title: Aerobic glucose limited chemostat with anaerobic factors (tween and ergosterol) -3

hyb_protocol: 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

label: Biotinylated UTP

label_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.

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 medium was supplemented with anaerobic factors (ergosterol and tween 80) as in anaerobic chemostat fermentation.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.

extract_protocol: Sampling and RNA Isolation—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).


title: Aerobic phosphorus limited chemostat with anaerobic factors (tween and ergosterol) -3

status: Public on Feb 05 2009

type: RNA

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.

data_processing:
Data Acquisition and Primary Analysis—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 dUTP - streptavidine Phycoerythrin (SAPE)

molecule: polyA RNA

organism: Saccharomyces cerevisiae

source_name: chemostat culture

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]. Medium was supplementes with anaerobic growth factors tween 90 and ergosterol. 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.
Media—
The defined mineral medium composition was based on that described by Verduyn et al [. Verduyn, C., Postma, E., Scheffers, W. A., and van Dijken, J. P. (1990) Microbiol.Rev. 58, 616–630]. The residual glucose concentration was targeted to 17 g_liter_1 to sustain glucose repression at the same level . For phosphorus-limited, the composition was 5.0 g of (NH4)2SO4, 1.9 g of K2SO4, 0.12 g of KH2PO4, 0.5 g of MgSO4_7H2O, and 59 g of glucose . Medium was supplementes with anaerobic growth factors tween 90 and ergosterol.

extract_protocol:
Sampling and RNA Isolation—
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: Liquid Nitrogen Quenching

tag: Strain and Maintenance—

tag: 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: Aerobic glucose limited chemostat with anaerobic factors (tween and ergosterol) -2

scan_protocol: Data Acquisition and Primary Analysis—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 5.0), 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.

data_processing: MAS 5.0

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]. Medium was supplemented with anaerobic factor tween and eergosterol.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.

treatment_protocol: Liquid N2 quenching

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: Anaerobic carbon limited with methionine as N-source -3

data_processing: Data Acquisition and Primary Analysis—Acquisition and quantification

label_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.

growth_protocol:
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. For anaerobic cultivation, the reservoir medium was supplemented with Tween 80 and ergosterol as described previously. Anaerobic conditions were maintained by sparging the medium reservoir and the fermentor with pure nitrogen gas (0.5 liter_min_1). Furthermore, Norprene tubing and butyl rubber septa were used to minimize oxygen 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.
Media—
The defined mineral medium composition was based on that described by Verduyn et al [. Verduyn, C., Postma, E., Scheffers, W. A., and van Dijken, J. P. (1990) Microbiol.Rev. 58, 616–630]. For carbon limited cultivation: 5.0 g of methionine 11.3 g of methionine, 3.0 g of KH2PO4, 0.5 g of MgSO4.7H2O, and 25 g of glucose.

treatment_protocol: Liquid nitrogen quenching


title: Aerobic phosphorus limited chemostat with anaerobic factors (tween and ergosterol) -1

scan_protocol: scanning with the Affymetrix GeneArray Scanner


title: Anaerobic carbon limited with methionine as N-source -2

scan_protocol: scanning with the Affymetrix GeneArray Scanner.


title: Anaerobic carbon limited with methionine as N-source

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. For anaerobic cultivation, the reservoir medium was supplemented with Tween 80 and ergosterol as described previously. Anaerobic conditions were maintained by sparging the medium reservoir and the fermentor with pure nitrogen gas (0.5 liter_min_1). Furthermore, Norprene tubing and butyl rubber septa were used to minimize oxygen 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.
Media—
The defined mineral medium composition was based on that described by Verduyn et al [. Verduyn, C., Postma, E., Scheffers, W. A., and van Dijken, J. P. (1990) Microbiol.Rev. 58, 616–630]. For carbon limited cultivation: 5.0 g of methionine 11.3 g of methionine, 3.0 g of KH2PO4, 0.5 g of MgSO4.7H2O, and 25 g of glucose.


title: N-lim Anaerobic chemostat dilution rate 0.03h-1 - 3

hyb_protocol: According to manufacturer's procedures

scan_protocol: Data acquisition was performed using the Affymetrix scanner 3000, quantification of array images and data filtering were performed with the Affymetrix software packages GCOS 1.2

data_processing: Chip file targetted to 150 GCOS (Affymetrix - version 1.2.0.037)

label: SAPE

molecule: total RNA

source_name: Chemostat sample

label_protocol:
Sampling of chemostat cultures, probe preparation and hybridization to Affymetrix GeneChip microarrays was performed as described previously (Piper et al., 2002), but with the following modifications. Double-stranded cDNA synthesis was carried out using 15 μg of total RNA and the components of the One Cycle cDNA Synthesis Kit (Affymetrix). The double-stranded cDNA was purified (Genechip Sample Cleanup Module, Qiagen) before in vitro transcription and labeling (GeneChip IVT Labeling Kit, Affymetrix). Finally, labeled cRNA was purified (GeneChip Sample Cleanup Module) prior to fragmentation and hybridization of 15 μg of biotinylated cRNA.
Piper MDW, Daran-Lapujade P, Bro C, Regenberg B, Knudsen S, Nielsen J & Pronk JT (2002) Reproducibility of oligonucleotide microarray transcriptome analyses. An interlaboratory comparison using chemostat cultures of Saccharomyces cerevisiae. J Biol Chem 277: 37001-37008.

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 Tai et al (2005) with nitrogen 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 0.03 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.
Tai SL, Boer VM, Daran-Lapujade P, de Winde JH, Daran JM, Pronk JT (2005) Two-dimensional transcriptome analysis in chemostat cultures: combinatorial effects of oxygen availability and macronutrient limitation in Saccharomyces cerevisiae, J. Biol. Chem., 280(1), 437-447.

extract_protocol:
Sampling of chemostat cultures and probe preparation was performed as described previously (Piper et al., 2002).
Piper MDW, Daran-Lapujade P, Bro C, Regenberg B, Knudsen S, Nielsen J & Pronk JT (2002) Reproducibility of oligonucleotide microarray transcriptome analyses. An interlaboratory comparison using chemostat cultures of Saccharomyces cerevisiae. J Biol Chem 277: 37001-37008.

treatment_protocol: liquid N2 quenching

tag: prototrophic Saccharomyces cerevisiae CEN.PK 113-7D (MATa)


title: Aerobic glucose limited chemostat with anaerobic factors (tween and ergosterol) -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]. medium supplemented with anaerobic factors tween 80 and ergosterol.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.


title: N-lim Anaerobic chemostat dilution rate 0.2h-1 - 4

scan_protocol: Hybridization protocol According to manufacturer's procedures Scan protocol Data acquisition was performed using the Affymetrix scanner 3000, quantification of array images and data filtering were performed with the Affymetrix software packages GCOS 1.2

data_processing: Data processing Chip file targetted to 150 GCOS (Affymetrix - version 1.2.0.037)

label_protocol:
Sampling of chemostat cultures, probe preparation and hybridization to Affymetrix GeneChip microarrays was performed as described previously (Piper et al., 2002), but with the following modifications. Double-stranded cDNA synthesis was carried out using 15 μg of total RNA and the components of the One Cycle cDNA Synthesis Kit (Affymetrix). The double-stranded cDNA was purified (Genechip Sample Cleanup Module, Qiagen) before in vitro transcription and labeling (GeneChip IVT Labeling Kit, Affymetrix). Finally, labeled cRNA was purified (GeneChip Sample Cleanup Module) prior to fragmentation and hybridization of 15 μg of biotinylated cRNA.
Piper MDW, Daran-Lapujade P, Bro C, Regenberg B, Knudsen S, Nielsen J & Pronk JT (2002) Reproducibility of oligonucleotide microarray transcriptome analyses. An interlaboratory comparison using chemostat cultures of Saccharomyces cerevisiae. J Biol Chem 277: 37001-37008

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 Tai et al (2005) with nitrogen 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 0.2 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.
Tai SL, Boer VM, Daran-Lapujade P, de Winde JH, Daran JM, Pronk JT (2005) Two-dimensional transcriptome analysis in chemostat cultures: combinatorial effects of oxygen availability and macronutrient limitation in Saccharomyces cerevisiae, J. Biol. Chem., 280(1), 437-447.

extract_protocol:
Extraction protocol Sampling of chemostat cultures and probe preparation was performed as described previously (Piper et al., 2002).
Piper MDW, Daran-Lapujade P, Bro C, Regenberg B, Knudsen S, Nielsen J & Pronk JT (2002) Reproducibility of oligonucleotide microarray transcriptome analyses. An interlaboratory comparison using chemostat cultures of Saccharomyces cerevisiae. J Biol Chem 277: 37001-37008.


title: Ethanol limited chemostat culture D=0.1/h -4

hyb_protocol: Sampling of cells from chemostats, probe preparation, and hybridization to Affymetrix GeneChip microarrays were performed as described previously (6) 6- Piper, M. D. W., Daran-Lapujade, P., Bro, C., Regenberg, B., Knudsen, S.,Nielsen, J., and Pronk, J. T. (2002) J. Biol. Chem. 277, 37001-37008

data_processing: MAS5.0 calculated intensity with global array targetting at 150

source_name: ethanol limited aerobic chemostat culture

growth_protocol:
Strain and Growth Conditions Wild-type S. cerevisiae strain CEN.PK113-7D (MATa) (1) was grown at 30 °C in 2-liter chemostats (Applikon), with a working volume of 1.0 liter as described in Ref. 2. Cultures were fed with a defined mineral medium that limited growth by glucose, ethanol with all other growth requirements in excess. The dilution rate was set at 0.10 h-1. The pH was measured on-line and kept constant at 5.0 by the automatic addition of 2 M KOH with the use of an Applikon ADI 1030 biocontroller. Stirrer speed was 800 rpm, and the airflow was 0.5 liters_min-1. Dissolved oxygen tension was measured online with an Ingold model 34-100-3002 probe, and was between 60 and 75% of air saturation. The off-gas was cooled by a condenser connected to a cryostat set at 2 Â°C and analyzed as previously described (3). Steady-state samples were taken after 10-14 volume changes to avoid strain adaptation caused by long term cu
ltivation (4).
Media The defined mineral medium composition was based on that described by Verduyn et al. (5). The carbon source was 256 mmol
of carbon/liter.
1. Pronk, J. T., Wenzel, T. J., Luttik, M. A. H., Klaassen, C. C. M., Scheffers, W. A., and van Dijken, J. P. (1994) Microbiology 140, 601-610
2. 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
3. van Maris, A. J. A., Luttik, M. A. H., Winkler, A. A., van Dijken, J. P., and Pronk, J. T. (2003) Appl. Environ. Microbiol. 69, 2094-2099
4. Ferea, T. L., Botstein, D., Brown, P. O., and Rosenzweig, R. F. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 9721-9726
5- Verduyn, C., Postma, E., Scheffers, W. A., and van Dijken, J. P. (1992) Yeast 8, 501-517

treatment_protocol: liquid nitrogen quenching

tag: Prototrophic S. cerevisiae strain CEN.PK113â€“7D (MATa)


title: N-lim Anaerobic chemostat dilution rate 0.2h-1 - 3

hyb_protocol: Hybridization protocol According to manufacturer's procedures


title: C-lim Anaerobic chemostat dilution rate 0.03h-1 - 3

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 0.03 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.
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.


