title: human cellular RNA_primary airway epithelial cells_tracheobronchial _H511_2h_0.4mM_HG-U133A_2_YCHSUP501

description: H511_2h_0.4mM_HG-U133A_2_YCHSUP501: Primary epithelial cells from airways of four different healthy individuals were cultivated and grown to full differentiation. Apical sides of the cells were treated with HCLO at different doses (0, 0.4,1, 4mM) for 2 and 6hrs respectively. Total RNA was subjuect to transcriptional analysis.


title: human cellular RNA_primary airway epithelial cells_tracheobronchial _H421_6h_0.4mM_HG-U133A_2_YCHSUP501

status: Public on Jun 03 2008

type: RNA

city: Research Triangle Park

first: Yin

last: Chen

state: NC

address: 6 Davis Dr.

department: Division of Translational Biology

country: USA

institute: Hamner Institutes for Health Sciences

description: H421_6h_0.4mM_HG-U133A_2_YCHSUP501: Primary epithelial cells from airways of four different healthy individuals were cultivated and grown to full differentiation. Apical sides of the cells were treated with HCLO at different doses (0, 0.4,1, 4mM) for 2 and 6hrs respectively. Total RNA was subjuect to transcriptional analysis.

hyb_protocol: standard Affymetrix procedures

scan_protocol: standard Affymetrix procedures

data_processing: Affymetrix GCOS 1.0

label: biotin

molecule: total RNA

organism: Homo sapiens

source_name: Sample Type: human cellular RNA, Cell Line: primary airway epithelial cells, Cell Type: tracheobronchial

label_protocol: standard Affymetrix protocol

growth_protocol: Growth Media: C media, Growth Conditions: 37C, 5%CO2

extract_protocol: Extraction Method: Rneasy mini-kit

tag: Additional Information

value: please return the leftover samples to PI.

tag: Array Type

tag: Amount to Core

tag: Units of Amount

value: ug

tag: Ratio


title: human cellular RNA_primary airway epithelial cells_tracheobronchial _H421_6h_Con_HG-U133A_2_YCHSUP501

description: H421_6h_Con_HG-U133A_2_YCHSUP501: Primary epithelial cells from airways of four different healthy individuals were cultivated and grown to full differentiation. Apical sides of the cells were treated with HCLO at different doses (0, 0.4,1, 4mM) for 2 and 6hrs respectively. Total RNA was subjuect to transcriptional analysis.


title: human cellular RNA_primary airway epithelial cells_tracheobronchial _H465_2h_1mM_HG-U133A_2_YCHSUP501

description: H465_2h_1mM_HG-U133A_2_YCHSUP501: Primary epithelial cells from airways of four different healthy individuals were cultivated and grown to full differentiation. Apical sides of the cells were treated with HCLO at different doses (0, 0.4,1, 4mM) for 2 and 6hrs respectively. Total RNA was subjuect to transcriptional analysis.


title: human cellular RNA_primary airway epithelial cells_tracheobronchial _H510_6h_Con_HG-U133A_2_YCHSUP501

description: H510_6h_Con_HG-U133A_2_YCHSUP501: Primary epithelial cells from airways of four different healthy individuals were cultivated and grown to full differentiation. Apical sides of the cells were treated with HCLO at different doses (0, 0.4,1, 4mM) for 2 and 6hrs respectively. Total RNA was subjuect to transcriptional analysis.


title: human cellular RNA_primary airway epithelial cells_tracheobronchial _H511_2h_4mM_HG-U133A_2_YCHSUP501

description: H511_2h_4mM_HG-U133A_2_YCHSUP501: Primary epithelial cells from airways of four different healthy individuals were cultivated and grown to full differentiation. Apical sides of the cells were treated with HCLO at different doses (0, 0.4,1, 4mM) for 2 and 6hrs respectively. Total RNA was subjuect to transcriptional analysis.


title: human cellular RNA_primary airway epithelial cells_tracheobronchial _H510_6h_1mM_HG-U133A_2_YCHSUP501

description: H510_6h_1mM_HG-U133A_2_YCHSUP501: Primary epithelial cells from airways of four different healthy individuals were cultivated and grown to full differentiation. Apical sides of the cells were treated with HCLO at different doses (0, 0.4,1, 4mM) for 2 and 6hrs respectively. Total RNA was subjuect to transcriptional analysis.


title: human cellular RNA_primary airway epithelial cells_tracheobronchial _H510_6h_0.4mM_HG-U133A_2_YCHSUP501

description: H510_6h_0.4mM_HG-U133A_2_YCHSUP501: Primary epithelial cells from airways of four different healthy individuals were cultivated and grown to full differentiation. Apical sides of the cells were treated with HCLO at different doses (0, 0.4,1, 4mM) for 2 and 6hrs respectively. Total RNA was subjuect to transcriptional analysis.


title: human cellular RNA_primary airway epithelial cells_tracheobronchial _H421_2h_Con_HG-U133A_2_YCHSUP501

description: H421_2h_Con_HG-U133A_2_YCHSUP501: Primary epithelial cells from airways of four different healthy individuals were cultivated and grown to full differentiation. Apical sides of the cells were treated with HCLO at different doses (0, 0.4,1, 4mM) for 2 and 6hrs respectively. Total RNA was subjuect to transcriptional analysis.

treatment_protocol: Treatment_2: in vitro Treatment: HCLO, Treatment Duration: 2, Units of Duration: hour


title: human cellular RNA_primary airway epithelial cells_tracheobronchial _H465_6h_Con_HG-U133A_2_YCHSUP501

description: H465_6h_Con_HG-U133A_2_YCHSUP501: Primary epithelial cells from airways of four different healthy individuals were cultivated and grown to full differentiation. Apical sides of the cells were treated with HCLO at different doses (0, 0.4,1, 4mM) for 2 and 6hrs respectively. Total RNA was subjuect to transcriptional analysis.


title: human cellular RNA_primary airway epithelial cells_tracheobronchial _H510_2h_0.4mM_HG-U133A_2_YCHSUP501

description: H510_2h_0.4mM_HG-U133A_2_YCHSUP501: Primary epithelial cells from airways of four different healthy individuals were cultivated and grown to full differentiation. Apical sides of the cells were treated with HCLO at different doses (0, 0.4,1, 4mM) for 2 and 6hrs respectively. Total RNA was subjuect to transcriptional analysis.


title: human cellular RNA_primary airway epithelial cells_tracheobronchial _H510_2h_4mM_HG-U133A_2_YCHSUP501

description: H510_2h_4mM_HG-U133A_2_YCHSUP501: Primary epithelial cells from airways of four different healthy individuals were cultivated and grown to full differentiation. Apical sides of the cells were treated with HCLO at different doses (0, 0.4,1, 4mM) for 2 and 6hrs respectively. Total RNA was subjuect to transcriptional analysis.


title: human cellular RNA_primary airway epithelial cells_tracheobronchial _H510_2h_1mM_HG-U133A_2_YCHSUP501

description: H510_2h_1mM_HG-U133A_2_YCHSUP501: Primary epithelial cells from airways of four different healthy individuals were cultivated and grown to full differentiation. Apical sides of the cells were treated with HCLO at different doses (0, 0.4,1, 4mM) for 2 and 6hrs respectively. Total RNA was subjuect to transcriptional analysis.


title: human cellular RNA_primary airway epithelial cells_tracheobronchial _H510_2h_Con_HG-U133A_2_YCHSUP501

description: H510_2h_Con_HG-U133A_2_YCHSUP501: Primary epithelial cells from airways of four different healthy individuals were cultivated and grown to full differentiation. Apical sides of the cells were treated with HCLO at different doses (0, 0.4,1, 4mM) for 2 and 6hrs respectively. Total RNA was subjuect to transcriptional analysis.


title: human cellular RNA_primary airway epithelial cells_tracheobronchial _H510_6h_4mM_HG-U133A_2_YCHSUP501

description: H510_6h_4mM_HG-U133A_2_YCHSUP501: Primary epithelial cells from airways of four different healthy individuals were cultivated and grown to full differentiation. Apical sides of the cells were treated with HCLO at different doses (0, 0.4,1, 4mM) for 2 and 6hrs respectively. Total RNA was subjuect to transcriptional analysis.


title: human cellular RNA_primary airway epithelial cells_tracheobronchial _H511_6h_Con_HG-U133A_2_YCHSUP501

description: H511_6h_Con_HG-U133A_2_YCHSUP501: Primary epithelial cells from airways of four different healthy individuals were cultivated and grown to full differentiation. Apical sides of the cells were treated with HCLO at different doses (0, 0.4,1, 4mM) for 2 and 6hrs respectively. Total RNA was subjuect to transcriptional analysis.


title: human cellular RNA_primary airway epithelial cells_tracheobronchial _H465_6h_4mM_HG-U133A_2_YCHSUP501

description: H465_6h_4mM_HG-U133A_2_YCHSUP501: Primary epithelial cells from airways of four different healthy individuals were cultivated and grown to full differentiation. Apical sides of the cells were treated with HCLO at different doses (0, 0.4,1, 4mM) for 2 and 6hrs respectively. Total RNA was subjuect to transcriptional analysis.


title: human cellular RNA_primary airway epithelial cells_tracheobronchial _H511_6h_4mM_HG-U133A_2_YCHSUP501

description: H511_6h_4mM_HG-U133A_2_YCHSUP501: Primary epithelial cells from airways of four different healthy individuals were cultivated and grown to full differentiation. Apical sides of the cells were treated with HCLO at different doses (0, 0.4,1, 4mM) for 2 and 6hrs respectively. Total RNA was subjuect to transcriptional analysis.


title: Streptococcus gordonii coaggregated 3 h replicate 1

status: Public on Jun 02 2008

city: Newcastle upon Tyne

first: Nicholas

middle: S

last: Jakubovics

address: Framlington Place

department: School of Dental Sciences

country: United Kingdom

institute: Newcastle University

description: Biological replicate 1 of 3. Raw data files are : 13044782.mev, monoculture (Cy3) vs coaggregate culture (Cy5) and 13044780.mev, moculture (Cy5) vs coaggregate culture (Cy3).

hyb_protocol: Microarray slides were prehybridized at 42°C for 1 h in 5x SSC, 0.1% SDS, 1% BSA. Slides were washed extensively in deionized water, washed once in isopropyl alcohol and dried. Slides were hybridized with mixtures of Cy3- and Cy5-labeled cDNAs in hybridization buffer (40% formamide, 5x SSC, 0.1% SDS, 0.6 mg/ml sheared salmon sperm DNA) at 42°C for 16 h. Following hybridization, slides were washed twice for 5 min at 25°C in each of three wash buffers: low stringency buffer (2x SSC, 0.1% SDS, 0.1 mM DTT preheated to 55°C), medium stringency buffer (0.1x SSC, 0.1% SDS, 0.1 mM DTT), and high stringency buffer (0.1x SSC, 0.1 mM DTT). Slides were rinsed several times in deionised water and air dried.

