title: Normal lamina cribrosa cells

city: Dublin

first: Ruaidhri

middle: Patrick

last: Kirwan

state: Leinster

address: Belfield

department: Conway Institute of Biomolecular and Biomedical Research & School of Medicine and Medical Science

country: Ireland

institute: University College Dublin

description: human glial fibrillary acid protein negative (GFAP) lamina cribrosa (LC) cells

hyb_protocol: cRNA fragmentation, hybridisation and microarray scanning cRNA was fragmented in a 5X fragmentation buffer (200mM Tris-acetate, pH 8.1, 500 mM KOAc, 150 mM MgOAc) at 94°C for 35 minutes. 15 μg of fragmented cRNA was mixed with 20X Gene Chip Eukaryotic Hybridization Cocktail containing a mixture of 4 biotin-labeled anti-sense fragmented control cRNAs (Bio B, Bio C, Bio D and CRE).  The mixture was hybridized to Affymetrix HG-U133A (human) arrays as per Affymetrix target hybridization protocol.  Subsequent washing and staining of the arrays was carried out using the Affymetrix GeneChip fluidics station protocol EukGE-WS2.

scan_protocol: Following washing and staining, the probe arrays were scanned twice at 532 nm with a filter of 570 nm and 3 µm resolution using the Affymetrix GeneChip System confocal scanning laser (Affymetix, Santa Clara, CA, USA).  Raw image *.cel files (cell intensity files) were obtained through Affymetrix GeneChip software (MAS 5.0) using the default settings. Once scanned by the confocal laser each probe cell signal intensity is divided into a 10 X 10 pixel grid from which the outer 36 pixels are removed to reduce noise.  The 75th percentile of the remaining 8 X 8 pixel signal value is then taken as the raw signal intensity for that probe (oligonucleotide) and stored in the *cel file.

data_processing:
Normalisation and filtering of microarray data
Robust multi-chip average (RMA)
Raw microarrray output data from MAS 5.0 was background adjusted and normalised using the method of Robust Multichip Average (RMA) (Irizarry et al. 2003).  RMA is based on the ‘R’ statistical programming language (http://www.r-project.org/ and www.bioconductor.org) which analyses directly from the Affymetrix MAS 5.0  *.cel image files.  It consists of three steps:
1. Background adjustment:
This compensates for non-specific cRNA/probe binding.  The average of the lowest 2% of probe cell values in a region of the microarray is taken as the background value for that region and subtracted from all values in that region.  There are 16 regions on each HG-U133A microarray.
2. Quantile normalisation:
This step unifies perfect match cRNA/target probe distributions across the arrays. The objective of quantile normalisation is to make the distribution of perfect match probe signal intensities the same for each array.  This is required in order to minimise the effects of variation in the amounts of RNA used, the rates of the microarray hybridisation reactions and the conditions of hybridisation within the Affymetrix hybridisation oven. The normalisation algorithm used by RMA is called a “complete data method” as it combines information from all arrays in an experiment to form the normalising relation (*.cel files are read and normalised consecutively) (Bolstad et al. 2003).
3. Summarisation:
This final step median polishes the Log Base2 normalised/transformed probe signal level data.
RMAExpress

RMAExpress (version 0.3) is a freely available graphic user interface (GUI) programme that can compute RMA measures. It is written for the Microsoft Windows operating system and for this thesis was used within Microsoft Virtual PC (version 6.1) for Macintosh operating system ten (OS X).  The RMAExpress GUI (http://stat-www.berkeley.edu/users/bolstad/RMAExpress/RMAExpress.html) allows consecutive uploading of *.cel files followed by the Affymetrix U133A chip description *.cdf file.  The *.cdf file provides the co-ordinate data and probeset identifier names to which raw signal intensity values have been assigned in the *.cel file.  The output data created by RMAExpress consisted of a tab delimited text file containing the Affymetrix probeset identifiers (IDs) in the left-most column followed by columns of the experimental and control arrays whose cells contained the probe set RMA log base2 transformed signal values (quantitative measures which reflect the absolute expression value of that mRNA transcript).

label_protocol:
Preparation of complimentary biotin labelled RNA (cRNA)
First and second strand cDNA synthesis
The starting amount of RNA for each microarray hybridization preparation was 5 μg of total RNA. This was then annealed to 1 ml of Affymetrix (Affymetrix, Santa Clara, CA, USA) T7-(dT)24 primer (sequence: 5’-GGCCAGTGAATTGTAATACGACTC-ACTATAGGGAGGCGG-(dT)24-3’) by incubation at 70OC for 10 minutes.  Following hybridisation, 4 μl of 5X first strand buffer cDNA buffer was added with 2 μl of DTT (0.1M) and 1 μl of dNTPs (10mM) and the mixture incubated at 42OC for 2 minutes.  First-strand cDNA synthesis was completed by incubating the mixture with 1 μL of Superscript II RT in a 20 μl volume, at 42°C for 1 hour.  Second-strand cDNA was synthesized by adding 30 μl of 5X second-strand buffer, 91 μl of DEPC H2O, 3 μl dNTPs (10mM), 1 μl of DNA ligase (10U), 4 μl DNA polymerase I (10U) and 1 μl of Rnase H (2U) to a final reaction volume of 150 μl.   The mixture was then incubated at 16OC for 2 hours.   2 μl (10U) of T4 DNA polymerase was added and the mixture was incubated at 16°C for 5 minutes followed by 10μL of 0.5M EDTA.  Samples were stored at -20OC until clean up procedure was commenced as described below.
Clean-up of double stranded cDNA
Double stranded cDNA was purified using cDNA cleanup columns from the Affymetrix GeneChip Sample Cleanup Module kit (Affymetrix, Santa Clara, CA, USA).  600 μl of cDNA binding buffer was added to the 162 μl of double stranded cDNA volume and the sample was vortexed for 3 seconds.   The mixture was then added to a cDNA cleanup spin column sitting in 2 ml collection tube and centrifuged at 12,000xg for 1 minute.   The flow through was discarded. The spin column was then transferred to a new 2 ml collection tube.  750 μl of cDNA wash buffer was applied to the column and centrifuged for 1 minute at 12,000xg. The flow through was discarded.  The spin column cap was then opened and the column centrifuged again for 5 minutes at maximum speed 20,000xg.  The flow through and collection tube were discarded.   The spin column was transferred to a new 1.5ml collection tube and 14 μl of cDNA elution buffer added carefully onto the column membrane.  The spin column was incubated at room temperature for 1 minute followed by centrifugation at 20,000xg for 1 minute to elute the cDNA sample.   The samples were then stored at -20OC until in-vitro transcription was commenced as described below.
In-vitro transcription and cRNA labeling
Synthesis of biotin labelled cRNA was performed by in an in-vitro transcription reaction (IVT) using the Bioarray High-Yield RNA Transcript Labeling Kit (Enzo, Farmingdale, NY, USA). 10 μl of double stranded DNA template sample was added (per reaction) to a master-mix solution containing 4 μl 10X HY reaction buffer, 4 μl 10X biotin labeled ribonucleotides, 4 μl 10X DTT, 4 μl 10X RNase inhibitor mix, 2 μl 20X T7 RNA polymerase and DEPC treated H2O to a total reaction volume of 40 μl. The reaction was mixed and incubated in a 370 C water bath for 4 hours, gently mixing the contents of the tube every 30 minutes.  Following the 4 hour incubation period the biotin labeled cRNA was immediately stored at -80OC until clean up and quantification were performed as described in section 2.4.1.4 below.
cRNA clean-up and spectroscopic measurement of cRNA concentration
The biotin labelled cRNA was purified using cRNA cleanup columns from the Affymetrix GeneChip Sample Cleanup Module kit (Affymetrix, Santa Clara, CA, USA).   60 μl of RNase-free H2O was added to the cRNA and mixed by vortexing for 1 minute followed by 350 μl of IVT cRNA binding buffer and an additional vortexing for 3 seconds.  250 μl of 100% ethanol was then added to the lysate and mixed by pipetting.  The mixture was then applied to an IVT cRNA cleanup spin column sitting in a 2 ml collection tube.  The spin column was then centrifuged at 10,000xg for 15 seconds and the flow through and collection tube were discarded.  The spin column was then transferred to a new 2 ml collection tube and 500 μl of cRNA wash buffer pipetted on to the spin column which was centrifuged for 15 seconds at 10,000xg. The flow through was discarded.  The cap of the spin column was opened and the column was centrifuged for 5 minutes at maximum speed 20,000xg. The flow through and collection tube were discarded.  The spin column was then transferred to a new 1.5 ml collection tube and 11 μl of Rnase-free H2O was pipetted carefully onto the column membrane followed by centrifugation at 20,000xg for 1 minute.  Another elution step was then performed with an additional 10 μl of Rnase-free H2O.  The RNA yield was analysed by spectrophotometry as described above.  As total RNA was as the starting material an adjusted cRNA yield was calculated using a spectrophotometer and the following formula:
 		Adjusted cRNA yield 		= 	RNAm – (total RNAi)(y)
Where:			RNAm 		= 	amount of cRNA measured after IVT (µg)
			total RNAi 	= 	starting amount of total RNA (µg)
			y 		= 	fraction of cDNA reaction used in IVT

extract_protocol:
Preparation of cellular RNA
Silica gel-based spin column protocol
Total RNA was isolated using RNeasy Minikit (Qiagen, Valencia, CA, USA). This technology combines the selective binding properties of a silica-gel-based membrane with the high speed microspin technology. Harvesting and lysis of the cells was achieved through the addition of 600 μl Buffer RLT.  Buffer RLT is a highly denaturing guanidine isothiocyanate-containing buffer, which immediately inactivates RNases to ensure isolation of intact RNA. The samples were then added to a QIAshredder spin column and centrifuged for 2 minutes at 12,000 rpm for homogenization.  This is necessary to reduce the viscosity of the cell lysates and shears the high-molecular-weight genomic DNA and other high molecular weight cellular components to create a homogenous lysate.  Incomplete homogenization results inefficient binding of RNA to the silica-gel membrane and therefore significantly reduced yields. 350 μl of 70% ethanol was then added to the homogenized lysate. The ethanol provides appropriate binding conditions before the addition of the sample to the RNeasy mini column. This is then centrifuged again at 12,000 rpm and the flow-through is discarded. 700 μl of Buffer RW1 (which contains ethanol) is then applied to wash the column and again the flow-through is discarded.  The samples were transferred to new 2 ml collection tubes and the columns were washed twice with 500 μl of Buffer RPE.  During the second wash the columns were spun for two minutes to dry the silica-gel membranes. To elute the RNA the minicolumns were transferred to a new sterile 1.5 ml collection tubes and 30 μl of Rnase-free water pipetted directly onto the silca-gel membranes.  This was then centrifuged for 2 minutes at 12,000 rpm. RNA concentration and purity was ascertained as described below.  Total RNA was stored at –80oC.
Spectroscopic measurement of RNA concentration
Quantification of total RNA was determined by measuring the absorbance of a 1/1000 RNA dilution in DEPC treated H2O at 260 nm in a spectrophotometer.  An absorbance of 1 unit at 260 nm corresponds to 40 μg of RNA per 1000 μl.  The ratio between absorbencies at 260 nm and 280 nm provides an estimate of the purity of the nucleic acid. Adequately pure preparations of RNA have a 260 / 280 absorbance ratio of 1.8 to 2.0.   The concentration of RNA in μg/μl was calculated using the following formula:

	O.D. reading (260nm) x ((40 μg/ 1000 μl )/ 1 O.D. unit ) x dilution factor
			= 	RNA concentration in μg/ μl
RNA agarose gel electrophoresis
Agarose gel electrophoresis was used to assess the integrity of total RNA samples. The RNA fragments were identified using the nucleic acid binding dye, ethidium bromide (EtBr). Electrophoresis was carried out in TAE buffer (40 mM Tris-acetate, 1 mM EDTA, pH 7.6) (Gibco, Paisley, UK).   Gels were prepared by adding agarose to TAE buffer to a final concentration of 1.5%. This mixture was heated in a microwave oven until the agarose had dissolved completely. The solution was allowed to cool to approximately 550C and EtBr added to a final concentration of 0.2 mg / ml from a 10 ng/ml stock solution.  The agarose solution was then poured into a gel box containing a comb for creating wells and allowed to set. The gel was placed in an electrophoresis tank to which TAE buffer had been added.  0.5mg samples of RNA were mixed with 2μl of 6X loading dye (0.1% xylene cyanol, 0.1% bromophenol blue and 30% glycerol) and DEPC H2O to a final volume of 12 mland loaded into the submerged wells of the gel. The gel was run at 80 volts until the dye front had migrated three quarters the length of the gel. The gel was then visualised under UV light and photographed using standard image capture software.  RNA integrity was confirmed by the presence of two discrete bands corresponding to the 28S and 18S ribosomal RNA in the total RNA sample.

