Comment[ArrayExpressAccession] E-GEOD-31381
Public Release Date 2011-11-10
Investigation Title Collection of mouse ES cell lines engineered for the forced induction of transcription factors
Comment[Submitted Name] Collection of mouse ES cell lines engineered for the forced induction of transcription factors
Experiment Description This SuperSeries is composed of the following subset Series: GSE30917: Collection of mouse ES cell lines engineered for the forced induction of transcription factors (part A) GSE31374: Collection of mouse ES cell lines engineered for the forced induction of transcription factors (part B) Refer to individual Series
Date of Experiment
Term Source Name EFO
Term Source Version
Term Source File http://www.ebi.ac.uk/efo/efo.owl
Person Last Name Ko
Person First Name Minoru
Person Mid Initials S.H.
Person Email kom@mail.nih.gov
Person Affiliation NIH
Person Phone 410-558-8359
Person Fax 410-558-8331
Person Address National Institute on Aging, NIH, 251 Bayview Blvd, Suite 100, 10C, Baltimore, MD, USA
Person Roles submitter
Person Roles Term Source REF
Person Roles Term Accession Number
Normalization Type
Normalization Term Accession Number
Normalization Term Source REF
Replicate Type
Replicate Term Accession Number
Replicate Term Source REF
Experimental Design
Experimental Design Term Accession Number
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Quality Control Type
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Protocol Name P-GSE31381-1 P-GSE31381-2 P-GSE31381-8 P-GSE31381-23 P-GSE31381-7 P-GSE31381-3 P-GSE31381-21 P-GSE31381-6 P-GSE31381-22 P-GSE31381-4 P-GSE31381-5 P-GSE31381-13 P-GSE31381-10 P-GSE31381-18 P-GSE31381-14 P-GSE31381-11 P-GSE31381-26 P-GSE31381-12 P-GSE31381-28 P-GSE31381-27 P-GSE31381-15 P-GSE31381-9 P-GSE31381-20 P-GSE31381-17 P-GSE31381-30 P-GSE31381-29 P-GSE31381-19 P-GSE31381-16 P-GSE31381-25 P-GSE31381-24
Protocol Description ID_REF = Feature Number (FeatureNum)
VALUE = Normalized value described in the Data Processing section ID_REF = Feature Number (FeatureNum).
VALUE = The normalized value described in the Data Processing section. Slides were scanned with an Agilent DNA Microarray Scanner (model G2505-64120) at 100% and 10% PMT in both channels, with a scan resolution of 5um. Slides were hybridized according to the manufacturer's protocol (Two-Color Microarray-Based Genr Expression Analysis Protocol, Product # G4140-90050, Version 5.0.1, August 2006). Slides were hybridized according to the manufacturer's protocol (Two-Color Microarray-Based Genr Expression Analysis Protocol, Product # G4140-90050, Version 5.0.1, August 2006). Day 2: Cells were plated in regular ES medium with Doxycycline (0.2ug/ml) and Puromycin (1.5ug/ml). Approximately 18 hours after plating, the cells were rinsed once with PBS, then the medium was replaced with warmed medium with Doxycycline. Three hours after the first medium change, the medium was changed again. Finally, at three hours after the second medium change, the medium was replaced for a final time. Thereafter, the medium was changed daily. At 48 hours after the final wash, the cells were harvested in Trizol. There was no medium change on the day of RNA harvest. Day 2: Cells were plated in regular ES medium with Doxycycline (0.2ug/ml) and Puromycin (1.5ug/ml). Approximately 18 hours after plating, the cells were rinsed once with PBS, then the medium was replaced with warmed medium without Doxycycline. Three hours after the first medium change, the medium was changed again. Finally, at three hours after the second medium change, the medium was replaced for a final time. Thereafter, the medium was changed daily. At 48 hours after the final wash, the cells were harvested in Trizol. There was no medium change on the day of RNA harvest. 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. The resulting URM RNA was mixed with 129ES cell RNA (Lif+) at 2:1 ratio and 2.5ug of mixed RNA was used for labeling in each tube Stratagene Absolute RNA extraction kit The samples were dissolved in the well with TRIzol reagent 1/2 mL. They were then transferred to Phase Lock Gel Heavy tubes (eppendorf) and 0.2 volumes of chloroform (Sigma-Aldrich) was added to each sample. They were mixed by hand and allowed to stand for 2-5 min at room temperature. The samples were then separated by centrifugation for 5 minutes at 15,300 rpm on an eppendorf centrifuge at 4C. The aqeous phase was transferred to another eppendorf tube, and 0.8 volumes of