 0.4% TritonX-100 for an additional 15 min.  After incubation in blocking buffer (PBS containing
2% bovine serum albumin and 0.1% Tween 20) for 16 h, coverslips were incubated in a 1:500 dilution of mouse anti-HuR antibody (Santa Cruz Biotechnology) in blocking buffer for 1 h.  Following washes in
blocking buffer, samples were incubated with a mixture of horse anti-mouse Texas Red (1:200; Jackson Immunoresearch Laboratories, West Grove, PA) and Hoechst 33342 (1:5,000; Molecular Probes/Life
Technologies) or DAPI nuclear staining (1 ug/ml, Roche Applied Science, Indianapolis, IN) for 1 h.  After washes with blocking buffer, coverslips were mounted in Vectashield (Vector Laboratories, Burlingame,
CA).  A Zeiss Axiovert 200 M microscope (63X lens) was used for visualization employing separate channels for the analysis of phase-contrast images, red fluorescence and blue fluorescence.  Images
were then processed with the Zeiss AxioVision program, version 3.0.

tag: agent

value: TNF (TNFα) at 50 ng/ml and IFN (IFNγ) at 50 ng/ml

tag: time

tag: antibody

value: HuR

tag: antibody vendor

value: R&D


title: BEAS-2B cells_TNF&IFN-treated 18h_HuR IP-rep4

description:
HuR antibody
TNF-IFN18h.HuR-set4

source_name: TNF and IFN, HuR IP was done using anti-HuR antibody, replicate 4


title: BEAS-2B cells_TNF&IFN-treated 18h_HuR IP-rep1

description:
HuR antibody
TNF-IFN18h.HuR-set1

source_name: TNF and IFN, HuR IP was done using anti-HuR antibody, replicate 1


title: BEAS-2B cells_TNF&IFN-treated 18h_IgG1 IP-rep4

description:
IgG1 control
TNF-IFN18h.IgG1-set4

source_name: TNF and IFN, IgG1 serves as the negative antibody control, replicate 4


title: BEAS-2B cells_untreated (PBS) 18h_HuR IP-rep1

description:
HuR antibody
Ctr18h.HuR-set1

source_name: BEAS-2B is a human cell line, untreated 18h , HuR IP was done using anti-HuR antibody, replicate 1

value: untreated (PBS)

value: Santa Cruz Biotechnology


title: BEAS-2B cells_untreated (PBS) 18h_IgG1 IP-rep4

description:
IgG1 control
Ctr18h.IgG1-set4

source_name: BEAS-2B is a human cell line, untreated 18h , IgG1 serves as the negative antibody control, replicate 4


title: BEAS-2B cells_untreated (PBS) 18h_HuR IP-rep4

description:
HuR antibody
Ctr18h.HuR-set4

source_name: BEAS-2B is a human cell line, untreated 18h , HuR IP was done using anti-HuR antibody, replicate 4


title: BEAS-2B cells_TNF&IFN-treated 18h_IgG1 IP-rep3

description:
IgG1 control
TNF-IFN18h.IgG1-set3

source_name: TNF and IFN, IgG1 serves as the negative antibody control, replicate 3


title: BEAS-2B cells_TNF&IFN-treated 18h_IgG1 IP-rep1

description:
IgG1 control
TNF-IFN18h.IgG1-set1

source_name: TNF and IFN, IgG1 serves as the negative antibody control, replicate 1


title: BEAS-2B cells_untreated (PBS) 18h_HuR IP-rep3

description:
HuR antibody
Ctr18h.HuR-set3

source_name: BEAS-2B is a human cell line, untreated 18h , HuR IP was done using anti-HuR antibody, replicate 3


title: BEAS-2B cells_TNF&IFN-treated 3h_HuR IP-rep3

description:
HuR antibody
TNF-IFN3h.HuR-set3


title: BEAS-2B cells_untreated (PBS) 18h_IgG1 IP-rep1

description:
IgG1 control
Ctr18h.IgG1-set1

source_name: BEAS-2B is a human cell line, untreated 18h , IgG1 serves as the negative antibody control, replicate 1


title: BEAS-2B cells_TNF&IFN-treated 3h_HuR IP-rep4

description:
HuR antibody
TNF-IFN3h.HuR-set4


title: Peripheral mononuclear blood cells in patients with vasculitis 7

source_name: Peripheral mononuclear blood cells from 16 healthy controls

tag: disease status

tag: age

source_name: Peripheral blood cells from vasculitis patient 7


title: BEAS-2B cells_untreated (PBS) 18h_IgG1 IP-rep3

description:
IgG1 control
Ctr18h.IgG1-set3

source_name: BEAS-2B is a human cell line, untreated 18h , IgG1 serves as the negative antibody control, replicate 3

tag: cell line


title: BEAS-2B cells_untreated (PBS) 3h_IgG1 IP-rep3

description:
IgG1 control
Ctr3h.IgG1-set3

source_name: BEAS-2B is a human cell line, untreated 3h , IgG1 serves as the negative antibody control, replicate 3


title: BEAS-2B cells_untreated (PBS) 3h_HuR IP-rep3

description:
HuR antibody
Ctr3h.HuR-set3

source_name: BEAS-2B is a human cell line, untreated 3h , HuR IP was done using anti-HuR antibody, replicate 3


title: BEAS-2B cells_TNF&IFN-treated 3h_IgG1 IP-rep1

description:
IgG1 control
TNF-IFN3h.IgG1-set1


title: BEAS-2B cells_untreated (PBS) 3h_HuR IP-rep4

description:
HuR antibody
Ctr3h.HuR-set4

source_name: BEAS-2B is a human cell line, untreated 3h , HuR IP was done using anti-HuR antibody, replicate 4


title: BEAS-2B cells_TNF&IFN-treated 3h_IgG1 IP-rep3

description:
IgG1 control
TNF-IFN3h.IgG1-set3


title: BEAS-2B cells_TNF&IFN-treated 3h_IgG1 IP-rep4

description:
IgG1 control
TNF-IFN3h.IgG1-set4


title: BEAS-2B cells_TNF&IFN-treated 3h_HuR IP-rep1

description:
HuR antibody
TNF-IFN3h.HuR-set1


title: BEAS-2B cells_untreated (PBS) 3h_HuR IP-rep1

description:
HuR antibody
Ctr3h.HuR-set1

source_name: BEAS-2B is a human cell line, untreated 3h , HuR IP was done using anti-HuR antibody, replicate 1


title: BEAS-2B cells_untreated (PBS) 3h_IgG1 IP-rep4

description:
IgG1 control
Ctr3h.IgG1-set4

source_name: BEAS-2B is a human cell line, untreated 3h , IgG1 serves as the negative antibody control, replicate 4


title: BEAS-2B cells_untreated (PBS) 3h_IgG1 IP-rep1

description:
IgG1 control
Ctr3h.IgG1-set1

source_name: BEAS-2B is a human cell line, untreated 3h , IgG1 serves as the negative antibody control, replicate 1


title: lincRNA-ST8SIA3 siRNA1 - gene expression, replicate 2

city: Cambridge

first: Nataly

middle: Moran

last: Cabili

state: MA

address: 7  Cambridge Center

institute: Broad Institute ;  Harvard University

description: lincRNA-ST8SIA3 knock-down experiment

hyb_protocol: Standard Affymetrix protocols for Human U133 Plus 2.0 Array

data_processing: RMA

source_name: hFib2-iPS5 cells

growth_protocol: iPSCs were grown on MEFs in standard human ESC media (DMEM/F12, 20% KOSR, 2mM L-Glutamine, 0.1mM NEAA, 0.1mM b-mercaptoethanol, 10ng/ml bFGF)