tag: Cultures of normal LC cells from human donors with no history of eye disease. Cultures were maintained in DMEM (Sigma, Dorset, UK) supplemented with 10% fetal calf serum, penicillin (100U/ml) and streptomycin (100(μg/ml) in a humidified incubator containing 5% CO2 / 95% air at 37oC.  All cells maintained their broad polygonal morphology during propagation in-vitro and were used between passages 4 and 6.  The normal LC cells and POAG LC cells were cultured until approximately 90% confluent on 100mm plates. The cells were then serum starved for 24 hours before being harvested for extraction of total cellular RNA.


title: POAG LC cells

status: Public on Jan 06 2009

type: RNA

description: human optic nerve GFAP negative lamina cribrosa cells

label: biotin

molecule: total RNA

organism: Homo sapiens

tag: Glial fibrillary acid protein negative lamina cribrosa cells (LC).Cultures were maintained in DMEM (Sigma, Dorset, UK) supplemented with 10% fetal calf serum, penicillin (100U/ml) and streptomycin (100(μg/ml) in a humidified incubator containing 5% CO2 / 95% air at 37oC.  All cells maintained their broad polygonal morphology during propagation in-vitro and were used between passages 4 and 6.  The normal LC cells and POAG LC cells were cultured until approximately 90% confluent on 100mm plates. The cells were then serum starved for 24 hours before being harvested for extraction of total cellular RNA.


title: 9417 TAF-tib48 rep3

status: Public on Jan 05 2009

city: South San Francisco

first: Josh

last: Kaminker

state: CA

address: 1 DNA Way

department: Bioinformatics and Computational Biology

country: USA

institute: Genentech

hyb_protocol: Agilent's In situ Hybridization kit

scan_protocol: Agilent DNA microarray scanner

data_processing: Rosetta Resolver Feature Extraction Version 8.5.1.1

organism: Mus musculus

label_protocol: Agilent's Low input RNA Linear Amp kit

extract_protocol: GE2-SSPE_95_Feb07 with FE version 9.5

source_name: Universal Mouse Reference RNA: Stratagene Catalog #740100

tag: Universal Mouse Reference RNA

value: Stratagene Catalog #740100


title: Glaucoma LC cell

description: gene expression analysis of LC cells from glaucomatous human donor.

hyb_protocol: The mixture was hybridized to Affymetrix HG-U133A (human) arrays as per Affymetrix target hybridization protocol. Subsequent washing and staining of the arrays was carried out using the Affymetrix GeneChip fluidics station protocol EukGE-WS2.

scan_protocol: Following washing and staining, the probe arrays were scanned twice at 532 nm with a filter of 570 nm and 3(m resolution using the Affymetrix GeneChip System confocal scanning laser(Affymetix, Santa Clara, CA, USA)(see Appendix A,D&E). Rawimage*.cel files(cell intensity files) were obtained through Affymetrix GeneChip software (MAS 5.0) using the default settings.

data_processing:
Robust multi-chip average (RMA)
Raw microarrray output data from MAS5.0 was background adjusted and normalised using the method of Robust Multichip Average (RMA) (Irizarryetal., 2003). RMA is based on the "R" statistical programming language (http://www.r-project.org and http:/ www.bioconductor.org) which analyses directly from the Affymetrix MAS 5.0 *.cel image files.  It consists of three steps:
1. Background adjustment
This compensates for non-specific cRNA/probe binding. The average of the lowest 2% of probe cell values in a region of the microarray is taken as the background value for that region and subtracted from all values in that region. There are 16 regions on each HG-U133A microarray.
2. Quantile normalisation
This step unifies perfect match cRNA/target probe distributions across the arrays. The
objective of quantile normalisation is to make the distribution of perfect match probe signal intensities the same for each array. This is required in order to minimise the effects of variation in the amounts of RNAu sed, the rates of the microarray hybridisation reactions and the conditions of hybridisation within the Affymetrix hybridisation oven. The normalisation algorithm used by RMA i scalled a“complete data method” as it combines information from all arrays in an experiment to form the normalising relation (*.cel files are read and normalised consecutively) (Bolstad et al., 2003).
3. Summarisation
This final step median polishes the Log Base 2 normalised/transformed probe signal level data.

source_name: GFAP negative lamina cribrosa cell (LC) RNA from a glaucomatous human donor

label_protocol: Biotin labeled cRNA was prepared from template cDNA followed by fragmentation and hybridisation to Affymetrix HG-U133A arrays as per the Affymetrix protocol (Affymetrix, CA, USA). cRNA was fragmented in a 5X fragmentation buffer (200 mMTris-acetate, pH8.1, 500mM KOAc, 150mMMgOAc) at 94°C for 35 minutes. 15 µg of fragmented cRNA was mixed with 20X GeneChip Eukaryotic Hybridization Cocktail containing a mixture of 4 biotin- labeled anti-sense fragmented control cRNAs (BioB, BioC, BioD and CRE).

growth_protocol: Cultures were maintained in DMEM (Sigma, Dorset, UK) supplemented with 10% fetal calf serum, penicillin (100 U/ml) and streptomycin (100 μg/ml) in a humidified incubator containing 5% CO2 / 95% air at 37oC.  All cells maintained their broad polygonal morphology during propagation in-vitro and were used between passages 4 and 6.  The normal LC cells and POAG LC cells were cultured until approximately 90% confluent on 100mm (p100) plates. The cells were then serum starved for 24 hours before being harvested for extraction of total cellular RNA.

extract_protocol: Total RNA was isolated from all LC cells using the RNeasy minicolumn protocol (Qiagen, CA, USA). cDNA was synthesised from total RNA using SuperScript Choice Kit (Invitrogen, Paisley, UK).

tag: Glial fribrillary acid protein negative cells from the human optic nerve head.


title: 9418 TAF-tib6 rep2


title: 9417 TAF-tib48 rep1


title: 9418 TAF-tib6 rep3


title: 9418 TAF-tib6 rep1


title: 9417 TAF-tib48 rep2


title: 9416 TAF-tib42 rep3


title: normal LC cell

description: gene expression analysis of LC cells from a normal (non glaucomatous) human donor.

source_name: GFAP negative lamina cribrosa cell (LC) RNA from a normal human donor

treatment_protocol: Cultures were maintained in DMEM (Sigma, Dorset, UK) supplemented with 10% fetal calf serum, penicillin (100 U/ml) and streptomycin (100 μg/ml) in a humidified incubator containing 5% CO2 / 95% air at 37oC.  All cells maintained their broad polygonal morphology during propagation in-vitro and were used between passages 4 and 6.  The normal LC cells and POAG LC cells were cultured until approximately 90% confluent on 100mm (p100) plates. The cells were then serum starved for 24 hours before being harvested for extraction of total cellular RNA.

tag: glial fibrillary acid protein negative lamina cribrosa cells.


title: 9415 TAF-el4 rep1


title: 9416 TAF-tib42 rep1


title: NotchICD embryos, 7-8 hours AEL, rep. 2

city: Boston

first: Gregory

middle: D.

last: Hurlbut

state: MA

address: 240 Longwood Ave,LHRRB-410A

department: Cell Biology

institute: Harvard Medical School

description: Transgenic Drosophila embryos expressing armadillo-GAL4 controlled activated Notch (Drosophila Notch intracellular domain), 7-8 hours after egg lay (AEL) (stage 11 mid-12 of embryonic development)

hyb_protocol: Following fragmentation, 10 ug of cRNA were hybridized for 16 hr at 45C on GeneChip Drosophila Genome Array 1. GeneChips were washed and stained in the Affymetrix Fluidics Station 400.

scan_protocol: GeneChips were scanned using the Hewlett-Packard GeneArray Scanner G2500A.

data_processing: The data were analyzed by GC-RMA as part of the LIMMA package in the R programming language environment

organism: Drosophila melanogaster

source_name: Drosophila embryos, NotchICD, 7-8 hours after egg lay (AEL)

label_protocol: Biotinylated cRNA were prepared according to the standard Affymetrix protocol from 8 ug total RNA (Expression Analysis Technical Manual, 2001, Affymetrix).

growth_protocol: Embryos of each type were collected on apple juice plates for one hour, incubated at 25°C for 5 hours and 30 minutes for the first time point and 6 hours and 45 minutes for the second time point; dechorionated by treatment with 50% Clorox bleach in dH2O at room temperature for 1 minute with agitation, washed with 0.5X PBS, 1% Tween, rinsed with ddH2O.  Embryos were then shock frozen and stored in liquid Nitrogen within 15 minutes. All collections were performed in parallel sets, which were slightly staggered to equalize the length of embryo submersion in liquid. Each collection contained approximately 500 embryos per 1.5 ml microcentrifuge tube.

extract_protocol: Frozen embryos were ground by pestle in the Trizol reagent (Invitrogen). RNA was extracted according to the manufacturer's instructions, with the inclusion of an additional Trizol extraction prior to RNA precipitation to increase RNA purity.

tag: Transgene

value: arm-GAL4, UAS-NotchICD

tag: Genotype

value: progeny of homozygous arm-GAL4 females crossed with homozygous UAS-NotchICD males

tag: Age

value: 7-8 hours after egg lay (developmental stage 11 mid-12)

tag: Reared at 25 degrees C


title: 9416 TAF-tib42 rep2


title: 9414 NSF rep3


title: 9414 NSF rep1


title: Biophenotypic leukemia patient #26

city: Memphis

first: Xueyuan

last: Cao

state: TN

address: 262 Danny Thomas Place

department: Biostatistics

institute: St Jude Children's Research Hospital

description: Gene expression data for pediatric patient diagnostic sample.

hyb_protocol: Following fragmentation, 10 ug of cRNA were hybridized for 16 hr at 45C on GeneChip Human Genome U133 Array Set HG-U133A. GeneChips were washed at 25°C with 6 X SSPE (0.9M NaCl, 60 mM NaH2PO4, 6 mM EDTA + 0.01% Tween 20) followed by a stringent wash at 50°C with 100 mM MES, 0.1M NaCl2, 0.01% Tween 20.

scan_protocol: Arrays were scanned using a laser confocal scanner (Agilent, Palo Alto, CA).

data_processing: The data were analyzed with Microarray Suite version 5.0 (MAS 5.0) using Affymetrix default analysis settings and global scaling as normalization method. The trimmed mean target intensity of each array was arbitrarily set to 500.

source_name: diagnostic BM aspirates or peripheral blood, patient #26

label_protocol: cRNA was synthesized and labeled with biotinylated UTP and CTP by in vitro transcription using the T7 promoter coupled double stranded cDNA as template and the T7 RNA Transcript Labeling Kit (ENZO Diagnostics Inc., Farmingdale NY).

extract_protocol: Trizol extraction of total RNA was performed according to the manufacturer's instructions.

