title: EE6.5 vs control, rep5

type: other

city: Cardiff

first: Fiona

middle: Catherine

last: Mansergh

state: Wales

address: Museum Avenue

department: Biosciences

country: United Kingdom

institute: Cardiff University

description: These array experiments were carried out in order to compare the transcriptional profiles of ES cells with various very early embryonic stages and compartements, including those from which ES cells can normally be derived. The pooled control was comprised of all the stages from which the NIA clone set, printed on the slides, was originally derived. Owing to the small size of most embryonic samples, two rounds of RNA amplification were used on the tiny amounts of total RNA extracted from the sample, prior to labelling for array hybridization

hyb_protocol: Array slides were incubated in blocking solution for 1 hour at 42oC (50% formamide, 5XSSC, 0.1% SDS, 1% BSA). The volume of the targets were adjusted to 18l via vacuum centrifugation and 1l Cot1 DNA and 1l poly A oligo were added as blocking agents. The targets were then denatured by heating to 95oC for 5 l minutes. 20 20xSSC,of 2x hybridisation buffer(49.9% de-ionised formamide, 49.9% 0.2% SDS) The labelled target was added to the microarray under C. Slides were Hybri-slips (Sigma) and hybridised for 18 hours at 42 on a levelthen placed plastic cover above some 1xSSC moistened tissue. Following hybridization, slides were washed once in Wash solution 1 (1X SSC, 2% SDS, filtered autoclaved ddH2O) for 20 minutes, then twice in Wash solution 2 (0.1X SSC, 0.2% SDS, filtered autoclaved distilled deionised H2O (ddH2O) for 20 minutes each. Slides were dipped 0.1X SSC, then dried with pressurised air.

scan_protocol: The arrays were scanned in a ScanArray® Express HT (PerkinElmer™) equipped with red ( run633nm) and green(543nm) lasers. The slides were scanned at a resolution of 5 powerat half speed. The pmt gain was set at 65% and the laser at 95%.

data_processing: Image analysis was carried out using the package Spot (CSIRO). The image files generated by Spot were input to either limma using limmaGUI version where appropriate and the data was normalised. The Bayesian statistics were generated in limma and tables of the highest differentially expressed genes were generated. The normalised M and A data was extracted to Excel files from which normalised, log2 red and green intensity values were calculated. This data was then input to the online NIA Array Analysis tool (http://stemcell.biosi.cf.ac.uk/ ). This suite of tools was used to carry out ANOVA and produce lists of differentially regulated genes as well as hierarchical clustering.

molecule: other

source_name: Pooled control

label_protocol: All RNA samples were subjected to two rounds of linear amplification using the RiboAmp™ Kit (Arcturus) according to the protocols published by the manufacturer, Version B prior to labelling. Three independent samples were collected for each tissue or cell type under investigation. 5ug of amplified RNA was labelled with Cy3 using the Atlas™ PowerScript™ Fluorescent Labelling Kit and 5μg of amplified RNA according to the manufacturer’s protocol (Promega). The RNA template was destroyed using NaOH instead of the RNaseH supplied with the kit. The quantity of cDNA and dye incorporation was assessed by spectrophotometry and the size spread and amount of residual unincorporated dye assessed by slide gel electrophoresis. Control and experimental samples were then combined and prepared for hybridization.

growth_protocol: Mice were maintained under Home Office licence under normal care and diet conditions for wild type animals (Lab Diet and water ad libitum). Mice of the strain 129SvEv were maintained on a 12h light/12h dark light cycle. Plugs were checked to time matings before embryo collections. Females were caged with males and the females checked each morning for the presence of a vaginal plug. As oestrus females usually ovulate at the mid-point of the dark period, embryo stages are represented as days post coitum (d.p.c.) assuming that fertilization occurred at midnight preceding the discovery of the vaginal plug. The morning of finding a plug is thus Day0.5 and from 12:00 noon is Day 1. Embryonic diapause g was induced by intraperitoneal injection of 10of tamoxifen (Sigma) and subcutaneous injection of 1mg of DepoProveraTM (Hunter and Evans 1999) on day 2.5-3 of gestation. Blastocysts were flushed from the uteri in OptiMEMTM-1 supplemented with 4mg/ml BSA (OptiMEM+ BSA). Early post-implantation embryos were dissected from the uterus and decidua.

extract_protocol: Pregnant females were sacrificed on days 3.5- 5 post coitum. Blastocysts were flushed from the uteri in OptiMEMTM-1 supplemented with 4mg/ml BSA(OptiMEM+ BSA). The inner cell masses of blastocysts were dissected by immumosurgery (Solter and Knowles 1975). Briefly, where necessary, the zona pellucida was removed from the embryos with acidic Tyrode’s solution (Nicolson et al. 1975). The embryos were then incubated in rabbit anti mouse spleen antibody diluted 1:10 in OptiMEM+BSA at room temperature for 20 minutes. The embryos were then washed through six drops of OptiMEM+BSA to remove all unbound antibody. The blastocysts were then incubated in Guinea pig complement diluted 1:5 in OptiMEM+BSA at 37oC for 20minutes. After washing through 3 drops of OptiMEM+BSA, the lysed trophectoderm was removed by pipetting through a finely drawn glass capillary The Reichart’s Membrane and ectoplacental cone were removed from post implantation stage embryos with tungsten needles and the embryonic portion was separated from the rest of the embryo. The visceral endoderm layer was removed by incubating the embryonic tissue in 0.25% pancreatin, 0.5% trypsin in DPBS-A at 4oC for 10min. The endoderm layer was then peeled off by pipetting through a glass capillary drawn to a diameter slightly smaller than the embryonic cylinder. Groups of embryo parts (approximately 30ICM and 2-3 epiblast and l of embryonic ectoderm) were lysed in 200 ZymoRNA Extraction buffer provided in the Mini RNA Isolation kit™ (Zymo) and RNA isolated according to the manufacturer’s protocol.

treatment_protocol: Oocytes were isolated between six and eight h.p.c from females which had been mated with vasectomised males. The oviducts were dissected from the mice into a dish of OptiMEM+BSA and the ampulla was opened with No 5 watchmaker’s forceps allowing the escape of the oocytes with their associated cumulus cells into the medium. The cumulus cells were separated from the oocytes by incubating in hyaluronidase (Sigma),0.3mg/ml in OptiMEM at room temperature until the cumulus cells drop off. The oocytes were then rinsed through several drops of OptiMEM+BSA to free them from cumulus cells, hyaluronidase and other debris. The oocytes were then transferred to an RNase free microcentrifuge tube in the smallest volume of medium before lysis in the buffer supplied in the Zymo Mini RNA Isolation kit™(Zymo) Fertilised eggs were collected from timed matings (8-10 h.p.c.) with fertile males in exactly the same manner as for oocytes The developmental stages from 2-cell to morula were isolated from the oviducts of timed matings with fertile males. The oviducts were dissected into dishes and embryos were isolated from the oviducts by inserting a 33gauge needle into the infindibulum of the oviduct and flushing with OptiMEM+BSA by means of a syringe. Blastocysts were flushed from the uteri in OptiMEMTM-1 supplemented with 4mg/ml BSA(OptiMEM+ BSA) on days 3.5- 5 post coitum. Pregnant females were sacrificed on days 6-7 post-coitum and the decidua were dissected free of the uteri. Embryos were carefully removed from the decidua with tungsten needles. In the case of day 7.5 embryos, the ovary/mesonephros isolated from Day 12.5 embryos and newborn ovary, groups of eight embryos and tissues fragments were collected and the RNA isolated as for the pre-implantation embryos. When amplifying these larger tissues, to avoid saturation of the second round of in vitro transcription, the amount of aRNA produced by the first round of amplification was measured by spectrophotometry. The second round of amplification was then initiated with 0.5μg of aRNA from the first round of amplification.

tag: Pooled control consisting of equal amounts of RNA from tissues listed below

value: hpc = hours post coitum Tissue:oocytes, Day of Gestation 0.5, Harvested hpc 7-7.5 1 cell zygote, Day of Gestation 0.5, Harvested hpc 7.5-8.5 2 cell embryo, Day of Gestation1.5, Harvested hpc 32-35 3-4 cell embryo, Day of Gestation2.0, Harvested hpc45-46.5 4-8cell embryo, Day of Gestation2.5, Harvested hpc 55.5-58 8-16 cell embryo, Day of Gestation 3, Harvested hpc 67-68 Late morulae- early blastocysts, Day of Gestation3.5, Harvested hpc80-84 Whole embryo, mid-streak stage, Day of Gestation7.5 Ovary+mesonephros, Day of Gestation12.5 Ovary, Newborn

source_name: Embryonic ectoderm dissected from day 6.5 embryos (EE6.5).

growth_protocol: Mice were maintained under Home Office licence under normal care and diet conditions for wild type animals (Lab Diet and water ad libitum). Mice of the strain 129SvEv were maintained on a 12h light/12h dark light cycle. Plugs were checked to time matings before embryo collections. Females were caged with males and the females checked each morning for the presence of a vaginal plug. As oestrus females usually ovulate at the mid-point of the dark period, embryo stages are represented as days post coitum (d.p.c.) assuming that fertilization occurred at midnight preceding the discovery of the vaginal plug. The morning of finding a plug is thus Day0.5 and from 12:00 noon is Day 1. Embryonic ectoderm was sampled from day 5.5 and day 6.5 embryos

extract_protocol: Pregnant females were sacrificed on days 5.5 and 6.5 post coitum for sampling of embryonic ectoderm. Early post-implantation embryos were dissected from the uterus and decidua. The Reichart’s Membrane and ectoplacental cone were removed from post implantation stage embryos with tungsten needles and the embryonic portion was separated from the rest of the embryo. The visceral endoderm layer was removed by incubating the embryonic tissue in 0.25% pancreatin, 0.5% trypsin in DPBS-A at 4oC for 10min. The endoderm layer was then peeled off by pipetting through a glass capillary drawn to a diameter slightly smaller than the embryonic cylinder. Groups of embryo parts (approximately 2-3 samples for embryonic ectoderm) were lysed in 200 ZymoRNA Extraction buffer provided in the Mini RNA Isolation kit™ (Zymo) and RNA isolated according to the manufacturer’s protocol.

treatment_protocol: Early post-implantation embryos were dissected from the uterus and decidua. The Reichart’s Membrane and ectoplacental cone were removed from post implantation stage embryos with tungsten needles and the embryonic portion was separated from the rest of the embryo. The visceral endoderm layer was removed by incubating the embryonic tissue in 0.25% pancreatin, 0.5% trypsin in DPBS-A at 4oC for 10min. The endoderm layer was then peeled off by pipetting through a glass capillary drawn to a diameter slightly smaller than the embryonic cylinder.  Groups of embryonic ectoderm samples were RNA extracted using the Zymo Mini RNA isolation kit, and RNA was amplified as described below

tag: Embryonic ectoderm dissected from post-implantation day 6.5 embryos (EE6.5). At this point in development, the isolation of ES cell lines is no longer possible, although other multipotent stem cell lines can be obtained


title: NSCLC tumor 110T sample

status: Public on Jul 22 2008

type: RNA

city: Madrid

first: Barbara

last: Angulo

address: Melchor Fernandez Almagro, 3

department: Molecular Pathology Program

country: Spain

institute: Spanish National Cancer Centre

description: Stratagene’s Universal Human Reference RNA is composed of total RNA from 10 human cell lines. The reference RNA is designed to  be used as a reference for microarray gene-profiling experiments. Equal quantities of DNase-treated total RNA from each cell line (adenocarcinoma, mammary gland; melanoma;  hepatoblastoma, liver; liposarcoma; adenocarcinoma, cervix; histiocytic lymphoma; macrophage; embryonal carcinoma, testis; lymphoblastic leukemia, T lymphoblast; glioblastoma, brain; plasmacytoma; myeloma; B lymphocyte) were pooled to make the Universal Human Reference RNA.

