title: TLR3-/- -2

status: Public on Feb 08 2009

city: Jerusalem

first: Reba

last: Condiotti

department: Goldyne Savad Institute of Gene Therapy

country: Israel

institute: Hadassah Medical Center

description: total liver RNA taken from untreated, 3 months old, TLR3-/-, triplicate-2

hyb_protocol: Affymetrix wp protocol

scan_protocol: Affymetrix wp protocol

data_processing: Array data were normalized by the RMA algorithm using Affymetrix Expression Console. Gene and Gene Ontology (GO) annotations were determined for each probe set according to the annotation files published in the Affymetrix website (www.affymetrix.com). Data was transformed to log 2 scale and a threshold of 5 was applied.

source_name: liver RNA TLR3-/-

label_protocol: Affymetrix wp protocol

growth_protocol: mice were self-breeded in SPF

extract_protocol: RNA was extracted using Tri-zol, according to protocol

tag: liver RNA taken from untreated, 3 months old TLR3-/-


title: Sample 66: untreated

status: Public on Feb 10 2009

type: RNA

city: Oklahoma City

first: Bart

last: Frank

state: OK

country: USA

institute: OMRF

hyb_protocol: cRNA was hybridized overnight on Illumina human Ref-8 version 3 BeadChips.  Microarray chips were washed to high stringency and labeled with streptavidin -Cy3 (Amersham Biosciences, Piscataway, NJ) prior to scanning on an Illumina BeadArray Reader.

scan_protocol: Microarrays were scanned on an Illumina BeadArray Reader

data_processing: Initial intensity values were obtained using with Illumina Bead Studio software with background subtraction. For normalization, negative values were replaced with 1, and all values were quantile normalized using Matlab software.

molecule: total RNA

organism: Homo sapiens

source_name: Bladder transitional cell carcinoma J82 was obtained from ATCC (HTB-1; Manassas, VA)

label_protocol: 250 ng of RNA from a single cell culture from each time point was labeled using the Illumina Total Prep RNA Amplification Kit following manufacturer’s directions (Ambion, Austin. TX)

growth_protocol: J82 cells were maintained MEM medium supplemented with 10% FBS, 0.1 mM non-essential amino acids, 1 mM sodium pyruvate, 2% MEM vitamin solution, and 100 units/ml penicillin-, and 100 µg/ml streptomycin.

extract_protocol: Total RNA was isolated at 0 hours (no treatment) or and at 0.5, 1, 2, and 3 hours following exposure to frankincense oil using RNeasy® Mini total RNA isolation kit based on manufacture’s procedures.

treatment_protocol: Cells untreated

tag: J86 cell line untreated


title: 16h anti-CD40 siRNA versus control replicate 2

city: Badalona

first: Lauro

last: Sumoy

state: Barcelona

address: Ctra. Can Ruti, Camí de les escoles s/n

department: High Content Genomics and Bioinformatics

country: Spain

institute: IGTP

description: 16h time point, biological replicate 2 of 2. Technical replicate 1 of 2. HUVEC cells treated with anti-CD40 siRNA activated by D1.1 T lymphocytes overexpressing the CD40L (CD40 ligand) compared to same transfected with control siRNA.

hyb_protocol: Two biological replicate experiments were performed, each comparing msiRNA-2-transfected with siRNA-2-transfected HUVEC. Each experimental pair of labelled samples was co-hybridized on two separate microarrays with dye swapping to correct for dye bias effects. Thus, up to four microarray hybridizations were processed for each of three EC-D1.1 Jurkat cell co-incubation time points, totalling 12 array datasets.

scan_protocol: Fluorescent images were obtained using an Agilent G2565BA scanner at 100% PMT 100% laser power settings and quantified through the GenePix 6.0 software (Axon, Molecular Devices, Sunnywale, CA) using the irregular feature finding option.

data_processing: Extracted raw data were filtered and normalized using the Limma package developed within the Bioconductor project in the R statistical programming environment. The two channels were balanced by lowess normalization using 0.3 as the span parameter with reduced weights in control and poor quality spots, followed by scaling across chips. An Empirical Bayes B statistic was computed taking into account dye swapping and the probes were ranked according to B value, although given the small number of replicates it was only taken as a ranking measure. Differentially expressed genes were chosen, unless otherwise stated, using as cut off criteria a Bayesian atatistic (B rank) percentile above 95% and absolute fold change (|FC|) above 1.2 in at least one of the time points. Moreover, we applied the following quality filter: GenePix flag status found in 6 of the 12 hybridizations and normalized background subtracted intensity in at least one of the two channels above 250 in at least 2 of the 4 hybridizations for each time point.

source_name: human umbilical vein endothelial cells (HUVEC)

label_protocol: 500 ng of total RNA were reverse transcribed, amplified, labelled by in vitro transcription using the Low Input Linear Amplification Kit (Agilent 5184-3523), and hybridized following the manufacturer’s instructions (SSC wash Agilent 60-mer oligo microarray processing protocol, Version 4.1, April 2004).

growth_protocol: HUVEC were maintained in endothelial growth medium EGM-Bullet kit (Clonetics-Cambrex, Walkersville, MD) at 37ºC in a 5% CO2 atmosphere. siRNA-transfected HUVEC were stimulated by co-incubation with Jurkat D1.1 (CD154+) cells (American Type Culture Collection (ATCC), Manassas, VA) (CRL-10915) (1:10 ratio) at 4, 10 or 16 h prior to analysis. At 48 h post-transfection, the co-cultures were extensively washed with warm PBS to release the attached Jurkat D1.1 cells from the HUVEC monolayer. Residual Jurkat D1.1 contamination of HUVEC monolayers was estimated to be less than 0.5% by flow cytometry (data not shown).

extract_protocol: Total RNA  from treated HUVEC were extracted using the RNeasy RNA Isolation kit (Qiagen) and incubated with RNase-free DNase I (Ambion, Austin, TX) following manufacturer's instructions. RNA quantification was performed through a Nanodrop spectrophotometer, and their quality was assessed using the Bioanalyzer 2100 nanoelectrophoresis Lab-on-a–chip system (Agilent, Wilmington, DE). All RNAs used had RIN (RNA integrity number) ranging between 9.3 and 10, and 28S/18S ratios between 1.67 and 2.07.

treatment_protocol: The siRNA duplex targeting human CD40 (siRNA-2) and its corresponding mismatched siRNA control (msiRNA-2) were chemically synthesized (Qiagen, Hilden, Germany). Specific sequences and characterization of their silencing effect in HUVEC have already been described [PMID: 15315968]. Incorporation of the siRNAs into exponentially growing HUVEC (passage 3-4) was achieved by electroporation using an ECM 830 Electropulse Generator System (BTX, San Diego, CA). Briefly, 400 µl of cell suspension containing 2x10E6 cells and 100 nM of siRNA in siPORT siRNA Electroporation Buffer (Ambion, Austin, TX), a low-conductivity electroporation pulse medium, were transferred into a 4-mm electroporation cuvette (BTX), incubated 10 min at 4 ºC, and pulsed for 70 msec at 160V. The electroporation parameters were previously optimized using a FITC-labeled siRNA, reaching 80-90% transfection efficiencies. After electroporation, ECs were recovered in the cuvette for 10 min at room temperature (RT) and transferred into a culture dish with 10 ml of pre-warmed complete growth medium.