title: Acetate limited chemostat culture D=0.1/h -4

source_name: Acetate limited aerobic chemostat culture

label_protocol:
Sampling of cells from chemostats, probe preparation, and hybridization to Affymetrix GeneChip microarrays were performed as described previously (6)
6- Piper, M. D. W., Daran-Lapujade, P., Bro, C., Regenberg, B., Knudsen, S.,Nielsen, J., and Pronk, J. T. (2002) J. Biol. Chem. 277, 37001-37008

growth_protocol:
Strain and Growth Conditions Wild-type S. cerevisiae strain CEN.PK113-7D (MATa) (1) was grown at 30 Â°C in 2-liter chemostats (Applikon), with a working volume of 1.0 liter as described in Ref. 2. Cultures were fed with a defined mineral medium that limited growth by glucose, ethanol, acetate, or maltose with all other growth requirements in excess. The dilution rate was set at 0.10 h-1. The pH was measured on-line and kept constant at 5.0 by the automatic addition of 2 M KOH with the use of an Applikon ADI 1030 biocontroller. Stirrer speed was 800 rpm, and the airflow was 0.5 liters_min-1. Dissolved oxygen tension was measured online with an Ingold model 34-100-3002 probe, and was between 60 and 75% of air saturation. The off-gas was cooled by a condenser connected to a cryostat set at 2 Â°C and analyzed as previously described (3). Steady-state samples were taken after 10-14 volume changes to avoid strain adaptation caused by long term cu
ltivation (4).
Media The defined mineral medium composition was based on that described by Verduyn et al. (5). The carbon source was 256 mmol
of carbon/liter.
1. Pronk, J. T., Wenzel, T. J., Luttik, M. A. H., Klaassen, C. C. M., Scheffers, W. A., and van Dijken, J. P. (1994) Microbiology 140, 601-610
2. 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
3. van Maris, A. J. A., Luttik, M. A. H., Winkler, A. A., van Dijken, J. P., and Pronk, J. T. (2003) Appl. Environ. Microbiol. 69, 2094-2099
4. Ferea, T. L., Botstein, D., Brown, P. O., and Rosenzweig, R. F. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 9721-9726
5- Verduyn, C., Postma, E., Scheffers, W. A., and van Dijken, J. P. (1992) Yeast 8, 501-517

tag: Prototrophic S. cerevisiae strain CEN.PK113-7D (MATa)


title: Aerobic pH6.5  C-lim chemostat culture-3

hyb_protocol: According to manufacturer's proceduresProbe 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 usersâ€™ 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 Â°C for 16 h as described in the Affymetrix usersâ€™ 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

label_protocol:
Probe Preparation and Hybridization to Arraysâ€”mRNA extraction, cDNA synthesis, cRNA synthesis and labeling, as well as array hybridization were performed as described in the Affymetrix usersâ€™ 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 Â°C for 16 h as described in the Affymetrix usersâ€™ 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

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 6.5.
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.

tag: Strain and Maintenance

tag: 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: C-lim Anaerobic chemostat dilution rate 0.03h-1 - 2

tag: Characteristics	prototrophic Saccharomyces cerevisiae CEN.PK 113-7D (MATa)


title: Aerobic pH6.5  C-lim chemostat culture-1

extract_protocol:
Probe Preparation and Hybridization to Arraysâ€”mRNA extraction, cDNA synthesis, cRNA synthesis and labeling, as well as array hybridization were performed as described in the Affymetrix usersâ€™ 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 Â°C for 16 h as described in the Affymetrix usersâ€™ 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: N-lim Anaerobic chemostat dilution rate 0.2h-1 - 1


title: N-lim Anaerobic chemostat dilution rate 0.2h-1 - 2


title: Aerobic pH3.5  C-lim chemostat culture-3

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 3.5.
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.


title: Aerobic pH3.5  C-lim chemostat culture-1


title: Aerobic pH6.5  C-lim chemostat culture-2


title: Aerobic pH3.5  C-lim chemostat culture-2


title: C-lim Anaerobic chemostat dilution rate 0.2h-1 - 2

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 0.2 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.
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.


title: C-lim Anaerobic chemostat dilution rate 0.2h-1 - 1


title: N-lim Anaerobic chemostat dilution rate 0.03h-1 - 2


title: C-lim Anaerobic chemostat dilution rate 0.03h-1 - 1


title: Aerobic galactose  C-lim chemostat culture-4

label_protocol:
Sampling of chemostat cultures, probe preparation and hybridization to Affymetrix GeneChip microarrays was performed as described previously (Piper et al., 2002)
Piper MDW, Daran-Lapujade P, Bro C, Regenberg B, Knudsen S, Nielsen J & Pronk JT (2002) Reproducibility of oligonucleotide microarray transcriptome analyses. An interlaboratory comparison using chemostat cultures of Saccharomyces cerevisiae. J Biol Chem 277: 37001-37008.

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 galactose 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 aerotion was maintained by sparging the fermentor with air gas at 500 ml min-1. 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.

tag: Haploid prototrophic Saccharomyces cerevisiae CEN.PK 113-7D (MATa)


title: Aerobic galactose  C-lim chemostat culture-5


title: Aerobic galactose  C-lim chemostat culture-3


title: Aerobic galactose  C-lim chemostat culture-2


title: Aerobic galactose  C-lim chemostat culture-6


title: Aerobic S-lim chemostat culture-2

label_protocol:
Sampling and RNA Isolation—
Sampling of chemostat cultures, probe preparation and hybridization to Affymetrix GeneChip microarrays was performed as described previously (Piper et al., 2002), but with the following modifications. Double-stranded cDNA synthesis was carried out using 15 μg of total RNA and the components of the One Cycle cDNA Synthesis Kit (Affymetrix). The double-stranded cDNA was purified (Genechip Sample Cleanup Module, Qiagen) before in vitro transcription and labeling (GeneChip IVT Labeling Kit, Affymetrix). Finally, labeled cRNA was purified (GeneChip Sample Cleanup Module) prior to fragmentation and hybridization of 15 μg of biotinylated cRNA.
Piper MDW, Daran-Lapujade P, Bro C, Regenberg B, Knudsen S, Nielsen J & Pronk JT (2002) Reproducibility of oligonucleotide microarray transcriptome analyses. An interlaboratory comparison using chemostat cultures of Saccharomyces cerevisiae. J Biol Chem 277: 37001-37008.

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.
Media—
The defined mineral medium composition was based on that described by Verduyn et al [. Verduyn, C., Postma, E., Scheffers, W. A., and van Dijken, J. P. (1990) Microbiol.Rev. 58, 616–630]. The residual glucose concentration was targeted to 17 g_liter_1 to sustain glucose repression at the same level . For sulfur-limited, the composition was 4.0 g of NH4Cl, 0.05 g of MgSO4_7H2O, 3.0 g of KH2PO4, 0.4g of MgCl2, and 42 g of glucose.


title: Aerobic S-lim chemostat culture-1


title: C-lim Anaerobic chemostat dilution rate 0.05h-1 - 3

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 0.05 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.
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.


title: Clcn1_null_1_Experiment 2_430B

city: Rochester

first: Stephen

last: Welle

state: NY

address: 601 Elmwood Avenue

country: USA

institute: University of Rochester

hyb_protocol: Hybridization, washes, and staining with phycoerythrin-streptavidin (including antibody enahncement step) were carried out exactly as recommended by Affymetrix for eukaryotic expression arrays, using hybridization oven and fluidics station purchased from Affymetrix.

scan_protocol: Scans were done with Affymetrix scanner exactly as recommended by Affymetrix.

data_processing: background subtraction and normalization were done with the GCRMA method.

label: biotin

organism: Mus musculus

source_name: quadriceps muscle

label_protocol: Biotinylated cRNA was prepared with kits from Enzo as recommended by supplier.

growth_protocol: Mice were reared under normal conditions. CUGexp mice were sacrificed at 6 months of age. Mbnl1-null and Clcn1-null mice were sacrificed at 3 months of age.

extract_protocol: Total RNA was extracted into Tri-Reagent as recommended by supplier.

treatment_protocol: No treatments other than constitutive genetic modifications

tag: Gender

value: male

tag: Age

value: 3 months old

tag: Genotype

value: Clcn1 knockout with BALB background


title: P10_RU10

city: Tromsø

first: Ruth

middle: Hracky

last: Paulssen

address: MH-Bygget Breivika

department: Institute of Clinical Medicine

country: Norway

institute: Universitetet i Tromsø

description: Condition group 4, biological replicate 5

hyb_protocol: Hybridizations were carried out in a TECAN HS4800 instrument using the formamide-based hybridization buffer from Genisphere containing 5% dextrane sulfate and 5.5 ng/μl COT1 DNA (GIBCO BRL Life Technologies) at 37º for 23 hours. 3DNA dendrimer hybridizations were carried out in formamide-based hybridization buffer alone. Post-hybridization washes were carried out at room temperature with 2xSSC for 1 min, 0.2% SDS /2xSSC for 1 min and finally with 0.2xSSC for 30 sec.

scan_protocol: The arrays were scanned with the GenePix 4000B scanner (Axon Instruments Inc).

data_processing: The features were extracted from the arrays using Genepix Pro 6.0 [Axon instruments Inc.). The background estimates were calculated using the morphological opening method. Spots that displayed a signal-to-noise ratio of less than 2, or that were significantly saturated (more than 20 % saturation among foreground pixels) were filtered out. The median was used as the averaging measure of the foreground pixels.  After quality control, genes that were present in less than 50 % of the arrays were filtered out. The arrays were normalized using the lowess method.