hyb_protocol: 1.- Slide pre-washing and denaturation: slide was incubated in SDS 0.1% at room temperature for 5min (x2). After rinse with miliQ-water, slide was transfered to 95º-100ºC water bath for 2min and then to miliQ-water at room temperature. 2.- Slide pre-hybridization: slide was sinked in a staining dish containing pre-warmed pre-hybridization buffer (4x SCC, 0.1% SDS and 0.002% BSA) and was incubated at 65ºC for 45min. After rinse with miliQ-water, slide was dried using centrifuge (300g, 3min). 3.- Hybridization: the probe (mix Cy3-labeled and Cy5-labeled cDNAs) was centrifuged at full speed for 5min at room temperature. Then it was applied (approx. 44µl, leave 2µl from the bottom of the tube behind in order to avoid transferring any sediment to the slide) to a clean cover-slip and place on the pre-hybridized microarray. The slide was placed in a hybridization chamber with the printed area and the cover-slip at the top, and was incubated overnight (15-20h) at 55ºC in an oven. 4.- Post-hybridization washes: low- and high-stringency wash buffers were prepared from 20x SCC and 10% SDS stocks solutions (wash buffer 1: 2x SSC, 0.5% SDS; wash buffer 2: 0.5x SSC, 0.5% SDS). Wash buffer 1 was pre-warmed and kept at the temperature used for the hybridization,  wash buffer 2 was kept at room temperature. Slide was placed in a falcon with wash buffer 1 until the cover-slip has fallen from the slide. Wash slide 3 times in pre-warmed wash buffer 1, 10min each, with shaking at room temeprature. Wash slide in wash buffer 2 with shaking, 15min for the first wash and 5min for the last ones. Slide was dried using a centrifuge (300g, 3min). Scanning as soon as possible and protect from direct light.

scan_protocol: Slides were scanned for Cy3 and Cy5 fluorescence on a Agilent G2565BA Microarray Scanner System. 1.- Place each slide in a cassette as follows: orient the slide so that it is array-side down and to the left; take cassette flat side down, place on flat surface and push down on the two buttons; with the buttons pressed, insert the slide (keeping everything flat); release buttons when slide has been fully inserted. 2.- Place cassettes into carousel. The etched side should now face to the left, and you should be looking at the back of the slide. Note: the cassette must start with the first slot. The scanner recognizes an empty slot as a sign to stop scanning. Close the scanner top. 3.- Double-click on the Agilent Scanner icon. When it opens, the scanner will go through some initialization steps. Also, if the lasers are not fully warm, there will be a warning sign that will give an approximate time that the lasers will be ready. 4.- Enter your name under Operator and check to see that the controls are set for the following: output directory is D:\niehs; both Red and Green channels are checked and are set to 100%; the size is 10 microns. 5.- Click on Options: choose to scan default slide area under Scan Region Options; specify slide area to largest area under Default Scan Region;  click on Instrument serial number under Automatic File Naming (it will then be highlighted). Choose and type in your identifier for that day. Leave the Slot Number as is. 6.- Fill out log in the Scanner Cassette Log with your name, date and the order you put your slides into the carousel. This is important to assure proper renaming after scanning is complete. Click on OK to close the Options window. 7.- Double check that all the settings are properly set and click on Scan. 8.- After scan is complete, you will need to change the names to format pxxsxx. To do this: with the right mouse button, click on the Start menu and choose Explorer. Navigate your way through the files by clicking on the D drive on the far left hand side of the screen. Double click on D drive then double click onniehs. This folder contains all of the scanned files. Locate your slides by looking for the identifier you used for the scan. Click once to highlight name and click once more to get a cursor in the file name. Type in the proper name using the log you filled out earlier. Once all of the names have been changed, highlight the newest slides and move them into the appropriate boxes on Witchfire. 9.- Once you are done, log out of the computer, take the cassettes out of the carousel and your slides out of the cassettes. Turn off scanner.

data_processing:
Cy3 and Cy5 fluorescent signals were quantified using GenePix Pro 5.0 (Axon Instruments Inc.). Substandard spots were manually flagged and discarded for further analysis.
The Cy5/Cy3 ratios were normalized to the median ratio value using the DNMAD tool, based on a print-tip loess method. The conditions for the normalization were: 1) use negative flags, that means that the flagged spots are not used for the normalization and are turned into missing values; 2) do not use background correction.
The normalized and semi-logarithmic transformed data were preprocessing using GEPAS software. Conditions for data pre-processing were: 1) merge replicates (the Cy5/Cy3 ratios of replicated spots were averaged); 2) filter missing values in order to exclude for further analysis any gene with data missing in more than 10% of the samples.
For unsupervised hierarchical clustering, we used a complete-linkage method with correlation distance. Genes whose expression levels varied by a factor of less than 3 across the samples were eliminated to leave only the mos variably expressed transcripts.The SOTARRAY Clustering Program was used for the hierarchical clustering of the samples and the Gene Cluster/Tree View program was used to view gene expression profiles.
To identify the genes that are important for distinguishing subgroups defined by histological type or gene alterations, we did t-tests with 200.000 random permutations to compute significance. To account for the effect of multiple hypothesis testing on the identification of significance, adjusted P-values were computed to control for the false discovery rate (FDR). The differentially expressed genes were functionaly classified manually on the basis of exhaustive searches in Pubmed an in Gene Ontology databases.

molecule: total RNA

organism: Homo sapiens

source_name: RNA from NSCLC tumor 110T labeled with Cy5

label_protocol:
1.- 2.5µg of amplified RNA from each sample (lung tumors, normal lung tissues and Universal Human Reference RNA) were primed with 3µl of random hexamers d(N)6 0.5mg/ml (Promega) at 70ºC for 10min. For each labeling reaction, a master mix was prepared: 5µl First Strand buffer 5x, 2.5µl DTT 0.1M, 0.5µl unlabeled dNTPs 50x (dATP/dCTP/dGTP:dTTP 25:5), 1.5µl Cy5-dUTP 10mM (for lung tumor or normal lung samples) or Cy3-dUTP 10mM (for Universal Human Reference RNA), 0.63µl RNasin Inhibitor and 1.25µl SuperScript II (200U/µl). The labeling reaction was incubated at room temperature for 10min and then incubated at 42ºC for 2h. Tubes were protected from direct light.
2.- Each fluorescent labeled cDNA was purified with CyScribe GFX Purification kit (Amersham) following manufacturer's instructions.
3.- The Cy3-labeled and Cy5-labeled cDNAs for each hybridization were combined into a single tube. 10µg of Human Cot-1 DNA (Invitrogen) were added.
4.- To precipitate each hybridization probe, 0.1 volumes of sodium acetate 0.1M and 2.5 volumes of absolute ethanol were added.
5.- Each probe was resuspended in 4µl of filtered EDTA 10mM ph 8 and denatured at 95ºC for 10min. 40µl of SlideHyb buffer 1x (Ambion), prewarmed at 65ºC, and blocking agents (mix of polyA and yeast tRNA, 10µg and 4µg in 2µl of final volume) were added.

extract_protocol:
1.- Total RNA  from lung tumors and normal lung tissues was isolated in two steps using TRIZOL reagent (Invitrogen), followed by purification with RNeasy mini kit (QIAGEN) and digestion with RNase-free DNase I (QIAGEN) following the manufacturer's instructions. Universal Human Reference RNA (Stratagene), the reference used in all the hybridizations, is provided in a solution of 70% ethanol and 0.1M sodium acetate; it was prepared by centrifugation and washing with 70% ethanol.
2.- cDNA was synthesized from 4µg of each total RNA using SuperScript II Rnase H- Reverse Transcriptase (Invitrogen). Subsequent amplification was carried out with MegaScript T7 Kit (Ambion) following the manufacturer's instruction for assembling the in vitro transcription reaction. The amplified RNA was cleaned up with RNeasy mini kit (QIAGEN).

tag: Tissue

value: lung

tag: Gender

value: male

tag: Smoking status

value: smoker

tag: Histological type

value: squamous cell carcinoma

tag: Genetic background

value: P53+ / PIK3CA-

tag: Degree of differentiation

value: moderate

tag: Tumor size

tag: Lymph node involvement

value: X

source_name: RNA from Stratagene's Universal Human Reference RNA labeled with Cy3

tag: no additional information


title: NSCLC tumor 114T sample

source_name: RNA from NSCLC tumor 114T labeled with Cy5


title: EE6.5 vs control, rep7

organism: Mus musculus


title: EE6.5 vs control, rep2

source_name: Embryonic ectoderm dissected from day 6.5 embryos (EE6.5)


title: EE6.5 vs control, rep3


title: NSCLC tumor 106T sample

source_name: RNA from NSCLC tumor 106T labeled with Cy5

value: adenocarcinoma

value: P53+ / KRAS- / LKB1- / EGFR- / PIK3CA- / BRAF-


title: EE6.5 vs control, rep1


title: EE5.5 vs control, rep6

source_name: Embryonic ectoderm dissected from day 5.5 embryos (EE5.5)

tag: Embryonic ectoderm dissected from post-implantation day 5.5 embryos (EE5.5). At this point in development, the isolation of ES cell lines is no longer possible, although other multipotent stem cell lines can be obtained


title: EE6.5 vs control, rep4


title: EE5.5 vs control, rep5


title: EE5.5 vs control, rep4


title: NSCLC tumor 102T sample

source_name: RNA from NSCLC tumor 102T labeled with Cy5

value: P53+ / KRAS+ / LKB1- / EGFR- / PIK3CA- / BRAF-

value: well


title: EE5.5 vs control, rep3


title: NSCLC tumor 104T sample

source_name: RNA from NSCLC tumor 104T labeled with Cy5

value: poor


title: EE5.5 vs control, rep1

tag: Embryonic ectoderm dissected from post-implantation day 5.5 embryos (EE5.5). At this pint in development, the isolation of ES cell lines is no longer possible, although other multipotent stem cell lines can be obtained


title: DICM180 vs control, rep2

source_name: Delayed embryonic inner cell mass, 180 hours post coitum

growth_protocol: Mice were maintained under Home Office licence under normal care and diet conditions for wild type animals (Lab Diet and water ad libitum). Mice of the strain 129SvEv were maintained on a 12h light/12h dark light cycle. Plugs were checked to time matings before embryo collections. Females were caged with males and the females checked each morning for the presence of a vaginal plug. As oestrus females usually ovulate at the mid-point of the dark period, embryo stages are represented as days post coitum (d.p.c.) assuming that fertilization occurred at midnight preceding the discovery of the vaginal plug. The morning of finding a plug is thus Day0.5 and from 12:00 noon is Day 1.  Embryonic diapause was induced by intraperitoneal injection of 10g of tamoxifen (Sigma) and subcutaneous injection of 1mg of DepoProveraTM  (Hunter and Evans 1999) on day 2.5-3 of gestation. Delayed blastocysts were flushed from the uteri in OptiMEMTM-1 supplemented with 4mg/ml BSA (OptiMEM+ BSA), at the appropriate number of hours post coitum

extract_protocol: Pregnant females were sacrificed on days 3.5- 5 post coitum. Blastocysts were flushed from the uteri in OptiMEMTM-1 supplemented with 4mg/ml BSA(OptiMEM+ BSA). The inner cell masses of blastocysts were dissected by immumosurgery (Solter and Knowles 1975). Briefly, where necessary, the zona pellucida was removed from the embryos with acidic Tyrode’s solution (Nicolson et al. 1975). The embryos were then incubated in rabbit anti mouse spleen antibody diluted 1:10 in OptiMEM+BSA at room temperature for 20 minutes. The embryos were then washed through six drops of OptiMEM+BSA to remove all unbound antibody. The blastocysts were then incubated in Guinea pig complement diluted 1:5 in OptiMEM+BSA at 37oC for 20minutes. After washing through 3 drops of OptiMEM+BSA, the lysed trophectoderm was removed by pipetting through a finely drawn glass capillary. Groups of embryo parts (approximately 30ICM and 2-3 epiblast and l of embryonic ectoderm) were lysed in 200 ZymoRNA Extraction buffer provided in the Mini RNA Isolation kit™ (Zymo) and RNA isolated according to the manufacturer’s protocol.