extract_protocol: Trizol

treatment_protocol: iPSCs were dissociated to single cells with accutase in the presence of 10µM  Y-27632, then plated at 35-50k per 24well onto hESC-qualified matrigel coated plates in the presence of 10µM  Y-27632. Cells were transfected with 100nM siRNAs targeting lincRNA-ST8SIA3 or a non-targeting control. Total RNA was harvested 72h post-transfection

tag: cell type

value: hFib2-iPS5 cells

tag: treatment

value: lincRNA-ST8SIA3 targeting siRNA1


title: lincRNA-ST8SIA3 siRNA4 - gene expression, replicate 2

description: lincRNA-ST8SIA3 knock-down experiment

scan_protocol: Standard Affymetrix protocols for Human U133 Plus 2.0 Array

value: lincRNA-ST8SIA3 targeting siRNA4


title: lincRNA-ST8SIA3 siRNA4 - gene expression, replicate 1

description: lincRNA-ST8SIA3 knock-down experiment

label_protocol: Standard Affymetrix protocols for Human U133 Plus 2.0 Array


title: lincRNA-ST8SIA3 siRNA1 - gene expression, replicate 1

description: lincRNA-ST8SIA3 knock-down experiment


title: ctrl siRNA1 - gene expression, replicate 2

description: lincRNA-ST8SIA3 knock-down experiment

value: non-targeting control siRNA,


title: ctrl siRNA1 - gene expression, replicate 1

description: lincRNA-ST8SIA3 knock-down experiment

value: non-targeting control siRNA


title: BJ1 fibroblasts - gene expression

description: Gene expression profiles of human fibroblasts

source_name: BJ1 fibroblasts

growth_protocol: ESCs and iPSCs were grown on MEFs in standard human ESC media (DMEM/F12, 20% KOSR, 2mM L-Glutamine, 0.1mM NEAA, 0.1mM b-mercaptoethanol, 10ng/ml bFGF), fibroblasts were grown in aMEM/10%FCS

treatment_protocol: differentiated cells were removed from ESC and iPSC cultures prior to RNA isolation

value: BJ1 fibroblasts


title: hFib2-derived iPSC line 5 - gene expression

description: Gene expression profiles of human  fibroblast-derived induced pluripotent stem cells

source_name: hFib2-derived iPSC line 5

value: hFib2-derived iPSC line 5


title: hFib2 fibroblasts, replicate 1 - gene expression

source_name: hFib2 fibroblasts, replicate 1

value: hFib2 fibroblasts


title: MSC fibroblasts - gene expression

source_name: MSC fibroblasts

value: MSC fibroblasts


title: hFib2-derived iPSC line 4 - gene expression

source_name: hFib2-derived iPSC line 4

value: hFib2-derived iPSC line 4


title: ctrl siRNA1 - gene expression, replicate 3

description: lincRNA-ST8SIA3 knock-down experiment


title: MRC5-derived iPSC line 7 - gene expression

source_name: MRC5-derived iPSC line 7

value: MRC5-derived iPSC line 7


title: MSC-derived iPSC line 1 - gene expression

source_name: MSC-derived iPSC line 1

value: MSC-derived iPSC line 1


title: hFib2 fibroblasts, replicate 2 - gene expression

source_name: hFib2 fibroblasts, replicate 2


title: MSC-derived iPSC line 3 - gene expression

source_name: MSC-derived iPSC line 3

value: MSC-derived iPSC line 3


title: BJ1-derived iPSC line 2 - gene expression

source_name: BJ1-derived iPSC line 2

value: BJ1-derived iPSC line 2


title: BG01 ESCs - gene expression

description: Gene expression profiles of human embryonic stem cells

source_name: BG01 ESCs

value: BG01 ESCs


title: MRC5-derived iPSC line 20 - gene expression

source_name: MRC5-derived iPSC line 20

value: MRC5-derived iPSC line 20


title: 4hr_LPS_control_rep3

city: Stevenage

first: Paul

middle: A

last: Wilson

state: Hertfordshire

address: GlaxoSmithKline Medicine Research Centre

department: Target Sciences

country: United Kingdom

institute: GSK

description: replicate 3

data_processing: Data analyses were completed using the R/Bioconductor BioC 2.5 suite of tool.  Briefly, raw expression data was quality assessed, and subjected to background adjustment and quantile normalisation using BeadArray.  Log2 scaled data was used for linear modelling, differential expression profiling and relevant graphical representations, using the LIMMA library.

source_name: LPS-induced macrophages

label_protocol: Biotinylated cRNA were prepared with the Ambion MessageAmp kit for Illumina arrays

growth_protocol: Standard culture techniques

extract_protocol: Qiagen RNeasy kit - followed manufacturer's protocol. Quality control was performed with Agilent Bioanalyser.

value: 6 weeks old

tag: sex

value: female

tag: tissue

value: bone marrow

value: macrophages

tag: treatment group


title: H9 ESCs - gene expression

source_name: H9 ESCs

value: H9 ESCs


title: 4hr_LPS_BET treat_rep3

description: replicate 3

treatment_protocol: Biotinylated cRNA were prepared with the Ambion MessageAmp kit for Illumina arrays

value: 4hr_LPS_BET treat


title: HUVECs  treated with 4 µg/ml vWF in Hank's buffered salt solution for 4 hours rep 3

city: Stony Brook

first: Sergey

middle: V.

last: Doronin

state: NY

address: Nicolls Rd

department: Physiology&Biophysics

institute: Stony Brook University

description: HUVECs activated with vWF

hyb_protocol: Following fragmentation, 10 µg of cRNA were hybridized for 16 hours at 45oC on GeneChip Human Genome U133 Plus 2.0 Array. GeneChips were washed and stained in the Affymetrix Fluidics Station 450.

scan_protocol: GeneChips were scanned using the Affymetrix 7G Scanner.

data_processing: The data were analyzed using Bioconductor software package

source_name: Human umbilical  vein endothelial cells  treated with vWF in HBSS

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

growth_protocol: HUVECs were maintained in EGM2 media (Lonza)

extract_protocol: Total RNA was extracted using the Qiagen RNeasy kit

treatment_protocol: Confluent HUVECs were treated for 4 hours with Hank's buffered salt solution (HBSS), von Willebrand factor (vWF) in HBSS, IL1-beta in EGM2, Actinomycin D in EGM2 or the combination of IL1-beta with Actinomycin D

value: human umbilical vein endothelial cell


title: HUVECs  treated with 4 µg/ml vWF in Hank's buffered salt solution for 4 hours rep 1

description: HUVECs activated with vWF


title: 2hr_LPS_control_rep3

description: replicate 3


title: MRC5 fibroblasts - gene expression

source_name: MRC5 fibroblasts

value: MRC5 fibroblasts


title: 1hr_LPS_BET treat_rep3

description: replicate 3

value: 1hr_LPS_BET treat


title: HUVECs  treated with Hank's buffered salt solution for 4 hours rep 3

description: Serum-deprived HUVECs

source_name: Human umbilical  vein endothelial cells  treated with HBSS


title: 1hr_LPS_control_rep3

description: replicate 3


title: 0hr_LPS_BET treat_rep3

description: replicate 3

value: 0hr_LPS_BET treat


title: 2hr_LPS_BET treat_rep3

description: replicate 3

value: 2hr_LPS_BET treat


title: HUVECs  treated with Hank's buffered salt solution for 4 hours rep 2

description: Serum-deprived HUVECs


title: HUVECs  treated with 4 µg/ml vWF in Hank's buffered salt solution for 4 hours rep 2