tag: Pediatric patient sample


title: 9415 TAF-el4 rep2


title: 2007-06-27 U112 gro383 T1

city: Stanford

first: Anna

last: Brotcke

department: Microbiology and Immunology

institute: Stanford University

description:
Simple annotation: Transcription, RNA
Image: http://smd.stanford.edu/MicroArray/gifs/2007-07/75960.gif

hyb_protocol: not provided

scan_protocol: Feature Extraction; Software and parameters for feature extraction.; Protocol Type = Feature Extraction; Parameter Image Origin = 1520, 15080; Parameter Datafile type = GenePix Results 3; Software: type: feature extraction; Performer: Anna,,Brotcke Scanning parameters - Channel 1; Parameters for laser scanning of a hybridized array in channel 1.; Protocol Type = Scanning parameters; Parameter Scanner Make = Axon Instruments (V1.00); Parameter Scan Temperature = 38.02; Parameter Scan Time = 09:45:52; Parameter PMT Volts = 620; Parameter Scan Date = 2007-07-12; Parameter Pixel Size = 10; Parameter Laser on time = 62820; Parameter Focus Position = 0; Parameter Scanner Model = GenePix 4000A [47902]; Parameter Laser Power = 2.11; Parameter Scan Power = 100; Parameter Scanning software version = 2.0.3.2; Parameter Scanning software = AxImageIO; Performer: Anna,,Brotcke Scanning parameters - Channel 2; Parameters for laser scanning of a hybridized array in channel 2.; Protocol Type = Scanning parameters; Parameter Scanner Make = Axon Instruments (V1.00); Parameter Scan Temperature = 38.02; Parameter Scan Time = 09:45:52; Parameter PMT Volts = 680; Parameter Scan Date = 2007-07-12; Parameter Pixel Size = 10; Parameter Laser on time = 135953; Parameter Focus Position = 0; Parameter Scanner Model = GenePix 4000A [47902]; Parameter Laser Power = 2.18; Parameter Scanning software version = 2.0.3.2; Parameter Scan Power = 100; Parameter Scanning software = AxImageIO; Performer: Anna,,Brotcke

data_processing: VALUE is Log (base 2) of the ratio of the median of Channel 2 (usually 635 nm) to Channel 1 (usually 532 nm)

organism: Francisella tularensis

source_name: Ref

label_protocol: not provided

extract_protocol: not provided

tag: Ref

source_name: Timepoints

tag: Timepoints


title: 9415 TAF-el4 rep3


title: 9414 NSF rep2


title: p1118_n

type: genomic

city: Washington

first: David

middle: A

last: Solomon

state: DC

address: 3970 Reservoir Rd, N.W.

department: Oncology

institute: Georgetown University School of Medicine

description: copy number analysis

hyb_protocol: The biotinylated fragments are added to a hybridization solution containing a biotinylated control oligonucletide (for quality control), and hybridized to a microarray chip overnight at 49°C. The chips are then transferred to a fluidics instrument that performs washes to remove DNA that has not hybridized to its complementary oligonucleotide probe. The bound DNA is then fluorescently labeled using phycoerythrin-conjugated streptavidin (SAPE) followed by biotinylated anti-streptavidin, followed by SAPE.

scan_protocol: Each DNA bound at its complementary oligonucleotide is excited using a confocal laser scanner, and the positions and intensities of the fluorescent emissions are captured. These measures provide the basis of subsequent biostatistical analysis.

data_processing: dChip was used for copy number analysis inferred by median smoothing with a window size of 3 and annotated according to the hg17 genome assembly.

molecule: genomic DNA

source_name: GBM primary tumor

label_protocol: One aliquot of 250 ng of DNA and is cleaved with NspI.  Linkers are ligated to the restricton fragments using T4 DNA ligase. The linkers provide a primer site for the subsequent PCR reaction. The fragments are amplified using Taq polymerase. The PCR products are purified from the primers and free nucleotides by ultrafiltration. They are then quantified spectrophotometrically, and are also assayed using size fractionation on a microfluidics device to determine whether the size distribution of products is as expected. The purified PCR products are fragmented by DNAse I in order to provide 3' hydroxyl groups for subsequent labeling, and to facilitate the subsequent hybridization step. The fragmented PCR products are labeled with a single biotin at each free 3'OH using terminal deoxynucleotidyl transferase and a dideoxy biotinylated nucleoside triphosphate.

growth_protocol: No growth, genomic DNA was prepared from tumors and xenografts snap-frozen at time of surgical resection.

extract_protocol: Tissue was lysed in SDS and digested with Proteinase K.  Genomic DNA was purified by phenol:chloroform extraction followed by ethanol precipitation.

tag: glioblastoma tumor


title: M059J_n

description: copy number analysis

source_name: GBM cell line


title: p1113_n

description: copy number analysis


title: 9N_KG-1-C

description: copy number analysis


title: 7N_SNB19

description: copy number analysis


title: p1108_n

description: copy number analysis


title: 8N_AM38

description: copy number analysis


title: DBTRG-05MG_n

description: copy number analysis


title: K60 vs G60 biol_replicate3_L

city: Goiania

first: Silvia Maria

last: Salem-Izacc

state: Goias

address: ICB, Campus 2

department: Bioquimica e Biologia Molecular

country: Brazil

institute: Universidade Federal de Goiás

description: Fluorescence mean intensity and surrounding median background from each spot were obtained with ArrayVision v6.0 (Imaging Research, Inc). Unreliable spots, having intensities too similar to local background or saturated were filtered out. Spots presenting mean intensity below 2 times the standard deviation of its background in Cy3 and Cy5 simultaneously were eliminated from the subsequent analysis since their expression ratio is meaningless,. Saturated signals [intensity greater than >990 fluorescence units] were also discarded. Normalization was carried out by LOWESS fitting on a M versus S plot, where M is the fluorescence log-ratio of the heat shock time-point relative to the control condition [M=log2(ratio)] and S is the log-mean fluorescence intensity [S = log2((cy3+cy5)/2)].

hyb_protocol: Cy3 and Cy5 fluorescent labeled cDNAs were resuspended in 13ul of water and mixed in a solution containing 25% of Hybridization Buffer (Amersham Biosciences) and 50% formamide, in a final volume of 56ul. Samples were denatured by heating at 95oC for 2 min and immediately chilled on ice. The targets were applied on the microarray slides, covered with a 24 x 60 mm coverslip (Corning) and hybridized at 42oC for 16h. Washing was performed once in 1xSSC, 0.1% SDS for 10 min, twice in 0.1xSSC, 0.01% SDS for 10 min and in 0.1xSSC for 1 min. The slides were shrinked in water for 10s and dried with nitrogen gas.

scan_protocol: Microarray slides were scanned with a Generation III DNA Scanner (Amersham Biosciences).

data_processing: filtering low intensity spots, LOWESS normalization

organism: Blastocladiella emersonii

source_name: 60 min of germination in nutrient medium DM3

label_protocol: 10ug of total RNA was reverse transcribed and labeled using CyScribe Post-labelling kit (GE Healthcare). Unincorporated dye was removed by purification in Millipore filters MAFB000 in guanidine buffer pH 4.8 followed by 4 washes with ethanol 80%. Labeled cDNA was eluted with 90 ul of Tris 10mM, pH8.0 and dried in speed vac.

extract_protocol: Total RNA was extracted using Trizol reagent (Invitrogen)

source_name: 60 min of germination induced by potassium


title: 6N_LN405

description: copy number analysis


title: K60 vs G60 biol_replicate3_R

tag: 60 min of germination in nutrient medium DM3

tag: 60 min of germination induced by potassium


title: A172_n

description: copy number analysis


title: U87MG_n

description: copy number analysis


title: p817_n

description: copy number analysis


title: UI 617

description: copy number analysis

source_name: GBM primary culture


title: Hs683_n

description: copy number analysis


title: p1016_n

description: copy number analysis


title: K60 vs G60 biol_replicate2_R


title: 645_n

description: copy number analysis

source_name: GBM xenograft


title: K60 vs G60 biol_replicate1_R


title: p819_n

description: copy number analysis


title: 398_n

description: copy number analysis


title: UI 645

description: copy number analysis


title: p1044_n

description: copy number analysis


title: 3N_DKMG

description: copy number analysis


title: 5N_GMS10

description: copy number analysis


title: H4_n

description: copy number analysis


title: U138MG_n

description: copy number analysis


title: 4N_GAMG

description: copy number analysis


title: K60 vs G60 biol_replicate1_L


title: 368_n

description: copy number analysis


title: UI 603

description: copy number analysis


title: K60 vs G60 biol_replicate2_L


title: UI 590

description: copy number analysis


title: A60 vs G60 biol_replicate3_R

source_name: 60 min of germination in nutrient medium

source_name: 60 min of germination induced by adenine


title: K30 vs G30 biol_replicate3_L

source_name: 30 min of germination induced by potassium

source_name: 30 min of germination in nutrient medium DM3


title: K30 vs G30 biol_replicate3_R

tag: 30 min of germination induced by potassium

tag: 30 min of germination in nutrient medium DM3


title: K30 vs G30 biol_replicate1_L


title: K30 vs G30 biol_replicate1_R


title: 259_n

description: copy number analysis


title: A60 vs G60 biol_replicate3_L

source_name: 60 min of germination in nutrient medium G60

tag: 60 min of germination induced by adenine


title: CCF-STTG1_n

description: copy number analysis


title: 245_n

description: copy number analysis


title: 329_n

description: copy number analysis


title: UI 583

description: copy number analysis


title: 31N_UI 540

description: copy number analysis


title: ATCC 25404 WT LB 5FU 7 h 37C biofilm cell (Rep2)

city: College Station

first: Can

last: Attila

state: TX

address: 524 Jack E. Brown Bldg.

department: Chemical Eng

institute: Texas A&M University

description:
RNA extracted from biofilm cells of E. coli K-12 wild type after 7 hours of growth in LB with 10 μM 5-FU (dissolved in DMF) with glasswool at 37C.
Biological replicate 2 of 2.

hyb_protocol: Following affymetrix protocol. Prepared hybridization cocktail for Single Probe Array (169 Format) including 1 X hybridization buffer, 50 pM B2 Control Oligo, 0.1 mg/mL Herring Sperm DNA, 0.5 mg/mL BSA, and at least 1 ug of fragmented and labelled cDNA. After loading of hybridization coctail in Affymetrix E. coli Genome 2.0 Array, the hybridization was performed at 45C, with 60 rpm for 16 hours. After hybridization, the probe array was washed and stained using Affymetrix Genechip Fluidics Station 450 and the software GenomeChipOperating Software (GCOS)

scan_protocol: Following affymetrix protocol. After washing and staining, the probe array was scanned using Affymetrix Genechip scanner GCS3000 7G system and the software GenomeChipOperating Software (GCOS)

data_processing: MAS 5.0 Expression Analysis Default Setting

organism: Escherichia coli

source_name: E. coli K-12 biofilm cells, LB 5FU

label_protocol: Following affymetrix protocol. cDNA was synthesized first using Promega M-MLV Reverse transcriptase (Cat#M1705). After removing RNA, DNA fragmentation was performed to obtain and 50-200 base cDNA fragments. The fragmented cDNA was labelled with Biotin-ddUTP using Enzo BioArray Terminal Labeling Kit (Affymetrix, P/N 900181)

extract_protocol: The overnight culture (1 mL of OD: 7.5) was used to inoculate 250 ml of fresh LB medium with 10 g of submerged glass wool (Corning Glass Works, Corning, NY) for forming biofilm. After incubation for 7h at 37C with shaking (250 rpm), the glass wool was carefully and quickly removed and rinsed with 100 ml of sterile 0.85% NaCl solution at 0C. Biofilm cells were removed by sonicating the glass wool in 200 ml of sterile 0.85% NaCl solution at 0C. After breaking the cells with a bead beater, and the total RNA was isolated with Qiagen RNeasy mini kit (Cat#74104)

tag: RNA extracted from biofilm cells of E. coli K-12 ATCC 25404 wild type after 7 hours of growth in LB with 10 μM 5-fluorouracil (dissolved in DMF) with glass wool at 37C


title: ATCC 25404 WT LB DMF 7 h 37C biofilm cell (Rep2)

description:
RNA extracted from biofilm cells of E. coli K-12 wild type after 7 hours of growth in LB DMF with glasswool at 37C.
Biological replicate 2 of 2.