tag: HUVEC transfected with anti-CD40 siRNA-2 for 48 hours, activated by co-culture with Jurkat D1.1 T cells (CD40L+) during the last 16 hours

tag: HUVEC transfected with control msiRNA-2 for 48 hours, activated by co-culture with Jurkat D1.1 T cells (CD40L+) during the last 16 hours


title: Sample 68: treated with frankincense oil for 1 hr

scan_protocol: Microarrays were scanned on an Illumina BeadArray Reader.

treatment_protocol: Cells untreated or Cells treated with 1/1000 dilution of frankincense oil (Young Living EWssential Oil, Lehi, Utah) for 1 hr prior to RNA extraction

tag: J86 cell line treated with frankincense oil for 1 hr


title: Adjuvant mice with MLIF, biological rep1

status: Public on Feb 07 2009

city: Zacatecas

first: Juan

last: Velazquez

state: Zacatecas

address: Alameda 45

country: Mexico

institute: Instituto Mexicano del Seguro Social

hyb_protocol: Following fragmentation, 5.5 mg of biotinilated RNA were hybridized for 16 hr at 45C into individual Affymetrix Gene Mouse Arrays 1.0 ST. GeneChips were washed and stained in the GeneChip® Fluidics Station 450 (Affymetrix) according to GeneChip® Whole Transcript (WT) Sense Target Labeling Assay manual (Affymetrix) instructions.

scan_protocol: Microarrays were scanned in a GeneChip® Scanner 3000 7G (Affymetrix) and quality control was evaluated by Affymetrix software (Expresion ConsoleTM).

data_processing: Analysis of generated cell files for each microarray included Benjamini and Hochberg corrections. Normalized dates from Gene arrays were obtained by utilizing GeneSifterTM (VizX Labs LLC, Seattle, WA, USA) (www.genesifter.net). Our scrutiny included the 28,833 genes of the Mouse Gene Array by performing comparison of CFA vs CIA, CFA vs CIA/MLIF, CFA vs MLIF, and CIA vs CIA/MLIF mice groups.

label: biotin

organism: Mus musculus

source_name: Articular tissue of mice treated with emulsified CFA, and MLIF, sacrified at four week after CII booster

label_protocol: Labeling protocol was carried out with 300 ng of purified RNA, with biotin, accord to Affymetrix Gene Mouse Arrays 1.0 ST protocol.

growth_protocol: DBA1/J male mice (Harlan México) maintained under standard conditions at the care facility and fed ad libitum. Mice aged 8-10 weeks and divided into four groups of 8 mice each, were employed.

extract_protocol: Excised mice paws were triturated in Trizol using a politron PT-200 and homogenized into Qiashredder columns. Total RNA extraction was carried out from homogenized material by acid-phenol:chloroform extraction and further purified on a column (QIAGEN). Samples were run on a RNA 6000 Nano Gel System for RNA quality determination.

treatment_protocol: CIA group, was immunized intradermally (i.d) at the base of the tail with 150 μg  of Chicken collagen II (CII) (Sigma Chemical Co., St Louis MO, USA), dissolved in 5mM acetic acid and emulsified (1:1) in complete Freund adjuvant (CFA) (Sigma). According to established protocols, 3 weeks afterward, a booster using this time incomplete Freund adjuvant (IFA) was administered [9] CIA/MLIF-group animals; in addition to CIA protocol, 50 mg of 98 % pure MLIF (EzBiolab, Inc. Westfield, IN, USA) dissolved in 10 ml sterile and pyroxenes-free PBS was applied nasally weekly from 1 week prior to the first CII injection until the end of the experiment. CFA group was immunized i.d. at the base of the tail with 60ml of CFA followed by a boost of IFA 3 weeks latter. MLIF group was treated as CFA group in addition to a weekly nasal application of 50mg of MLIF during 9 weeks.

tag: strain

tag: gender

value: Male

tag: age

value: 8 weeks older

tag: tissue

value: paw articular


title: 4h anti-CD40 siRNA versus control replicate 1

description: 4h time point, biological replicate 1 of 2. Technical replicate 1 of 2. HUVEC cells treated with anti-CD40 siRNA activated by D1.1 T lymphocytes overexpressing the CD40L (CD40 ligand) compared to same transfected with control siRNA.

tag: HUVEC transfected with anti-CD40 siRNA-2 for 48 hours, activated by co-culture with Jurkat D1.1 T cells (CD40L+) during the last 4 hours

tag: HUVEC transfected with control msiRNA-2 for 48 hours, activated by co-culture with Jurkat D1.1 T cells (CD40L+) during the last 4 hours


title: MeCP2 WT (Cy3) versus S80A (Cy5)

city: Los Angeles

first: Yi

last: Sun

state: CA

address: 635, Charles E. Young Dr. South

department: Molecular and Medical Pharmacology

institute: UCLA

description: MeCP2 WT (Cy3) versus S80A (Cy5)

hyb_protocol: Agilent standard procedures

scan_protocol: Agilent Scanner (5nm, PMT 100% for both channels)

data_processing: Agilent Feature Extraction 9.5 (Lowess normalization)

source_name: Mecp2-/y cortical neurons rescued with wild-type MeCP2

label_protocol: Agilent Low RNA Input Fluorescuent Linear Amplification Kit

extract_protocol: Trizol

value: Embryonic day 15.5

source_name: Mecp2-/y cortical neurons rescued with mutant MeCP2 (S80A)


title: TLR3-/- -3

description: total liver RNA taken from untreated, 3 months old, TLR3-/-, triplicate-3


title: TLR3wt -3

description: total liver RNA taken from untreated, 3 months old, TLR3wt C57Bl/6, triplicate-3

source_name: liver RNA C57Bl/6

tag: liver RNA taken from untreated, 3 months old C57Bl/6


title: Adjuvant mice, biological rep1

description: Adjuvant mice, biological rep1

source_name: Articular tissue of mice treated with emulsified CFA, sacrified at four week after CII booster


title: MeCP2 S80A (Cy3) versus WT (Cy5)

description: MeCP2 S80A (Cy3) versus WT (Cy5)

source_name: Mecp2-/y cortical neurons rescued with mutant MeCP2 (S80A)_replicate 2 (dye swap)

source_name: Mecp2-/y cortical neurons rescued with wild-type MeCP2_replicate 2 (dye swap)


title: Adjuvant mice with MLIF, biological rep2


title: TLR3wt -1

description: total liver RNA taken from untreated, 3 months old, TLR3wt C57Bl/6, triplicate-1


title: TLR3wt -2

description: total liver RNA taken from untreated, 3 months old, TLR3wt C57Bl/6, triplicate-2


title: TLR3-/- -1

description: total liver RNA taken from untreated, 3 months old, TLR3-/-, triplicate-1


title: CIA mice with MLIF, biological rep2

description: CIA mice with MLIF, biological rep2

source_name: Articular tissue of mice treated with CII emulsified in CFA, and MLIF, sacrified at four week after CII booster


title: 10h anti-CD40 siRNA versus control replicate 2

description: 10h time point, biological replicate 2 of 2. Technical replicate 1 of 2. HUVEC cells treated with anti-CD40 siRNA activated by D1.1 T lymphocytes overexpressing the CD40L (CD40 ligand) compared to same transfected with control siRNA.

