|
| Status |
Public on Oct 12, 2005 |
| Title |
Triple-Fusion Transfected Embryonic Stem Cells Replicate 3 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Total RNA from murine ES-D3 triple-transfected embryonic stem cells labeled with Cyanine-5 (red).
|
| Organism |
Mus musculus |
| Characteristics |
ES-D3 cell line (CRL-1934)
Transfected with pUb-fluc-mrfp-ttk triple fusion reporter gene.
Age: day 4
Tissue: blastocytes
Strain: 129/Sv mice
|
| Treatment protocol |
PCR amplification and standard cloning techniques were used to insert fluc and mrfp genes from plasmids pCDNA 3.1-CMV-fluc (Promega, Madison, WI) and pCDNA3.1-CMV-mrfp in frame with the ttk gene into the pCDNA3.1-truncated sr39tk. This triple fusion (TF) reporter gene fragment (3.3 kbp) was released from the plasmid with Not1 and BamH1 restriction enzymes before blunt-end ligation into the multiple cloning site of lentiviral transfer vector, FUG, driven by the human ubiquitin-C promoter. Self-inactivating (SIN) lentivirus was prepared by transient transfection of 293T cells. Briefly, pFUG-TF containing the triple fusion reporter gene was co-transfected into 293T cells with HIV-1 packaging vector (δ8.9) and vesicular stomatitis virus G glycoprotein-pseudotyped envelop vector (pVSVG). Lentivirus supernatant was concentrated by sediment centrifugation using a SW29 rotor at 50,000 x g for two hours. Concentrated virus was titered on 293T cells. Murine ES cells were transfected with LV-pUb-fluc-mrfp-ttk at a multiplicity of infection (MOI) of 10.
|
| Growth protocol |
ES cells were kept in an undifferentiated, pluripotent state by using 1000 IU/ml leukemia inhibitory factor (LIF; Chemicon, ESGRO, ESG1107), and grown on top of murine embryonic fibroblasts feeder layer inactivated by 10 ug/ml of mitomycin C (Sigma, St. Louis). ES cells were cultured on 0.1% gelatin-coated plastic dishes in ES medium containing Dulbecco modified Eagle medium supplemented with 15% fetal calf serum, 0.1 mM beta-mercaptoethanol, 2 mM glutamine, and 0.1 mN non-essential amino acids.
|
| Extracted molecule |
total RNA |
| Label |
Cy5
|
| Label protocol |
10 µg of total RNA were primed with 2 µl of 100 µM T16N2 DNA primer at 70°C for 10 min, then reversed transcribed at 42°C for 1 h in the presence of 400 U SuperScript II RTase (Invitrogen), and 100 µM each dATP, dTTP, dGTP, with 25 µM dCTP, 25 µM Cy5-labeled dCTP (NEN Life Science, Boston, MA), and RNase inhibitor (Invitrogen). RNA was then degraded with RNase A, and labeled cDNAs were purified using QIAquick PCR columns (Qiagen).
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| |
|
| Channel 2 |
| Source name |
Total RNA from pooled whole mouse embryos e17.5, labeled with Cyanine-3 (green).
|
| Organism |
Mus musculus |
| Characteristics |
Strain: C57BL/6
Age: e17.5 d
Tissue: whole embryo
|
| Extracted molecule |
total RNA |
| Label |
Cy3
|
| Label protocol |
10 µg of total RNA were primed with 2 µl of 100 µM T16N2 DNA primer at 70°C for 10 min, then reversed transcribed at 42°C for 1 h in the presence of 400 U SuperScript II RTase (Invitrogen), and 100 µM each dATP, dTTP, dGTP, with 25 µM dCTP, 25 µM Cy3-labeled dCTP (NEN Life Science, Boston, MA), and RNase inhibitor (Invitrogen). RNA was then degraded with RNase A, and labeled cDNAs were purified using QIAquick PCR columns (Qiagen).