source_name: E. coli K-12 biofilm cells, LB DMF

tag: RNA extracted from biofilm cells of E. coli K-12 ATCC 25404 wild type after 7 hours of growth in LB DMF with glasswool at 37C


title: p782_n

description: copy number analysis


title: 2N_42MGBA

description: copy number analysis


title: K30 vs G30 biol_replicate2_L


title: UI 581

description: copy number analysis


title: UI 520

description: copy number analysis


title: A60 vs G60 biol_replicate2_R

tag: 60 min of germination in nutrient medium


title: UI 519

description: copy number analysis


title: A60 vs G60 biol_replicate1_R


title: A60 vs G60 biol_replicate2_L


title: p667_n

description: copy number analysis


title: 1N_8MGBA

description: copy number analysis


title: A60 vs G60 biol_replicate1_L


title: UI 541

description: copy number analysis


title: 566_n

description: copy number analysis


title: 561_n

description: copy number analysis


title: K30 vs G30 biol_replicate2_R

source_name: 30 min of germination in nutrient medium


title: 661_n

description: copy number analysis


title: A30 vs G30 biol_replicate2_L

source_name: 30 min of germination induced by adenine


title: T98G_n

description: copy number analysis


title: 298_n

description: copy number analysis


title: p1155_n

description: copy number analysis


title: 317_n

description: copy number analysis


title: 409_n

description: copy number analysis


title: A30 vs G30 biol_replicate3_R

tag: 30 min of germination induced by adenine


title: G90 vs ZSP biol_replicate3_R

description: Fluorescence mean intensity and surrounding median background from each spot were obtained with ArrayVision v6.0 (Imaging Research, Inc). Unreliable spots, having intensities too similar to local background or saturated were filtered out. Spots presenting mean intensity below 2 times the standard deviation of its background in Cy3 and Cy5 simultaneously were eliminated from the subsequent analysis since their expression ratio is meaningless,. Saturated signals [intensity greater than >990 fluorescence units] were also discarded. Normalization was carried out by LOWESS fitting on a M versus S plot, where M is the fluorescence log-ratio of the heat shock time-point relative to the control condition [M=log2(ratio)] and S is the log-mean fluorescence intensity [S = log2((cy3+cy5)/2)]

source_name: 90 min of germination in nutrient medium DM3

source_name: ZSP (zoospores; time = 0 of germination)


title: G90 vs ZSP biol_replicate3_L

tag: 90 min of germination in nutrient medium DM3

tag: ZSP (zoospores; time = 0 of germination)


title: A30 vs G30 biol_replicate1_R


title: A30 vs G30 biol_replicate1_L


title: p1151_n

description: copy number analysis

source_name: GBM primary tumor


title: 395_n

description: copy number analysis


title: A30 vs G30 biol_replicate2_R


title: 11N_xenograft#3

description: copy number analysis


title: LN229_n

description: copy number analysis

source_name: GBM cell line


title: G90 vs ZSP biol_replicate2_R


title: 10N_NMC-G1

description: copy number analysis


title: LN18_n

description: copy number analysis


title: p1125_n

description: copy number analysis


title: OBF-1 overexpression BCS-2 (EPLM)

city: Basel

first: Edward

middle: James

last: Oakeley

address: Maulbeerstrasse 66

country: Switzerland

institute: Friedrich Miescher Institute for Biomedical Research

description: EPLM from an OBF-1 overexpressing mouse

hyb_protocol: RNA was hybridised using the standard Affymetrix hybridisation protocol as per manufacturer's instructions (45°C over night)

scan_protocol: The chips were scanned using an Affymetrix genechip scanner as per manufacturer's instructions

data_processing: Expression values were estimated using the GCRMA algorithm implemented in Genedata's Refiner 4.5. Further data anaylsis was performed using Genedata's Analyst 4.5

label: Biotin

source_name: EPLM

label_protocol: RNA was amplified and labelled using the Affymetrix 1-cycle 3'IVT protocol

growth_protocol: Mice were grown for seven weeks in our mouse facility

extract_protocol: RNA was purified using the Qiagen RNA easy protocol according to the manufacturer's insructions

treatment_protocol: EPLM and Large PreB cells were FACS sorted

tag: Strain

value: B6CF1 x C57BL/6, Tissue:Bone marrow, Age:7 weeks


title: G90 vs ZSP biol_replicate1_R


title: G90 vs ZSP biol_replicate2_L


title: OBF-1 overexpression BCS-3 (EPLM)

description: EPLM from an OBF-1 overexpressing mouse


title: carbon-limited chemostat of Penicillium chrysogenum DS50661 (cluster free strain) in presence of adipic acid - 3

city: Delft

first: Jean-Marc

last: Daran

address: Julianalaan 67

department: Department of Biotechnology

country: Netherlands

institute: Delft University of Technology

hyb_protocol: Probe preparation and Target hybridization Double stranded cDNA synthesis was carried out using 10 ug of total RNA and the components of the One Cycle cDNA Synthesis Kit (Affymetrix, Santa Clara, USA). The double-stranded cDNA was purified with the GeneChip® Sample Cleanup Module (Affymetrix/Qiagen) followed by in vitro transcription and labeling using the GeneChip® IVT labeling Kit (Affymetrix). Finally, labeled cRNA was purified (GeneChip® Sample Cleanup Module, Affymetrix/Qiagen) prior to fragmentation. 15 μg of fragmented, biotinylated cRNA was hybridized to the P.chrysogenum MicroArrays at 45 °C for 16 h as described in the Affymetrix users'manual. Washing and staining of arrays were performed using the GeneChip® Fluidics Station 400 and scanning with the Affymetrix GeneArray Scanner 3000

data_processing:
Data analysis Acquisition and quantification of array images were performed using Affymetrix GeneChip® Operating Software (GCOS version 1.2). Before comparison, all arrays were globally scaled to a target value of 100 using the average signal from all
gene features. To the 15,531 transcript features on the arrays, a filter was applied to extract 13,925 open reading frames.

label: SAPE

organism: Penicillium chrysogenum

source_name: Chemostat culture

growth_protocol:
Aerobic glucose-limited chemostat cultivation was performed at 25ºC in 3 - L turbine stirred bioreactors (Applikon, Schiedam, The Netherlands) with a working volume of 1.8 L. The pH was maintained at 6.5 via automated addition of 2 M NaOH (ADI 1030 biocontroller, Applikon, Schiedam, The Netherlands). The fermenter was sparged with air at a flow rate of 0.9 L•min-1 using a Brooks mass-flow controller (Brooks Instruments, Hatfield, USA) and stirred at 750 rpm. The dissolved-oxygen concentration was continuously monitored with an oxygen electrode (Applisens, Schiedam, The Netherlands). Continuous cultivation was initiated after 50-60 hours of batch cultivation. The feed medium was supplied continuously by a peristaltic pump (Masterflex, Cole Parmer, USA) and the dilution rate was set at 0.03 h-1 for all chemostat experiments. Effluent was removed discontinuously by means of a special overflow device. The time interval between effluent removals was fixed in such a way that each time approximately 1 % of the culture volume was removed. To prevent excessive foaming, silicone antifoam (10 % vol/vol, BDH) was discontinuously added at timed intervals. The offgas was cooled by a condensor at 4ºC after which it was dried with a Perma Pure dryer (type MD-110-48P-4, Perma Pure, Toms River USA). Oxygen and carbon dioxide concentrations in the offgas were determined with a NGA 2000 analyzer (Rosemount Analytical, Orville, USA). Off-gas flow rates were determined from an average of 10 measurements using a SAGA digital flow meter (Ion Science, Cambridge, UK). Specific rates of carbon dioxide and oxygen consumption were calculated.
Chemostats cultures were grown on a mineral medium, which was prepared and contained per litre of demineralised water 7.5 g glucose, 3.5 g (NH4)2SO4, 0.8 g KH2PO4, 0.5 g MgSO4•7H2O, 10 mL of a trace element solution. The trace element solution contained 15 g•L-1 Na2EDTA•2H2O, 0.5 g•L-1 Cu2SO4•5H2O, 2 g•L-1 ZnSO4•7H2O, 2 g•L-1 MnSO4•H2O, 4 g•L-1 FeSO4•7H2O, and 0.5 g•L-1 CaCl2•2H2O.
Production of ad7-ACCCA was induced by adding 5.0 g•L-1 adipic acid (ADA) to the medium.

extract_protocol:
Sampling and RNA extraction procedures 60 mL of culture broth was sampled and rapidly filtered over a glass fiber filter (Type A/E, Pall Life Sciences, East Hills, USA). The filter with mycelium was wrapped in aluminum foil, quenched in liquid nitrogen and subsequently stored at -80 °C until further use. For total RNA extraction half of the pellet was ground by mortar and pestle under constant cooling with liquid nitrogen. The powder was taken up in 5 mL of Trizol reagent (Invitrogen) and 1 mL chloroform (Sigma) and mixed well. The two phases were separated by centrifugation (4600 g, 15 minutes).
Total RNA was further purified by extraction with acid phenol/chloroform/isoamyl alcohol (5:1, pH 4.8, Ambion), followed by a chloroform extraction. Each time the phases were separated by centrifugation (4600 g, 15 minutes). Total RNA was precipitated for 30
minutes at -20°C in 96% ethanol and 0.3 M sodium acetate. After centrifugation at 23000 g for 15 minutes the RNA was dissolved in RNAse free H2O.

treatment_protocol: liquid nitrogen quenching

tag: DS50661 lacks the penicillin biosynthesis cluster and was constructed from DS17690

tag: To enable studies on the genome-wide impact of penicillin-G biosynthesis in Penicillium chrysogenum, penicillin gene clusters (pcbAB-pcbC-penDE) were removed from the penicillin-G high-producing strain P. chrysogenum DS17690. Since the Î²-lactam gene amplifications are in direct repeats of a larger amplified region on the same chromosome spontaneous recombinations between different repeats can result in loss of gene cluster copies. Spontaneous recombinations were induced via protoplast formation and subsequent sporulation of regenerating colonies  27 random isolates were selected for Southern analysis to estimate the relative gene copy numbers of the penicillin biosynthesis gene pcbC and the single-copy niaA gene. The same strains were tested for penicillin-V production in shake-flask cultures grown on mineral medium with the side-chain precursor phenoxyacetic acid. All  strains with reduced pcbC:niaA ratioâ€™s also showed reduced penicillin-V titres. One selected strain was

tag: subjected to a second round of protoplast formation and screening. Again, 27 random isolates were selected and analysed. Several putative single-copy penicillin gene cluster candidates (indicated by arrows in Figure 1, panel B) were identified based on a similar pcbC:niaA ratio to that of the single-copy Wisconsin54-1255 strain [8,27,55,78]. Three of these strains were tested in for penicillin-V production, with the parental P. chrysogenum DS17690 parent, the intermediate parent DS47274, the laboratory strain Wisconsin54-1255 and a non-producing isolate of the latter strain, npe10, as controls. All three isolates showed a drastically reduced penicillin-V titre, comparable to that of the single-copy strain Wisconsin54-1255. It was therefore assumed that these three isolates are derivatives of P. chrysogenum DS17690 that carry a single copy of the penicillin biosynthesis gene cluster. To delete the last remaining copy of the penicillin gene cluster, a double homologous recomb  ination strategy was applied. Sequences adjacent to the Î²-lactam biosynthetic gene cluster (3â€™ from the pcbAB gene and 3â€™ from the penDE gene) were used as homologous flanking sequences to target the amdS selection marker to this locus and to delete the entire 17 kb region containing the cluster of Î²-lactam biosynthetic genes (pcbAB-pcbC-penDE) in the same integration event. Homologous integration by double crossover will generate transformants able to use acetamide as the sole carbon source (due to the presence of the amdS gene) but unable to produce any penicillins. Moreover, genomic DNA of strains that have undergone a successful double crossover should not hybridise to Î²-lactam gene specific probes. Of 27,076 transformants tested, 22 (0.08%) gave no inhibition zone when overlaid with a penicillin sensitive E. coli strain and were selected for further analyses. All 22 putative mutants were negative in the diagnostic PCR for the Î²-lactam biosynthetic gene penDE. Tw  o of these strains gave no signal for amdS, suggesting that they had spontaneously lost marker gene. Southern analysis of the obtained P. chrysogenum strains was subsequently carried out. As a probe an internal DNA fragment of 300 nucleotides from the pcbAB sequence was amplified. All strains with intact penicillin-gene amplicon sequences (i.e. the industrial parent strain as well as the 1 amplicon copy strain) show an 8.1 kb DNA hybridising fragment. Putative Î²-lactam cluster free DS17690 derivatives showed no hybridisation with the probe. To confirm their inability to biosynthesise Î²-lactams all 22 mutants were inoculated in liquid mineral medium with phenylacetic acid as precursor. Indeed, none of the mutants was capable of producing penicillin-G . It was concluded that all 22 strains are derivatives of the P. chrysogenum DS17690 strain in which all copies of the penicillin gene cluster have been deleted. Isolate DS50661 was used as cluster-free strain in this study.