organism: Homo sapiens

source_name: 10 μM progesterone, 4 hrs

label_protocol: 5 μg total RNA and Arabidopsis thaliana mRNA (Stratagene Spot reporter) was reverse transcribed and labelled with Cy3 or Cy5-attached dendrimers using the Genisphere 3DNA 350HS kit (Genisphere) as described in the manufacturer's protocol.

growth_protocol: Ishikawa cells, originally derived from a well-differentiated endometrial adenocarcinoma, were obtained from the European Collection of Cell Cultures (ECACC #99040201) (Sigma, UK). Monolayer cultures were established in RPMI 1640 medium (Sigma,St.Louis) with 10 % dialyzed fetal calf serum (PAA-laboratories, Austria) without phenol red to avoid potential steroid-mimicking effects and without antibiotics. Cells were seeded at a density of 1 x 105 cells/ml in 25 cm2 plastic flasks in humidified atmosphere (5 % CO2) at 37 °C with daily change of supplemented growth medium.

extract_protocol: Total RNA extraction with TriZol (GIBCO BRL Life technologies, NY).

treatment_protocol: Ten independent hormone- and glucocorticoid antagonist- treatment experiments were carried out such that all cells were harvested simultaneously at the end of an experiment. Progesterone and/or glucocorticoid antagonist Mifepristone (Ru-486) were added from an ethanol stock to the cell culture medium to obtain a final concentration of 10 μg/ml. The final ethanol concentration in the culture medium was 0.2 %. The cells were incubated for four hours before harvest. Parallel controls (no treatment) were run in medium containing 0.2% ethanol. The medium was always changed at the start of the experiment. All experiments were carried out using cells in the exponential phase of growth.

tag: cell type

value: Ishikawa endometrial cancer cell line ECACC#99040201

source_name: 10 μM Mifepristone, 4 hrs


title: Clcn1_WT_1_Experiment 2_430B

value: BALB wild type


title: P9_RU9

description: Condition group 4, biological replicate 4


title: Clcn1_WT_2_Experiment 2_430B


title: P8_RU8

description: Condition group 4, biological replicate 3


title: Clcn1_null_3_Experiment 2_430B


title: FVB wild type 2_Experiment 1, 430B

description: FVB wild type 2_Experiment 1, 430B

value: FVB wild type

value: 6 months old


title: Line 20b 1_Experiment 1, 430B

description: Line 20b 1_Experiment 1, 430B

value: CUGexp transgenic Line 20b


title: FVB wild type 3_Experiment 1, 430B

description: FVB wild type 3_Experiment 1, 430B


title: Line 20b 2_Experiment 1, 430B

description: Line 20b 2_Experiment 1, 430B


title: Line 41 6_Experiment 1, 430B

description: Line 41 6_Experiment 1, 430B

value: CUGexp transgenic Line 41


title: P7_RU7

description: Condition group 4, biological replicate 2


title: FVB wild type 6_Experiment 1, 430B

description: FVB wild type 6_Experiment 1, 430B


title: C-lim Anaerobic chemostat dilution rate 0.05h-1 - 2


title: FVB wild type 1_Experiment 1, 430B

description: FVB wild type 1_Experiment 1, 430B


title: P6_RU6

description: Condition group 4, biological replicate 1


title: Clcn1_null_2_Experiment 2_430B


title: Line 41 4_Experiment 1, 430B

description: Line 41 4_Experiment 1, 430B


title: Aerobic galactose  C-lim chemostat culture-1


title: FVB wild type 5_Experiment 1, 430B

description: FVB wild type 5_Experiment 1, 430B


title: Line 20b 6_Experiment 1, 430B

description: Line 20b 6_Experiment 1, 430B


title: PRU5_RU5

description: Condition group 3, biological replicate 5

source_name: 10 μM progesterone plus 10 μM Mifepristone, 4 hrs


title: Line 41 5_Experiment 1, 430B

description: Line 41 5_Experiment 1, 430B


title: C-lim Anaerobic chemostat dilution rate 0.05h-1 - 1


title: Clcn1_WT_3_Experiment 2_430B


title: FVB wild type 4_Experiment 1, 430B

description: FVB wild type 4_Experiment 1, 430B


title: Line 20b 3_Experiment 1, 430B

description: Line 20b 3_Experiment 1, 430B


title: Line 20b 5_Experiment 1, 430B

description: Line 20b 5_Experiment 1, 430B


title: Line 20b 4_Experiment 1, 430B

description: Line 20b 4_Experiment 1, 430B


title: PRU3_RU3

description: Condition group 3, biological replicate 3


title: PRU10_K10

description: Condition group 2, biological replicate 5

source_name: Untreated


title: Line 41 1_Experiment 1, 430B

description: Line 41 1_Experiment 1, 430B


title: Line 41 3_Experiment 1, 430B

description: Line 41 3_Experiment 1, 430B


title: PRU2_RU2

description: Condition group 3, biological replicate 2


title: PRU4_RU4

description: Condition group 3, biological replicate 4


title: P3_K3

description: Condition group 1, biological replicate 3


title: Line 41 2_Experiment 1, 430B

description: Line 41 2_Experiment 1, 430B


title: PRU6_K6

description: Condition group 2, biological replicate 1


title: P4_K4

description: Condition group 1, biological replicate 4


title: PRU9_K9

description: Condition group 2, biological replicate 4


title: PRU7_K7

description: Condition group 2, biological replicate 2


title: P1_K1

description: Condition group 1, biological replicate 1


title: P5_K5

description: Condition group 1, biological replicate 5


title: PRU8_K8

description: Condition group 2, biological replicate 3


title: 96-INVS

status: Public on Feb 04 2009

city: San Diego

first: Xiao-Jun

last: Ma

state: CA

address: 11025 Roselle St, Suite 200

institute: bioTheranostics, Inc.

description: breast cancer

hyb_protocol: Hybridization, washing and staining performed per manufacturer's instructions (Affymetrix)

scan_protocol: GeneChips were scannned using GeneChip Scanner 3000.

data_processing: Expression measures were extracted using the RMA algorithm with default parameters for background subtraction and quantile normalization.

source_name: breast cancer biopsy

label_protocol: Biotinylated cRNA were prepared after two-rounds of T7-based amplification using the RiboAmp kit (Arcturus BioScience)

growth_protocol: fresh frozen primary breast cancer biopsy

extract_protocol: Picopure RNA extraction kit (Arcturus BioScience, Inc.)

treatment_protocol: laser capture microdissected

tag: invasive stroma, tumor grade:III


title: 96-NSS

description: breast cancer

tag: normal stroma


title: 79-N

description: breast cancer

tag: normal epithelium


title: 96-IS

description: breast cancer

tag: DCIS epithelium, tumor grade:III


title: 44-INVS

description: breast cancer


title: 96-N

description: breast cancer


title: 44-ISS

description: breast cancer

tag: DCIS stroma, tumor grade:III


title: 79-NSS

description: breast cancer


title: 45-INVS

description: breast cancer

tag: invasive stroma, tumor grade:I


title: 45-INV

description: breast cancer

tag: invasive epithelium, tumor grade:I


title: 44-N

description: breast cancer


title: 44-NSS

description: breast cancer


title: 44-IS

description: breast cancer


title: 96-INV

description: breast cancer

tag: invasive epithelium, tumor grade:III


title: 79-INV

description: breast cancer


title: 79-INVS

description: breast cancer


title: 79-ISS

description: breast cancer

tag: DCIS stroma, tumor grade:I


title: 45-NSS

description: breast cancer


title: 96-ISS

description: breast cancer


title: 44-INV

description: breast cancer


title: 45-N

description: breast cancer


title: 180-ISS

description: breast cancer


title: 45-ISS

description: breast cancer


title: 180-NSS

description: breast cancer


title: 179-NSS

description: breast cancer


title: 178-ISS

description: breast cancer


title: 179-ISS

description: breast cancer


title: 180-IS

description: breast cancer

tag: DCIS epithelium, tumor grade:I


title: 153-N

description: breast cancer


title: 148-NSS

description: breast cancer


title: 153-INVS

description: breast cancer


title: 180-N

description: breast cancer


title: 169-NSS

description: breast cancer


title: 133-ISS

description: breast cancer


title: 179-IS

description: breast cancer


title: 148-INVS

description: breast cancer

tag: invasive stroma, tumor grade:II


title: 169-INVS

description: breast cancer


title: 148-N

description: breast cancer


title: 178-IS

description: breast cancer


title: 169-N

description: breast cancer


title: 153-INV

description: breast cancer


title: 178-NSS

description: breast cancer


title: 179-N

description: breast cancer


title: 178-N

description: breast cancer


title: 148-INV

description: breast cancer

tag: invasive epithelium, tumor grade:II


title: 133-NSS

description: breast cancer


title: 153-NSS

description: breast cancer


title: 169-INV

description: breast cancer


title: Müller glia at 24 hpl, biological rep3

city: Ann Arbor

first: Zhao

last: Qin

state: MI

address: 830 North University

department: Molecular, Cellular and Developmental Biology

institute: University of Michigan

description: Gene expression data of zebrafish Müller glia isolated from 24 hour post-lesion retinas

hyb_protocol: 2.75 µg of biotin-labeled, fragmented cDNA was hybridized to a GeneChip Zebrafish Genome Array. GeneChips were washed and stained in the Affymetrix Fluidics Station 450.

scan_protocol: GeneChips were scanned using the Affymetrix GeneChip Scanner 3000.

data_processing: The Robust Multichip Average (RMA) method was used for background correction, quantile normalization, and summarization of expression scores.

organism: Danio rerio

label_protocol: 20 ng of total RNA was used for linear amplification and biotin-labeling with Ovation Biotin Labeling System (NuGEN).

extract_protocol: Total RNA was extracted using the RNAqueous-4PCR kit (Ambion).