scan_protocol: Microarrays were scanned using GenePix Pro v5.0 with a Axon 4000B GenePix scanner (Molecular Devices, Sunnyvale, CA). The intensities of the two dyes at each spot were quantified using TIGR spotfinder.

data_processing: Data were analyzed using the TIGR TM4 Microarray Software Suite. Processing included in-slide replicate analysis and flip dye pair consistency checking in Microarray Data Analysis System (MIDAS). These processes output the geometric means of in-slide replicates and flip dye pairs. Statistical analyses were conducted using Multiexperiment Viewer (MEV).

organism: Streptococcus gordonii str. Challis

source_name: Streptococcus gordonii monoculture

label_protocol: Aminoallyl-labeled cDNA was synthesized using Powerscript reverse transcriptase (Clontech, Moutain View, Cal.), random hexamer primers (Invitrogen) with 5-(3-aminoallyl)-dUTP (Sigma) included in the reaction. Following removal of unincorporated nucleotides on Qiagen MinElute columns, the cDNAs were labeled with Cy3 dye (Amersham Biosciences, Piscataway, NJ) and re-purified on Qiagen MinElute columns. Labeling efficiencies were quantified using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, Del.). Labeled cDNAs were denatured at 95°C for 5 min prior to use in hybridization reactions.

growth_protocol: Cells were cultured for 16 h at 37°C in TYEG medium containing 1% Bacto-tryptone, 0.5% yeast extract, 0.3% K2HPO4 and 0.2% D-glucose, adjusted to pH 7.5. Approximately 2.5 x 10(9) cells were harvested and resuspended in 0.6 ml of a chemically defined medium (CDM), based on Tereleckyj’s FMC with minor modifications (Jakubovics et al., 2008). Samples were vortex mixed for 10 s, diluted to 15 ml (1 x 10(8) cells/ml) and incubated at 37°C for 3 h. Two volumes of RNAprotect reagent (Qiagen, Valencia, Cal.) were added to one volume of culture and vortex mixed for 5 s to stabilize RNA. Cells were harvested, the supernatant was discarded and the pellet was stored at -70°C for up to 48 h prior to RNA extraction.

extract_protocol: Total RNA was extracted using Trizol reagent (Invitrogen, Carlsbad, Cal.). Cell pellets were resuspended in 1 ml Trizol and transferred to Eppendorf tubes containing lysing matrix B (Qbiogene, Morgan Irvine, Cal.). Cells were homogenized in a FastPrep bead beater (Qbiogene) and incubated at 20°C for 10 min. Chloroform (0.2 ml) was added and RNA was purified according to the instructions supplied with Trizol reagent. Extracted RNA was treated with RQ1 DNaseI (Promega, Madison, Wisc.) at 37°C for 1 h and re-purified on Qiagen RNeasy MinElute columns. To ensure that RNA had not degraded during extraction, an aliquot of each sample was analyzed by capillary electrophoresis using an RNA 6000 Nano chip in a Bioanalyzer 2100 system (Agilent Technologies, Santa Clara, Cal.). Purified RNA was stored at -70°C.

treatment_protocol: Total RNA from cell cultures was extracted with Invitrogen Trizol reagent and bead beating, and purified on Qiagen RNeasy columns.

tag: Streptococcus gordonii Challis DL1

source_name: Streptococcus gordonii coaggregated with Actinomyces naeslundii.

growth_protocol: S. gordonii and A. naeslundii cells were cultured 16 h at 37°C in TYEG medium. Approximately 2.5 x 10(9) cells of each species were harvested and resuspended in 0.3 ml of CDM. The cultures were combined and vortex mixed for 10 s to stimulate formation of coaggregates. The mixed culture was diluted to 15 ml (i.e. 1 x 10(8) S. gordonii cells/ml) and incubated at 37°C for 3 h. Two volumes of RNAprotect reagent (Qiagen) were added to one volume of culture and vortex mixed for 5 s to stabilize RNA. Cells were harvested, the supernatant was discarded and the pellet was stored at -70°C for up to 48 h prior to RNA extraction.

extract_protocol: Total RNA was extracted using Trizol reagent (Invitrogen). Cell pellets were resuspended in 1 ml Trizol and transferred to Eppendorf tubes containing lysing matrix B (Qbiogene). Cells were homogenized in a FastPrep bead beater (Qbiogene) and incubated at 20°C for 10 min. Chloroform (0.2 ml) was added and RNA was purified according to the instructions supplied with Trizol reagent. Extracted RNA was treated with RQ1 DNaseI (Promega) at 37°C for 1 h and re-purified on Qiagen RNeasy MinElute columns. To ensure that RNA had not degraded during extraction, an aliquot of each sample was analyzed by capillary electrophoresis using an RNA 6000 Nano chip in a Bioanalyzer 2100 system (Agilent Technologies) RNA was stored at -70°C.


title: Streptococcus gordonii coaggregated 3 h replicate 2

description: Biological replicate 2 of 3. Raw data files are : 13044941.mev, monoculture (Cy3) vs coaggregate culture (Cy5) and 13044793.mev, moculture (Cy5) vs coaggregate culture (Cy3).


title: Streptococcus gordonii coaggregated 3 h replicate 3

description: Biological replicate 3 of 3. Raw data files is: 13044783.mev, monoculture (Cy3) vs coaggregate culture (Cy5).

data_processing: Data were analyzed using the TIGR TM4 Microarray Software Suite. Processing included in-slide replicate analysis in Microarray Data Analysis System (MIDAS). This process output the geometric means of in-slide replicates. Statistical analyses were conducted using Multiexperiment Viewer (MEV).


title: human cellular RNA_primary airway epithelial cells_tracheobronchial _H511_6h_1mM_HG-U133A_2_YCHSUP501

description: H511_6h_1mM_HG-U133A_2_YCHSUP501: Primary epithelial cells from airways of four different healthy individuals were cultivated and grown to full differentiation. Apical sides of the cells were treated with HCLO at different doses (0, 0.4,1, 4mM) for 2 and 6hrs respectively. Total RNA was subjuect to transcriptional analysis.


title: human cellular RNA_primary airway epithelial cells_tracheobronchial _H511_6h_0.4mM_HG-U133A_2_YCHSUP501

description: H511_6h_0.4mM_HG-U133A_2_YCHSUP501: Primary epithelial cells from airways of four different healthy individuals were cultivated and grown to full differentiation. Apical sides of the cells were treated with HCLO at different doses (0, 0.4,1, 4mM) for 2 and 6hrs respectively. Total RNA was subjuect to transcriptional analysis.


title: SitkaSpruce_BC12_BC42_needles

status: Public on Jun 01 2008

city: Vancouver

first: Joerg

last: Bohlmann

state: BC

department: Michael Smith Laboratories

country: Canada

institute: University of British Columbia

hyb_protocol: Following pooling of the appropriate cDNAs, samples were precipitated with linear acrylamide and resuspended in a 45 μL hybridization solution consisting of 0.25M NaPO4, 0.5% SDS, 1x SSC, 2x Denhardt's solution, 1 mM EDTA, 2.75 μL LNA d(T) blocker, 2 μg sheared salmon testes DNA (Invitrogen) and 0.3 μL of Cy5-labeled GFP cDNA (Cy5-dUTP and Ready-To-Go labeling beads, Amersham Pharmacia Biotech). Immediately prior to use, arrays were pre-washed 2x in 0.1% SDS at room temperature for 5 min each, followed by two washes in MilliQ-H2O for 2 min each, 3 min at 95ºC in MilliQ-H2O, and dried by centrifugation (3 min at 2000 rpm in an IEC Centra CL2 centrifuge with rotor IEC 2367-00 in 50 mL conical tube). The cDNA probe was heat denatured at 80ºC for 10 min, then maintained at 65ºC prior to adding to a microarray slide heated to 55ºC, covered with a 22 x 60 x 1.5 mm glass coverslip (Fisher Scientific), and incubated for 16 h at 60ºC. Arrays were washed in 2x SSC, 0.2% SDS at room temperature for 5 min to remove the coverslip, followed by 15 min at 65ºC in the same solution, then three washes of 5 min in 2x SSC at room temperature, and three washes of 5 min in 0.2x SSC at room temperature, and dried by centrifugation. The Cy3 and Cy5 3DNA capture reagent (Genisphere) were then hybridized to the bound cDNA on the microarray in a 45 μL volume consisting of 0.25M NaPO4, 0.5% SDS, 1x SSC, 2x Denhardt's solution, 1 mM EDTA, 2.5 μL Cy3 capture reagent and 2.5 μL Cy5 capture reagent. The 3DNA capture reagent is bound to its complementary cDNA capture sequence on the Cy3 or Cy5 oligo d(T) primers. The second hybridization was performed for 3 h at 60ºC, then washed and dried as before.

scan_protocol: Fluorescent images of hybridized arrays were acquired by using ScanArray Express (PerkinElmer, Foster City, USA). The Cy3 and Cy5 cyanine fluors were excited at 543 nm and 633 nm, respectively. All scans were performed at the same laser power (90%), but with the photomultiplier tube settings for the two channels adjusted such that the ratio of the mean signal intensities was ~1, and the percentage of saturated array elements was < 0.5% but > 0%, while minimizing background fluorescence. Fluorescent intensity data were extracted by using the ImaGene 5.5 software (Biodiscovery, El Segundo, USA).

data_processing: To correct for background intensity, the lowest 10% of median foreground intensities was subtracted from the median foreground intensities. Data were then normalized by variance stabilizing normalization (VSN) to compensate for nonlinearity of intensity distributions (Huber et al., 2002) To identify significant changes in gene expression, a linear mixed-effects model was fit to the normalized intensities in the Cy3 and Cy5 channels of the 32 microarray slides. The model contained an adjustment for dye bias, an array effect indicating which Cy5/Cy3 pair was on each array, a treatment effect indicating sample population and time point, and a random effect to adjust for repeated measures on the same biological sample (Kerr et al., 2000). P-values were computed for each gene-by-treatment effect and Q-values were calculated to adjust for the false discovery rate (FDR) (Storey & Tibshirani, 2003). All statistical analyses were done using the R statistical package (www.r-project.org/).

organism: Picea sitchensis

source_name: Sitka spruce seedlings originating from Prince Rupert, British Columbia, pooled from four trees

label_protocol: Hybridizations were performed using the Genisphere Array350 kit (Genisphere, Hatfield, USA) following manufacturer's instructions. 40 micrograms total RNA was reverse transcribed using Superscript II reverse transcriptase (Invitrogen) and oligo d(T18) primers with a 5' unique sequence overhang specific to either the Cy3 or Cy5 labeling reactions. The RNA strand of the resulting cDNA:RNA hybrid was hydrolyzed in 0.075 M NaOH / 0.0075 M EDTA at 65ºC for 15 min followed by neutralization in 0.175 M Tris-HCl (pH 8.0).

growth_protocol: Trees were grown from seed under greenhouse conditions and transferred after one year to an outdoor common garden at Vancouver, British Columbia, Canada.