title: 263_n

description: copy number analysis


title: G60 vs ZSP 3000148375_700_R

source_name: ZSP (zoospores; time = 0 of germination) DM3


title: G90 vs ZSP biol_replicate1_L


title: G60 vs ZSP 3000148375_700_L

source_name: 60 min of germination in nutrient medium DM3


title: WT-3 (EPLM)

description: Control EPLM


title: G60 vs ZSP 3000148373_630_R


title: G60 vs ZSP 3000148373_630_L


title: carbon-limited chemostat of Penicillium chrysogenum DS50661 (cluster free strain) in presence of adipic acid - 2

scan_protocol: Probe preparation and Target hybridization Double stranded cDNA synthesis was carried out using 10 ug of total RNA and the components of the One Cycle cDNA Synthesis Kit (Affymetrix, Santa Clara, USA). The double-stranded cDNA was purified with the GeneChip® Sample Cleanup Module (Affymetrix/Qiagen) followed by in vitro transcription and labeling using the GeneChip® IVT labeling Kit (Affymetrix). Finally, labeled cRNA was purified (GeneChip® Sample Cleanup Module, Affymetrix/Qiagen) prior to fragmentation. 15 μg of fragmented, biotinylated cRNA was hybridized to the P.chrysogenum MicroArrays at 45 °C for 16 h as described in the Affymetrix users'manual. Washing and staining of arrays were performed using the GeneChip® Fluidics Station 400 and scanning with the Affymetrix GeneArray Scanner 3000


title: WT-2 (EPLM)

description: Control EPLM


title: WT-1 (EPLM)

description: Control EPLM


title: A30 vs G30 biol_replicate3_L

tag: 30 min of germination in nutrient medium


title: Carbon-limited chemostat of Penicillium chrysogenum DS50661 (cluster free strain) in presence of adipic acid - 1

label_protocol:
Probe preparation and Target hybridization Double stranded cDNA synthesis was carried out using 10 ug of total RNA and the components of the One Cycle cDNA Synthesis Kit (Affymetrix, Santa Clara, USA). The double-stranded cDNA was purified with the GeneChip® Sample Cleanup Module (Affymetrix/Qiagen) followed by in vitro transcription and labeling using the GeneChip® IVT labeling Kit (Affymetrix). Finally,
labeled cRNA was purified (GeneChip® Sample Cleanup Module, Affymetrix/Qiagen) prior to fragmentation. 15 μg of fragmented, biotinylated cRNA was hybridized to the P.chrysogenum MicroArrays at 45 °C for 16 h as described in the Affymetrix users'manual. Washing and staining of arrays were performed using the GeneChip® Fluidics
Station 400 and scanning with the Affymetrix GeneArray Scanner 3000


title: G60 vs ZSP 3000148365_630_R


title: G30 vc ZSP 3000148370_630_R


title: Carbon-limited chemostat of Penicillium chrysogenum DS50661 (cluster free strain) in absence of side chain -3

growth_protocol:
Aerobic glucose-limited chemostat cultivation was performed at 25ºC in 3 - L turbine stirred bioreactors (Applikon, Schiedam, The Netherlands) with a working volume of 1.8 L. The pH was maintained at 6.5 via automated addition of 2 M NaOH (ADI 1030 biocontroller, Applikon, Schiedam, The Netherlands). The fermenter was sparged with air at a flow rate of 0.9 L•min-1 using a Brooks mass-flow controller (Brooks Instruments, Hatfield, USA) and stirred at 750 rpm. The dissolved-oxygen concentration was continuously monitored with an oxygen electrode (Applisens, Schiedam, The Netherlands). Continuous cultivation was initiated after 50-60 hours of batch cultivation. The feed medium was supplied continuously by a peristaltic pump (Masterflex, Cole Parmer, USA) and the dilution rate was set at 0.03 h-1 for all chemostat experiments. Effluent was removed discontinuously by means of a special overflow device. The time interval between effluent removals was fixed in such a way that each time approximately 1 % of the culture volume was removed. To prevent excessive foaming, silicone antifoam (10 % vol/vol, BDH) was discontinuously added at timed intervals. The offgas was cooled by a condensor at 4ºC after which it was dried with a Perma Pure dryer (type MD-110-48P-4, Perma Pure, Toms River USA). Oxygen and carbon dioxide concentrations in the offgas were determined with a NGA 2000 analyzer (Rosemount Analytical, Orville, USA). Off-gas flow rates were determined from an average of 10 measurements using a SAGA digital flow meter (Ion Science, Cambridge, UK). Specific rates of carbon dioxide and oxygen consumption were calculated.
Chemostats cultures were grown on a mineral medium, which was prepared and contained per litre of demineralised water 7.5 g glucose, 3.5 g (NH4)2SO4, 0.8 g KH2PO4, 0.5 g MgSO4•7H2O, 10 mL of a trace element solution. The trace element solution contained 15 g•L-1 Na2EDTA•2H2O, 0.5 g•L-1 Cu2SO4•5H2O, 2 g•L-1 ZnSO4•7H2O, 2 g•L-1 MnSO4•H2O, 4 g•L-1 FeSO4•7H2O, and 0.5 g•L-1 CaCl2•2H2O.

tag: To enable studies on the genome-wide impact of penicillin-G biosynthesis in Penicillium chrysogenum, penicillin gene clusters (pcbAB-pcbC-penDE) were removed from the penicillin-G high-producing strain P. chrysogenum DS17690. Since the beta-lactam gene amplifications are in direct repeats of a larger amplified region on the same chromosome spontaneous recombinations between different repeats can result in loss of gene cluster copies. Spontaneous recombinations were induced via protoplast formation and subsequent sporulation of regenerating colonies  27 random isolates were selected for Southern analysis to estimate the relative gene copy numbers of the penicillin biosynthesis gene pcbC and the single-copy niaA gene. The same strains were tested for penicillin-V production in shake-flask cultures grown on mineral medium with the side-chain precursor phenoxyacetic acid. All  strains with reduced pcbC:niaA ratio's also showed reduced penicillin-V titres. One selected strain was

tag: subjected to a second round of protoplast formation and screening. Again, 27 random isolates were selected and analysed. Several putative single-copy penicillin gene cluster candidates (indicated by arrows in Figure 1, panel B) were identified based on a similar pcbC:niaA ratio to that of the single-copy Wisconsin54-1255 strain [8,27,55,78]. Three of these strains were tested in for penicillin-V production, with the parental P. chrysogenum DS17690 parent, the intermediate parent DS47274, the laboratory strain Wisconsin54-1255 and a non-producing isolate of the latter strain, npe10, as controls. All three isolates showed a drastically reduced penicillin-V titre, comparable to that of the single-copy strain Wisconsin54-1255. It was therefore assumed that these three isolates are derivatives of P. chrysogenum DS17690 that carry a single copy of the penicillin biosynthesis gene cluster. To delete the last remaining copy of the penicillin gene cluster, a double homologous recomb  ination strategy was applied. Sequences adjacent to the beta-lactam biosynthetic gene cluster (3' from the pcbAB gene and 3' from the penDE gene) were used as homologous flanking sequences to target the amdS selection marker to this locus and to delete the entire 17 kb region containing the cluster of beta-lactam biosynthetic genes (pcbAB-pcbC-penDE) in the same integration event. Homologous integration by double crossover will generate transformants able to use acetamide as the sole carbon source (due to the presence of the amdS gene) but unable to produce any penicillins. Moreover, genomic DNA of strains that have undergone a successful double crossover should not hybridise to beta-lactam gene specific probes. Of 27,076 transformants tested, 22 (0.08%) gave no inhibition zone when overlaid with a penicillin sensitive E. coli strain and were selected for further analyses. All 22 putative mutants were negative in the diagnostic PCR for the beta-lactam biosynthetic gene penDE. Two of these strains gave no signal for amdS, suggesting that they had spontaneously lost marker gene. Southern analysis of the obtained P. chrysogenum strains was subsequently carried out. As a probe an internal DNA fragment of 300 nucleotides from the pcbAB sequence was amplified. All strains with intact penicillin-gene amplicon sequences (i.e. the industrial parent strain as well as the 1 amplicon copy strain) show an 8.1 kb DNA hybridising fragment. Putative beta-lactam cluster free DS17690 derivatives showed no hybridisation with the probe. To confirm their inability to biosynthesise beta-lactams all 22 mutants were inoculated in liquid mineral medium with phenylacetic acid as precursor. Indeed, none of the mutants was capable of producing penicillin-G . It was concluded that all 22 strains are derivatives of the P. chrysogenum DS17690 strain in which all copies of the penicillin gene cluster have been deleted. Isolate DS50661 was used as cluster-free strain in this study.


title: G60 vs ZSP 3000148365_630_L

tag: 60 min of germination in nutrient medium DM3


title: carbon-limited chemostat of Penicillium chrysogenum DS49834 in presence of adipic acid - 3

tag: Construction of a Penicillium chrysogenum strain DS49834 producing ad7-ACCCA

tag: In order to produce carbamoylated cephalosporins with P. chrysogenum, the penicillinG high-producing strain DS17690 was transformed with various expression constructs. The cmcH carbamoyltransferase gene of Streptomyces clavuligerus  and cefEF expandase/hydroxylase gen of Acremonium chrysogenum were introduced via co-transformation with the amdS selection marker. The integration of the amdS marker enables the P. chrysogenum transformants to grow on selection medium containing acetamide as the sole nitrogen source. Acetamide-positive clones were transferred to a second acetamide-containing plate and subsequently transferred to induce sporulation.


title: Carbon-limited chemostat of Penicillium chrysogenum DS50661 (cluster free strain) in absence of side chain -1

tag: To enable studies on the genome-wide impact of penicillin-G biosynthesis in Penicillium chrysogenum, penicillin gene clusters (pcbAB-pcbC-penDE) were removed from the penicillin-G high-producing strain P. chrysogenum DS17690. Since the beta-lactam gene amplifications are in direct repeats of a larger amplified region on the same chromosome spontaneous recombinations between different repeats can result in loss of gene cluster copies. Spontaneous recombinations were induced via protoplast formation and subsequent sporulation of regenerating colonies  27 random isolates were selected for Southern analysis to estimate the relative gene copy numbers of the penicillin biosynthesis gene pcbC and the single-copy niaA gene. The same strains were tested for penicillin-V production in shake-flask cultures grown on mineral medium with the side-chain precursor phenoxyacetic acid. All  strains with reduced pcbC:niaA ratio’s also showed reduced penicillin-V titres. One selected strain was subjected to a second round of protoplast formation and screening. Again, 27 random isolates were selected and analysed. Several putative single-copy penicillin gene cluster candidates (indicated by arrows in Figure 1, panel B) were identified based on a similar pcbC:niaA ratio to that of the single-copy Wisconsin54-1255 strain [8,27,55,78]. Three of these strains were tested in for penicillin-V production, with the parental P. chrysogenum DS17690 parent, the intermediate parent DS47274, the laboratory strain Wisconsin54-1255 and a non-producing isolate of the latter strain, npe10, as controls. All three isolates showed a drastically reduced penicillin-V titre, comparable to that of the single-copy strain Wisconsin54-1255. It was therefore assumed that these three isolates are derivatives of P. chrysogenum DS17690 that carry a single copy of the penicillin biosynthesis gene cluster. To delete the last remaining copy of the penicillin gene cluster, a double homologous recombination strategy was applied. Sequences adjacent to the beta-lactam biosynthetic gene cluster (3’ from the pcbAB gene and 3’ from the penDE gene) were used as homologous flanking sequences to target the amdS selection marker to this locus and to delete the entire 17 kb region containing the cluster of beta-lactam biosynthetic genes (pcbAB-pcbC-penDE) in the same integration event. Homologous integration by double crossover will generate transformants able to use acetamide as the sole carbon source (due to the presence of the amdS gene) but unable to produce any penicillins. Moreover, genomic DNA of strains that have undergone a successful double crossover should not hybridise to beta-lactam gene specific probes. Of 27,076 transformants tested, 22 (0.08%) gave no inhibition zone when overlaid with a penicillin sensitive E. coli strain and were selected for further analyses. All 22 putative mutants were negative in the diagnostic PCR for the beta-lactam biosynthetic gene penDE. Two of these strains gave no signal for amdS, suggesting that they had spontaneously lost marker gene. Southern analysis of the obtained P. chrysogenum strains was subsequently carried out. As a probe an internal DNA fragment of 300 nucleotides from the pcbAB sequence was amplified. All strains with intact penicillin-gene amplicon sequences (i.e. the industrial parent strain as well as the 1 amplicon copy strain) show an 8.1 kb DNA hybridising fragment. Putative beta-lactam cluster free DS17690 derivatives showed no hybridisation with the probe. To confirm their inability to biosynthesise beta-lactams all 22 mutants were inoculated in liquid mineral medium with phenylacetic acid as precursor. Indeed, none of the mutants was capable of producing penicillin-G . It was concluded that all 22 strains are derivatives of the P. chrysogenum DS17690 strain in which all copies of the penicillin gene cluster have been deleted. Isolate DS50661 was used as cluster-free strain in this study.