tag: zebrafish line

tag: age

value: 5- to 6-month old


title: Müller glia at 24 hpl, biological rep2

source_name: zebrafish Müller glia isolated from 24 hour post-lesion retinas


title: Müller glia at 16 hpl, biological rep1

description: Gene expression data of zebrafish Müller glia isolated from 16 hour post-lesion retinas


title: Müller glia at 36 hpl, biological rep1

description: Gene expression data of zebrafish Müller glia isolated from 36 hour post-lesion retinas


title: 131-IS

description: breast cancer

tag: DCIS epithelium, tumor grade:II


title: 133-N

description: breast cancer


title: Müller glia at 8 hpl, biological rep3

description: Gene expression data of zebrafish Müller glia isolated from 8 hour post-lesion retinas


title: 133-IS

description: breast cancer


title: Müller glia at 24 hpl, biological rep1


title: Müller glia at 8 hpl, biological rep2

source_name: zebrafish Müller glia isolated from 8 hour post-lesion retinas


title: Müller glia at 0 hpl, biological rep3

description: Gene expression data of zebrafish Müller glia isolated from unlesioned retinas


title: Müller glia at 36 hpl, biological rep2

source_name: zebrafish Müller glia isolated from 36 hour post-lesion retinas


title: Müller glia at 16 hpl, biological rep2

source_name: zebrafish Müller glia isolated from 16 hour post-lesion retinas


title: 131-ISS

description: breast cancer

tag: DCIS stroma, tumor grade:II


title: Müller glia at 0 hpl, biological rep1

source_name: zebrafish Müller glia isolated from unlesioned retinas


title: 121-INVS

description: breast cancer


title: Müller glia at 8 hpl, biological rep1


title: RSPA16 treated with 12 ug/mL Ramoplanin replicate 3

city: Durham

first: John

middle: W.

last: Schmidt

state: NC

department: Chemistry

institute: Duke University

description: See above.

hyb_protocol: According to Affymetrix recommendations for antisense prokaryotic arrays.

data_processing: For each GeneChip values corresponding to the level of expression (abundance of transcript) were calculated using the Affymetrix GeneChip Operating System (GCOS) software.  Different experiments were normalized to each other using RMAExpress, using background adjustment and quantile normalization (Version 1.0) (http://rmaexpress.bmbolstad.com).  The median normalized signal intensities from three biological replicates for each condition were calculated.  Genes determined to increase or decrease in expression 2-fold or more between data sets and were significantly different (two tailed Student’s t-test; P cut-off value of 0.10) were considered to be regulated.  In cases where a gene corresponded to more than one Affymetrix probe set, the median normalized signal intensities for theses probe sets were averaged.  Additionally, for each GeneChip the GCOS software determined if the signal for each probe was distinguishable from background variability, the “GCOS Call”.  If a signal was distinguishable from background it was declared “Present”, otherwise it was declared “Marginal” or “Absent”.  Genes were also considered upregulated and labeled as “Up” if the signal was “Absent” or “Marginal” for more than one untreated sample for a particular probe set and the signals for all ramoplanin treated samples were “Present” for that probe set.  Genes were considered downregulated and was labeled as “Down” if the signal was “Absent” or “Marginal” for more than one sample treated with ramoplanin for a particular probe set and the signals for the untreated samples were all “Present” for that probe set.

label: Biotin

organism: Staphylococcus aureus

source_name: Staphylococcus aureus RSPA16

label_protocol: Labeled according to Affymetrix recommendations for antisense prokaryotic arrays.

growth_protocol:
Ramoplanin resistant S. aureus was isolated by step pressure.  Isolated colonies of S. aureus NCTC 8325-4 were inoculated into 5 mL aliquots of Cation Adjusted Muller Hinton Broth II (Becton, Dickinson and Company) supplemented with 0.02% BSA (CAMHB2+BSA) containing ramoplanin at concentrations of 0.1 to 10 μg/mL.  The cultures were incubated at 37 °C with aeration for 48 hours.  At 48 hours growth was observed in the culture containing 0.1 μg/mL ramoplanin.  This culture was used to inoculate 5 mL CAMHB2+BSA containing ramoplanin at concentrations of 0.1 to 5 μg/mL at a final conc of approx 1e6 CFU/mL.  These cultures were incubated for 24 to 72 hours at 37 °C with aeration.  The culture with growth in the highest concentration of ramoplanin was used to inoculate another series.  Passage of the culture in this fashion was continued.  Following the 16th series growth was observed in a culture containing 5 μg/mL ramoplanin.  A sample from this culture struck on Trptic Soy Agar (TSA, Becton, Dickinson and Company) with no antibiotic and incubated at 37 °C overnight.  An isolated colony was selected and restruck onto TSA and grown overnight at 37 °C twice.  An isolated colony was then selected and named RSPA16 for “Reduced Susceptibility to Peptide Antimicrobials 16th series.”
A 100 mL tryptic soy broth culture in a 250 mL flask was inoculated a small amount of overnight culture.  Incubated at 37C shaking at 200 rpm until OD620 = 0.4

extract_protocol: Cultures were combined with an equal volume ice cold ethanol:acetone (1:1) and flash frozen in liquid nitrogen.  Frozen cultures were thawed on wet ice and pelleted by centrifugation at 5,000 × g at 4 oC for 10 min. Total RNA was isolated using a RiboPure-Bacteria kit (Ambion) according to the manufacturer’s instructions.  Each RNA sample was electrophoresed to assess RNA integrity. Reverse transcription, cDNA fragmentation and terminal labeling of cDNA fragments with biotin were prepared and processed according to Affymetrix recommendations for antisense prokaryotic arrays.

treatment_protocol: Ramoplanin was added to a final concentration of 12 ug/mL to a culture at OD620 = 0.4. The culture was then incubated at 37C shaking at 200 rpm for 30 minutes.

tag: Staphylococcus aureus RSPA16 ("Reduced Susceptibility to Peptide Antimicrobials 16th series")


title: Müller glia at 0 hpl, biological rep2


title: Müller glia at 16 hpl, biological rep3


title: 131-N

description: breast cancer


title: Müller glia at 36 hpl, biological rep3


title: RSPA16 treated with 12 ug/mL Ramoplanin replicate 2

description: See above.

scan_protocol: According to Affymetrix recommendations for antisense prokaryotic arrays.


title: RSPA16 treated with 12 ug/mL Ramoplanin replicate 1

description: See above.


title: RSPA16 untreated replicate 1

description: See above.

treatment_protocol:
No Treatment, Control.
A culture at OD620 = 0.4 was incubated at 37C shaking at 200 rpm for 30 minutes.


title: 121-ISS

description: breast cancer


title: NCTC 8325-4 treated with 4 ug/mL Ramoplanin replicate 2

description: See above.

source_name: Staphylococcus aureus NCTC 8325-4

growth_protocol: A 100 mL tryptic soy broth culture in a 250 mL flask was inoculated a small amount of overnight culture.  Incubated at 37C shaking at 200 rpm until OD620 = 0.4

treatment_protocol: Ramoplanin was added to a final concentration of 4 ug/mL to a culture at OD620 = 0.4. The culture was then incubated at 37C shaking at 200 rpm for 30 minutes.

tag: Strain obtained from NARSA (Network on Antimicrobial Resistance in S. aureus). NARSA strain designations NRS135.  AKA RN0450.


title: NCTC 8325-4 treated with 4 ug/mL Ramoplanin replicate 3

description: See above.


title: 121-INV

description: breast cancer


title: 121-N

description: breast cancer


title: NCTC 8325-4 untreated replicate 3

description: See above.


title: NCTC 8325-4 untreated replicate 2

description: See above.


title: RSPA16 untreated replicate 3

description: See above.


title: 121-NSS

description: breast cancer

tag: normal stroma


title: NCTC 8325-4 untreated replicate 1

description: See above.


title: 102-INVS

description: breast cancer


title: 102-N

description: breast cancer

tag: normal epithelium


title: 121-IS

description: breast cancer


title: 102-IS

description: breast cancer


title: 102-ISS

description: breast cancer


title: 102-INV

description: breast cancer


title: NCTC 8325-4 treated with 4 ug/mL Ramoplanin replicate 1

description: See above.


title: App_sero5a_rep1

status: Public on Feb 03 2009

type: genomic

city: Ottawa

middle: H

last: Nash

state: Ontario

address: 100 Sussex Drive

department: Institute for Biological Sciences

country: Canada

institute: National Research Council

hyb_protocol: Equivalent amounts (2 ug) of labeled tester and control samples were pooled, lyophilized, and then re-suspended in 42 ul of hybridization buffer [1 x DIGEasy hybridization solution (Roche Applied Science); 0.5 ug/ul of Torulla yeast tRNA (Invitrogen); 0.5 ug/ul of salmon sperm genomic DNA (Invitrogen)]. Labelled gDNA was denatured at 65°C for 5 min and applied to the microarray. Hybridizations were performed overnight at 37°C under 22 x 40-mm glass cover slips in a high-humidity chamber. Microarrays were washed 2 x 5 min at 50°C in 1 x SSC with 0.1% SDS, then 2 x 5 min at 50°C in 0.5 x SSC, and 1 x 5 min at 50°C in 0.1 x SSC.

scan_protocol: After hybridization with labelled gDNA, microarray slides of the 15 reference serovars were scanned using a Chipreader laser scanner (BioRad, Mississauga, ON) according to the manufacturer's recommendations.

data_processing: Spot quantification, signal normalization and data visualization were performed using ArrayPro Analyzer v4.5 (Media Cybernetics, Silver Spring, MD). Net signal intensities were obtained by performing local-ring background subtraction.

molecule: genomic DNA

organism: Actinobacillus pleuropneumoniae

source_name: App_serotype: 5a

label_protocol: Five ug of fragmented DNA were fluorescently labelled using direct chemical coupling with the Label-IT (Mirus Corp., Madison, WI) cyanine dyes Cy3 and Cy5 as recommended by the manufacturer.

growth_protocol: All A. pleuropneumoniae strains were inoculated into Brain-Heart Infusion (BHI, Difco Laboratories, Detroit, MI) medium supplemented with NAD: either 15 ug/ml in agar or 5 ug/ml in broth.  Cultures were grown at 37°C for 16-18 hours before genomic DNA isolation.

extract_protocol: A. pleuropneumoniae strains were harvested after growth on agar plates for 16-18 h, resuspended in H2O, and treated with lysozyme (Roche, Laval, QC) and RNase A (Qiagen, Mississauga, ON) for 10 min at room temperature. The cell suspensions were then digested with proteinase K (MBI Fermentas, Burlington, ON) for 1 h at 37°C, and complete lysis was obtained by addition of sodium dodecyl sulfate to a final concentration of 0.1% (wt/vol). Genomic DNA, extracted from the cell lysates by two extractions with phenol-chloroform-isoamyl alcohol (25:24:1) and two extractions with chloroform, was precipitated in ethanol.