extract_protocol:
Total RNA was isolated according to procedures described in Kolosova et al. (2004) Biotechniques 36: 821-824.
Total RNA was quantified and quality checked by spectrophotometer and agarose gel. RNA was also evaluated for integrity and the presence of contaminants using reverse transcription with Superscript II reverse transcriptase (Invitrogen, Carlsbad, USA) with an oligo d(T18) primer and _P32 dGTP incorporation. After removal of unincorporated nucleotides using gel filtration columns (Microspin S-300 HR columns, Amersham Pharmacia Biotech, Buckinghamshire, UK) the resulting cDNA smear was resolved using a vertical 1% agarose alkaline gel and visualized using a Storm 860 phosphorimager (Amersham Pharmacia Biotech).

treatment_protocol: Time point one of five during autumn, 2004; replicate two.

tag: Four year old trees from an outdoor common garden. Needle tissue from current year upper lateral shoots, separated from stem and bark, was collected on August 30, 2004

treatment_protocol: Time point four of five during autumn, 2004; replicate two.

tag: Four year old trees from an outdoor common garden. Needle tissue from current year upper lateral shoots, separated from stem and bark, was collected on December 1, 2004


title: SitkaSpruce_AK23_CA22_needles

source_name: Sitka spruce seedlings originating from Valdez, Alaska, pooled from four trees

treatment_protocol: Time point one of two during autumn, 2004; replicate one.

tag: Four year old trees from an outdoor common garden. Needle tissue from current year upper lateral shoots, separated from stem and bark, was collected on October 18, 2004

source_name: Sitka spruce seedlings originating from Redwood, California, pooled from four trees


title: SitkaSpruce_BC32_BC42_needles

treatment_protocol: Time point three of five during autumn, 2004; replicate two.

tag: Four year old trees from an outdoor common garden. Needle tissue from current year upper lateral shoots, separated from stem and bark, was collected on November 22, 2004


title: SitkaSpruce_CA43_BC43_needles

treatment_protocol: Time point two of two during autumn, 2004; replicate one.


title: SitkaSpruce_BC41_BC11_needles

treatment_protocol: Time point four of five during autumn, 2004; replicate one.

treatment_protocol: Time point one of five during autumn, 2004; replicate one.


title: SitkaSpruce_BC21_BC51_needles

treatment_protocol: Time point two of five during autumn, 2004; replicate one.

treatment_protocol: Time point five of five during autumn, 2004; replicate one.

tag: Four year old trees from an outdoor common garden. Needle tissue from current year upper lateral shoots, separated from stem and bark, was collected on December 13, 2004


title: SitkaSpruce_BC32_BC12_needles


title: SitkaSpruce_CA44_BC44_needles

treatment_protocol: Time point two of two during autumn, 2004; replicate two.


title: SitkaSpruce_BC22_BC42_needles

treatment_protocol: Time point two of five during autumn, 2004; replicate two.


title: SitkaSpruce_BC24_AK24_needles

treatment_protocol: Time point one of two during autumn, 2004; replicate two.


title: SitkaSpruce_BC31_BC21_needles

treatment_protocol: Time point three of five during autumn, 2004; replicate one.


title: SitkaSpruce_BC41_BC21_needles


title: SitkaSpruce_BC43_AK43_needles


title: SitkaSpruce_BC44_AK44_needles


title: SitkaSpruce_CA24_BC24_needles


title: SitkaSpruce_BC12_BC22_needles


title: SitkaSpruce_BC52_BC22_needles

treatment_protocol: Time point five of five during autumn, 2004; replicate two.

tag: Four year old trees from an outdoor common garden. Needle tissue from current year upper lateral shoots, separated from stem and bark, was collected on December 13, 2004.

tag: Four year old trees from an outdoor common garden. Needle tissue from current year upper lateral shoots, separated from stem and bark, was collected on October 18, 2004.


title: SitkaSpruce_AK44_CA44_needles


title: SitkaSpruce_BC51_BC41_needles


title: SitkaSpruce_BC11_BC51_needles


title: SitkaSpruce_AK43_CA43_needles

tag: Four year old trees from an outdoor common garden. Needle tissue from current year upper lateral shoots, separated from stem and bark, was collected on December 1, 2004.


title: SitkaSpruce_BC22_BC32_needles

tag: Four year old trees from an outdoor common garden. Needle tissue from current year upper lateral shoots, separated from stem and bark, was collected on November 22, 2004.


title: SitkaSpruce_BC41_BC31_needles


title: SitkaSpruce_AK24_CA24_needles


title: SitkaSpruce_BC42_BC52_needles


title: SitkaSpruce_BC52_BC32_needles


title: SitkaSpruce_CA22_BC23_needles

tag: Four year old trees from an outdoor common garden. Needle tissue from current year upper lateral shoots, separated from stem and bark, was collected on August 30, 2004.


title: SitkaSpruce_BC31_BC51_needles


title: SitkaSpruce_BC23_AK23_needles


title: field-grown juvenile leaf - slideB - rep 2

city: Stanford

first: John

middle: F

last: Fernandes

state: CA

address: 385 Serra Mall

department: Bio Sci

institute: Stanford University

description: total RNA extracted from pools of 10 siblings

hyb_protocol: Arrays were hybridized for 14 hours at 42 C in a hybridization buffer containing 50% formamide, 5X SSC, and 0.1% SDS.  After hybridization, arrays were initially washed at 42 C in 2X SSC, 01% SDS for 5 minutes and then subject to two, five minute washes in 0.1X SSC at room temperature.  Complete details are available at http://www.maizearray.org/files/Hybridization_Protocol_For_cRNA_Targets.pdf

scan_protocol: Arrays were scanned on an Axon 4100 AL scanner (Molecular Devices Corporation, Sunnyvale CA) at 95% laser power.  Raw data was extracted from the resulting images using the GenePix 6.0 software. (Molecular Devices Corporation, Sunnyvale CA)

data_processing: Each sample was hybridized 4 times to different other samples. The 2 dye intensities were imported into R and normalized with limma ( (lowess for within array, quantile for between arrays). Then the individual intensities were calculated and the 4 measurements for each sample were averaged..

organism: Zea mays

source_name: field-grown juvenile leaf

label_protocol: Total RNA (~1.5 ug) was labeled with either Cy3 or Cy5 using the Ambion Message Amp II target labeling kit (Ambion Cat # 1751) according to the manufacturers recommendations using reduced reaction reagents and volumes in order to conserve reagents.  Complete details are available at http://www.maizearray.org/files/cRNA_Target_Production_Using_RNA_Amplification.pdf

growth_protocol: field grown

extract_protocol: Trizol reagent (Invitrogen), purified by ethanol precipitation.  Samples were concentrated using Qiagen (Valencia CA) MinElute columns

treatment_protocol: field grown, no treatment

tag: Strain

value: B73, Tissue:juvenile leaf, Age:21 days


title: cold - slideB - rep 2

source_name: cold seedling

growth_protocol: planted 2 cm deep in 5 cm of sterilized vermiculite in plastic trays with bottom drainage holes to prevent water-logging.  Trays were saturated with distilled, deionized water then placed under fluorescent illumination (16 h 350 uE m-2) on racks in a walk-in 26ºC room.

treatment_protocol: 10ºC for 24 h, with the final 8 h in the dark

value: B73, Tissue:whole seedling, Age:7 days


title: etiolated, dark - slideB - rep 2

source_name: etiolated, dark seedling

treatment_protocol: tray covered with 3 layers of heavy duty aluminum foil for 7 days, watered only on days 3 and 6


title: heat - slideB - rep 1

source_name: heat seedling

treatment_protocol: 45ºC for 4 h during the light treatment corresponding to the last four hours of the 84 h regime


title: Sitka_spruce_BC11_BC31_needles


title: supplemental UV-B - slideB - rep 2

source_name: supplemental UV-B seedling

treatment_protocol: 8 h supplementary UV-B during the final light illumination


title: Yeast mitochondrial associated RNA with puromycin, prep02 (puro2) Dye swap replicate 2

type: mixed

city: Paris

first: Stéphane

last: LE CROM

address: 46 rue d'Ulm

department: Biology Institute - IBENS

country: France

institute: École normale supérieure

description: Biological replicate 2 of 2, S. cerevisiae Wild Type strain grown on galactose medium at 28°C in presence of 2,1 mM puromycin.

hyb_protocol: 1.5µg of labelled cDNA is mixed with one volume of 2X GE buffer (Agilent) and 10% of blocking agent. The hybridization sample volume is dispense on the selected microarray and incubated overnight at 65¡C in a hybridization oven (Agilent). The array slides are washed 1 minute in "wash 1 buffer" at room temperature and one minute in "wash 2 buffer" at 37¡C. The array slides are then air dryed and ready for scaning.

scan_protocol: Scanned on a genepix 4000B scanner (Axon)

data_processing:
Scanning software: GenePix Pro 6.1
Normalization using Goulphar WebTool http://transcriptome.ens.fr/goulphar/
Genepix flags are removed from the analysis. Spots are considered as saturating spot above 50000 and removed. Background is not subtracted from the main signal. Global Lowess normalisation method.

VALUE column reports log2(Mitochondrial associated RNA/Total RNA) ratios.

molecule: other

organism: Saccharomyces cerevisiae

source_name: Mitochondrial associated RNA from WT (CW252) strain extracted in presence of 2,1mM puromycin 1M KCllabeled with Cyanine-5 (red).

label_protocol: 1.5 µg RNA are retro transcribed using Superscript III retro transcriptase. The cDNA are purified using QiaQuick column (Quiagen) and incubated 1 hour at room temperature in presence of 0,05M NaBicarbonate and NHS-ester Cy5.The coupling is stoped by addition of hydroxylamine to a final concentration of 0,6M. Labeled cDNA are purified using QiaQuick column.

growth_protocol: Cell growth overnight at 28°C in galactose rich medium (1% bactopeptone, 1% yeast extract, 2% galactose, 0.1% KH2PO4 and 0.12% (NH4)2SO4) to an OD = 1.0

extract_protocol: Spheroplastic cell are fractionated by differential centrifugation in presence of 2,1 mM puromycin and 1M KCl. The pellet corresponding to mitochondria is store at -80°C. RNA associated to mitochondria are then extracted using RNEasy Mini Kit (Quiagen)

treatment_protocol: Spheroplastic cells are incubated 10 minutes in presence of  2,1 mM puromycin before being harvested

tag: CW252 WT strain

tag: Mitochondria extracted from CW252 spheroplastic cells

source_name: Total RNA extracted from WT (CW252) in presence of 2,1mM puromycin strain labeled with with Cyanine-3 (green).

label_protocol: 1.5 µg RNA are retro transcribed using Superscript III retro transcriptase. The cDNA are purified using QiaQuick column (Quiagen) and incubated 1 hour at room temperature in presence of 0,05M NaBicarbonate and NHS-ester Cy3.The coupling is stoped by addition of hydroxylamine to a final concentration of 0,6M. Labeled cDNA are purified using QiaQuick column.