title: Carbon-limited chemostat of Penicillium chrysogenum DS50661 (cluster free strain) in absence of side chain -2


title: G30 vc ZSP 3000148370_630_L

tag: 30 min of germination in nutrient medium DM3


title: OBF-1 overexpression BCS-1 (EPLM)

description: EPLM from an OBF-1 overexpressing mouse


title: G30 vs ZSP 3000148367_700_R


title: carbon-limited chemostat of Penicillium chrysogenum DS49834 in presence of adipic acid - 2


title: carbon-limited chemostat of Penicillium chrysogenum DS49834 in presence of adipic acid - 4


title: G30 vs ZSP 3000148361_600_R


title: G30 vc ZSP 3000148367_700_L


title: G30 vc ZSP 3000148361_600_L


title: carbon-limited chemostat of Penicillium chrysogenum DS49834 in absence of side chain - 1

tag: P, chrysogenum DS49834:


title: carbon-limited chemostat of Penicillium chrysogenum DS49834 in absence of side chain - 2


title: MIX (5-100) - 4

status: Public on Jan 03 2009

city: Vicksburg

first: Natalia

last: Vinas

state: MS

address: 3909 Halls Ferry Rd

institute: Mississippi State University

description: Four replicates consisting of four different individuals were analyzed for each of the treatments (solvent (TEG) control, non-solvent control, EE2, ZM, EE2 /ZM).  The cDNA synthesis, cRNA labeling and hybridization were performed following the manufacturer’s kits and protocols (Agilent Low RNA Input Fluorescent Linear Amplification Kit and Agilent 60-mer oligo microarray processing protocol; Agilent, Palo Alto, CA). The gonad samples were labeled with Cy5 while the reference sample was labeled with Cy3. Once the labeling was complete, samples were hybridized to the microarray using conditions recommended by the manufacturer.  After hybridizing for 17 h, microarrays were washed and then scanned with a laser-based detection system (Agilent, Palo Alto, CA).

hyb_protocol: Standard Agilent two-color protocol (Agilent 60-mer oligo microarray processing protocol)

scan_protocol: Microarrays were scanned with a laser-based detection system (Agilent, Palo Alto, CA).

data_processing: Microarray image processing and data pre-processing were performed using Agilent's Feature Extraction software v 9.5. The intensity of each spot was summarized by the median pixel intensity. A log10 transformed signal ratio between the experimental (Cy5) channel and the reference (Cy3) channel was calculated for each spot, followed by within-array lowess transformation.

organism: Pimephales promelas

source_name: Fathead minnows testis exposed to a mix of EE2 and ZM189,154

label_protocol: cDNA synthesis, cRNA labelling, amplification and hybridization were performed following the manufacturer's kits and protocols (Agilent Low RNA Input Fluorescent Linear Amplification Kit)

growth_protocol: Reproductively-mature, pond-reared FHM were purchased from Andersen Minnow Farm, AR, 4 days prior to starting the experiment. Upon arrival, the fish were treated for parasites and bacteria by a prophylactic salt-water dip (3%, 1 min). Males were separated from the population the following day, and acclimated in the treatment aquaria for 48 h.  The water used for this study was carbon-filtered, dechlorinated tap water. The exposure system consisted of 40 L glass aquaria.  Each exposure was conducted in quadruplicate and each aquarium contained eight male fathead minnows in 25 L of treatment water.    Test chemicals for each treatment group (100L for 4 aquaria) were prepared in separate (by treatment) 250 L fiberglass tanks the day of exposure.   Aquaria were equilibrated with test chemicals for 24 h prior to the introduction of fish.  Test solutions were renewed to 90% of the 25L exposure volume after 24 h and the exposure was ended at 48 h. The positions of the treatment tanks were randomized and test initiation times were staggered to ensure an exposure/sampling interval of 48 h. The fish were not fed the day before and during the experiment.  Temperature was maintained at 25oC with a photoperiod of 16 h light: 8 h dark.

extract_protocol: Total RNA was isolated from gonadal tissue with the RNA Stat-60 reagent (Tel-test, Friendswood, TX).

treatment_protocol: Fathead minnow ovary exposed for 48h to 5 ng/L ethinylestradiol, a mix of 5 ng/L ethinylestradiol and 100 ng/L ZM189,154, or control (triethylene glycol, TEG)

tag: RNA from fathead minnow testis exposed toa mix of 5 ng/L ethinylestradiol and 100 ng/L ZM189,154

source_name: Reference Pool

tag: The reference pool consisted of equal amounts of RNA from control fathead minnow female and male tissues (liver, brain and gonad).


title: MIX (5-100) - 1

tag: RNA from fathead minnow testis exposed to a mix of 5 ng/L ethinylestradiol and 100 ng/L ZM189,154


title: EE2_5 - 2

source_name: Fathead minnows testis treated with 5 ng/L ethinylestradiol

tag: RNA from fathead minnow testis exposed to 5 ng/L ethinylestradiol


title: MIX (5-100) - 3


title: carbon-limited chemostat of Penicillium chrysogenum DS49834 in absence of side chain - 3


title: MIX (5-100) - 2


title: TEG (EE2_5) - 2

source_name: Fathead minnows control testis

tag: RNA from fathead minnow control testis


title: EE2_5 - 1


title: EE2_5 - 4


title: carbon-limited chemostat of Penicillium chrysogenum DS49834 in presence of adipic acid - 1


title: EE2_5 - 3


title: TEG (EE2_5) - 4


title: TEG (EE2_5) - 3


title: TEG (EE2_5) - 1


title: CONTROL (EE2_10) - 3

treatment_protocol: Fathead minnow ovary exposed for 48h to 10 ng/L ethinylestradiol or control


title: ZM - 3

source_name: Fathead minnows testis treated with 100 ng/L ZM189,154

treatment_protocol: Fathead minnow ovary exposed for 48h to EE2, ZM189,154, a mix of EE2/ZM, or control (non-vehicle control or 70% triethylene glycol, TEG)

tag: RNA from fathead minnow testis exposed to 100 ng/L ZM189,154


title: EE2_10 - 1

source_name: Fathead minnows testis treated with 10 ng/L ethinylestradiol

tag: RNA from fathead minnow testis exposed to 10 ng/L ethinylestradiol


title: EE2_10 - 4


title: EE2_10 - 3


title: EE2_10 - 2


title: CONTROL (EE2_10) - 2


title: ZM - 2


title: CONTROL (EE2_10) - 4


title: ZM - 1


title: TEG - 4

source_name: Fathead minnows vehicle control testis

tag: RNA from fathead minnow vehicle control testis (triethylene glycol)


title: TEG - 1


title: F3-vinclozolin treated adult female rat hippocampus, replica 2

city: Pullman

first: Michael

middle: K

last: Skinner

state: WA

address: Abelson 507

department: SBS

institute: WSU

description: Gene expression data for adult female hippocampus from F3 vinclozolin-treated generation.

hyb_protocol: Standard Affymetrix procedures.

scan_protocol: Chips were scanned using Affymetrix Gene Chip Scanner 3000.

data_processing: The data were analyzed with Affymetrix GeneChip Operating Software using default normalization method. Target signal was set to 125. MAS-5 preprocessing was used.

organism: Rattus norvegicus

source_name: adult female rat hippocampus, vinclozolin-treated

label_protocol: Standard Affymetrix procedures.

extract_protocol: Tissue from one animal was used to make each RNA sample.  RNA was extracted using the Trizol reagent according to manufacturer's instructions (Sigma, St. Louis, MO).

treatment_protocol: Pregnant rats were given intraperitoneal injections of vinclozolin (100 mg/kg/day) in vehicle suspense (sesame oil and DMSO) from pregnancy day 8 to 14. F1-generation male and female from different control lines were mated to produce F2 generation animals which were mated again to produce F3-generation. F3-generation adult female rats were euthanized and hippocampi were removed according to IACUC-approved animal use protocols.

tag: Hippocampus was dissected from adult female of F3-vinclozolin-treated Sprague-Dawley rat.


title: TEG - 3


title: MIX (50-100) - 4

tag: RNA from fathead minnow testis exposed to a mix of 50 ng/L ethinylestradiol and 100 ng/L ZM189,154


title: ZM - 4


title: F3-control adult female rat hippocampus, replica 2

description: Gene expression data for adult female hippocampus from F3 control generation.

source_name: adult female rat hippocampus, control

treatment_protocol: Pregnant rats were given intraperitoneal injections of vehicle suspense (sesame oil and DMSO) from pregnancy day 8 to 14. F1-generation male and female from different control lines were mated to produce F2 generation animals which were mated again to produce F3-generation. F3-generation adult female rats were euthanized and hippocampi were removed according to IACUC-approved animal use protocols.

tag: Hippocampus was dissected from adult female of F3-control Sprague-Dawley rat.


title: F3-control adult female rat hippocampus, replica 1


title: F3-vinclozolin treated adult female rat hippocampus, replica 1


title: CONTROL (EE2_10) - 1


title: MIX (50-100) - 1


title: F3-control adult female rat amygdala, replica 1

description: Gene expression data for adult female amygdala from F3 control generation.

source_name: adult female rat amygdala, control

treatment_protocol: Pregnant rats were given intraperitoneal injections of vehicle suspense (sesame oil and DMSO) from pregnancy day 8 to 14. F1-generation male and female from different control lines were mated to produce F2 generation animals which were mated again to produce F3-generation. F3-generation adult female rats were euthanized and amygdalas were removed according to IACUC-approved animal use protocols.

tag: Amygdala was dissected from adult female of F3-control Sprague-Dawley rat.


title: F3-control adult female rat amygdala, replica 2


title: TEG - 2


title: MIX (50-100) - 2


title: F3-vinclozolin treated adult male rat hippocampus, replica 2

description: Gene expression data for adult male hippocampus from F3 vinclozolin-treated generation.

source_name: adult male rat hippocampus, vinclozolin-treated

treatment_protocol: Pregnant rats were given intraperitoneal injections of vinclozolin (100 mg/kg/day) in vehicle suspense (sesame oil and DMSO) from pregnancy day 8 to 14. F1-generation male and female from different control lines were mated to produce F2 generation animals which were mated again to produce F3-generation. F3-generation adult male rats were euthanized and hippocampi were removed according to IACUC-approved animal use protocols.

tag: Hippocampus was dissected from adult male of F3-vinclozolin-treated Sprague-Dawley rat.


title: F3-control adult male rat hippocampus, replica 2

description: Gene expression data for adult male hippocampus from F3 control generation.

source_name: adult male rat hippocampus, control

treatment_protocol: Pregnant rats were given intraperitoneal injections of vehicle suspense (sesame oil and DMSO) from pregnancy day 8 to 14. F1-generation male and female from different control lines were mated to produce F2 generation animals which were mated again to produce F3-generation. F3-generation adult male rats were euthanized and hippocampi were removed according to IACUC-approved animal use protocols.

tag: Hippocampus was dissected from adult male of F3-control Sprague-Dawley rat.


title: EE2_50 - 4

source_name: Fathead minnows testis treated with 50 ng/L ethinylestradiol

tag: RNA from fathead minnow testis exposed to 50 ng/L ethinylestradiol


title: F3-vinclozolin treated adult female rat amygdala, replica 1

description: Gene expression data for adult female amygdala from F3 vinclozolin-treated generation.

source_name: adult female rat amygdala, vinclozolin-treated

tag: Amygdala was dissected from adult female of F3-vinclozolin-treated Sprague-Dawley rat.


title: MIX (50-100) - 3


title: EE2_50 - 3


title: EE2_50 - 2


title: Control NV_3


title: F3-vinclozolin treated adult female rat amygdala, replica 2


title: Control NV_2


title: F3-vinclozolin treated adult male rat amygdala, replica 1

description: Gene expression data for adult male amygdala from F3 vinclozolin-treated generation.

source_name: adult male rat amygdala, vinclozolin-treated

treatment_protocol: Pregnant rats were given intraperitoneal injections of vinclozolin (100 mg/kg/day) in vehicle suspense (sesame oil and DMSO) from pregnancy day 8 to 14. F1-generation male and female from different control lines were mated to produce F2 generation animals which were mated again to produce F3-generation. F3-generation adult male rats were euthanized and amygdalas were removed according to IACUC-approved animal use protocols.

