title: Sham Day 5,# 1

description:
Male C57Bl/6 mice (6 week old) were sham-operated and olfactory epithelium tissue collected 5 days post-operation. Total RNA was extracted from the olfactory epithelium tissue.  Total RNA (5µg each) was pooled from 3 mice. Target cRNA was prepared and hybridized to Mouse Genome U74Av2 GeneChip (GPL81) as per standard, commercially available Affymetrix GeneChips protocol.
Keywords = olfaction
Keywords = neurogenesis
Keywords = apoptosis
Keywords = gene silencing
Keywords = microarray, transcription factor


title: Gene expression profiling of C2C12 MyoD-silenced clone at 3 days of myogenic differentiation (B)

status: Public on Jul 28 2005

type: RNA

city: Padova

first: Gerolamo

last: Lanfranchi

address: Via U. Bassi, 58/B

department: CRIBI - Biotechnology Center and Biology Department

country: Italy

institute: University of Padova

description: RNA purified from C2C12 MyoD(-)cl.A8 at three myogenic differentiation time-points was compared with RNA from C2C12 control cells at the corresponding differentiation stages. These samples were labeled and hybridized to muscle specific microarrays produced by our group (Human Array 1.0).

hyb_protocol: Microarray hybridisation was carried out in a dual slide chamber (HybChamber, Gene Machines, San Carlos, CA, USA) humidified with 100 µl of 3 x SSC. Labeled cDNA was dissolved in 40 µl of hybridisation buffer, denatured at 90°C for 2 min in a thermal cycler and applied directly to the slides. Microarrays were covered with 22 x 40 mm cover slip and hybridized overnight at 65°C by immersion in a high precision water bath (W28, Grant, Cambridge, UK). Post-hybridization washing was performed according to the MICROMAX TSA Detection kit (PerkinElmer).

scan_protocol: Digital images were generated in a GSI Lumonics LITE dual confocal laser scanner (ScanArray Microarray Analysis Software) and processed with QuantArray Analysis Software (GSI Lumonics, Ottawa, Canada).

data_processing: The gene expression data analysis tool SNOMAD (http://pevsnerlab.kennedykrieger.org/snomadinput.html) was used for microarray data normalization and standardization. Log2 transformation was performed for each normalized expression value.

molecule: total RNA

organism: Mus musculus

source_name: C2C12 control cells (Reference)

label_protocol: Total RNA was retro-transcribed and labeled using a MICROMAX TSA Labeling Kit (PerkinElmer). Two ug of total RNA were used in each reaction but only half of the labeled cDNA was actually hybridized to the microarrays.

extract_protocol: Total RNA was purified from C2C12 control cells following the standard Trizol protocol. The RNA 6000 LabChip kit (Agilent Technologies) was used for RNA quantification and quality control in conjunction with an Agilent Bioanalyzer 2001.

tag: - C2C12 cell line

tag: - Species

value: mouse, C3H; Tissue:muscle

tag: - Morphology

value: Myoblast

tag: - Depositor

value: Obtained from ATCC, USA

tag: - Properties

value: differentiation; muscle proteins express; myogenesis

tag: - Growing medium

value: DMEM + 2mM Glutamine + 10% Fetal Bovine Serum (FBS)

tag: - Differentiating medium

value: DMEM + 2mM Glutamine + 2% Horse Serum (HS)

source_name: C2C12 MyoD-silenced cells (Test)

extract_protocol: Total RNA was purified from C2C12 MyoD(-) cells (MyoD(-)cl.A8) after three days of myogenic differentiation following the standard Trizol protocol. The RNA 6000 LabChip kit (Agilent Technologies) was used for RNA quantification and quality control in conjunction with an Agilent Bioanalyzer 2001.

tag: - C2C12 MyoD-silenced cells

value: the mouse MyoD transcript was cloned in pcDNA 3.0 mammalian expression vector in antisense orientation and the construct was transfected in C2C12 cells. The cells were selected for ten days and the G-418 resistant clones were tested for MyoD silencing.

tag: - MyoD knock-down clone


title: Gene expression profiling of C2C12 MyoD-silenced clone at 6 days of myogenic differentiation (A)

description: RNA purified from C2C12 MyoD(-)cl.A8 at six myogenic differentiation time-points was compared with RNA from C2C12 control cells at the corresponding differentiation stages. These samples were labeled and hybridized to muscle specific microarrays produced by our group (Human Array 1.0).

extract_protocol: Total RNA was purified from C2C12 MyoD(-) cells (MyoD(-)cl.A8) after six days of myogenic differentiation following the standard Trizol protocol. The RNA 6000 LabChip kit (Agilent Technologies) was used for RNA quantification and quality control in conjunction with an Agilent Bioanalyzer 2001.


title: Gene expression profiling of C2C12 MyoD-silenced clone at 3 days of myogenic differentiation (A)


title: WT treated replicate 3

status: Public on Jul 27 2005

city: Cambridge

first: Jennifer

last: Robbins

state: MA

address: 77 Massachusetts Ave.

department: Biological Engineering

country: USA

institute: Massachusetts Institute of Technology

description: Overnight culture was diluted 1000-fold with fresh LB medium and cultured further. Log phase culture was diluted to a cell density of 2 X 108 cells/ml in M9 salts containing 150 uM cisplatin and incubated at 37°C for two hours, after which it was resuspended in LB broth for 90 minutes. Total RNA was isolated from cells by extraction using the MasterPure RNA Purification Kit (Epicentre Technologies) followed by DNAse digestion according to the manufacturer’s protocol. The isolated total RNA was quantitated by absorption at 260 nm (typical yield from a 15 ml culture was 250-500 µg of total RNA), and the purity was determined by the ratio of absorption values at 260/280nm. RNA quality was determined by formaldehyde agarose gel electrophoresis (1.2% agarose in FA Buffer pH 7.0 (20 mM 3-[N-morpholino]propanesulfonic acid, 5 mM sodium acetate, 1 mM ethylenadiaminetetraacetic acide (EDTA))) or by analysis on an Agilent 2100 Bioanalyzer. All samples visualized by gel electrophoresis or by the bioanalyzer electropherogram showed clear distinct bands correlating to 16S and 23S ribosomal RNA, indicating that no detectable RNA degradation occurred and that RNA integrity was maintained throughout the RNA isolation procedure. mRNA was enriched from total RNA as described in the Affymetrix GeneChip Expression Analysis Technical Manual for GeneChip E. coli Sense Genome Arrays.

data_processing: GCRMA normalization using the Array Analyzer module version 2.0.2 in S-Plus version 6.2 (Insightful)

label: PEO-iodeacetyl-Biotin

organism: Escherichia coli

source_name: E. coli WT strain AB1157

label_protocol: Affymetrix protocol for E. coli sense genome arrays

extract_protocol: Epicentre MasterPure RNA Purification kit

tag: F- thr-1 araC14 leuB6(Am)Δ (gpt-proA)62 lacY1 tsx-33 supE44(AS) galK2(Oc) hisG4(Oc) rfbD1 mgl-51 rpoS396(Am) rpsL31(StrR) kdgK51 xylA5 mtl-1 argE3(Oc) thi-1


title: WT treated replicate 2

source_name: E. coli wildtype strain AB1157

growth_protocol: Overnight culture was diluted 1000-fold with fresh LB medium and cultured further. Log phase culture was diluted to a cell density of 2 X 108 cells/ml in M9 salts containing 150 uM cisplatin and incubated at 37°C for two hours, after which it was resuspended in LB broth for 90 minutes.


title: WT treated replicate 1


title: mutS treated replicate 3

source_name: E. coli mutS mutant strain GM5555

tag: As AB1157 but mutS215::Tn10


title: Gene expression profiling of C2C12 MyoD-silenced clone at 1 day of myogenic differentiation (B)

description: RNA purified from C2C12 MyoD(-)cl.A8 at one myogenic differentiation time-points was compared with RNA from C2C12 control cells at the corresponding differentiation stages. These samples were labeled and hybridized to muscle specific microarrays produced by our group (Human Array 1.0).

extract_protocol: Total RNA was purified from C2C12 MyoD(-) cells (MyoD(-)cl.A8) after one day of myogenic differentiation following the standard Trizol protocol. The RNA 6000 LabChip kit (Agilent Technologies) was used for RNA quantification and quality control in conjunction with an Agilent Bioanalyzer 2001.


title: mutS treated replicate 2


title: WT mock replicate 2

description: Overnight culture was diluted 1000-fold with fresh LB medium and cultured further. Log phase culture was diluted to a cell density of 2 X 108 cells/ml in M9 salts and incubated at 37°C for two hours, after which it was resuspended in LB broth for 90 minutes. This treatment served as a mock treatment for cultures incubated with a chemical (cisplatin). Total RNA was isolated from cells by extraction using the MasterPure RNA Purification Kit (Epicentre Technologies) followed by DNAse digestion according to the manufacturer’s protocol. The isolated total RNA was quantitated by absorption at 260 nm (typical yield from a 15 ml culture was 250-500 µg of total RNA), and the purity was determined by the ratio of absorption values at 260/280nm. RNA quality was determined by formaldehyde agarose gel electrophoresis (1.2% agarose in FA Buffer pH 7.0 (20 mM 3-[N-morpholino]propanesulfonic acid, 5 mM sodium acetate, 1 mM ethylenadiaminetetraacetic acide (EDTA))) or by analysis on an Agilent 2100 Bioanalyzer. All samples visualized by gel electrophoresis or by the bioanalyzer electropherogram showed clear distinct bands correlating to 16S and 23S ribosomal RNA, indicating that no detectable RNA degradation occurred and that RNA integrity was maintained throughout the RNA isolation procedure. mRNA was enriched from total RNA as described in the Affymetrix GeneChip Expression Analysis Technical Manual for GeneChip E. coli Sense Genome Arrays.


title: mutS mock replicate 1

data_processing: GCRMA normalization using Array Analyzer version 2.0.2 in S-Plus version 6.2 (Insightful)

label_protocol: Affymetrix protocol for E. coli sense arrays

growth_protocol: Overnight cultures were diluted 1000-fold with fresh LB medium and cultured further.  Log phase cultures were diluted to a cell density of 2 X 108 cells/ml in M9 salts and incubated at 37°C for two hours, after which they were resuspended in LB broth for 90 minutes.

extract_protocol: Epicentre MasterPure RNA purification kit


title: dam treat replicate 1

source_name: E. coli dam mutant strain GM3819

tag: dam-16::Kan thr-1 leuB6 thi-1 argE3 hisG4 proA2 lacY1 galK2 mtl-1 xyl-5 ara-14 rpsL31 tsx-33 supE44 rfbD1 kdgK51


title: dam treat replicate 2


title: dam mutS treat replicate 1

source_name: E. coli dam mutS mutant strain GM5556

tag: As GM3819  dam-16::Kan but mutS215::Tn10


title: mutS mock replicate 2


title: dam mutS treat replicate 3


title: WT mock replicate 1

source_name: WT E. coli strain AB1157

growth_protocol: Overnight culture was diluted 1000-fold with fresh LB medium and cultured further. Log phase culture was diluted to a cell density of 2 X 108 cells/ml in M9 salts and incubated at 37°C for two hours, after which it was resuspended in LB broth for 90 minutes.


title: WT mock replicate 3


title: dam treat replicate 3


title: mutS treated replicate 1


title: dam mutS mock replicate 3


title: dam mutS treat replicate 2


title: dam mock replicate 3


title: dam mutS mock replicate 1


title: mutS mock replicate 3


title: dam mock replicate 1


title: dam mock replicate 2


title: Cell Culture Exposure (3MAC ISO3)

status: Public on Jul 23 2005

city: Philadelphia

first: Roderic

last: Eckenhoff

state: PA

institute: University of Pennsylvania

description: Fetal brains were obtained from 3 pregnant rats at pregnant day 17. Primary cortical neurons were isolated and pooled separately from each mother and plated onto 100mm ´ dishes, for a total of 4 plates from each pregnant rat.  At Day in Vitro (DIV)  16, 3 of the 4 platesfrom the same mother, were exposed to either 1 or 3 MAC halothane (about 1% or 3%) or 3 MAC isoflurane (about 4%) in a gas tight chamber inside the culture incubator for 6 hr.  The 4th plate from each mother treated with air were used (inside the same incubator, but outside the chamber) as controls.  Isoflurane at 1 MAC was not conducted because of the complete lack of transcriptional response in the animals exposed to 1 MAC of isoflurane.  The cells were recovered for an additional 6 hr, harvested, frozen by liquid nitrogen, and stored at –80oC.