extract_protocol: Hot phenol extract procedure followed by RNEasy Mini Kit purification step (Quiagen)

tag: Total RNAs


title: Yeast mitochondrial associated RNA prep 02 (puf02) replicate 2 dye swap

description: Biological replicate 2 of 2, S. cerevisiae delta puf3 strain grown on galactose medium at 28°C.

source_name: Mitochondrial associated RNA from delta puf3 () strain labeled with Cyanine-5 (red).

growth_protocol: Cell grown overnight at 28°C in galactose rich medium (1% bactopeptone, 1% yeast extract, 2% galactose, 0.1% KH2PO4 and 0.12% (NH4)2SO4) to an OD = 1.0

extract_protocol: Spheroplastic cell are fractionated by differential centrifugation. The pellet corresponding to mitochondria is store at -80°C. RNA associated to mitochondria are then extracted using RNEasy Mini Kit (Quiagen)

tag: delta puf3 strain

tag: Mitochondria extracted from  spheroplastic cells

source_name: Total RNA extracted from delta puf3 () strain labeled with Cyanine-3 (green).

extract_protocol: Hot phenol extract procedure and RNEasy Mini Kit (Quiagen)


title: heat - slideB - rep 2


title: salt - slideB - rep 2

source_name: salt seedling

treatment_protocol: watered on the final two days with 250 mM NaCl to saturation (48 h exposure)


title: Yeast mitochondrial associated RNA with puromycin, prep02 (puro2) replicate 2

source_name: Total RNA extracted from WT (CW252) in presence of 2,1mM puromycin  strain labeled with Cyanine-5 (red).

tag: spheroplatic CW252 cells

source_name: Mitochondrial associated RNA from WT (CW252) strain extracted in presence of 2,1mM puromycin 1M KCllabeled with Cyanine-3 (green).

tag: Mitochondria extracted from CW252 spheroplastic cells in presence of 2,1mM puromycin 1M KCl.


title: desiccation - slideB - rep 2

source_name: desiccation seedling

treatment_protocol: watered with 11% w/v mannitol to saturation on the final two days (48 h exposure)


title: cold - slideB - rep 1


title: SitkaSpruce_FB3-425_Bark_UntreatedControl_SelfHyb3

description: Not applicable.

data_processing:
Before data normalization, the lowest 10% of median foreground intensities was subtracted from the median foreground intensities to correct background intensity. After quantification of the signal intensities, data were normalized to compensate for non-linearity of intensity distributions using the variance stabilizing normalization (VSN) method of Huber et al. (2002) Bioinformatics 18: S96-S104.
To assess performance of the spruce 21.8K microarray, a model containing a dye effect and a treatment effect for untreated control (C) minus C' was fit using data from four microarray slides. The C minus C' effect was computed by assigning two of the self-hybridized arrays to be Cy5-Cy3 and two to be Cy3-Cy5. Three analyses were done such that each self-hybridized array was paired with each other self-hybridized array only once, either as a Cy5-Cy3 or a Cy3-Cy5 combination. Expression variance was derived from technical variance between slides. The ratio of the C minus C' parameter estimate to the standard error was used to calculate a t statistic, from which a P value was obtained. The Q value for each effect and gene was calculated for each of the two models to adjust for the false discovery rate [Storey & Tibshirani (2003) Proc. Natl. Acad. Sci. USA. 100: 9440-9445].  All statistical analyses were performed within the R statistical package (www.r-project.org/).

source_name: Sitka spruce FB3-425 untreated control bark pooled from 4 trees

label_protocol: Hybridizations were performed using the Genisphere Array900 kit (Genisphere, Hatfield, USA) following manufacturer's instructions. Ten micrograms total RNA was reverse transcribed using Superscript II reverse transcriptase (Invitrogen) and oligo d(T18) primers with a 5' unique sequence overhang specific to either the Cy3 or Cy5 labeling reactions. The RNA strand of the resulting cDNA:RNA hybrid was hydrolyzed in 0.075 M NaOH / 0.0075 M EDTA at 65ºC for 15 min followed by neutralization in 0.175 M Tris-HCl (pH 8.0).

growth_protocol: Trees were grown under standard greenhouse conditions. See Ralph et al (2006) Plant Cell & Environment 29: 1545-1570 for further details.

extract_protocol:
Total RNA was isolated according to procedures described in Kolosova et al. (2004) Biotechniques 36: 821-824.
Total RNA was quantified and quality checked by spectrophotometer and agarose gel. Poly(A)+ RNA was isolated by oligo d(T) cellulose using the Poly(A) Pure kit (Ambion, Austin, USA), following manufacturer's instructions. RNA was also evaluated for integrity and the presence of contaminants using reverse transcription with Superscript II reverse transcriptase (Invitrogen, Carlsbad, USA) with an oligo d(T18) primer and _P32 dGTP incorporation. After removal of unincorporated nucleotides using gel filtration columns (Microspin S-300 HR columns, Amersham Pharmacia Biotech, Buckinghamshire, UK) the resulting cDNA smear was resolved using a vertical 1% agarose alkaline gel and visualized using a Storm 860 phosphorimager (Amersham Pharmacia Biotech).

treatment_protocol: Untreated control.

tag: Two year old clonal trees grown under greenhouse conditions. Bark, excluding the green portion in the apical leader, was harvested by peeling away the outer tissue from the inner stem. Bark tissue includes cortex, phloem and cambium.


title: light control - slideB - rep 2

source_name: light control seedling

treatment_protocol: 16 h light, adequate watering


title: Yeast mitochondrial associated RNA with puromycin, prep01 (puro1)Dye swap replicate 1

description: Biological replicate 1 of 2, S. cerevisiae Wild Type strain grown on galactose medium at 28°C in presence of 2,1 mM puromycin.


title: SitkaSpruce_BC21_BC11_needles

source_name: Sitka spruce seedlings originating from Prince Rupert, British Columbia, pooled from four trees

treatment_protocol: Time point two of five during autumn, 2004; replicate one.

treatment_protocol: Time point one of five during autumn, 2004; replicate one.


title: Yeast mitochondrial associated RNA prep 01 (puf01) replicate 1

description: Biological replicate 1 of 2, S. cerevisiae delta puf3 strain grown on galactose medium at 28°C.

source_name: Total RNA extracted from delta puf3 () strain labeled with Cyanine-5 (red).

tag: spheroplatic  cells

source_name: Mitochondrial associated RNA from delta puf3 () strainlabeled with Cyanine-3 (green).


title: Yeast mitochondrial associated RNA with puromycin, prep01 (puro1) replicate 1


title: SitkaSpruce_BC52_BC12_needles

treatment_protocol: Time point three of five during autumn, 2004; replicate two.


title: SitkaSpruce_FB3-425_Bark_UntreatedControl_SelfHyb4

description: Not applicable.


title: SitkaSpruce_FB3-425_Bark_UntreatedControl_SelfHyb2

description: Not applicable.

data_processing:
Before data normalization, the lowest 10% of median foreground intensities was subtracted from the median foreground intensities to correct background intensity.  After quantification of the signal intensities, data were normalized to compensate for non-linearity of intensity distributions using the variance stabilizing normalization (VSN) method of Huber et al. (2002) Bioinformatics 18: S96-S104.
To assess performance of the spruce 21.8K microarray, a model containing a dye effect and a treatment effect for untreated control (C) minus C' was fit using data from four microarray slides. The C minus C' effect was computed by assigning two of the self-hybridized arrays to be Cy5-Cy3 and two to be Cy3-Cy5. Three analyses were done such that each self-hybridized array was paired with each other self-hybridized array only once, either as a Cy5-Cy3 or a Cy3-Cy5 combination. Expression variance was derived from technical variance between slides. The ratio of the C minus C' parameter estimate to the standard error was used to calculate a t statistic, from which a P value was obtained. The Q value for each effect and gene was calculated for each of the two models to adjust for the false discovery rate [Storey & Tibshirani (2003) Proc. Natl. Acad. Sci. USA. 100: 9440-9445].  All statistical analyses were performed within the R statistical package (www.r-project.org/).


title: field-grown juvenile leaf - slideB - rep 1

source_name: field-grown juvenile leaf


title: Yeast mitochondrial associated RNA prep 01 (puf01) replicate 1 dye swap


title: SitkaSpruce_FB3-425_Bark_UntreatedControl_SelfHyb1

description: Not applicable.

hyb_protocol: Following pooling of the appropriate cDNAs, samples were precipitated with linear acrylamide and resuspended in a 45 μL hybridization solution consisting of 0.25M NaPO4, 0.5% SDS, 1x SSC, 2x Denhardt's solution, 1 mM EDTA, 2.75 μL LNA d(T) blocker, 2 μg sheared salmon testes DNA (Invitrogen) and 0.3 μL of Cy5-labeled GFP cDNA (Cy5-dUTP and Ready-To-Go labeling beads, Amersham Pharmacia Biotech).  Immediately prior to use, arrays were pre-washed 2x in 0.1% SDS at room temperature for 5 min each, followed by two washes in MilliQ-H2O for 2 min each, 3 min at 95ºC in MilliQ-H2O, and dried by centrifugation (3 min at 2000 rpm in an IEC Centra CL2 centrifuge with rotor IEC 2367-00 in 50 mL conical tube). The cDNA probe was heat denatured at 80ºC for 10 min, then maintained at 65ºC prior to adding to a microarray slide heated to 55ºC, covered with a 22 x 60 x 1.5 mm glass coverslip (Fisher Scientific), and incubated for 16 h at 60ºC.  Arrays were washed in 2x SSC, 0.2% SDS at room temperature for 5 min to remove the coverslip, followed by 15 min at 65ºC in the same solution, then three washes of 5 min in 2x SSC at room temperature, and three washes of 5 min in 0.2x SSC at room temperature, and dried by centrifugation.  The Cy3 and Cy5 3DNA capture reagent (Genisphere) were then hybridized to the bound cDNA on the microarray in a 45 μL volume consisting of 0.25M NaPO4, 0.5% SDS, 1x SSC, 2x Denhardt's solution, 1 mM EDTA, 2.5 μL Cy3 capture reagent and 2.5 μL Cy5 capture reagent.  The 3DNA capture reagent is bound to its complementary cDNA capture sequence on the Cy3 or Cy5 oligo d(T) primers.  The second hybridization was performed for 3 h at 60ºC, then washed and dried as before.

scan_protocol: Fluorescent images of hybridized arrays were acquired by using ScanArray Express (PerkinElmer, Foster City, USA).  The Cy3 and Cy5 cyanine fluors were excited at 543 nm and 633 nm, respectively.  All scans were performed at the same laser power (90%), but with the photomultiplier tube settings for the two channels adjusted such that the ratio of the mean signal intensities was ~1, and the percentage of saturated array elements was < 0.5% but > 0%, while minimizing background fluorescence.  Fluorescent intensity data were extracted by using the ImaGene 5.5 software (Biodiscovery, El Segundo, USA).