tag: Amygdala was dissected from adult male of F3-vinclozolin-treated Sprague-Dawley rat.


title: F3-vinclozolin treated adult male rat hippocampus, replica 1


title: EE2_50 - 1


title: F3-control adult male rat hippocampus, replica 1


title: Control NV_4


title: F3-control adult male rat amygdala, replica 1

description: Gene expression data for adult male amygdala from F3 control generation.

source_name: adult male rat amygdala, control

treatment_protocol: Pregnant rats were given intraperitoneal injections of vehicle suspense (sesame oil and DMSO) from pregnancy day 8 to 14. F1-generation male and female from different control lines were mated to produce F2 generation animals which were mated again to produce F3-generation. F3-generation adult male rats were euthanized and amygdalas were removed according to IACUC-approved animal use protocols.

tag: Amygdala was dissected from adult male of F3-control Sprague-Dawley rat.


title: F3-vinclozolin treated adult male rat amygdala, replica 2


title: F3-control adult male rat amygdala, replica 2


title: OPM1_Dex_DAC

city: Tokyo

first: Masanori

last: Nojima

state: Tokyo

address: 4-6-1, Shirokanedai, Minato-ku

department: The Institution of Medical Science

country: Japan

institute: The University of Tokyo

description: OPM1 is one of dexamethasone-resistant multiple myeloma cell lines.

hyb_protocol: Agilent protocol

scan_protocol: Agilent scanner and Agilent scan control software

data_processing: Feature Extraction ver 9.5

source_name: Human multiple myeloma cell line, OPM1

label_protocol: Agilent protocol

extract_protocol: Trizol and RNeasy Mini Elute Cleanup kit

treatment_protocol: Cell lines were incubated for 72 h with 0.1 µM 5-aza-2'-deoxycytidine (DAC; Sigma), and were then incubated for 24 h with 1 µM Dexamethasone (Dex; EMD bioscience).

tag: OPM1 with Dex and DAC treatment


title: F3-vinclozolin treated adult male rat whole brain, replica 1

description: Gene expression data for adult male whole brain from F3 vinclozolin-treated generation.

source_name: adult male rat whole brain, vinclozolin-treated

treatment_protocol: Pregnant rats were given intraperitoneal injections of vinclozolin (100 mg/kg/day) in vehicle suspense (sesame oil and DMSO) from pregnancy day 8 to 14. F1-generation male and female from different control lines were mated to produce F2 generation animals which were mated again to produce F3-generation. F3-generation adult male rats were euthanized and whole brains were removed according to IACUC-approved animal use protocols.

tag: Whole brain was dissected from adult male of F3-vinclozolin-treated Sprague-Dawley rat.


title: Control NV_1

source_name: Reference Pool

tag: The reference pool consisted of equal amounts of RNA from control fathead minnow female and male tissues (liver, brain and gonad).


title: F3-vinclozolin treated adult male rat whole brain, replica 2


title: OPM1_Dex

treatment_protocol: Cell lines were incubated for 24 h with 1 µM Dexamethasone (Dex; EMD bioscience).

tag: OPM1 with Dex treatment


title: F3-control adult male rat whole brain, replica 2

description: Gene expression data for adult male whole brain from F3 control generation.

source_name: adult male rat whole brain, control

treatment_protocol: Pregnant rats were given intraperitoneal injections of vehicle suspense (sesame oil and DMSO) from pregnancy day 8 to 14. F1-generation male and female from different control lines were mated to produce F2 generation animals which were mated again to produce F3-generation. F3-generation adult male rats were euthanized and whole brains were removed according to IACUC-approved animal use protocols.

tag: Whole brain was dissected from adult male of F3-control Sprague-Dawley rat.


title: OPM1_mock

tag: OPM1 with no treatment (control)


title: F3-control adult male rat whole brain, replica 1


title: ATCC 25404 WT LB 5FU 7 h 37C biofilm cell

status: Public on Jan 02 2009

description:
RNA extracted from biofilm cells of E. coli K-12 wild type after 7 hours of growth in LB with 10 μM 5-FU (dissolved in DMF) with glasswool at 37C.
Biological replicate 1 of 2.

hyb_protocol: Following affymetrix protocol.  Prepared hybridization cocktail for Single Probe Array (169 Format) including 1 X hybridization buffer, 50 pM B2 Control Oligo, 0.1 mg/mL Herring Sperm DNA, 0.5 mg/mL BSA, and at least 1 ug of fragmented and labelled cDNA. After loading of hybridization coctail in Affymetrix E. coli Genome 2.0 Array, the hybridization was performed at 45C, with 60 rpm for 16 hours. After hybridization, the probe array was washed and stained using Affymetrix Genechip Fluidics Station 450 and the software GenomeChipOperating Software (GCOS)

scan_protocol: Following affymetrix protocol.  After washing and staining, the probe array was scanned using Affymetrix Genechip scanner GCS3000 7G system and the software GenomeChipOperating Software (GCOS).

source_name: E. coli K-12 biofilm cells, LB 5FU

label_protocol: Following affymetrix protocol.  cDNA was synthesized first using Promega M-MLV Reverse transcriptase (Cat#M1705).  After removing RNA, DNA fragmentation was performed to obtain and 50-200 base cDNA fragments.  The fragmented cDNA was labelled with Biotin-ddUTP using Enzo BioArray Terminal Labeling Kit (Affymetrix, P/N 900181)

extract_protocol: The overnight culture (1 mL of OD: 7.5) was used to inoculate 250 ml of fresh LB medium with 10 g of submerged glass wool (Corning Glass Works, Corning, NY) for forming biofilm.  After incubation for 7h at 37C with shaking (250 rpm), the glass wool was carefully and quickly removed and rinsed with 100 ml of sterile 0.85% NaCl solution at 0C.  Biofilm cells were removed by sonicating the glass wool in 200 ml of sterile 0.85% NaCl solution at 0C.  After breaking the cells with a bead beater, and the total RNA was isolated with Qiagen RNeasy mini kit (Cat#74104)

tag: RNA extracted from biofilm cells of E. coli K-12 ATCC 25404 wild type after 7 hours of growth in LB with 10 μM 5-fluorouracil (dissolved in DMF) with glass wool at 37C


title: OPM1_DAC

treatment_protocol: Cell lines were incubated for 72 h with 0.1 µM 5-aza-2'-deoxycytidine (DAC; Sigma)

tag: OPM1 with DAC treatment


title: ATCC 25404 WT LB DMF 7 h 37C biofilm cell

description:
RNA extracted from biofilm cells of E. coli K-12 wild type after 7 hours of growth in LB DMF with glasswool at 37C.
Biological replicate 1 of 2.

source_name: E. coli K-12 biofilm cells, LB DMF

extract_protocol: The overnight culture (1 mL of OD: 7.5) was used to inoculate 250 ml of fresh LB medium with 10 g of submerged glass wool (Corning Glass Works, Corning, NY) for forming biofilm.  After incubation for 7h at 37C with shaking (250 rpm), the glass wool was carefully and quickly removed and rinsed with 100 ml of sterile 0.85% NaCl solution at 0C.  Biofilm cells were removed by sonicating the glass wool in 200 ml of sterile 0.85% NaCl solution at 0C.  After breaking the cells with a bead beater, the total RNA was isolated with Qiagen RNeasy mini kit (Cat#74104)

tag: RNA extracted from biofilm cells of E. coli K-12 ATCC 25404 wild type after 7 hours of growth in LB DMF with glasswool at 37C


title: Mycobacterium bovis BCG_no treatment_0min_replicate3

status: Public on Dec 01 2008

city: ft. meade

first: Hyeung-Jin

last: Jang

state: MD

address: 701 Mapes Road

department: OPP/BEAD

institute: USEPA

description: No treatment

hyb_protocol: Standard Affymetrix procedures

scan_protocol: Standard Affymetrix procedures (target signal value was 500)

data_processing: Affymetrix GeneChip Opererating Software (GCOS) version 1.2

organism: Mycobacterium tuberculosis variant bovis BCG str. Pasteur 1173P2

source_name: Mycobacterium bovis BCG_no treatment

label_protocol: Approximately 10 µg of total RNA was processed to produce biotinylated cDNA targets.

growth_protocol: (i) we initiated M. bovis cultures at 37°C with shaking at 250 rpm using sterilized Middlebrook 7H9 broth for 5days, (ii) Mycobacteria were harvested, washed and resuspended in sterilized Lurria-Bertani (LB) broth with 0.1% (v/v) Tween 80. Then the cultures were incubated for further 24 hours shaking at 37°C. To see the efficacy of an antimicrobial against mycobacteria, several concentrations of sodium hypochlorite were added to the cells.

extract_protocol: Total RNA was isolated using the RiboPure – Bacteria kit (Ambion, Inc., Austin, TX)[16]. The treated and untreated mycobacterial cells were harvested and resuspended in PBS buffer. For stabilize of RNA, we used for breaking the micobaterium cell, we used bead beater, 5 times for 1min beating and 2min on ice.

treatment_protocol: No treatment

tag: strain Pasteur 1173P2 Wild type


title: Mycobacterium bovis BCG_no treatment_0min_replicate1

description: No treatment


title: Mycobacterium bovis BCG_no treatment_0min_replicate2

description: No treatment


title: NusG ChIP-chip in E. coli K-12 MG1655 HA3::nusG cells (Dataset 62263)

city: Madison

first: Rachel

middle: Anne

last: Mooney

state: WI

address: 1550 Linden Drive

department: Biochemistry

institute: University of Wisconsin-Madison

description: NusG ChIP-chip in E. coli K-12 MG1655 HA3::nusG cells using monoclonal antibody 12CA5.

hyb_protocol: Samples were hybridized to arrays by Nimblegen according to established protocols (http://www.nimblegen.com/products/lit/lit.html).

scan_protocol: Arrays were scanned by Nimblegen according to established protocols (http://www.nimblegen.com/products/lit/lit.html).

source_name: NusG ChIP in E. coli K-12 MG1655 HA3::nusG cells

label_protocol: Samples were labeled by Nimblegen according to established protocols (http://www.nimblegen.com/products/lit/lit.html).

growth_protocol: Strain MG1655 or isogenic strain MG1655 HA3::nusG cells were grown in MOPS minimal medium supplemented with 0.2% glucose (www.genome.wisc.edu/resources/protocols/mopsminimal.htm).

extract_protocol: Cell pellets (from initial 50 ml of culture) were thawed and resuspended in 250ul of IP buffer (100 mM Tris pH 8, 300 mM NaCl, 2% TritonX-100) and sonicated using a microtip sonicator set at 10% output for 20 second intervals with periods of cooling in between. Cells were then treated for one hour at 4 °C with RNase A (2 ng/ml) micrococcal nuclease (50 units), 20 μM CaCl2,1.2 mM KCl, 0.3 mM NaCl, 6 mM sucrose, and 10 μM DTT. After treatment, a distribution of DNA fragments ranging from 200-600 bp was detected by agarose-gel electrophoretic separation of a small sample that was de-crosslinked by incubation at 65 °C for >4 hr. EDTA was added to 10 mM to stop the micrococcal nuclease and the samples were spun down to remove cell debris. The lysate was then incubated with a 50/50 slurry of Sepharose protein A beads and protein G beads in IP buffer for 2-3 hours at 4 °C. The beads were removed by centrifugation and antibody was added to the pre-cleared lysate for an overnight incubation. The next day, 30 μl of a 50/50 slurry of Sepharose protein A and G beads in IP buffer was added to the lysate to capture antibody-protein-DNA complex for one hour at 4 °C. Beads were then washed once with 1 ml of 250 mM LiCl wash buffer (100 mM Tris pH 8, 250 mM LiCl, 2% TritonX-100), twice with 600 mM NaCl wash buffer (100 mM Tris pH 8, 600 mM NaCl, 2%SDS), twice with 300 mM NaCl wash buffer (100 mM Tris pH 8, 300 mM NaCl, 2%SDS), and twice with TE. Elution buffer (50 mM Tris pH 8, 10 mM EDTA, 1% SDS) was added after the final wash step, and beads were incubated at 65 °C for 30 minutes toremove the crosslinked protein-DNA complexes from the beads. After centrifugation to remove the beads, the samples were incubated overnight at 65 °C to reverse the protein-DNA formaldehyde crosslinks. DNA was purified using Qiagen’s PCR Purification kit and eluted to a final volume of 50ul with EB.

