title: 0204_WTH_D9_R(20-1)_G(N)

source_name: Patient 20_1

tag: Patient 20 before CCRT treatment

source_name: common reference (Normal)


title: 0204_WTH_D9_R(19-6)_G(N)

status: Public on Oct 20 2007

type: RNA

city: TauYau

first: Yun-Shien

last: Lee

state: Taiwan

address: TauYau

department: Dept. Biotechnology

country: Taiwan

institute: Ming Chuan Univ

description: a) Normalization strategy total array b) Normalization algorithm Lowess (local weighted regression),

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

scan_protocol: Axon GenePix4000A

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

molecule: total RNA

organism: Homo sapiens

source_name: Patient 19_6

label_protocol: Protocol Genisphere 3DNA-Random Primer Submicro Expression Array Detection Kit (category # W300130/W300140);

extract_protocol: Extract Protocol Total RNA was extracted from 2.5 ml of whole blood using the Paxgene Blood RNA System (PreAnalytiX, Hombrechtikon, CH) according to the manufacturer’s instruction. The quality and quantity of RNA was analyzed with Bioanalyzer 2100 (Agilent, Palo Alto, CA)

tag: Patient 19 one month after the treatment

tag: Blood samples from 11 women with benign gynecological diseases at out-patient clinic served as normal control individuals. Pooled RNA from these samples was used as a common reference for microarray analyses.


title: 1104_CCRT_D9_R(62-3)_G(N)

description:
a) Normalization strategy
total array
b) Normalization algorithm
Lowess (local weighted regression),

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

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

source_name: Patient 62-3

label_protocol: Genisphere 3DNA-Random Primer Submicro Expression Array Detection Kit  (category # W300130/W300140);

extract_protocol: Total RNA was extracted from 2.5 ml of whole blood using the Paxgene Blood RNA System (PreAnalytiX, Hombrechtikon, CH) according to the manufacturer’s instruction. The quality and quantity of RNA was analyzed with Bioanalyzer 2100 (Agilent, Palo Alto, CA)

tag: Patient 62 one week after completion of CCRT


title: 1012_CCRT_D9_R(57-4)_G(N)

source_name: Patient 57-4

tag: Patient 57 one month after the treatment


title: 0204_WTH_D9_R(20-3)_G(N)

source_name: Patient 20_3

tag: Patient 20 one week after completion of CCRT


title: 0204_WTH_D9_R(20-2)_G(N)

source_name: Patient 20_2

tag: Patient 20 one week after the first course of chemotherapy and initiation of radiotherapy


title: 1021_CCRT_D9_R(56-4)_G(N)

source_name: Patient 56_4

tag: Patient 56 one month after the treatment


title: 0204_WTH_D9_R(N)_G(19-6)


title: 0204_WTH_D9_R(N)_G(20-1)


title: 0923_CCRT_D9_R(55-3)_G(N)

source_name: Patient 55_3

tag: Patient 55 one week after completion of CCRT


title: breast tumor part_IPC_rep1_1595

city: Taipei

first: Hsin-Chieh

last: Yao

address: 1,  Rd. Roosevelt,  Sec. 4

department: CHE

institute: National Taiwan University

description: Hybridized with Buffer1

scan_protocol: Agilent Oligo Microarray Kit, hybridization protocol version 5.0 with Agilent scanner and Feature Extraction 6.0.

data_processing: Agilent Two-Color Microarray-Based Gene Expression Analysis, version 4.0

source_name: Intracystic Papillary Carcinoma (IPC)

label_protocol: Agilent Low RNA Input Fluorescent Linear Amplification Kit, protocol version 2.0

growth_protocol: The fresh samples used for this study were immediately frozen by liquid nitrogen without any additional growth procedure applied before RNA extraction.

extract_protocol: Trizol reagent combined with Rneasy mini kit

treatment_protocol: The surgical removed tumors and non-tumor part of breast specimens were excised, collected and snap-frozen in liquid nitrogen immediately  before RNA extraction.

tag: gender

value: female

tag: tissue

value: primary tumor

source_name: Stratagene's human common reference RNA

tag: reference

value: Stratagene's human common reference RNA


title: 0128_WTH_D9_R(N)_G(12_2)

source_name: Patient 12_2

tag: Patient 12 one week after the first course of chemotherapy and initiation of radiotherapy


title: 0128_WTH_D9_R(N)_G(12_1)

source_name: Patient 12_1

tag: Patient 12 before CCRT treatment


title: 0128_WTH_D9_R(N)_G(12_3)

source_name: Patient 12_3

tag: Patient 12 one week after completion of CCRT


title: breast nontumor part_N_rep1_1593

description: Hybridized with Buffer1

hyb_protocol: Agilent Oligo Microarray Kit, hybridization protocol version 5.0

source_name: Nontumor part (also called normal(N))

value: non-tumor part of breast


title: breast tumor part_IPC_rep1_1585

description: Hybridized with Buffer1


title: 0128_WTH_D9_R(12_2)_G(N)


title: Gamma-ray exposured Replicate 2

city: sendai-city

first: Taisuke

last: Baba

state: Miyagi

address: seiryo-machi 4-1

country: Japan

institute: Tohoku Univ. IDAC

description: Biological replicate 2 of 2.

hyb_protocol: Hybridization were carried out on UltraGAPS coated slides in accordance with the manufacturers manual (Corning, NY)

scan_protocol: The image was captured in a GenePix 4000B (Axon Instruments, Inc. CA) The quantification of gene expression arrays was performed by Array Vision software (Imaging Research Inc., Ontario, Canada).

data_processing: LOWESS normalized, background subtracted VALUE data obtained from log of processed Red signal/processed Green signal.

organism: Mus musculus

source_name: Total RNA from irradiated mouse(Gamma-ray) with Cyanine-5 (red).

label_protocol: Poly-A RNA was separated using dT(25)-coupled magnetic beads (Dynal Biotech, Oslo, Norway).  Complementary DNA (cDNA) probes were generated starting with 1 ƒÊg of polyA RNA using a CyScribe First-Strand cDNA Labelling kit (Amersham Biosciences Corp., Piscataway, NJ).  cDNA from irradiated mice was labeled with Cy5.

extract_protocol: TriZol procedure

tag: Age

value: 6 weeks

tag: Tissue

value: liver

tag: Strain

tag: Sex

value: Male

source_name: Total RNA from pooled non-irradiated mice with Cyanine-3 (green).

label_protocol: Poly-A RNA was separated using dT(25)-coupled magnetic beads (Dynal Biotech, Oslo, Norway).  Complementary DNA (cDNA) probes were generated starting with 1 ƒÊg of polyA RNA using a CyScribe First-Strand cDNA Labelling kit (Amersham Biosciences Corp., Piscataway, NJ).  cDNA from non-irradiated mice was labeled with Cy3.

value: 6 weels


title: 0128_WTH_D9_R(N)_G(12_4)

tag: Blood samples from 11 women with benign gynecological diseases at out-patient clinic served as normal control individuals. Pooled RNA from these samples was used as a common reference for microarray analyses.

source_name: Patient 12_4

tag: Patient 12 one month after the treatment


title: 0128_WTH_D9_R(12_1)_G(N)

source_name: common reference (Normal)


title: breast nontumor part_N_rep1_1541

description: Hybridized with Buffer1


title: breast nontumor part_N_rep1_1543

description: Hybridized with Buffer1


title: breast nontumor part_N_rep1_1539

description: Hybridized with Buffer1


title: 0128_WTH_D9_R(12_3)_G(N)


title: 0126_WTH_D9_R(N)_G(6_2)

source_name: Patient 6_2

tag: Patient 6 one week after the first course of chemotherapy and initiation of radiotherapy


title: 0128_WTH_D9_R(12_4)_G(N)


title: 0126_WTH_D9_R(N)_G(6_3)

source_name: Patient 6_3

tag: Patient 6 one week after completion of CCRT


title: 0126_WTH_D9_R(N)_G(6_4)

source_name: Patient 6_4

tag: Patient 6 one month after the treatment


title: breast tumor part_IDC_rep1_1537

description: Hybridized with Buffer1

source_name: Infiltrating Ductal Carcinoma (IDC)

value: recurrent tumor

tag: estrogen receptor status


title: 0126_WTH_D9_R(6_3)_G(N)


title: breast tumor part_IDC_rep1_1535

description: Hybridized with Buffer1

tag: progesterone receptor status


title: Gamma-ray exposured Replicate 1

description: Biological replicate 1 of 2.


title: 0126_WTH_D9_R(6_2)_G(N)

description:
a) Normalization strategy total array
b) Normalization algorithm Lowess (local weighted regression),

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

source_name: Patient 6_T2

tag: Patient 6 one week after the first course of chemotherapy and initiation of radiotherapy,


title: breast nontumor part_N_rep1_1403

description: Hybridized with Buffer1

tag: gender

tag: tissue


title: 0126_WTH_D9_R(N)_G(6_1)

hyb_protocol: Hybridization Protocol Hybridization buffer: Supply by Genisphere 3DNA kits. Slide blocking: 3x SSC, 0.1% SDS, 0.1 mg/mL BSA. Prehybridization for 30 min at 50°C. wash procedure: 1x 10 min with 2x SSC, 0.2% SDS at 60oC; 1x 10 min with 2x SSC, at room temperature; 1x 10 min with 0.2x SSC at room temperature; spin-dried at 250 x g for 3 min. quantity of labeled target used 2 μg of total RNA (Genisphere 3DNA-Random

source_name: Patient 6_1

tag: Patient 6 before CCRT treatment


title: breast nontumor part_N_rep1_1401

description: Hybridized with Buffer1

tag: reference


title: breast tumor part_MC_rep1_1351

description: Hybridized with Buffer1

source_name: Mucinous Carcinoma (MC)


title: breast tumor part_ILC_rep1_1259

description: Hybridized with Buffer1

source_name: Infiltrating Lobular Carcinoma (ILC)


title: 0126_WTH_D9_R(6_1)_G(N)


title: Drosophila RasV12 sample 3

status: Public on Oct 19 2007

city: Columbus

first: Amanda

last: Simcox

state: OH

address: 484 W. 12th Avenue

department: Molecular Genetics

country: USA

institute: The Ohio State University

description: Affymetrix standard reagents and protocols were used for labeling, hybridizing and scanning.

hyb_protocol: Affymetrix standard protocol

scan_protocol: Affymetrix standard protocol

data_processing: R and bioconductor were used to preprocess and analyze the data

label: biotin

organism: Drosophila melanogaster

source_name: Drosophila cell culture

label_protocol: Affymetrix standard protocol

extract_protocol: Qiagen RNeasy

tag: Drosophila cell line generated from primary cells using RasV12


title: Drosophila RasV12 sample 1

tag: Cell line from primary culture, Drosophila


title: Drosophila RasV12 sample 2


title: 0126_WTH_D9_R(6_4)_G(N)


title: Strain LR128 (three biological replicates)

status: Public on Oct 18 2007

city: Seattle

first: Lakshmi

last: Rajagopal

state: WA

address: 1900 9th Ave

department: Pediatric Infectious Disease

institute: Seattle Children's Hospital Research Institute

description: Catalog design for Streptococcus agalactiae A909 (Taxonomy Id: 205921) covering NC_007432. Probes selected for 1996/1996 sequences. Median number of probes/sequence is 18 with an average of 18.00. Each probe will be replicated 5 times on the chip. Probes are randomly distributed over the surface of the array. Unused features have been filled with randomly generated probes of comparable GC content.

hyb_protocol: Hybridization was performed by NimbleGen Inc.

scan_protocol: Each chip was scanned in 5 technical replicates by NimbleGen Inc., with the average scan values reported here.

data_processing: The raw data were re-scaled to account for the differences in individual hybridization intensities by NimbleGen Inc.

organism: Streptococcus agalactiae

label_protocol: Labeling was performed by NimbleGen Inc. using Cy3.

extract_protocol: Total prokaryotic RNA was extracted from late log-phase (OD600=0.6) cultures of GBS grown in TSB at 37C and 5% CO2. Three biological replicates were used, on three separate chips, with average values reported here.

tag: covR deletion mutant


title: MCF7_E2_replicate2

city: Dallas

first: W. Lee

last: Kraus

state: TX

address: 5323 Harry Hines Blvd.

institute: UT Southwestern Medical Center

hyb_protocol: Standard hybridization to Affymetrix U133A 2.0 microarray.

scan_protocol: Standard scanning protocol

data_processing: The raw data were processed by Affymetrix GCOS software to obtain detection calls and signal values, and then normalized by scaling.