tag: HUVEC transfected with anti-CD40 siRNA-2 for 48 hours, activated by co-culture with Jurkat D1.1 T cells (CD40L+) during the last 10 hours

tag: HUVEC transfected with control msiRNA-2 for 48 hours, activated by co-culture with Jurkat D1.1 T cells (CD40L+) during the last 10 hours


title: CIA mice, biological rep2

description: CIA mice, biological rep2

source_name: Articular tissue of mice treated with CII emulsified in CFA, sacrified at time of clinical severity, four week after CII booster


title: Adjuvant mice, biological rep2

description: Adjuvant mice, biological rep2


title: CIA mice, biological rep1

description: CIA mice, biological rep1


title: retina_time 0_CD133-_donorB

city: BRISTOL

first: DEBRA

middle: ANN

last: CARTER

address: BRISTOL EYE HOSPITAL, LOWER MAUDLIN STREET

department: CLINICAL SCIENCE @ SOUTH BRISTOL

country: United Kingdom

institute: BRISTOL UNIVERSITY

hyb_protocol: using the genechip hybridisation, wash and stain kit

scan_protocol: Array washing and staining as described in chp 6 of above manual using the GeneChip Fluidics station 450 (fluidics script FS450-007).  Arrays were scanned on a GeneChip scanner 3000 7G using default scanner settings. Affymetrix GeneChip® Command Console® (AGCC) was used to process all Affymetrix arrays through fluidics and scanning.

data_processing: Gene level probe set summarisation (signal value), calculation of QC metrics and CHP file generation were performed using GeneChip® Expression Console software using the Robust Multichip Analysis (RMA) algorithm

label_protocol: GeneChip Whole Transcript (WT) sense target labeling assay manual, version 4 -  starting with 300ng RNA and following the Total RNA labeling protocol to prepare targets for GeneChip gene 1.0 ST Arrays(omitting RiboMinus procedure).

growth_protocol: human retina was disected from post mortem eyes (with research consent)

extract_protocol: 300ng of RNA were isolated from magnetically sorted cells using RNeasy mini® kit according to the manufacturers instructions

treatment_protocol: Retina was dissociated to form a single cell population and CD133+ & CD133- cells were isolated using magnetic beads

value: retina


title: retina_time 0_CD133-_donorC


title: retina_time 0_CD133+_donorB


title: retina_time 0_CD133+_donorC


title: retina_time 0_CD133+_donorA


title: retina_time 0_CD133-_donorA


title: Rat_PFOS_3wks_trd_171-JG

city: RTP

first: Hongzu

last: Ren

state: NC

address: 109 TW Alexander Dr

institute: US Environmental Protection Agency

description: gene expression data from rat liver treated with PFOS

hyb_protocol: Following hybridization and washing, staining and scanning procedures were performed in the Genomics facility on Michigan State University campus.

data_processing: The intensity data files (.cel files) were loaded to Rosetta Resolver (Rosetta Biosoftware, Seattle, WA) for analysis.

label: biotin-streptavidin-R-phycoerythrin

organism: Rattus norvegicus

source_name: Sprague-Dawley rats (female only)

label_protocol: First and second strand cDNA was synthesized from total RNA samples (16 µg) using the SuperScript Choice System (Gibco BRL life Technologies).  The rest of sample labeling procedures followed Affymetrix recommended protocols.

extract_protocol: Total RNA from rat liver samples were extracted using TriPure Isolation Reagent (Boehringer Ingelheim, Germany) using the manufacturers recommended procedures.

treatment_protocol:
Sixty-day-old rats were exposed to PFOS for 3 days or 21 days by gavage at a rate of 1 ml/kg body weight.  Liver samples were collected and immediately placed in TriReagent (Boehringer Ingelheim, Germany) and processed immediately for RNA isolation.
Rats received PFOS (5 mg/kg) or vehicle control (0.5% Tween-20) daily by oral gavage.  At the end of exposure, animals were sacrificed by decapitation and liver samples were collected.

value: Sprague-Dawley


title: retina_time 0_CD133+_donor1


title: PFOS-Rat-Liver-10mg-Dix-R95479

description: gene expression data from rat liver treated with PFOA and PFOS

hyb_protocol: Hybridization, washing and scanning were performed according to Affymetrix protocols.

scan_protocol: The fluorescence intensities were measured with Affymetrix GeneChip scanner

source_name: Male Sprague-Dawley (10 weeks old) were obtained from Charles River Laboratories (Hollister, CA).

label_protocol: Total RNA was labeled with Affymetrix protocol.

extract_protocol: Total RNA was isolated using Trizol and RNeasy protocols.

treatment_protocol:
The liver samples were harvested and rapidly frozen in liquid nitrogen, and stored at  80°C until they were processed.
Rats received 20 and 10 mg/kg/day of PFOA and PFOS respectively by oral gavage.
PFOA ammonium salt (CAS# 3825-26-1; > 98% purity) and PFOS potassium salt (CAS# 2795-29-3; > 98% purity) were obtained from Fluka (Steinheim, Switzerland).  Dosing vehicle 15% Alkamuls EL-620 CAS# 61791-12-6), a gift of Rhodia Inc.,West Point, GA.


title: PFOA-PFOS-Rat-Liver-0mg-Dix-_R95434


title: PFOA-Rat-Liver-20mg-Dix-R95470@T2


title: PFOS-Rat-Liver-10mg-Dix-R95481


title: retina_time 0_CD133+_donor3


title: Rat_PFOS_3wks_trd_153-JG

scan_protocol: scanning procedures were performed in the Genomics facility on Michigan State University campus.


title: retina_time 0_CD133+_donor2


title: PFOS-Rat-Liver-10mg-Dix-R95480

treatment_protocol:
Rats received 20 and 10 mg/kg/day of PFOA and PFOS respectively by oral gavage.
The liver samples were harvested and rapidly frozen in liquid nitrogen, and stored at  80°C until they were processed.
PFOA ammonium salt (CAS# 3825-26-1; > 98% purity) and PFOS potassium salt (CAS# 2795-29-3; > 98% purity) were obtained from Fluka (Steinheim, Switzerland).  Dosing vehicle 15% Alkamuls EL-620 CAS# 61791-12-6), a gift of Rhodia Inc.,West Point, GA.


title: PFOA-Rat-Liver-20mg-Dix-R95471


title: PFOA-PFOS-Rat-Liver-0mg-Dix-_R95436


title: PFOA-Rat-Liver-20mg-Dix-R95472


title: retina_time 0_CD133+_donor4


title: Rat_PFOS_3wks_ctr_170-JG


title: PFOA-PFOS-Rat-Liver-0mg-Dix-_R95435


title: Rat_PFOS_3wks_ctr_152-JG


title: Nrf2-D3T-Sutter-Rat-liver--HDD3T-2

description: gene expression data from rat liver treated with D3T

hyb_protocol: Forty g of transcripts, fragmented to 50–150 nt size, were placed in a solution of 1.0 M NaCl, 10 mM Tris-HCl (pH 7.6), and 0.005% Triton X-100, containing herring sperm DNA (0.1 mg/ml) and 5 nM control oligonucleotides.  Targets were hybridized for 16 h at 45°C, and the arrays were washed at 50°C with 6  SSET [0.9 M NaCl, 60 mM NaH2PO4, 6 mM EDTA, and 0.005% Triton X-100 (pH 7.6)] and then at 40°C with 0.5  SSET.

scan_protocol: The fluorescence intensities were measured with a laser confocal scanner (Agilent/Affymetrix).

source_name: Female Sprague Dawley rats, 4 weeks old, were obtained from Harlan Sprague-Dawley, Inc. (Indianapolis, IN).

label_protocol: Total RNA (5 µg) and the T7-polyT primer were used to synthesize cDNA (Ambion Megascript System) by in vitro transcription in the presence of biotinylated UTP and CTP (Enzo Diagnostics).