tag: Strain

source_name: control serotype: 5b


title: App_sero9_rep2

source_name: App_serotype: 9


title: App_sero9_rep1


title: App_sero8_rep2

source_name: App_serotype: 8


title: App_sero5a_rep2


title: App_sero8_rep1


title: App_sero7_ds

source_name: App_serotype: 7


title: App_sero7_rep1


title: App_sero6_rep1

source_name: App_serotype: 6

value: Fem¸


title: App_sero6_rep2


title: App_sero7_rep2


title: App_sero4_rep2

source_name: App_serotype: 4


title: App_sero3_rep2

source_name: App_serotype: 3


title: App_sero4_rep1


title: App_sero2_ds

source_name: App_serotype: 2


title: App_sero3_rep1


title: App_sero2_rep2


title: App_sero2_rep1


title: App_sero1_rep1

source_name: App_serotype: 1


title: App_sero1_ds


title: App_sero15_rep2

source_name: App_serotype: 15


title: App_sero15_rep1


title: 102-NSS

description: breast cancer

source_name: breast cancer biopsy


title: App_sero1_rep2


title: App_sero14_rep1

source_name: App_serotype: 14


title: App_sero14_rep2


title: App_sero13_rep2

source_name: App_serotype: 13


title: App_sero13_rep1


title: App_sero12_rep2

source_name: App_serotype: 12


title: App_sero10_rep1

source_name: App_serotype: 10


title: App_sero10_rep2


title: App_sero11_rep2

source_name: App_serotype: 11


title: App_sero11_rep1


title: App_sero12_rep1


title: ASP cy5 - 4 days - low pmt - repeat 3 - mAdbID:78047

city: Bethesda

first: Vincent

last: Hearing

state: MD

address: Bldg 37  Rm 2132

department: National Cancer Institute

institute: National Institutes of Health

description: mAdb experiment ID: 78047

hyb_protocol: Hybridization Protocol Other: For pre-hybrization, apply 45 ul of pre-hybridization buffer (5X SSC, 0.1% SDS, 1% BSA) to the array and incubate at 42 °C at least 30 minutes and up to an hour. Wash off the pre-hybridization solution by rapidly plunging the slide in distilled water for 2 minutes, then transfer slide to 100% isopropanol for 2 minutes. Allow slide to air dry completely prior to use. For hybridization, combine Cy3 and Cy5 labeled targets together (~9 ul recovered for each). Add 1 µl COT-1 DNA (8-10 ug/ul) and 1 µl poly A (8-10 ug/ul). Denature target at 100°C 1 minute, then snap cool on ice. (Final volume should be about 20 ul.) Make fresh 2X Formamide hybridization buffer (50% formamide, 10X SSC, 0.2% SDS) and warm to 42°C before adding to samples. Add 25 µl of 2X F-hyb buffer to samples, warm at 70 °C. Load 40 ul sample onto microarray. Add 20 ul of 3X SSC to wells in hyb chamber to maintain humidity. Incubate overnight (12-16 hours) at 42°C water bath. After hybridization of slides, wash slides for 2 minute in 2X SSC with 0.1% SDS (with occasional plunging), for 2 minute in 1X SSC (with occasional plunging), for 2 minutes in 0.2X SSC (with occasional plunging), for 1 minute in 0.05X SSC, and spin for 3 minutes at 650 rpm to dry. (Refer to NCI Microarray Manual)

scan_protocol: Creator: GenePix Pro 5.0.1.24 Scanner: GenePix 4000A [54243] ScanPower: 100;; 100 LaserPower: 2.35;; 1.54 Temperature: 38.04

data_processing:
mAdb Data Processing Protocol (v. 1)
Calculation Method: After background correction, ratios were median centered based on signals greater than 100 in both channels and linear transformed to obtain the log and linear values given in the data table.

source_name: Untreated melanocytes - Cy3

label_protocol:
Cy3 Sample Labeling Protocol
Other: Sample was labeled with cy3 (Amersham Pharmacia Biotech, Piscataway, NJ) in an indirect mannerusing manufacturer's instructions.

extract_protocol:
Sample Extraction Protocol
Other: Used an RNeasy mini kit (Qiagen, Valencia, CA.

tag: Cell line

value: Melan-a

tag: Cell type

value: melanocytes

source_name: ASP - 4 days - Cy5

label_protocol:
Cy5 Sample Labeling Protocol
Other: Sample was labeled with cy5 (Amersham Pharmacia Biotech, Piscataway, NJ) in an indirect manner using manufacturer's instructions.

treatment_protocol:
Treatment type: compound
Agent: recombinant murine Agouti signaling protein (ASP)
Treatment dose: 10 nM
Treatment time: 4 days
Treatment temperature: 37 C


title: ASP cy3 - 4 days - low pmt - rev repeat 5 - mAdbID:78049

description: mAdb experiment ID: 78049

scan_protocol: Creator: GenePix Pro 5.0.1.24 Scanner: GenePix 4000A [54243] ScanPower: 100;; 100 LaserPower: 2.53;; 1.41 Temperature: 39.86

source_name: Untreated melanocytes - Cy5

source_name: ASP - 4 days - Cy3


title: ASP cy5 - 4 days - low pmt - repeat 2 - mAdbID:78046

description: mAdb experiment ID: 78046

scan_protocol: Creator: GenePix Pro 5.0.1.24 Scanner: GenePix 4000A [54243] ScanPower: 100;; 100 LaserPower: 2.51;; 1.6 Temperature: 39.58


title: ASP cy3 - 4 days - low pmt - rev repeat 4 - mAdbID:78048

description: mAdb experiment ID: 78048

scan_protocol: Creator: GenePix Pro 5.0.1.24 Scanner: GenePix 4000A [54243] ScanPower: 100;; 100 LaserPower: 2.56;; 1.6 Temperature: 40.82


title: ASP cy3 - 3 days - low pmt - rev repeat 5 - mAdbID:78043

description: mAdb experiment ID: 78043

scan_protocol: Creator: GenePix Pro 5.0.1.24 Scanner: GenePix 4000A [54243] ScanPower: 100;; 100 LaserPower: 2.35;; 1.66 Temperature: 36.68

source_name: ASP - 3 days - Cy3

treatment_protocol:
Treatment type: compound
Agent: recombinant murine Agouti signaling protein (ASP)
Treatment dose: 10 nM
Treatment time: 3 days
Treatment temperature: 37 C


title: ASP cy3 - 3 days - low pmt - rev repeat 6 - mAdbID:78044

description: mAdb experiment ID: 78044

scan_protocol: Creator: GenePix Pro 5.0.1.24 Scanner: GenePix 4000A [54243] ScanPower: 100;; 100 LaserPower: 2.43;; 1.21 Temperature: 39.72


title: ASP cy3 - 4 days - low pmt - rev repeat 6 - mAdbID:78050

description: mAdb experiment ID: 78050

scan_protocol: Creator: GenePix Pro 5.0.1.24 Scanner: GenePix 4000A [54243] ScanPower: 100;; 100 LaserPower: 2.43;; 1.53 Temperature: 38.67


title: ASP cy5 - 4 days - low pmt - repeat 1 - mAdbID:78045

description: mAdb experiment ID: 78045

scan_protocol: Creator: GenePix Pro 5.0.1.24 Scanner: GenePix 4000A [54243] ScanPower: 100;; 100 LaserPower: 2.51;; 1.62 Temperature: 40.27


title: ASP cy3 - 2 days - low pmt - rev repeat 5 - mAdbID:78038

description: mAdb experiment ID: 78038

scan_protocol: Creator: GenePix Pro 5.0.1.24 Scanner: GenePix 4000A [54243] ScanPower: 100;; 100 LaserPower: 2.75;; 1.32 Temperature: 45.34

source_name: ASP - 2 days - Cy3

treatment_protocol:
Treatment type: compound
Agent: recombinant murine Agouti signaling protein (ASP)
Treatment dose: 10 nM
Treatment time: 2 days
Treatment temperature: 37 C


title: ASP cy3 - 2 days - low pmt - rev repeat 3 - mAdbID:78036

description: mAdb experiment ID: 78036

scan_protocol: Creator: GenePix Pro 5.0.1.24 Scanner: GenePix 4000A [54243] ScanPower: 100;; 100 LaserPower: 2.72;; 1.28 Temperature: 44.7


title: ASP cy5 - 3 days - low pmt - repeat 1 - mAdbID:78039

description: mAdb experiment ID: 78039

scan_protocol: Creator: GenePix Pro 5.0.1.24 Scanner: GenePix 4000A [54243] ScanPower: 100;; 100 LaserPower: 2.36;; 1.6 Temperature: 33.1

source_name: ASP - 3 days - Cy5


title: ASP cy5 - 2 days - low pmt - repeat 2 - mAdbID:78035

description: mAdb experiment ID: 78035

scan_protocol: Creator: GenePix Pro 5.0.1.24 Scanner: GenePix 4000A [54243] ScanPower: 100;; 100 LaserPower: 2.76;; 1.31 Temperature: 45.12

source_name: ASP - 2 days - Cy5


title: ASP cy3 - 2 days - low pmt - rev repeat 4 - mAdbID:78037

description: mAdb experiment ID: 78037

scan_protocol: Creator: GenePix Pro 5.0.1.24 Scanner: GenePix 4000A [54243] ScanPower: 100;; 100 LaserPower: 2.77;; 1.3 Temperature: 45.05


title: ASP cy3 - 3 days - low pmt - rev repeat 4 - mAdbID:78042

description: mAdb experiment ID: 78042

scan_protocol: Creator: GenePix Pro 5.0.1.24 Scanner: GenePix 4000A [54243] ScanPower: 100;; 100 LaserPower: 2.37;; 1.54 Temperature: 33.97


title: ASP cy5 - 1 day - low pmt - repeat 3 - mAdbID:78033

description: mAdb experiment ID: 78033

scan_protocol: Creator: GenePix Pro 5.0.1.24 Scanner: GenePix 4000A [54243] ScanPower: 100;; 100 LaserPower: 2.4;; 1.48 Temperature: 33.74

source_name: ASP - 1 day - Cy5

treatment_protocol:
Treatment type: compound
Agent: recombinant murine Agouti signaling protein (ASP)
Treatment dose: 10 nM
Treatment time: 1 day
Treatment temperature: 37 C


title: ASP cy5 - 2 days - low pmt - repeat 1 - mAdbID:78034

description: mAdb experiment ID: 78034

scan_protocol: Creator: GenePix Pro 5.0.1.24 Scanner: GenePix 4000A [54243] ScanPower: 100;; 100 LaserPower: 2.75;; 1.28 Temperature: 44.76