data_processing:
Before data normalization, the lowest 10% of median foreground intensities was subtracted from the median foreground intensities to correct background intensity.  After quantification of the signal intensities, data were normalized to compensate for non-linearity of intensity distributions using the variance stabilizing normalization (VSN) method of Huber et al. (2002) Bioinformatics 18: S96-S104.
To assess performance of the spruce 21.8K microarray, a model containing a dye effect and a treatment effect for untreated control (C) minus C' was fit using data from four microarray slides. The C minus C' effect was computed by assigning two of the self-hybridized arrays to be Cy5-Cy3 and two to be Cy3-Cy5. Three analyses were done such that each self-hybridized array was paired with each other self-hybridized array only once, either as a Cy5-Cy3 or a Cy3-Cy5 combination.  Expression variance was derived from technical variance between slides. The ratio of the C minus C' parameter estimate to the standard error was used to calculate a t statistic, from which a P value was obtained. The Q value for each effect and gene was calculated for each of the two models to adjust for the false discovery rate [Storey & Tibshirani (2003) Proc. Natl. Acad. Sci. USA. 100: 9440-9445].  All statistical analyses were performed within the R statistical package (www.r-project.org/).

label_protocol: Hybridizations were performed using the Genisphere Array900 kit (Genisphere, Hatfield, USA) following manufacturer's instructions.  Ten micrograms total RNA was reverse transcribed using Superscript II reverse transcriptase (Invitrogen) and oligo d(T18) primers with a 5' unique sequence overhang specific to either the Cy3 or Cy5 labeling reactions.  The RNA strand of the resulting cDNA:RNA hybrid was hydrolyzed in 0.075 M NaOH / 0.0075 M EDTA at 65ºC for 15 min followed by neutralization in 0.175 M Tris-HCl (pH 8.0).

growth_protocol: Trees were grown under standard greenhouse conditions.  See Ralph et al (2006) Plant Cell & Environment 29: 1545-1570 for further details.

extract_protocol:
Total RNA was isolated according to procedures described in Kolosova et al. (2004) Biotechniques 36: 821-824.
Total RNA was quantified and quality checked by spectrophotometer and agarose gel.  Poly(A)+ RNA was isolated by oligo d(T) cellulose using the Poly(A) Pure kit (Ambion, Austin, USA), following manufacturer's instructions. RNA was also evaluated for integrity and the presence of contaminants using reverse transcription with Superscript II reverse transcriptase (Invitrogen, Carlsbad, USA) with an oligo d(T18) primer and _P32 dGTP incorporation.  After removal of unincorporated nucleotides using gel filtration columns (Microspin S-300 HR columns, Amersham Pharmacia Biotech, Buckinghamshire, UK) the resulting cDNA smear was resolved using a vertical 1% agarose alkaline gel and visualized using a Storm 860 phosphorimager (Amersham Pharmacia Biotech).

tag: Two year old clonal trees grown under greenhouse conditions.  Bark, excluding the green portion in the apical leader, was harvested by peeling away the outer tissue from the inner stem.  Bark tissue includes cortex, phloem and cambium.


title: supplemental UV-B - slideB - rep 1


title: Yeast mitochondrial associated RNA prep 02 (pu0f2) replicate 2

extract_protocol: Hot phenol extract procedure followed by RNEasy Mini Kit purification (Quiagen)


title: Yeast mitochondrial associated RNA with cycloheximide, prep02 (puro2) Dye swap replicate 2

description: Biological replicate 2 of 2, S. cerevisiae Wild Type strain grown on galactose medium at 28°C in presence of 200µg/ml cycloheximide.

source_name: Mitochondrial associated RNA from WT (CW252) strain extracted in presence of 200µg/ml cycloheximidelabeled with Cyanine-5 (red).

extract_protocol: Spheroplastic cell are fractionated by differential centrifugation in presence of 200µg/ml cycloheximide. The pellet corresponding to mitochondria is store at -80°C. RNA associated to mitochondria are then extracted using RNEasy Mini Kit (Quiagen)

treatment_protocol: Spheroplastic cells are incubated 10 minutes in presence of  200µg/ml cycloheximide before being harvested

source_name: Total RNA extracted from WT (CW252) in presence of 200µg/ml cycloheximide strain labeled with with Cyanine-3 (green).


title: desiccation - slideB - rep 1


title: Yeast mitochondrial associated RNA prep 03 replicate 2 dye swap

description: Biological replicate 2 of 3 (6?), S. cerevisiae Wild Type strain grown on galactose medium at 28°C.

source_name: Mitochondrial associated RNA from WT (CW252) strain labeled with Cyanine-5 (red).

source_name: Total RNA extracted from WT (CW252) strain labeled with Cyanine-3 (green).


title: Yeast mitochondrial associated RNA prep 04 replicate 3 dye swap

description: Biological replicate 3 of 3 (6?), S. cerevisiae Wild Type strain grown on galactose medium at 28°C.


title: Yeast mitochondrial associated RNA with cycloheximide, prep02 (puro2) replicate 2

source_name: Total RNA extracted from WT (CW252) in presence of 200µg/ml cycloheximide  strain labeled with Cyanine-5 (red).

source_name: Mitochondrial associated RNA from WT (CW252) strain extracted in presence of 200µg/ml cycloheximidelabeled with Cyanine-3 (green).

tag: Mitochondria extracted from CW252 spheroplastic cells in presence of 200µg/ml cycloheximide.


title: Yeast mitochondrial associated RNA prep 05 replicate 1 dye swap

description: Biological replicate 1 of 3, S. cerevisiae Wild Type strain grown on galactose medium at 28°C.


title: light control - SlideB - rep 4


title: heat - SlideB - rep 4

source_name: heat seedling


title: field-grown juvenile leaf - SlideB - rep 4


title: supplemental UV-B - SlideB - rep 4

source_name: supplemental UV-B seedling


title: cold - SlideB - rep 4

source_name: cold seedling


title: Yeast mitochondrial associated RNA prep 04 replicate 3

source_name: Total RNA extracted from WT (CW252) strain labeled with Cyanine-5 (red).

source_name: Mitochondrial associated RNA from WT (CW252) strainlabeled with Cyanine-3 (green).


title: etiolated, dark - slideB - rep 1

source_name: etiolated, dark seedling


title: Yeast mitochondrial associated RNA with cycloheximide, prep01 (cyclo1) replicate 1

description: Biological replicate 1 of 2, S. cerevisiae Wild Type strain grown on galactose medium at 28°C in presence of 200µg/ml cycloheximide.

source_name: Total RNA extracted from WT (CW252) in presence of 200µg/ml cycloheximide strain labeled with Cyanine-5 (red).


title: Yeast mitochondrial associated RNA prep 05 replicate 1


title: heat - SlideB - rep 3


title: supplemental UV-B - SlideB - rep 3


title: Yeast mitochondrial associated RNA prep 03 replicate 2


title: desiccation - SlideB - rep 4

source_name: desiccation seedling


title: cold - SlideB - rep 3


title: light control - SlideB - rep 3

source_name: light control seedling


title: salt - SlideB - rep 4

source_name: salt seedling


title: salt - slideB - rep 1


title: desiccation - SlideB - rep 3


title: light control - slideB - rep 1


title: etiolated, dark - SlideB - rep 4


title: salt - SlideB - rep 3


title: salt - SlideA - rep 4

tag: Treatment

value: 250 mM NaCl


title: light control  - SlideA - rep 4

value: growth chamber


title: cold - SlideA - rep 4

value: 10ºC for 24 h


title: heat - SlideA - rep 4

value: 45ºC for 4 h


title: etiolated, dark - SlideB - rep 3


title: field-grown juvenile leaf - SlideA - rep 4

source_name: field-grown juvenile leaf

value: no treatment


title: light control  - SlideA - rep 3


title: supplemental UV-B  - SlideA - rep 3

value: UV-B


title: supplemental UV-B - SlideA - rep 2


title: etiolated, dark - SlideA - rep 2

value: covered with 3 layers of heavy duty aluminum foil for 7 days


title: supplemental UV-B  - SlideA - rep 4


title: cold - SlideA - rep 3


title: field-grown juvenile leaf - SlideA - rep 2


title: salt - SlideA - rep 3


title: salt - SlideA - rep 2


title: field-grown juvenile leaf - SlideA - rep 3


title: field-grown juvenile leaf - SlideB - rep 3


title: heat - SlideA - rep 3


title: etiolated, dark - SlideA - rep 3


title: heat - SlideA - rep 2


title: light control  - SlideA - rep 2


title: heat - SlideA - rep 1


title: desiccation - SlideA - rep 4

value: 11% w/v mannitol


title: desiccation - SlideA - rep 3


title: desiccation - SlideA - rep 2


title: cold - SlideA - rep 2


title: cold - SlideA - rep 1


title: desiccation - SlideA - rep 1


title: supplemental UV-B - SlideA - rep 1


title: etiolated, dark - SlideA - rep 1


title: field-grown juvenile leaf - SlideA - rep 1


title: light control - SlideA - rep 1

data_processing: Each sample was hybridized 4 times to different other samples. The 2 dye intensities were imported into R and normalized with limma ( (lowess for within array, quantile for between arrays). Then the individual intensities were calculated and the 4 measurements for each sample were averaged..  .


title: salt - SlideA - rep 1


title: Biological Replicate 3 Technical Replicate 2 HOG1-CCAAX time 0

city: Berkeley

first: Jesse

middle: C

last: Patterson

address: 528 Barker Hall

department: Dept of Molecular and Cell Biology

institute: UC-Berkeley

description: Arrays were post proccessed according to the protocol found at: http://cat.ucsf.edu/pdfs/hybCourseProtocols_v2.6.pdf

hyb_protocol: Sample 7.5 ul (2.5 ug), Pool 7.5 ul (2.5 ug), 20X SSC 3 ul, PolyAdenlylic acid 1.5 ul, 1M HEPES 0.5 ul, 10% SDS 0.5 ul. Bring to 46 ul, for use in AO MAUI mixers. boil for 1:30 mins, let cool to room temperature. Apply to arrays pre-warmed in the MAUI hybridization chamber. Hybridize at 63C in MAUI for 24 hrs as per the MAUI hybridization protocol.

scan_protocol: Scanned with Axon GenePix 4000B scanner.

data_processing: This technical replicate represents unique spots on an array in which each feature was printed in duplicate. Biological replicate 3 technical replicate 1 are values from duplicated spots on the same array from which technical replicate 2 data is derived from. Unnormalized GPR files from GenePix were converted to .mev files by TM4 express converter. .mev files were then normalized by TM4 MIDAS v2.20 beta using LOWESS and SD regularization algorithms.  Values correspond to the log2 ratios of the normalized data. All data analysis was done using TIGR TM4 MeV v4.0.01. The un-normalized .gpr files are attached as supplementary files in biological replicate 3 technical replicate 1.

source_name: Reference pool

label_protocol: 2.5 ug aRNA in 0.1 M Sodium Bicarbonate and 50% DMSO, clean in Zymo Research RNA clean up columns.