extract_protocol: Total RNA was isolated using the methods described in Chomczynski and Sacchi (Anal. Biochem.,162: 156–169, 1987)

treatment_protocol:
4 weeks old rats, were randomly assigned to treatment groups (n=4/group) and fed Harlan Teklad Rodent Diet, 4% mash diet (control), or the same formulation containing D3T (3H-1,2-dithiole-3-thione, 600 mg/kg diet).
The rats were sacrificed with carbon dioxide. The livers were harvested within 3 min, rapidly frozen in liquid nitrogen, and stored at  80°C until they were processed.

value: Sprague Dawley rats


title: Nrf2-D3T-Sutter-Rat-liver--HDD3T-1


title: Nrf2-D3T-Sutter-Rat-liver-Control-1


title: Nrf2-D3T-Sutter-Rat-liver--Control-4


title: Nrf2-D3T-Sutter-Rat-liver-Control-3


title: Nrf2-D3T-Sutter-Rat-liver--Control-2


title: Nrf2-D3T-Sutter-Rat-liver--HDD3T-4


title: Nrf2-D3T-Sutter-Rat-liver--HDD3T-3


title: 5515_female_rat_liver_PCN_5day

description: gene expression data from phenobarbital or PCN treated rat liver

hyb_protocol: Samples were hybridized according to the Affymetrix protocol

scan_protocol: scanned with the Hewlett Packard GeneArray Scanner

data_processing: Affymetrix scan data (.cel files) were imported into Rosetta Resolver for analysis

source_name: Male and female Sprague-Dawley rats were dosed orally by gavage with PCN (100 mg/kg), PB (100 mg/kg), or vehicle (0.5% aqueous methylcellulose with 1% Tween 80

label_protocol: Double-stranded cDNA was synthesized, purified, and used to transcribe biotin-labeled cRNA with the BioArray High Yield Transcript Labeling Kit (ENZO Life Sciences, Farmingdale, NY)

extract_protocol: Total RNA was isolated using the Trizole reagent (Invitrogen Corporation, Carlsbad, CA) and the Qiagen Rneasy mini RNA cleanup protocol (Qiagen, Valencia, CA)

treatment_protocol: Animals were housed individually in suspended wire cages and maintained on a diet of Certified Rodent Chow.  Male and female rats (n= 5 per sex) were dosed orally by gavage with PCN (100 mg/kg), PB (100 mg/kg), or vehicle (0.5% aqueous methylcellulose with 1% Tween 80). Livers were collected at 6 and 24 h after a single dose or 24 h after 5 consecutive days of dosing (n = 5 animals per sex per time point), immediately flash frozen in liquid N2, and stored at approximately -70 C.

value: Sprague-Dawley rats


title: 5513_female_rat_liver_PCN_5day


title: 5511_female_rat_liver_PCN_5day


title: 5507_female_rat_liver_PCN_24hr


title: 5512_female_rat_liver_PCN_5day


title: 5505_female_rat_liver_PCN_6hr


title: 5504_female_rat_liver_PCN_6hr


title: 5508_female_rat_liver_PCN_24hr


title: 5514_female_rat_liver_PCN_5day


title: 5509_female_rat_liver_PCN_24hr


title: 5510_female_rat_liver_PCN_24hr


title: 5506_female_rat_liver_PCN_24hr


title: 5501_female_rat_liver_PCN_6hr


title: 5502_female_rat_liver_PCN_6hr


title: 5014_male_rat_liver_PCN_5day


title: 5011_male_rat_liver_PCN_5day


title: 5503_female_rat_liver_PCN_6hr


title: 5013_male_rat_liver_PCN_5day


title: 5012_male_rat_liver_PCN_5day


title: 5015_male_rat_liver_PCN_5day


title: 5008_male_rat_liver_PCN_24hr


title: 5007_male_rat_liver_PCN_24hr


title: 5009_male_rat_liver_PCN_24hr


title: 5004_male_rat_liver_PCN_6hr


title: 5003_male_rat_liver_PCN_6hr


title: 5001_male_rat_liver_PCN_6hr


title: 4512_female_rat_liver_PB_5day


title: 4515_female_rat_liver_PB_5day


title: 5006_male_rat_liver_PCN_24hr


title: 5005_male_rat_liver_PCN_6hr


title: 4514_female_rat_liver_PB_5day


title: 4511_female_rat_liver_PB_5day


title: 4505_female_rat_liver_PB_6hr


title: 4509_female_rat_liver_PB_24hr


title: 5002_male_rat_liver_PCN_6hr


title: 4504_female_rat_liver_PB_6hr


title: 4506_female_rat_liver_PB_24hr


title: 4508_female_rat_liver_PB_24hr


title: 4502_female_rat_liver_PB_6hr


title: 4501_female_rat_liver_PB_6hr


title: 5010_male_rat_liver_PCN_24hr


title: 4503_female_rat_liver_PB_6hr


title: 4012_male_rat_liver_PB_5day


title: 4507_female_rat_liver_PB_24hr


title: 4510_female_rat_liver_PB_24hr


title: 4513_female_rat_liver_PB_5day


title: 4011_male_rat_liver_PB_5day


title: 4013_male_rat_liver_PB_5day


title: 4010_male_rat_liver_PB_24hr


title: 4015_male_rat_liver_PB_5day


title: 4009_male_rat_liver_PB_24hr


title: 4006_male_rat_liver_PB_24hr


title: 4014_male_rat_liver_PB_5day


title: 4008_male_rat_liver_PB_24hr


title: 4002_male_rat_liver_PB_6hr


title: 4005_male_rat_liver_PB_6hr


title: 4003_male_rat_liver_PB_6hr


title: 4004_male_rat_liver_PB_6hr


title: 4001_male_rat_liver_PB_6hr


title: Wy_7d_Ellinger 14

description: gene expression data from rat liver treated with Wy-14643.

hyb_protocol: following Affymetrix protocol

source_name: Male Wistar Hanover rats were from Charles River Laboratories Inc. (Raleigh, NC)

label_protocol: Total RNA (5 ug) and the T7-polyT primer were used to synthesize cDNA by in vitro transcription in the presence of biotinylated UTP and CTP.

extract_protocol: RNA isolation with Qiagen RNAeasy 96 well kits and quality control using RNA 6000 Nano chips on an Agilent 2001 Bioanalyzer (Agilent Technologies GmbH, Germany) were performed.

treatment_protocol:
Wy-14643 were administered for 1, 3, 7, and 14 days.
Following treatment, the first surviving five animals/dose/test group were sacrificed during the same time period the morning following the last dose.
Wy-14643 was from TCI America (Portland, OR; CAS 50892-23-4; purity 100%; 60 mg/kg/day).  Carboxymethyl cellulose was used as vehicle.

value: Wistar Hanover rats


title: Wy_7d_Ellinger 13


title: 1514_female_rat_liver_methylcellulose_5day


title: 1513_female_rat_liver_methylcellulose_5day


title: 1515_female_rat_liver_methylcellulose_5day


title: Ctr_7d_Ellinger 35


title: 4007_male_rat_liver_PB_24hr


title: 1511_female_rat_liver_methylcellulose_5day


title: Wy_7d_Ellinger 12


title: Ctr_7d_Ellinger 34


title: Ctr_7d_Ellinger 33


title: CIA mice with MLIF, biological rep1

description: CIA mice with MLIF, biological rep1


title: 1512_female_rat_liver_methylcellulose_5day


title: Wy_3d_Ellinger 08


title: Wy_3d_Ellinger 07


title: Wy_3d_Ellinger 06

treatment_protocol:
Wy-14643 were administered for 1, 3, 7, and 14 days.
Following treatment, the first surviving five animals/dose/test group were sacrificed during the same time period the morning following the last dose.