description: HUVECs activated with vWF


title: 0hr_LPS_control_rep3

description: replicate 3


title: 4hr_LPS_BET treat_rep2

description: replicate 2


title: 4hr_LPS_control_rep2

description: replicate 2


title: HUVECs  treated with Hank's buffered salt solution for 4 hours rep 1

description: Serum-deprived HUVECs


title: HUVECs  maintained in EGM2 media, the control rep 2

description: HUVECs in the basal state

source_name: Human umbilical  vein endothelial cells


title: HUVECs  maintained in EGM2 media, the control rep 3

description: HUVECs in the basal state


title: HUVECs  maintained in EGM2 media, the control rep 1

description: HUVECs in the basal state


title: 1hr_LPS_BET treat_rep2

description: replicate 2


title: 0hr_LPS_BET treat_rep2

description: replicate 2


title: S23

description: Mouse_Wnt1Cre-R26R_YFP+ vs. YFP-_E11.5 embryos_cells from 5 embryos pooled

source_name: DSK1_Mouse_Wnt1Cre-R26R_YFP+ cells from 5 embryos pooled

extract_protocol:
Equal number of Wnt1Cre,R26R YFP+ and Wnt1Cre,R26R YFP- were harvested at E11.5.  After removal from the embryonic sac, the region from just above the eyes to just below pharyngeal arch 6 was dissected out and placed in ice-cold PBS (Ca2+ and Mg2+ free).  The tissue was then sonicated to break up the tissue.  The tissue was then treated with 0.05% Trypsin/EDTA to generate single cells.  The cells were spun, resuspended in DMEM, and passed through a 40um nylon filter.   Cells from five E11.5 embryos were sorted into YFP+ and YFP- populations and pooled.
Total RNA was isolated from sorted cells using Trizol following the manufacturer's recommended protocols.

value: Mixed genders

value: Mixed background

tag: developmental state

value: The entire region from just above the eye to below pharyngeal arch 6 was dissected out and subjected to FACS and YFP+ cells were pooled from 5 embryos

source_name: DSK2_Mouse_Wnt1Cre-R26R_YFP- cells from 5 embryos pooled

value: The entire region from just above the eye to below pharyngeal arch 6 was dissected out and subjected to FACS and YFP- cells were pooled from 5 embryos


title: 2hr_LPS_BET treat_rep2

description: replicate 2


title: 4hr_LPS_BET treat_rep1

description: replicate 1


title: 0hr_LPS_control_rep2

description: replicate 2


title: 2hr_LPS_control_rep2

description: replicate 2


title: 2hr_LPS_BET treat_rep1

description: replicate 1


title: HUVECs  treated with Actinomycin D (5 µg/ml) and IL1-beta (10 ng/ml) in EGM2 media

description: Test that HUVEC activation by the inflammatory factor occurs via de novo RNA synthesis

source_name: Human umbilical  vein endothelial cells  treated with IL1-beta and Actinomycin D


title: 4hr_LPS_control_rep1

description: replicate 1


title: 0hr_LPS_BET treat_rep1

description: replicate 1


title: 1hr_LPS_control_rep1

description: replicate 1


title: 1hr_LPS_control_rep2

description: replicate 2


title: 1hr_LPS_BET treat_rep1

description: replicate 1


title: 2hr_LPS_control_rep1

description: replicate 1


title: 0hr_LPS_control_rep1

description: replicate 1


title: HUVECs  treated with 10 ng/ml IL1-beta for 4 hours in EGM2 media

description: Test for HUVEC activation by the inflammatory factor

source_name: Human umbilical vein endothelial cells  treated with IL1-beta


title: Wild-type cremasteric sac, rep3

city: Miami

first: Alexander

middle: I

last: Agoulnik

state: FL

address: 11200 SW 8th Street, HLSI 419B

department: Herbert Wertheim College of Medicine

institute: Florida International University

description: Replicate 3.

hyb_protocol: Standard Illumina hybridization protocol.

scan_protocol: Standard Illumina scanning protocol.

data_processing: The data were normalized using quantile normalization with GenomeStudioV2010.2 Gene expression module V1.7.0.

source_name: Mouse cremasteric sac, wild-type

label_protocol: Biotinylated cRNA were prepared with the Ambion MessageAmp kit for Illumina arrays.

extract_protocol: RNA was extracted with Trizol reagent, followed by clean-up and DNase I treatment with the QIAGEN RNeasy mini kit in accordance with the prescribed protocol provided with the kit. Quality control was performed with the Agilent Bioanalyser.

value: mixed

tag: genotype

value: wild-type

value: male

value: 1 month

value: cremasteric sac


title: Cryptorchid cremasteric sac, rep2

description: Replicate 2.

source_name: Mouse cremasteric sac, cryptorchid

tag: phenotype

value: cryptorchidism

tag: gender


title: Cryptorchid cremasteric sac, rep3

description: Replicate 3.

tag: strain


title: HUVECs  treated with 5 µg/ml Actinomycin D for 4 hours in EGM2 media

description: Test for the inhibition of RNA synthesis

source_name: Human umbilical vein endothelial cells  treated with Actinomycin D


title: Cryptorchid cremasteric sac, rep1

description: Replicate 1.


title: Wild-type cremasteric sac, rep1

description: Replicate 1.


title: Control siRNA 1

city: Duarte

first: keqiang

last: zhang

address: 1500 East Duarte Road

department: Molecular Pharmacology

institute: City of Hope National Medical Center

description: Gene expression data from KB cancer cells transfected with control siRNA

hyb_protocol: Following fragmentation, 6 ug of cRNA were hybridized for 16 hr at 45C on GeneChip Drosophila Genome Array. GeneChips were washed and stained in the Affymetrix Fluidics Station 400.

scan_protocol: Arrays were scanned at 5um resolution with Affymetrix scanner 3000 7G.

data_processing: All data processing was done using Bioconductor or APT tools; GCBG using background probes; Sketch Quantile to standardize arrays. Transformation: Log2 with offset of 1 to prevent negative numbers.

source_name: control siRNA

label_protocol: Biotinylated cRNA were prepared according to the standard Affymetrix protocol from 100 ng total RNA (Expression Analysis Technical Manual, 2001, Affymetrix).

growth_protocol: 10% FBS DMEM cell culture medium

extract_protocol: RNA-Easy kit (Qiagen) of total RNA was performed according to the manufacturer's instructions.

treatment_protocol: 2.5 x 105/well KB cells in 6 well-plates were transfected with 60 pmol p53R2 siRNA or scramble siRNA by Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer's instructions.

value: oropharyngeal carcinoma KB


title: Wild-type cremasteric sac, rep2

description: Replicate 2.


title: Control siRNA 2


title: JARID1B MCF-7

city: Beijing

first: Shang

last: Yongfeng

address: Hai dian district,38# Xue Yuan Road

department: Biochemistry and Molecular Biology

country: China

institute: Peking University

description: whole genome target analysis for JARID1B

hyb_protocol: standard AVIVA method

scan_protocol: CapitalBio manual

data_processing:
After background correction and removal of bad spots, expression ratio was linear normalized using 50 percentile rank
software: R version 2.10.1 (2009-12-14)
Copyright (C) 2009 The R Foundation for Statistical Computing
ISBN 3-900051-07-0

source_name: enriched DNA using anti-JARID1B

label_protocol: primer labeled with fluorescence molecules, protocol for labelling the extract follows the "Complete Product Handbook for The Aviva Systems Biology ChIP-GLAS System" (in attachment), IX. Detailed Protocol, Step 5. PCR Amplification and Labeling of GLAS Products

extract_protocol: common cell lysate

value: MCF-7 breast cancer cell line

value: santa cruz

tag: antibody lot/bach#

tag: antibody catalog#

source_name: Input DNA


title: IgG MCF-7

description: control of the experiment

source_name: enriched DNA using normal IgG

tag: antibody

value: IgG


title: p53r2 siRNA 2

description: Gene expression data from KB cancer cells transfected with p53R3 specific siRNA to knockdown its expression

source_name: p53R2 attenuated


title: p53r2 siRNA 1

description: Gene expression data from KB cancer cells transfected with p53R2 specific siRNA to knockdown its expression