hyb_protocol: Agilent

scan_protocol: Agilent

data_processing: GeneSpring7.0 and Partek Pro.

organism: Rattus norvegicus

source_name: Pooled Control

label_protocol: Agilent

growth_protocol: Agilent

extract_protocol: Agilent

treatment_protocol: Agilent

source_name: 3MAC ISO3


title: Cell Culture Exposure (3MAC ISO2)

source_name: 3MAC ISO2


title: Cell Culture Exposure (3MAC ISO1)

source_name: 3MAC ISO1


title: dam mutS mock replicate 2


title: Cell Culture Exposure (1MAC Halo3)

source_name: 1MAC Halo3


title: Cell Culture Exposure (3MAC Halo3)

source_name: 3MAC Halo3


title: Cell Culture Exposure (control3)

source_name: Control 3


title: HEK293_HNF4a2_4-

status: Public on Jul 21 2005

city: Essen

first: Ludger

last: Klein-Hitpass

address: Virchowstr. 173

department: Universitaetsklinikum

country: Germany

institute: Institut fuer Zellbiologie

description:
Analysis of gene expression in human embryonic kidney cells HEK293 overexpressing the transcription factors HNF4a2 or mutant forms HNF4a2 C106R and HNF4a2 R154X, which were introduced by FRT/FLP recombination. The analysis was performed 24 hours following 1µg/ml tetracycline treatment to induce the expression of the genes. Results identify HNF4a regulated genes in kidney cells being several of these genes deregulated in renal cell carcinoma.
Keywords = HEK293
Keywords = embryonic kidney cells
Keywords = HNF4a2
Keywords = tetracycline
Keywords = FRT

organism: Homo sapiens

source_name: HEK293 cell line #4 with noninduced HNF4a2 wild type


title: wt1 filter1

city: Burjassot

first: Jose

middle: E.

last: Perez-Ortin

state: Valencia

address: Dr. Moliner 50

department: Bioquimica y Biologia Molecular

country: Spain

institute: Universitat de Valencia

description:
Total RNA isolated from a exponentially growing culture of wild type yeast cells. cDNA labelling using oligo(dT)15VN and 33P-dCTP
Keywords = yeast
Keywords = HAT

organism: Saccharomyces cerevisiae

source_name: exponential growing wild type yeast strain


title: Cell Culture Exposure (3MAC Halo1)

source_name: 3MAC Halo1


title: Cell Culture Exposure (1MAC Halo2)

source_name: 1MAC Halo2


title: Cell Culture Exposure (control2)

source_name: Control 2


title: HEK293_HNF4a2_1+

source_name: HEK293 cell line #1 with induced HNF4a2 wild type


title: Cell Culture Exposure (1MAC Halo1)

source_name: 1MAC Halo1


title: HEK293_HNF4a2_4+

source_name: HEK293 cell line #4 with induced HNF4a2 wild type


title: HEK293_HNF4a2_R154X_6+

source_name: HEK293 cell line #6 with induced HNF4a2 mutant R154X


title: WTCy3/Rv2869c Cy5 with Tween #1

status: Public on Jul 22 2005

city: New York

first: Michael

last: Glickman

state: NY

institute: Sloan Kettering Institute for Cancer Research

description:
Protocol for preparing the hybridization extract:

15ml of liquid culture described above were collected by centrifugation at 3000g, and cell pellets frozen on dry ice. Frozen pellets were resuspended in 1ml of Trizol reagent (Invitrogen) and lysed by mechanical disruption using .7mm Zirconia beads for 3 x 1min in a Mini bead beater 8 (Biospec products) at maximum speed. The remainder of the procedure was according to the Trizol manufacturers instructions with the addition of the optional high salt precipitation step. RNA was subsequently cleaned using Qiagen RNAeasy columns according ot the manufacturers RNA cleanup protocol.

Labeling protocol(s).
5-7.5micrograms of total RNA was labeled using the Fairplay microrray labeling kit (Stratagene) with substitution of Random Hexamer primers. Aminoallyl labeled CDNA was coupled to Cy3/Cy5 using Post labeling reactive dye packs (Amersham). Excess dye was removed using Qiaquick nucleotide removal columns according to the manufacturer’s instructions.

data_processing: Intensity dependent normalization in GenePix 4.0

organism: Mycobacterium tuberculosis

source_name: WT Mtb grown with Tween

tag: Wild type Mtb erdman grown with .05% Tween80

source_name: M. tuberculosis Rv2869c null mutant

tag: Rv2869c null mutant grown in vitro with .05% Tween.


title: HEK293_HNF4a2_R154X_6-

source_name: HEK293 cell line #6 with noninduced HNF4a2 mutant R154X


title: Cell Culture Exposure

source_name: Pooled Control

tag: Cy3 label

source_name: Control 1


title: HEK293_HNF4a2_C106R_1-

source_name: HEK293 cell line #1 with noninduced HNF4a2 mutant C106R


title: WT Cy5/Rv2869c mutant Cy3 no Tween #2

description:
Protocol for preparing the hybridization extract:

15ml of liquid culture described above were collected by centrifugation at 3000g, and cell pellets frozen on dry ice. Frozen pellets were resuspended in 1ml of Trizol reagent (Invitrogen) and lysed by mechanical disruption using .7mm Zirconia beads for 3 x 1min in a Mini bead beater 8 (Biospec products) at maximum speed. The remainder of the procedure was according to the Trizol manufacturers instructions with the addition of the optional high salt precipitation step. RNA was subsequently cleaned using Qiagen RNAeasy columns according ot the manufacturers RNA cleanup protocol.

Labeling protocol(s).
5-7.5micrograms of total RNA was labeled using the Fairplay microrray labeling kit (Stratagene) with substitution of Random Hexamer primers. Aminoallyl labeled CDNA was coupled to Cy3/Cy5 using Post labeling reactive dye packs (Amersham). Excess dye was removed using Qiaquick nucleotide removal columns according to the manufacturer’s instructions

source_name: Rv2869c mutant without tween

tag: see below for channel 2

source_name: Wild type M. tuberculosis Erdman

tag: o All RNA was from the indicated strain of M. tuberculosis (either wild type M. tuberculosis Erdman or the Mtb Rv2869c null mutant

tag: •RNA was isolated from logarithmic phase cultures at OD600 .3. For (+Tween) conditions, the media was Middlebrook 7H9 with 10% OADC supplement, .5% glycerol, and .05% Tween 80. For (-Tween) conditions, bacilli were transferred to identical media without Tween 80 and harvested for RNA preparation 18hours later.


title: B20 MtIFS1-expressing line replicate 3

status: Public on Jul 20 2005

city: Ardmore

first: Bettina

last: Deavours

state: OK

institute: Samuel Roberts Noble Foundation

description:
PolyA RNA was isolated from 400 ug of total leaf RNA using the Poly(A)Purist MAG kit from Ambion (Austin, TX).  Two ug of polyA was transcribed into cDNA and fluorescently labeled with Cy3 and Cy5 dyes using the SuperScript Indirect cDNA Labeling System (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. Labeled cDNA for experimental (Cy5) and reference (Cy3) samples were combined, ethanol precipitated, and resuspended in 10 ul of water.  The cDNA was heated at 100°C for 3 min before the addition of 70 ul of SlideHyb Glass Array Hybridization Buffer #1 (Ambion, Austin, TX).  The hybridization solution was pipetted onto slides covered with a gapped coverslip (LifterSlips, Erie Scientific Company, Portsmouth, NH).  Slides were incubated overnight at 42°C in Corning hybridization chambers, then washed in 1x SSC, 0.1% (w/v) SDS for 5 min at 42°C followed by 5 min washes in 0.1x SSC, 0.1% SDS (w/v); and 0.05x SSC at room temperature.  Slides were scanned using the ScanArray 4000 Microarray Analysis System.  Based on an initial low resolution (50 um) scan, laser power settings were manually adjusted to balance the intensities between the two channels and slides were rescanned at 10 um resolution.  Typical laser settings were: Cy3 laser power=100, PMT=80; Cy5:  laser power=85, PMT=75.  GPR files were generated using GenePix Pro 4.1 software and the mean pixel intensities for each spot were used in subsequent data analysis.  Data files were imported into GeneTraffic Duo Version 2.6 and data for each hybridization were normalized using the Lowess Sub-Grid method.  Spots flagged by the program under the default settings (spots with a raw pixel intensity less than 100 or lower than the average background and spots with an intensity/background intensity ratio <1) were removed from further analysis.
Keywords = Medicago
Keywords = alfalfa
Keywords = isoflavone synthase
Lot batch = Batch 1_9

organism: Medicago truncatula

source_name: B20, Cy5, leaf poly_A RNA

source_name: vector control line VB2, Cy3, leaf poly_A RNA


title: B20 MtIFS1-expressing line replicate 2


title: C22 MtIFS1-expressing line replicate 3

source_name: C22, Cy5, leaf poly_A RNA


title: B20 MtIFS1-expressing line replicate 1


title: C22 MtIFS1-expressing line replicate 2


title: Cell Culture Exposure (3MAC Halo2)

source_name: 3MAC Halo2


title: HEK293_HNF4a2_C106R_1+

source_name: HEK293 cell line #1 with induced HNF4a2 mutant C106R


title: n5t0n

city: St Lucia

first: Darrin

middle: F

last: Taylor

state: Qld

address: Research Rd

department: Genomics and Bioinformatics

country: Australia

institute: Institute for Molecular Bioscience, Uni of Qld

description: Naïve cells (RPMI 1640 for 48 h).  Total RNA was isolated using RNeasy Mini Kit and Qiashredder column.  Targets were produced using standard Affymetrix procedures from 10 micrograms of total RNA.

source_name: U937 cells; Human monoblastoid line devoid of guanylate cyclase


title: VC11 vector control line replicate 3

source_name: VC11, Cy5, leaf poly_A RNA


title: C22 MtIFS1-expressing line replicate 1


title: VC11 vector control line replicate 2


title: VC11 vector control line replicate 1


title: SiHa17v13

status: Public on Jul 17 2005

city: Seattle

first: Pavel

last: Sova

state: WA

address: 325 9th Avenue

department: Department of Pathology

institute: University of Washington

description: SiHa were plated in 10-cm dishes at approximately 25% confluence. After attachment (8 or 16 hours after plating), 5-aza-2’-deoxycytidine was added at 200 nM concentration.  Twenty-four hours later, medium was exchanged with fresh 200 nM 5-aza-2’-deoxycytidine.  Twenty four hours later, medium was exchanged with fresh 200 nM 5-aza-2’-deoxycytidine and 300 nM trichostatin A.  Mock-treated cultures were handled identically except that only solvents (PBS and EtOH, respectively) were added in media instead of drugs.  Twenty four hours later (72 hours after starting the treatment) cells were harvested by brief trypsinization followed by pelleting of the cells.  All RNA isolation was accomplished with Qiagen RNeasy Mini Kit reagents. The RNA (25 ug) was annealed with 5 ug oligo dT12-18, and reverse-transcribed into cDNA with Superscript II reverse transcriptase for 2h at 42oC in the presence of 0.5mM dGTP, 0.5mM dCTP, 0.5mM dATP, 0.3mM dTTP, 0.2mM amino-allyl dUTP. After hydrolysis of RNA in 0.2M NaOH, Tris was removed from the reaction with a Microcon-30 concentrator. The cDNA from drug-treated and mock-treated SiHa was covalently coupled separately with Cy3 and Cy5 monoreactive fluors, respectively, in 50mM sodium bicarbonate, pH 9.0, followed by quenching with 2.7M hydroxylamine. The Cy5 and Cy3 labelled cDNAs were combined and purified with a QIAquick PCR purification kit. Fluorescent array images were collected for both Cy3 and Cy5 with a GenePix 4000A fluorescent scanner and image intensity data were extracted and analyzed with GenePix Pro 3.0 analysis software. After background correction, removal of flagged values, normalization (LOWESS), log base 2 fluorescence signals were subtracted (Cy5 channel minus Cy3 channel) to provide the ratio (VALUE).  This is a dye-flip experiment to SiHa13v17 (GSM37898).