treatment_protocol: Blastocysts were flushed from the uteri in OptiMEMTM-1 supplemented with 4mg/ml BSA(OptiMEM+ BSA) on days 3.5- 5 post coitum (7 days post coitum in the case of DICM180 samples, owing to induced delay in implantation). Samples were then immunodissected to free the inner cell mass, then groups of inner cell masses were RNA extracted using the Zymo Mini RNA isolation kit, and RNA was amplified as described below

tag: inner cell mass (ICM) isolated from delayed blastocysts flushed from the uterus approximately 180 hours post coitum. This is one of the embryonic tissues from which ES cells can normally be isolated. "delayed" refers to the use of induced diapause, which prevents implantation at a time when this would normally have occurred


title: DICM180 vs control, rep1


title: DICM180 vs control, rep3


title: DICM136 vs control, rep5

source_name: Delayed embryonic inner cell mass, 136 hours post coitum

growth_protocol: Mice were maintained under Home Office licence under normal care and diet conditions for wild type animals (Lab Diet and water ad libitum). Mice of the strain 129SvEv were maintained on a 12h light/12h dark light cycle. Plugs were checked to time matings before embryo collections. Females were caged with males and the females checked each morning for the presence of a vaginal plug. As oestrus females usually ovulate at the mid-point of the dark period, embryo stages are represented as days post coitum (d.p.c.) assuming that fertilization occurred at midnight preceding the discovery of the vaginal plug. The morning of finding a plug is thus Day0.5 and from 12:00 noon is Day 1.  Embryonic diapause was induced by intraperitoneal injection of 10g of tamoxifen (Sigma) and subcutaneous injection of 1mg of DepoProveraTM  (Hunter and Evans 1999) on day 2.5-3 of gestation. Blastocysts were flushed from the uteri in OptiMEMTM-1 supplemented with 4mg/ml BSA (OptiMEM+ BSA).

treatment_protocol: Blastocysts were flushed from the uteri in OptiMEMTM-1 supplemented with 4mg/ml BSA(OptiMEM+ BSA) on days 3.5- 5 post coitum. Samples were then immunodissected to free the inner cell mass, then groups of inner cell masses were RNA extracted using the Zymo Mini RNA isolation kit, and RNA was amplified as described below

tag: inner cell mass (ICM) isolated from delayed blastocysts flushed from the uterus approximately 136 hours post coitum. This is one of the embryonic tissues from which ES cells can normally be isolated. "delayed" refers to the use of induced diapause, which prevents implantation at a time when this would normally have occurred


title: DICM136 vs control, rep3


title: DICM180 vs control, rep4


title: ICM105 vs control, rep1

source_name: Embryonic inner cell mass, 105 hours post coitum (ICM105)

growth_protocol: Mice were maintained under Home Office licence under normal care and diet conditions for wild type animals (Lab Diet and water ad libitum). Mice of the strain 129SvEv were maintained on a 12h light/12h dark light cycle. Plugs were checked to time matings before embryo collections. Females were caged with males and the females checked each morning for the presence of a vaginal plug. As oestrus females usually ovulate at the mid-point of the dark period, embryo stages are represented as days post coitum (d.p.c.) assuming that fertilization occurred at midnight preceding the discovery of the vaginal plug. The morning of finding a plug is thus Day0.5 and from 12:00 noon is Day 1. Blastocysts were flushed from the uteri in OptiMEMTM-1 supplemented with 4mg/ml BSA (OptiMEM+ BSA).

tag: inner cell mass (ICM) isolated from blastocysts flushed from the uterus approximately 105 hours post coitum. This is one of the embryonic tissues from which ES cells can normally be derived


title: ICM88 vs control, rep5

hyb_protocol: Array slides were incubated in blocking solution for 1 hour at 42oC (50% formamide, 5XSSC, 0.1% SDS, 1% BSA). The volume of the targets were adjusted to l poly A oligo were addedl Cot1 DNA and 1l via vacuum centrifugation and 118 as blocking agents. The targets were then denatured by heating to 95oC for 5 l of 2x hybridisation buffer(49.9% de-ionised formamide, 49.9%minutes. 20 20xSSC, 0.2% SDS) The labelled target was added to the microarray under C. Slides were then placedHybri-slips (Sigma) and hybridised for 18 hours at 42 on a level plastic cover above some 1xSSC moistened tissue. Following hybridization, slides were washed once in Wash solution 1 (1X SSC, 2% SDS, filtered autoclaved ddH2O) for 20 minutes, then twice in Wash solution 2 (0.1X SSC, 0.2% SDS, filtered autoclaved distilled deionised H2O (ddH2O) for 20 minutes each. Slides were dipped 0.1X SSC, then dried with pressurised air.

scan_protocol: The arrays were scanned in a ScanArray® Express HT (PerkinElmer™) equipped 543nm) lasers. The slides were scanned at a633nm) and green(with red (  run at half speed. The pmt gain was set at 65% and the laserresolution of 5 power at 95%.

growth_protocol: Mice were maintained under Home Office licence under normal care and diet conditions for wild type animals (Lab Diet and water ad libitum). Mice of the strain 129SvEv were maintained on a 12h light/12h dark light cycle. Plugs were checked to time matings before embryo collections. Females were caged with males and the females checked each morning for the presence of a vaginal plug. As oestrus females usually ovulate at the mid-point of the dark period, embryo stages are represented as days post coitum (d.p.c.) assuming that fertilization occurred at midnight preceding the discovery of the vaginal plug. The morning of finding a plug is thus Day0.5 and from 12:00 noon is Day 1. Embryonic diapause g of tamoxifen (Sigma) andwas induced by intraperitoneal injection of 10 subcutaneous injection of 1mg of DepoProveraTM (Hunter and Evans 1999) on day 2.5-3 of gestation. Blastocysts were flushed from the uteri in OptiMEMTM-1 supplemented with 4mg/ml BSA (OptiMEM+ BSA). Early post-implantation embryos were dissected from the uterus and decidua.

extract_protocol: Pregnant females were sacrificed on days 3.5- 5 post coitum. Blastocysts were flushed from the uteri in OptiMEMTM-1 supplemented with 4mg/ml BSA(OptiMEM+ BSA). The inner cell masses of blastocysts were dissected by immumosurgery (Solter and Knowles 1975). Briefly, where necessary, the zona pellucida was removed from the embryos with acidic Tyrode’s solution (Nicolson et al. 1975). The embryos were then incubated in rabbit anti mouse spleen antibody diluted 1:10 in OptiMEM+BSA at room temperature for 20 minutes. The embryos were then washed through six drops of OptiMEM+BSA to remove all unbound antibody. The blastocysts were then incubated in Guinea pig complement diluted 1:5 in OptiMEM+BSA at 37oC for 20minutes. After washing through 3 drops of OptiMEM+BSA, the lysed trophectoderm was removed by pipetting through a finely drawn glass capillary The Reichart’s Membrane and ectoplacental cone were removed from post implantation stage embryos with tungsten needles and the embryonic portion was separated from the rest of the embryo. The visceral endoderm layer was removed by incubating the embryonic tissue in 0.25% pancreatin, 0.5% trypsin in DPBS-A at 4oC for 10min. The endoderm layer was then peeled off by pipetting through a glass capillary drawn to a diameter slightly smaller than the embryonic cylinder. Groups of embryo parts (approximately 30ICM and 2-3 epiblast and l of RNA Extraction buffer provided in theembryonic ectoderm) were lysed in 200 Zymo Mini RNA Isolation kit™ (Zymo) and RNA isolated according to the manufacturer’s protocol.

source_name: Embryonic inner cell mass, 88 hours post coitum (ICM88)

extract_protocol: Pregnant females were sacrificed on days 3.5- 5 post coitum. Blastocysts were flushed from the uteri in OptiMEMTM-1 supplemented with 4mg/ml BSA(OptiMEM+ BSA). The inner cell masses of blastocysts were dissected by immumosurgery (Solter and Knowles 1975). Briefly, where necessary, the zona pellucida was removed from the embryos with acidic Tyrode’s solution (Nicolson et al. 1975). The embryos were then incubated in rabbit anti mouse spleen antibody diluted 1:10 in OptiMEM+BSA at room temperature for 20 minutes. The embryos were then washed through six drops of OptiMEM+BSA to remove all unbound antibody. The blastocysts were then incubated in Guinea pig complement diluted 1:5 in OptiMEM+BSA at 37oC for 20minutes. After washing through 3 drops of OptiMEM+BSA, the lysed trophectoderm was removed by pipetting through a finely drawn glass capillary. Groups of embryo parts (approximately 30ICM and 2-3 epiblast and l of RNA Extraction buffer provided in theembryonic ectoderm) were lysed in 200 Zymo Mini RNA Isolation kit™ (Zymo) and RNA isolated according to the manufacturer’s protocol.

tag: inner cell mass (ICM) isolated from blastocysts flushed from the uterus approximately 88 hours post coitum.


title: DICM136 vs control, rep1

tag: Pooled control consisting of equal amounts of RNA from tissues listed below


title: EE5.5 vs control, rep2


title: ICM105 vs control, rep3

source_name: Embryonic inner cell mass, 105 hours post coitum

tag: inner cell mass (ICM) isolated from blastocysts flushed from the uterus approximately 105 hours post coitum. This is one of the embryonic tissues from which ES cells can normally be isolated


title: ICM105 vs control, rep2


title: ICM105 vs control, rep4


title: DICM136 vs control, rep6


title: DICM136 vs control, rep4


title: ICM105 vs control, rep5


title: ICM88 vs control, rep1

source_name: embryonic inner cell mass, 88 hours post coitum (ICM88)

tag: Inner cell masses, isolated from pregnant female 129Sv/Ev mice, 88 hours post coitum. Inner cell masses from various stages including this one, represent common sources for embryonic stem cell lines


title: ICM105 vs control, rep6


title: IMT11 vs control, rep6

source_name: IMT11 embryonic stem cells

growth_protocol: ES cells were maintained in their undifferentiated state in the medium MEM-Alpha (GibcoTM, Invitrogen Ltd, Paisley, Renfrewshire, UK) supplemented with -Mercaptoethanol (Merck KGaA, 64293 Darmstadt, Germany), 2mM glutamine10-4M  and 10-3 U/ml murine LIF (ESGROTM, Invitrogen, Ltd, Paisley, Renfrewshire, UK), 10% FBS and 10% NBS (selected batches, PAA Laboratories GmbH, Linz, A-4020 Austria). The cells were maintained at 37oC in a humidified atmosphere with 5% CO2 on 0.1% gelatin (Stem Cell Technologies) coated tissue culture grade plastic ware (NUNCTM, Fisher Scientific, Loughborough, Leics, UK).