title: BJ1-iPS2 - lincRNA expression

description: LincRNA expression profiles on tiling arrays

hyb_protocol: Nimblegen ChIP hybridization protocol (http://www.nimblegen.com)

scan_protocol: Arrays were scanned using AXON 5uM scanner

data_processing: Intensities were computed using the Nimblescan software package

source_name: BJ1-derived iPSC line 2

label_protocol: Nimblegen ChIP labeling protocol (http://www.nimblegen.com)


title: hFib2 - lincRNA expression

source_name: hFib2 fibroblasts, replicate 1


title: hFib2a - lincRNA expression

source_name: hFib2 fibroblasts, replicate 2


title: CD34+ derived iPSC line 4 - lincRNA expression, replicate 1

data_processing: Probe signal intensity (ie, Sample VALUEs) obtained using the NimbleScan software provided by Nimblegen. The .gff files (linked below as supplementary files) were also generated with NimbleScan

source_name: CD34+ derived iPSC line 4

growth_protocol: ESCs and iPSCs were grown on MEFs in standard human ESC media (DMEM/F12, 20% KOSR, 2mM L-Glutamine, 0.1mM NEAA, 0.1mM b-mercaptoethanol, 10ng/ml bFGF), CD34+ cells: mobilized peripheral blood CD34+ cells (AllCells, mBP014F). Cells were thawed and cultured as described (Loh, Y.H., et al. Blood 113, 5476-5479 (2009)) for two days before RNA isolation


title: BG01 ESC - lincRNA expression, replicate 1

source_name: BG01 ESCs

value: BG01 ESCs


title: CD34+ derived iPSC line 4 - lincRNA expression, replicate 2


title: Repeat of grnA morphants at 3 dpf

city: Taipei

first: Yen-Hsing

last: Li

address: ICOB, Academia Sinica, Nankang

country: Taiwan

institute: Academia Sinica

description:
The commercial zebrafish 14K oligonucleotides set (MWG
Biotech AG, Ebersbach, Germany) were obtained and were printed on UltraGAPS Coated slide (Corning, New York, NY ) with use of the OmniGrid 100 microarrayer (Genomic Solutions, Ann Arbor, MI) according to the manufacture’s instructions. The 14,067 oligonucleotides represent 9666 genes (7009 singlet genes and 2657 redundant genes), and the redundancy of this chip is 31 %. The detailed description of the oligonucleotides information can be obtained on the Ocimun Biosolution website (http://www.ocimumbio.com/web/default.asp).

hyb_protocol: The zebrafish 14K OciChip array chip was pretreated with 1% bovine serum albumin (BSA) (fraction V), 4x saline-sodium citrate (SSC), and 1% sodium dodecylsulfate (SDS) at 42 °C for 45 min, and then hybridized overnight in a cocktail containing 5x Denhardt's solution, 6x SSC, 0.5% SDS, 50% formamide, 50 mM sodium phosphate, and 2 µg/µl yeast tRNA. Slides were washed with 2x SSC and 0.1% SDS (5 min),0.5x SSC (5 min), and twice with 0.1x SSC (2 min each).

scan_protocol: Scanning was performed with a Scanarray Gx scanner (PerkinElmer, Waltham, MA). The acquired images were analyzed using ScanArray Express 3.0 (PerkinElmer, Waltham, MA)and Genespring software (Aglient Technologies, Foster City, CA).

data_processing: The measurements of spots were filtered by flags, and the lowest normalization was performed after subtraction of the median background. The VALUE indicated the transformation algorithm of Cy3/Cy5 normalized log2 ratio.

organism: Danio rerio

source_name: 3 dpf grnA morphants

label_protocol: cDNA probes were synthesized by reverse transcription of 20 μg total RNA using a SuperScript indirect cDNA labeling system (Invitrogen).The amine-modified DNA then was incubated with resuspend one aliquot of Cy5 Dye (Amersham Bioscience, Buckinghamshire, UK) at RT in the dark for overnight. Finally, the probe was purified by kit and was eluted with elution EB buffer.

growth_protocol: Organisms were originally obtained from the University of Oregon, and were kept in the zebrafish stock center at Academia Sinica, Taipei, Taiwan.

extract_protocol: Embryos were pooled as a sample and then homogenized in 5 ml Trizol reagent (Invitrogen, Carlsbad, CA). After chloroform extraction, RNA precipitation and ethanol washing, the RNA samples were purified and treated with DNase1 to remove the genomic DNA by using RNeasy Mini Kit (Qiagen, Huntsville, Alabama).The quantity and quality of total RNA were assessed by spectrophotometry and agarose gel electrophoresis, respectively.

treatment_protocol: Embryos were injected by MO-grnA at 1-2 cell stage.

tag: developmental stage

value: 3 dpf

tag: genome/variation

value: grnA morphant

value: AB

source_name: 3 dpf control embryos

label_protocol: cDNA probes were synthesized by reverse transcription of 20 μg total RNA using a SuperScript indirect cDNA labeling system (Invitrogen).The amine-modified DNA then was incubated with resuspend one aliquot of Cy3 Dye (Amersham Bioscience, Buckinghamshire, UK) at RT in the dark for overnight. Finally, the probe was purified by kit and was eluted with elution EB buffer.

treatment_protocol: Embryos were injected by MO-control at 1-2 cell stage.

value: control


title: hFib2-iPS5 - lincRNA expression

source_name: hFib2-derived iPSC line 5


title: ZYNPCipS3-RPE

city: Los Angeles

first: Kevin

last: Huang

address: Gonda BLDG Rm. 6554, P.O.Box 957088

department: Human Genetics

institute: UCLA

description: Gene expression at confluency

hyb_protocol: 1000ng of cyanine 3-labeled cRNA was fragmented and hybridized on Agilent 44K Whole Human genome arrays (G4112A; Agilent Technologies) and incubated at 65 °C for 17 hours using the Agilent Gene Expression Hybridization Kit.

scan_protocol: After washing, the processed microarrays were immediately scanned with the Agilent DNA microarray scanner immediately after washing to prevent ozone degradation and data were extracted with Agilent Feature Extraction software.

data_processing: Scanned data was analyzed using Bioconductor (R) and Matlab.

source_name: iPS-derived RPE

label_protocol: Sample labeling and microarray processing was performed as detailed in the One-Color Microarray-Based Gene Expression Analysis protocol. The labeling reactions were performed using the Agilent Low RNA Input Linear Amplification Kit in the presence of cyanine 3-CTP (PerkinElmer Life and Analytical Sciences). Fluorescent labeled probes were purified using the Qiagen RNeasy Mini kit (Qiagen) as described by the manufacturer.

growth_protocol: After formation, pigmented foci were excised manually using the needles of 2 syringes. This approach was only possible after foci had reached at least 1 mm in diameter. During this procedure, we avoided the surrounding, nonpigmented material and placed the pigmented foci into 24-well culture dishes coated with growth factor-reduced Matrigel (BD Biosciences, diluted 1:30). Alternatively, pigmented foci were placed in Millicell-HA culture wells (Millipore) coated with Matrigel. Two to 3 pigmented foci were placed in each well. The hESC-RPE and hiPSC-RPE were allowed to expand on Matrigel for a further 3 months in bFGF-deficient basic hESC medium (media changes every 2–3 days). This time frame was sufficient to yield monolayer sheets of pigmented cells that could be studied further.

extract_protocol: RNA was purified using RNeasy Kit (Qiagen) following the manufacturer’s instruction

value: iPS-derived retinal pigmented epithelium


title: BJ-fiboblast

description: Gene expression at confluency

source_name: somatic fibroblast

growth_protocol: Fibroblasts were grown following standard protocol.