|
| |
|
| |
| Hybridization protocol |
Oligoarray control targets and hybridization buffer (Agilent In Situ Hybridization Kit Plus) were added, and samples were applied to microarrays enclosed in Agilent SureHyb-enabled hybridization chambers. After hybridization, slides were washed sequentially with 6x SSC/0.005% Triton X-102 and 0.1x SSC/0.005% Triton X-102 before scanning. Slides were hybridized for 17 h at 60°C in a rotating oven, and washed.
|
| Scan protocol |
Scanned on an Agilent G2565AA scanner.
|
| Description |
Biological replicate 3 of 3. Stable triple-fusion-reporter-gene transfected embryonic stem cells, harvested after several passages.
|
| Data processing |
LOWESS normalized, background subtracted VALUE data obtained from log of processed Red signal/processed Green signal.
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| |
|
| Submission date |
Oct 10, 2005 |
| Last update date |
Oct 28, 2005 |
| Contact name |
Joshua M. Spin |
| E-mail(s) |
[email protected]
|
| Phone |
650-498-6353
|
| Organization name |
Stanford University
|
| Department |
Internal Medicine/Cardiovascular Division
|
| Lab |
Philip S. Tsao Lab
|
| Street address |
300 Pasteur Drive
|
| City |
Stanford |
| State/province |
CA |
| ZIP/Postal code |
94305 |
| Country |
USA |
| |
|
| Platform ID |
GPL1307 |
| Series (1) |
| GSE3432 |
Murine ES Cells: Control vs. Triple-Fusion Transfected |
|
| Data table header descriptions |
| ID_REF |
|
| PositionX |
Found X coordinate of feature centroid in pixels. |
| PositionY |
Found Y coordinate of feature centroid in pixels. |
| VALUE |
log(REDsignal/GREENsignal) per feature (processed signals used). |
| LogRatioError |
error of the log ratio calculated according to the error model chosen. |
| PValueLogRatio |
Significance level of the Log Ratio computed for a feature. |
| gSurrogateUsed |
The green surrogate value used. |
| rSurrogateUsed |
The red surrogate value used. |
| gIsFound |
A boolean used to flag found (strong) features.The flag is applied independently in each channel. A feature is considered found if the found spot centroid is within the bounds of the spot deviation limit with respect to corresponding nominal centroid. |
| rIsFound |
A boolean used to flag found (strong) features.The flag is applied independently in each channel. A feature is considered found if the found spot centroid is within the bounds of the spot deviation limit with respect to corresponding nominal centroid. |
| gProcessedSignal |
Dye-normalized signal after surrogate "algorithm," green "channel," used for computation of log ratio. |
| rProcessedSignal |
Dye-normalized signal after surrogate "algorithm," red "channel," used for computation of log ratio. |
| gProcessedSigError |
Standard error of propagated feature "signal," green channel. |
| rProcessedSigError |
Standard error of propagated feature "signal," red channel. |
| gNumPixOLHi |
Number of outlier pixels per feature with intensity > upper threshold set via the pixel outlier rejection method. The number is computed independently in each channel. These pixels are omitted from all subsequent calculations. |
| rNumPixOLHi |
Number of outlier pixels per feature with intensity > upper threshold set via the pixel outlier rejection method. The number is computed independently in each channel. These pixels are omitted from all subsequent calculations. |
| gNumPixOLLo |
Number of outlier pixels per feature with intensity < lower threshold set via the pixel outlier rejection method. The number is computed independently in each channel. NOTE: The pixel outlier method is the ONLY step that removes data in Feature Extraction. |
| rNumPixOLLo |
Number of outlier pixels per feature with intensity < lower threshold set via the pixel outlier rejection method. The number is computed independently in each channel. NOTE: The pixel outlier method is the ONLY step that removes data in Feature Extraction. |
| gNumPix |
Total number of pixels used to compute feature statistics; ie.total number of inlier pixels/per spot; same in both channels. |
| rNumPix |
Total number of pixels used to compute feature statistics; ie.total number of inlier pixels/per spot; same in both channels. |
| gMeanSignal |
Raw mean signal of feature in green channel (inlier pixels). |