extract_protocol: ES cells were washed twice with DPBS-A (Invitrogen Ltd., Paisley, UK and 20 colonies were picked using a finely drawn glass capillary in a minimal volume of DPBS-A into a sterile microcentrifuge tube. The cells were lysed by the addition l of RNA Extraction buffer provided in the Zymo Mini RNA Isolation kit™of 200 (Zymo) and RNA isolated according to the manufacturer’s protocol.

treatment_protocol: See growth protocol. These ES cell lines were maintained normally, as the purpose of this experiment was to detect differences between different ES cell lines under normal tissue culture conditions. All cell lines were at passage 16 when sampled.

tag: Amplified RNA from individually picked IMT-11 colonies.\ ES cell line IMT-11, derived from inner cell mass (ICM) isolated from blastocysts flushed from the uterus approximately 88 and 105hpc, ICM isolated from blastocysts undergoing experimentally induced diapause at approximately 134 and 180hpc, epiblast dissected from early post implantation embryos approximately 132 and 150hpc.


title: ICM88 vs control, rep3

source_name: Pooled control


title: ICM88 vs control, rep2


title: ICM88 vs control, rep6


title: DICM136 vs control, rep2


title: ICM88 vs control, rep4

source_name: embryonic inner cell mass, 88 hours post coitum


title: IMT11 vs control, rep5


title: IMT11 vs control, rep2


title: IMT11 vs control, rep3


title: time 0.5 h, reference: Cy5, NA: Cy3

status: Public on Jul 20 2008

city: Bremen

first: Christine

last: Klockow

address: Celsiusstr. 1

country: Germany

institute: Max Planck Institute for Marine Microbiology

description: Influence of nalidixic acid on gene expression in E. coli

hyb_protocol: The differently labelled cDNAs of both samples are hybridized (42°C over night) simultaneously in one experiment to the same array (E.coli K12 V2 array whole genomearrays, MWG, Ebersberg, Germany). After hybridization, the unbound and unspecific fixed cDNA is removed by stringent washing from the array (2 x SSC, 0.1 % SDS; 1 x SSC; 0.5 x SSC).

scan_protocol: The array is scanned with an Affymetrix 428 Fluorescence Laser Scanner (Affymetrix, St. Clara, USA) for the two distinct fluorescent dyes (Cy3 and Cy5) of the cDNA derived from differently treated cells.

data_processing: Mean (local background) was subtracted from Mean (signal); Spots with a bad shape were deleted from data set, signal values for spots, which were lower than background or noise  were set to a value of 1  (quality of spot: 0= okay, 2= low signal, 3= bad shape of spot, 4= background higher than signal), an array-to-array standardisation was carried out by global scaling; Cy3 and Cy5 values belonging to the same sample were added. Growth curve effects were eliminated.

organism: Escherichia coli

source_name: Escherichia coli, grown in shaking flask cultivations in Luria-Bertaini-medium

label_protocol: First-strand cDNA synthesis was performed using LabelStar reverse transcriptase (Qiagen, Hilden, Germany), 5 pmol oligo(dT)18 primer and 0.02 mM cyanine 3-dCTP or cyanine 5-dCTP. In total, 150 µg RNA were used for each dye.

growth_protocol: The cells were platted on agar with Luria-Bertaini-medium and ampicillin. For the pre-culture 50 mL LB-medium with 75 mg/mL ampicillin were inoculated with these cells and incubated for 20 h (37°C, 180 rpm). As soon as an optical density of 0.4 was reached, the pre-culture was divided an filled into two separated shaking flasks. 75 mg/mL ampicillin are added to both flasks. Nalidixic acid is added to the test-flask (final concentration 0.25-0.3 mg/L). Both cultures are incubated 37°C, 180 rpm for eight hours.

extract_protocol: The cell disruption was carried out with FastPrep FP120 (Bio101, ThermoSavant, Waltham, USA). Total RNA was isolated using Qiagen RNeasy Midi Kit (Qiagen, Hilden, Germany) and Qiagen PCR Purification Kit according to the manufacturer’s protocol.

treatment_protocol: Nalidixic acid (0.25 – 0.3 mg/L) was added to the medium. Cells grown without nalidixic acid served as reference.

tag: Strain

value: Escherichia coli K12GFP-UTL2


title: IMT11 vs control, rep1

hyb_protocol: Array slides were incubated in blocking solution for 1 hour at 42oC (50% formamide, 5XSSC, 0.1% SDS, 1% BSA). The volume of the targets were adjusted to 18l via vacuum centrifugation and 1l Cot1 DNA and 1l poly A oligo were added as blocking agents. The targets were then denatured by heating to 95oC for 5 minutes. 20l of 2x hybridisation buffer(49.9% de-ionised formamide, 49.9% 20xSSC, 0.2% SDS) The labelled target was added to the microarray under Hybri-slips (Sigma) and hybridised for 18 hours at 42C. Slides were then placed on a level plastic cover above some 1xSSC moistened tissue. Following hybridization, slides were washed once in Wash solution 1 (1X SSC, 2% SDS, filtered autoclaved ddH2O) for 20 minutes, then twice in Wash solution 2 (0.1X SSC, 0.2% SDS, filtered autoclaved distilled deionised H2O (ddH2O) for 20 minutes each. Slides were dipped 0.1X SSC, then dried with pressurised air.

scan_protocol: The arrays were scanned in a ScanArray® Express HT (PerkinElmer™) equipped with red (633nm) and green(543nm) lasers. The slides were scanned at a resolution of 5 run at half speed. The pmt gain was set at 65% and the laser power at 95%.

data_processing:
Image analysis was carried out using the package Spot (CSIRO). The image files generated by Spot were input to either limma using limmaGUI version where appropriate and the data was normalised. The Bayesian statistics were generated in limma and tables of the highest differentially expressed genes were generated.
The normalised M and A data was extracted to Excel files from which normalised, log2 red and green intensity values were calculated. This data was then input to the online NIA Array Analysis tool (http://stemcell.biosi.cf.ac.uk/ ). This suite of tools was used to carry out ANOVA and produce lists of differentially regulated genes as well as hierarchical clustering.

label_protocol:
All RNA samples were subjected to two rounds of linear amplification using the RiboAmp™ Kit (Arcturus) according to the protocols published by the manufacturer, Version B prior to labelling.
Three independent samples were collected for each tissue or cell type under investigation. 5ug of amplified RNA was labelled with Cy3 using the Atlas™ PowerScript™ Fluorescent Labelling Kit and 5μg of amplified RNA according to the manufacturer’s protocol (Promega). The RNA template was destroyed using NaOH instead of the RNaseH supplied with the kit. The quantity of cDNA and dye incorporation was assessed by spectrophotometry and the size spread and amount of residual unincorporated dye assessed by slide gel electrophoresis.  Control and experimental samples were then combined and prepared for hybridization.

growth_protocol:
Mice were maintained under Home Office licence under normal care and diet conditions for wild type animals (Lab Diet and water ad libitum).  Mice of the strain 129SvEv were maintained on a 12h light/12h dark light cycle.  Plugs were checked to time matings before embryo collections. Females were caged with males and the females checked each morning for the presence of a vaginal plug. As oestrus females usually ovulate at the mid-point of the dark period, embryo stages are represented as days post coitum (d.p.c.) assuming that fertilization occurred at midnight preceding the discovery of the vaginal plug. The morning of finding a plug is thus Day0.5 and from 12:00 noon is Day 1.  Embryonic diapause was induced by intraperitoneal injection of 10g of tamoxifen (Sigma) and subcutaneous injection of 1mg of DepoProveraTM  (Hunter and Evans 1999) on day 2.5-3 of gestation.
Blastocysts were flushed from the uteri in OptiMEMTM-1 supplemented with 4mg/ml BSA (OptiMEM+ BSA).
Early post-implantation embryos were dissected from the uterus and decidua.

extract_protocol:
Pregnant females were sacrificed on days 3.5- 5 post coitum. Blastocysts were flushed from the uteri in OptiMEMTM-1 supplemented with 4mg/ml BSA(OptiMEM+ BSA).
The inner cell masses of blastocysts were dissected by immumosurgery (Solter and Knowles 1975). Briefly, where necessary, the zona pellucida was removed from the embryos with acidic Tyrode’s solution (Nicolson et al. 1975). The embryos were then incubated in rabbit anti mouse spleen antibody diluted 1:10 in OptiMEM+BSA at room temperature for 20 minutes. The embryos were then washed through six drops of OptiMEM+BSA to remove all unbound antibody. The blastocysts were then incubated in Guinea pig complement diluted 1:5 in OptiMEM+BSA at 37oC for 20minutes. After washing through 3 drops of OptiMEM+BSA, the lysed trophectoderm was removed by pipetting through a finely drawn glass capillary
The Reichart’s Membrane and ectoplacental cone were removed from post implantation stage embryos with tungsten needles and the embryonic portion was separated from the rest of the embryo. The visceral endoderm layer was removed by incubating the embryonic tissue in 0.25% pancreatin, 0.5% trypsin in DPBS-A at 4oC for 10min. The endoderm layer was then peeled off by pipetting through a glass capillary drawn to a diameter slightly smaller than the embryonic cylinder.
Groups of embryo parts (approximately 30ICM and 2-3 epiblast and embryonic ectoderm) were lysed in 200l  of RNA Extraction buffer provided in the Zymo Mini RNA Isolation kit™ (Zymo) and RNA isolated according to the manufacturer’s protocol.

treatment_protocol:
Oocytes were isolated between six and eight h.p.c from females which had been mated with vasectomised males. The oviducts were dissected from the mice into a dish of OptiMEM+BSA and the ampulla was opened with No 5 watchmaker’s forceps allowing the escape of the oocytes with their associated cumulus cells into the medium. The cumulus cells were separated from the oocytes by incubating in hyaluronidase (Sigma),0.3mg/ml in OptiMEM at room temperature until the cumulus cells drop off. The oocytes were then rinsed through several drops of OptiMEM+BSA to free them from cumulus cells, hyaluronidase and other debris. The oocytes were then transferred to an RNase free microcentrifuge tube in the smallest volume of medium before lysis in the buffer supplied in the Zymo Mini RNA Isolation kit™(Zymo)
Fertilised eggs were collected from timed matings (8-10 h.p.c.) with fertile males in exactly the same manner as for oocytes
The developmental stages from 2-cell to morula were isolated from the oviducts of timed matings with fertile males. The oviducts were dissected into dishes and embryos were isolated from the oviducts by inserting a 33gauge needle into the infindibulum of the oviduct and flushing with OptiMEM+BSA by means of a syringe.