source_name: drug-treated SiHa cells

source_name: mock-treated SiHa cells


title: Six4 target genes in mk4 cells

city: Minamikawachi

first: Shigeru

last: Sato

state: Tochigi

address: Yakushiji

department: Division of Biology

country: Japan

institute: Jichi Medical School

description: To identify Six4 target genes, we chose the mK4 cell line that represents later metanephric mesenchyme of the embryonic kidney. The cells were infected with adenovirus vectors (AxCAwt) overexpressing VP16-Six4wt or VP16-Six4W263R and cultured for 24h. Total RNA was extracted using Isogen reagent (Nippon Gene), treated with RNase-free DNase (Roche Diagnostics) and purified with RNeasy Midi Kit (QIAGEN). Poly A+ RNA was selected using Oligotex-dT30 <super> latex beads (TaKaRa). Poly A+ RNA (20 µg) was reverse transcribed using an oligo(dT) primer in the presence of aminoallyl dUTP without the addition of spike-in controls. Then, single-stranded cDNAs were coupled with Cy3 (AxCAwt VP16-Six4wt infected sample) or Cy5 (AxCAwt VP16-Six4W263R infected sample) dyes, and the labeled probes were purified with QIAquick PCR Purification kit (QIAGEN). Hybridization to the microarray was carried out at 65°C for 17h according to the manufacturer's instruction. The arrays were washed, dried and scanned using ScanArray 5000 (GSI Lumonics) at two different PMT voltage settings (high and low) to avoid signal saturation. Cy3 and Cy5 intensities for each spot on the array were determined by QuantArray software (Version 3.0.0.0, GSI Lumonics). The raw data thus obtained was processed and the Cy3 to Cy5 ratios were calculated as follows: 1) subtraction of the fluorescence intensity of negative control spots as background from the intensity of each of the Cy3 and Cy5 spots, 2) normalization of the entire data set using the global normalization method, 3) elimination of spots with high background intensity for either dye, and 4) determination of the Cy3 to Cy5 ratios.

source_name: mk4 cells overexpressing VP16-Six4wt

source_name: mk4 cells overexpressing VP16-Six4W263R


title: SiHa14v16

description: SiHa were plated in 10-cm dishes at approximately 25% confluence. After attachment (8 or 16 hours after plating), 5-aza-2’-deoxycytidine was added at 200 nM concentration.  Twenty-four hours later, medium was exchanged with fresh 200 nM 5-aza-2’-deoxycytidine.  Twenty four hours later, medium was exchanged with fresh 200 nM 5-aza-2’-deoxycytidine and 300 nM trichostatin A.  Mock-treated cultures were handled identically except that only solvents (PBS and EtOH, respectively) were added in media instead of drugs.  Twenty four hours later (72 hours after starting the treatment) cells were harvested by brief trypsinization followed by pelleting of the cells.  All RNA isolation was accomplished with Qiagen RNeasy Mini Kit reagents. The RNA (5 ug) was amplified and labeled with aa-UTP using Amino Allyl Message Amp aRNA kit (Ambion, Austin, TX). The aRNA from mock-treated and drug-treated SiHa cells was covalently coupled separately with Cy3 and Cy5 monoreactive fluors, respectively, in 50mM sodium bicarbonate, pH 9.0, followed by quenching with 2.7M hydroxylamine. The Cy5 and Cy3 labelled aRNAs were combined and purified with a QIAquick PCR purification kit.  Fluorescent array images were collected for both Cy3 and Cy5 with a GenePix 4000A fluorescent scanner and image intensity data were extracted and analyzed with GenePix Pro 3.0 analysis software. After background correction, removal of flagged values, normalization (LOWESS), log base 2 fluorescence signals were subtracted (Cy5 channel minus Cy3 channel) to provide the ratio (VALUE).  This is a dye-flip experiment to SiHa16v14 (GSM901).


title: SiHa15v13

description: SiHa were plated in 10-cm dishes at approximately 25% confluence. After attachment (8 or 16 hours after plating), 5-aza-2’-deoxycytidine was added at 200 nM concentration.  Twenty-four hours later, medium was exchanged with fresh 200 nM 5-aza-2’-deoxycytidine.  Twenty four hours later, medium was exchanged with fresh 200 nM 5-aza-2’-deoxycytidine and 300 nM trichostatin A.  Mock-treated cultures were handled identically except that only solvents (PBS and EtOH, respectively) were added in media instead of drugs.  Twenty four hours later (72 hours after starting the treatment) cells were harvested by brief trypsinization followed by pelleting of the cells.  All RNA isolation was accomplished with Qiagen RNeasy Mini Kit reagents. The RNA (25 ug) was annealed with 5 ug oligo dT12-18, and reverse-transcribed into cDNA with Superscript II reverse transcriptase for 2h at 42oC in the presence of 0.5mM dGTP, 0.5mM dCTP, 0.5mM dATP, 0.3mM dTTP, 0.2mM amino-allyl dUTP. After hydrolysis of RNA in 0.2M NaOH, Tris was removed from the reaction with a Microcon-30 concentrator. The cDNA from drug-treated and mock-treated SiHa was covalently coupled separately with Cy3 and Cy5 monoreactive fluors, respectively, in 50mM sodium bicarbonate, pH 9.0, followed by quenching with 2.7M hydroxylamine. The Cy5 and Cy3 labelled cDNAs were combined and purified with a QIAquick PCR purification kit. Fluorescent array images were collected for both Cy3 and Cy5 with a GenePix 4000A fluorescent scanner and image intensity data were extracted and analyzed with GenePix Pro 3.0 analysis software. After background correction, removal of flagged values, normalization (LOWESS), log base 2 fluorescence signals were subtracted (Cy5 channel minus Cy3 channel) to provide the ratio (VALUE).  This is a dye-flip experiment to SiHa13v15 (GSM37897).


title: CaSki1v4

description: CaSki were plated in 10-cm dishes at approximately 25% confluence. After attachment (8 or 16 hours after plating), 5-aza-2’-deoxycytidine was added at 200 nM concentration.  Twenty-four hours later, medium was exchanged with fresh 200 nM 5-aza-2’-deoxycytidine.  Twenty four hours later, medium was exchanged with fresh 200 nM 5-aza-2’-deoxycytidine and 300 nM trichostatin A.  Mock-treated cultures were handled identically except that only solvents (PBS and EtOH, respectively) were added in media instead of drugs.  Twenty four hours later (72 hours after starting the treatment) cells were harvested by brief trypsinization followed by pelleting of the cells.  All RNA isolation was accomplished with Qiagen RNeasy Mini Kit reagents. The RNA (5 ug) was amplified and labeled with aa-UTP using Amino Allyl Message Amp aRNA kit (Ambion, Austin, TX). The aRNA from drug-treated and mock-treated CaSki cells was covalently coupled separately with Cy3 and Cy5 monoreactive fluors, respectively, in 50mM sodium bicarbonate, pH 9.0, followed by quenching with 2.7M hydroxylamine. The Cy5 and Cy3 labelled aRNAs were combined and purified with a QIAquick PCR purification kit.  Fluorescent array images were collected for both Cy3 and Cy5 with a GenePix 4000A fluorescent scanner and image intensity data were extracted and analyzed with GenePix Pro 3.0 analysis software. After background correction, removal of flagged values, normalization (LOWESS), log base 2 fluorescence signals were subtracted (Cy5 channel minus Cy3 channel) to provide the ratio (VALUE).  This is a dye-flip experiment to CaSki4v1 (GSM895).

source_name: mock-treated CaSki cells

source_name: drug-treated CaSki cells


title: SiHa13v17

description: SiHa were plated in 10-cm dishes at approximately 25% confluence. After attachment (8 or 16 hours after plating), 5-aza-2’-deoxycytidine was added at 200 nM concentration.  Twenty-four hours later, medium was exchanged with fresh 200 nM 5-aza-2’-deoxycytidine.  Twenty four hours later, medium was exchanged with fresh 200 nM 5-aza-2’-deoxycytidine and 300 nM trichostatin A.  Mock-treated cultures were handled identically except that only solvents (PBS and EtOH, respectively) were added in media instead of drugs.  Twenty four hours later (72 hours after starting the treatment) cells were harvested by brief trypsinization followed by pelleting of the cells.  All RNA isolation was accomplished with Qiagen RNeasy Mini Kit reagents. The RNA (5 ug) was amplified and labeled with aa-UTP using Amino Allyl Message Amp aRNA kit (Ambion, Austin, TX). The aRNA from mock-treated and drug-treated SiHa cells was covalently coupled separately with Cy3 and Cy5 monoreactive fluors, respectively, in 50mM sodium bicarbonate, pH 9.0, followed by quenching with 2.7M hydroxylamine. The Cy5 and Cy3 labelled aRNAs were combined and purified with a QIAquick PCR purification kit.  Fluorescent array images were collected for both Cy3 and Cy5 with a GenePix 4000A fluorescent scanner and image intensity data were extracted and analyzed with GenePix Pro 3.0 analysis software. After background correction, removal of flagged values, normalization (LOWESS), log base 2 fluorescence signals were subtracted (Cy5 channel minus Cy3 channel) to provide the ratio (VALUE).  This is a dye-flip experiment to SiHa17v13 (GSM902).


title: CaSki4v1

description: CaSki were plated in 10-cm dishes at approximately 25% confluence. After attachment (8 or 16 hours after plating), 5-aza-2’-deoxycytidine was added at 200 nM concentration.  Twenty-four hours later, medium was exchanged with fresh 200 nM 5-aza-2’-deoxycytidine.  Twenty four hours later, medium was exchanged with fresh 200 nM 5-aza-2’-deoxycytidine and 300 nM trichostatin A.  Mock-treated cultures were handled identically except that only solvents (PBS and EtOH, respectively) were added in media instead of drugs.  Twenty four hours later (72 hours after starting the treatment) cells were harvested by brief trypsinization followed by pelleting of the cells.  All RNA isolation was accomplished with Qiagen RNeasy Mini Kit reagents. The RNA (25 ug) was annealed with 5 ug oligo dT12-18, and reverse-transcribed into cDNA with Superscript II reverse transcriptase for 2h at 42oC in the presence of 0.5mM dGTP, 0.5mM dCTP, 0.5mM dATP, 0.3mM dTTP, 0.2mM amino-allyl dUTP. After hydrolysis of RNA in 0.2M NaOH, Tris was removed from the reaction with a Microcon-30 concentrator. The cDNA from drug-treated and mock-treated CaSki was covalently coupled separately with Cy3 and Cy5 monoreactive fluors, respectively, in 50mM sodium bicarbonate, pH 9.0, followed by quenching with 2.7M hydroxylamine. The Cy5 and Cy3 labelled cDNAs were combined and purified with a QIAquick PCR purification kit. Fluorescent array images were collected for both Cy3 and Cy5 with a GenePix 4000A fluorescent scanner and image intensity data were extracted and analyzed with GenePix Pro 3.0 analysis software. After background correction, removal of flagged values, normalization (LOWESS), log base 2 fluorescence signals were subtracted (Cy5 channel minus Cy3 channel) to provide the ratio (VALUE).  This is a dye-flip experiment to CaSki1v4 (GSM37893).


title: CaSki2v5

description: CaSki were plated in 10-cm dishes at approximately 25% confluence. After attachment (8 or 16 hours after plating), 5-aza-2’-deoxycytidine was added at 200 nM concentration.  Twenty-four hours later, medium was exchanged with fresh 200 nM 5-aza-2’-deoxycytidine.  Twenty four hours later, medium was exchanged with fresh 200 nM 5-aza-2’-deoxycytidine and 300 nM trichostatin A.  Mock-treated cultures were handled identically except that only solvents (PBS and EtOH, respectively) were added in media instead of drugs.  Twenty four hours later (72 hours after starting the treatment) cells were harvested by brief trypsinization followed by pelleting of the cells.  All RNA isolation was accomplished with Qiagen RNeasy Mini Kit reagents. The RNA (5 ug) was amplified and labeled with aa-UTP using Amino Allyl Message Amp aRNA kit (Ambion, Austin, TX). The aRNA from drug-treated and mock-treated CaSki cells was covalently coupled separately with Cy3 and Cy5 monoreactive fluors, respectively, in 50mM sodium bicarbonate, pH 9.0, followed by quenching with 2.7M hydroxylamine. The Cy5 and Cy3 labelled aRNAs were combined and purified with a QIAquick PCR purification kit.  Fluorescent array images were collected for both Cy3 and Cy5 with a GenePix 4000A fluorescent scanner and image intensity data were extracted and analyzed with GenePix Pro 3.0 analysis software. After background correction, removal of flagged values, normalization (LOWESS), log base 2 fluorescence signals were subtracted (Cy5 channel minus Cy3 channel) to provide the ratio (VALUE).  This is a dye-flip experiment to CaSki5v2 (GSM896).