ice-cold isopropanol was added. They were allowed to incubate for a minimum of 10 minutes at room temperature. If longer, they were placed at 4C. The sample was centrifuged again at the same conditions for 15 minutes. After centrifugation, the liquid was dumped out of the tubes and the pellet was washed with 1 mL of ice-cold 70% ethanol. The tube was spun for 5 minutes, then the ethanol was dumped out. Excess was removed with a pipette and the pellet was allowed to air dry until no liquid remained, but no longer. ddH2O was then added in the appropriate volume, the sample was allowed to dissolve for 5 minutes at 55C and then transferred to -80C for storage. Total RNA was labeled using the Agilent Low RNA Input Fluorescent Linear Amplification kit according to the manufacturer's instructions. (2) Take average of Xri's for the oligo among 28 arrays in the series: AverXr = average(Xri). where x(i,j) is logintensities for i-th CTT and replication j, N = number of CTT. Then we estimate z-value for each CTT: z = (x(i,1)-x(i,2))/sqrt(ASD). If abs(z) > 4 then one of the replications is an outlier. The value that is farther away from the median expression value is considered an outlier and it is replaced with the value from another replication. (3) For each array estimate Yi = Xgi-Xri+AverXr (adjust to UMR) The data is normalized with the following method: (2) in each column estimate 15 quantiles that correspond to ratios: 1/30, 3/30, ... 29/30. (1) Take values from the columns gDyeNormSignal,rDyeNormSignal in the raw files and log-transform them using: log10(max(x,10)). These values will be referenced below as Xgi and Xri where i is array number. (4) transform data using a piece-linear function that converts actual quantiles in each column into target quantiles. (3) estimate 15 target quantiles as average quantiles across all columns. (4) Remove outliers. Each cell/treatment type (CTT) is characterized by 2 replications, all are log-transformed (log10). Here we check if any of these values is an outlier. Data are extracted with Agilent Feature Extraction Software. The data were further processed with NIA ANOVA tool utilities. See http://lgsun.grc.nia.nih.gov/ANOVA for details. More detailed information with error correction can be found at http://lgsun.grc.nia.nih.gov/ANOVA/help.html#arrayjoin ASD = SUM[(x(i,1)-x(i,2))^2]/N/2 (1) Take values from the columns gDyeNormSignal in the raw files and log-transform them using: log10(max(x,10)). The data is then combined into a matrix. (5) back-transform data with exponent function. The result is used as the normalized VALUE. First we estimate the average square difference (ASD) between replication pairs: (1) Take values from the columns rDyeNormSignal in the raw files and log-transform them using: log10(max(x,10)). The data is then combined into a matrix. Data are extracted with Agilent Feature Extraction Software.The data were further processed with NIA ANOVA tool utilities.See http://lgsun.grc.nia.nih.gov/ANOVA for details.
Protocol Software
Protocol Hardware
Protocol Contact
Protocol Type bioassay_data_transformation bioassay_data_transformation image_aquisition hybridization hybridization specified_biomaterial_action specified_biomaterial_action nucleic_acid_extraction nucleic_acid_extraction nucleic_acid_extraction labeling feature_extraction feature_extraction feature_extraction feature_extraction feature_extraction feature_extraction feature_extraction feature_extraction feature_extraction feature_extraction feature_extraction feature_extraction feature_extraction feature_extraction feature_extraction feature_extraction feature_extraction feature_extraction feature_extraction
Protocol Term Source REF
Protocol Term Accession Number
Experimental Factor Name TRANSCRIPTION FACTOR CULTURE CONDITION INDIVIDUAL IDENTIFIER TRANSCRIPT FACTOR CELL TYPE BIOSOURCEPROVIDER
Experimental Factor Type transcription factor culture condition individual identifier transcript factor cell type BioSourceProvider
Experimental Factor Term Source REF
Experimental Factor Term Accession Number
Publication Title
Publication Author List
PubMed ID
Publication DOI
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Publication Status Term Accession Number
Comment[SecondaryAccession] GSE31381
Comment[GEOLastUpdateDate] 2011-11-16
Comment[AEExperimentType] transcription profiling by array
Comment[GEOReleaseDate] 2011-11-10
Comment[ArrayExpressSubmissionDate] 2011-08-14
SDRF File E-GEOD-31381.sdrf.txt