Blastocysts were flushed from the uteri in OptiMEMTM-1 supplemented with 4mg/ml BSA(OptiMEM+ BSA) on days 3.5- 5 post coitum.
Pregnant females were sacrificed on days 6-7 post-coitum and the decidua were dissected free of the uteri. Embryos were carefully removed from the decidua with tungsten needles.
In the case of day 7.5 embryos, the ovary/mesonephros isolated from Day 12.5 embryos and newborn ovary, groups of eight embryos and tissues fragments were collected and the RNA isolated as for the pre-implantation embryos. When amplifying these larger tissues, to avoid saturation of the second round of in vitro transcription, the amount of aRNA produced by the first round of amplification was measured by spectrophotometry. The second round of amplification was then initiated with 0.5μg of aRNA from the first round of amplification.

value: hpc = hours post coitum

tag: Tissue

value: oocytes, Day of Gestation 0.5, Harvested hpc 7-7.5

tag: 1 cell zygote, Day of Gestation 0.5, Harvested hpc 7.5-8.5

tag: 2 cell embryo, Day of Gestation1.5, Harvested hpc 32-35

tag: 3-4 cell embryo, Day of Gestation2.0,  Harvested hpc45-46.5

tag: 4-8cell embryo, Day of Gestation2.5, Harvested hpc 55.5-58

tag: 8-16 cell embryo, Day of Gestation 3, Harvested hpc 67-68

tag: Late morulae- early blastocysts, Day of Gestation3.5, Harvested hpc80-84

tag: Whole embryo, mid-streak stage, Day of Gestation7.5

tag: Ovary+mesonephros, Day of Gestation12.5

tag: Ovary, Newborn

growth_protocol: ES cells were maintained in their undifferentiated state in the medium MEM-Alpha (GibcoTM, Invitrogen Ltd, Paisley, Renfrewshire, UK) supplemented with 10-4M -Mercaptoethanol (Merck KGaA, 64293 Darmstadt, Germany), 2mM glutamine and 10-3 U/ml murine LIF (ESGROTM, Invitrogen, Ltd, Paisley, Renfrewshire, UK), 10% FBS and 10% NBS (selected batches, PAA Laboratories GmbH, Linz, A-4020 Austria). The cells were maintained at 37oC in a humidified atmosphere with 5% CO2 on 0.1% gelatin (Stem Cell Technologies) coated tissue culture grade plastic ware (NUNCTM, Fisher Scientific, Loughborough, Leics, UK).

extract_protocol: ES cells were washed twice with DPBS-A (Invitrogen Ltd., Paisley, UK and 20 colonies were picked using a finely drawn glass capillary in a minimal volume of DPBS-A into a sterile microcentrifuge tube.  The cells were lysed by the addition of 200l of RNA Extraction buffer provided in the Zymo Mini RNA Isolation kit™ (Zymo) and RNA isolated according to the manufacturer’s protocol.

tag: Amplified RNA from individually picked IMT-11 colonies.ES cell line IMT-11, derived from inner cell mass (ICM) isolated from blastocysts flushed from the uterus approximately 88 and 105hpc, ICM isolated from blastocysts undergoing experimentally induced diapause at approximately 134 and 180hpc, epiblast dissected from early post implantation  embryos approximately 132 and 150hpc.


title: time 8 h, reference: Cy5, NA: Cy3

tag: strain


title: time 1 h, reference: Cy3, NA: Cy5


title: time 0.25 h NA Cy3 and 4 h NA Cy5


title: time 8 h, reference: Cy3, NA: Cy5


title: time 2 h, reference: Cy5, NA: Cy3


title: time 1 h, reference: Cy5, NA: Cy3


title: time 2 h, reference: Cy3, NA: Cy5


title: time 4 h NA Cy3 and 0.25 h NA Cy5


title: time 0 h, reference: Cy3, NA: Cy5

growth_protocol: The cells were platted on agar with Luria-Bertaini-medium and ampicillin. For the pre-culture 50 mL LB-medium with 75 mg/mL ampicillin were inoculated with these cells and incubated for 20 h (37°C, 180 rpm). As soon as an optical density of 0.4 was reached, the pre-culture was divided an filled into two separated shaking flasks. 75 mg/mL ampicillin are added to both flasks. Both cultures are incubated 37°C, 180 rpm for eight hours.

treatment_protocol: no treatment - Reference


title: time 0.5 h, reference: Cy3, NA: Cy5


title: time 0 h, reference: Cy5, NA: Cy3


title: tumor-07

status: Public on Jul 16 2008

type: genomic

city: Houston

first: Ralf

last: Krahe

state: TX

address: 1515 Holcombe Blvd., Unit #1010

department: Cancer Genetics

country: USA

institute: University of Texas M.D. Anderson Cancer Center

hyb_protocol: Following fragmentation, 45 ug of DNA were hybridized for 16 hr at 48C on 50K Xba mapping Array. GeneChips were washed and stained in the Affymetrix Fluidics Station 450.

scan_protocol: GeneChips were scanned using the Affymetrix Scanner 7000.

data_processing: The data were analyzed with CNAG v2.0, AsCNAR and dCHIP.

label: biotin

molecule: genomic DNA

source_name: Human tumor tissue

label_protocol: Biotinylated DNA were prepared according to the standard Affymetrix protocol from 45 ug total DNA (100K Mapping Asssay Technical Manual, 2005, Affymetrix).

extract_protocol: Gentra_sold out DNA extraction was performed according to the manufacturer's instructions.

tag: Male, LFS_osteosarcoma


title: tumor-08

tag: Male, LFS_soft tissue sarcoma


title: SitkaSpruce_FB3-425_Bark_PissodesStrobi_HerbivoryHyb4

status: Public on Jul 18 2008

city: Vancouver

first: Joerg

last: Bohlmann

state: BC

department: Michael Smith Laboratories

country: Canada

institute: University of British Columbia

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

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

data_processing:
Before data normalization, the lowest 10% of median foreground intensities was subtracted from the median foreground intensities to correct background intensity. After quantification of the signal intensities, data were normalized to compensate for non-linearity of intensity distributions using the variance stabilizing normalization (VSN) method of Huber et al. (2002) Bioinformatics 18: S96-S104.
In experiments with four independent biological replicates, RNA isolated from each of the four individual weevil-treated trees were compared against four individual untreated control trees using two hybridizations with a dye flip for each pair of four treatment and control replicates (eight slides total). In order to assess the biological response to weevil herbivory (H), a mixed effects model containing a dye effect and a treatment effect for H minus C was fit using data from eight microarray slides derived from four pairs of H versus C trees with a dye flip for each pair. Expression variance was obtained from two sources, biological and technical. The variance between hybridizations from the same H versus C tree pair is solely technical, whereas the variance between hybridizations in different tree pairs is a combination of biological and technical. Dye and H minus C effects, as well as biological and technical variation were estimated using a mixed effects model where the error term for the H minus C and dye effects was computed by pooling the biological and technical variation. Since all eight slides contained technical variation but only four pairs have biological variation, the biological term was given twice the weight of the technical term in the pooled estimate. Next, the ratio of the H minus C parameter estimate to the standard error was used to calculate a t statistic and P value. The Q value for each effect and gene was calculated for each of the two models to adjust for the false discovery rate [Storey & Tibshirani (2003) Proc. Natl. Acad. Sci. USA. 100: 9440-9445]. All statistical analyses were performed within the R statistical package (www.r-project.org/).

organism: Picea sitchensis

source_name: Sitka spruce FB3-425 bark, tree #3, untreated control

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

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

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

treatment_protocol: Untreated control.

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

source_name: Sitka spruce FB3-425 bark, tree #2, weevil feeding for 48 hours

treatment_protocol: For insect treatment, adult weevils (Pissodes strobi) were kept without food on moist filter paper for 48 hours prior to placing weevils on trees. Ten weevils for each of four individual trees were caged under mesh bags on the upper two-thirds of the stem.


title: SitkaSpruce_FB3-425_Bark_PissodesStrobi_HerbivoryHyb3


title: tumor-10

tag: Female, LFS_soft tissue sarcoma


title: SitkaSpruce_FB3-425_Bark_PissodesStrobi_HerbivoryHyb7

source_name: Sitka spruce FB3-425 bark, tree #5, weevil feeding for 48 hours

source_name: Sitka spruce FB3-425 bark, tree #5, untreated control


title: SitkaSpruce_FB3-425_Bark_PissodesStrobi_HerbivoryHyb8


title: tumor-09


title: SitkaSpruce_FB3-425_Bark_PissodesStrobi_HerbivoryHyb6

source_name: Sitka spruce FB3-425 bark, tree #4, untreated control

source_name: Sitka spruce FB3-425 bark, tree #4, weevil feeding for 48 hours


title: SitkaSpruce_FB3-425_Bark_PissodesStrobi_HerbivoryHyb1

data_processing:
Before data normalization, the lowest 10% of median foreground intensities was subtracted from the median foreground intensities to correct background intensity. After quantification of the signal intensities, data were normalized to compensate for non-linearity of intensity distributions using the variance stabilizing normalization (VSN) method of Huber et al. (2002) Bioinformatics 18: S96-S104.
In experiments with four independent biological replicates, RNA isolated from each of the four individual weevil-treated trees were compared against four individual untreated control trees using two hybridizations with a dye flip for each pair of four treatment and control replicates (eight slides total).  In order to assess the biological response to weevil herbivory (H), a mixed effects model containing a dye effect and a treatment effect for H minus C was fit using data from eight microarray slides derived from four pairs of H versus C trees with a dye flip for each pair. Expression variance was obtained from two sources, biological and technical. The variance between hybridizations from the same H versus C tree pair is solely technical, whereas the variance between hybridizations in different tree pairs is a combination of biological and technical. Dye and H minus C effects, as well as biological and technical variation were estimated using a mixed effects model where the error term for the H minus C and dye effects was computed by pooling the biological and technical variation. Since all eight slides contained technical variation but only four pairs have biological variation, the biological term was given twice the weight of the technical term in the pooled estimate. Next, the ratio of the H minus C parameter estimate to the standard error was used to calculate a t statistic and P value. The Q value for each effect and gene was calculated for each of the two models to adjust for the false discovery rate [Storey & Tibshirani (2003) Proc. Natl. Acad. Sci. USA. 100: 9440-9445]. All statistical analyses were performed within the R statistical package (www.r-project.org/).

source_name: Sitka spruce FB3-425 bark, tree #1, weevil feeding for 48 hours

treatment_protocol: For insect treatment, adult weevils (Pissodes strobi) were kept without food on moist filter paper for 48 hours prior to placing weevils on trees.  Ten weevils for each of four individual trees were caged under mesh bags on the upper two-thirds of the stem.

source_name: Sitka spruce FB3-425 bark, tree #1, untreated control


title: tumor-06

tag: Female, LFS_sarcomatoid carcinoma


title: SitkaSpruce_FB3-425_Bark_PissodesStrobi_HerbivoryHyb2


title: SitkaSpruce_FB3-425_Bark_PissodesStrobi_HerbivoryHyb5


title: Crypt LCM LOI(+) rep3

city: Baltimore

first: Andrew

last: Feinberg

state: MD

address: 720 Rutland Ave., 1064 Ross

department: Medicine

institute: Johns Hopkins University

hyb_protocol: Agilent 60-mer oligo microarray processing protocol (SSC Wash/SureHyb Chamber set-up) V4.1, April 2004.  Agilent Publication Number:  G4140-90030 V4.1 APRIL 2004.

scan_protocol: Slides were scanned with an Agilent DNA Microarray Scanner model G2505-64120 at 100% PMT in both channels, with a scan resolution of 10um.  A scan window of 61 x 21.6 mm was used, then images were cropped to size and saved in a modified two-color TIFF format.  Images were examined visually for evidence of foreign debris or major failures. Agilent Microarray Scanner User Manual (6.3)

data_processing: Data are extracted with Agilent Feature Extraction Software.The data were further processed with NIA ANOVA tool utilities.See NIA Array Analysis website for details.

label_protocol: Total RNA was labeled using Agilents Fluorescent Linear Amplification kit, according to manufacturers instructions (Product Number G2554A or P/N G2556-66002, Version 3.0, June 2002).

extract_protocol: Total RNA extracted from microdissected crypts using RNeasy as directed by manufacturer. Samples QCs on an Agilent Bioanalyzer for RNA quality and quantity.

treatment_protocol: The mouse was euthanized by CO2 inhalation and the small intestine was collected and washed with PBS. Pieces of the middle part of the small intestine were collected and frozen at -80C, embedded in OCT, sectioned at 10um, and fixed with 70% EtOH on the slides. Slides were stained with hematoxylin, dehydraded and used for LCM within one week. 5,000 to 13,000 intestinal crypts were dissected by LCM, and RNA was extracted using RNeasy (QIAGEN).