title: Six1 target genes in mk4 cells

description: To identify Six1 target genes, we chose the mK4 cell line that represents later metanephric mesenchyme of the embryonic kidney. The cells were infected with adenovirus vectors (AxCAwt) overexpressing VP16-Six1wt or VP16-Six1W171R and cultured for 24h. Total RNA was extracted using Isogen reagent (Nippon Gene), treated with RNase-free DNase (Roche Diagnostics) and purified with RNeasy Midi Kit (QIAGEN). Poly A+ RNA was selected using Oligotex-dT30 <super> latex beads (TaKaRa). Poly A+ RNA (20 µg) was reverse transcribed using an oligo(dT) primer in the presence of aminoallyl dUTP without the addition of spike-in controls. Then, single-stranded cDNAs were coupled with Cy3 (AxCAwt VP16-Six1wt infected sample) or Cy5 (AxCAwt VP16-Six1W171R infected sample) dyes, and the labeled probes were purified with QIAquick PCR Purification kit (QIAGEN). Hybridization to the microarray was carried out at 65°C for 17h according to the manufacturer's instruction. The arrays were washed, dried and scanned using ScanArray 5000 (GSI Lumonics) at two different PMT voltage settings (high and low) to avoid signal saturation. Cy3 and Cy5 intensities for each spot on the array were determined by QuantArray software (Version 3.0.0.0, GSI Lumonics). The raw data thus obtained was processed and the Cy3 to Cy5 ratios were calculated as follows: 1) subtraction of the fluorescence intensity of negative control spots as background from the intensity of each of the Cy3 and Cy5 spots, 2) normalization of the entire data set using the global normalization method, 3) elimination of spots with high background intensity for either dye, and 4) determination of the Cy3 to Cy5 ratios.

source_name: mk4 cells overexpressing VP16-Six1wt

source_name: mk4 cells overexpressing VP16-Six1W171R


title: SiHa16v14

description: SiHa were plated in 10-cm dishes at approximately 25% confluence. After attachment (8 or 16 hours after plating), 5-aza-2’-deoxycytidine was added at 200 nM concentration.  Twenty-four hours later, medium was exchanged with fresh 200 nM 5-aza-2’-deoxycytidine.  Twenty four hours later, medium was exchanged with fresh 200 nM 5-aza-2’-deoxycytidine and 300 nM trichostatin A.  Mock-treated cultures were handled identically except that only solvents (PBS and EtOH, respectively) were added in media instead of drugs.  Twenty four hours later (72 hours after starting the treatment) cells were harvested by brief trypsinization followed by pelleting of the cells.  All RNA isolation was accomplished with Qiagen RNeasy Mini Kit reagents. The RNA (25 ug) was annealed with 5 ug oligo dT12-18, and reverse-transcribed into cDNA with Superscript II reverse transcriptase for 2h at 42oC in the presence of 0.5mM dGTP, 0.5mM dCTP, 0.5mM dATP, 0.3mM dTTP, 0.2mM amino-allyl dUTP. After hydrolysis of RNA in 0.2M NaOH, Tris was removed from the reaction with a Microcon-30 concentrator. The cDNA from drug-treated and mock-treated SiHa was covalently coupled separately with Cy3 and Cy5 monoreactive fluors, respectively, in 50mM sodium bicarbonate, pH 9.0, followed by quenching with 2.7M hydroxylamine. The Cy5 and Cy3 labelled cDNAs were combined and purified with a QIAquick PCR purification kit. Fluorescent array images were collected for both Cy3 and Cy5 with a GenePix 4000A fluorescent scanner and image intensity data were extracted and analyzed with GenePix Pro 3.0 analysis software. After background correction, removal of flagged values, normalization (LOWESS), log base 2 fluorescence signals were subtracted (Cy5 channel minus Cy3 channel) to provide the ratio (VALUE).  This is a dye-flip experiment to SiHa14v16 (GSM37899).


title: CaSki5v2

description: CaSki were plated in 10-cm dishes at approximately 25% confluence. After attachment (8 or 16 hours after plating), 5-aza-2’-deoxycytidine was added at 200 nM concentration.  Twenty-four hours later, medium was exchanged with fresh 200 nM 5-aza-2’-deoxycytidine.  Twenty four hours later, medium was exchanged with fresh 200 nM 5-aza-2’-deoxycytidine and 300 nM trichostatin A.  Mock-treated cultures were handled identically except that only solvents (PBS and EtOH, respectively) were added in media instead of drugs.  Twenty four hours later (72 hours after starting the treatment) cells were harvested by brief trypsinization followed by pelleting of the cells.  All RNA isolation was accomplished with Qiagen RNeasy Mini Kit reagents. The RNA (25 ug) was annealed with 5 ug oligo dT12-18, and reverse-transcribed into cDNA with Superscript II reverse transcriptase for 2h at 42oC in the presence of 0.5mM dGTP, 0.5mM dCTP, 0.5mM dATP, 0.3mM dTTP, 0.2mM amino-allyl dUTP. After hydrolysis of RNA in 0.2M NaOH, Tris was removed from the reaction with a Microcon-30 concentrator. The cDNA from drug-treated and mock-treated CaSki was covalently coupled separately with Cy3 and Cy5 monoreactive fluors, respectively, in 50mM sodium bicarbonate, pH 9.0, followed by quenching with 2.7M hydroxylamine. The Cy5 and Cy3 labelled cDNAs were combined and purified with a QIAquick PCR purification kit. Fluorescent array images were collected for both Cy3 and Cy5 with a GenePix 4000A fluorescent scanner and image intensity data were extracted and analyzed with GenePix Pro 3.0 analysis software. After background correction, removal of flagged values, normalization (LOWESS), log base 2 fluorescence signals were subtracted (Cy5 channel minus Cy3 channel) to provide the ratio (VALUE).  This is a dye-flip experiment to CaSki2v5 (GSM37894).


title: HeLa4v1

description: HeLa were plated in 10-cm dishes at approximately 25% confluence. After attachment (8 or 16 hours after plating), 5-aza-2’-deoxycytidine was added at 200 nM concentration.  Twenty-four hours later, medium was exchanged with fresh 200 nM 5-aza-2’-deoxycytidine.  Twenty four hours later, medium was exchanged with fresh 200 nM 5-aza-2’-deoxycytidine and 300 nM trichostatin A.  Mock-treated cultures were handled identically except that only solvents (PBS and EtOH, respectively) were added in media instead of drugs.  Twenty four hours later (72 hours after starting the treatment) cells were harvested by brief trypsinization followed by pelleting of the cells.  All RNA isolation was accomplished with Qiagen RNeasy Mini Kit reagents. The RNA (25 ug) was annealed with 5 ug oligo dT12-18, and reverse-transcribed into cDNA with Superscript II reverse transcriptase for 2h at 42oC in the presence of 0.5mM dGTP, 0.5mM dCTP, 0.5mM dATP, 0.3mM dTTP, 0.2mM amino-allyl dUTP. After hydrolysis of RNA in 0.2M NaOH, Tris was removed from the reaction with a Microcon-30 concentrator. The cDNA from drug-treated and mock-treated HeLa was covalently coupled separately with Cy3 and Cy5 monoreactive fluors, respectively, in 50mM sodium bicarbonate, pH 9.0, followed by quenching with 2.7M hydroxylamine. The Cy5 and Cy3 labelled cDNAs were combined and purified with a QIAquick PCR purification kit. Fluorescent array images were collected for both Cy3 and Cy5 with a GenePix 4000A fluorescent scanner and image intensity data were extracted and analyzed with GenePix Pro 3.0 analysis software. After background correction, removal of flagged values, normalization (LOWESS), log base 2 fluorescence signals were subtracted (Cy5 channel minus Cy3 channel) to provide the ratio (VALUE).  This is a dye-flip experiment to HeLa1v4 (GSM37829).

source_name: drug-treated HeLa cells

source_name: mock-treated HeLa cells


title: HeLa6v3

description: HeLa were plated in 10-cm dishes at approximately 25% confluence. After attachment (8 or 16 hours after plating), 5-aza-2’-deoxycytidine was added at 200 nM concentration.  Twenty-four hours later, medium was exchanged with fresh 200 nM 5-aza-2’-deoxycytidine.  Twenty four hours later, medium was exchanged with fresh 200 nM 5-aza-2’-deoxycytidine and 300 nM trichostatin A.  Mock-treated cultures were handled identically except that only solvents (PBS and EtOH, respectively) were added in media instead of drugs.  Twenty four hours later (72 hours after starting the treatment) cells were harvested by brief trypsinization followed by pelleting of the cells.  All RNA isolation was accomplished with Qiagen RNeasy Mini Kit reagents. The RNA (25 ug) was annealed with 5 ug oligo dT12-18, and reverse-transcribed into cDNA with Superscript II reverse transcriptase for 2h at 42oC in the presence of 0.5mM dGTP, 0.5mM dCTP, 0.5mM dATP, 0.3mM dTTP, 0.2mM amino-allyl dUTP. After hydrolysis of RNA in 0.2M NaOH, Tris was removed from the reaction with a Microcon-30 concentrator. The cDNA from drug-treated and mock-treated HeLa was covalently coupled separately with Cy3 and Cy5 monoreactive fluors, respectively, in 50mM sodium bicarbonate, pH 9.0, followed by quenching with 2.7M hydroxylamine. The Cy5 and Cy3 labelled cDNAs were combined and purified with a QIAquick PCR purification kit. Fluorescent array images were collected for both Cy3 and Cy5 with a GenePix 4000A fluorescent scanner and image intensity data were extracted and analyzed with GenePix Pro 3.0 analysis software. After background correction, removal of flagged values, normalization (LOWESS), log base 2 fluorescence signals were subtracted (Cy5 channel minus Cy3 channel) to provide the ratio (VALUE).  This is a dye-flip experiment to HeLa3v6 (GSM37831).


title: HeLa2v5

description: HeLa were plated in 10-cm dishes at approximately 25% confluence. After attachment (8 or 16 hours after plating), 5-aza-2’-deoxycytidine was added at 200 nM concentration.  Twenty-four hours later, medium was exchanged with fresh 200 nM 5-aza-2’-deoxycytidine.  Twenty four hours later, medium was exchanged with fresh 200 nM 5-aza-2’-deoxycytidine and 300 nM trichostatin A.  Mock-treated cultures were handled identically except that only solvents (PBS and EtOH, respectively) were added in media instead of drugs.  Twenty four hours later (72 hours after starting the treatment) cells were harvested by brief trypsinization followed by pelleting of the cells.  All RNA isolation was accomplished with Qiagen RNeasy Mini Kit reagents. The RNA (25 ug) was annealed with 5 ug oligo dT12-18, and reverse-transcribed into cDNA with Superscript II reverse transcriptase for 2h at 42oC in the presence of 0.5mM dGTP, 0.5mM dCTP, 0.5mM dATP, 0.3mM dTTP, 0.2mM amino-allyl dUTP. After hydrolysis of RNA in 0.2M NaOH, Tris was removed from the reaction with a Microcon-30 concentrator. The cDNA from mock-treated and drug-treated HeLa was covalently coupled separately with Cy3 and Cy5 monoreactive fluors, respectively, in 50mM sodium bicarbonate, pH 9.0, followed by quenching with 2.7M hydroxylamine. The Cy5 and Cy3 labelled cDNAs were combined and purified with a QIAquick PCR purification kit. Fluorescent array images were collected for both Cy3 and Cy5 with a GenePix 4000A fluorescent scanner and image intensity data were extracted and analyzed with GenePix Pro 3.0 analysis software. After background correction, removal of flagged values, normalization (LOWESS), log base 2 fluorescence signals were subtracted (Cy5 channel minus Cy3 channel) to provide the ratio (VALUE).  This is a flip experiment to HeLa5v2.


title: WTCy3/Rv2869c Cy5 no Tween #1

description:
o	All RNA was from the indicated strain of M. tuberculosis (either wild type M. tuberculosis Erdman or the Mtb Rv2869c null mutant
•RNA was isolated from logarithmic phase cultures at OD600 .3.  For (+Tween) conditions, the media was Middlebrook 7H9 with 10% OADC supplement, .5% glycerol, and .05% Tween 80.  For (-Tween) conditions, bacilli were transferred to identical media without Tween 80 and harvested for RNA preparation 18hours later.

Protocol for preparing the hybridization extract:

15ml of liquid culture described above were collected by centrifugation at 3000g, and cell pellets frozen on dry ice.  Frozen pellets were resuspended in 1ml of Trizol reagent (Invitrogen) and lysed by mechanical disruption using .7mm Zirconia beads for 3 x 1min in a Mini bead beater 8 (Biospec products) at maximum speed. The remainder of the procedure was according to the Trizol manufacturers instructions with the addition of the optional high salt precipitation step.  RNA was subsequently cleaned using Qiagen RNAeasy columns according ot the manufacturers RNA cleanup protocol.