title: 1503_female_rat_liver_methylcellulose_6hr


title: 1508_female_rat_liver_methylcellulose_24hr


title: 1504_female_rat_liver_methylcellulose_6hr


title: 1510_female_rat_liver_methylcellulose_24hr


title: Ctr_3d_Ellinger 29


title: 1011_male_rat_liver_methylcellulose_5day


title: 1506_female_rat_liver_methylcellulose_24hr


title: 1509_female_rat_liver_methylcellulose_24hr


title: 1502_female_rat_liver_methylcellulose_6hr


title: 1501_female_rat_liver_methylcellulose_6hr


title: 1507_female_rat_liver_methylcellulose_24hr


title: Ctr_3d_Ellinger 28


title: Ctr_3d_Ellinger 26


title: Wy_1d_Ellinger 05


title: Wy_1d_Ellinger 04


title: 1007_male_rat_liver_methylcellulose_24hr


title: 1014_male_rat_liver_methylcellulose_5day


title: 1015_male_rat_liver_methylcellulose_5day


title: 1012_male_rat_liver_methylcellulose_5day


title: 1505_female_rat_liver_methylcellulose_6hr


title: 1013_male_rat_liver_methylcellulose_5day


title: 1006_male_rat_liver_methylcellulose_24hr


title: 1009_male_rat_liver_methylcellulose_24hr


title: Wy_1d_Ellinger 03


title: 1010_male_rat_liver_methylcellulose_24hr


title: 1008_male_rat_liver_methylcellulose_24hr


title: Ctr_1d_Ellinger 23


title: Ctr_1d_Ellinger 22


title: 1005_male_rat_liver_methylcellulose_6hr


title: 1002_male_rat_liver_methylcellulose_6hr


title: 1003_male_rat_liver_methylcellulose_6hr


title: 1001_male_rat_liver_methylcellulose_6hr


title: 37.6wk preeclampsia basal plate biopsy_U133A

status: Public on Feb 06 2009

city: SAN FRANCISCO

first: Susan

last: Fisher

address: 513 PARNASSUS AVE

department: Obstretrics, Gynecology and Reproductive Sciences

institute: UCSF

description: Gene expression data.

hyb_protocol: Following fragmentation, 15 ug of cRNA were hybridized on Human GeneChip U133A.

scan_protocol: GeneChips were scanned using a  GeneArray Scanner.

data_processing: RMA normalized using RMAExpress.

source_name: human placenta, preeclampsia

label_protocol: Double-stranded cDNAs were generated from total RNA samples by using SuperScript II reverse transcriptase (Invitrogen) and a T7-oligo primer (QIAGEN). Biotin-labeled cRNA was synthesized by in vitro transcription using an Enzo Bioassay RNA labeling kit.

extract_protocol: RNA was isolated from snap-frozen basal plate specimens using a modified Trizol method (keeping specimens cold).

tag: condition

value: 37.6wk preeclamptic


title: 37.6wk preeclampsia basal plate biopsy_U133B

description: Gene expression data.

hyb_protocol: Following fragmentation, 15 ug of cRNA were hybridized on Human GeneChip U133B.


title: 35.9wk preeclampsia basal plate biopsy_U133B

description: Gene expression data.

value: 35.9wk preeclamptic


title: 35.9wk preeclampsia basal plate biopsy_U133A

description: Gene expression data.


title: 34.0wk preeclampsia basal plate biopsy_U133B

description: Gene expression data.

value: 34.0wk preeclamptic


title: 1004_male_rat_liver_methylcellulose_6hr


title: 34.0wk preeclampsia basal plate biopsy_U133A

description: Gene expression data.


title: Ctr_1d_Ellinger 24


title: 32.0wk preeclampsia basal plate biopsy_U133A

description: Gene expression data.

value: 32.0wk preeclamptic


title: 33.0wk preeclampsia basal plate biopsy_U133B

description: Gene expression data.

value: 33.0wk preeclamptic


title: 31.9wk preeclampsia basal plate biopsy_U133A

description: Gene expression data.

value: 31.9wk preeclamptic


title: 32.9wk preeclampsia basal plate biopsy_U133A

description: Gene expression data.

value: 32.9wk preeclamptic


title: 33.0wk preeclampsia basal plate biopsy_U133A

description: Gene expression data.


title: 32.0wk preeclampsia basal plate biopsy_U133B

description: Gene expression data.


title: 31.7wk preeclampsia basal plate biopsy_U133A

description: Gene expression data.

value: 31.7wk preeclamptic


title: 32.9wk preeclampsia basal plate biopsy_U133B

description: Gene expression data.


title: 31.1wk preeclampsia basal plate biopsy_U133B

description: Gene expression data.

value: 31.1wk preeclamptic


title: 31.7wk preeclampsia basal plate biopsy_U133B

description: Gene expression data.


title: 30.6wk preeclampsia basal plate biopsy_U133A

description: Gene expression data.

value: 30.6wk preeclamptic


title: 36.6wk preterm basal plate biopsy_U133A

description: Gene expression data.

source_name: human placenta, preterm labor

value: 36.6wk preterm


title: 24.1wk preeclampsia basal plate biopsy_U133B

description: Gene expression data.

value: 24.1wk preeclamptic


title: 30.6wk preeclampsia basal plate biopsy_U133B

description: Gene expression data.


title: 31.9wk preeclampsia basal plate biopsy_U133B

description: Gene expression data.


title: 36.6wk preterm basal plate biopsy_U133B

description: Gene expression data.


title: 35.9wk preterm basal plate biopsy_U133B

description: Gene expression data.

value: 35.9wk preterm


title: 24.1wk preeclampsia basal plate biopsy_U133A

description: Gene expression data.


title: 35.7wk preterm basal plate biopsy_U133A

description: Gene expression data.

value: 35.7wk preterm


title: 35.9wk preterm basal plate biopsy_U133A

description: Gene expression data.


title: 30.4wk preeclampsia basal plate biopsy_U133A

description: Gene expression data.

value: 30.4wk preeclamptic


title: 35.7wk preterm basal plate biopsy_U133B

description: Gene expression data.


title: 33.9wk preterm basal plate biopsy_U133A

description: Gene expression data.

value: 33.9wk preterm


title: 31.1wk preeclampsia basal plate biopsy_U133A

description: Gene expression data.


title: 33.0wk preterm basal plate biopsy_U133A

description: Gene expression data.

value: 33.0wk preterm


title: 30.4wk preeclampsia basal plate biopsy_U133B

description: Gene expression data.


title: 33.0wk preterm basal plate biopsy_U133B

description: Gene expression data.


title: 33.9wk preterm basal plate biopsy_U133B

description: Gene expression data.


title: 33.3wk preterm basal plate biopsy_U133B

description: Gene expression data.

value: 33.3wk preterm


title: 29.9wk preterm basal plate biopsy_U133A

description: Gene expression data.

value: 29.9wk preterm


title: 33.3wk preterm basal plate biopsy_U133A

description: Gene expression data.


title: 27.1wk preterm basal plate biopsy_U133A

description: Gene expression data.

value: 27.1wk preterm


title: 25.6wk preterm basal plate biopsy_U133B

description: Gene expression data.

value: 25.6wk preterm


title: 29.9wk preterm basal plate biopsy_U133B

description: Gene expression data.


title: 25.4wk preterm basal plate biopsy_U133B

description: Gene expression data.

value: 25.4wk preterm


title: 25.6wk preterm basal plate biopsy_U133A

description: Gene expression data.


title: 24.7wk preterm basal plate biopsy_U133A

description: Gene expression data.

value: 24.7wk preterm


title: 25.4wk preterm basal plate biopsy_U133A

description: Gene expression data.


title: 24.7wk preterm basal plate biopsy_U133B

description: Gene expression data.


title: Clcn1_WT_1_Experiment 2_430A

status: Public on Feb 05 2009

city: Rochester

first: Stephen

last: Welle

state: NY

address: 601 Elmwood Avenue

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.