value: somatic fibroblast


title: BG01 ESC - lincRNA expression, replicate 2


title: Whole embryo_3dpf_grnA_morphants


title: HSF1-RPE

description: Gene expression at confluency

source_name: ES-derived RPE

value: ES-derived retinal pigmented epithelium

tag: cell line source


title: CD34+ derived iPSC line 8 - lincRNA expression, replicate 1

source_name: CD34+ derived iPSC line 8


title: MRC5-iPS7 - lincRNA expression

source_name: MRC5-derived iPSC line 7


title: hFib2-iPS4 - lincRNA expression

source_name: hFib2-derived iPSC line 4


title: CD34+ cells - lincRNA expression, replicate 2

source_name: CD34+ cells

value: CD34+ cell


title: HSF1

description: Gene expression at confluency

source_name: embryonic stem cell

growth_protocol: The human embryonic stem cell lines H9, H1, HSF1, HSF6, HUES12, and HUES15 were maintained in cell culture dishes coated with 0.2% gelatin and seeded with gamma-irradiated CF1 mouse embryonic fibroblast (MEF) feeders. Cells were maintained in Dulbecco’s modified Eagles medium (DMEM)/F12 with 20% Knockout serum replacement (Invitrogen), 1% nonessential amino acid solution, 1 mM l-glutamine (Invitrogen), 100U/ml Penicillin-Streptomycin (VWR), 4 ng/ml human bFGF (Invitrogen), and 0.1 mM β-mercaptoethanol (Sigma). The human induced pluripotent stem cell lines HiPS2, HiPS18, ZY-NPC-iPS#3, YIN-NPC-iPS#10, and HSF6-NPC-iPS were maintained in similar conditions with a 1:1 ratio of hESC medium to conditioned medium. Conditioned medium was prepared by incubation of hESC medium overnight with MEF cells.

value: embryonic stem cell

tag: cell line


title: MRC5-iPS20 - lincRNA expression

source_name: MRC5-derived iPSC line 20

tag: cell type


title: hIPS2-RPE

description: Gene expression at confluency


title: MSC-iPS3 - lincRNA expression

source_name: MSC-derived iPSC line 3


title: BJ1 - lincRNA expression

source_name: BJ1 fibroblasts


title: BG01 ESC - lincRNA expression


title: CD34+ cells - lincRNA expression, replicate 1


title: H9

description: Gene expression at confluency


title: MSC-iPS1 - lincRNA expression

source_name: MSC-derived iPSC line 1


title: MRC5 - lincRNA expression

source_name: MRC5 fibroblasts


title: fRPE-15w

description: Gene expression at confluency

source_name: primary RPE, 15 week

growth_protocol: We followed the culture method for human fetal RPE cells (fRPE) as described previously (4-5).  The tenets of the Declaration of Helsinki were followed, and the donors (or their guardians) gave consent for donation of the tissue. The approval of the Institutional Human Experimentation Committee was obtained for the use of human eyes. We dissected and collected fRPE cells from human abortuses (16-21 weeks gestation) and grew them in low-calcium Chee’s essential modified medium until they proliferated to confluence and released cells into the medium. These nonattached cells were collected and grown for 4 to 8 months on permeable Millicell-HA culture wells (Millipore). The culture medium was changed to a 1:1 mixture of normal calcium Chee’s essential modified medium and Eagle’s minimum essential medium, with bovine retinal extract and 1% heat-inactivated calf serum. Cell cultures were maintained up to 3 months, by which time a homogenous monolayer of cells had formed. Alternatively, human fRPE cells were maintained in cell culture with RPE cell culture medium in accordance with the described protocol (6). RPE cell culture medium was prepared by using 500 ml of MEM–Alpha (Sigma), 15% fetal bovine serum (Invitrogen), 5 ml of N1 Supplement (Sigma), 5 ml of glutamine-penicillin-streptomycin, 5 ml of nonessential amino acids (Sigma), 125 mg of Taurine (Sigma), 10 μg of hydrocortisone (Sigma), and 0.0065 μg of Triiodo-thyronin (Sigma). Cells settled and adhered to the cell culture dish and were maintained for 2 weeks in RPE culture medium.

value: primary retinal pigmented epithelium

tag: age

value: 15 wk


title: fRPE-14w

description: Gene expression at confluency

source_name: primary RPE, 14 week

value: 14 wk


title: fRPE-18w

description: Gene expression at confluency

source_name: primary RPE, 18 week

value: 18 wk


title: MSC - lincRNA expression

source_name: MSC fibroblasts

value: MSC


title: transfected anti-K10a replicate 1

city: Bethesda

first: Joseph

middle: M

last: Ziegelbauer

address: 10 Center Dr. 6N110

institute: NIH / National Cancer Institute

description:
BCBL-1 cells transfected with inhibitor anti-K10a. Biological replicate 1 of 1.
a10a-rep1

hyb_protocol: Samples were prepared with Gene Expression Hybridization Kit (Agilent) and placed in Agilent microarray hybridization chambers and rack. Hybridization occurred for 17 hr. at 65°.

scan_protocol: Scanned on Axon Genepix 4000B scanner. Images were processed with Agilent Feature Extraction Software v9.1.1.

data_processing:
Normalization: Background substraction and linear LOWESS normalization was used in Feature Extraction software.

Filtering:
Performed with Genespring gx which uses information from agilent feature extraction software. To summarize, a 'present' flag means the feature on the array is: positive, significant, uniform, above background, not saturated and is not a population outlier.

Flags:
By default, GeneSpring GX11 uses the spot information generated by Feature Extractor in the data file to flag the data. It uses 5 spot problems which are given below and flags the probes accordingly.

Problem --> Flag
If Feature is not positive and significant --> M
If Feature is not uniform --> A
If Feature is not above background --> M
If Feature is saturated --> A
If Feature is a population outlier --> A

This flagging is applicable for each probe present on the array. As there will be replicates for each probe on the array, the flag values are further summarized to report one value for a probeset.

Summarization:
Treatment of on-chip replicates: For each replicate with multiple flags, the order of importance is Absent(A)>Marginal(M)>Present(P). If there is even one A, then the resultant flag is 'A'. If there is no A, but M and P, then M is assigned. If there are only Ps then only the resultant flag is assigned as 'P'. To get the overall flag for all replicates, GeneSpring GX excludes 'A' flag and assigns the majority considering the remaining ones. If there are only 'A' flags, only then the overall flag becomes 'A'.

The flag value of a particular probeset is dependant on the flag values of the probes in it. If a probeset contains a probe which is marked as Present (P), the probeset is marked as P irrespective of the other flag values. The order of importance for flag values is Present>Marginal>Absent.

source_name: Stratagene Universal Reference RNA

label_protocol: 500 ng of total RNA was labeled using Low RNA Input Fluor Linear Amp Kit PLUS (Agilent).

growth_protocol: Cells were split to 2x10^5 cells per ml every 48 hr.

extract_protocol: Cells were harvested with RNA-bee (Tel-test). RNA integrity was assayed using a Bioanalyzer 2100 (Agilent) and quantitated with a ND1000 spectrophotometer (Nanodrop).

treatment_protocol: BJAB and BCBL-1 cells were electroporated with siPORT system (Ambion) at 30 nM for mimics and 100 nM for inhibitors (RNA harvested 48 and 72 hr. after transfection, respectively)

tag: reference

value: Pooled cell line RNA

source_name: transfected BCBL-1 cells

tag: protocol (transfection)

value: inhibitor anti-K10a

tag: time

value: 72 hr. post transfection


title: transfected anti-neg replicate 1

description:
BCBL-1 cells transfected with control inhibitor anti-neg. Biological replicate 1 of 1.
aneg-rep1

value: control inhibitor anti-neg


title: fRPE

description: Gene expression at confluency

source_name: cultured fetal RPE

tag: tissue

value: cultured fetal retinal pigmented epithelium


title: transfected miR-neg replicate 1

description:
BJAB cells transfected with miR-neg. Biological replicate 1 of 2.
pneg-rep1

source_name: transfected BJAB cells

value: BJAB

value: miR-neg

value: 48 hr. post transfection


title: Ume1.2

status: Public on Nov 09 2010

city: Leicester

first: Eran

last: Tauber

address: University Road

department: Genetics

institute: University of Leicester

hyb_protocol: Affymetrix Eukaryotic Target Hybridization protocol

scan_protocol: Affymetrix protocol.