label: Biotin

source_name: MCF7 cells, treated for 3 hours with EtOH or 17beta-estradiol (E2)

label_protocol: Seven μg of total RNA were subjected to One-Cycle Target Labeling Assay (Affymetrix) to generate biotinylated cRNA targets for hybridization to Affymetrix U133A 2.0 microarray.

extract_protocol: Total RNA was prepared using Trizol Reagent according to the manufacturer’s instructions (Invitrogen) and was further purified using RNeasy columns (Qiagen).

tag: MCF-7 cells were grown as described above and treated with ethanol or 100 nM E2 for 3h.


title: strain GR1373 replicate 2

city: Stockholm

first: Suvi

middle: Tuulia

last: Asikainen

state: Huddinge

address: Karolinska University Hospital

department: CLINTEC

country: Sweden

institute: Karolinska Institutet

description: GR1373 independent biological replicate 2. Asikainen et al., (2007) Whole genome microarray analysis of rrf-3 and eri-1 mutant C.elegans.

hyb_protocol: According to Affymetrix protocol

scan_protocol: According to Affymetrix protocol

data_processing: Robust Multichip Average (RMA) -algorithm was used for normalization and statistical processing (Bolstad et al., 2003).

organism: Caenorhabditis elegans

source_name: Whole C.elegans, larva 4 stage

label_protocol: According to Affymetrix protocol

growth_protocol: fed with OP50 E.coli

extract_protocol: Ribopure Total RNA isolation kit

treatment_protocol: no treatment

tag: eri-1 mutant strain

value: GR1373 (mg366). Whole C.elegans, larva 4 stage


title: Strain A909 WT (three biological replicates)

source_name: A909 WT

tag: wild type


title: strain NL2099 replicate 3

description: NL2099 independent biological replicate 3. Asikainen et al., (2007) Whole genome microarray analysis of rrf-3 and eri-1 mutant C.elegans.

tag: rrf-3 mutant strain

value: NL2099 (pk1426). Whole C.elegans, larva 4 stage


title: strain GR1373 replicate 3

description: GR1373 independent biological replicate 3. Asikainen et al., (2007) Whole genome microarray analysis of rrf-3 and eri-1 mutant C.elegans.


title: strain NL2099 replicate 1

description: NL2099 independent biological replicate 1. Asikainen et al., (2007) Whole genome microarray analysis of rrf-3 and eri-1 mutant C.elegans.

treatment_protocol: No treatment


title: N2 replicate 2

description: N2 replicate 2. Asikainen et al., (2007) Whole genome microarray analysis of rrf-3 and eri-1 mutant C.elegans.

source_name: Whole C.elegans, Larva 4 stage

tag: N2 strain. Whole C.elegans, larva 4 stage.


title: N2 replicate 3

description: N2 replicate 3. Asikainen et al., (2007) Whole genome microarray analysis of rrf-3 and eri-1 mutant C.elegans.

tag: N2 strain. Whole C.elegans, larva 4 stage


title: strain GR1373 replicate 1

description: GR1373 independent biological replicate 1. Asikainen et al., (2007) Whole genome microarray analysis of rrf-3 and eri-1 mutant C.elegans.


title: MCF7_E2_replicate3


title: N2 replicate 1

description: N2 replicate 1. Asikainen et al. (2007): Whole genome microarray analysis of rrf-3 and eri-1 mutant C.elegans.

data_processing: Robust Multichip Average (RMA) -algorithm was used for normalization and statistical processing (Bolstad et al.).


title: strain NL 2099 replicate 2

description: NL2099 independent biological replicate 2. Asikainen et al., (2007) Whole genome microarray analysis of rrf-3 and eri-1 mutant C.elegans.


title: HFP-N2

city: shanghai

first: zensun

last: biology

address: shanghai zhangjiang

country: China

institute: zensun biology

description: HFP-N2:vehicle rat teated with rhNRG-1(recombinant human neuregulin-1), number 2

hyb_protocol: The cRNA was fragmented and hybridized to the chips

scan_protocol: probe arrays were scanned under the control of Affymetrix GeneChip 5.0 software.

data_processing: Data sets on each GeneChip were normalized by scaling total chip fluorescence intensities to a common value of 500 before comparison via GCOS1.3 software (Affymetrix). Selected and clustered candidate genes had to be present on all of the three chips in vehicle, rhNRG-1 treated or sham operated groups, with at least a 1.5-fold change in expression level compared with any another groups as implemented in GeneSpring 7.31(Agilent). 1-Way ANOVA Tukey analysis was done at the same time, all of the clustered genes were p-value <0.05

organism: Rattus norvegicus

source_name: Rat left ventricle non-infarction area

label_protocol: After reverse transcription, an in vitro transcription (IVT) reaction was then done to produce biotin-labeled cRNA from the cDNA.

extract_protocol: The total RNA was extracted from non-infarcted region of left ventricle by trizol reagent (Invitrogen)


title: MCF7_EtOH_replicate3


title: HFP-F1

description: HFP-F1:vehicle rat, number F1

tag: HFP-F1:vehicle rat, number F1


title: NOR-F8

description: NOR-F8:sham operated rats, number F8

tag: NOR-F8:sham operated rats, number F8


title: HFP-N3

description: HFP-N3:vehicle rat teated with rhNRG-1(recombinant human neuregulin-1), number 3


title: MCF7_EtOH_replicate2


title: HFP-N4

description: HFP-N4:vehicle rat teated with rhNRG-1(recombinant human neuregulin-1), number 4


title: HFP-F2

description: HFP-F2:vehicle rat, number F2

tag: HFP-F2:vehicle rat, number F2


title: Carbon-limited anaerobic chemostat with benzoate -1

status: Public on Oct 17 2007

city: Delft

first: Jean-Marc

last: Daran

address: Julianalaan 67

department: Department of Biotechnology

country: Netherlands

institute: Delft University of Technology

hyb_protocol: Probe Preparation and Hybridization to Arrays—mRNA extraction, cDNA synthesis, cRNA synthesis and labeling, as well as array hybridization were performed as described in the Affymetrix users’ manual (1). Briefly, poly(A)_ RNA was enriched from total RNA in a single round using the Qiagen Oligotex kit. Double-stranded cDNA synthesis was carried out incorporating the T7 RNA-polymerase promoter in the first round. This cDNA was then used as template for in vitro transcription (ENZO BioArray High Yield IVT kit), which amplifies the RNA pool and incorporates biotinylated ribonucleotides required for the staining procedures after hybridization. 15 mg of fragmented, biotinylated cRNA was hybridized to Affymetrix yeast S98 arrays at 45 °C for 16 h as described in the Affymetrix users’ manual (1). Washing and staining of arrays were performed using the GeneChip Fluidics Station 400 and scanning with the Affymetrix GeneArray Scanner. (1) Affymetrix (2000) Affymetrix GeneChip Expression Analysis Technical Manual, Santa Clara, CA

data_processing: Acquisition and quantification of array images as well as primary data analysis were performed using the Affymetrix software packages: Microarray Suite version 4.0.1, MicroDB version 2.0, and Data Mining Tool version 2.0. Microsoft Excel was used for further statistical analyses. All arrays were globally scaled to a target value of 150 using the average signal from all gene features using Microarray Suite version 4.0.1. When pairwise comparisons were performed (using Microarray Suite version 4.0.1), a transcript was considered “changed” when a call of Increase or Decrease was made, the -fold change was at least 2, and the higher of the two average difference values was called present.

label: Biotinylated-UTP SAPE-Phycoerythrin

molecule: polyA RNA

organism: Saccharomyces cerevisiae

source_name: Chemostat sample

growth_protocol:
The laboratory reference strain CEN.PK 113-7D (MATa) was grown at 30 °C in 2-L chemostat fermentors (Applikon, Schiedam, The Netherlands) with a working volume of 1-L using an electronic level sensor to maintain a constant volume. All cultures, including the reference, were fed with minimal medium as described by Verduyn et al. (1992) with 25 g L-1 glucose as the limiting nutrient and 0.15 ml L-1 silicone antifoam (BDH, Poole, England) to prevent excessive foaming. The dilution rate was set to 0.10 h-1 and the pH was controlled at 5.0 with the automatic addition (ADI 1031 bio controller, Applikon) of 2 M KOH. The stirrer speed was set at 800 RPM and anaerobicity was maintained by sparging the fermentor with N2 gas at 500 ml min-1. To prevent diffusion of oxygen, the fermentor was equipped with Norprene tubing and Viton O-rings and the medium vessel was also flushed with N2 gas. A comparable degree of weak acid uncoupling was ensured by decreasing the biomass yield to approximately 50% of the reference condition (no organic acids added) with the addition of the appropriate concentration of acetic acid, sodium benzoate, propionic acid or potassium sorbate to the reservoir media.
Verduyn C, Postma E, Scheffers WA & van Dijken JP (1992) Effect of benzoic acid on metabolic fluxes in yeast: a continuous-culture study on the regulation of respiration and alcoholic fermentation. Yeast 8: 501-517.

extract_protocol: Samples from the chemostat cultures were taken as rapidly as possible to limit any potential changes in transcript profiles during the procedure. 40–60 ml of culture broth was sampled directly from the chemostat into a beaker containing 200 ml of liquid nitrogen. With vigorous stirring, the sample froze instantly. The frozen sample was then broken into small fragments and transferred to a 50-ml centrifuge tube. The sample was then thawed at room temperature, ensuring that it remained as close to zero as possible. Cells were pelleted (5000 rpm at 0 °C for 4 min), resuspended in 2 ml of ice-cold AE buffer (50 mM sodium acetate, 10 mM EDTA, pH 5.0) and aliquoted into 5 Eppendorf tubes. This corresponded to _20 mg of dry weight per tube. For each array, total RNA was extracted from a single tube using the hot-phenol method (32) or the FastRNA kit, Red (BIO 101, Inc., Vista, CA).

treatment_protocol: N2 quenching

tag: Saccharomyces cerevisiae CENPK113-7D (MATa)


title: HFP-F4

description: HFP-F4:vehicle rat, number F4

tag: HFP-F4:vehicle rat, number F4


title: Carbon-limited anaerobic chemostat with benzoate -3

label_protocol:
mRNA extraction, cDNA synthesis, cRNA synthesis and labeling, as well as array hybridization were performed as described in the Affymetrix users’ manual (1). Briefly, poly(A)_ RNA was enriched from total RNA in a single round using the Qiagen Oligotex kit. Double-stranded cDNA synthesis was carried out incorporating the T7 RNA-polymerase promoter in the first round. This cDNA was then used as template for in vitro transcription (ENZO BioArray High Yield IVT kit), which amplifies the RNA pool and incorporates biotinylated ribonucleotides required for the staining procedures after hybridization. 15 mg of fragmented, biotinylated cRNA was hybridized to Affymetrix yeast S98 arrays at 45 °C for 16 h as described in the Affymetrix users’ manual (1). Washing and staining of arrays were performed using the GeneChip Fluidics Station 400 and scanning with the Affymetrix GeneArray Scanner.
(1) Affymetrix (2000) Affymetrix GeneChip Expression Analysis Technical Manual, Santa Clara, CA


title: PBMC-CRR-vs-BTB-infected-3

status: Public on Oct 16 2007

city: Dublin

first: David

middle: E

last: MacHugh

address: University College Dublin, Belfield

department: College of Agriculture, Food Science and Veterinary Medicine

country: Ireland

institute: University College Dublin

description: No additional information.

hyb_protocol: Labelled samples were combined (either an infected or a control sample combined with a CRR sample) and co-hybridized on the BOTL-5 microarrays using SlideHyb Glass Array Hybridization Buffer #3 (Ambion Ltd.). Microarray hybridizations were performed using a Tecan HS400 hybridisation station (Tecan Ltd.) with the following protocol – Step 1: 75°C, wash 10 s, soak 20 s, 1 cycle; probe injection: 85°C; denaturation: 95°C, 2 min; hybridization cycle 1: 65°C, time 35 min, agitation frequency medium; hybridization cycle 2: 55°C, time 35 min, agitation frequency medium; hybridization cycle 3: 50°C, time 2 h 30m, agitation frequency medium; wash cycle 1: 42°C, wash 10 s, soak 20 s, 2 cycles; wash cycle 2: 33°C, wash 15 s, soak 30 s, 2 cycles; wash cycle 3: 33°C, wash 20 s, soak 40 s, 2 cycles; slide drying: 30°C, 1 min 30 s.

scan_protocol: Microarrays were scanned immediately using a GenePix 4000B microarray scanner (Molecular Devices Ltd.). Data was captured using GenePix Pro version 5.0 software (Molecular Devices Ltd.).