tag: Strain

tag: Gender

tag: Age

value: 120 days

tag: Development stage

value: adult

tag: Individual Identifier

source_name: Universal Mouse Reference RNA

growth_protocol: The UMRR is provided in a solution of 70% ethanol and 0.1 M sodium acetate. Prepare the UMRR for use as follows: 1.Centrifuge the tube at 12,000 x g for 15 minutes at 4C.  2.Carefully remove the supernatant. 3.Wash the pellet in 70% ethanol. 4.Centrifuge the tube at 12,000 x g for 15 minutes at 4C.  5.Carefully remove the supernatant and air-dry the pellet at room temperature for 30 minutes to remove retained ethanol. 6.Resuspend the pellet in RNase-free water to the desired concentration.

extract_protocol: Total RNA was extracted from cultured cells using a Stratagene Absolutely RNA kit. DNase-treated total RNA samples from 11 cultured cell lines derived from embryo, embryo fibroblast, kidney, liver/hepatocyte, lung/alveolar macrophage, B-lymphocyte, T-lymphocyte (thymus), mammary gland, muscle myoblast, skin, and testis were pooled in equal parts

tag: Universale Mouse Reference


title: Crypt LCM LOI(-) rep1


title: tumor-05


title: Crypt LCM LOI(+) rep2


title: tumor-04

tag: Female, LFS_osteosarcoma


title: Crypt LCM LOI(+) rep1


title: Crypt LCM LOI(-) rep2


title: LBCL-10

description: no additional information

hyb_protocol: Hybridization buffer (Spectral Genomics) were added, and samples were applied to microarrays enclosed in sealed hybridization chambers and hybdizied at 37C for 16h. After hybridization, slides were washed according to manufactur's protocol.

scan_protocol: Scanned on an GenPix 4000B scanner.

data_processing: Global linear regressionnormalized, background subtracted value data obtained from log2 of processed Red signal/processed Green signal.

source_name: normal DNA

label_protocol: 2 µg tumor DNA were labeled by Cy5, 2 µg control DNA were labeled with Cy3 for first chip; and for second chip vice verca

extract_protocol: DNA extracted using Gentra following manufacturer's instructions

tag: from female, LFS_soft tissue sarcoma

source_name: sex matching normal pool DNA

tag: same sex with tumor sample


title: tumor-08

description: no additonal

data_processing: Global normalized, background subtracted value data obtained from log2 of processed Red signal/processed Green signal.

source_name: tumor DNA

label_protocol: 2 µg tumor DNA were labeled by Cy3, 2 µg control DNA were labeled with Cy5 for first chip; and for second chip vice verca


title: tumor-10

description: no additonal


title: tumor-09

description: no additonal


title: FBCL-09

description: no additional information

tag: from male, LFS_soft tissue sarcoma


title: SarZ mutant biological rep 2

city: Hamilton

first: Dan

last: Sturdevant

state: MT

address: 903 S 4th street

department: NIAID

institute: NIH

description: Gene expression data from_SarZ mutant biological rep 2

hyb_protocol: Following fragmentation, 10 ug of cRNA were hybridized for 16 hr at 40C on our custom Array. GeneChips were washed and stained in the Affymetrix Fluidics Station 450.

scan_protocol: GeneChips were scanned using the Affymetrix GeneChip Scanner 7GPlus.

data_processing: The data were analyzed with GCOS 1.4 using Affymetrix default analysis with a scale filter for Se genes setting and global scaling as normalization method. The trimmed mean target intensity of each array was arbitrarily set to 500.

organism: Staphylococcus epidermidis

source_name: SarZ mutant biological rep 2

label_protocol: Biotinylated cRNA were prepared according to the standard Affymetrix protocol from 10 ug total RNA (Expression Analysis Technical Manual, 2001, Affymetrix).

growth_protocol: For microarray experiments, overnight cultures were diluted 1:100 into 50 ml of TSB and incubated at 37℃ with shaking at 180rpm until grown to the late exponential phase.

extract_protocol: Total RNA was isolated by use of an RNeasy Mini Kit (Qiagen) recommended by a standard protocol.

tag: SarZ mutant


title: tumor-07

description: no additonal


title: SarZ mutant biological rep 1

description: Gene expression data from_SarZ mutant biological rep 1

source_name: SarZ mutant biological rep 1


title: SarZ mutant biological rep 3

description: Gene expression data from_SarZ mutant biological rep 3

source_name: SarZ mutant biological rep 3


title: wild type biological rep 2

description: Gene expression data from_wild type biological rep 2

source_name: wild type biological rep 2

tag: 1457 strain


title: FBCL-08

description: no additional information


title: tumor-06

description: no additonal


title: FBCL-05

description: no additional information

tag: from male, LFS_osteosarcoma


title: Crypt LCM LOI(-) rep3


title: LBCL-06

description: no additional information

tag: from female, LFS_sarcomatoid carcinoma


title: tumor-05

description: no additonal

tag: Male, LFS_osteosarcoma


title: LBCL-04

description: no additional information

tag: from female, LFS_osteosarcoma


title: tumor-03

source_name: Human tumor tissue


title: wild type biological rep 1

description: Gene expression data from_wild type biological rep 1

source_name: wild type biological rep 1


title: tumor-04

description: no additonal


title: tumor-02

description: no additonal

tag: Female, LFS_soft tissue sarcoma


title: LBCL-02

description: no additional information


title: LBCL-07

description: no additional information


title: LBCL-03

description: no additional information


title: tumor-01

description: no additonal


title: tumor-03

description: no additonal


title: LBCL-01

description: no additional information


title: 6XIMO 3

status: Public on Jul 15 2008

city: Valhalla

first: Xiaoping

last: Liu

state: NY

address: BSB 127, 95 Grassland RD

department: Biochemistry and Molecular biology

institute: New York Medical College

description: Adrenal medulla brain tissue dissected from mouse stressed daily for 2-hours

hyb_protocol: Total RNA (> 4 μg) from each group was converted to cDNA by using superscript reverse transcriptase and the T7-Oligo (dT) promoter primer kit (Affymetrix, Inc). Following RNase H-mediated second-strand cDNA synthesis, the double-stranded cDNA were purified and served as a template in the subsequent in vitro transcription reaction (Affymetrix, Inc.). The in vitro transcription reaction was carried by T7 RNA polymerase and a biotinylated nucleotide analog/ribonucleotide mix (Affymetrix, Inc.). The biotinylated cRNA targets were cleaned up and fragmented. Each cRNA was hybridized to an individual Affymetrix GeneChip Rat Array Expression 230 2.0 (RAE 230 2.0 array).

scan_protocol: The hybridized cRNA was subsequently processed for washing and staining with the antibody stain solution with streptavidin phycoerythrin and the arrays were scanned on the GeneChip Scanner 3000.

data_processing: The raw pixel data were uploaded into GeneTraffic (GeneTraffic version 3.2, Iobion Informatics, La Jolla, CA), and all subsequent analyses were performed on a GeneTraffic Server at the Functional Genomics Core Facility of New York Medical College.

organism: Rattus norvegicus

source_name: Adrenal medulla brain tissue from stressed mouse

label_protocol: performed by Affymetrix

extract_protocol:
RNA was extracted using Absolutely RNA Miniprep Kit (Stratagene, La
Jolla, CA). The integrity of the RNA was assessed by the A260/A280 ratio which was close to 2.0 and by electrophoresis (Agilent
Bioanalyzer 2100)

treatment_protocol: Repeated immobilization stressed

tag: 2-hour immobilization stress daily for 6 days


title: 1XIMO 1

description: Adrenal medulla brain tissue dissected from mouse stressed once for 2-hours

treatment_protocol: Single immobilization stressed

tag: 2-hour immobilization stress once


title: 6XIMO 2


title: wild type biological rep 3

description: Gene expression data from_wild type biological rep 3

source_name: wild type biological rep 3


title: 1XIMO 2


title: 6XIMO 1


title: Control 1

description: Adrenal medulla brain tissue dissected from unstressed mouse

source_name: Adrenal medulla brain tissue from control mouse

treatment_protocol: Unstressed

tag: Control animal


title: Control 3


title: 1XIMO 3


title: Cap_Dkk1_rep3

first: Kathryn

last: Maddison

department: Genetics

description: Embryo injected at 1 cell stage with Dkk1, cap cut at stage 9 and incubated with CHX and Dex, harvested after 2.5 hours.

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

scan_protocol: Scanned using manufacturer's protocols.

data_processing: Data were normalised and analysed using Genespring 7.0 using default settings.

organism: Xenopus laevis

source_name: Dkk1 injected animal cap_CHX_Dex_2.5 hours

growth_protocol: Embryos were injected with 10ng of each mRNA at the one cell stage and allowed to reach stage 9, whereupon animal caps were excised and cultured in .75 NAM for 2.5 hours in the presence of cycloheximide and dexamethasone.

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

treatment_protocol: 10ng of inducible mRNA construct was injected into one cell embryos at around 20 degrees off the animal pole.


title: Cap_Gata5-GR_rep1

description: Embryo injected at 1 cell stage with Gata5-GR, cap cut at stage 9 and incubated with CHX and Dex, harvested after 2.5 hours.

source_name: Gata5_GR injected animal cap_CHX+Dex_2.5 hours


title: Cap_Dkk1_rep1

tag: Embryo injected at 1 cell stage with Dkk1, cap cut at stage 9 and incubated with CHX and Dex, harvested after 2.5 hours.


title: Cap_Gata4_GR+Dkk1_rep3

description: Embryo injected at 1 cell stage with Gata4-GR+Dkk1, cap cut at stage 9 and incubated with CHX and Dex, harvested after 2.5 hours.

source_name: Gata4+Dkk1 injected animal cap_CHX+Dex_2.5 hours.


title: Cap_Gata4-GR_rep3

description: Embryo injected at 1 cell stage with Gata4-GR, cap cut at stage 9 and incubated with CHX and Dex, harvested after 2.5 hours.

source_name: Gata4-GR injected animal cap+CHX+Dex_2.5 hours


title: Cap_Gata5_GR_rep4

tag: Embryo injected at 1 cell stage with Gata5-GR, cap cut at stage 9 and incubated with CHX and Dex, harvested after 2.5 hours.


title: Cap_Gata4-GR_rep4

tag: Embryo injected at 1 cell stage with Gata4-GR, cap cut at stage 9 and incubated with CHX and Dex, harvested after 2.5 hours.


title: Cap_Gata5-GR_rep2


title: Control 2


title: Cap_Gata4-GR+Dkk1_rep2

tag: Embryo injected at 1 cell stage with Gata4-GR+Dkk1, cap cut at stage 9 and incubated with CHX and Dex, harvested after 2.5 hours.