Labeling protocol(s).
5-7.5micrograms of total RNA was labeled using the Fairplay microrray labeling kit (Stratagene) with substitution of Random Hexamer primers.  Aminoallyl labeled CDNA was coupled to Cy3/Cy5 using Post labeling reactive dye packs (Amersham). Excess dye was removed using Qiaquick nucleotide removal columns according to the manufacturer’s instructions.

source_name: Rv2869c mutant

source_name: Wild Type M. tuberculosis


title: HeLa1v4

description: HeLa were plated in 10-cm dishes at approximately 25% confluence. After attachment (8 or 16 hours after plating), 5-aza-2’-deoxycytidine was added at 200 nM concentration.  Twenty-four hours later, medium was exchanged with fresh 200 nM 5-aza-2’-deoxycytidine.  Twenty four hours later, medium was exchanged with fresh 200 nM 5-aza-2’-deoxycytidine and 300 nM trichostatin A.  Mock-treated cultures were handled identically except that only solvents (PBS and EtOH, respectively) were added in media instead of drugs.  Twenty four hours later (72 hours after starting the treatment) cells were harvested by brief trypsinization followed by pelleting of the cells.  All RNA isolation was accomplished with Qiagen RNeasy Mini Kit reagents. The RNA (25 ug) was annealed with 5 ug oligo dT12-18, and reverse-transcribed into cDNA with Superscript II reverse transcriptase for 2h at 42oC in the presence of 0.5mM dGTP, 0.5mM dCTP, 0.5mM dATP, 0.3mM dTTP, 0.2mM amino-allyl dUTP. After hydrolysis of RNA in 0.2M NaOH, Tris was removed from the reaction with a Microcon-30 concentrator. The cDNA from mock-treated and drug-treated HeLa was covalently coupled separately with Cy3 and Cy5 monoreactive fluors, respectively, in 50mM sodium bicarbonate, pH 9.0, followed by quenching with 2.7M hydroxylamine. The Cy5 and Cy3 labelled cDNAs were combined and purified with a QIAquick PCR purification kit. Fluorescent array images were collected for both Cy3 and Cy5 with a GenePix 4000A fluorescent scanner and image intensity data were extracted and analyzed with GenePix Pro 3.0 analysis software. After background correction, removal of flagged values, normalization (LOWESS), log base 2 fluorescence signals were subtracted (Cy5 channel minus Cy3 channel) to provide the ratio (VALUE).  This is a flip experiment to HeLa4v1.


title: HeLa3v6

description: HeLa were plated in 10-cm dishes at approximately 25% confluence. After attachment (8 or 16 hours after plating), 5-aza-2’-deoxycytidine was added at 200 nM concentration.  Twenty-four hours later, medium was exchanged with fresh 200 nM 5-aza-2’-deoxycytidine.  Twenty four hours later, medium was exchanged with fresh 200 nM 5-aza-2’-deoxycytidine and 300 nM trichostatin A.  Mock-treated cultures were handled identically except that only solvents (PBS and EtOH, respectively) were added in media instead of drugs.  Twenty four hours later (72 hours after starting the treatment) cells were harvested by brief trypsinization followed by pelleting of the cells.  All RNA isolation was accomplished with Qiagen RNeasy Mini Kit reagents. The RNA (25 ug) was annealed with 5 ug oligo dT12-18, and reverse-transcribed into cDNA with Superscript II reverse transcriptase for 2h at 42oC in the presence of 0.5mM dGTP, 0.5mM dCTP, 0.5mM dATP, 0.3mM dTTP, 0.2mM amino-allyl dUTP. After hydrolysis of RNA in 0.2M NaOH, Tris was removed from the reaction with a Microcon-30 concentrator. The cDNA from mock-treated and drug-treated HeLa was covalently coupled separately with Cy3 and Cy5 monoreactive fluors, respectively, in 50mM sodium bicarbonate, pH 9.0, followed by quenching with 2.7M hydroxylamine. The Cy5 and Cy3 labelled cDNAs were combined and purified with a QIAquick PCR purification kit. Fluorescent array images were collected for both Cy3 and Cy5 with a GenePix 4000A fluorescent scanner and image intensity data were extracted and analyzed with GenePix Pro 3.0 analysis software. After background correction, removal of flagged values, normalization (LOWESS), log base 2 fluorescence signals were subtracted (Cy5 channel minus Cy3 channel) to provide the ratio (VALUE).  This is a flip experiment to HeLa6v3.


title: C4I9v12

description: C4I were plated in 10-cm dishes at approximately 25% confluence. After attachment (8 or 16 hours after plating), 5-aza-2’-deoxycytidine was added at 200 nM concentration.  Twenty-four hours later, medium was exchanged with fresh 200 nM 5-aza-2’-deoxycytidine.  Twenty four hours later, medium was exchanged with fresh 200 nM 5-aza-2’-deoxycytidine and 300 nM trichostatin A.  Mock-treated cultures were handled identically except that only solvents (PBS and EtOH, respectively) were added in media instead of drugs.  Twenty four hours later (72 hours after starting the treatment) cells were harvested by brief trypsinization followed by pelleting of the cells.  All RNA isolation was accomplished with Qiagen RNeasy Mini Kit reagents. The RNA (5 ug) was amplified and labeled with aa-UTP using Amino Allyl Message Amp aRNA kit (Ambion, Austin, TX). The aRNA from mock-treated and drug-treated C4I cells was covalently coupled separately with Cy3 and Cy5 monoreactive fluors, respectively, in 50mM sodium bicarbonate, pH 9.0, followed by quenching with 2.7M hydroxylamine. The Cy5 and Cy3 labelled aRNAs were combined and purified with a QIAquick PCR purification kit. Fluorescent array images were collected for both Cy3 and Cy5 with a GenePix 4000A fluorescent scanner and image intensity data were extracted and analyzed with GenePix Pro 3.0 analysis software. After background correction, removal of flagged values, normalization (LOWESS), log base 2 fluorescence signals were subtracted (Cy5 channel minus Cy3 channel) to provide the ratio (VALUE).  This is a dye flip experiment to C4I12v9 (GSM37822).

source_name: mock-treated C4I cells

source_name: drug-treated C4I cells


title: C4I8v11

description: C4I were plated in 10-cm dishes at approximately 25% confluence. After attachment (8 or 16 hours after plating), 5-aza-2’-deoxycytidine was added at 200 nM concentration.  Twenty-four hours later, medium was exchanged with fresh 200 nM 5-aza-2’-deoxycytidine.  Twenty four hours later, medium was exchanged with fresh 200 nM 5-aza-2’-deoxycytidine and 300 nM trichostatin A.  Mock-treated cultures were handled identically except that only solvents (PBS and EtOH, respectively) were added in media instead of drugs.  Twenty four hours later (72 hours after starting the treatment) cells were harvested by brief trypsinization followed by pelleting of the cells.  All RNA isolation was accomplished with Qiagen RNeasy Mini Kit reagents. The RNA (5 ug) was amplified and labeled with aa-UTP using Amino Allyl Message Amp aRNA kit (Ambion, Austin, TX). The aRNA from mock-treated and drug-treated C4I cells was covalently coupled separately with Cy3 and Cy5 monoreactive fluors, respectively, in 50mM sodium bicarbonate, pH 9.0, followed by quenching with 2.7M hydroxylamine. The Cy5 and Cy3 labelled aRNAs were combined and purified with a QIAquick PCR purification kit. Fluorescent array images were collected for both Cy3 and Cy5 with a GenePix 4000A fluorescent scanner and image intensity data were extracted and analyzed with GenePix Pro 3.0 analysis software. After background correction, removal of flagged values, normalization (LOWESS), log base 2 fluorescence signals were subtracted (Cy5 channel minus Cy3 channel) to provide the ratio (VALUE).  This is a dye flip experiment to C4I11v8 (GSM37821).


title: HeLa5v2

description: HeLa were plated in 10-cm dishes at approximately 25% confluence. After attachment (8 or 16 hours after plating), 5-aza-2’-deoxycytidine was added at 200 nM concentration.  Twenty-four hours later, medium was exchanged with fresh 200 nM 5-aza-2’-deoxycytidine.  Twenty four hours later, medium was exchanged with fresh 200 nM 5-aza-2’-deoxycytidine and 300 nM trichostatin A.  Mock-treated cultures were handled identically except that only solvents (PBS and EtOH, respectively) were added in media instead of drugs.  Twenty four hours later (72 hours after starting the treatment) cells were harvested by brief trypsinization followed by pelleting of the cells.  All RNA isolation was accomplished with Qiagen RNeasy Mini Kit reagents. The RNA (25 ug) was annealed with 5 ug oligo dT12-18, and reverse-transcribed into cDNA with Superscript II reverse transcriptase for 2h at 42oC in the presence of 0.5mM dGTP, 0.5mM dCTP, 0.5mM dATP, 0.3mM dTTP, 0.2mM amino-allyl dUTP. After hydrolysis of RNA in 0.2M NaOH, Tris was removed from the reaction with a Microcon-30 concentrator. The cDNA from drug-treated and mock-treated HeLa was covalently coupled separately with Cy3 and Cy5 monoreactive fluors, respectively, in 50mM sodium bicarbonate, pH 9.0, followed by quenching with 2.7M hydroxylamine. The Cy5 and Cy3 labelled cDNAs were combined and purified with a QIAquick PCR purification kit. Fluorescent array images were collected for both Cy3 and Cy5 with a GenePix 4000A fluorescent scanner and image intensity data were extracted and analyzed with GenePix Pro 3.0 analysis software. After background correction, removal of flagged values, normalization (LOWESS), log base 2 fluorescence signals were subtracted (Cy5 channel minus Cy3 channel) to provide the ratio (VALUE).  This is a dye-flip experiment to HeLa2v5 (GSM37830).


title: C4I12v9

description: C4I were plated in 10-cm dishes at approximately 25% confluence. After attachment (8 or 16 hours after plating), 5-aza-2’-deoxycytidine was added at 200 nM concentration.  Twenty-four hours later, medium was exchanged with fresh 200 nM 5-aza-2’-deoxycytidine.  Twenty four hours later, medium was exchanged with fresh 200 nM 5-aza-2’-deoxycytidine and 300 nM trichostatin A.  Mock-treated cultures were handled identically except that only solvents (PBS and EtOH, respectively) were added in media instead of drugs.  Twenty four hours later (72 hours after starting the treatment) cells were harvested by brief trypsinization followed by pelleting of the cells.  All RNA isolation was accomplished with Qiagen RNeasy Mini Kit reagents. The RNA (5 ug) was amplified and labeled with aa-UTP using Amino Allyl Message Amp aRNA kit (Ambion, Austin, TX). The aRNA from drug-treated and mock-treated C4I cells was covalently coupled separately with Cy3 and Cy5 monoreactive fluors, respectively, in 50mM sodium bicarbonate, pH 9.0, followed by quenching with 2.7M hydroxylamine. The Cy5 and Cy3 labelled aRNAs were combined and purified with a QIAquick PCR purification kit. Fluorescent array images were collected for both Cy3 and Cy5 with a GenePix 4000A fluorescent scanner and image intensity data were extracted and analyzed with GenePix Pro 3.0 analysis software. After background correction, removal of flagged values, normalization (LOWESS), log base 2 fluorescence signals were subtracted (Cy5 channel minus Cy3 channel) to provide the ratio (VALUE).  This is a flip experiment to C4I9v12.


title: 0-9 Cr(III) untreated

status: Public on Jul 15 2005

city: Ocean Springs

first: Jonathan

last: Roling

state: MS

address: 703 East Beach Dr

department: Department of Coastal Sciences

institute: University of Southern Mississippi

description: liver from adult male Fundulus heteroclitus

hyb_protocol: Probes are heated to 100C for 10 minutes, and then chilled on ice.  Arrays are prehybridized with 5 mL of ExpressHyb Hybridization Solution (Clontech, Palo Alto, CA) and 1 mg sheared salmon sperm (denatured at 95C for 10 minutes then chilled on ice) at 68C for greater than 4 hours.  Arrays were hybridized overnight at 68C.  The blots were washed four times for 15 minutes at 64C using 2x SSC/0.5% SDS, then four times for 15 minutes at 64C using 0.5x SSC/0.5% SDS.