data_processing:
The working signal intensities were generated using the mean foreground intensity values minus the median background intensity values as outputted from the GenePix Pro 5.0 results file. Two methods of data pre-processing were used to flag unreliable data. If the signal intensity in one channel was less than 100 and if the signal intensity for the other channel is less than 200, the spot was flagged. If the signal intensity in one channel was less than 100 and the signal intensity was larger than 200 in the second channel, 100 was assigned to the intensity of the first channel.
Median-based normalization corrects the data such that all arrays have the same median. The median value is less likely to be influenced by outlying values. A normalization factor was calculated by summing the intensities in both channels and adjusts both ratios to ensure a median of 1.0. Therefore, the median log expression ratio for all features on the array was adjusted to zero (corresponding to an expression ratio of 1.0).

organism: Bos taurus

source_name: Peripheral blood mononuclear cells (PBMC) sampled from 13 animals across a tuberculin stimulation time course

label_protocol: Each labelling reaction contained a total of 8 µg RNA per sample and 10 µg RNA from the CRR. Total RNA was used as the template in reverse transcription reactions (BD Atlas Powerscript fluorescent labelling system, BD Biosciences: www.bdbeurope.com) using oligo(dT) 15-18 as primer and incorporating amino-modified dUTPs. To provide a control for cDNA synthesis and labelling efficiency, hybridisation and slide orientation, 650 pg of synthetic lambda Q gene RNA containing an engineered poly A tail was spiked into each cDNA synthesis reaction. Test and CRR samples were differentially labelled using N-hydroxysuccinimide-(NHS) activated fluorescent Cy3 or Cy5 dyes (Amersham Biosciences/GE Healthcare: www.amershambiosciences.com). The dye couples to the cDNA by reacting with the amino functional groups on the incorporated amino-tagged dUTPs. Labelled cDNAs were purified to remove unincorporated dyes using a QIAquick purification kit and concentrated using Microcon® centrifugal filter devices according to the manufacturer’s instructions (Millipore Corp: www.millipore.com).

growth_protocol:
A Trypan exclusion test was used to estimate cell numbers: 10 µl of the cell solution was added to 90 µl Trypan (100 ml trypan blue solution 0.4% - T8154, Sigma-Aldrich Ireland Ltd: www.sigmaaldrich.com) in a 96-well microtitre plate and left for 5 mins. Viable cells were counted by pipetting 10 µl of solution under the coverslip of a haemocytometer, using a light microscope.
Cells from each animal were seeded at no less than 10^7 for each of the two treatments and cultured in RPMI 1640 culture medium supplemented with 5% FBS, 0.1% mercaptoethanol and 0.1% gentamycin. PBMC were cultured in 75-ml culture flasks at 37 °C in a humidified atmosphere with 5% CO2. PBMC cultures from the eight animals were stimulated using 50 µg/ml bovine purified protein derivative (PPD-bovine) and unstimulated control cultures from the same animals were run in parallel. PBMC were harvested from all treatments at 0. 3 and 12 h post-stimulation and stored at -80°C in 1 ml of Trizol® reagent (Invitrogen Corp: www.invitrogen.com).

extract_protocol: Total RNA was extracted from PBMC using a combined TriReagent®, DNase treatment and Qiagen RNeasy® method (Qiagen Ltd: www.qiagen.com). Cells were removed from the -80°C freezer and thawed quickly on ice, 400 µl of chloroform was added and shaken for 10 s. After a 2 min room temperature incubation, tubes were centrifuged on maximum speed for one min to separate the phases. The upper aqueous phase was transferred to a new 1.5 ml tube and an equal volume of isopropanol was added. After shaking and incubating at room temperature for 20 mins, the samples were centrifuged on maximum speed at 4°C for 20 mins. The liquid was then removed and the RNA pellet washed with 1 ml 70% ethanol (stored at -20°C) by centrifugation at 4°C for 6 mins. The ethanol was removed by careful aspiration and the pellet was allowed to air dry for 10 mins after which it was resuspended in 100 µl of nuclease free water and incubated at 55°C to ensure complete resuspension. To remove any residual DNA, 5 µl DNase (1 U/µl) was added and incubated at 37°C for 15 mins. The extracted RNA was then ready for further purification using the Qiagen RNeasy® mini kit according to the manufacturer’s instructions and eluted twice, in 40 µl and 30 µl of nuclease free water, and stored at -80°C.

tag: Source

value: PBMC from eight M. bovis-infected cattle and five non-infected cattle. PBMC were stimulated with purified protein derivative (PPD) of Mycobacterium bovis (tuberculin) and sampled across a time course of T0 (no stimulation) T3 (3 hours post-stimulation) and T12 (12 hours post-stimulation). A total of 39 RNA samples were combined to form the common RNA reference (CRR) pool

growth_protocol: PBMC from each animal (control and infected) were seeded at 10^7 per culture plate and cultured in RPMI 1640 culture medium supplemented with 5% FBS, 0.1% mercaptoethanol and 0.1% gentamycin. PBMC were cultured in 75-ml culture flasks at 37 °C in a humidified atmosphere with 5% CO2. Overnight culture was carried out to minimise noise in gene expression measurements potentially introduced by the mechanical disruption of cells associated with PBMC isolation.

treatment_protocol: No treatment other than overnight culture.

tag: PBMC from a Mycobacterium bovis-infected animal cultured overnight and then used for total RNA extraction.


title: PBMC-CRR-vs-BTB-infected-5

description: No additional information.

treatment_protocol: PBMC cultures from the eight animals were stimulated using 50 µg/ml bovine purified protein derivative (PPD-bovine) and unstimulated control cultures from the same animals were run in parallel.


title: PBMC-CRR-vs-BTB-infected-4

description: No additional information.


title: NOR-F4

description: NOR-F4:sham operated rats,number F4

tag: NOR-F4:sham operated rats,number F4


title: PBMC-CRR-vs-BTB-infected-6

description: No additional information.


title: PBMC-CRR-vs-BTB-infected-2

description: No additional information.


title: PBMC-CRR-vs-control-5

description: No additional information.

tag: PBMC from a control non-infected animal cultured overnight and then used for total RNA extraction.


title: PBMC-CRR-vs-control-4

description: No additional information.


title: Carbon-limited anaerobic chemostat with benzoate -2

scan_protocol: scanning with the Affymetrix GeneArray Scanner.


title: PBMC-CRR-vs-control-6

description: No additional information.


title: PBMC-CRR-vs-control-2

description: No additional information.


title: PBMC-CRR-vs-BTB-infected-1

description: No additional information.


title: PBMC-CRR-vs-control-3

description: No additional information.


title: PBMC-CRR-vs-control-1

description: No additional information.

value: PBMC from eight M. bovis-infected cattle and five non-infected cattle. PBMC were stimulated with purified protein derivative (PPD) of Mycobacterium bovis (tuberculin) and sampled across a time course of T0 (no stimulation) T3 (3 hours post-stimulation) and T12 (12 hours post-stimulation). A total of 39 RNA samples were combined to form the common RNA reference (CRR) pool.


title: Nitrogen-limited Aerobic chemostat culture-3

hyb_protocol: Microarray analysis. Sampling of cells from chemostats and total RNA extraction was performed as previously described in Abbott et al. (2007). Probe preparation and hybridization to Affymetrix Genechip® microarrays were performed following Affymetrix instructions. The one-cycle eukaryotic target labelling assay was g of total RNA. The quality of total RNA, cDNA, cRNA andused, starting with 15  fragmented cRNA were checked using the Agilent Bioanalyzer 2100 (Agilent Technologies). Results for each growth condition were derived from three independent culture replicates. Abbott DA, Knijnenburg TA, de Poorter LM, Reinders MJ, Pronk JT, van Maris AJ. (2007) Generic and specific transcriptional responses to different weak organic acids in anaerobic chemostat cultures of Saccharomyces cerevisiae. FEMS Yeast Res. doi:10.1111/j.1567-1364.2007.00242.x

scan_protocol: Affymetrix scanner 3000

data_processing: GCOS

label: SAPE

source_name: Chemostat culture

label_protocol:
EukGE-ws2v4
Microarray analysis. Sampling of cells from chemostats and total RNA extraction was performed as previously described in Abbott et al. (2007). Probe preparation and hybridization to Affymetrix Genechip® microarrays were performed following Affymetrix instructions. The one-cycle eukaryotic target g of total RNA. The quality of totallabelling assay was used, starting with 15  RNA, cDNA, cRNA and fragmented cRNA were checked using the Agilent Bioanalyzer 2100 (Agilent Technologies). Results for each growth condition were derived from three independent culture replicates.
Abbott DA, Knijnenburg TA, de Poorter LM, Reinders MJ, Pronk JT, van Maris AJ. (2007) Generic and specific transcriptional responses to different weak organic acids in anaerobic chemostat cultures of Saccharomyces cerevisiae. FEMS Yeast Res. doi:10.1111/j.1567-1364.2007.00242.x

growth_protocol:
Chemostat Cultivation—Steady-state chemostat cultures were grown in Applikon laboratory fermentors of 1-liter working volume as described in detail elsewhere [van den Berg, M. A., de Jong-Gubbels, P., Kortland, C. J., van Dijken, J. P., Pronk, J. T., and Steensma, H. Y. (1996) J. Biol. Chem. 271, 28953–28959]. In brief, the cultures were fed with a defined mineral medium containing glucose as the growth-limiting nutrient [. Verduyn, C., Postma, E., Scheffers, W. A., and van Dijken, J. P. (1990) Microbiol.Rev. 58, 616–630]. The dilution rate (which equals the specific growth rate) in the steady-state cultures was 0.10 h_1, the temperature was 30 °C, and the culture pH was 5.0.Aerobic conditions were maintained by sparging the cultures with air (0.5 liter_min_1). The dissolved oxygen concentration, which was continuously monitored with an Ingold model 34-100-3002 probe, remained above 80% of air saturation.
The synthetic media composition was based on that described by Verduyn (1992). In all chemostats except for those limited by carbon, residual glucose concentration was targeted to 17 g.liter-1 in order to sustain glucose repression at the same level. The media composition for aerobic cultivations was the following: for nitrogen-limited cultivation, 1.0 g.liter-1 (NH4)2SO4, 3.0 g.liter-1 KH2PO4, 0.5 g.liter-1 MgSO4•7H2O, 59 g.liter-1 glucose and 4.5 mg.liter-1 ZnSO4•7H2O; for zinc-limited cultivation, 5.0 g.liter-1 (NH4)2SO4, 3.0 g.liter-1 KH2PO4, 0.5 g.liter-1 MgSO4•7H2O, 66 g.liter-1 glucose and 0.014 mg.liter-1 ZnSO4•7H2O.
van den Berg, M. A., P. de Jong-Gubbels, C. J. Kortland, J. P. van Dijken, J. T. Pronk, and H. Y. Steensma. 1996. The two acetyl-coenzyme A synthetases of Saccharomyces cerevisiae differ with respect to kinetic properties and transcriptional regulation. J. Biol. Chem. 271:28953-28959.
van Dijken, J. P., J. Bauer, L. Brambilla, P. Duboc, J. M. Francois, C. Gancedo, M. L. Giuseppin, J. J. Heijnen, M. Hoare, H. C. Lange, E. A. Madden, P. Niederberger, J. Nielsen, J. L. Parrou, T. Petit, D. Porro, M. Reuss, R. N. van, M. Rizzi, H. Y. Steensma, C. T. Verrips, J. Vindelov, and J. T. Pronk. 2000. An interlaboratory comparison of physiological and genetic properties of four Saccharomyces cerevisiae strains. Enzyme Microb. Technol. 26:706-714.
Verduyn, C., E. Postma, W. A. Scheffers, and J. P. van Dijken. 1990. Energetics of Saccharomyces cerevisiae in anaerobic glucose-limited chemostat cultures. J. Gen. Microbiol. 136:405-412.
Verduyn, C., E. Postma, W. A. Scheffers, and J. P. van Dijken. 1992. Effect of benzoic acid on metabolic fluxes in yeasts: a continuous-culture study on the regulation of respiration and alcoholic fermentation. Yeast 8:501-517.
Visser, W., W. A. Scheffers, Batenburg-van der Vegte WH, and J. P. van Dijken. 1990. Oxygen requirements of yeasts. Appl. Environ. Microbiol. 56:3785-3792.
Boer, V. M., J. H. de Winde, J. T. Pronk, and M. D. Piper. 2003. The genome-wide transcriptional responses of Saccharomyces cerevisiae grown on glucose in aerobic chemostat cultures limited for carbon, nitrogen, phosphorus, or sulfur. J. Biol. Chem. 278:3265-3274.
Ferea, T. L., D. Botstein, P. O. Brown, and R. F. Rosenzweig. 1999. Systematic changes in gene expression patterns following adaptive evolution in yeast. Proc. Natl. Acad. Sci. U. S. A 96:9721-9726.
Jansen, M. L., P. Daran-Lapujade, J. H. de Winde, M. D. Piper, and J. T. Pronk. 2004. Prolonged maltose-limited cultivation of Saccharomyces cerevisiae selects for cells with improved maltose affinity and hypersensitivity. Appl. Environ. Microbiol. 70:1956-1963.