title: Cap_Gata4_GR+Dkk1_rep4


title: Cap_Gata4-GR+Dkk1_rep1


title: Cap_uinjected_rep 2

description: cap cut from uninjected embryo at stage 9, CHX and Dex added, harvested after 2.5 hours

source_name: uninjected animal cap+CHX+Dex_2.5hours


title: Cap_Dkk1_rep4


title: Cap_Gata5_GR_rep3


title: Cap_uninjected_rep3

tag: cap cut from uninjected embryo at stage 9, CHX and Dex added, harvested after 2.5 hours


title: Cap_uninjected_rep4


title: Cap_Gata4-GR_rep1


title: IMT11 vs control, rep4

description: These array experiments were carried out in order to compare the transcriptional profiles of ES cells with various very early embryonic stages and compartements, including those from which ES cells can normally be derived. The pooled control was comprised of all the stages from which the NIA clone set, printed on the slides, was originally derived. Owing to the small size of most embryonic samples, two rounds of RNA amplification were used on the tiny amounts of total RNA extracted from the sample, prior to labelling for array hybridization

source_name: Pooled control

tag: Pooled control consisting of equal amounts of RNA from tissues listed below

value: hpc = hours post coitum Tissue:oocytes, Day of Gestation 0.5, Harvested hpc 7-7.5 1 cell zygote, Day of Gestation 0.5, Harvested hpc 7.5-8.5 2 cell embryo, Day of Gestation1.5, Harvested hpc 32-35 3-4 cell embryo, Day of Gestation2.0, Harvested hpc45-46.5 4-8cell embryo, Day of Gestation2.5, Harvested hpc 55.5-58 8-16 cell embryo, Day of Gestation 3, Harvested hpc 67-68 Late morulae- early blastocysts, Day of Gestation3.5, Harvested hpc80-84 Whole embryo, mid-streak stage, Day of Gestation7.5 Ovary+mesonephros, Day of Gestation12.5 Ovary, Newborn

source_name: IMT11 embryonic stem cells

tag: Amplified RNA from individually picked IMT-11 colonies.\ ES cell line IMT-11, derived from inner cell mass (ICM) isolated from blastocysts flushed from the uterus approximately 88 and 105hpc, ICM isolated from blastocysts undergoing experimentally induced diapause at approximately 134 and 180hpc, epiblast dissected from early post implantation embryos approximately 132 and 150hpc.


title: Cap_Dkk1_rep2


title: TbDRBD3 RNAi, biological replicate #2, -tet is Cy3, + tet is Cy5

city: Armilla

first: Antonio

middle: M

last: Estevez

state: Granada

address: Avda. del Conocimiento 17

department: Lopez-Neyra

institute: CSIC

hyb_protocol: Slides were pre-incubated in 5xSSC, 0.1% SDS and 1% BSA for 1 h at 42°C, dipped in water then isopropanol and allowed to dry. Hybridization with mixed Cy3- and Cy5-labelled cDNAs was in 50% deionised formamide, 5% dextran sulfate, 3xSSC, 1% SDS and 5x Denhardts solution at 42°C for 16h, using 24-60mm cover slips and glass array hybridisation cassettes (Ambion).  Slides were then washed for 5 min each in 1x SSC, 0.2% SDS, 0.1x SSC, 0.2% SDS, and 0.1x SSC at room temperature.

scan_protocol: ScanArray 5000 laser-scanner (Packard BioScience, Germany)

data_processing: The resulting images were analyzed using GenePix (Axon Instruments, USA) and MCHIPS software (PMID 11535808, PMID 11934741) essentially as described (PMID 12270627, PMID 15664651). Only those clones satisfying the following parameters - fitted intensities: at least 150,000; ratio: ± 2; min/max-separation: 0.1 - were selected. Regulated clones were sequenced from one end and identified in the T. brucei genome database (http://www.genedb.org/genedb/tryp/).

organism: Trypanosoma brucei

source_name: Uninduced RNAi TbDRBD3 cells (- tet)

label_protocol: 15 µg of RNA were reverse-transcribed in the presence of 100 ng of oligo(dT)12-18, 0.25 mM dATP, 0.25 mM dTTP, 0.25 mM dGTP, 0.05 mM dCTP, 0.05 mM Cy3-dCTP, 50 mM Tris-HCl, pH 8.3, 75 mM KCl, 3 mM MgCl2, 5 mM DTT, 40 U of RNaseOUT (Invitrogen) and 400 U of Superscript III reverse transcriptase (Invitrogen). Reactions were incubated for 3 hours at 50ºC, stopped by heating at 70ºC for 15 min and treated with 5 U of RNase H for 20 min at 37ºC. cDNA was purified using the MinElute kit (Qiagen), ethanol precipitated and resuspended in hybridization buffer.

extract_protocol: Trizol, as per manufacturer's instructions

tag: Procyclic TbDRBD3 RNAi cell line minus tetracycline

source_name: Induced RNAi TbDRBD3 cells (+ tet)

label_protocol: 0.25 mM dATP, 0.25 mM dTTP, 0.25 mM dGTP, 0.05 mM dCTP, 0.05 mM Cy5-dCTP, 50 mM Tris-HCl, pH 8.3, 75 mM KCl, 3 mM MgCl2, 5 mM DTT, 40 U of RNaseOUT (Invitrogen) and 400 U of Superscript III reverse transcriptase (Invitrogen). Reactions were incubated for 3 hours at 50ºC, stopped by heating at 70ºC for 15 min and treated with 5 U of RNase H for 20 min at 37ºC. cDNA was purified using the MinElute kit (Qiagen), ethanol precipitated and resuspended in hybridization buffer.

tag: Procyclic TbDRBD3 RNAi cell line plus tetracycline


title: TbDRBD3 RNAi, biological replicate #1, -tet is Cy3, + tet is Cy5

tag: Procyclic TbDRBD3 RNAi cell line without tetracycline


title: TbDRBD3 RNAi, biological replicate #1, -tet is Cy5, + tet is Cy3 (dye swap)


title: TbDRBD3 RNAi, biological replicate #2, -tet is Cy5, + tet is Cy3 (dye swap)


title: Individual PT, healthy control, replicate 2

status: Public on Jul 11 2008

city: Zurich

first: Thierry

last: Hennet

department: Institute of physiology

country: Switzerland

institute: University of Zurich

hyb_protocol: 15 mg of fragmented biotin-labeled cRNA was hybridized  to HG-U133A chips for 16 h at 45°C. Gene chips were washed and stained in the Affymetrix Fluidistic station 400.

scan_protocol: Gene Chips were scanned using the Gene-Array scanner G2500 (Agilent)

data_processing: 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 wasnormalized to a target intensity arbitrarily set to 500.

source_name: forearm skin biopsy

label_protocol: Biotinilated cRNA was in vitro transcribed using the High Yield Transcription kit (ENZO diagnostic) from 15 mg of total RNA

growth_protocol: Primary fibroblasts from healthy control subjects and from CDG-I patients were grown in F12 Dulbecco's Modified Eagle's Medium (DMEM, Invitrogen) supplemented with 4.5 g/l glucose and 10% fetal calf serum. Cells were passaged when reached 70-80% confluence.

extract_protocol: TRIZOL extraction of Total RNA according to manufacturer's protocol followed by on column RNA clean-up (Rneasy mini Kit QIAGEN)

tag: primary skin fibroblasts


title: Individual PT, healthy control, replicate 3


title: Cap_uninjected_rep 1


title: Individual MG, healthy control, replicate 1


title: Individual HK, healthy control, replicate 3


title: Individual HK, healthy control, replicate 2


title: CDG Ig patient MA, biological replicate 3


title: CDG Ig patient MA, biological replicate 2


title: Individual MG, healthy control, replicate 2


title: Individual PT, healthy control, replicate 1


title: CDG Ig patient HA, biological replicate 1


title: Individual MG, healthy control, replicate 3


title: CDG Ig patient HA, biological replicate 2


title: CDG Ig patient HA, biological replicate 3


title: CDG Ig patient MA, biological replicate 1


title: CDG Ie patient OR, biological replicate 2


title: CDG Ie patient OR, biological replicate 3


title: CDG Ie patient YU, biological replicate 3


title: Individual HK, healthy control, replicate 1


title: CDG Ie patient YU, biological replicate 2


title: CDG Ie patient CA, biological replicate 3


title: CDG Ie patient OR, biological replicate 1


title: CDG Ie patient CA, biological replicate 2


title: CDG Ie patient YU, biological replicate 1


title: CDG Ie patient CA, biological replicate 1


title: CDG Ic patient VB, biological replicate 3


title: CDG Ic patient ME, biological replicate 3


title: CDG Ic patient BO, biological replicate 1


title: 13602893 - Ea wt 24h set2 vs Ea dspA/E 24h set2

status: Public on Jul 10 2008

city: evry

first: Soubigou-Taconnat

last: Ludivine

address: 2 rue gaston crémieux

department: URGV

country: France

institute: INRA

description: Identification of Arabidopsis genes regulated by the type three effector Dspa/E of Erwinia amylovora.

hyb_protocol: Ea dspA/E 24h set2 Cy5 / Ea wt 24h set2 Cy3 : 30pmol. Hybridization Protocol: CATMA slides (Corning Microarray Technology, CORNING) are pretreated in the prehybridisation solution (1 % BSA, 0.1 SDS, 5X SSC ) at 42°C for 60 min. They are dipped a couple of times in distilled water at room temperature, then in isopropanol and dried immediately by compressed nitrogen stream. Slides were placed in Corning hybridization chambers with a 25x60 lifterslip and 10ul of distilled water for each groove. The target was diluted to a final volume of 60 µL as follows 15µl of purified, labeled cDNA, 15 µl of 4X Hybridization Buffer ( 20X SDS, 0.4 % SDS), 30 µL formamide. The target mixture is heated for 3 min at 95°C, put on ice for 30 sec and centrifuged to remove dust for 1 min. The target mixture was put on the chip as quickly as possible. The microarray is sealed in a chamber and submerged in a 42°C water bath for approximately 16 h. The microarray is washed for 4 min in 1xSSC, 0.2% SDS (42°C); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC (RT); dipped a few times in distilled water and dried immediately by compressed nitrogen stream.

scan_protocol: GenePix Pro 6.0, Cy3:pmt voltage 532nm,500V,laser power 30%, Cy5:635nm,pmt voltage 500V,laser power 30%

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

organism: Arabidopsis thaliana

source_name: Ea dspA/E 24h set2

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

growth_protocol: leaf - Media = soil	 hygrometry = 70%	 Temperature = 21degreeC	 Light = 8Hlight/16Hdark (900 lux)

extract_protocol: Ea dspA/E 24h set2:15ug.

treatment_protocol: Name:Erwinia amylovora dspA/E 24h - environmental treatment - pathogen infection,erwinia amylovora dspa/e 24h:quantity 108cfu/ml time 24hour . A bacterial suspension of each strain is prepared in sterile water at an optical density (600 nm) of 0,1. Arabidopsis leaves of independent plants are syringe-infiltrated with the different bacterial strains. Infected leaves are collected 6 or 24 hours post inoculation.

tag: Arabidopsis thaliana (columbia)  - dev.stage (Boyes et al. Plant Cell 2001):boyes = 1.10

source_name: Ea wt 24h set2

extract_protocol: Ea wt 24h set2:15ug.

treatment_protocol: Name:Erwinia amylovora 24h - environmental treatment - pathogen infection,erwinia amylovora:quantity 108cfu/ml time 24hour . A bacterial suspension of each strain is prepared in sterile water at an optical density (600 nm) of 0,1. Arabidopsis leaves of independent plants are syringe-infiltrated with the different bacterial strains. Infected leaves are collected 6 or 24 hours post inoculation.


title: 13602885 - Ea wt 6h set2 vs Ea sec 6h set2

hyb_protocol: Ea sec 6h set2 Cy5 / Ea wt 6h set2 Cy3 : 30pmol. Hybridization Protocol: CATMA slides (Corning Microarray Technology, CORNING) are pretreated in the prehybridisation solution (1 % BSA, 0.1 SDS, 5X SSC ) at 42°C for 60 min. They are dipped a couple of times in distilled water at room temperature, then in isopropanol and dried immediately by compressed nitrogen stream. Slides were placed in Corning hybridization chambers with a 25x60 lifterslip and 10ul of distilled water for each groove. The target was diluted to a final volume of 60 µL as follows 15µl of purified, labeled cDNA, 15 µl of 4X Hybridization Buffer ( 20X SDS, 0.4 % SDS), 30 µL formamide. The target mixture is heated for 3 min at 95°C, put on ice for 30 sec and centrifuged to remove dust for 1 min. The target mixture was put on the chip as quickly as possible. The microarray is sealed in a chamber and submerged in a 42°C water bath for approximately 16 h. The microarray is washed for 4 min in 1xSSC, 0.2% SDS (42°C); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC (RT); dipped a few times in distilled water and dried immediately by compressed nitrogen stream.