scan_protocol: Blots are wrapped in plastic wrap and exposed to a phosphoscreen at room temperature, visualized on BioRad FX Imager (Hercules, CA), and quantified with Invitrogen ResGen Pathways software (Carlsbad, CA).

data_processing: Data normalized to immediate background using ResGen Pathways software.

label: radioactive phosphorus 33 - [33P] dATP

organism: Fundulus heteroclitus

source_name: Fundulus heteroclitus adult male liver

label_protocol: 2 ug RNA is diluted into 10 uL of DEPC water. 10 uL of 0.5 uM poly T primers are added and incubated at 65C for 5 minutes and cooled to room temperature for two minutes.  Radiolabelled cDNA is made by adding 30 uL of a mix containing 10 uL 5x reverse transcription buffer, 6 uL 10 mM dNTPs (without dATP), 10 uCi [33P]dATP, and 200 units MMLV-RT and incubating for 2 hours at 37C.  Probes are cleaned of short nucleotide sequences through ProbeQuant G-50 Micro Columns (Amersham Biosciences, Piscataway, NJ) and counted on scintillation counter.

extract_protocol: RNA isolation performed by TriReagent according to manufacture?s protocols, followed by DNAse treatment.

treatment_protocol: Laboratory acclimated fish were exposed in individual tanks to chromium chloride hexahydrate at 0 mg/L Cr(III) for 7 days with water exchanges every other day.  Fish maintained at a 14:10 light:dark cycle at 25C in salinity of 18 parts per thousand.  Fish were fed daily Tetramin flake fish food.  At the conclusion of the exposure, livers were excised and frozen at -80C.

tag: Adult (>4cm in length) male liver excised immediately upon euthanasia


title: 0-8 Cr(III) untreated


title: C4I11v8

description: C4I were plated in 10-cm dishes at approximately 25% confluence. After attachment (8 or 16 hours after plating), 5-aza-2’-deoxycytidine was added at 200 nM concentration.  Twenty-four hours later, medium was exchanged with fresh 200 nM 5-aza-2’-deoxycytidine.  Twenty four hours later, medium was exchanged with fresh 200 nM 5-aza-2’-deoxycytidine and 300 nM trichostatin A.  Mock-treated cultures were handled identically except that only solvents (PBS and EtOH, respectively) were added in media instead of drugs.  Twenty four hours later (72 hours after starting the treatment) cells were harvested by brief trypsinization followed by pelleting of the cells.  All RNA isolation was accomplished with Qiagen RNeasy Mini Kit reagents. The RNA (5 ug) was amplified and labeled with aa-UTP using Amino Allyl Message Amp aRNA kit (Ambion, Austin, TX). The aRNA from drug-treated and mock-treated C4I cells was covalently coupled separately with Cy3 and Cy5 monoreactive fluors, respectively, in 50mM sodium bicarbonate, pH 9.0, followed by quenching with 2.7M hydroxylamine. The Cy5 and Cy3 labelled aRNAs were combined and purified with a QIAquick PCR purification kit. Fluorescent array images were collected for both Cy3 and Cy5 with a GenePix 4000A fluorescent scanner and image intensity data were extracted and analyzed with GenePix Pro 3.0 analysis software. After background correction, removal of flagged values, normalization (LOWESS), log base 2 fluorescence signals were subtracted (Cy5 channel minus Cy3 channel) to provide the ratio (VALUE).  This is a dye-flip experiment to C4I8v11 (GSM37825).


title: 0-7 Cr(III) untreated


title: 0-6 Cr(III) untreated


title: C4I7v10

description: C4I were plated in 10-cm dishes at approximately 25% confluence. After attachment (8 or 16 hours after plating), 5-aza-2’-deoxycytidine was added at 200 nM concentration.  Twenty-four hours later, medium was exchanged with fresh 200 nM 5-aza-2’-deoxycytidine.  Twenty four hours later, medium was exchanged with fresh 200 nM 5-aza-2’-deoxycytidine and 300 nM trichostatin A.  Mock-treated cultures were handled identically except that only solvents (PBS and EtOH, respectively) were added in media instead of drugs.  Twenty four hours later (72 hours after starting the treatment) cells were harvested by brief trypsinization followed by pelleting of the cells.  All RNA isolation was accomplished with Qiagen RNeasy Mini Kit reagents. The RNA (5 ug) was amplified and labeled with aa-UTP using Amino Allyl Message Amp aRNA kit (Ambion, Austin, TX). The aRNA from mock-treated and drug-treated C4I cells was covalently coupled separately with Cy3 and Cy5 monoreactive fluors, respectively, in 50mM sodium bicarbonate, pH 9.0, followed by quenching with 2.7M hydroxylamine. The Cy5 and Cy3 labelled aRNAs were combined and purified with a QIAquick PCR purification kit. Fluorescent array images were collected for both Cy3 and Cy5 with a GenePix 4000A fluorescent scanner and image intensity data were extracted and analyzed with GenePix Pro 3.0 analysis software. After background correction, removal of flagged values, normalization (LOWESS), log base 2 fluorescence signals were subtracted (Cy5 channel minus Cy3 channel) to provide the ratio (VALUE).  This is a dye-flip experiment to C4I10v7 (GSM37748).


title: SCD1 +/+ liver rep5

status: Public on Jul 14 2005

city: Madison

first: Alan

middle: D

last: Attie

state: WI

address: 433 Babcock Drive

department: Biochemistry

institute: University of Wisconsin-Madison

description: Loss of stearoyl–CoA desaturase-1 function protects mice against adiposity

data_processing: dChip

label: SCD+/+ rep 5

source_name: liver, SCD+/+

tag: Genetic background

value: C57BL/6J; Age:6 weeks old; Gender:female; Diet:chow; Non-fasted; Genetic modification:None (Scd1 +/+; wide type control).


title: mutS mutant replicate 2

description: Overnight culture was diluted 1000-fold with fresh LB medium and cultured further.  Log phase culture was diluted to a cell density of 2 X 108 cells/ml in M9 salts and incubated at 37°C for two hours, after which it was resuspended in LB broth for 90 minutes.  This treatment served as a mock treatment for cultures incubated with a chemical in a parallel study. Total RNA was isolated from cells by extraction using the MasterPure RNA Purification Kit (Epicentre Technologies) followed by DNAse digestion according to the manufacturer’s protocol.  The isolated total RNA was quantitated by absorption at 260 nm (typical yield from a 15 ml culture was 250-500 µg of total RNA), and the purity was determined by the ratio of absorption values at 260/280nm.  RNA quality was determined by formaldehyde agarose gel electrophoresis (1.2% agarose in FA Buffer pH 7.0 (20 mM 3-[N-morpholino]propanesulfonic acid, 5 mM sodium acetate, 1 mM ethylenadiaminetetraacetic acide (EDTA))) or by analysis on an Agilent 2100 Bioanalyzer.  All samples visualized by gel electrophoresis or by the bioanalyzer electropherogram showed clear distinct bands correlating to 16S and 23S ribosomal RNA, indicating that no detectable RNA degradation occurred and that RNA integrity was maintained throughout the RNA isolation procedure. mRNA was enriched from total RNA as described in the Affymetrix GeneChip Expression Analysis Technical Manual for GeneChip E. coli Sense Genome Arrays.

hyb_protocol: Affymetrix

scan_protocol: Affymetrix

data_processing: RMA Version 0.1 Release

source_name: E. coli mutS mutant Strain GM5555

label_protocol: Affymetrix E. coli Sense arrays

growth_protocol: Exponentially growing culture held in M9 minimal salts for 120 min. then allowed to recover in LB for 90 min., after which total RNA was isolated.

extract_protocol: Epicentre MasterPure RNA Purification kit and Affymetrix protocol for E. coli Sense arrays (mRNA enrichment protocol)

tag: As AB1157  but  mutS215::Tn10


title: SCD1 +/+ liver rep4

label: SCD+/+ rep 4


title: C4I10v7

description: C4I were plated in 10-cm dishes at approximately 25% confluence. After attachment (8 or 16 hours after plating), 5-aza-2’-deoxycytidine was added at 200 nM concentration.  Twenty-four hours later, medium was exchanged with fresh 200 nM 5-aza-2’-deoxycytidine.  Twenty four hours later, medium was exchanged with fresh 200 nM 5-aza-2’-deoxycytidine and 300 nM trichostatin A.  Mock-treated cultures were handled identically except that only solvents (PBS and EtOH, respectively) were added in media instead of drugs.  Twenty four hours later (72 hours after starting the treatment) cells were harvested by brief trypsinization followed by pelleting of the cells.  All RNA isolation was accomplished with Qiagen RNeasy Mini Kit reagents. The RNA (5 ug) was amplified and labeled with aa-UTP using Amino Allyl Message Amp aRNA kit (Ambion, Austin, TX). The aRNA from drug-treated and mock-treated C4I cells was covalently coupled separately with Cy3 and Cy5 monoreactive fluors, respectively, in 50mM sodium bicarbonate, pH 9.0, followed by quenching with 2.7M hydroxylamine. The Cy5 and Cy3 labelled aRNAs were combined and purified with a QIAquick PCR purification kit. Fluorescent array images were collected for both Cy3 and Cy5 with a GenePix 4000A fluorescent scanner and image intensity data were extracted and analyzed with GenePix Pro 3.0 analysis software. After background correction, removal of flagged values, normalization (LOWESS), log base 2 fluorescence signals were subtracted (Cy5 channel minus Cy3 channel) to provide the ratio (VALUE).  This is a dye-flip experiment to C4I7v10 (GSM37823).


title: Aldosterone-responsive genes in kidney cortical collecting duct cell line, mpkCCDc14 (1 hour treatment)

city: Belmont

first: Ting Ting

last: Zhang

address: 115 Millman st

department: Mclean Hospital

institute: Harvard University

description: Array data were collected with Genepix 4.0 scanner.  The quality of array was examined with RBioconductor.   First, the uniformity of spot and labeling, and the intensity of labeling were checked with MA-plot (M=log2(Cy5/Cy3), A=log2((Cy5*Cy3)/2).  Quantitative assessment such as signal to noise ratio and the median signal intensity were also performed.  Only these arrays which have the reasonable qualities would be used for further analysis.  The significantly different expressed genes are selected with SAM using the one class response with data (log2 Cy5/Cy3) normalized in RBioconductor.  Hierarchical clustering is performed with Acuity 4.0 (Axon Instruments).

hyb_protocol: After cleanup Cy3 or Cy5-labeled cDNA with CyScribe GFX purification kit (Amersham Biosciences), the labeled cDNA was hybridized with microarray slide at 50oC for >40 hours.

data_processing: data (log2 Cy5/Cy3)  is normalized in RBioconductor

source_name: Kidney cortical collecting duct cell line (mpkCCDc14)

label_protocol: Microarray analysis was performed following the protocol from UCSF genomics core laboratories (original document can be found at www.arrays.ucsf.edu).  Briefly, the first strand cDNA was synthesized with 20 µg total RNA, 1.0 µg oligo dT, 830 µM each dATP, dCTP, dGTP, 166 µM dTTP, 666 µM 5-(3-aminoallyl)- dUTP (Ambion),0.1 m DTT, 1 µl RNase inhibitor ( Fisher), 600 U RT, 10x RT buffer and H2O to final 30 ul for 2 hours at 42oC.  After hydrolysis with 10 µl 1N NaOH and 10 µl 0.5 M EDTA, cDNA was purified and recovered with Microcon 30. The Cy5 (Amersham Biosciences) was then coupled with cDNA in 0.05 M NaBiocarbonate (pH 9.0) in the dark at the room temperature for more than 1.5 hours.

growth_protocol: mpkCCD cells were cultured on 30 mm collagen-coated 6-transwell plates at a density of 1.5-2.0 x 106 cells/well in a modified medium described previously with 1% Pen-strep.  Fresh medium was changed every other day. 2-3 days later, while transepithelial cell  resistance reach ~2 KΩ, cell medium was replaced with dexamethasone-free and charcoal-treated steroid-free FBS for another 2-3 days.  Fresh medium was changed everyday.

extract_protocol: Total RNAs were isolated with RNA STAT-60 (Tel-Test, Inc).   The contaminated genomic DNA in the total RNA was removed with DNase treatment & removal reagents (Ambion).

treatment_protocol: Cells were treated with 10-6M aldosteron for 1 hr.