treatment_protocol: Liquid nitrogen

tag: Strain and Maintenance—This study was performed with the prototrophic laboratory strain S. cerevisiae CEN.PK113-7D (MATa) [van Dijken, J. P., Bauer, J., Brambilla, L., Duboc, P., Francois, J. M., Gancedo, C., Giuseppin, M. L. F., Heinen, J. J., Hoare, M., Lange, H. C., Madden, E. A., Niederberger, P., Nielsen, J., Parrou, J. L., Petit, T., Porro, D., Reuss, M., van Riel, N., Rizzi, M., Steensma, H. Y., Verrips, C. T., Vindelov, J., and Pronk, J. T. (2000) Enz. Microb. Technol. 26, 706–714]


title: Nitorgen-limited Anaerobic chemostat culture-1

growth_protocol:
Chemostat Cultivation—Steady-state chemostat cultures were grown in Applikon laboratory fermentors of 1-liter working volume as described in detail elsewhere [van den Berg, M. A., de Jong-Gubbels, P., Kortland, C. J., van Dijken, J. P., Pronk, J. T., and Steensma, H. Y. (1996) J. Biol. Chem. 271, 28953–28959]. In brief, the cultures were fed with a defined mineral medium containing glucose as the growth-limiting nutrient [. Verduyn, C., Postma, E., Scheffers, W. A., and van Dijken, J. P. (1990) Microbiol.Rev. 58, 616–630]. The dilution rate (which equals the specific growth rate) in the steady-state cultures was 0.10 h_1, the temperature was 30 °C, and the culture pH was 5.0.Aerobic conditions were maintained by sparging the cultures with air (0.5 liter_min_1). The dissolved oxygen concentration, which was continuously monitored with an Ingold model 34-100-3002 probe, remained above 80% of air saturation.
The synthetic media composition was based on that described by Verduyn (1992). In all chemostats except for those limited by carbon, residual glucose concentration was targeted to 17 g.liter-1 in order to sustain glucose repression at the same level. For anaerobic cultivations, Tween 80 (420 mg.l-1) and ergosterol (10 mg.l-1) were supplemented to the media as described previously to compensate for the inability of S. cerevisiae to synthesise fatty acids in the absence of oxygen. These media contained the following components: for nitrogen-limited cultivation, 0.65 g.liter-1 (NH4)2SO4, 3.0 g.liter-1 KH2PO4, 0.5 g.liter-1 MgSO4•7H2O, 46 g.liter-1 glucose, and 4.5 mg.liter-1 ZnSO4•7H2O.
van den Berg, M. A., P. de Jong-Gubbels, C. J. Kortland, J. P. van Dijken, J. T. Pronk, and H. Y. Steensma. 1996. The two acetyl-coenzyme A synthetases of Saccharomyces cerevisiae differ with respect to kinetic properties and transcriptional regulation. J. Biol. Chem. 271:28953-28959.
van Dijken, J. P., J. Bauer, L. Brambilla, P. Duboc, J. M. Francois, C. Gancedo, M. L. Giuseppin, J. J. Heijnen, M. Hoare, H. C. Lange, E. A. Madden, P. Niederberger, J. Nielsen, J. L. Parrou, T. Petit, D. Porro, M. Reuss, R. N. van, M. Rizzi, H. Y. Steensma, C. T. Verrips, J. Vindelov, and J. T. Pronk. 2000. An interlaboratory comparison of physiological and genetic properties of four Saccharomyces cerevisiae strains. Enzyme Microb. Technol. 26:706-714.
Verduyn, C., E. Postma, W. A. Scheffers, and J. P. van Dijken. 1990. Energetics of Saccharomyces cerevisiae in anaerobic glucose-limited chemostat cultures. J. Gen. Microbiol. 136:405-412.
Verduyn, C., E. Postma, W. A. Scheffers, and J. P. van Dijken. 1992. Effect of benzoic acid on metabolic fluxes in yeasts: a continuous-culture study on the regulation of respiration and alcoholic fermentation. Yeast 8:501-517.
Visser, W., W. A. Scheffers, Batenburg-van der Vegte WH, and J. P. van Dijken. 1990. Oxygen requirements of yeasts. Appl. Environ. Microbiol. 56:3785-3792.
Boer, V. M., J. H. de Winde, J. T. Pronk, and M. D. Piper. 2003. The genome-wide transcriptional responses of Saccharomyces cerevisiae grown on glucose in aerobic chemostat cultures limited for carbon, nitrogen, phosphorus, or sulfur. J. Biol. Chem. 278:3265-3274.
Ferea, T. L., D. Botstein, P. O. Brown, and R. F. Rosenzweig. 1999. Systematic changes in gene expression patterns following adaptive evolution in yeast. Proc. Natl. Acad. Sci. U. S. A 96:9721-9726.
Jansen, M. L., P. Daran-Lapujade, J. H. de Winde, M. D. Piper, and J. T. Pronk. 2004. Prolonged maltose-limited cultivation of Saccharomyces cerevisiae selects for cells with improved maltose affinity and hypersensitivity. Appl. Environ. Microbiol. 70:1956-1963.

extract_protocol:
Microarray analysis. Sampling of cells from chemostats and total RNA extraction was performed as previously described in Abbott et al. (2007). Probe preparation and hybridization to Affymetrix Genechip® microarrays were performed following Affymetrix instructions. The one-cycle eukaryotic target labelling assay was g of total RNA. The quality of total RNA, cDNA, cRNA andused, starting with 15  fragmented cRNA were checked using the Agilent Bioanalyzer 2100 (Agilent Technologies). Results for each growth condition were derived from three independent culture replicates.
Abbott DA, Knijnenburg TA, de Poorter LM, Reinders MJ, Pronk JT, van Maris AJ. (2007) Generic and specific transcriptional responses to different weak organic acids in anaerobic chemostat cultures of Saccharomyces cerevisiae. FEMS Yeast Res. doi:10.1111/j.1567-1364.2007.00242.x


title: Nitrogen-limited Anaerobic chemostat culture-2


title: Nitrogen-limited Anaerobic chemostat culture-3


title: Carbon-limited Aerobic chemostat culture -1

growth_protocol:
Chemostat Cultivation—Steady-state chemostat cultures were grown in Applikon laboratory fermentors of 1-liter working volume as described in detail elsewhere [van den Berg, M. A., de Jong-Gubbels, P., Kortland, C. J., van Dijken, J. P., Pronk, J. T., and Steensma, H. Y. (1996) J. Biol. Chem. 271, 28953–28959]. In brief, the cultures were fed with a defined mineral medium containing glucose as the growth-limiting nutrient [. Verduyn, C., Postma, E., Scheffers, W. A., and van Dijken, J. P. (1990) Microbiol.Rev. 58, 616–630]. The dilution rate (which equals the specific growth rate) in the steady-state cultures was 0.10 h_1, the temperature was 30 °C, and the culture pH was 5.0.Aerobic conditions were maintained by sparging the cultures with air (0.5 liter_min_1). The dissolved oxygen concentration, which was continuously monitored with an Ingold model 34-100-3002 probe, remained above 80% of air saturation.
The synthetic media composition was based on that described by Verduyn (1992). The media composition for aerobic cultivations was the following: for carbon-limited cultivation, 5.0 g.liter-1 (NH4)2SO4, 3.0 g.liter-1 KH2PO4, 0.5 g.liter-1 MgSO4•7H2O, 7.5 g.liter-1 glucose and 4.5 mg.liter-1 ZnSO4•7H2O.
van den Berg, M. A., P. de Jong-Gubbels, C. J. Kortland, J. P. van Dijken, J. T. Pronk, and H. Y. Steensma. 1996. The two acetyl-coenzyme A synthetases of Saccharomyces cerevisiae differ with respect to kinetic properties and transcriptional regulation. J. Biol. Chem. 271:28953-28959.
van Dijken, J. P., J. Bauer, L. Brambilla, P. Duboc, J. M. Francois, C. Gancedo, M. L. Giuseppin, J. J. Heijnen, M. Hoare, H. C. Lange, E. A. Madden, P. Niederberger, J. Nielsen, J. L. Parrou, T. Petit, D. Porro, M. Reuss, R. N. van, M. Rizzi, H. Y. Steensma, C. T. Verrips, J. Vindelov, and J. T. Pronk. 2000. An interlaboratory comparison of physiological and genetic properties of four Saccharomyces cerevisiae strains. Enzyme Microb. Technol. 26:706-714.
Verduyn, C., E. Postma, W. A. Scheffers, and J. P. van Dijken. 1990. Energetics of Saccharomyces cerevisiae in anaerobic glucose-limited chemostat cultures. J. Gen. Microbiol. 136:405-412.
Verduyn, C., E. Postma, W. A. Scheffers, and J. P. van Dijken. 1992. Effect of benzoic acid on metabolic fluxes in yeasts: a continuous-culture study on the regulation of respiration and alcoholic fermentation. Yeast 8:501-517.
Visser, W., W. A. Scheffers, Batenburg-van der Vegte WH, and J. P. van Dijken. 1990. Oxygen requirements of yeasts. Appl. Environ. Microbiol. 56:3785-3792.
Boer, V. M., J. H. de Winde, J. T. Pronk, and M. D. Piper. 2003. The genome-wide transcriptional responses of Saccharomyces cerevisiae grown on glucose in aerobic chemostat cultures limited for carbon, nitrogen, phosphorus, or sulfur. J. Biol. Chem. 278:3265-3274.
Ferea, T. L., D. Botstein, P. O. Brown, and R. F. Rosenzweig. 1999. Systematic changes in gene expression patterns following adaptive evolution in yeast. Proc. Natl. Acad. Sci. U. S. A 96:9721-9726.
Jansen, M. L., P. Daran-Lapujade, J. H. de Winde, M. D. Piper, and J. T. Pronk. 2004. Prolonged maltose-limited cultivation of Saccharomyces cerevisiae selects for cells with improved maltose affinity and hypersensitivity. Appl. Environ. Microbiol. 70:1956-1963.


title: Nitrogen-limited Aerobic chemostat culture-2


title: NOR-F1

description: NOR-F1:sham operated rats, number F1

tag: NOR-F1:sham operated rats, number F1


title: Carbon-limited Aerobic chemostat culture-2


title: Carbon-limited Aerobic chemostat culture-3


title: Patient 15

city: Sydney

first: Leon

middle: Edward

last: Wescombe

state: New South Wales

address: Nepean Hospital, Derby Street, Penrith

department: Medicine

country: Australia

institute: University of Sydney

description: Total RNA with RNA Integrity Number (RIN) > 6.8 was reverse transcribed, amplified, and hybridised to Illumina HumanRef-8 BeadChips.

hyb_protocol: aRNA was reverse transcribed and hybridised to Illumina Sentrix HumanRef-8 following the manufacturer's protocols at the Westmead Millenium Institute, Sydney, Australia.

scan_protocol: Arrays were scanned and quantified using an Illumina BeadStation 500 GX.

data_processing: Data was processed using Illumina BeadStudio version 3.0.19 software using background subtraction, cubic spline normalisation, and Illumina custom differential expression error model.

source_name: thyroid

label_protocol: RNA integrity was analysed using an Agilent Bioanalyzer and each RNA sample showed a RIN number above 7.4. RNA samples were reverse transcribed, amplified and labelled following the Amino Allyl MessageAmpTM II aRNA Amplification Kit (Ambion Cat #1753).

extract_protocol: Thyroid tissue was collected from hyperthyroid patients post-thyroidectomy and stored in RNAlater solution for 24 hours at 4C, then swab-dried and stored at -70C. RNA was extracted from tissue by Promega Pronto Plus Indirect System (Cat #TM261) (techserv@promega.com).

tag: DISEASE STATE

value: hyperthyroidism - eye disease

tag: AGE

tag: SEX

value: Female

tag: BEADCHIP BARCODE

tag: BEADCHIP POSITION

value: C


title: Patient 18

value: H


title: Nitrogen-limited Aerobic chemostat culture-1


title: 13302316 - RA-H1 vs RC-H1

city: Evry

first: Frederique

last: Bitton

address: 2 rue Gaston Cremieux

country: France

institute: INRA

description: What are the transcriptomic plant responses to urea nitrogen supply ?