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: BALB wild type


title: 27.1wk preterm basal plate biopsy_U133B

description: Gene expression data.


title: Clcn1_null_3_Experiment 2_430A

value: Clcn1 knockout with BALB background


title: Clcn1_null_2_Experiment 2_430A


title: Clcn1_WT_2_Experiment 2_430A


title: FVB wild type 3_Experiment 1, 430A

description: FVB wild type 3_Experiment 1, 430A

value: FVB wild type

value: 6 months old


title: Line 20b 5_Experiment 1, 430A

description: Line 20b 5_Experiment 1, 430A

value: CUGexp transgenic Line 20b


title: FVB wild type 1_Experiment 1, 430A

description: FVB wild type 1_Experiment 1, 430A


title: Line 20b 4_Experiment 1, 430A

description: Line 20b 4_Experiment 1, 430A


title: Clcn1_WT_3_Experiment 2_430A


title: Line 41 6_Experiment 1, 430A

description: Line 41 6_Experiment 1, 430A

value: CUGexp transgenic Line 41


title: FVB wild type 6_Experiment 1, 430A

description: FVB wild type 6_Experiment 1, 430A


title: Line 20b 6_Experiment 1, 430A

description: Line 20b 6_Experiment 1, 430A


title: Line 20b 1_Experiment 1, 430A

description: Line 20b 1_Experiment 1, 430A


title: Line 20b 3_Experiment 1, 430A

description: Line 20b 3_Experiment 1, 430A


title: Line 20b 2_Experiment 1, 430A

description: Line 20b 2_Experiment 1, 430A


title: FVB wild type 5_Experiment 1, 430A

description: FVB wild type 5_Experiment 1, 430A


title: Clcn1_null_1_Experiment 2_430A


title: Line 41 5_Experiment 1, 430A

description: Line 41 5_Experiment 1, 430A


title: Line 41 2_Experiment 1, 430A

description: Line 41 2_Experiment 1, 430A


title: Line 41 4_Experiment 1, 430A

description: Line 41 4_Experiment 1, 430A


title: FVB wild type 2_Experiment 1, 430A

description: FVB wild type 2_Experiment 1, 430A


title: Line 41 3_Experiment 1, 430A

description: Line 41 3_Experiment 1, 430A


title: FVB wild type 4_Experiment 1, 430A

description: FVB wild type 4_Experiment 1, 430A


title: CENPK113-7D glucose/ethanol (19mM) limited chemostat -3

city: Delft

first: Jean-Marc

last: Daran

address: Julianalaan 67

department: Department of Biotechnology

country: Netherlands

institute: Delft University of Technology

hyb_protocol: 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

scan_protocol: Scan protocol Affymetrix scanner 3000

data_processing: Data processing Affymetrix GCOS version 2.1 CHIP file targetted at 150

label: Biotinylated dUTP - streptavidine Phycoerythrin (SAPE)

organism: Saccharomyces cerevisiae

source_name: Chemostat culture

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 19.80 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 CEN.PK 113-7D (MATa, SUC2, MAL2-8 c)


title: C-lim Aerobic chemostat with maltose as C-source 1

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

molecule: polyA RNA

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

treatment_protocol: liquid nitrogen quenching

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


title: Line 41 1_Experiment 1, 430A

description: Line 41 1_Experiment 1, 430A


title: CENPK113-7D glucose/ethanol (19mM) limited chemostat -2

label_protocol:
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: C-lim aerobic chemostat with 25mM formate

hyb_protocol: Hybridization protocol According to manufacturer's procedures

scan_protocol: 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: Label Biotinylated dUTP - streptavidine Phycoerythrin (SAPE)

label_protocol:
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 carbon 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 0.1 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  Formate was added to reached a concentration of 25mM in the reactor.
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: CENPK113-7D glucose/ethanol (19mM) limited chemostat -1

extract_protocol:
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: Aerobic sulfur limited culture (cDNA from total RNA)-2

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

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

growth_protocol:
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.

extract_protocol:
Extracted molecule polyA RNA Extraction 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: Characteristics 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 25 mM formate-1

hyb_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)

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

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 and 25 mM formate [. 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.

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 nitrogen limited chemostat 0.03h-1 -1

hyb_protocol: Sampling of cells from chemostats, probe preparation, and hybridization to Affymetrix Genechip® microarrays were performed as recommended by Affymetrix

scan_protocol: Affymetrix scanner 3000

data_processing: GCOS V 1.2

source_name: 30C anaerobic N-lim chemostat

growth_protocol:
The S. cerevisiae prototrophic haploid reference strain CEN.PK113-7D (MATa) was grown at a dilution rate (D) of 0.03 h-1 at 30oC in 2-liter chemostats (Applikon, Schiedam, The Netherlands) with a working volume of 1.0 liter as described in {van den Berg, 1996;Tai, 2005}. A temperature probe connected to a cryostat controlled cultures grown at 30oC. Cultures were grown in a defined synthetic medium that was limited by nitrogen with all other growth requirements in excess as described by {Tai, 2005}. The dilution rate was set at 0.03 hr-1 with pH measured online and kept constant at 5.0 by automatic addition of 2 M KOH using an Applikon ADI 1030 Biocontroller. The stirrer speed was set to 600 rpm. Anaerobic growth and steady state conditions were maintained as described {Tai, 2005}. Biomass dry weight, metabolite, dissolved oxygen, and gas profiles were constant at least three volume changes prior to sampling.
Tai, S. L., Boer, V. M., Daran-Lapujade, P., Walsh, M. C., de Winde, J. H., Daran, J. M. & Pronk, J. T. (2005) J. Biol. Chem. 280, 437-447.
van den Berg, M. A., de Jong-Gubbels, P., Kortland, C. J., van Dijken, J. P., Pronk, J. T. & Steensma, H. Y. (1996) J. Biol. Chem. 271, 28953-28959.

treatment_protocol: Culture quenched in liquid nitrogen

tag: Characteristics	S. cerevisiae prototrophic haploid reference strain CEN.PK113-7D (MATa)


title: Aerobic glucose limited chemostat with leucine as nitrogen source-2

hyb_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). 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..

scan_protocol: Scan protocol 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.