extract_protocol: Total RNA extracted according to the Ambion RiboPure(TM)-Yeast kit protocol. 1 ug total RNA was then used to amplify amino-allyl UTP antisense RNA accoding to the Ambion Amino Allyl Message AMP(TM) II aRNA Amplification Kit protocol.

tag: Pooled aRNA from all 6 samples in biological replicate 3

source_name: B3 T2 HOG1-CCAAX 0 mins

tag: Cy3 labelled aRNA created from total RNA isolated from the HOG1-GFP strain at 0 mins hyperosomotic shock with 1 M sorbitol


title: Biological Replicate 3 Technical Replicate 2 HOG1-CCAAX time 60

source_name: B3 T2 HOG1-CCAAX 60 mins

tag: Cy3 labelled aRNA created from total RNA isolated from the HOG1-CCAAX strain at 60 mins hyperosomotic shock with 1 M sorbitol


title: Biological Replicate 3 Technical Replicate 2 HOG1-GFP time 60

source_name: B3 T1 HOG1-GFP 60 mins

tag: Cy3 labelled aRNA created from total RNA isolated from the HOG1-GFP strain at 60 mins hyperosomotic shock with 1 M sorbitol


title: Biological Replicate 3 Technical Replicate 1 HOG1-CCAAX time 0

data_processing: This technical replicate represents unique spots on an array in which each feature was printed in duplicate. Biological replicate 2 technical replicate 2 are values from duplicated spots on the same array from which technical replicate 1 data is derived from. Unnormalized GPR files from GenePix were converted to .mev files by TM4 express converter. .mev files were then normalized by TM4 MIDAS v2.20 beta using LOWESS and SD regularization algorithms. Values correspond to the log2 ratios of the normalized data. All data analysis was done using TIGR TM4 MeV v4.0.01. The un-normalized .gpr files are attached as supplementary files.

source_name: B3 T1 HOG1-CCAAX 0 mins

tag: Cy3 labelled aRNA created from total RNA isolated from the HOG1-CCAAX strain at 0 mins hyperosomotic shock with 1 M sorbitol


title: Biological Replicate 2 Technical Replicate 2 HOG1-CCAAX time 0

data_processing: This technical replicate represents unique spots on an array in which each feature was printed in duplicate. Biological replicate 2 technical replicate 1 are values from duplicated spots on the same array. Unnormalized GPR files from GenePix were converted to .mev files by TM4 express converter. .mev files were then normalized by TM4 MIDAS v2.20 beta using LOWESS and SD regularization algorithms. Values correspond to the log2 ratios of the normalized data. All data analysis was done using TIGR TM4 MeV v4.0.01. The un-normalized .gpr files are attached as supplementary files with Biological replicate 2 technical replicate 1 samples.

tag: Pooled aRNA from all 6 samples in biological replicate 2

source_name: B2 T2 HOG1-CCAAX 0 mins


title: Biological Replicate 3 Technical Replicate 2 hog1 delete time 0

source_name: B3 T2 hog1del 0 mins

tag: Cy3 labelled aRNA created from total RNA isolated from the hog1 delete strain at 0 mins hyperosomotic shock with 1 M sorbitol


title: Biological Replicate 3 Technical Replicate 2 HOG1-GFP time 0

scan_protocol: Scanned with Axon GenePix 4000B scanner.  High and Low PMT scans were taken in order to increase the dynamic range of data collection.

data_processing: This technical replicate represents unique spots on an array in which each feature was printed in duplicate. Biological replicate 3 technical replicate 1 are values from duplicated spots on the same array from which technical replicate 2 data is derived from. Unnormalized GPR files from GenePix were converted to .mev files by TM4 express converter. .mev files were then normalized by TM4 MIDAS v2.20 beta using LOWESS and SD regularization algorithms. If the median intensity of either channel in the high PMT scan was above 50,0000, then the value from the low PMT scan was used.  If the signal to noise ratio was lower then 50 in either channel in the low scan, the value from the high PMT scan was used.  If niether was the case, the values were averaged.  Values correspond to the log2 ratios of the normalized data. All data analysis was done using TIGR TM4 MeV v4.0.01. The un-normalized .gpr files are attached as supplementary files in biological replicate 3 technical replicate 1.

source_name: B3 T2 HOG1-GFP 0 mins


title: Biological Replicate 3 Technical Replicate 2 hog1 delete time 60

source_name: B3 T2 hog1del 60 mins

tag: Cy3 labelled aRNA created from total RNA isolated from the hog1 delete strain at 60 mins hyperosomotic shock with 1 M sorbitol


title: Biological Replicate 3 Technical Replicate 1 HOG1-CCAAX time 60

source_name: B3 T1 HOG1-CCAAX 60 mins


title: Biological Replicate 2 Technical Replicate 2 hog1 delete time 60

source_name: B2 T2 hog1del 60 mins


title: Biological Replicate 2 Technical Replicate 1 HOG1-CCAAX time 0

data_processing: This technical replicate represents unique spots on an array in which each feature was printed in duplicate. Biological replicate 2 technical replicate 2 are values from duplicated spots on the same array. Unnormalized GPR files from GenePix were converted to .mev files by TM4 express converter. .mev files were then normalized by TM4 MIDAS v2.20 beta using LOWESS and SD regularization algorithms. Values correspond to the log2 ratios of the normalized data. All data analysis was done using TIGR TM4 MeV v4.0.01. The un-normalized .gpr files are attached as supplementary files.

source_name: B2 T1 HOG1-CCAAX 0 mins


title: Biological Replicate 3 Technical Replicate 1 hog1 delete time 60

source_name: B3 T1 hog1del 60 mins


title: Biological Replicate 3 Technical Replicate 1 HOG1-GFP time 60

scan_protocol: Scanned with Axon GenePix 4000B scanner.  Both high and low PMT scans were take to increase the dynamic range of data collection.

data_processing: This technical replicate represents unique spots on an array in which each feature was printed in duplicate. Biological replicate 2 technical replicate 2 are values from duplicated spots on the same array. Unnormalized GPR files from GenePix were converted to .mev files by TM4 express converter. .mev files were then normalized by TM4 MIDAS v2.20 beta using LOWESS and SD regularization algorithms. If the high PMT scan had a median intensity of above 50,000 in either channel, the value from the low intensity scan was used.  If the signal to noise ratio was below 50 in either channel in the low PMT scan, the value from the high PMT scan was used.  If niether was the case, the values were averaged.  Values correspond to the log2 ratios of the normalized data. All data analysis was done using TIGR TM4 MeV v4.0.01. The un-normalized .gpr files are attached as supplementary files.


title: Biological Replicate 2 Technical Replicate 2 HOG1-CCAAX time 60

source_name: B2 T2 HOG1-CCAAX 60 mins


title: Biological Replicate 2 Technical Replicate 2 hog1 delete time 0

source_name: B2 T2 hog1del 0 mins


title: Biological Replicate 3 Technical Replicate 1 hog1 delete time 0

source_name: B3 T1 hog1del 0 mins


title: Biological Replicate 3 Technical Replicate 1 HOG1-GFP time 0

source_name: B3 T1 HOG1-GFP 0 mins


title: Orange02 glucose/ethanol limited chemostat -2

city: Delft

first: Jean-Marc

last: Daran

address: Julianalaan 67

department: Department of Biotechnology

country: Netherlands

institute: Delft University of Technology

hyb_protocol: Microarray analysis Sampling of strain Orange02 from aerobic C-limited chemostats with glucose/ethanol mixture, 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). 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. The results for each growth condition were derived from three or more independently cultured replicates. Piper, M. D., P. Daran-Lapujade, C. Bro, B. Regenberg, S. Knudsen, J. Nielsen, and J. T. Pronk. 2002. Reproducibility of oligonucleotide microarray transcriptome analyses. An interlaboratory comparison using chemostat cultures of Saccharomyces cerevisiae. J Biol Chem. 277:37001-8

scan_protocol: Affymetrix scanner 3000

data_processing: Affymetrix GCOS version 2.1 CHIP file targetted at 150

label: SAPE (Phycoerythrin)

source_name: carbon limited chemostat 0.1h-1

growth_protocol:
Chemostat cultivation
Strains Orange02 were grown at 30°C in 2 liter (L) chemostats (Applikon) with a working volume of 1.0 L. Cultures were fed with a defined mineral medium that limited growth by glucose (C-limited) with all other growth requirements in excess and at a constant residual concentration. Strain Orange02 was grown on synthetic medium, containing 7,5 g L-1 glucose supplemented with 9.38 mM ethanol to avoid metabolic oscillations.. The dilution rate was set at 0.10 h-1. The pH was measured online and kept constant at 5.0 by the automatic addition of 2 M KOH with the use of an Applikon ADI 1030 bio controller. Stirrer speed was 800 rpm, and the airflow was 0.5 L min-1. Dissolved oxygen tension was measured on line with an Ingold model 34-100-3002 probe and was above 50% of air saturation. The off-gas was cooled by a condenser connected to a cryostat set at 2°C, and oxygen and carbon dioxide were measured off-line with an NGA 2000 Rosemount gas analyzer. Steady-state samples were taken after ~10-14 volume changes to avoid strain adaptation due to long term cultivation. Dry weight, glucose and ethanol levels, dissolved oxygen and gas profiles had to be constant over at least 3 volume changes before sampling for RNA extraction.

treatment_protocol: fermentation sample quenched in liquid nitrogen

tag: Saccharomyces cerevisiae Orange 02	(CEN.PK 113-5D ura3-52::PTDH3-crtYB-TCYC1; PTDH3-crtI-TCYC1; PTDH3-crtE-TCYC1  (URA3))


title: CENPK113-7D glucose/ethanol limited chemostat -4

hyb_protocol: Microarray analysis Sampling of strain CEN.PK113-7D from aerobic C-limited chemostats with glucose/ethanol mixture, 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). 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. The results for each growth condition were derived from three or more independently cultured replicates. Piper, M. D., P. Daran-Lapujade, C. Bro, B. Regenberg, S. Knudsen, J. Nielsen, and J. T. Pronk. 2002. Reproducibility of oligonucleotide microarray transcriptome analyses. An interlaboratory comparison using chemostat cultures of Saccharomyces cerevisiae. J Biol Chem. 277:37001-8

growth_protocol:
Chemostat cultivation
Strains CEN.PK113-7D were grown at 30°C in 2 liter (L) chemostats (Applikon) with a working volume of 1.0 L. Cultures were fed with a defined mineral medium that limited growth by glucose (C-limited) with all other growth requirements in excess and at a constant residual concentration. Strain Orange02 was grown on synthetic medium, containing 7,5 g L-1 glucose supplemented with 9.38 mM ethanol to avoid metabolic oscillations.. The dilution rate was set at 0.10 h-1. The pH was measured online and kept constant at 5.0 by the automatic addition of 2 M KOH with the use of an Applikon ADI 1030 bio controller. Stirrer speed was 800 rpm, and the airflow was 0.5 L min-1. Dissolved oxygen tension was measured on line with an Ingold model 34-100-3002 probe and was above 50% of air saturation. The off-gas was cooled by a condenser connected to a cryostat set at 2°C, and oxygen and carbon dioxide were measured off-line with an NGA 2000 Rosemount gas analyzer. Steady-state samples were taken after ~10-14 volume changes to avoid strain adaptation due to long term cultivation. Dry weight, glucose and ethanol levels, dissolved oxygen and gas profiles had to be constant over at least 3 volume changes before sampling for RNA extraction.