data_processing: R/Bioconductor affylmGUI package (version 1.8.0)

organism: Saccharomyces cerevisiae

label_protocol: Affymetrix GeneChip® IVT Labeling Kit, according to manufacturer's protocol

growth_protocol: SC –Ura GAL (2%) cultures (12 ml) were inoculated at OD600 0.2 and incubated with shaking at 30º C until reaching an OD600 of ~1.0.

extract_protocol: Cells were harvested and lysed with acid-washed glass beads. Total RNA was isolated with Qiagen RNeasy Midi kit

treatment_protocol: Htt103Q is a galactose (GAL)-inducible,

tag: genotype/variation

value: MATa (BY4741)


title: Ume1.3


title: mbf1.3


title: Htt103Q rep.3

value: mutant htt fragment


title: transfected miR-neg replicate 2

description:
BJAB cells transfected with miR-neg. Biological replicate 2 of 2.
pneg-rep2


title: H9 ESC - lincRNA expression

source_name: H9 ESCs

value: H9 ESCs


title: bna4rep.1


title: transfected miR-K10a replicate 2

description:
BJAB cells transfected with miR-K10a. Biological replicate 2 of 2.
p10a-rep2


title: H9-RPE

description: Gene expression at confluency

source_name: ES-derived RPE

value: ES-derived retinal pigmented epithelium


title: bna4rep.2


title: Htt103Q rep.2


title: Htt25Q rep.1

value: wild-type


title: Htt25Q rep.2


title: Htt103Q rep.1


title: mbf1.1


title: transfected miR-K10a replicate 1

description:
BJAB cells transfected with miR-K10a. Biological replicate 1 of 2.
p10a-rep1


title: Htt25Q rep.3


title: mbf1.2


title: CD34+ derived iPSC line 8 - lincRNA expression, replicate 2

source_name: CD34+ derived iPSC line 8


title: bna4rep.3


title: control_48h_rep.2

city: Seidai

first: NAO

last: Hirota

state: Miyagi

address: Aoba-ku, Seiryo-machi 2-1

department: Respratory medicine

institute: Tohoku university

description: Gene expression in no treatments cell line

hyb_protocol: 1.65 ug of Cy3-labelled cRNA was fragmented at 60°C for 30 minutes in a reaction volume of 110 ml containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 110 ml of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent Whole Human Genome Oligo Microarrays (G4112F) for 17 hours at 65Â°C in a rotating Agilent hybridization oven (G2545A).  After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37Â°C GE Wash buffer 2 (Agilent), then dried immediately by brief centrifugation.

scan_protocol: Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2565AA) using one color scan setting for 4x44k array slides (Scan Area 61x21.6 mm, Scan resolution 5μm, Dye channel is set to Green and Green PMT is set to 100%).

data_processing: The scanned images were analyzed with Feature Extraction Software 9.5.3 (Agilent) using default parameters (protocol GE1-v5_95_Feb07 and Grid: 014850_D_20060807) to obtain background subtracted and spatially detrended Processed Signal intensities.  Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded.

source_name: MRC5 cell extract, 48h, control, replicate 2

label_protocol: Cyanine-3 (Cy3) labeled cRNA was prepared from 1 ug RNA using the One-Color Low RNA Input Linear Amplification PLUS kit (Agilent) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN).  Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.

growth_protocol: The human fetal lung fibroblast cell line (MRC5) were maintained in tissue culture 75cm2 flasks in DMEM supplemented with 10% FBS at 37°C in a humidified atmosphere containing 95% air and 5% CO2. Cells were seeded at 5×104 cells/ml placed into 100mm tissue culture plate (Falcon) 24 hours before treatment resulting in ~70 % confluency at treatment.

extract_protocol: RNA was prepared using RNeasy mini kit (QIAGEN, ) following the manufacturer's recommendations.  The protocol includes an on-column DNase digestion.  RNA was quantified using a NanoDrop-1000 spectrophotometer and quality was monitored with the Agilent 2100 Bioanalyzer (Agilent Technologies).

treatment_protocol: After 24 hours serum starvation, fibroblasts were treated with 3 ng/ml TGF-β1 and were treated with or without 2μg/ml Cyclosporine. The effects were examined at 48 hours after treatment.

value: embryonal lung, normal diploid fibroblast

tag: genetics

value: diploid, male


title: control_48h_rep.1

source_name: MRC5 cell extract, 48h, control, replicate 1


title: TGF_48h_rep.3

description: Gene expression after 48hr in TGF-added cell line

source_name: MRC5 cell extract, 48h, TGF, replicate 3


title: TGF+CsA_48h_rep.3

description: Gene expression after 48hr in TGF + CsA-added cell line

source_name: MRC5 cell extract, 48h, TGF+CsA, replicate 3


title: TGF_48h_rep.1

source_name: MRC5 cell extract, 48h, TGF, replicate 1


title: TGF_48h_rep.2

source_name: MRC5 cell extract, 48h, TGF, replicate 2


title: control_48h_rep.3

source_name: MRC5 cell extract, 48h, control, replicate 3


title: TGF+CsA_48h_rep.2

source_name: MRC5 cell extract, 48h, TGF+CsA, replicate 2


title: B-cell EBV_transformed rep18

city: Richmond

first: Mikhail

last: Dozmorov

state: VA

address: 830 E Main St

department: Biostatistics

institute: Virginia Commonwealth University

description:
Replicate 18
Array18

hyb_protocol: Labeled cRNA probes were hybridized to Illumina arrays

scan_protocol: Images were obtained on an Illumina Beadchip scanner.

data_processing:
We used Illumina BeadStudio 3.0.19.0 to obtain raw data values. Raw data values were quantile-normalized with affy package, a part of Bioconductor, written in R language.
The images and raw data from each chip were transferred automatically to the microarray database using one of the specified microarray core servers.

source_name: Epstein-Barr Virus-transformed B cells collected from normal healthy donors

label_protocol: Illumina Whole Genome Human Ref-8 v2.0 arrays containing ~24,000 probes were handled according to standard protocols established by the manufacturer. Briefly, 200ng of total RNA from each sample were used to generate biotin-labeled cRNA probes using an Illumina MessageAmp cRNA labeling kit protocol (Ambion).

extract_protocol: RNA was isolated using Ambion’s RNAqueous RNA isolation kit (Ambion Inc., TX) according to the manufacturer’s protocol. After purification, RNA concentration was determined with a Nanodrop scanning spectrophotometer.

tag: individual

value: normal healthy donor

value: B cell

tag: transformation

value: Epstein-Barr Virus


title: B-cell EBV_transformed rep20

description:
Replicate 20
Array20


title: TGF+CsA_48h_rep.1

source_name: MRC5 cell extract, 48h, TGF+CsA, replicate 1


title: B-cell EBV_transformed rep19

description:
Replicate 19
Array19


title: CsA_48h_rep.1

description: Gene expression after 48hr in CsA-added cell line

source_name: MRC5 cell extract, 48h, CsA, replicate 1


title: B-cell EBV_transformed rep17

description:
Replicate 17
Array17


title: CsA_48h_rep.2

source_name: MRC5 cell extract, 48h, CsA, replicate 2


title: B-cell EBV_transformed rep13

description:
Replicate 13
Array13


title: Ormes series (in vitro), biological rep4

city: Detroit

first: Carol

state: Michigan

address: 275 East Hancock Street

department: Physiology and Obstetrics and Gynecology

institute: Wayne State University

hyb_protocol: Affymetrix Chromatin Immunoprecipitation Assay Protocol

data_processing: The data were analyzed with ChipInspector software from Genomatix using default analysis settings: Minimum number of probes that overlap with the genes is 3, fold change as threshold is 2 for identifying up/down regulated genes. ChipInspector interprets Affymetrix data with alternative way. It doesn't show each sample's signal intensity. Instead, ChipInspector gives the log2 based fold change ratio between two group samples for each gene. In our case, the log2 base fold change between in vitro and in vivo was given.