title: ASP cy5 - 3 days - low pmt - repeat 2 - mAdbID:78040

description: mAdb experiment ID: 78040

scan_protocol: Creator: GenePix Pro 5.0.1.24 Scanner: GenePix 4000A [54243] ScanPower: 100;; 100 LaserPower: 2.34;; 1.66 Temperature: 35.68


title: ASP cy3 - 1 day - low pmt - rev repeat 5 - mAdbID:78031

description: mAdb experiment ID: 78031

scan_protocol: Creator: GenePix Pro 5.0.1.24 Scanner: GenePix 4000A [54243] ScanPower: 100;; 100 LaserPower: 2.37;; 1.69 Temperature: 36.18

source_name: ASP - 1 day - Cy3


title: ASP cy3 - 1 day - low pmt - rev repeat 4 - mAdbID:78030

description: mAdb experiment ID: 78030

scan_protocol: Creator: GenePix Pro 5.0.1.24 Scanner: GenePix 4000A [54243] ScanPower: 100;; 100 LaserPower: 2.71;; 1.29 Temperature: 44.34


title: ASP cy5 - 1 day - low pmt - repeat 2 - mAdbID:78032

description: mAdb experiment ID: 78032

scan_protocol: Creator: GenePix Pro 5.0.1.24 Scanner: GenePix 4000A [54243] ScanPower: 100;; 100 LaserPower: 2.37;; 1.46 Temperature: 35.53


title: ASP cy5 - 3 hr - low pmt - repeat 3 - mAdbID:78024

description: mAdb experiment ID: 78024

scan_protocol: Creator: GenePix Pro 5.0.1.24 Scanner: GenePix 4000A [54243] ScanPower: 100;; 100 LaserPower: 2.4;; 1.28 Temperature: 36.09

source_name: ASP - 3 hr - Cy5

treatment_protocol:
Treatment type: compound
Agent: recombinant murine Agouti signaling protein (ASP)
Treatment dose: 10 nM
Treatment time: 3 hours
Treatment temperature: 37 C


title: ASP cy5 - 3 hr - low pmt - repeat 2 - mAdbID:78023

description: mAdb experiment ID: 78023

scan_protocol: Creator: GenePix Pro 5.0.1.24 Scanner: GenePix 4000A [54243] ScanPower: 100;; 100 LaserPower: 2.47;; 1.65 Temperature: 39.41


title: ASP cy3 - 3 hr - low pmt - rev repeat 4 - mAdbID:78025

description: mAdb experiment ID: 78025

scan_protocol: Creator: GenePix Pro 5.0.1.24 Scanner: GenePix 4000A [54243] ScanPower: 100;; 100 LaserPower: 2.5;; 1.62 Temperature: 38.93

source_name: ASP - 3 hr - Cy3


title: MSH cy3 - 2 days - low pmt - rev repeat 6 - mAdbID:72495

description: mAdb experiment ID: 72495

scan_protocol: Creator: GenePix Pro 5.0.1.24 Scanner: GenePix 4000A [54243] ScanPower: 100;; 100 LaserPower: 2.46;; 1.39 Temperature: 40

source_name: alpha-MSH - 2 day - Cy3

treatment_protocol:
Treatment type: compound
Agent: alpha-melanocyte stimulating hormone (sigma)
Treatment dose: 100 nM
Treatment time: 2 days
Treatment temperature: 37 C


title: MSH cy3 - 1 day - low pmt - rev repeat 6 - mAdbID:74665

description: mAdb experiment ID: 74665

scan_protocol: Creator: GenePix Pro 5.0.1.24 Scanner: GenePix 4000A [54243] ScanPower: 100;; 100 LaserPower: 2.54;; 1.59 Temperature: 39.75

source_name: alpha-MSH - 1 day - Cy3

treatment_protocol:
Treatment type: compound
Agent: alpha-melanocyte stimulating hormone (sigma)
Treatment dose: 100 nM
Treatment time: 1 day
Treatment temperature: 37 C


title: MSH cy5 - 3 hr - low pmt - repeat 2 - mAdbID:74662

description: mAdb experiment ID: 74662

scan_protocol: Creator: GenePix Pro 5.0.1.24 Scanner: GenePix 4000A [54243] ScanPower: 100;; 100 LaserPower: 2.5;; 1.6 Temperature: 35.15

source_name: alpha-MSH - 3 hr - Cy5

treatment_protocol:
Treatment type: compound
Agent: alpha-melanocyte stimulating hormone (sigma)
Treatment dose: 100 nM
Treatment time: 3 hours
Treatment temperature: 37 C


title: ASP cy5 - 3 hr - low pmt - repeat 1 - mAdbID:77981

description: mAdb experiment ID: 77981

scan_protocol: Creator: GenePix Pro 5.0.1.24 Scanner: GenePix 4000A [54243] ScanPower: 100;; 100 LaserPower: 2.46;; 1.65 Temperature: 41.05


title: MSH cy3 - 3 days - low pmt - rev repeat 6 - mAdbID:72494

description: mAdb experiment ID: 72494

scan_protocol: Creator: GenePix Pro 5.0.1.24 Scanner: GenePix 4000A [54243] ScanPower: 100;; 100 LaserPower: 2.44;; 1.57 Temperature: 38.57

source_name: alpha-MSH - 3 day - Cy3

treatment_protocol:
Treatment type: compound
Agent: alpha-melanocyte stimulating hormone (sigma)
Treatment dose: 100 nM
Treatment time: 3 days
Treatment temperature: 37 C


title: MSH cy5 - 1 day - low pmt - repeat 3 - mAdbID:74664

description: mAdb experiment ID: 74664

scan_protocol: Creator: GenePix Pro 5.0.1.24 Scanner: GenePix 4000A [54243] ScanPower: 100;; 100 LaserPower: 2.5;; 1.62 Temperature: 39.53

source_name: alpha-MSH - 1 day - Cy5


title: ASP cy3 - 3 hr - low pmt - rev repeat 5 - mAdbID:78026

description: mAdb experiment ID: 78026

scan_protocol: Creator: GenePix Pro 5.0.1.24 Scanner: GenePix 4000A [54243] ScanPower: 100;; 100 LaserPower: 2.39;; 1.69 Temperature: 36.68


title: MSH cy3 - 2 days - low pmt - rev repeat 5 - mAdbID:72493

description: mAdb experiment ID: 72493

scan_protocol: Creator: GenePix Pro 5.0.1.24 Scanner: GenePix 4000A [54243] ScanPower: 100;; 100 LaserPower: 2.4;; 1.66 Temperature: 34.79


title: MSH cy3 - 3 hr - low pmt - rev repeat 6 - mAdbID:74663

description: mAdb experiment ID: 74663

scan_protocol: Creator: GenePix Pro 5.0.1.24 Scanner: GenePix 4000A [54243] ScanPower: 100;; 100 LaserPower: 2.47;; 1.32 Temperature: 37.19

source_name: alpha-MSH - 3 hr - Cy3


title: MSH cy3 - 1 day - low pmt - rev repeat 5 - mAdbID:72487

description: mAdb experiment ID: 72487

scan_protocol: Creator: GenePix Pro 5.0.1.24 Scanner: GenePix 4000A [54243] ScanPower: 100;; 100 LaserPower: 2.57;; 1.49 Temperature: 43.31


title: ASP cy3 - 3 hr - low pmt - rev repeat 6 - mAdbID:78027

description: mAdb experiment ID: 78027

scan_protocol: Creator: GenePix Pro 5.0.1.24 Scanner: GenePix 4000A [54243] ScanPower: 100;; 100 LaserPower: 2.43;; 1.34 Temperature: 34.76


title: MSH cy5 - 3 days - low pmt - repeat 3 - mAdbID:72491

description: mAdb experiment ID: 72491

scan_protocol: Creator: GenePix Pro 5.0.1.24 Scanner: GenePix 4000A [54243] ScanPower: 100;; 100 LaserPower: 2.45;; 1.57 Temperature: 38.16

source_name: Untreated melanocytes - Cy3

source_name: alpha-MSH - 3 day - Cy5


title: MSH cy5 - 1 day - low pmt - repeat 2 - mAdbID:72488

description: mAdb experiment ID: 72488

scan_protocol: Creator: GenePix Pro 5.0.1.24 Scanner: GenePix 4000A [54243] ScanPower: 100;; 100 LaserPower: 2.58;; 1.49 Temperature: 42.97


title: MSH cy3 - 4 days - low pmt - rev repeat 6 - mAdbID:72486

description: mAdb experiment ID: 72486

scan_protocol: Creator: GenePix Pro 5.0.1.24 Scanner: GenePix 4000A [54243] ScanPower: 100;; 100 LaserPower: 2.57;; 1.49 Temperature: 42.78

source_name: Untreated melanocytes - Cy5

source_name: alpha-MSH - 4 day - Cy3

treatment_protocol:
Treatment type: compound
Agent: alpha-melanocyte stimulating hormone (sigma)
Treatment dose: 100 nM
Treatment time: 4 days
Treatment temperature: 37 C


title: ASP cy5 - 1 day - low pmt - repeat 1 - mAdbID:78029

description: mAdb experiment ID: 78029

scan_protocol: Creator: GenePix Pro 5.0.1.24 Scanner: GenePix 4000A [54243] ScanPower: 100;; 100 LaserPower: 2.5;; 1.31 Temperature: 34.28


title: Kidney_1K_SBH6 (male)

city: Ashkelon

first: Yoram

last: Yagil

address: Hahistadrut

country: Israel

institute: Ben-Gurion University, Barzilai Medical Center Campus

description: Expression data from male rat kidney

hyb_protocol: Following fragmentation, we hybridized cRNA overnight at 450C on GeneChip 230 2.0 Array. We washed the GeneChips and stained them in an Affymetrix Fluidics Station.

scan_protocol: We scanned the GeneChips using the GenChip Scanner 3000Hewlett-Packard GeneArray Scanner G2500A.