hyb_protocol: RC-H1 Cy5 / RA-H1 Cy3 : 30pmol. Hybridization Protocol: CATMA slides (Corning Microarray Technology, CORNING) are pretreated in the prehybridisation solution (1 % BSA, 0.1 SDS, 5X SSC ) at 42°C for 60 min. They are dipped a couple of times in distilled water at room temperature, then in isopropanol and dried immediately by compressed nitrogen stream. Slides were placed in Corning hybridization chambers with a 25x60 lifterslip and 10ul of distilled water for each groove. The target was diluted to a final volume of 60 µL as follows 15µl of purified, labeled cDNA, 15 µl of 4X Hybridization Buffer ( 20X SDS, 0.4 % SDS), 30 µL formamide. The target mixture is heated for 3 min at 95°C, put on ice for 30 sec and centrifuged to remove dust for 1 min. The target mixture was put on the chip as quickly as possible. The microarray is sealed in a chamber and submerged in a 42°C water bath for approximately 16 h. The microarray is washed for 4 min in 1xSSC, 0.2% SDS (42°C); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC (RT); dipped a few times in distilled water and dried immediately by compressed nitrogen stream.

scan_protocol: GenePix Pro 3.0, Cy3:pmt voltage 532nm,650V,laser power 100%, Cy5:635nm,pmt voltage 620V,laser power 100%

data_processing: The raw data comprised the logarithm of median feature pixel intensity at wavelength 635 nm (red) and 532 nm (green). No background was subtracted. An array-by-array normalization was performed to remove systematic biases. First, we excluded spots that were considered badly formed features by the experimenter (Flags=-100). Then we performed a global intensity-dependent normalization using the loess procedure (see Yang et al., 2002) to correct the dye bias. Finally, on each block, the log-ratio median is subtracted from each value of the log-ratio of the block to correct a print-tip effect on each metablock.

organism: Arabidopsis thaliana

label_protocol: labelling Cy3 and Cy5 indirect, amplification=yes, DNA 5 ug. Labelling protocol: 5 µg of aRNA (8µl) mixed with 2 µl of random nonamers 1µg/µl and 0.5 µl of RNAse Out 40 U/µl, denatured 10 min at 70 °C and chilled on ice. The followig components were added to the sample 4 µl of first strand buffer 5 X, 1 µl of 10 mM dNTP/2mM dCTP, 2 µl of 0.1 M DTT, 1.5 µl of Cy3-dCTP or Cy5-dCTP (Amersham Pharmacia Biotech, 25nmol tube, PA5502), 1 µl of SuperScript II RT 200U/µl. Then it was incubated at 42°C for 2.5 hours and chilled on ice. The sample was denaturated by adding 2 µl of NaOH 2.5M and incubated at 37 °C for exactly 15 min, then was added 10 µl of 2M MOPS and put on ice. The dyes were purified with the QIAquick PCR Purification Kit (QIAGEN).

growth_protocol: root - NH4NO3 0.5 mM MgSO4/KH2PO4 1 mM K2SO4 2.5 mM CaCl2 2.2 mM Fer Sequestrene 10 mg.L-1 Oligo 1 ml.L-1 hygro 80%	 Temp : Day 21degreeC/Night 17degreeC, light : Day 8 h/night 16h

extract_protocol: RC-H1:9ug. Grind samples in liquide nitrogen, Heat 500 µl phenol pH4+500 µl TLES at 80°C, homogenize samples in hot phenol+TLES, vortex 30 '', heat again at 80°C 1', add 500 µl chloroforme/isoamylalcool (24:1), vortex 30'', transfer in safelock 2 ml and put on ice, centrifuge 10' 15000 rpm 4°C, transfer supernatant in eppendorf and add the same volume of LiCl 4M (about 400 µl), precipitate on ice overnight at 4°C. Centrifuge 30' 14500 rpm 4°C, remove supernatant, resuspend the pellet in 250 µl RNase-free water, add 1/10 volume of sodium acetate 2.5M pH 5.6 and 2 volumes of 100% ethanol, precipitate overnight at - 20°C. Centrifuge 20' 15000 rpm 4°C, wash the RNA pellet with 500 µl of 70% ethanol, centrifuge 10' 15000 rpm 4°C, briefly dry the pellet (air dry for 5-10'), resuspend in 30 µl RNase-free water.

treatment_protocol: Name:C-Urea - compound based treatment - compound addition,urea:quantity 1mM time 7day . 7 days on : Media :	 Urea		1 mM MgSO4/KH2PO4	1 mM K2SO4		2.5 mM CaCl2		2.2 mM Fer Sequestrene	10 mg.L-1 Oligo		1 ml.L-1 hygrometry : 80% Temperature : Day 21degre Celsius/Night 17degre Celsius Light : 8 h/1h

tag: Arabidopsis thaliana (columbia)  - dev.stage (Boyes et al. Plant Cell 2001):boyes

extract_protocol: RA-H1:6ug. Grind samples in liquide nitrogen, Heat 500 µl phenol pH4+500 µl TLES at 80°C, homogenize samples in hot phenol+TLES, vortex 30 '', heat again at 80°C 1', add 500 µl chloroforme/isoamylalcool (24:1), vortex 30'', transfer in safelock 2 ml and put on ice, centrifuge 10' 15000 rpm 4°C, transfer supernatant in eppendorf and add the same volume of LiCl 4M (about 400 µl), precipitate on ice overnight at 4°C. Centrifuge 30' 14500 rpm 4°C, remove supernatant, resuspend the pellet in 250 µl RNase-free water, add 1/10 volume of sodium acetate 2.5M pH 5.6 and 2 volumes of 100% ethanol, precipitate overnight at - 20°C. Centrifuge 20' 15000 rpm 4°C, wash the RNA pellet with 500 µl of 70% ethanol, centrifuge 10' 15000 rpm 4°C, briefly dry the pellet (air dry for 5-10'), resuspend in 30 µl RNase-free water.

treatment_protocol: Name:A-Ammonitrate - compound based treatment - compound addition,ammonitrate:quantity 1mM time 7day . 7 days on : Media :	 NH4NO3		1 mM MgSO4/KH2PO4		1 mM K2SO4			2.5 mM CaCl2			2.2 mM Fer Sequestrene		10 mg.L-1 Oligo			1 ml.L-1 hygrometry : 80% Temperature : Day 21degre Celsius/Night 17degre Celsius Light : 8 h/1h


title: 13301734 - SC-H2 vs SA-H2

hyb_protocol: SA-H2 Cy5 / SC-H2 Cy3 : 30pmol. Hybridization Protocol: CATMA slides (Corning Microarray Technology, CORNING) are pretreated in the prehybridisation solution (1 % BSA, 0.1 SDS, 5X SSC ) at 42°C for 60 min. They are dipped a couple of times in distilled water at room temperature, then in isopropanol and dried immediately by compressed nitrogen stream. Slides were placed in Corning hybridization chambers with a 25x60 lifterslip and 10ul of distilled water for each groove. The target was diluted to a final volume of 60 µL as follows 15µl of purified, labeled cDNA, 15 µl of 4X Hybridization Buffer ( 20X SDS, 0.4 % SDS), 30 µL formamide. The target mixture is heated for 3 min at 95°C, put on ice for 30 sec and centrifuged to remove dust for 1 min. The target mixture was put on the chip as quickly as possible. The microarray is sealed in a chamber and submerged in a 42°C water bath for approximately 16 h. The microarray is washed for 4 min in 1xSSC, 0.2% SDS (42°C); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC (RT); dipped a few times in distilled water and dried immediately by compressed nitrogen stream.

extract_protocol: RC-H2:17ug. Grind samples in liquide nitrogen, Heat 500 µl phenol pH4+500 µl TLES at 80°C, homogenize samples in hot phenol+TLES, vortex 30 '', heat again at 80°C 1', add 500 µl chloroforme/isoamylalcool (24:1), vortex 30'', transfer in safelock 2 ml and put on ice, centrifuge 10' 15000 rpm 4°C, transfer supernatant in eppendorf and add the same volume of LiCl 4M (about 400 µl), precipitate on ice overnight at 4°C. Centrifuge 30' 14500 rpm 4°C, remove supernatant, resuspend the pellet in 250 µl RNase-free water, add 1/10 volume of sodium acetate 2.5M pH 5.6 and 2 volumes of 100% ethanol, precipitate overnight at - 20°C. Centrifuge 20' 15000 rpm 4°C, wash the RNA pellet with 500 µl of 70% ethanol, centrifuge 10' 15000 rpm 4°C, briefly dry the pellet (air dry for 5-10'), resuspend in 30 µl RNase-free water.

growth_protocol: leaf - NH4NO3 0.5 mM MgSO4/KH2PO4 1 mM K2SO4 2.5 mM CaCl2 2.2 mM Fer Sequestrene 10 mg.L-1 Oligo 1 ml.L-1 hygro 80%	 Temp : Day 21degreeC/Night 17degreeC, light : Day 8 h/night 16h

treatment_protocol: Name:B-Ammonitrate+Urea - compound based treatment - compound addition,ammonitrate+urea:quantity 1mM time 7day . 7 days on : Media :	 NH4NO3		0.5 mM Urea		0.5 mM MgSO4/KH2PO4	1 mM K2SO4		2.5 mM CaCl2		2.2 mM Fer Sequestrene	10 mg.L-1 Oligo		1 ml.L-1 hygrometry : 80% Temperature : Day 21degre Celsius/Night 17degre Celsius Light : 8 h/1h


title: 13301735 - SA-H2 vs SC-H2

hyb_protocol: SC-H2 Cy5 / SA-H2 Cy3 : 30pmol. Hybridization Protocol: CATMA slides (Corning Microarray Technology, CORNING) are pretreated in the prehybridisation solution (1 % BSA, 0.1 SDS, 5X SSC ) at 42°C for 60 min. They are dipped a couple of times in distilled water at room temperature, then in isopropanol and dried immediately by compressed nitrogen stream. Slides were placed in Corning hybridization chambers with a 25x60 lifterslip and 10ul of distilled water for each groove. The target was diluted to a final volume of 60 µL as follows 15µl of purified, labeled cDNA, 15 µl of 4X Hybridization Buffer ( 20X SDS, 0.4 % SDS), 30 µL formamide. The target mixture is heated for 3 min at 95°C, put on ice for 30 sec and centrifuged to remove dust for 1 min. The target mixture was put on the chip as quickly as possible. The microarray is sealed in a chamber and submerged in a 42°C water bath for approximately 16 h. The microarray is washed for 4 min in 1xSSC, 0.2% SDS (42°C); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC (RT); dipped a few times in distilled water and dried immediately by compressed nitrogen stream.


title: Patient 12

value: F


title: 13301339 - RA-H2 vs RB-H2

hyb_protocol: RB-H2 Cy5 / RA-H2 Cy3 : 30pmol. Hybridization Protocol: CATMA slides (Corning Microarray Technology, CORNING) are pretreated in the prehybridisation solution (1 % BSA, 0.1 SDS, 5X SSC ) at 42°C for 60 min. They are dipped a couple of times in distilled water at room temperature, then in isopropanol and dried immediately by compressed nitrogen stream. Slides were placed in Corning hybridization chambers with a 25x60 lifterslip and 10ul of distilled water for each groove. The target was diluted to a final volume of 60 µL as follows 15µl of purified, labeled cDNA, 15 µl of 4X Hybridization Buffer ( 20X SDS, 0.4 % SDS), 30 µL formamide. The target mixture is heated for 3 min at 95°C, put on ice for 30 sec and centrifuged to remove dust for 1 min. The target mixture was put on the chip as quickly as possible. The microarray is sealed in a chamber and submerged in a 42°C water bath for approximately 16 h. The microarray is washed for 4 min in 1xSSC, 0.2% SDS (42°C); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC (RT); dipped a few times in distilled water and dried immediately by compressed nitrogen stream.

extract_protocol: RB-H2:12ug. Grind samples in liquide nitrogen, Heat 500 µl phenol pH4+500 µl TLES at 80°C, homogenize samples in hot phenol+TLES, vortex 30 '', heat again at 80°C 1', add 500 µl chloroforme/isoamylalcool (24:1), vortex 30'', transfer in safelock 2 ml and put on ice, centrifuge 10' 15000 rpm 4°C, transfer supernatant in eppendorf and add the same volume of LiCl 4M (about 400 µl), precipitate on ice overnight at 4°C. Centrifuge 30' 14500 rpm 4°C, remove supernatant, resuspend the pellet in 250 µl RNase-free water, add 1/10 volume of sodium acetate 2.5M pH 5.6 and 2 volumes of 100% ethanol, precipitate overnight at - 20°C. Centrifuge 20' 15000 rpm 4°C, wash the RNA pellet with 500 µl of 70% ethanol, centrifuge 10' 15000 rpm 4°C, briefly dry the pellet (air dry for 5-10'), resuspend in 30 µl RNase-free water.