organism: Macaca mulatta

source_name: ESC lines generated from in vitro derived embryos

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

growth_protocol: ESC were grown on mitotically inactivated mouse embryonic fibroblast (MEF) feeder cells in Dulbecco’s Modified Eagle Medium (DMEM/F12) (Invitrogen, Grand Island, NY) supplemented with 15% fetal bovine serum (FBS) (Hyclone, Logan, UT), 0.1 mM ß-mercaptoethanol, 1% nonessential amino acids (Invitrogen), 2 mM L-glutamine (Invitrogen), and 4 ng/ml FGF2 (Sigma), at 37°C under a 5% CO2-balance air atmosphere.

extract_protocol: Total RNA was isolated from embryonic stem cell colonies using TRIZOL reagent (Invitrogen), followed by further purification with a RNeasy MinElute Cleanup Kit (Qiagen).


title: B-cell EBV_transformed rep15

description:
Replicate 15
Array15


title: B-cell EBV_transformed rep11

description:
Replicate 11
Array11


title: B-cell EBV_transformed rep16

description:
Replicate 16
Array16


title: B-cell EBV_transformed rep14

description:
Replicate 14
Array14


title: B-cell EBV_transformed rep12

description:
Replicate 12
Array12


title: R series (in vivo), biological rep4

scan_protocol: Affymetrix Chromatin Immunoprecipitation Assay Protocol

source_name: ESC lines generated from in vivo derived embryos


title: R series (in vivo), biological rep3

value: ESC lines generated from in vivo derived embryos


title: Ormes series (in vitro), biological rep3

value: ESC lines generated from in vitro derived embryos


title: CsA_48h_rep.3

source_name: MRC5 cell extract, 48h, CsA, replicate 3


title: B-cell EBV_transformed rep10

description:
Replicate 10
Array10


title: B-cell EBV_transformed rep9

description:
Replicate 9
Array9


title: hfPSMC_CTRLvsMix2_biolrep2_techrepl2

city: Rome

first: Sabrina

last: Tait

state: Italy

address: Viale Regina Elena 299

department: Dpt. Infectious Diseases

country: Italy

institute: Istituto Superiore di Sanità

hyb_protocol: Cy3 and Cy5 labelled samples, to be hybridized on same slides, were mixed and added with Agilent Fragmentation Buffer following manufaturer's instructions. Following 30 min incubation at 60 °C, Agilent GEx Hybridization Buffer was added, volumes loaded on gasket slides into the Agilent SureHyb chamber, array slides placed over and chamber closed. Arrays were incubated for 17h at 65°C with a 10rpm rotation in an Agilent hybridization oven. Arrays were then washed with Agilent Gene Expression Buffers 1 and 2.

scan_protocol: Scanned on an Agilent G2505B Microarray Scanner System. Data extraction and array quality control were performed through the Agilent Feature Extraction Software v9.5.

data_processing: The limma package, part of Bioconductor within the R environment, was used for background subtraction, Global Loess normalization and Aquantile between-arrays normalization.

source_name: hfPSMC, PCB-Mix2 treatment, biolrepl2

label_protocol: cDNA and cRNA synthesis, amplification and labelling was performed using the Quick Amp Labelling Kit, Two Colors (Agilent, Santa Clara, CA, USA), with spike-in internal controls (Agilent) following manufacturer’s instructions, with 1 µg of total RNA as input

growth_protocol: hfPSMC were grown in DMEM/F12 medium without phenol red and supplemented with 10% FBS. After 24h, medium was replaced with fresh medium containing Mix1, Mix2, Mix3 or medium alone as control. After 72h cells were trypsinized and collected.

extract_protocol: Total RNA was extracted by the RNAeasy Kit (QIAGEN) following manufacturer's instructions

tag: established cells

value: hfPSMC

value: 11-12 wks gestation fetus

tag: gender

value: male

value: penile smooth muscle

source_name: hfPSMC, pooled control replicates


title: hfPSMC_Mix2vsMix3_biolrep3_techrepl1

source_name: hfPSMC, PCB-Mix2 treatment, biolrepl3

source_name: hfPSMC, PCB-Mix3 treatment, biolrepl3


title: B-cell EBV_transformed rep8

description:
Replicate 8
Array8


title: B-cell EBV_transformed rep7

description:
Replicate 7
Array7


title: hfPSMC_CTRLvsMix1_biolrep3_techrepl1

source_name: hfPSMC, PCB-Mix1 treatment, biolrepl3


title: Ormes series (in vitro), biological rep2


title: hfPSMC_CTRLvsMix3_biolrep2_techrepl2

source_name: hfPSMC, PCB-Mix3 treatment, biolrepl2


title: hfPSMC_CTRLvsMix2_biolrep2_techrepl1


title: hfPSMC_CTRLvsMix3_biolrep3_techrepl2


title: R series (in vivo), biological rep2


title: B-cell EBV_transformed rep4

description:
Replicate 4
Array4


title: hfPSMC_CTRLvsMix1_biolrep2_techrepl2

source_name: hfPSMC, PCB-Mix1 treatment, biolrepl2


title: B-cell EBV_transformed rep3

description:
Replicate 3
Array3


title: B-cell EBV_transformed rep1

description:
Replicate 1
Array1


title: B-cell EBV_transformed rep5

description:
Replicate 5
Array5


title: hfPSMC_Mix1vsMix2_biolrep3_techrepl2


title: Ormes series (in vitro), biological rep1


title: R series (in vivo), biological rep1


title: B-cell EBV_transformed rep6

description:
Replicate 6
Array6


title: hfPSMC_CTRLvsMix3_biolrep2_techrepl1


title: hfPSMC_CTRLvsMix2_biolrep1_techrepl2

source_name: hfPSMC, PCB-Mix2 treatment, biolrepl1


title: hfPSMC_CTRLvsMix1_biolrep1_techrepl2

source_name: hfPSMC, PCB-Mix1 treatment, biolrepl1


title: hfPSMC_CTRLvsMix3_biolrep1_techrepl1

source_name: hfPSMC, PCB-Mix3 treatment, biolrepl1


title: hfPSMC_CTRLvsMix1_biolrep2_techrepl1


title: B-cell EBV_transformed rep2

description:
Replicate 2
Array2

source_name: Epstein-Barr Virus-transformed B cells collected from normal healthy donors


title: hfPSMC_CTRLvsMix3_biolrep1_techrepl2


title: hfPSMC_CTRLvsMix2_biolrep1_techrepl1


title: hfPSMC_CTRLvsMix1_biolrep1_techrepl1


title: cont. vs. CsA

status: Public on Nov 08 2010

city: Okayama city

first: Atsushi

last: Fujimura

institute: Okayama university

description:
PHK: cont. vs. CsA
US22502561_251239147799_S02_GE2_44k_1205.txt

hyb_protocol: According to manufacturer's protocol (Agilent).

scan_protocol: Scanned on an Agilent Microarray scanner G2565BA.

data_processing:
For calculating the ratio, we used the following protocol; Feature Extraction Software (v8.5.1.1) protocol GE2_22k_1205 (Agilent Technologies).
For normalization, the data were normalized log10 ratio (Cy5/Cy3) representing test/reference, following background subtraction and LOWESS normalization.