data_processing: The data were analyzed with the GeneChip Operating Software (GCOS); the arrays were normalized by the log scale Robust Multiaray Analysis (RMA)

organism: Rattus norvegicus

label_protocol: We used the Genechip Affymetrix labeling kit and the standard protocol recommended by Affymetrix

growth_protocol: We fed the animals standard rat chow and provided water ad libitum

extract_protocol: We extracted total RNA using the RNeasy mini-kit from QIAGEN (Cat #74104)

tag: Strain:SBH/y after right uninephrectomy Gender:Male Age:4 months Diet:Regular rat chow


title: MSH cy5 - 2 days - low pmt - repeat 3 - mAdbID:72492

description: mAdb experiment ID: 72492

scan_protocol: Creator: GenePix Pro 5.0.1.24 Scanner: GenePix 4000A [54243] ScanPower: 100;; 100 LaserPower: 2.45;; 1.36 Temperature: 39.47

source_name: alpha-MSH - 2 day - Cy5


title: Estradiol and Tamoxifen 2

city: Winston-Salem

first: Charles

last: Wood

address: Medical Center Blvd

department: Pathology

institute: Wake Forest University School of Medicine

description: Data were extracted from scanned images using GeneChip Operating Software(Affymetrix, Inc., Santa Clara,CA.

hyb_protocol: Standard Affymetrix protocol for Eukaryotic samples.  For each sample, ten µg of biotinylated cRNA spiked with bioB, bioC, bioD and cre (Hybridization Control) was hybridized to a Rhesus Macaque Microarray for 16 hours at 45ºC.  Following hybridization, all arrays were washed and stained in an Affymetrix GeneChip Fluidics Station.

scan_protocol: Stained arrays were scanned with an Affymetrix GeneChip® Scanner 3000.

data_processing: Quality checks and data analyses were carried out using Affymetrix GeneChip Operating Software (GCOS) and Expression Console.  The samples were analyzed using the MAS5 algorithm in Affymetrix Expression Console.

label: Biotin dye

organism: Macaca fascicularis

source_name: postmenopausal endometrium, Estradiol and Tamoxifen 2

label_protocol: Enzo Bioarray kit.  Biotinylated cRNA samples were prepared according to the standard Enzo BioarrayTM protocol (Enzo Life Sciences). In brief, one ug of total RNA from each sample was converted to double-stranded cDNA with the BioarrayTM Single-Round RNA Amplification and Labeling Kit (Enzo Life Sciences).  After second-strand synthesis, the cDNA was purified with the cDNA Purification Kit (Enzo Life Sciences).  The resulting double-stranded DNA was then used to generate multiple copies of biotinylated cRNA by in vitro transcription with the BioarrayTM HighYieldTM RNA Transcript Labeling Kit (Enzo Life Sciences).

extract_protocol: Total RNA was extracted from frozen endometrial samples using Tri Reagent (Molecular Research Center, Cincinnati,OH); purified using RNeasy Mini Kit (Qiagen, Valencia,CA); and quantified using NanoDrop ND1000 UV-vis spectrophotometer(NanoDrop, Wilmington,DE). RNA intactness and quality was confirmed using an Agilent 2100 Bioanalyzer (Wilmington,DE).

tag: Adult female ovariectomized macaque

tag: normal endometrium


title: Kidney_2K_SBH5 (male)

description: Expression data from male rat kidney

tag: Strain:SBH/y with 2 indwelling kidneys Gender:Male Age:4 months Diet:Regular rat chow


title: MSH cy3 - 4 days - low pmt - rev repeat 5 - mAdbID:72484

description: mAdb experiment ID: 72484

scan_protocol: Creator: GenePix Pro 5.0.1.24 Scanner: GenePix 4000A [54243] ScanPower: 100;; 100 LaserPower: 2.53;; 1.49 Temperature: 41.94


title: Kidney_1K_SBH2 (male)

description: Expression data from male rat kidney

source_name: Strain:SBH/y after right uninephrectomy Gender:Male


title: Kidney_2K_SBH6 (male)

description: Expression data from male rat kidney

source_name: Strain:SBH/y with 2 indwelling kidneys Gender:Male


title: MSH cy5 - 4 days - low pmt - repeat 2 - mAdbID:72485

description: mAdb experiment ID: 72485

scan_protocol: Creator: GenePix Pro 5.0.1.24 Scanner: GenePix 4000A [54243] ScanPower: 100;; 100 LaserPower: 2.53;; 1.49 Temperature: 42.34

source_name: alpha-MSH - 4 day - Cy5


title: MSH cy5 - 4 days - low pmt - repeat 3 - mAdbID:72483

description: mAdb experiment ID: 72483

scan_protocol: Creator: GenePix Pro 5.0.1.24 Scanner: GenePix 4000A [54243] ScanPower: 100;; 100 LaserPower: 2.52;; 1.49 Temperature: 41.16


title: Kidney_1K_SBH3 (male)

description: Expression data from male rat kidney


title: MSH cy5 - 3 hr - low pmt - repeat 1 - mAdbID:72481

description: mAdb experiment ID: 72481

scan_protocol: Creator: GenePix Pro 5.0.1.24 Scanner: GenePix 4000A [54243] ScanPower: 100;; 100 LaserPower: 2.5;; 1.29 Temperature: 39.62


title: Estradiol and Tamoxifen 3

source_name: postmenopausal endometrium, Estradiol and Tamoxifen 3


title: MSH cy3 - 3 hr - low pmt - rev repeat 5 - mAdbID:72482

description: mAdb experiment ID: 72482

scan_protocol: Creator: GenePix Pro 5.0.1.24 Scanner: GenePix 4000A [54243] ScanPower: 100;; 100 LaserPower: 2.52;; 1.27 Temperature: 41


title: Estradiol and Tamoxifen 4

source_name: postmenopausal endometrium, Estradiol and Tamoxifen 4


title: Estradiol and Tamoxifen 1

source_name: postmenopausal endometrium, Estradiol and Tamoxifen 1


title: MSH cy5 - 2 days - low pmt - repeat 2 - mAdbID:72489

description: mAdb experiment ID: 72489

scan_protocol: Creator: GenePix Pro 5.0.1.24 Scanner: GenePix 4000A [54243] ScanPower: 100;; 100 LaserPower: 2.41;; 1.42 Temperature: 33.29


title: Kidney_2K_SBH4 (male)

description: Expression data from male rat kidney


title: ASP cy3 - 1 day - low pmt - rev repeat 6 - mAdbID:78028

description: mAdb experiment ID: 78028

scan_protocol: Creator: GenePix Pro 5.0.1.24 Scanner: GenePix 4000A [54243] ScanPower: 100;; 100 LaserPower: 2.46;; 1.5 Temperature: 34.84

source_name: ASP - 1 day - Cy3


title: Kidney_1K_SBH5 (male)

description: Expression data from male rat kidney


title: Kidney_2K_SBH3 (male)

description: Expression data from male rat kidney


title: Kidney_1K_SBH4 (male)

description: Expression data from male rat kidney


title: Kidney_1K_SBN5 (male)

description: Expression data from male rat kidney

tag: Strain:SBN/y after right uninephrectomy Gender:Male Age:4 months Diet:Regular rat chow


title: Tamoxifen 4

source_name: postmenopausal endometrium, Tamoxifen 4


title: Kidney_2K_SBH1 (male)

description: Expression data from male rat kidney


title: Kidney_2K_SBH2 (male)

description: Expression data from male rat kidney


title: Kidney_1K_SBN6 (male)

description: Expression data from male rat kidney

source_name: Strain:SBN/y after right uninephrectomy Gender:Male


title: MSH cy3 - 3 days - low pmt - rev repeat 5 - mAdbID:72480

description: mAdb experiment ID: 72480

scan_protocol: Creator: GenePix Pro 5.0.1.24 Scanner: GenePix 4000A [54243] ScanPower: 100;; 100 LaserPower: 2.49;; 1.55 Temperature: 40.12

source_name: alpha-MSH - 3 day - Cy3


title: Kidney_1K_SBN3 (male)

description: Expression data from male rat kidney


title: Kidney_1K_SBN4 (male)

description: Expression data from male rat kidney


title: MSH cy5 - 3 days - low pmt - repeat 2 - mAdbID:72479

description: mAdb experiment ID: 72479

scan_protocol: Creator: GenePix Pro 5.0.1.24 Scanner: GenePix 4000A [54243] ScanPower: 100;; 100 LaserPower: 2.47;; 1.47 Temperature: 33.06


title: Tamoxifen 2

source_name: postmenopausal endometrium, Tamoxifen 2


title: Kidney_1K_SBN2 (male)

description: Expression data from male rat kidney


title: MSH cy5 - 3 hr - low pmt - repeat 3 - mAdbID:71781

description: mAdb experiment ID: 71781

scan_protocol: Creator: GenePix Pro 5.0.1.24 Scanner: GenePix 4000A [54243] ScanPower: 100;; 100 LaserPower: 2.41;; 1.36 Temperature: 38.82

source_name: Untreated melanocytes - Cy3

source_name: alpha-MSH - 3 hr - Cy5


title: Tamoxifen 1

source_name: postmenopausal endometrium, Tamoxifen 1


title: MSH cy3 - 3 hr - low pmt - rev repeat 4 - mAdbID:71782

description: mAdb experiment ID: 71782

scan_protocol: Creator: GenePix Pro 5.0.1.24 Scanner: GenePix 4000A [54243] ScanPower: 100;; 100 LaserPower: 2.43;; 1.38 Temperature: 39.24

source_name: Untreated melanocytes - Cy5

source_name: alpha-MSH - 3 hr - Cy3


title: Tamoxifen 3

source_name: postmenopausal endometrium, Tamoxifen 3


title: Estradiol 4

source_name: postmenopausal endometrium, Estradiol 4


title: Kidney_2K_SBN3 (male)

description: Expression data from male rat kidney

tag: Strain:SBN/y with 2 indwelling kidneys Gender:Male Age:4 months Diet:Regular rat chow


title: Kidney_2K_SBN4 (male)

description: Expression data from male rat kidney

source_name: Strain:SBN/y with 2 indwelling kidneys Gender:Male


title: Estradiol 3

source_name: postmenopausal endometrium, Estradiol 3


title: MSH cy3 - 4 days - low pmt - rev repeat 4 - mAdbID:71778

description: mAdb experiment ID: 71778

scan_protocol: Creator: GenePix Pro 5.0.1.24 Scanner: GenePix 4000A [54243] ScanPower: 100;; 100 LaserPower: 2.39;; 1.63 Temperature: 38.35

source_name: alpha-MSH - 4 day - Cy3


title: Kidney_2K_SBN5 (male)

description: Expression data from male rat kidney


title: MSH cy3 - 3 days - low pmt - rev repeat 4 - mAdbID:71775

description: mAdb experiment ID: 71775

scan_protocol: Creator: GenePix Pro 5.0.1.24 Scanner: GenePix 4000A [54243] ScanPower: 100;; 100 LaserPower: 2.34;; 1.43 Temperature: 36.67


title: 011104A Liver Stage 42 #1

city: Chiyoda-ku

first: TADAYOSHI

last: HAYATA

address: 2-3-10 Kandasurugadai

department: Medical Research Institute

country: Japan

institute: Tokyo Medical and Dental University

hyb_protocol: Manufacturer's protocols (Affymetrix, Santa Clara, CA).