label_protocol: Microarray analysis was performed following the protocol from UCSF genomics core laboratories (original document can be found at www.arrays.ucsf.edu).  Briefly, the first strand cDNA was synthesized with 20 µg total RNA, 1.0 µg oligo dT, 830 µM each dATP, dCTP, dGTP, 166 µM dTTP, 666 µM 5-(3-aminoallyl)- dUTP (Ambion),0.1 m DTT, 1 µl RNase inhibitor ( Fisher), 600 U RT, 10x RT buffer and H2O to final 30 ul for 2 hours at 42oC.  After hydrolysis with 10 µl 1N NaOH and 10 µl 0.5 M EDTA, cDNA was purified and recovered with Microcon 30. The Cy3 (Amersham Biosciences) was then coupled with cDNA in 0.05 M NaBiocarbonate (pH 9.0) in the dark at the room temperature for more than 1.5 hours.

treatment_protocol: Cells were treated with vehicle (ethanol)for 1 hr.


title: SCD1 +/+ liver rep3

label: SCD+/+ rep 3


title: mutS mutant replicate 3


title: mutS mutant replicate 1


title: SCD1 -/- liver rep5

label: SCD-/- rep 5

source_name: liver, SCD-/-

value: mixed 129 and C57BL/6J; Age:6 weeks old; Gender:female; Diet:chow; Non-fasted; Genetic modification:Scd1 -/-.


title: SiHa13v15

description: SiHa were plated in 10-cm dishes at approximately 25% confluence. After attachment (8 or 16 hours after plating), 5-aza-2’-deoxycytidine was added at 200 nM concentration.  Twenty-four hours later, medium was exchanged with fresh 200 nM 5-aza-2’-deoxycytidine.  Twenty four hours later, medium was exchanged with fresh 200 nM 5-aza-2’-deoxycytidine and 300 nM trichostatin A.  Mock-treated cultures were handled identically except that only solvents (PBS and EtOH, respectively) were added in media instead of drugs.  Twenty four hours later (72 hours after starting the treatment) cells were harvested by brief trypsinization followed by pelleting of the cells.  All RNA isolation was accomplished with Qiagen RNeasy Mini Kit reagents. The RNA (5 ug) was amplified and labeled with aa-UTP using Amino Allyl Message Amp aRNA kit (Ambion, Austin, TX). The aRNA from mock-treated and drug-treated SiHa cells was covalently coupled separately with Cy3 and Cy5 monoreactive fluors, respectively, in 50mM sodium bicarbonate, pH 9.0, followed by quenching with 2.7M hydroxylamine. The Cy5 and Cy3 labelled aRNAs were combined and purified with a QIAquick PCR purification kit.  Fluorescent array images were collected for both Cy3 and Cy5 with a GenePix 4000A fluorescent scanner and image intensity data were extracted and analyzed with GenePix Pro 3.0 analysis software. After background correction, removal of flagged values, normalization (LOWESS), log base 2 fluorescence signals were subtracted (Cy5 channel minus Cy3 channel) to provide the ratio (VALUE).  This is a dye-flip experiment to SiHa15v13 (GSM900).

source_name: mock-treated SiHa cells

source_name: drug-treated SiHa cells


title: dam mutS mutant replicate 1

source_name: E. coli dam mutS mutant Strain GM5556

tag: As GM3819 dam-16 ::Kan but mutS215::Tn10


title: dam mutS mutant replicate 3

tag: As GM3819  dam-16::Kan   but mutS215::Tn10


title: dam mutant replicate 1

source_name: E. coli dam-16::Kan


title: dam mutant replicate 3

source_name: E. coli dam mutant strain GM3819


title: SCD1 -/- liver rep3

label: SCD-/- rep 3


title: SCD1 -/- liver rep4

label: SCD-/- rep 4


title: SCD1 +/+ liver rep1

label: SCD+/+ rep 1


title: SCD1 +/+ liver rep2

label: SCD+/+ rep 2


title: Wildtype replicate 3

source_name: E. coli AB1157 wildtype

tag: Escherichia coli strain AB1157 wildtype


title: Wildtype replicate 1

source_name: E.coli wildtype AB1157

tag: E. coli AB1157 wildtype


title: SCD1 -/- liver rep2

label: SCD-/- rep 2


title: dam mutant replicate 2


title: SCD1 -/- liver rep1

label: SCD-/- rep 1

value: mixed 129 and C57BL/6J; Age:6 weeks old; Gender:female; Diet:chow, non-fasted; Genetic modification:Scd1 -/-.


title: HEK293_HNF4a2_1-

description:
Analysis of gene expression in human embryonic kidney cells HEK293 overexpressing the transcription factors HNF4a2 or mutant forms HNF4a2 C106R and HNF4a2 R154X, which were introduced by FRT/FLP recombination. The analysis was performed 24 hours following 1µg/ml tetracycline treatment to induce the expression of the genes. Results identify HNF4a regulated genes in kidney cells being several of these genes deregulated in renal cell carcinoma.
Keywords = HEK293
Keywords = embryonic kidney cells
Keywords = HNF4a2
Keywords = tetracycline
Keywords = FRT

source_name: HEK293 cell line #1 with noninduced HNF4a2 wild type


title: N/SM5

status: Public on Jul 10 2005

city: Berlin

first: Oliver

last: Hummel

department: Experimental Genetics of Cardiovascular Diseases

institute: Max-Delbrück-Center

description:
Female C57BL/6 mice were purchased from Charles River Laboratories (Sulzfeld, Germany) at age 4-6 weeks and kept in a pathogen free environment. The microarray experiments were performed in mice 32 - 34 weeks of age. RNA was extracted from the right quadriceps muscle of 5 C57BL/6 mice using TRIzol reagent (Invitrogen, Life Technologies, Carlsbad, USA). Total RNA was purified following the TRIzol standard protocol. Total RNA was treated by deoxyribonuclease I (Invitrogen) and purified using the RNeasy Mini Kit (Qiagen GmbH, Hilden, Germany) according to the manufacture's protocol. RNA integrity was checked by denaturating agarose gel electrophoresis, and only RNA with absorbance ratios 260 nm/280 nm between 1.9 and 2.1 was used. 8 µg RNA were transcribed in double-stranded cDNA by cDNA Synthesis System (Roche Diagnostics GmbH, Mannheim, Germany). cRNA was produced by MEGAscript TM High Yield Transcription Kit (Ambion, Austin, USA) and labelled with biotin-11-CTP and biotin-16-UTP nucleotides (Perkin Elmer Life Sciences, Boston, USA). 5 quardiceps biological replicates were analyzed using standard Affymetrix procedures.
Keywords = Mus musculus,  C57BL/6, skeletal muscle, quardiceps, effect of deleted gene dysferlin

source_name: skeletal muscle, quadriceps


title: N/SM4


title: N/SM3


title: N/SM2


title: S/SM4

description:
Female SJL/J mice were purchased from Charles River Laboratories (Sulzfeld, Germany) at age 4-6 weeks and kept in a pathogen free environment. The microarray experiments were performed in mice 32 - 34 weeks of age. The SJL/J mouse strain has been derived from non-inbred Swiss Websterstock by brother-sister mating for 30 generations (Murphy, ED. Proc Am Assoc Cancer Res 4:46, 1963) and is a naturally occurring model for autosomal recessive muscular dystrophy (Bittner, RE et al. Nature Genet 23, 141-142, 1999). The SJL/J mice develop a spontaneously occurring myopathy resulting in muscle weakness (Weller, AH et al. Muscle Nerve 20, 72-82, 1997). This is due to a 171-bp deletion in the dysferlin gene of SJL mice resulting in an in-frame deletion and removal of 57amino acids (Bittner, RE et al. Nature Genet 23, 141-142, 1999). RNA was extracted from the left ventricle of heart of 5 SJL/J mice using TRIzol reagent (Invitrogen, Life Technologies, Carlsbad, USA). Total RNA was purified following the TRIzol standard protocol. Total RNA was treated by deoxyribonuclease I (Invitrogen) and purified using the RNeasy Mini Kit (Qiagen GmbH, Hilden, Germany) according to the manufacture's protocol. RNA integrity was checked by denaturating agarose gel electrophoresis, and only RNA with absorbance ratios 260 nm/280 nm between 1.9 and 2.1 was used. 8 µg RNA were transcribed in double-stranded cDNA by cDNA Synthesis System (Roche Diagnostics GmbH, Mannheim, Germany). cRNA was produced by MEGAscript TM High Yield Transcription Kit (Ambion, Austin, USA) and labelled with biotin-11-CTP and biotin-16-UTP nucleotides (Perkin Elmer Life Sciences, Boston, USA). 5 left ventricle biological replicates were analyzed using standard Affymetrix procedures.
Keywords = Mus musculus,  SJL/J, skeletal muscle, quadriceps, effect of deleted gene dysferlin


title: N/SM1


title: S/SM5


title: Wildtype replicate 2

source_name: E. coli wildtype AB1157

tag: Strain AB1157 wildtype


title: S/SM2


title: S/SM3

description:
Female SJL/J mice were purchased from Charles River Laboratories (Sulzfeld, Germany) at age 4-6 weeks and kept in a pathogen free environment. The microarray experiments were performed in mice 32 - 34 weeks of age. The SJL/J mouse strain has been derived from non-inbred Swiss Websterstock by brother-sister mating for 30 generations (Murphy, ED. Proc Am Assoc Cancer Res 4:46, 1963) and is a naturally occurring model for autosomal recessive muscular dystrophy (Bittner, RE et al. Nature Genet 23, 141-142, 1999). The SJL/J mice develop a spontaneously occurring myopathy resulting in muscle weakness (Weller, AH et al. Muscle Nerve 20, 72-82, 1997). This is due to a 171-bp deletion in the dysferlin gene of SJL mice resulting in an in-frame deletion and removal of 57amino acids (Bittner, RE et al. Nature Genet 23, 141-142, 1999). RNA was extracted from the left ventricle of heart of 5 SJL/J mice using TRIzol reagent (Invitrogen, Life Technologies, Carlsbad, USA). Total RNA was purified following the TRIzol standard protocol. Total RNA was treated by deoxyribonuclease I (Invitrogen) and purified using the RNeasy Mini Kit (Qiagen GmbH, Hilden, Germany) according to the manufacture's protocol. RNA integrity was checked by denaturating agarose gel electrophoresis, and only RNA with absorbance ratios 260 nm/280 nm between 1.9 and 2.1 was used. 8 µg RNA were transcribed in double-stranded cDNA by cDNA Synthesis System (Roche Diagnostics GmbH, Mannheim, Germany). cRNA was produced by MEGAscript TM High Yield Transcription Kit (Ambion, Austin, USA) and labelled with biotin-11-CTP and biotin-16-UTP nucleotides (Perkin Elmer Life Sciences, Boston, USA). 5 left ventricle biological replicates were analyzed using standard Affymetrix procedures.
Keywords = Mus musculus,  SJL/J,  skeletal muscle, quadriceps, effect of deleted gene dysferlin


title: S/LV5

description:
Female SJL/J mice were purchased from Charles River Laboratories (Sulzfeld, Germany) at age 4-6 weeks and kept in a pathogen free environment. The microarray experiments were performed in mice 32 - 34 weeks of age. The SJL/J mouse strain has been derived from non-inbred Swiss Websterstock by brother-sister mating for 30 generations (Murphy, ED. Proc Am Assoc Cancer Res 4:46, 1963) and is a naturally occurring model for autosomal recessive muscular dystrophy (Bittner, RE et al. Nature Genet 23, 141-142, 1999). The SJL/J mice develop a spontaneously occurring myopathy resulting in muscle weakness (Weller, AH et al. Muscle Nerve 20, 72-82, 1997). This is due to a 171-bp deletion in the dysferlin gene of SJL mice resulting in an in-frame deletion and removal of 57amino acids (Bittner, RE et al. Nature Genet 23, 141-142, 1999). RNA was extracted from the left ventricle of heart of 5 SJL/J mice using TRIzol reagent (Invitrogen, Life Technologies, Carlsbad, USA). Total RNA was purified following the TRIzol standard protocol. Total RNA was treated by deoxyribonuclease I (Invitrogen) and purified using the RNeasy Mini Kit (Qiagen GmbH, Hilden, Germany) according to the manufacture's protocol. RNA integrity was checked by denaturating agarose gel electrophoresis, and only RNA with absorbance ratios 260 nm/280 nm between 1.9 and 2.1 was used. 8 µg RNA were transcribed in double-stranded cDNA by cDNA Synthesis System (Roche Diagnostics GmbH, Mannheim, Germany). cRNA was produced by MEGAscript TM High Yield Transcription Kit (Ambion, Austin, USA) and labelled with biotin-11-CTP and biotin-16-UTP nucleotides (Perkin Elmer Life Sciences, Boston, USA). 5 left ventricle biological replicates were analyzed using standard Affymetrix procedures.
Keywords = Mus musculus,  SJL/J, cardiac muscle, left ventricle, effect of deleted gene dysferlin