source_name: Ea sec 6h set2

extract_protocol: Ea sec 6h set2:15ug.

treatment_protocol: Name:Erwinia amylovora sec mutant 6h - environmental treatment - pathogen infection,erwinia amylovora sec mutant:quantity 108cfu/ml time 6hour . A bacterial suspension of each strain is prepared in sterile water at an optical density (600 nm) of 0,1. Arabidopsis leaves of independent plants are syringe-infiltrated with the different bacterial strains. Infected leaves are collected 6 or 24 hours post inoculation.

source_name: Ea wt 6h set2

extract_protocol: Ea wt 6h set2:15ug.

treatment_protocol: Name:Erwinia amylovora 6h - environmental treatment - pathogen infection,erwinia amylovora:quantity 108cfu/ml time 6hour . A bacterial suspension of each strain is prepared in sterile water at an optical density (600 nm) of 0,1. Arabidopsis leaves of independent plants are syringe-infiltrated with the different bacterial strains. Infected leaves are collected 6 or 24 hours post inoculation.


title: 13602894 - Ea dspA/E 24h set2 vs Ea wt 24h set2

hyb_protocol: Ea wt 24h set2 Cy5 / Ea dspA/E 24h set2 Cy3 : 30pmol. Hybridization Protocol: CATMA slides (Corning Microarray Technology, CORNING) are pretreated in the prehybridisation solution (1 % BSA, 0.1 SDS, 5X SSC ) at 42°C for 60 min. They are dipped a couple of times in distilled water at room temperature, then in isopropanol and dried immediately by compressed nitrogen stream. Slides were placed in Corning hybridization chambers with a 25x60 lifterslip and 10ul of distilled water for each groove. The target was diluted to a final volume of 60 µL as follows 15µl of purified, labeled cDNA, 15 µl of 4X Hybridization Buffer ( 20X SDS, 0.4 % SDS), 30 µL formamide. The target mixture is heated for 3 min at 95°C, put on ice for 30 sec and centrifuged to remove dust for 1 min. The target mixture was put on the chip as quickly as possible. The microarray is sealed in a chamber and submerged in a 42°C water bath for approximately 16 h. The microarray is washed for 4 min in 1xSSC, 0.2% SDS (42°C); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC (RT); dipped a few times in distilled water and dried immediately by compressed nitrogen stream.


title: 13602883 - Ea sec 6h set1 vs Ea wt 6h set1

hyb_protocol: Ea wt 6h set1 Cy5 / Ea sec 6h set1 Cy3 : 30pmol. Hybridization Protocol: CATMA slides (Corning Microarray Technology, CORNING) are pretreated in the prehybridisation solution (1 % BSA, 0.1 SDS, 5X SSC ) at 42°C for 60 min. They are dipped a couple of times in distilled water at room temperature, then in isopropanol and dried immediately by compressed nitrogen stream. Slides were placed in Corning hybridization chambers with a 25x60 lifterslip and 10ul of distilled water for each groove. The target was diluted to a final volume of 60 µL as follows 15µl of purified, labeled cDNA, 15 µl of 4X Hybridization Buffer ( 20X SDS, 0.4 % SDS), 30 µL formamide. The target mixture is heated for 3 min at 95°C, put on ice for 30 sec and centrifuged to remove dust for 1 min. The target mixture was put on the chip as quickly as possible. The microarray is sealed in a chamber and submerged in a 42°C water bath for approximately 16 h. The microarray is washed for 4 min in 1xSSC, 0.2% SDS (42°C); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC (RT); dipped a few times in distilled water and dried immediately by compressed nitrogen stream.

source_name: Ea wt 6h set1

extract_protocol: Ea wt 6h set1:15ug.

source_name: Ea sec 6h set1

extract_protocol: Ea sec 6h set1:15ug.


title: CDG Ic patient AE, biological replicate 3


title: CDG Ic patient AE, biological replicate 1


title: 13602892 - Ea wt 24h set1 vs Ea dspA/E 24h set1

hyb_protocol: Ea dspA/E 24h set1 Cy5 / Ea wt 24h set1 Cy3 : 30pmol. Hybridization Protocol: CATMA slides (Corning Microarray Technology, CORNING) are pretreated in the prehybridisation solution (1 % BSA, 0.1 SDS, 5X SSC ) at 42°C for 60 min. They are dipped a couple of times in distilled water at room temperature, then in isopropanol and dried immediately by compressed nitrogen stream. Slides were placed in Corning hybridization chambers with a 25x60 lifterslip and 10ul of distilled water for each groove. The target was diluted to a final volume of 60 µL as follows 15µl of purified, labeled cDNA, 15 µl of 4X Hybridization Buffer ( 20X SDS, 0.4 % SDS), 30 µL formamide. The target mixture is heated for 3 min at 95°C, put on ice for 30 sec and centrifuged to remove dust for 1 min. The target mixture was put on the chip as quickly as possible. The microarray is sealed in a chamber and submerged in a 42°C water bath for approximately 16 h. The microarray is washed for 4 min in 1xSSC, 0.2% SDS (42°C); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC (RT); dipped a few times in distilled water and dried immediately by compressed nitrogen stream.

source_name: Ea dspA/E 24h set1

extract_protocol: Ea dspA/E 24h set1:15.4ug.

source_name: Ea wt 24h set1

extract_protocol: Ea wt 24h set1:15ug.


title: 13602891 - Ea dspA/E 6h set2 vs Ea wt 6h set2

hyb_protocol: Ea wt 6h set2 Cy5 / Ea dspA/E 6h set2 Cy3 : 30pmol. Hybridization Protocol: CATMA slides (Corning Microarray Technology, CORNING) are pretreated in the prehybridisation solution (1 % BSA, 0.1 SDS, 5X SSC ) at 42°C for 60 min. They are dipped a couple of times in distilled water at room temperature, then in isopropanol and dried immediately by compressed nitrogen stream. Slides were placed in Corning hybridization chambers with a 25x60 lifterslip and 10ul of distilled water for each groove. The target was diluted to a final volume of 60 µL as follows 15µl of purified, labeled cDNA, 15 µl of 4X Hybridization Buffer ( 20X SDS, 0.4 % SDS), 30 µL formamide. The target mixture is heated for 3 min at 95°C, put on ice for 30 sec and centrifuged to remove dust for 1 min. The target mixture was put on the chip as quickly as possible. The microarray is sealed in a chamber and submerged in a 42°C water bath for approximately 16 h. The microarray is washed for 4 min in 1xSSC, 0.2% SDS (42°C); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC (RT); dipped a few times in distilled water and dried immediately by compressed nitrogen stream.

source_name: Ea dspA/E 6h set2

extract_protocol: Ea dspA/E 6h set2:15ug.

treatment_protocol: Name:Erwinia amylovora dspA/E 6h - environmental treatment - pathogen infection,erwinia amylovora dspa/e :quantity 108cfu/ml time 6hour . A bacterial suspension of each strain is prepared in sterile water at an optical density (600 nm) of 0,1. Arabidopsis leaves of independent plants are syringe-infiltrated with the different bacterial strains. Infected leaves are collected 6 or 24 hours post inoculation.


title: CDG Ic patient VB, biological replicate 1


title: 13602902 - Ea dspA/E 24h set1 vs Ea wt 24h set1

hyb_protocol: Ea wt 24h set1 Cy5 / Ea dspA/E 24h set1 Cy3 : 30pmol. Hybridization Protocol: CATMA slides (Corning Microarray Technology, CORNING) are pretreated in the prehybridisation solution (1 % BSA, 0.1 SDS, 5X SSC ) at 42°C for 60 min. They are dipped a couple of times in distilled water at room temperature, then in isopropanol and dried immediately by compressed nitrogen stream. Slides were placed in Corning hybridization chambers with a 25x60 lifterslip and 10ul of distilled water for each groove. The target was diluted to a final volume of 60 µL as follows 15µl of purified, labeled cDNA, 15 µl of 4X Hybridization Buffer ( 20X SDS, 0.4 % SDS), 30 µL formamide. The target mixture is heated for 3 min at 95°C, put on ice for 30 sec and centrifuged to remove dust for 1 min. The target mixture was put on the chip as quickly as possible. The microarray is sealed in a chamber and submerged in a 42°C water bath for approximately 16 h. The microarray is washed for 4 min in 1xSSC, 0.2% SDS (42°C); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC (RT); dipped a few times in distilled water and dried immediately by compressed nitrogen stream.


title: CDG Ic patient BO, biological replicate 3


title: CDG Ic patient AE, biological replicate 2


title: 13602889 - Ea dspA/E 6h set1 vs Ea wt 6h set1

hyb_protocol: Ea wt 6h set1 Cy5 / Ea dspA/E 6h set1 Cy3 : 30pmol. Hybridization Protocol: CATMA slides (Corning Microarray Technology, CORNING) are pretreated in the prehybridisation solution (1 % BSA, 0.1 SDS, 5X SSC ) at 42°C for 60 min. They are dipped a couple of times in distilled water at room temperature, then in isopropanol and dried immediately by compressed nitrogen stream. Slides were placed in Corning hybridization chambers with a 25x60 lifterslip and 10ul of distilled water for each groove. The target was diluted to a final volume of 60 µL as follows 15µl of purified, labeled cDNA, 15 µl of 4X Hybridization Buffer ( 20X SDS, 0.4 % SDS), 30 µL formamide. The target mixture is heated for 3 min at 95°C, put on ice for 30 sec and centrifuged to remove dust for 1 min. The target mixture was put on the chip as quickly as possible. The microarray is sealed in a chamber and submerged in a 42°C water bath for approximately 16 h. The microarray is washed for 4 min in 1xSSC, 0.2% SDS (42°C); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC (RT); dipped a few times in distilled water and dried immediately by compressed nitrogen stream.

source_name: Ea dspA/E 6h set1

extract_protocol: Ea dspA/E 6h set1:15ug.


title: CDG Ic patient VB, biological replicate 2


title: CDG Ic patient ME, biological replicate 2


title: 13561085 - Ea wt 6h set1 vs Ea dspA/E 6h set1

hyb_protocol: Ea dspA/E 6h set1 Cy5 / Ea wt 6h set1 Cy3 : 30pmol. Hybridization Protocol: CATMA slides (Corning Microarray Technology, CORNING) are pretreated in the prehybridisation solution (1 % BSA, 0.1 SDS, 5X SSC ) at 42°C for 60 min. They are dipped a couple of times in distilled water at room temperature, then in isopropanol and dried immediately by compressed nitrogen stream. Slides were placed in Corning hybridization chambers with a 25x60 lifterslip and 10ul of distilled water for each groove. The target was diluted to a final volume of 60 µL as follows 15µl of purified, labeled cDNA, 15 µl of 4X Hybridization Buffer ( 20X SDS, 0.4 % SDS), 30 µL formamide. The target mixture is heated for 3 min at 95°C, put on ice for 30 sec and centrifuged to remove dust for 1 min. The target mixture was put on the chip as quickly as possible. The microarray is sealed in a chamber and submerged in a 42°C water bath for approximately 16 h. The microarray is washed for 4 min in 1xSSC, 0.2% SDS (42°C); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC (RT); dipped a few times in distilled water and dried immediately by compressed nitrogen stream.


title: Cap_Gata4-GR_rep2

source_name: Gata4-GR injected animal cap+CHX+Dex_2.5 hours