source_name: PHK: cont.

label_protocol: Labeled with Cy-3 or Cy-5 using an Agilent Low RNA Input Fluorescent Linear Amplification Kit (Agilent).

growth_protocol: PHK cells were kept in EpiLife medium (Cascade bio.)

extract_protocol: Total RNA extracted using Trizol.

treatment_protocol: untreated, CsA (1uM) and 11R-VIVIT (3uM)

value: PHK keratinocyte cell line

tag: agent

value: control

source_name: PHK: CsA

value: CsA


title: cont. vs. 11R-VIVIT

description:
PHK: cont. vs. 11R-VIVIT
US22502561_251239147798_S01_GE2_44k_1205.txt

source_name: PHK: 11R-VIVIT


title: S2-cells_no methoxyfenozide_1 vs S2_cells_untreated_2

city: Ghent

first: Guy

last: Smagghe

address: Coupure Links

department: Crop Protection

country: Belgium

institute: Ghent University

description: no extra information to be added

hyb_protocol: Prehybridisation: The slides were incubated in pre-warmed prehybridisation solution (5x SSC, 0.1% SDS, 1% BSA) at 42°C for 1hr. Next, they were washed five times in MilliQ water and once in isopropanol. The slides were dried by centrifugation and hybridization of the samples was performed immediately afterwards. Hybridisation: 75ul of pre-warmed (42°C) hybridisation buffer (50% formamide, 10x SSC, 0.2% SDS) was added to each Alexa Fluor 647 sample, which was dissolved by gently pipetting. Subsequently, the dissolved sample was added to the corresponding Alexa Fluor 555 sample, which was also dissolved by gently pipetting. Next, the samples were denaturated at 95°C during 3min. Immediately afterwards, the samples were applied onto the microarray slides. Hybridisation was performed in a SlideBooster System (Advalytix) at 42°C for 20hr. Wash: The slides were successively washed in 1x SSC, 0.2% SDS (42°C, 5min), 0.1x SSC, 0.2% SDS (room temperature, 5min) and 0,1x SSC (room temperature, 5min), and dried by centrifugation.

scan_protocol: Scanning of the microarray slides was performed at wavelengths of 635nm and 532nm by means of a Genepix Personal 4100A confocal scanner (MDS Analytical Technologies). The photomultiplier tube (PMT) voltage gain was adjusted for each array in order to obtain a count ratio close to one. Image analysis was performed by using the Genepix Pro 6.0 software (MDS Analytical Technologies) and consisted of spot identification and extraction of the fluorescent intensity data.

data_processing: Data were imported in a MIAME (Minimum Information About a Microarray Experiment)-compliant database (www.islab.ua.ac.be/base/), developed by the Intelligent Systems Laboratory (University of Antwerp, Antwerp, Belgium). For each spot the median intensity was calculated (which is the median of intensities measured for each pixel within the spot) and background (BG) corrected. Spots for which the median foreground (FG) signal was smaller than that of the median BG + 2SD (SD: standarddeviation of the background signal) were filtered out. The Cy5/Cy3-ratio was calculated for each spot, log2-transformed and normalized by using the 'variation stabilization normalization' (vsn) method.

label: Alexa Fluor 647 (Invitrogen)

organism: Drosophila melanogaster

source_name: methoxyfenozide treated - again cultured without methoxyfenozide

label_protocol:
The samples were labeled in an indirect manner: aminoallyl-dUTPs were incorporated during the reverse transcription step and subsequently, amine-reactive Alexa Fluor 647 or 555 carboxylic acid succinimidyl ester dyes (Invitrogen) were coupled to the aminoallyl groups. Aminoallyl-dUTPs (aa-dUTPs, Sigma Aldrich) were dissolved in 0,1M KPO4 (pH 7.5) to a final concentration of 100mM. 5ul dATP, 5ul dCTP, 5ul dGTP, 3ul dTTP and 2ul aa-dUTP (each 100mM) were mixed together to obtain the 50x aminoallyl-dNTP mix with a 2:3 aa-dUTP:dTTP ratio. For each sample, 10ug of total RNA was used. 2ug poly-dT oligo (dT18, Sigma Aldrich) and 3ug random hexamers (Sigma Aldrich) were added to the total RNA and the volume was adjusted to 18.5ul with di-methyl-propyl carbonate (DMPC)-treated MilliQ water. Subsequently, the mix was incubated at 70°C during 10min, and immediately afterwards placed on ice. 0.6ul 50x aminoallyl-dNTP mix, 6ul 5x first strand buffer, 3ul DTT (0,1 M), 400U SuperScript III RT (SuperScript III Reverse Transcriptase kit, Invitrogen) and 20U RNaseOut Recombinant Ribonuclease Inhibitor (Invitrogen) were added and the mix was incubated at 46°C for 2hr. Upon reverse transcription the RNA was hydrolized by adding 10ul NaOH (1M) and 10ul EDTA (0.5M). In order to obtain a neutral pH 10ul HCl (1M) and 10ul NaAc (3M) were added. The resulting aminoallyl-labeled cDNA was purified by means of the QIAQuick PCR Purification Kit (Qiagen) as described by the manufacturer, except that not the wash and elution buffers from the kit were used as these contain free amines which interfere with the dye coupling reaction. Instead phosphate wash buffer (5mM KPO4, pH 8.0; 80% EtOH) and elution buffer (4mM KPO4, pH 8.5) were used. Subsequently, the samples were dried by evaporation in a SpeedVac.
Each portion of Alexa Fluor 647 or 555 carboxylic acid succinimidyl ester dye was dissolved in 9ul Na2CO3 (0.1M, pH 9.0) and added to the dry cDNA. The mix was then incubated at room temperature for 1hr. The Alexa Fluor labeled samples were purified by means of the QIAQuick PCR Purification Kit (Qiagen) as described by the manufacturer. Quantification and quality control of the labeled samples were performed with the ND-1000 UV-Vis Spectrophotometer (Thermoscientific, NanoDrop Products). Subsequently, the samples were dried by evaporation in a SpeedVac.

growth_protocol: Schneider 2 (S2) cells (S2-Mt-Dl) were cultured in HyQ SFX-Insect™ medium (Perbio Science, Erembodegem, Belgium). A subpopulation of these untreated S2 cells was cultured in HyQ SFX-Insect™ medium to which increasing amounts of methoxyfenozide were added (cfr. Treatment Protocol). A subpopulation of the methoxyfenozide-resistant S2 was again cultured in normal medium.

extract_protocol: RNeasy Mini Kit (Qiagen)

treatment_protocol:
Cells were exposed to increasing concentrations of methoxyfenozide starting from 0.1 µM up to 100 µM. Gradually increasing amounts of methoxyfenozide (until the highest concentration, 100 µM) were applied for over 4 months. Methoxyfenozide-caused cessation of cell proliferation was used as a critrerium to select resistant S2 cells (as previously described in Mosallanejad, H., Soin, T. and Smagghe, G. (2008) Selection for resistance to methoxyfenozide and 20-hydroxyecdysone in cells of the beet armyworm, Spodoptera exigua. Archives of Insect Biochemistry and Physiology 67, 36-49).
The cell susceptibility of obtained methoxyfenozide-resistant cells was determined by an in vitro bioassay by using the MTT technique. After reaching the highest exposure concentration, we continued subculturing the resistant cells under this pressure.
In order to determine the stability of the observed resistance, a subpopulation of the resistant cells was propagated for four passages in the absence of methoxyfenozide.

value: Schneider 2 cells (S2-Mt-Dl)

value: embryo

label: Alexa Fluor 555 (Invitrogen)

source_name: untreated

treatment_protocol: untreated