source_name: cardiac muscle, left ventricle


title: S/SM1


title: S/LV3

description:
Female SJL/J mice were purchased from Charles River Laboratories (Sulzfeld, Germany) at age 4-6 weeks and kept in a pathogen free environment. The microarray experiments were performed in mice 32 - 34 weeks of age. The SJL/J mouse strain has been derived from non-inbred Swiss Websterstock by brother-sister mating for 30 generations (Murphy, ED. Proc Am Assoc Cancer Res 4:46, 1963) and is a naturally occurring model for autosomal recessive muscular dystrophy (Bittner, RE et al. Nature Genet 23, 141-142, 1999). The SJL/J mice develop a spontaneously occurring myopathy resulting in muscle weakness (Weller, AH et al. Muscle Nerve 20, 72-82, 1997). This is due to a 171-bp deletion in the dysferlin gene of SJL mice resulting in an in-frame deletion and removal of 57amino acids (Bittner, RE et al. Nature Genet 23, 141-142, 1999). RNA was extracted from the left ventricle of heart of 5 SJL/J mice using TRIzol reagent (Invitrogen, Life Technologies, Carlsbad, USA). Total RNA was purified following the TRIzol standard protocol. Total RNA was treated by deoxyribonuclease I (Invitrogen) and purified using the RNeasy Mini Kit (Qiagen GmbH, Hilden, Germany) according to the manufacture's protocol. RNA integrity was checked by denaturating agarose gel electrophoresis, and only RNA with absorbance ratios 260 nm/280 nm between 1.9 and 2.1 was used. 8 µg RNA were transcribed in double-stranded cDNA by cDNA Synthesis System (Roche Diagnostics GmbH, Mannheim, Germany). cRNA was produced by MEGAscript TM High Yield Transcription Kit (Ambion, Austin, USA) and labelled with biotin-11-CTP and biotin-16-UTP nucleotides (Perkin Elmer Life Sciences, Boston, USA). 5 left ventricle biological replicates were analyzed using standard Affymetrix procedures.
Keywords = Mus musculus,  SJL/J,  cardiac muscle, left ventricle, effect of deleted gene dysferlin


title: N/LV5

description:
Female C57BL/6 mice were purchased from Charles River Laboratories (Sulzfeld, Germany) at age 4-6 weeks and kept in a pathogen free environment. The microarray experiments were performed in mice 32 - 34 weeks of age. RNA was extracted from the left ventricle of heart of 5 C57BL/6 mice using TRIzol reagent (Invitrogen, Life Technologies, Carlsbad, USA). Total RNA was purified following the TRIzol standard protocol. Total RNA was treated by deoxyribonuclease I (Invitrogen) and purified using the RNeasy Mini Kit (Qiagen GmbH, Hilden, Germany) according to the manufacture's protocol. RNA integrity was checked by denaturating agarose gel electrophoresis, and only RNA with absorbance ratios 260 nm/280 nm between 1.9 and 2.1 was used. 8 µg RNA were transcribed in double-stranded cDNA by cDNA Synthesis System (Roche Diagnostics GmbH, Mannheim, Germany). cRNA was produced by MEGAscript TM High Yield Transcription Kit (Ambion, Austin, USA) and labelled with biotin-11-CTP and biotin-16-UTP nucleotides (Perkin Elmer Life Sciences, Boston, USA). 5 left ventricle biological replicates were analyzed using standard Affymetrix procedures.
Keywords = Mus musculus,  C57BL/6, cardiac muscle, left ventricle, effect of deleted gene dysferlin


title: N/LV4


title: S/LV2


title: N/LV1


title: S/LV4


title: S/LV1


title: N/LV3

description:
Female C57BL/6 mice were purchased from Charles River Laboratories (Sulzfeld, Germany) at age 4-6 weeks and kept in a pathogen free environment. The microarray experiments were performed in mice 32 - 34 weeks of age. RNA was extracted from the left ventricle of heart of 5 C57BL/6 mice using TRIzol reagent (Invitrogen, Life Technologies, Carlsbad, USA). Total RNA was purified following the TRIzol standard protocol. Total RNA was treated by deoxyribonuclease I (Invitrogen) and purified using the RNeasy Mini Kit (Qiagen GmbH, Hilden, Germany) according to the manufacture's protocol. RNA integrity was checked by denaturating agarose gel electrophoresis, and only RNA with absorbance ratios 260 nm/280 nm between 1.9 and 2.1 was used. 8 µg RNA were transcribed in double-stranded cDNA by cDNA Synthesis System (Roche Diagnostics GmbH, Mannheim, Germany). cRNA was produced by MEGAscript TM High Yield Transcription Kit (Ambion, Austin, USA) and labelled with biotin-11-CTP and biotin-16-UTP nucleotides (Perkin Elmer Life Sciences, Boston, USA). 5 left ventricle biological replicates were analyzed using standard Affymetrix procedures.
Keywords = Mus musculus,  C57BL/6,  cardiac muscle, left ventricle, effect of deleted gene dysferlin


title: N/LV2

description:
Female C57BL/6 mice were purchased from Charles River Laboratories (Sulzfeld, Germany) at age 4-6 weeks and kept in a pathogen free environment. The microarray experiments were performed in mice 32 - 34 weeks of age. RNA was extracted from the left ventricle of heart of 5 C57BL/6 mice using TRIzol reagent (Invitrogen, Life Technologies, Carlsbad, USA). Total RNA was purified following the TRIzol standard protocol. Total RNA was treated by deoxyribonuclease I (Invitrogen) and purified using the RNeasy Mini Kit (Qiagen GmbH, Hilden, Germany) according to the manufacture's protocol. RNA integrity was checked by denaturating agarose gel electrophoresis, and only RNA with absorbance ratios 260 nm/280 nm between 1.9 and 2.1 was used. 8 µg RNA were transcribed in double-stranded cDNA by cDNA Synthesis System (Roche Diagnostics GmbH, Mannheim, Germany). cRNA was produced by MEGAscript TM High Yield Transcription Kit (Ambion, Austin, USA) and labelled with biotin-11-CTP and biotin-16-UTP nucleotides (Perkin Elmer Life Sciences, Boston, USA). 5 left ventricle biological replicates were analyzed using standard Affymetrix procedures.
Keywords = Mus musculus
Keywords = C57BL/6
Keywords = cardiac muscle
Keywords = left ventricle
Keywords = effect of deleted gene dysferlin


title: Transdetermination study; regenerating discs vs reference_5-129

status: Public on Jul 08 2005

first: Ansgar

last: Klebes

address: Chausseestr. 117

department: Biology

institute: Humboldt University Berlin

description: These experiments are described in Klebes et al. (Development, 2005, in press).  Comparisons – category #9,  slide 5-129, regenerating disc – probe 1 vs. reference sample – probe 2 (ratios are shown as probe2/probe1). In this experiment, regenerating disc cells were compared to the common leg disc reference sample. These comparisons were intended to establish the unique expression profiles of regenerating leg imaginal cells in response to fragmentation. Regenerating cells were obtained from disc fragments that had been cultured in the abdomen of female flies to promote proliferation. To minimize possible contamination with transdetermined cells, we did not use 1st leg discs, but instead examined the ‘3/4 fragment’ of 2nd leg discs (Sustar and Schubiger, 2005). This fragment regenerates missing parts, and in contrast to 1st leg discs, does not transdetermine (G.S., unpublished). The disc fragments were collected after 3-5 days of culture. The common reference sample was generated by pooling amplified RNA of male and female 1st, 2nd and 3rd leg imaginal discs in equal proportions. Discs and fragments were dissected in PBS and carefully washed several times to reduce the concentration of contaminating fat body cells.  RNA was extracted from imaginal disc fragments using the Mini RNA isolation kit (Zymo research). Linear RNA amplification was performed essentially as described (Klebes et al., 2002). In brief, total RNA was used for a first round of reverse transcription and in vitro transcription using T7 RNA polymerase. Subsequently, the amplified RNA product was subjected to a 2nd round of reverse transcription and in vitro transcription yielding 10,000-fold amplification or more. Probes were indirectly labeled with amino-allyl modified dUTP in a reverse transcription reaction and coupled to reactive Cy3 or Cy5 dyes (Amersham). Correspondingly labeled Cy3 and Cy5 cDNA products were combined and purified on PCR purification columns (Qiagen), and diluted in hybridization buffer. Probes were added to the slides and incubated in a metal hybidization chamber (DieTech) in a waterbath at 65°C over night. Microarrays were washed and scanned on a GenePix 4000A scanner (Axon Instruments, Foster City, CA). Signal intensities were initially stored and analyzed using NOMAD (www.microarrays.org). Data was extracted as the global median normalized ratios (net median ratios) of the intensitites and log2 transformed. Signals with intensity values of the sum of median (foreground minus background) smaller than 300 were eliminated.  Each comparison was performed in replicates with independently isolated and processed probes. Within a set of replicate experiments, dyes were reversed to avoid bias in about half of the experiments. To represent all experiments with the same dye orientation the values of the log2 transformed expression ratios were reversed if necessary.

data_processing: log2 transformed global median normalized ratios (probe2/probe1)

organism: Drosophila melanogaster

source_name: Regenerating leg imaginal disc

tag: Cultivated 3/4 fragment of a mesothoracic (2nd) leg imaginal disc, 2 rounds of linear RNA amplification

source_name: Leg disc reference sample

tag: pooled amplified RNA of 1st, 2nd and 3rd leg discs of third instar male and female wildtype larvae


title: Transdetermination study; D(wt) cells vs V(wt) cells_7-059

description: These experiments are described in Klebes et al. (Development, 2005, in press).  Comparisons – category #8,  slide 7-059, D(wt), dorsal prothoracic leg disc cells (wildtype) – probe 1 vs. V(wt) ventral cells from the same discs– probe 2 (ratios are shown as probe 2/probe 1).  Dorsal cells (D(wt) of prothoracic leg imaginal discs were compared to ventral cells (V(wt)). Both cell populations were isolated from the same discs. For this experiment third instar larvae of Drosophila melanogaster strain OregonR (OR) were staged by feeding 1% bromphenol blue which allows to observe the gut content. Single male wandering late third instar larvae (half empty gut) were used to isolate leg discs. These comparisons were intended to establish the unique expression profiles of dorsal and ventral leg imaginal cells to distinguish them from genes identified in the transdeterminating cell population of Wingless over-expressing leg discs. Dorsal cells of wildtype prothoracic leg discs (D(wt)) were approximately separated from ventral cells (V(wt)) by microdissection. Discs and fragments were dissected in PBS and carefully washed several times to reduce the concentration of contaminating fat body cells.   RNA was extracted from imaginal disc fragments using the Mini RNA isolation kit (Zymo research). Linear RNA amplification was performed essentially as described (Klebes et al., 2002). In brief, total RNA was used for a first round of reverse transcription and in vitro transcription using T7 RNA polymerase. Subsequently, the amplified RNA product was subjected to a 2nd round of reverse transcription and in vitro transcription yielding 10,000-fold amplification or more.   Probes were indirectly labeled with amino-allyl modified dUTP in a reverse transcription reaction and coupled to reactive Cy3 or Cy5 dyes (Amersham). Correspondingly labeled Cy3 and Cy5 cDNA products were combined and purified on PCR purification columns (Qiagen), and diluted in hybridization buffer. Probes were added to the slides and incubated in a metal hybidization chamber (DieTech) in a waterbath at 65°C over night. Microarrays were washed and scanned on a GenePix 4000A scanner (Axon Instruments, Foster City, CA). Signal intensities were initially stored and analyzed using NOMAD (www.microarrays.org). Data was extracted as the global median normalized ratios (net median ratios) of the intensitites and log2 transformed. Signals with intensity values of the sum of median (foreground minus background) smaller than 300 were eliminated.  Each comparison was performed in replicates with independently isolated and processed probes. Within a set of replicate experiments, dyes were reversed to avoid bias in about half of the experiments. To represent all experiments with the same dye orientation the values of the log2 transformed expression ratios were reversed if necessary.

source_name: Dorsal cells (D(wt))

tag: microdissected dorsal cells from prothoracic leg imaginal discs (D(wt)) isolated from wildtype larvae, 2 rounds of linear amplification

source_name: Ventral cells (V(wt))

tag: microdissected ventral cells from prothoracic leg imaginal discs (V(wt)) isolated from wildtype larvae, 2 rounds of linear amplification