tag: Saccharomyces cerevisiae CEN.PK 113-7D (MATa, SUC2, MAL2-8 c)


title: Orange02 glucose/ethanol limited chemostat -3

label_protocol:
Microarray analysis
Sampling of strain Orange02 from aerobic C-limited chemostats with glucose/ethanol mixture, 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). 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. The results for each growth condition were derived from three or more independently cultured replicates.
Piper, M. D., P. Daran-Lapujade, C. Bro, B. Regenberg, S. Knudsen, J. Nielsen, and J. T. Pronk. 2002. Reproducibility of oligonucleotide microarray transcriptome analyses. An interlaboratory comparison using chemostat cultures of Saccharomyces cerevisiae. J Biol Chem. 277:37001-8

tag: Saccharomyces cerevisiae Orange 02 (CEN.PK 113-5D ura3-52::PTDH3-crtYB-TCYC1; PTDH3-crtI-TCYC1; PTDH3-crtE-TCYC1  (URA3))


title: Biological Replicate 2 Technical Replicate 2 HOG1-GFP time 0

source_name: Reference pool

source_name: B2 T2 HOG1-GFP 0 mins


title: CENPK-113-7D glucose/ethanol limited chemostat -3

label_protocol:
Microarray analysis
Sampling of strain CEN.PK113-7D from aerobic C-limited chemostats with glucose/ethanol mixture, 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). 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. The results for each growth condition were derived from three or more independently cultured replicates.
Piper, M. D., P. Daran-Lapujade, C. Bro, B. Regenberg, S. Knudsen, J. Nielsen, and J. T. Pronk. 2002. Reproducibility of oligonucleotide microarray transcriptome analyses. An interlaboratory comparison using chemostat cultures of Saccharomyces cerevisiae. J Biol Chem. 277:37001-8

tag: Saccharomyces cervisiae CEN.PK 113-7D (MATa, SUC2, MAL2-8 c)


title: Orange02 glucose/ethanol chemostat -1

extract_protocol:
Microarray analysis
Sampling of strain Orange02 from aerobic C-limited chemostats with glucose/ethanol mixture, 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). 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. The results for each growth condition were derived from three or more independently cultured replicates.
Piper, M. D., P. Daran-Lapujade, C. Bro, B. Regenberg, S. Knudsen, J. Nielsen, and J. T. Pronk. 2002. Reproducibility of oligonucleotide microarray transcriptome analyses. An interlaboratory comparison using chemostat cultures of Saccharomyces cerevisiae. J Biol Chem. 277:37001-8

tag: Saccharomyces cerevisea Orange 02 (CEN.PK 113-5D ura3-52::PTDH3-crtYB-TCYC1; PTDH3-crtI-TCYC1; PTDH3-crtE-TCYC1  (URA3))


title: Biological Replicate 2 Technical Replicate 2 HOG1-GFP time 60

tag: Total RNA extracted according to the Ambion RiboPure(TM)-Yeast kit protocol. 1 ug total RNA was then used to amplify amino-allyl UTP antisense RNA accoding to the Ambion Amino Allyl Message AMP(TM) II aRNA Amplification Kit protocol.

source_name: B2 T2 HOG1-GFP 60 mins


title: Biological Replicate 2 Technical Replicate 1 HOG1-CCAAX time 60

data_processing: This technical replicate represents unique spots on an array in which each feature was printed in duplicate. Biological replicate 2 technical replicate 2 are values from duplicated spots on the same array. Unnormalized GPR files from GenePix were converted to .mev files by TM4 express converter. .mev files were then normalized by TM4 MIDAS v2.20 beta using LOWESS and SD regularization algorithms. Values correspond to the log2 ratios of the normalized data. All data analysis was done using TIGR TM4 MeV v4.0.01. The un-normalized .gpr files are attached as supplementary files

source_name: B2 T1 HOG1-CCCAAX 60 mins


title: CENPK113-7D glucose/ethanol limited chemostat -2

extract_protocol:
Microarray analysis
Sampling of strain CEN.PK113-7D from aerobic C-limited chemostats with glucose/ethanol mixture, 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). 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. The results for each growth condition were derived from three or more independently cultured replicates.
Piper, M. D., P. Daran-Lapujade, C. Bro, B. Regenberg, S. Knudsen, J. Nielsen, and J. T. Pronk. 2002. Reproducibility of oligonucleotide microarray transcriptome analyses. An interlaboratory comparison using chemostat cultures of Saccharomyces cerevisiae. J Biol Chem. 277:37001-8


title: CENPK113-7D glucose/ethanol limited chemostat -1


title: Biological Replicate 2 Technical Replicate 1 hog1 delete time 60

data_processing: This technical replicate represents unique spots on an array in which each feature was printed in duplicate. Biological replicate 2 technical replicate 2 are values from duplicated spots on the same array. Unnormalized GPR files from GenePix were converted to .mev files by TM4 express converter. .mev files were then normalized by TM4 MIDAS v2.20 beta using LOWESS and SD regularization algorithms. Values correspond to the log2 ratios of the normalized data.  All data analysis was done using TIGR TM4 MeV v4.0.01. The un-normalized .gpr files are attached as supplementary files.

source_name: B2 T1 hog1del 60 mins


title: Orange01 glucose limited chemostat -3

hyb_protocol: Sampling ofOrange01 from aerobic C-limited chemostats with glucose as sole carbon source, probe preparation, and hybridization to Affymetrix Genechip® microarrays were performed as described previously by Piper et al. (2002). The results for each growth condition were derived from three or more independently cultured replicates. Piper, M. D., P. Daran-Lapujade, C. Bro, B. Regenberg, S. Knudsen, J. Nielsen, and J. T. Pronk. 2002. Reproducibility of oligonucleotide microarray transcriptome analyses. An interlaboratory comparison using chemostat cultures of Saccharomyces cerevisiae. J Biol Chem. 277:37001-8.

label_protocol:
Sampling of Orange01from aerobic C-limited chemostats with glucose as sole carbon source, probe preparation, and hybridization to Affymetrix Genechip® microarrays were performed as described previously by Piper et al. (2002). The results for each growth condition were derived from three or more independently cultured replicates.
Piper, M. D., P. Daran-Lapujade, C. Bro, B. Regenberg, S. Knudsen, J. Nielsen, and J. T. Pronk. 2002. Reproducibility of oligonucleotide microarray transcriptome analyses. An interlaboratory comparison using chemostat cultures of Saccharomyces cerevisiae. J Biol Chem. 277:37001-8.

growth_protocol:
Chemostat cultivation
Strains orange01 was grown at 30°C in 2 liter (L) chemostats (Applikon) with a working volume of 1.0 L. Cultures were fed with a defined mineral medium that limited growth by glucose (C-limited) with all other growth requirements in excess and at a constant residual concentration. Strains Orange 01 was grown on synthetic medium containing 7,5 g L-1 glucose to maintain carbon limitation. The dilution rate was set at 0.10 h-1. The pH was measured online and kept constant at 5.0 by the automatic addition of 2 M KOH with the use of an Applikon ADI 1030 bio controller. Stirrer speed was 800 rpm, and the airflow was 0.5 L min-1. Dissolved oxygen tension was measured on line with an Ingold model 34-100-3002 probe and was above 50% of air saturation. The off-gas was cooled by a condenser connected to a cryostat set at 2°C, and oxygen and carbon dioxide were measured off-line with an NGA 2000 Rosemount gas analyzer. Steady-state samples were taken after ~10-14 volume changes to avoid strain adaptation due to long term cultivation. Dry weight, glucose and ethanol levels, dissolved oxygen and gas profiles had to be constant over at least 3 volume changes before sampling for RNA extraction.

extract_protocol:
Sampling of Orange01 from aerobic C-limited chemostats with glucose as sole carbon source, probe preparation, and hybridization to Affymetrix Genechip® microarrays were performed as described previously by Piper et al. (2002). The results for each growth condition were derived from three or more independently cultured replicates.
Piper, M. D., P. Daran-Lapujade, C. Bro, B. Regenberg, S. Knudsen, J. Nielsen, and J. T. Pronk. 2002. Reproducibility of oligonucleotide microarray transcriptome analyses. An interlaboratory comparison using chemostat cultures of Saccharomyces cerevisiae. J Biol Chem. 277:37001-8.

tag: Saccharomyces cerevisiae Orange 01 (CEN.PK 113-5D ura3-52::PTDH3-crtYB-TCYC1; PTDH3-crtI-TCYC1; PTDH3-BTS1-TCYC1 (URA3))


title: Orange01 glucose limited chemostat -2


title: CENPK113-7D glucose limited chemostat-1

hyb_protocol: Sampling of CEN.PK113-7D from aerobic C-limited chemostats with glucose as sole carbon source, probe preparation, and hybridization to Affymetrix Genechip® microarrays were performed as described previously by Piper et al. (2002). The results for each growth condition were derived from three or more independently cultured replicates. Piper, M. D., P. Daran-Lapujade, C. Bro, B. Regenberg, S. Knudsen, J. Nielsen, and J. T. Pronk. 2002. Reproducibility of oligonucleotide microarray transcriptome analyses. An interlaboratory comparison using chemostat cultures of Saccharomyces cerevisiae. J Biol Chem. 277:37001-8.

source_name: carbon limited chemostat culture

growth_protocol:
Chemostat cultivation
Strains CEN.PK113-7D was grown at 30°C in 2 liter (L) chemostats (Applikon) with a working volume of 1.0 L. Cultures were fed with a defined mineral medium that limited growth by glucose (C-limited) with all other growth requirements in excess and at a constant residual concentration. Strains CEN.PK113-7D was grown on synthetic medium containing 7,5 g L-1 glucose to maintain carbon limitation. The dilution rate was set at 0.10 h-1. The pH was measured online and kept constant at 5.0 by the automatic addition of 2 M KOH with the use of an Applikon ADI 1030 bio controller. Stirrer speed was 800 rpm, and the airflow was 0.5 L min-1. Dissolved oxygen tension was measured on line with an Ingold model 34-100-3002 probe and was above 50% of air saturation. The off-gas was cooled by a condenser connected to a cryostat set at 2°C, and oxygen and carbon dioxide were measured off-line with an NGA 2000 Rosemount gas analyzer. Steady-state samples were taken after ~10-14 volume changes to avoid strain adaptation due to long term cultivation. Dry weight, glucose and ethanol levels, dissolved oxygen and gas profiles had to be constant over at least 3 volume changes before sampling for RNA extraction.