treatment_protocol: Cells were grown with vigorous shaking at 37 °C to mid-log (light scattering at 600 nm equivalent to 0.4 OD). Sodium phosphate (1/100 vol. of 1M, pH 7.6; 10 mM final) was added to the mid-log cultures followed by formaldehyde to 1% final, and shaking was continued for 5 min. Cold 2.5 M glycine was added to 100mM and the mixture was incubated at 4 °C with agitation for 30 minutes to stop the crosslinking. Cells were spun at 5000 x g, and washed repeatedly with phosphate buffered saline before being frozen at -80 °C.

tag: Monoclonal antibody for HA (12CA5)

source_name: Genomic DNA control from E. coli K-12 MG1655 HA3::nusG cells

tag: Genomic DNA control


title: psar slide 176815 (TIGR4)

status: Public on Nov 19 2008

city: Groningen

first: Anne

middle: de

last: Jong

address: Nijenborgh 7

department: Molecular Genetics

institute: university of Groningen

description: Microarray analysis was performed essentially as described (Hendriksen et al., 2007 [PMID: 17085554]; Hendriksen et al., 2008 [PMID: 18024519]). In short, 500 ml of CDM was inoculated with 10-20 colonies from agar plates, and these cultures were statically grown at 37°C. Samples for RNA isolation were taken when the cultures reached an optical density (OD600) of 0.2 (mid-log growth). RNA was isolated and purified using the High Pure RNA isolation kit (Roche diagnostics) as described (Hendriksen et al., 2007; Hendriksen et al., 2008a). Contaminating genomic DNA was removed by treatment with RNase-free DNase I (Roche diagnostics). RNA was isolated from three replicate cultures. Synthesis, subsequent labeling of cDNA, and microarray hybridization was performed as described (Hendriksen et al., 2007; Kloosterman et al., 2006 [16787930]).

hyb_protocol: The protocol was performed as described in Hendriksen et al., 2008 (PMID: 18024519)

scan_protocol: Images were acquired with GeneTac LS IV confocal laser scanner (Genomics Solutions).

data_processing: Dual-channel array images were analyzed with ArrayPro 4.5 software (Media Cybernetics Inc.). Spots were screened visually to identify those of low quality. Slide data were processed with MicroPreP as previously described (15, 18, 49). Prior to analysis, automatically and manually flagged spots and spots with very low background subtracted signal intensity (5% of the weakest spots [sum of Cy3 and Cy5 net signals]) were filtered out of all data sets.Net signal intensities were calculated by grid-based background subtraction. A grid-based Lowess transformation was performed for slide normalization, negative and empty values were removed, and outliers were removed by the deviation test. Expression ratios of mutant strain over the wild-type strain were calculated. Further analysis was performed with a Cyber-T Student t test for paired data.

organism: Streptococcus pneumoniae

source_name: TIGR4 wild type

label_protocol: Total RNA was incubated for 16 h at 42°C in the presence of 400 U Superscript III RNase H reverse transcriptase (Invitrogen), 0.2 mM aminoallyl dUTP (Amersham), 0.5 mM dATP, 0.5 mM dCTP, 0.5 mM dGTP, 0.3 mM dTTP, and 3.2 g random nonamers. The synthesized cDNA was then labeled by coupling either Cy3 or Cy5 to the dUTP aminoallyl reactive group (CyScribe postlabeling kit; Amersham Biosciences) and was purified using a GFX PCR, DNA, and gel band purification kit (Amersham Biosciences).

tag: Cells grown statically in CDM (37°C) and harvested at OD600=0.2.

source_name: TIGR4 psaR mutant


title: psar slide 176819 (TIGR4)

extract_protocol: RNA was isolated and purified using the High Pure RNA isolation kit (Roche diagnostics) as described (Hendriksen et al., 2007; Hendriksen et al., 2008a). Contaminating genomic DNA was removed by treatment with RNase-free DNase I (Roche diagnostics). RNA was isolated from three replicate cultures.


title: psar slide 176823 (TIGR4)


title: psar slide 176807

source_name: D39 psaR mutant

source_name: D39 wild type


title: psar slide 176803

source_name: D39 psar mutant


title: psar slide 176811


title: IL-2+IL-21 (Cy5) versus IL-2(Cy3)

status: Public on Dec 20 2008

city: Nantes

first: Emmanuel

last: Scotet

address: 9 quai moncousu

country: France

institute: INSERM

description: PIQOR Immunology Microarray Service from Miltenyi Biotec

hyb_protocol: Hybridization performed overnight using the a-Hyb Hybridization station according to the PIQOR User Manual

scan_protocol: Fluorescence signals detected using the laser scanner ScanArray Lite (PerkinElmer Life Sciences)

data_processing:
Mean signal and mean local background intensities were obtained for each spot of the microarray images using the ImaGene's software (Biodiscovery). Low-quality spots were flagged and excluded from data analysis. Unflagged spots were analysed with the PIQORTM Analyzer software. The PIQOR Analyzer allows automated data processing of the raw datatext files derived from the ImaGene software. This includes background subtraction to obtain the net signal intensity, data normalization, and calculation of the Cy5/Cy3 ratios. As an additional quality filtering step, only spots/genes are taken into account for the calculation of the Cy5/Cy3 ratio that have at least in one channel a signal intensity that is at least 2-fold higher than the mean background.
The PIQOR Analyzer calculates all normalized mean Cy5/Cy3 ratios of the four replicates per gene.

label_protocol: Sample labelling performed according to the PIQOR User Manual

growth_protocol: Cells grown in RPMI supp. 10 % FCS

extract_protocol:
RNA extacted by using Nucleospin RNA II from Macherey-Nagel
Amplification T7-based (1 microgram total RNA)
Quantity/Integrity checked by using Agilent 2100 Bioanalyzer

tag: Human T cells Vgamma9Vdelta2

tag: Amplified from human fresh PBMC for 3 weeks with IL-2 (60 ng/ml) and IL-21 (30 ng/ml) and soluble phospoantigen

tag: T cells purified by using a MACS pan gamma delta kit (positive selection)

tag: Amplified from human fresh PBMC for 3 weeks with IL-2 (60 ng/ml) and soluble phospoantigen


title: Patient no. 2: treated

status: Public on Oct 06 2008

city: Essen

first: Ludger

last: Klein-Hitpass

address: Virchowstr. 173

department: Universitaetsklinikum

country: Germany

institute: Institut fuer Zellbiologie

description: Gene expression data from primary liposarcom cultures incubated with doxorubicin (0.5 µg/ml) for 24 h

hyb_protocol: Following fragmentation, 10 ug of cRNA were hybridized for 16 hr at 45C on GeneChip Human Genome U133A  Array. GeneChips were washed and stained in the Affymetrix Fluidics Station 400.

data_processing: The data were analyzed with GCOS 1.4 using Affymetrix default analysis settings and global scaling as normalization method. The trimmed mean target intensity of each array was arbitrarily set to 1000.

source_name: Liposarcoma culture from tumor 337, doxorubicin treated

label_protocol: Biotinylated cRNA were prepared according to the standard Affymetrix protocol from 5 ug total RNA (Expression Analysis Technical Manual, 2001, Affymetrix).

growth_protocol: Primary human liposarcoma cultures were isolated by dissecting the tumor and digesting the minced samples enzymatically with 10 ml each of collagenase and dispase (10 mg/ml). The single cell suspension was depleted of red blood cells and cellular debris by centrifugation through a Ficoll-Hypaque density gradient. Liposarcioma cells were diluted and cultured during the whole experiment with Leibovitz's L-15 medium, supplemented with 2.0 mM glutamine and 10% fetal bovine serum in a humidified atmosphere in free air exchange with atmospheric air. Cells were seeded at a density of 2.00E+06 in 25 cm2 flasks.

treatment_protocol: 24 h later, after having grown to a subconfluent layer, cell cultures were incubated with doxorubicin (0.5 ug/ml) for 24 h or an equal volume of PBS as control

tag: Sex

value: female

tag: Age at operation

value: 74 years

tag: Site

value: thigh

tag: Size in cm

tag: Histological Subtype

value: myxoid/roundcell liposarcoma

tag: Responder type

value: high

tag: Grading

tag: Specimen character

value: primary tumor

tag: Previous radiation

value: no

tag: Previous chemotherapy


title: Rho ChIP-chip in E. coli K-12 MG1655 cells (Dataset 3421602)

description: Rho ChIP-chip in E. coli K-12 MG1655 cells using anti-Rho polyclonal antibody.

source_name: Rho ChIP in E. coli K-12 MG1655 cells

tag: Polyclonal antibody for Rho (gift from Jeff Roberts)

source_name: Genomic DNA control from E. coli K-12 MG1655 cells


title: Patient no. 6: treated

source_name: Liposarcoma culture from tumor 379, doxorubicin treated

value: 58 years

value: pelvis retroperitoneal

value: metastasis


title: Patient no. 8: treated

source_name: Liposarcoma culture from tumor 386, doxorubicin treated

value: 85 years

value: lower arm

value: pleomorphic liposarcoma


title: Patient no. 2: control

description: Gene expression data from primary liposarcom cultures incubated with PBS as control for 24 h

source_name: Liposarcoma culture from tumor 337, untreated


title: Patient no. 16: treated

source_name: Liposarcoma culture from tumor 398, doxorubicin treated

value: male

value: 60 years


title: Patient no. 16: control

source_name: Liposarcoma culture from tumor 398, untreated


title: Patient no. 11: control

source_name: Liposarcoma culture from tumor 389, untreated

value: 57 years

value: thorax

value: local recurrence


title: Sigma70 ChIP-chip in E. coli K-12 MG1655 cells (Dataset 64194)

description: Sigma70 ChIP-chip in E. coli K-12 MG1655 cells using monoclonal antibody 2G10.

source_name: Sigma70 ChIP in E. coli K-12 MG1655 cells

tag: Monoclonal antibody for Sigma70 (2G10)

tag: Genomic DNA control


title: Sigma70 ChIP-chip in E. coli K-12 MG1655 cells treated with 100ug/ml rifampicin (Dataset 84847)

description: Sigma70 ChIP-chip in E. coli K-12 MG1655 cells treated with 100ug/ml rifampicin using monoclonal antibody 2G10.

source_name: Sigma70 ChIP in E. coli K-12 MG1655 cells treated with 100ug/ml rifampicin

source_name: Genomic DNA control from E. coli K-12 MG1655 cells treated with 100ug/ml rifampicin


title: RNA polymerase (Beta') ChIP-chip in E. coli K-12 MG1655 cells (Dataset 100040)

description: RNA polymerase (Beta') ChIP-chip in E. coli K-12 MG1655 cells using monoclonal antibody NT73.

source_name: RNA polymerase (Beta') ChIP in E. coli K-12 MG1655 cells

tag: Monoclonal antibody for RNA polymerase Beta' subunit (NT73)


title: Sigma70 ChIP-chip in E. coli K-12 MG1655 cells (Dataset 62214)


title: RNA polymerase (Beta’) ChIP-chip in E. coli K-12 MG1655 cells (Dataset 62215)

description: RNA polymerase (Beta’) ChIP-chip in E. coli K-12 MG1655 cells using monoclonal antibody NT73.

source_name: RNA polymerase (Beta’) ChIP in E. coli K-12 MG1655 cells


title: Patient no. 11: treated

source_name: Liposarcoma culture from tumor 389, doxorubicin treated

value: female


title: NusA ChIP-chip in E. coli K-12 MG1655 cells (Dataset 61392)

description: NusA ChIP-chip in E. coli K-12 MG1655 cells using monoclonal antibody 1NA1.

source_name: NusA ChIP in E. coli K-12 MG1655 cells

tag: Monoclonal antibody for NusA (1NA1)


title: Patient no. 8: control

source_name: Liposarcoma culture from tumor 386, untreated


title: NusA ChIP-chip in E. coli K-12 MG1655 cells (Dataset 64131)


title: Patient no. 5: control

source_name: Liposarcoma culture from tumor 377, untreated

value: 38 years

value: knee

value: myxoid liposarcoma

value: yes


title: Patient no. 10: treated

source_name: Liposarcoma culture from tumor 388, doxorubicin treated

value: 76 years

value: thigh

value: dedifferentiated liposarcoma

value: high

value: no