data_processing: Data were normalized by Genedata Refiner  (RMA) and Expressionist.

organism: Xenopus laevis

source_name: Embryonic liver

extract_protocol: Total RNA was isolated using TRIZOL (invitrogen) according to manufacture's instruction.

tag: Stage

tag: Tissue

value: liver

tag: Gender

value: mixed


title: Kidney_2K_SBN2 (male)

description: Expression data from male rat kidney


title: 030604A Liver Stage 42 #2

scan_protocol: Manufacturer's protocols (Affymetrix, Santa Clara, CA).


title: MSH cy5 - 4 days - low pmt - repeat 1 - mAdbID:71777

description: mAdb experiment ID: 71777

scan_protocol: Creator: GenePix Pro 5.0.1.24 Scanner: GenePix 4000A [54243] ScanPower: 100;; 100 LaserPower: 2.36;; 1.43 Temperature: 37.58

source_name: alpha-MSH - 4 day - Cy5


title: Kidney_2K_SBN1 (male)

description: Expression data from male rat kidney

scan_protocol: We scanned the GeneChips using the GenChip Scanner 3000Hewlett-Packard GeneArray Scanner G2500A.


title: 011104A Stomach Stage 42 #1

source_name: Embryonic stomach

label_protocol: Manufacturer's protocols (Affymetrix, Santa Clara, CA).

value: stomach


title: Estradiol 2

scan_protocol: Stained arrays were scanned with an Affymetrix GeneChip® Scanner 3000.

source_name: postmenopausal endometrium, Estradiol 2


title: MSH cy5 - 3 days - low pmt - repeat 1 - mAdbID:71774

description: mAdb experiment ID: 71774

scan_protocol: Creator: GenePix Pro 5.0.1.24 Scanner: GenePix 4000A [54243] ScanPower: 100;; 100 LaserPower: 2.32;; 1.65 Temperature: 35.64

source_name: alpha-MSH - 3 day - Cy5


title: MSH cy3 - 1 day - low pmt - rev repeat 4 - mAdbID:71570

description: mAdb experiment ID: 71570

scan_protocol: Creator: GenePix Pro 5.0.1.24 Scanner: GenePix 4000A [54243] ScanPower: 100;; 100 LaserPower: 2.37;; 1.34 Temperature: 35.53

source_name: alpha-MSH - 1 day - Cy3


title: Estradiol 1

source_name: postmenopausal endometrium, Estradiol 1


title: Control 3

source_name: postmenopausal endometrium, Control 3

tag: adult female ovariectomized macaque


title: Transient expression of vector control in 293HEK cells duplicate

city: New York

first: Brett

middle: S

last: Carver

address: 1275 York Ave

department: Surgery

institute: Memorial Sloan-Kettering Cancer Center

description: 293 HEK cells transiently transfected with ERG or vector control

hyb_protocol: Following fragmentation 12 ug of cRNA were hybridized to Affymetrix HG-U133A chip according to standard protocol

scan_protocol: GeneChips were scanned using the Hewlett-Packard GeneArray scanner

data_processing: The data were processed into CHP and CEL files and analyed using GeneSpring software.

source_name: 293HEK cells vector control

label_protocol: Biotinylated cRNA were prepared from 2ug total RNA

extract_protocol: Cells were incubated with Trizol and RNA was harvested according to the standard Trizol protocol.  RNA was then treated with DNAse.

tag: 293HEK cells expressing vector control duplicate


title: Control 4

source_name: postmenopausal endometrium, Control 4


title: Transient expression of vector control in 293HEK cells

source_name: 293 HEK cells

tag: Transient transfection of 293 HEK cells with vector CMV-Tg2b


title: 030604A Stomach Stage 42 #2


title: 030604A Pancreas Stage 42 #2

hyb_protocol: Manufacturer's protocols (Affymetrix, Santa Clara, CA)

data_processing: Data were normalized by Genedata Refiner (RMA) and Expressionist.

source_name: Embryonic pancreas bud

extract_protocol: Total RNA was extracted using TRIZOL (Invitrogen) according to the manufacture's instruction.

value: pancreas bud

value: Mixed


title: 011104A Pancreas Stage 42 #1

scan_protocol: Manufacturer's protocols (Affymetrix, Santa Clara, CA)

data_processing: Data were normalized by Genedata Refiner (RMA) and expressionist.

extract_protocol: Total RNA was extracted using TRIZOL (Invitrogen) according to manufacture's instruction.


title: Kidney_1K_SBN1 (male)

description: Expression data from male rat kidney

source_name: Strain:SBN/y after right uninephrectomy Gender:Male


title: MSH cy5 - 2 days - low pmt - repeat 1 - mAdbID:71557

description: mAdb experiment ID: 71557

scan_protocol: Creator: GenePix Pro 5.0.1.24 Scanner: GenePix 4000A [54243] ScanPower: 100;; 100 LaserPower: 2.39;; 1.61 Temperature: 41.24

source_name: Untreated melanocytes - Cy3

source_name: alpha-MSH - 2 day - Cy5


title: Transient ERG expression in 293 HEK cells

source_name: Cells 293 HEK

tag: Transient transfection of CMV-Tg2b ERG or vector control, cells harvested 24 hours post-transfection, experiment conducted in independent duplication and RNA processed


title: MSH cy5 - 1 day - low pmt - repeat 1 - mAdbID:71569

description: mAdb experiment ID: 71569

scan_protocol: Creator: GenePix Pro 5.0.1.24 Scanner: GenePix 4000A [54243] ScanPower: 100;; 100 LaserPower: 2.35;; 1.54 Temperature: 34.39

source_name: alpha-MSH - 1 day - Cy5


title: Sample 97 - Ovary exposed to 7 mg O2/L for 14 d

status: Public on Feb 02 2009

city: Duluth

first: Dalma

last: Martinovic

state: MN

address: 6201 Congdon Blvd

department: ORD-NHEERL

institute: U.S. EPA

description: Mention of trade names or commercial products does not constitute endorsement or recommendation for use.

hyb_protocol: All of the labeling and post labeling procedures were conducted in ozone-free enclosure to ensure the integrity of the label.  Labeled materials were set up for the fragmentation reaction according to Agilent protocol described in their processing manual, and hybridized overnight in the rotating oven at 65°C in an ozone-free room.  The Agilent Oligo Microarray Zebrafish slide, with approximately 21, 000 genes represented was used for the gene expression analyses (4 x 44K format, product 015064).  Labeled samples (825 ng) were co-hybridized using sureHyb chambers from Agilent.  Wash conditions used were as outlined in the Agilent processing manual (protocol version 4.0).

scan_protocol: The arrays were scanned using Agilent scanner (G2505B). Agilent feature extraction software was used for extracting array data; the initial data quality analyses were done using Rosetta Luminator software (Rosetta Biosoftware, Seattle, WA).

data_processing: To generate lists of differentially expressed genes, output from Agilent’s feature extraction software was imported using GeneSpring GX 7.3.1 software (Agilent Technologies). The enhanced Agilent FE Import function was used to flag low quality spots based on feature saturation, uniformity, pixel population consistency and signal-to-noise ratio using procedures described in the Agilent Technologies Training Manual (version 4.5, May 2006). The ratios of treatment and reference raw intensity data then were log transformed and normalized using locally weighted least squares regression (lowess) normalization, available in GeneSpring GX 7.3.1.

label_protocol: RNA from each condition of interest (treatment) was labeled with a Cy5 dye and hybridized onto arrays together with Cy3 labeled RNA taken from a standard reference pool of RNA extracted from sexually mature male and female gonads and brains, and female livers. For the microarray analyses, total RNA was amplified using Agilent low input linear amplification kit according to the process outlined by the manufacturer (Agilent Technologies). Amplified target cRNA (1-5 µg) was labeled with either cyanine-5 or cyanine-3 using ULS aRNA Flurorescent Labeling Kits from Kreatech according to the manufacturers protocol (Kreatech Biotechnology, Amsterdam, The Netherlands). Concentrations of labeled cRNA and the label incorporation was determined using Nanodrop-1000 spectrophotometer.

growth_protocol: 25°C, 16:8 light:dark photoperiod, fed live Artemia

extract_protocol: Gonads were thawed, homogenized and total RNA extracted with TRI Reagent® (Sigma-Aldrich, St. Louis, MO) following the manufacturer’s protocol. Total RNA was quantified using a nanodrop ND-100 spectrophotometer (Nanodrop technologies, Wilmington, DE). Integrity and purity of RNA samples was evaluated using an Agilent 2100 Bioanalyzer (Agilent Technologies, Paulo Alto, CA) and an RNA Pico Chip Kit (Agilent).

treatment_protocol: fish were humanely euthanized in buffered tricaine methanesulfonate (Finquel, Argent, Redmond, WA, USA).

value: Female

tag: Age

value: 7 months

value: ab wild-type strain

value: Gonad

tag: Sample

value: Total RNA

source_name: Reference - pool of gonads and brains from males and females, and livers from females

extract_protocol: Tissues were thawed, homogenized and total RNA extracted with TRI Reagent® (Sigma-Aldrich, St. Louis, MO) following the manufacturer’s protocol. Total RNA was quantified using a nanodrop ND-100 spectrophotometer (Nanodrop technologies, Wilmington, DE). Integrity and purity of RNA samples was evaluated using an Agilent 2100 Bioanalyzer (Agilent Technologies, Paulo Alto, CA) and an RNA Pico Chip Kit (Agilent).

value: Females and Males

value: gonads and brains from males and females, livers from females


title: MSH cy3 - 2 days - low pmt - rev repeat 4 - mAdbID:71556

description: mAdb experiment ID: 71556

scan_protocol: Creator: GenePix Pro 5.0.1.24 Scanner: GenePix 4000A [54243] ScanPower: 100;; 100 LaserPower: 2.4;; 1.61 Temperature: 41.76

source_name: Untreated melanocytes - Cy5

tag: Strain

tag: Cell line

source_name: alpha-MSH - 2 day - Cy3