extract_protocol: RA-H2:20ug. Grind samples in liquide nitrogen, Heat 500 µl phenol pH4+500 µl TLES at 80°C, homogenize samples in hot phenol+TLES, vortex 30 '', heat again at 80°C 1', add 500 µl chloroforme/isoamylalcool (24:1), vortex 30'', transfer in safelock 2 ml and put on ice, centrifuge 10' 15000 rpm 4°C, transfer supernatant in eppendorf and add the same volume of LiCl 4M (about 400 µl), precipitate on ice overnight at 4°C. Centrifuge 30' 14500 rpm 4°C, remove supernatant, resuspend the pellet in 250 µl RNase-free water, add 1/10 volume of sodium acetate 2.5M pH 5.6 and 2 volumes of 100% ethanol, precipitate overnight at - 20°C. Centrifuge 20' 15000 rpm 4°C, wash the RNA pellet with 500 µl of 70% ethanol, centrifuge 10' 15000 rpm 4°C, briefly dry the pellet (air dry for 5-10'), resuspend in 30 µl RNase-free water.


title: 13365187 - RC-H2 vs RA-H2

hyb_protocol: RA-H2 Cy5 / RC-H2 Cy3 : 30pmol. Hybridization Protocol: CATMA slides (Corning Microarray Technology, CORNING) are pretreated in the prehybridisation solution (1 % BSA, 0.1 SDS, 5X SSC ) at 42°C for 60 min. They are dipped a couple of times in distilled water at room temperature, then in isopropanol and dried immediately by compressed nitrogen stream. Slides were placed in Corning hybridization chambers with a 25x60 lifterslip and 10ul of distilled water for each groove. The target was diluted to a final volume of 60 µL as follows 15µl of purified, labeled cDNA, 15 µl of 4X Hybridization Buffer ( 20X SDS, 0.4 % SDS), 30 µL formamide. The target mixture is heated for 3 min at 95°C, put on ice for 30 sec and centrifuged to remove dust for 1 min. The target mixture was put on the chip as quickly as possible. The microarray is sealed in a chamber and submerged in a 42°C water bath for approximately 16 h. The microarray is washed for 4 min in 1xSSC, 0.2% SDS (42°C); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC (RT); dipped a few times in distilled water and dried immediately by compressed nitrogen stream.


title: Patient 14

value: hyperthyroidism + eye disease

value: B


title: 13365188 - RA-H2 vs RC-H2

hyb_protocol: RC-H2 Cy5 / RA-H2 Cy3 : 30pmol. Hybridization Protocol: CATMA slides (Corning Microarray Technology, CORNING) are pretreated in the prehybridisation solution (1 % BSA, 0.1 SDS, 5X SSC ) at 42°C for 60 min. They are dipped a couple of times in distilled water at room temperature, then in isopropanol and dried immediately by compressed nitrogen stream. Slides were placed in Corning hybridization chambers with a 25x60 lifterslip and 10ul of distilled water for each groove. The target was diluted to a final volume of 60 µL as follows 15µl of purified, labeled cDNA, 15 µl of 4X Hybridization Buffer ( 20X SDS, 0.4 % SDS), 30 µL formamide. The target mixture is heated for 3 min at 95°C, put on ice for 30 sec and centrifuged to remove dust for 1 min. The target mixture was put on the chip as quickly as possible. The microarray is sealed in a chamber and submerged in a 42°C water bath for approximately 16 h. The microarray is washed for 4 min in 1xSSC, 0.2% SDS (42°C); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC (RT); dipped a few times in distilled water and dried immediately by compressed nitrogen stream.


title: Patient 16

value: D


title: Patient 17

value: G


title: 13302315 - RC-H1 vs RA-H1

hyb_protocol: RA-H1 Cy5 / RC-H1 Cy3 : 30pmol. Hybridization Protocol: CATMA slides (Corning Microarray Technology, CORNING) are pretreated in the prehybridisation solution (1 % BSA, 0.1 SDS, 5X SSC ) at 42°C for 60 min. They are dipped a couple of times in distilled water at room temperature, then in isopropanol and dried immediately by compressed nitrogen stream. Slides were placed in Corning hybridization chambers with a 25x60 lifterslip and 10ul of distilled water for each groove. The target was diluted to a final volume of 60 µL as follows 15µl of purified, labeled cDNA, 15 µl of 4X Hybridization Buffer ( 20X SDS, 0.4 % SDS), 30 µL formamide. The target mixture is heated for 3 min at 95°C, put on ice for 30 sec and centrifuged to remove dust for 1 min. The target mixture was put on the chip as quickly as possible. The microarray is sealed in a chamber and submerged in a 42°C water bath for approximately 16 h. The microarray is washed for 4 min in 1xSSC, 0.2% SDS (42°C); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC (RT); dipped a few times in distilled water and dried immediately by compressed nitrogen stream.


title: Patient 13

value: A


title: NSCB_CGH_390K_HSF1_1

type: genomic

city: Madison

first: Melodee

last: Patterson

state: WI

address: 510 Charmany Dr Ste 58 & 59

institute: WiCell

description: This CGH experiment is designed to detect deletion, insertation, amplication, etc. in the sample relative to the reference.

hyb_protocol: 1. Incubate at 95°C (in heat block), 5 min., spin briefly 2. Place at 42°C (in Maui machine), 5-15 min. 3. Prepare microarray: A)pre-heat array on Maui station B) put array into array-holder (with number facing up going in first) carefully remove glue-sticker from lid and put lid onto array C) take array w/ lid out of array-holder thoroughly attach lid using thick slide place array onto Maui station, approx. 2 min. make sure lid is completely attached D) Add 40 µl hyb solution onto microarray and allow to settle (approx. 15 sec.), dry lid thoroughly 4. Place stickers onto lid, press down on both at the same time, make sure are thoroughly attached 5. Squeeze bubbles away from center of array 6. Wet humid cover and place on Maui station 7. Incubate on Maui at 42°C overnight (17 hours) 8. Remove microarray w/lid from Maui and put in array-holder in Wash 1 (at 42°C) 9. Peel lid off chip while submerged 10. Incubate 10-15 sec. w/ gentle agitation 11. Transfer slide into a slide rack in a new container of Wash 1 (r.t.) Incubate 2 min. w/ gentle agitation 12. Transfer to Wash 2 13. Incubate 1 min. w/ gentle agitation 14. Transfer to Wash 3 15. Incubate 15 sec. w/ gentle agitation 16. Spin approx. 1 min. in Array-Go-Round (place arrays such that numbers are facing to the right when rack is placed in front part of wheel) (the rod on slide holder should be on the inside) 17. Blow off any residual liquid 18. Keep slides protected from light and scan as soon as possible

scan_protocol: NimbleGen scan protocol

data_processing:
1. Image extraction:
NimbleScan provided by NimbleGen is used to extract signal data from image files
(*.TFF).   Signals from the scan for Cy3 (green channel) are saved in a file named as chipID_532.pair while signals from the scan for Cy5 (red channel) are saved in a file named as chipID_635.pair.
2. Normalization:
Signals from Cy3/Cy5 channels are transformed by logarithms to base 2 and normalized with loess or qspline (Workman et al., 2002) algorithms by R to resolve the systematic errors and bias introduced by the microarray experimental platform. The log ratios of the signals are obtained by subtracting the log transformed signals from one channel by the corresponding ones from another channel.
3. Data smoothing and break-points identification:
Ratios of the normalized data are plotted by the corresponding genome locations. The data are smoothed by an adaptive weights smoothing algorithm (GLAD R-package, Wang et al., 2005), break-points are identified by a binary segmentation algorithm (DNAcopy R-package, Olshen et al., 2004) or by the segmentation algorithm available within Nimbelgen’s Nimblescan software.
4. Data visualization:
CGH results can be saved as SignalMap GFF files and can be visualized by SignalMap provided by NimbleGen. CGH results can also be visualized by other software, such as aCGHViewer (Shankar et al., 2006), CHARMView (Myers et al., 2005), etc. However, different software requires different input format.

molecule: genomic DNA

label_protocol:
1. Dry the sample in the Speed Vac set at 30°C.
2. Resuspend the sample in 4.5μl 0.1 M Na2CO3, pH 9.0.
3. Resuspend the Cy3 NHS ester in 4.5μl DMSO
4. Add Cy3 NHS ester to sample
5. Incubate at 22°C in a heat block for 1 hour. Protect the heat block and sample from light.
6. Purify the sample using a Qiaquick PCR purification Kit:
A) Add 35μl 100 mM NaOAc, pH 5.2 to the sample.
B) Add 250μl PB buffer to the sample and load the sample on the column.
C) Spin at 13.000 rpm for 1 minute and remove the flow through.
D) Add 750μl PE buffer to the column.
E) Spin at 13.000 rpm for 1 minute and remove the flow through.
F) Spin at 13.000 rpm for 1 minute.
G) Transfer the column to new tube.
H) Add 30μl H20 and incubate for 1 minute on the column.
I) Elute once by centrifuging at 13.000 rpm for 1 minute.

extract_protocol:
1. Determine When to Harvest hES cells for DNA Extraction
A) At least 1x10^6 hES cells should be harvested for DNA extraction, depending on the quantity
required.
B) Yields of genomic DNA will vary from sample to sample depending on the amount and type of cells processed. In addition, the quality of starting material will affect DNA yield.
C) Estimated yield for hES cells: 5pg of DNA per cell (5μg DNA per 10^6 cells).  One well of a 6-well plate will normally have between 1-4x106 hES cells by day 5 after passage.

2. Trypsinize the Cells

A) The following steps should be performed in a sterile biosafety cabinet while wearing personal protective equipment.
B) Add 1.5ml of 1X trypsin-EDTA working solution (pre-warmed to 37oC) to each well of a 6- well plate.
C) Return the plate to the 37C incubator for 2-3 minutes.
D) Remove the plate from the incubator and observe cell detachment with an inverted phase contrast microscope.  Proceed to next step when colony edges begin to lift off the plate.
E) Using a sterile glass serological pipet, gently pipet up and down to detach the cells and break up the cell clumps.
F) Pool the detached cells in a 15ml centrifuge tube containing 5ml of trypsin stop solution within 5-7 minutes of adding the trypsin-EDTA solution.
G) Gently pipet the solution up and down to mix thoroughly.
H) Centrifuge the tube for 5 minutes at 1000 RPM.
3. RNA Digestion
A) Aspirate the supernatant and gently flick the tube until the pellet is resuspended.
B) Slowly add 200μl PBS to the cells while tapping gently on the tube to ensure good mixing.
C) Add 4μl RNase A (100mg/ml) and incubate at room temperature (RT) for 2 minutes.

4. Protein Digestion
A) Add 20μl Proteinase K and 200μl Buffer AL to the sample, mixing thoroughly by
vortexing. It is important that the solution is immediately and thoroughly mixed by
vortexing and pipetting to yield a homogenous solution.
NOTE: Do not add proteinase K directly to buffer AL.
B) Incubate the cell solution in a 70°C waterbath for 10 minutes.

5. DNA Precipitation and Separation of Cellular Components
A) Add 200μl ethanol (96-100%) to the sample, and mix thoroughly by vortexing.
B) A white precipitate may form on addition of ethanol.  This precipitate does not interfere with the DNeasy procedure.
C) Pipet the mixture (including all of the precipitate) into the DNeasy Mini spin column placed in a 2ml collection tube.  Centrifuge at ≥6000 x g (8000 rpm) for 1 minute.  Discard flowthrough and collection tube.
NOTE: In order to obtain optimum DNA yield and quality, it is important not to overload
the spin column. The maximum number of hES cells that should be used in a DNA
extraction is 5x106 (normally, 2 wells of a 6-well plate).  For larger amounts of starting material use multiple spin columns.