title: Biological Replicate 2 Technical Replicate 1 hog1 delete time 0

source_name: B2 T1 hog1del 0 mins


title: Yeast mitochondrial associated RNA with cycloheximide, prep01 (puro1)Dye swap replicate 1

source_name: Mitochondrial associated RNA from WT (CW252) strain extracted in presence of 200µg/ml cycloheximide labeled with Cyanine-5 (red).

tag: CW252 WT strain

tag: Mitochondria extracted from CW252 spheroplastic cells

source_name: Total RNA extracted from WT (CW252) in presence of 200µg/ml cycloheximide strain labeled with with Cyanine-3 (green).

tag: Total RNAs


title: CEN-PK113-7D glucose limited chemostat-2

label_protocol:
Sampling of CEN.PK113-7D from aerobic C-limited chemostats with glucose as sole carbon source, probe preparation, and hybridization to Affymetrix Genechip® microarrays were performed as described previously by Piper et al. (2002). The results for each growth condition were derived from three or more independently cultured replicates.
Piper, M. D., P. Daran-Lapujade, C. Bro, B. Regenberg, S. Knudsen, J. Nielsen, and J. T. Pronk. 2002. Reproducibility of oligonucleotide microarray transcriptome analyses. An interlaboratory comparison using chemostat cultures of Saccharomyces cerevisiae. J Biol Chem. 277:37001-8.


title: Biological Replicate 1 Technical Replicate 1 hog1 delete time 0

hyb_protocol: Sample 	             7.5 ul    (2.5 ug), Pool 		7.5 ul  (2.5 ug), 20X SSC	             3 ul, PolyAdenlylic acid	1.5 ul, 1M HEPES	0.5 ul, 10% SDS	             0.5 ul. boil for 1:30 mins, let cool to room temperature. Apply to arrays, in metal hybridization chambers, hyb at 63C for 24 hours.

data_processing: Unnormalized GPR files from GenePix were converted to .mev files by TM4 express converter.  .mev files were then normalized by TM4 MIDAS v2.20 beta using LOWESS and SD regularization algorithms.  Values correspond to the log2 ratios of the normalized data.  All data analysis was done using TIGR TM4 MeV v4.0.01.  The un-normalized .gpr files are attached as supplementary files.

extract_protocol: Total RNA extracted according to the Ambion RiboPure(TM)-Yeast kit protocol.  1 ug total RNA was then used to amplify amino-allyl UTP antisense RNA accoding to the Ambion Amino Allyl Message AMP(TM) II aRNA Amplification Kit protocol.

tag: Pooled aRNA from all 6 samples in technical replicate 1

source_name: B1 T1 hog1del 0

tag: Cy3 labelled aRNA created from total RNA isolated from the delete strain at 0 mins hyperosomotic shock


title: Orange 01 glucose limited chemostat -1


title: Biological Replicate 1 Technical Replicate 3 hog1 delete time 0

tag: Pooled aRNA from all 6 samples in technical replicate 3

source_name: B1 T3 hog1del 0 mins


title: Biological Replicate 2 Technical Replicate 1 HOG1-GFP time 60

scan_protocol: Scanned with Axon GenePix 4000B scanner. Took high and low intensity scans by adjusting Photon multiplier tube(PMT) values.

data_processing: This technical replicate represents unique spots on an array in which each feature was printed in duplicate. Biological replicate 2 technical replicate 2 are values from duplicated spots on the same array. Unnormalized GPR files from GenePix were converted to .mev files by TM4 express converter. .mev files were then normalized by TM4 MIDAS v2.20 beta using LOWESS and SD regularization algorithms. Values correspond to the log2 ratios of the normalized data. High and low scans were used to obtain data on a wider range of intensities. If the median intensity was higher then 50,000 in the high PMT scan, the low intensity scan was used. If the signal to noise value was lower then 50 in the low PMT scan, the value from the high PMT value was used. If niether was the case, the values were averaged. All data analysis was done using TIGR TM4 MeV v4.0.01. The un-normalized .gpr files are attached as supplementary files.

source_name: B2 T1 HOG1-GFP 60 mins

tag: Cy3 labelled aRNA created from total RNA isolated from the HOG1-GFP strain at 60 mins hyperosomotic shock with 1 M sorbitol


title: Biological Replicate 2 Technical Replicate 1 HOG1-GFP time 0

hyb_protocol: Sample 7.5 ul (2.5 ug), Pool 7.5 ul (2.5 ug), 20X SSC 3 ul, PolyAdenlylic acid 1.5 ul, 1M HEPES 0.5 ul, 10% SDS 0.5 ul. Bring to 46 ul, for use in AO MAUI mixers. boil for 1:30 mins, let cool to room temperature. Apply to arrays pre-warmed in the MAUI hybridization chamber.  Hybridize at 63C in MAUI for 24 hrs as per the MAUI hybridization protocol.

scan_protocol: Scanned with Axon GenePix 4000B scanner.  Took high and low intensity scans by adjusting Photon multiplier tube(PMT) values.

data_processing: This technical replicate represents unique spots on an array in which each feature was printed in duplicate.  Biological replicate 2 technical replicate 2 are values from duplicated spots on the same array.  Unnormalized GPR files from GenePix were converted to .mev files by TM4 express converter. .mev files were then normalized by TM4 MIDAS v2.20 beta using LOWESS and SD regularization algorithms. Values correspond to the log2 ratios of the normalized data.  High and low scans were used to obtain data on a wider range of intensities.  If the median intensity was higher then 50,000 in the high PMT scan, the low intensity scan was used.  If the signal to noise value was lower then 50 in the low PMT scan, the value from the high PMT value was used.  If niether was the case, the values were averaged.  All data analysis was done using TIGR TM4 MeV v4.0.01. The un-normalized .gpr files are attached as supplementary files.

tag: Pooled aRNA from all 6 samples in biological replicate 2

source_name: B2 T1 HOG1-GFP 0 mins

tag: Cy3 labelled aRNA created from total RNA isolated from the HOG1-CCAAX strain at 0 mins hyperosomotic shock with 1 M sorbitol


title: CENPK113-7D glucose limited chemostat

extract_protocol:
Sampling of CEN.PK113-7D from aerobic C-limited chemostats with glucose as sole carbon source, probe preparation, and hybridization to Affymetrix Genechip® microarrays were performed as described previously by Piper et al. (2002). The results for each growth condition were derived from three or more independently cultured replicates.
Piper, M. D., P. Daran-Lapujade, C. Bro, B. Regenberg, S. Knudsen, J. Nielsen, and J. T. Pronk. 2002. Reproducibility of oligonucleotide microarray transcriptome analyses. An interlaboratory comparison using chemostat cultures of Saccharomyces cerevisiae. J Biol Chem. 277:37001-8.


title: Biological Replicate 1 Technical Replicate 3 HOG1-CCAAX time 60

source_name: B1 T3 HOG1-CCAAX 60 mins

tag: Cy3 labelled aRNA created from total RNA isolated from the HOG1-CCAAX strain at 60 mins hyperosomotic shock with 1 M sorbitol


title: Biological Replicate 1 Technical Replicate 2 HOG1-GFP time 60

tag: Pooled aRNA from all 6 samples in technical replicate 2

source_name: B1 T2 HOG1-GFP 60 mins


title: Rat Prostate Cancer Cells Over-expressing Ndrg-1

city: Sydney

first: Zaklina

last: Kovacevic

state: NSW

address: Level 5 Blackburn Building D06, University of Sydney

department: Pathology

country: Australia

institute: University of Sydney

description: Standard Affymetrix Protocol on total RNA extracted from AT6.1 cell line

hyb_protocol: Standard Affymetrix Protocol

scan_protocol: Standard Affymetrix Protocol, GeneChip Scanner 3000

data_processing: GeneChip Scanner 3000, GCOS v1.3

label: Biotin

organism: Rattus norvegicus

source_name: Rat Prostate (AT6.1) cells

label_protocol: Standard Affymetrix Protocol

growth_protocol: The cells were cultured in RPMI 1640 with 10% FCS. Streptomycin (100 μg/ml), penicillin (100 μg /ml), minimum non-essential amino acids (0.1 mM), sodium pyruvate (110 μg/mL) and (fungazone (0.28 ng/mL). The selection marker, G418, was used at a concentration of (0.3 mg/mL) to maintain selective pressure on transfected cells.

extract_protocol: Standard TRIzol RNA extraction protocol

tag: Cell line

value: AT6.1 rat prostate cancer cells

tag: Expression vector

tag: Inserted gene

value: N-myc downstream regulated gene-1


title: Biological Replicate 1 Technical Replicate 1 hog1 delete time 60

source_name: B1 T1 hog1del 60

tag: Cy3 labelled aRNA created from total RNA isolated from the hog1 delete strain at 60 mins hyperosomotic shock


title: Biological Replicate 1 Technical Replicate 2 hog1 delete time 60

source_name: Reference pool

source_name: B1 T2 hog1del 60 mins


title: Biological Replicate 1 Technical Replicate 1 HOG1-CCAAX time 60

source_name: B1 T1 HOG1-CCAAX 60 mins


title: Biological Replicate 1 Technical Replicate 3 hog1 delete time 60

source_name: B1 T3 hog1del 60 mins

tag: Cy3 labelled aRNA created from total RNA isolated from the hog1 delete strain at 60 mins hyperosomotic shock with 1 M sorbitol


title: Biological Replicate 1 Technical Replicate 3 HOG1-CCAAX time 0

source_name: B1 T3 HOG1-CCAAX 0 mins


title: Biological Replicate 1 Technical Replicate 2 HOG1-GFP time 0

source_name: B1 T2 HOG1-GFP 0 mins

tag: Cy3 labelled aRNA created from total RNA isolated from the HOG1-GFP strain at 0 mins hyperosomotic shock with 1 M sorbitol


title: Biological Replicate 1 Technical Replicate 1 HOG1-CCAAX time 0

source_name: B1 T1 HOG1-CCAAX 0 mins

tag: Cy3 labelled aRNA created from total RNA isolated from the HOG1-CCAAX strain at 0 mins hyperosomotic shock


title: Biological Replicate 1 Technical Replicate 2 hog1 delete time 0

source_name: B1 T2 hog1del 0 mins

tag: Cy3 labelled aRNA created from total RNA isolated from the hog1 delete strain at 0 mins hyperosomotic shock with 1 M sorbitol


title: Biological Replicate 1 Technical Replicate 2 HOG1-CCAAX time 0

source_name: B1 T2 HOG1-CCAAX 0 mins


title: CENPK113-7D glucose limited chemostat-4

source_name: carbon limited chemostat 0.1h-1

tag: Saccharomyces cerevisiae CEN.PK 113-7D (MATa, SUC2, MAL2-8 c)


title: Biological Replicate 1 Technical Replicate 2 HOG1-CCAAX time 60

source_name: B1 T2 HOG1-CCAAX 60 mins


title: Rat Prostate Cancer Cells Vector Control


title: Biological Replicate 1 Technical Replicate 3 HOG1-GFP time 0

source_name: B1 T3 HOG1-GFP 0 mins