growth_protocol:
Chemostat Cultivation—Steady-state chemostat cultures were grown in Applikon laboratory fermentors of 1-liter working volume as described in detail elsewhere [van den Berg, M. A., de Jong-Gubbels, P., Kortland, C. J., van Dijken, J. P., Pronk, J. T., and Steensma, H. Y. (1996) J. Biol. Chem. 271, 28953–28959]. In brief, the cultures were fed with a defined mineral medium containing glucose as the growth-limiting nutrient [. Verduyn, C., Postma, E., Scheffers, W. A., and van Dijken, J. P. (1990) Microbiol.Rev. 58, 616–630]. The dilution rate (which equals the specific growth rate) in the steady-state cultures was 0.10 h_1, the temperature was 30 °C, and the culture pH was 5.0.Aerobic conditions were maintained by sparging the cultures with air (0.5 liter_min_1). The dissolved oxygen concentration, which was continuously monitored with an Ingold model 34-100-3002 probe, remained above 80% of air saturation.
The synthetic media composition was based on that described by Verduyn (1992). In all chemostats except for those limited by carbon, residual glucose concentration was targeted to 17 g.liter-1 in order to sustain glucose repression at the same level. The media composition for aerobic cultivations was the following: for nitrogen-limited cultivation, 1.90 g.liter-1 leucine, 3.0 g.liter-1 KH2PO4, 0.5 g.liter-1 MgSO4•7H2O, 59 g.liter-1 glucose and 4.5 mg.liter-1 ZnSO4•7H2O; for zinc-limited cultivation, 5.0 g.liter-1 (NH4)2SO4, 3.0 g.liter-1 KH2PO4, 0.5 g.liter-1 MgSO4•7H2O, 66 g.liter-1 glucose and 0.014 mg.liter-1 ZnSO4•7H2O.
van den Berg, M. A., P. de Jong-Gubbels, C. J. Kortland, J. P. van Dijken, J. T. Pronk, and H. Y. Steensma. 1996. The two acetyl-coenzyme A synthetases of Saccharomyces cerevisiae differ with respect to kinetic properties and transcriptional regulation. J. Biol. Chem. 271:28953-28959.
van Dijken, J. P., J. Bauer, L. Brambilla, P. Duboc, J. M. Francois, C. Gancedo, M. L. Giuseppin, J. J. Heijnen, M. Hoare, H. C. Lange, E. A. Madden, P. Niederberger, J. Nielsen, J. L. Parrou, T. Petit, D. Porro, M. Reuss, R. N. van, M. Rizzi, H. Y. Steensma, C. T. Verrips, J. Vindelov, and J. T. Pronk. 2000. An interlaboratory comparison of physiological and genetic properties of four Saccharomyces cerevisiae strains. Enzyme Microb. Technol. 26:706-714.
Verduyn, C., E. Postma, W. A. Scheffers, and J. P. van Dijken. 1990. Energetics of Saccharomyces cerevisiae in anaerobic glucose-limited chemostat cultures. J. Gen. Microbiol. 136:405-412.
Verduyn, C., E. Postma, W. A. Scheffers, and J. P. van Dijken. 1992. Effect of benzoic acid on metabolic fluxes in yeasts: a continuous-culture study on the regulation of respiration and alcoholic fermentation. Yeast 8:501-517.
Visser, W., W. A. Scheffers, Batenburg-van der Vegte WH, and J. P. van Dijken. 1990. Oxygen requirements of yeasts. Appl. Environ. Microbiol. 56:3785-3792.
Boer, V. M., J. H. de Winde, J. T. Pronk, and M. D. Piper. 2003. The genome-wide transcriptional responses of Saccharomyces cerevisiae grown on glucose in aerobic chemostat cultures limited for carbon, nitrogen, phosphorus, or sulfur. J. Biol. Chem. 278:3265-3274.
Ferea, T. L., D. Botstein, P. O. Brown, and R. F. Rosenzweig. 1999. Systematic changes in gene expression patterns following adaptive evolution in yeast. Proc. Natl. Acad. Sci. U. S. A 96:9721-9726.
Jansen, M. L., P. Daran-Lapujade, J. H. de Winde, M. D. Piper, and J. T. Pronk. 2004. Prolonged maltose-limited cultivation of Saccharomyces cerevisiae selects for cells with improved maltose affinity and hypersensitivity. Appl. Environ. Microbiol. 70:1956-1963.


title: Iron limited chemostat cultures with ethanol as a carbon source

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

source_name: Aerobic ethanol limited chemostat iron limited

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 iron 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 °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 cultivation (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: Anaerobic glucose limited chemostat

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

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]. The residual glucose concentration was targeted to 17 g_liter_1 to sustain glucose repression at the same level . For carbon limited cultivation: 5.0 g of (NH4)2SO4, 3.0 g of KH2PO4, 0.5 g of MgSO4.7H2O, and 25 g of glucose.

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).

tag: Strain and Maintenance—


title: Aerobic glucose limited chemostat with Phenylalanine as N-source-2

source_name: chemostat culture with phenylalanine as N-source

label_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).
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:
The prototrophic S. cerevisiae strain CEN.PK113-7D (MATa) (1,2) was grown at 30°C in 1.0-liter working volume chemostats (3). Cultures were fed with a synthetic medium (4) that supported glucose-limited growth. The glucose concentration in the reservoir medium was 7.5 g•l-1. The nitrogen source was used, at the following concentration: L-phenylalanine, 5.0 g•l-1. When an amino acid served as nitrogen source, the synthetic medium was supplemented with 6.6 g•l-l K2SO4. 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 biocontroller. Stirrer speed was 800 rpm and the airflow was 0.5 l.min-1. Dissolved oxygen tension was measured online with an Ingold model 34 100 3002 probe, and was above 50% air saturation. A condenser connected to a cryostat set at 2 °C cooled the off-gas, and oxygen and carbon dioxide were measured off-line. Steady-state samples for arrays were taken after 10 to 14 volume changes to minimize the impact of evolutionary adaptation that occurs after long term cultivation (5,6). Dry weight, metabolite-, and gas profiles were constant over at least 3 volume changes prior to sampling for RNA extraction.
(1) van Dijken et al. 2000. An interlaboratory comparison of physiological and genetic properties of four Saccharomyces cerevisiae strains. Enzyme Microb Technol 26(9,10): 706-714.
(2) Daran-Lapujade P et al. 2003. Comparative genotyping of the Saccharomyces cerevisiae laboratory strains S288C and CEN.PK113-7D using oligonucleotide microarrays. FEMS Yeast Res 4: 285-296.
(3) van den Berg et al. 1996. The two acetyl-coenzyme A synthetases of Saccharomyces cerevisiae differ with respect to kinetic properties and transcriptional regulation. J Biol Chem 271: 28953-28959.
(4) Verduyn et al. 1992. Effect of benzoic acid on metabolic fluxes in yeasts: a continuous-culture study on the regulation of respiration and alcoholic fermentation. Yeast 8: 501-17.
(5) Ferea et al. 1999. Systematic changes in gene expression patterns following adaptive evolution in yeast. Proc Natl Acad Sci U S A 96: 9721-9726.
(6) Jansen et al. 2004. Prolonged maltose-limited cultivation of Saccharomyces cerevisiae selects for cells with improved maltose affinity and hypersensitivity. Appl Environ Microbiol 70: 1956-1963.