6. Wash the DNA
A) Place the DNeasy spin column in a new collection tube and add 500μl of Buffer AW1.
B) Centrifuge at ≥6000 x g (8000rpm) for 1 minute. Discard flow-through and collection tube.
C)Place the DNeasy spin column in a new collection tube and add 500μl of buffer AW2.
D) Centrifuge at 20,000 x g (14,000 rpm) for 3 minutes to dry the DNeasy membrane. Discard flow-through and collection tube.
NOTE: It is important to dry the membrane of the DNeasy Mini spin column, since
residual ethanol may interfere with subsequent reactions. This centrifugation step ensures that no residual ethanol will be carried over during the following elution.
E) Following the centrifugation step, remove the DNeasy Mini spin column carefully so the column does not come into contact with the flow-through, since this will result in carryover of ethanol. If carryover of ethanol occurs, place the spin column in a new collection tube and centrifuge again for 1 minute at 20,000 x g (14,000 rpm). Again discard the flow-through and collection tube.
7. Elution
A) Place the DNeasy Mini spin column in a clean 1.5ml or 2ml microcentrifuge tube and pipet 50-200μl Buffer AE directly onto the DNeasy membrane.  Incubate the column at room temperature for 1 minute and then centrifuge for 1 minute at ≥6000 x g (8000rpm) to elute.
NOTE: Elution with less than 200μl of Buffer AE increases the final DNA concentration in the elute, but also decreases the overall DNA yield.
B) Repeat elution step as described in 7 A).
NOTE: A new microcentrifuge tube can be used for the second elution step to prevent dilution of the first elute. Alternatively, to combine the two elutes, the microcentrifuge tube from step 4.7.1 can be reused for the second elution step.
NOTE:  More than 200μl should not be eluted into a 1.5 ml microcentrifuge tube because the DNeasy spin column will come into contact with the elute.

tag: cell line

value: human embryonic stem (hES) cell line H9 [reference]


title: Patient 9


title: Patient 10


title: NSCB_CGH_390K_ES03_68

value: DNA001 (reference)


title: Patient 11

value: E


title: NSCB_CGH_390K_WA09_24

data_processing:
1. Image extraction:
NimbleScan provided by NimbleGen is used to extract signal data from image files
(*.TFF). Signals from the scan for Cy3 (green channel) are saved in a file named as chipID_532.pair while signals from the scan for Cy5 (red channel) are saved in a file named as chipID_635.pair.
2. Normalization:
Signals from Cy3/Cy5 channels are transformed by logarithms to base 2 and normalized with loess or qspline (Workman et al., 2002) algorithms by R to resolve the systematic errors and bias introduced by the microarray experimental platform. The log ratios of the signals are obtained by subtracting the log transformed signals from one channel by the corresponding ones from another channel.
3. Data smoothing and break-points identification:
Ratios of the normalized data are plotted by the corresponding genome locations. The data are smoothed by an adaptive weights smoothing algorithm (GLAD R-package, Wang et al., 2005), break-points are identified by a binary segmentation algorithm (DNAcopy R-package, Olshen et al., 2004) or by the segmentation algorithm available within Nimbelgen’s Nimblescan software.
4. Data visualization:
CGH results can be saved as SignalMap GFF files and can be visualized by SignalMap provided by NimbleGen. CGH results can also be visualized by other software, such as aCGHViewer (Shankar et al., 2006), CHARMView (Myers et al., 2005), etc. However, different software requires different input format.

extract_protocol:
1. Determine When to Harvest hES cells for DNA Extraction
A) At least 1x10^6 hES cells should be harvested for DNA extraction, depending on the quantity
required.
B) Yields of genomic DNA will vary from sample to sample depending on the amount and type of cells processed. In addition, the quality of starting material will affect DNA yield.
C) Estimated yield for hES cells: 5pg of DNA per cell (5μg DNA per 10^6 cells). One well of a 6-well plate will normally have between 1-4x106 hES cells by day 5 after passage.
2. Trypsinize the Cells
A) The following steps should be performed in a sterile biosafety cabinet while wearing personal protective equipment.
B) Add 1.5ml of 1X trypsin-EDTA working solution (pre-warmed to 37oC) to each well of a 6- well plate.
C) Return the plate to the 37C incubator for 2-3 minutes.
D) Remove the plate from the incubator and observe cell detachment with an inverted phase contrast microscope. Proceed to next step when colony edges begin to lift off the plate.
E) Using a sterile glass serological pipet, gently pipet up and down to detach the cells and break up the cell clumps.
F) Pool the detached cells in a 15ml centrifuge tube containing 5ml of trypsin stop solution within 5-7 minutes of adding the trypsin-EDTA solution.
G) Gently pipet the solution up and down to mix thoroughly.
H) Centrifuge the tube for 5 minutes at 1000 RPM.
3. RNA Digestion
A) Aspirate the supernatant and gently flick the tube until the pellet is resuspended.
B) Slowly add 200μl PBS to the cells while tapping gently on the tube to ensure good mixing.
C) Add 4μl RNase A (100mg/ml) and incubate at room temperature (RT) for 2 minutes.
4. Protein Digestion
A) Add 20μl Proteinase K and 200μl Buffer AL to the sample, mixing thoroughly by
vortexing. It is important that the solution is immediately and thoroughly mixed by
vortexing and pipetting to yield a homogenous solution.
NOTE: Do not add proteinase K directly to buffer AL.
B) Incubate the cell solution in a 70°C waterbath for 10 minutes.
5. DNA Precipitation and Separation of Cellular Components
A) Add 200μl ethanol (96-100%) to the sample, and mix thoroughly by vortexing.
B) A white precipitate may form on addition of ethanol. This precipitate does not interfere with the DNeasy procedure.
C) Pipet the mixture (including all of the precipitate) into the DNeasy Mini spin column placed in a 2ml collection tube. Centrifuge at ≥6000 x g (8000 rpm) for 1 minute. Discard flowthrough and collection tube.
NOTE: In order to obtain optimum DNA yield and quality, it is important not to overload
the spin column. The maximum number of hES cells that should be used in a DNA
extraction is 5x106 (normally, 2 wells of a 6-well plate). For larger amounts of starting material use multiple spin columns.
6. Wash the DNA
A) Place the DNeasy spin column in a new collection tube and add 500μl of Buffer AW1.
B) Centrifuge at ≥6000 x g (8000rpm) for 1 minute. Discard flow-through and collection tube.
C)Place the DNeasy spin column in a new collection tube and add 500μl of buffer AW2.
D) Centrifuge at 20,000 x g (14,000 rpm) for 3 minutes to dry the DNeasy membrane. Discard flow-through and collection tube.
NOTE: It is important to dry the membrane of the DNeasy Mini spin column, since
residual ethanol may interfere with subsequent reactions. This centrifugation step ensures that no residual ethanol will be carried over during the following elution.
E) Following the centrifugation step, remove the DNeasy Mini spin column carefully so the column does not come into contact with the flow-through, since this will result in carryover of ethanol. If carryover of ethanol occurs, place the spin column in a new collection tube and centrifuge again for 1 minute at 20,000 x g (14,000 rpm). Again discard the flow-through and collection tube.
7. Elution
A) Place the DNeasy Mini spin column in a clean 1.5ml or 2ml microcentrifuge tube and pipet 50-200μl Buffer AE directly onto the DNeasy membrane. Incubate the column at room temperature for 1 minute and then centrifuge for 1 minute at ≥6000 x g (8000rpm) to elute.
NOTE: Elution with less than 200μl of Buffer AE increases the final DNA concentration in the elute, but also decreases the overall DNA yield.
B) Repeat elution step as described in 7 A).
NOTE: A new microcentrifuge tube can be used for the second elution step to prevent dilution of the first elute. Alternatively, to combine the two elutes, the microcentrifuge tube from step 4.7.1 can be reused for the second elution step.
NOTE: More than 200μl should not be eluted into a 1.5 ml microcentrifuge tube because the DNeasy spin column will come into contact with the elute.

value: WA09_24 embryonic stem cell


title: 13281479 - SB-H2 vs SA-H2

hyb_protocol: SA-H2 Cy5 / SB-H2 Cy3 : 30pmol. Hybridization Protocol: CATMA slides (Corning Microarray Technology, CORNING) are pretreated in the prehybridisation solution (1 % BSA, 0.1 SDS, 5X SSC ) at 42°C for 60 min. They are dipped a couple of times in distilled water at room temperature, then in isopropanol and dried immediately by compressed nitrogen stream. Slides were placed in Corning hybridization chambers with a 25x60 lifterslip and 10ul of distilled water for each groove. The target was diluted to a final volume of 60 µL as follows 15µl of purified, labeled cDNA, 15 µl of 4X Hybridization Buffer ( 20X SDS, 0.4 % SDS), 30 µL formamide. The target mixture is heated for 3 min at 95°C, put on ice for 30 sec and centrifuged to remove dust for 1 min. The target mixture was put on the chip as quickly as possible. The microarray is sealed in a chamber and submerged in a 42°C water bath for approximately 16 h. The microarray is washed for 4 min in 1xSSC, 0.2% SDS (42°C); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC (RT); dipped a few times in distilled water and dried immediately by compressed nitrogen stream.


title: 13302403 - RB-H2 vs RA-H2

hyb_protocol: RA-H2 Cy5 / RB-H2 Cy3 : 30pmol. Hybridization Protocol: CATMA slides (Corning Microarray Technology, CORNING) are pretreated in the prehybridisation solution (1 % BSA, 0.1 SDS, 5X SSC ) at 42°C for 60 min. They are dipped a couple of times in distilled water at room temperature, then in isopropanol and dried immediately by compressed nitrogen stream. Slides were placed in Corning hybridization chambers with a 25x60 lifterslip and 10ul of distilled water for each groove. The target was diluted to a final volume of 60 µL as follows 15µl of purified, labeled cDNA, 15 µl of 4X Hybridization Buffer ( 20X SDS, 0.4 % SDS), 30 µL formamide. The target mixture is heated for 3 min at 95°C, put on ice for 30 sec and centrifuged to remove dust for 1 min. The target mixture was put on the chip as quickly as possible. The microarray is sealed in a chamber and submerged in a 42°C water bath for approximately 16 h. The microarray is washed for 4 min in 1xSSC, 0.2% SDS (42°C); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC (RT); dipped a few times in distilled water and dried immediately by compressed nitrogen stream.


title: 13279242 - SB-H1 vs SA-H1

hyb_protocol: SA-H1 Cy5 / SB-H1 Cy3 : 30pmol. Hybridization Protocol: CATMA slides (Corning Microarray Technology, CORNING) are pretreated in the prehybridisation solution (1 % BSA, 0.1 SDS, 5X SSC ) at 42°C for 60 min. They are dipped a couple of times in distilled water at room temperature, then in isopropanol and dried immediately by compressed nitrogen stream. Slides were placed in Corning hybridization chambers with a 25x60 lifterslip and 10ul of distilled water for each groove. The target was diluted to a final volume of 60 µL as follows 15µl of purified, labeled cDNA, 15 µl of 4X Hybridization Buffer ( 20X SDS, 0.4 % SDS), 30 µL formamide. The target mixture is heated for 3 min at 95°C, put on ice for 30 sec and centrifuged to remove dust for 1 min. The target mixture was put on the chip as quickly as possible. The microarray is sealed in a chamber and submerged in a 42°C water bath for approximately 16 h. The microarray is washed for 4 min in 1xSSC, 0.2% SDS (42°C); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC (RT); dipped a few times in distilled water and dried immediately by compressed nitrogen stream.

extract_protocol: SA-H1:12ug. Grind samples in liquide nitrogen, Heat 500 µl phenol pH4+500 µl TLES at 80°C, homogenize samples in hot phenol+TLES, vortex 30 '', heat again at 80°C 1', add 500 µl chloroforme/isoamylalcool (24:1), vortex 30'', transfer in safelock 2 ml and put on ice, centrifuge 10' 15000 rpm 4°C, transfer supernatant in eppendorf and add the same volume of LiCl 4M (about 400 µl), precipitate on ice overnight at 4°C. Centrifuge 30' 14500 rpm 4°C, remove supernatant, resuspend the pellet in 250 µl RNase-free water, add 1/10 volume of sodium acetate 2.5M pH 5.6 and 2 volumes of 100% ethanol, precipitate overnight at - 20°C. Centrifuge 20' 15000 rpm 4°C, wash the RNA pellet with 500 µl of 70% ethanol, centrifuge 10' 15000 rpm 4°C, briefly dry the pellet (air dry for 5-10'), resuspend in 30 µl RNase-free water.

extract_protocol: SB-H1:13ug. Grind samples in liquide nitrogen, Heat 500 µl phenol pH4+500 µl TLES at 80°C, homogenize samples in hot phenol+TLES, vortex 30 '', heat again at 80°C 1', add 500 µl chloroforme/isoamylalcool (24:1), vortex 30'', transfer in safelock 2 ml and put on ice, centrifuge 10' 15000 rpm 4°C, transfer supernatant in eppendorf and add the same volume of LiCl 4M (about 400 µl), precipitate on ice overnight at 4°C. Centrifuge 30' 14500 rpm 4°C, remove supernatant, resuspend the pellet in 250 µl RNase-free water, add 1/10 volume of sodium acetate 2.5M pH 5.6 and 2 volumes of 100% ethanol, precipitate overnight at - 20°C. Centrifuge 20' 15000 rpm 4°C, wash the RNA pellet with 500 µl of 70% ethanol, centrifuge 10' 15000 rpm 4°C, briefly dry the pellet (air dry for 5-10'), resuspend in 30 µl RNase-free water.


title: Patient 8


title: 13279237 - RB-H1 vs RA-H1

hyb_protocol: RA-H1 Cy5 / RB-H1 Cy3 : 30pmol. Hybridization Protocol: CATMA slides (Corning Microarray Technology, CORNING) are pretreated in the prehybridisation solution (1 % BSA, 0.1 SDS, 5X SSC ) at 42°C for 60 min. They are dipped a couple of times in distilled water at room temperature, then in isopropanol and dried immediately by compressed nitrogen stream. Slides were placed in Corning hybridization chambers with a 25x60 lifterslip and 10ul of distilled water for each groove. The target was diluted to a final volume of 60 µL as follows 15µl of purified, labeled cDNA, 15 µl of 4X Hybridization Buffer ( 20X SDS, 0.4 % SDS), 30 µL formamide. The target mixture is heated for 3 min at 95°C, put on ice for 30 sec and centrifuged to remove dust for 1 min. The target mixture was put on the chip as quickly as possible. The microarray is sealed in a chamber and submerged in a 42°C water bath for approximately 16 h. The microarray is washed for 4 min in 1xSSC, 0.2% SDS (42°C); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC (RT); dipped a few times in distilled water and dried immediately by compressed nitrogen stream.

extract_protocol: RB-H1:7ug. Grind samples in liquide nitrogen, Heat 500 µl phenol pH4+500 µl TLES at 80°C, homogenize samples in hot phenol+TLES, vortex 30 '', heat again at 80°C 1', add 500 µl chloroforme/isoamylalcool (24:1), vortex 30'', transfer in safelock 2 ml and put on ice, centrifuge 10' 15000 rpm 4°C, transfer supernatant in eppendorf and add the same volume of LiCl 4M (about 400 µl), precipitate on ice overnight at 4°C. Centrifuge 30' 14500 rpm 4°C, remove supernatant, resuspend the pellet in 250 µl RNase-free water, add 1/10 volume of sodium acetate 2.5M pH 5.6 and 2 volumes of 100% ethanol, precipitate overnight at - 20°C. Centrifuge 20' 15000 rpm 4°C, wash the RNA pellet with 500 µl of 70% ethanol, centrifuge 10' 15000 rpm 4°C, briefly dry the pellet (air dry for 5-10'), resuspend in 30 µl RNase-free water.


title: Patient 7


title: NSCB_CGH_390K_HSF1

source_name: HSF1 Genomic DNA

value: DNA003 (reference)


title: 13279238 - RA-H1 vs RB-H1

hyb_protocol: RB-H1 Cy5 / RA-H1 Cy3 : 30pmol. Hybridization Protocol: CATMA slides (Corning Microarray Technology, CORNING) are pretreated in the prehybridisation solution (1 % BSA, 0.1 SDS, 5X SSC ) at 42°C for 60 min. They are dipped a couple of times in distilled water at room temperature, then in isopropanol and dried immediately by compressed nitrogen stream. Slides were placed in Corning hybridization chambers with a 25x60 lifterslip and 10ul of distilled water for each groove. The target was diluted to a final volume of 60 µL as follows 15µl of purified, labeled cDNA, 15 µl of 4X Hybridization Buffer ( 20X SDS, 0.4 % SDS), 30 µL formamide. The target mixture is heated for 3 min at 95°C, put on ice for 30 sec and centrifuged to remove dust for 1 min. The target mixture was put on the chip as quickly as possible. The microarray is sealed in a chamber and submerged in a 42°C water bath for approximately 16 h. The microarray is washed for 4 min in 1xSSC, 0.2% SDS (42°C); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC (RT); dipped a few times in distilled water and dried immediately by compressed nitrogen stream.


title: 13281456 - SC-H1 vs SA-H1

hyb_protocol: SA-H1 Cy5 / SC-H1 Cy3 : 30pmol. Hybridization Protocol: CATMA slides (Corning Microarray Technology, CORNING) are pretreated in the prehybridisation solution (1 % BSA, 0.1 SDS, 5X SSC ) at 42°C for 60 min. They are dipped a couple of times in distilled water at room temperature, then in isopropanol and dried immediately by compressed nitrogen stream. Slides were placed in Corning hybridization chambers with a 25x60 lifterslip and 10ul of distilled water for each groove. The target was diluted to a final volume of 60 µL as follows 15µl of purified, labeled cDNA, 15 µl of 4X Hybridization Buffer ( 20X SDS, 0.4 % SDS), 30 µL formamide. The target mixture is heated for 3 min at 95°C, put on ice for 30 sec and centrifuged to remove dust for 1 min. The target mixture was put on the chip as quickly as possible. The microarray is sealed in a chamber and submerged in a 42°C water bath for approximately 16 h. The microarray is washed for 4 min in 1xSSC, 0.2% SDS (42°C); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC (RT); dipped a few times in distilled water and dried immediately by compressed nitrogen stream.

extract_protocol: SC-H1:16ug. Grind samples in liquide nitrogen, Heat 500 µl phenol pH4+500 µl TLES at 80°C, homogenize samples in hot phenol+TLES, vortex 30 '', heat again at 80°C 1', add 500 µl chloroforme/isoamylalcool (24:1), vortex 30'', transfer in safelock 2 ml and put on ice, centrifuge 10' 15000 rpm 4°C, transfer supernatant in eppendorf and add the same volume of LiCl 4M (about 400 µl), precipitate on ice overnight at 4°C. Centrifuge 30' 14500 rpm 4°C, remove supernatant, resuspend the pellet in 250 µl RNase-free water, add 1/10 volume of sodium acetate 2.5M pH 5.6 and 2 volumes of 100% ethanol, precipitate overnight at - 20°C. Centrifuge 20' 15000 rpm 4°C, wash the RNA pellet with 500 µl of 70% ethanol, centrifuge 10' 15000 rpm 4°C, briefly dry the pellet (air dry for 5-10'), resuspend in 30 µl RNase-free water.


title: Patient 4


title: Bradyrhizobium japonicum mutant strain 613, induced with genistein, biological replicate 2

status: Public on Oct 15 2007

city: Dresden

first: Michael

last: Göttfert

address: Helmholtzstrasse 10

department: Biology

country: Germany

institute: Dresden University of Technology

description: Gene expression data of Bradyrhizobium japonicum grown with genistein for 8 hours.

hyb_protocol: Modification of Affymetrix protocol used for Escherichia coli. 1.8-2.5 µg of labeled cDNA was used.

scan_protocol: Standard Affymetrix protocol

data_processing: GeneChip Operating Software (GCOS) version 1.2 (Affymetrix), global scaling to a target intensity of 500.

organism: Bradyrhizobium japonicum

source_name: Bradyrhizobium japonicum 613 grown with genistein for 8 hours

label_protocol: 3 µg cDNA were labeled with biotin.

growth_protocol: After addition of genistein cells were grown for 8 hours at 28 °C.

extract_protocol: RNA was isolated as described by Lang et al. (2007) Mol Gen Genomics, submitted.

treatment_protocol: Genistein (1 µM end concentration) was added at an OD600 of 0.4-0.6.

tag: Genotype

value: nodW mutant

tag: Growth condition

value: AG medium supplemented with genistein

tag: Induction time

value: 8 hours


title: Bradyrhizobium japonicum mutant strain 613, induced with genistein, biological replicate 1


title: Bradyrhizobium japonicum mutant strain Δ901, induced with genistein, biological replicate 3

source_name: Bradyrhizobium japonicum Δ901 grown with genistein for 8 hours

value: nodW mutant, nwsA mutant, nwsB overexpressed


title: 13301722 - SA-H1 vs SC-H1

hyb_protocol: SC-H1 Cy5 / SA-H1 Cy3 : 30pmol. Hybridization Protocol: CATMA slides (Corning Microarray Technology, CORNING) are pretreated in the prehybridisation solution (1 % BSA, 0.1 SDS, 5X SSC ) at 42°C for 60 min. They are dipped a couple of times in distilled water at room temperature, then in isopropanol and dried immediately by compressed nitrogen stream. Slides were placed in Corning hybridization chambers with a 25x60 lifterslip and 10ul of distilled water for each groove. The target was diluted to a final volume of 60 µL as follows 15µl of purified, labeled cDNA, 15 µl of 4X Hybridization Buffer ( 20X SDS, 0.4 % SDS), 30 µL formamide. The target mixture is heated for 3 min at 95°C, put on ice for 30 sec and centrifuged to remove dust for 1 min. The target mixture was put on the chip as quickly as possible. The microarray is sealed in a chamber and submerged in a 42°C water bath for approximately 16 h. The microarray is washed for 4 min in 1xSSC, 0.2% SDS (42°C); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC (RT); dipped a few times in distilled water and dried immediately by compressed nitrogen stream.


title: Patient 5

hyb_protocol: aRNA was reverse transcribed and hybridised to Illumina Sentrix HumanRef-8 following the manufacturer's protocols at the Westmead Millenium Institute, Sydney, Australia.

value: Female


title: 13279243 - SA-H1 vs SB-H1

hyb_protocol: SB-H1 Cy5 / SA-H1 Cy3 : 30pmol. Hybridization Protocol: CATMA slides (Corning Microarray Technology, CORNING) are pretreated in the prehybridisation solution (1 % BSA, 0.1 SDS, 5X SSC ) at 42°C for 60 min. They are dipped a couple of times in distilled water at room temperature, then in isopropanol and dried immediately by compressed nitrogen stream. Slides were placed in Corning hybridization chambers with a 25x60 lifterslip and 10ul of distilled water for each groove. The target was diluted to a final volume of 60 µL as follows 15µl of purified, labeled cDNA, 15 µl of 4X Hybridization Buffer ( 20X SDS, 0.4 % SDS), 30 µL formamide. The target mixture is heated for 3 min at 95°C, put on ice for 30 sec and centrifuged to remove dust for 1 min. The target mixture was put on the chip as quickly as possible. The microarray is sealed in a chamber and submerged in a 42°C water bath for approximately 16 h. The microarray is washed for 4 min in 1xSSC, 0.2% SDS (42°C); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC (RT); dipped a few times in distilled water and dried immediately by compressed nitrogen stream.


title: Patient 1


title: Patient 6

value: H


title: 13279244 - SA-H2 vs SB-H2

hyb_protocol: SB-H2 Cy5 / SA-H2 Cy3 : 30pmol. Hybridization Protocol: CATMA slides (Corning Microarray Technology, CORNING) are pretreated in the prehybridisation solution (1 % BSA, 0.1 SDS, 5X SSC ) at 42°C for 60 min. They are dipped a couple of times in distilled water at room temperature, then in isopropanol and dried immediately by compressed nitrogen stream. Slides were placed in Corning hybridization chambers with a 25x60 lifterslip and 10ul of distilled water for each groove. The target was diluted to a final volume of 60 µL as follows 15µl of purified, labeled cDNA, 15 µl of 4X Hybridization Buffer ( 20X SDS, 0.4 % SDS), 30 µL formamide. The target mixture is heated for 3 min at 95°C, put on ice for 30 sec and centrifuged to remove dust for 1 min. The target mixture was put on the chip as quickly as possible. The microarray is sealed in a chamber and submerged in a 42°C water bath for approximately 16 h. The microarray is washed for 4 min in 1xSSC, 0.2% SDS (42°C); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC (RT); dipped a few times in distilled water and dried immediately by compressed nitrogen stream.


title: Bradyrhizobium japonicum mutant strain Δ901, induced with genistein, biological replicate 2


title: Patient 2

value: hyperthyroidism - eye disease

value: C


title: Bradyrhizobium japonicum 110spc4, biological replicate 1

source_name: Bradyrhizobium japonicum grown with methanol for 8 hours

growth_protocol: After addition of methanol cells were grown for 8 hours at 28 °C.

treatment_protocol: Methanol (as solvent for genistein) was added at an OD600 of 0.4-0.6.

value: wild type

value: AG medium supplemented with methanol


title: Bradyrhizobium japonicum mutant strain 613, induced with genistein, biological replicate 3


title: Bradyrhizobium japonicum 110spc4, induced with genistein, biological replicate 1

source_name: Bradyrhizobium japonicum grown with genistein for 8 hours


title: Patient 3

value: hyperthyroidism + eye disease

value: D


title: Bradyrhizobium japonicum 110spc4, biological replicate 2


title: Bradyrhizobium japonicum 110spc4, induced with genistein, biological replicate 2


title: Bradyrhizobium japonicum mutant strain Δ901, induced with genistein, biological replicate 1