tag: The prototrophic S. cerevisiae strain CEN.PK113-7D (MATa) (1)


title: Aerobic glucose limited chemostat with leucine as nitrogen source-1

growth_protocol:
Chemostat Cultivation—Steady-state chemostat cultures were grown in Applikon laboratory fermentors of 1-liter working volume as described in detail elsewhere [van den Berg, M. A., de Jong-Gubbels, P., Kortland, C. J., van Dijken, J. P., Pronk, J. T., and Steensma, H. Y. (1996) J. Biol. Chem. 271, 28953–28959]. In brief, the cultures were fed with a defined mineral medium containing glucose as the growth-limiting nutrient [. Verduyn, C., Postma, E., Scheffers, W. A., and van Dijken, J. P. (1990) Microbiol.Rev. 58, 616–630]. The dilution rate (which equals the specific growth rate) in the steady-state cultures was 0.10 h_1, the temperature was 30 °C, and the culture pH was 5.0.Aerobic conditions were maintained by sparging the cultures with air (0.5 liter_min_1). The dissolved oxygen concentration, which was continuously monitored with an Ingold model 34-100-3002 probe, remained above 80% of air saturation.
The synthetic media composition was based on that described by Verduyn (1992). In all chemostats except for those limited by carbon, residual glucose concentration was targeted to 17 g.liter-1 in order to sustain glucose repression at the same level. The media composition for aerobic cultivations was the following: for nitrogen-limited cultivation, 1.9 g.liter-1 Leucine, 3.0 g.liter-1 KH2PO4, 0.5 g.liter-1 MgSO4•7H2O, 59 g.liter-1 glucose and 4.5 mg.liter-1 ZnSO4•7H2O; for zinc-limited cultivation, 5.0 g.liter-1 (NH4)2SO4, 3.0 g.liter-1 KH2PO4, 0.5 g.liter-1 MgSO4•7H2O, 66 g.liter-1 glucose and 0.014 mg.liter-1 ZnSO4•7H2O.
van den Berg, M. A., P. de Jong-Gubbels, C. J. Kortland, J. P. van Dijken, J. T. Pronk, and H. Y. Steensma. 1996. The two acetyl-coenzyme A synthetases of Saccharomyces cerevisiae differ with respect to kinetic properties and transcriptional regulation. J. Biol. Chem. 271:28953-28959.
van Dijken, J. P., J. Bauer, L. Brambilla, P. Duboc, J. M. Francois, C. Gancedo, M. L. Giuseppin, J. J. Heijnen, M. Hoare, H. C. Lange, E. A. Madden, P. Niederberger, J. Nielsen, J. L. Parrou, T. Petit, D. Porro, M. Reuss, R. N. van, M. Rizzi, H. Y. Steensma, C. T. Verrips, J. Vindelov, and J. T. Pronk. 2000. An interlaboratory comparison of physiological and genetic properties of four Saccharomyces cerevisiae strains. Enzyme Microb. Technol. 26:706-714.
Verduyn, C., E. Postma, W. A. Scheffers, and J. P. van Dijken. 1990. Energetics of Saccharomyces cerevisiae in anaerobic glucose-limited chemostat cultures. J. Gen. Microbiol. 136:405-412.
Verduyn, C., E. Postma, W. A. Scheffers, and J. P. van Dijken. 1992. Effect of benzoic acid on metabolic fluxes in yeasts: a continuous-culture study on the regulation of respiration and alcoholic fermentation. Yeast 8:501-517.
Visser, W., W. A. Scheffers, Batenburg-van der Vegte WH, and J. P. van Dijken. 1990. Oxygen requirements of yeasts. Appl. Environ. Microbiol. 56:3785-3792.
Boer, V. M., J. H. de Winde, J. T. Pronk, and M. D. Piper. 2003. The genome-wide transcriptional responses of Saccharomyces cerevisiae grown on glucose in aerobic chemostat cultures limited for carbon, nitrogen, phosphorus, or sulfur. J. Biol. Chem. 278:3265-3274.
Ferea, T. L., D. Botstein, P. O. Brown, and R. F. Rosenzweig. 1999. Systematic changes in gene expression patterns following adaptive evolution in yeast. Proc. Natl. Acad. Sci. U. S. A 96:9721-9726.
Jansen, M. L., P. Daran-Lapujade, J. H. de Winde, M. D. Piper, and J. T. Pronk. 2004. Prolonged maltose-limited cultivation of Saccharomyces cerevisiae selects for cells with improved maltose affinity and hypersensitivity. Appl. Environ. Microbiol. 70:1956-1963.

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).
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..


title: Anaerobic nitrogen limited chemostat with methionine as nitrogen and sulfur source-2

data_processing:
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_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:
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]. The residual glucose concentration was targeted to 17 g_liter_1 to sustain glucose repression at the same level . For nitrogen-limited cultivation: 1.5g g of methionine, 10 g of KCl, 3.0 g of KH2PO4, 0.5 g of MgCl.7H2O and 46 g of glucose.

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

tag: Pronk, J. T. (2000) Enz. Microb. Technol. 26, 706–714]


title: Anaerobic nitrogen limited chemostat with methionine as nitrogen source-3

scan_protocol: scanning with the Affymetrix GeneArray Scanner

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]. The residual glucose concentration was targeted to 17 g_liter_1 to sustain glucose repression at the same level . For nitrogen-limited cultivation: 1.5g g of methionine, 5.75 g of K2SO4, 3.0 g of KH2PO4, 0.5 g of MgSO4.7H2O and 46 g of glucose.


title: Anaerobic nitrogen limited chemostat with methionine as nitrogen source-2


title: Anaerobic nitrogen limited chemostat with methionine as nitrogen and sulfur source-3


title: Anaerobic nitrogen limited chemostat with methionine as nitrogen and sulfur source-1


title: Anaerobic sulfur limited chemostat -1

growth_protocol:
Chemostat Cultivation—
Steady-state chemostat cultures were grown in Applikon laboratory fermentors of 1-liter working volume as described in detail elsewhere [van den Berg, M. A., de Jong-Gubbels, P., Kortland, C. J., van Dijken, J. P., Pronk, J. T., and Steensma, H. Y. (1996) J. Biol. Chem. 271, 28953–28959]. In brief, the cultures were fed with a defined mineral medium containing glucose as the growth-limiting nutrient [. Verduyn, C., Postma, E., Scheffers, W. A., and van Dijken, J. P. (1990) Microbiol.Rev. 58, 616–630]. The dilution rate (which equals the specific growth rate) in the steady-state cultures was 0.10 h_1, the temperature was 30 °C, and the culture pH was 5.0. Aerobic conditions were maintained by sparging the cultures with air (0.5 liter_min_1). The dissolved oxygen concentration, which was continuously monitored with an Ingold model 34-100-3002 probe, remained above 80% of air saturation.
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: Iron limited chemostat cultures with ethanol as a carbon source-2

scan_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

source_name: chemostat


title: Anaerobic glucose limited chemostat with methionine as sulfur source-2

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:
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]. The residual glucose concentration was targeted to 17 g_liter_1 to sustain glucose repression at the same level . For carbon limited cultivation: 10.0 g of NH4Cl, 3.0 g of KH2PO4, 0.5 g of MgCl2.7H2O, and 25 g of glucose. Methionine was used as sulphur source.

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: Anaerobic glucose limited chemostat with methionine as sulfur source-3


title: Anaerobic nitrogen limited chemostat with methionine as nitrogen source-1


title: Aerobic glucose limited chemostat with Phenylalanine as N-source-1

tag: (1) van Dijken et al. 2000. An interlaboratory comparison of physiological and genetic properties of four Saccharomyces cerevisiae strains. Enzyme Microb Technol 26(9,10)


title: Aerobic phosphorus limited chemostat with anaerobic factors (tween and ergosterol) -2

source_name: 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.


title: Anaerobic glucose limited chemostat with methionine as sulfur source-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]. Ammonium sulfate was replaced by ammonium chloride, magnesium sulfate was replaced by magnesium chloride, potassium sulfate was replaced  by potassium chlorideand the sulfur source was added  as methionine in mol equivalent of sulfate. 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 10.0 g of NH4Cl, 3.8 g of KCl, 0.12 g of KH2PO4, 0.5 g of MgCl2_7H2O, and 59 g of glucose . Medium was supplemented with methionine as sulfur source.

tag: Chemostat Cultivation—

tag: 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.

tag: Media—

tag: 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 carbon limited cultivation

value: 10.0 g of NH4Cl, 3.0 g of KH2PO4, 0.5 g of MgCl2.7H2O, and 25 g of glucose. Methionine was used as sulphur source.