| rMeanSignal |
Raw mean signal of feature in red channel (inlier pixels). |
| gMedianSignal |
Raw median signal of feature in green channel (inlier pixels). |
| rMedianSignal |
Raw median signal of feature in red channel (inlier pixels). |
| gPixSDev |
Standard deviation of all inlier pixels per feature; this is computed independently in each channel. |
| rPixSDev |
Standard deviation of all inlier pixels per feature; this is computed independently in each channel. |
| gBGNumPix |
Total Number of pixels used to compute Local BG statistics per spot; ie.total number of BG inlier pixels; same in both channels. |
| rBGNumPix |
Total Number of pixels used to compute Local BG statistics per spot; ie.total number of BG inlier pixels; same in both channels. |
| gBGMeanSignal |
Mean local background signal (local to corresponding feature) computed per channel (inlier pixels). |
| rBGMeanSignal |
Mean local background signal (local to corresponding feature) computed per channel (inlier pixels). |
| gBGMedianSignal |
Median local background signal (local to corresponding feature) computed per channel (inlier pixels). |
| rBGMedianSignal |
Median local background signal (local to corresponding feature) computed per channel (inlier pixels). |
| gBGPixSDev |
Standard deviation of all inlier pixels per Local BG of each "feature," computed independently in each channel. |
| rBGPixSDev |
Standard deviation of all inlier pixels per Local BG of each "feature," computed independently in each channel. |
| gNumSatPix |
Total number of saturated pixels per "feature," computed per channel. |
| rNumSatPix |
Total number of saturated pixels per "feature," computed per channel. |
| gIsSaturated |
Boolean flag indicating if a feature is saturated or not. |
| rIsSaturated |
Boolean flag indicating if a feature is saturated or not. |
| PixCorrelation |
Ratio of estimated feature covariance in RedGreen space to product of feature Standard Deviation in Red Green space. |
| BGPixCorrelation |
Ratio of estimated background covariance in RedGreen space to product of background Standard Deviation in Red Green space. |
| gIsFeatNonUnifOL |
Boolean flag indicating if a feature is NonUniformity Outlier or not Green Channel. |
| rIsFeatNonUnifOL |
Boolean flag indicating if a feature is NonUniformity Outlier or not Red Channel. |
| gIsBGNonUnifOL |
Boolean flag indicating if background is NonUniformity Outlier or not Green Channel. |
| rIsBGNonUnifOL |
Boolean flag indicating if background is NonUniformity Outlier or not Red Channel. |
| gIsFeatPopnOL |
Boolean flag indicating if a feature is a Population Outlier or not for Green Channel. Probes with replicate features on a microarray are examined using population statistics. |
| rIsFeatPopnOL |
Boolean flag indicating if a feature is a Population Outlier or not for Red Channe. Probes with replicate features on a microarray are examined using population statistics. |
| gIsBGPopnOL |
Boolean flag indicating if background is a Population Outlier or not for Green Channel. |
| rIsBGPopnOL |
Boolean flag indicating if background is a Population Outlier or not for Red Channel. |
| IsManualFlag |
Manual Flag. |
| gBGSubSignal |
The net g signal following the subtraction of the background from the raw mean g signal. |
| rBGSubSignal |
The net r signal following the subtraction of the background from the raw mean r signal. |
| gBGSubSigError |
Propagated standard error as computed on net g background subtracted signal. |
| rBGSubSigError |
Propagated standard error as computed on net r background subtracted signal. |
| BGSubSigCorrelation |
Ratio of estimated background subtracted feature signal covariance in RG space to product of background subtracted feature Standard Deviation in RG space. |
| gIsPosAndSignif |
Boolean flag indicating if the mean signal of a feature is greater than the corresponding background (selected by user) and if this difference is significant. |
| rIsPosAndSignif |
Boolean flag indicating if the mean signal of a feature is greater than the corresponding background (selected by user) and if this difference is significant. |
| gPValFeatEqBG |
P-value from t-test of significance between g Mean signal and g background (selected by user). |
| rPValFeatEqBG |
P-value from t-test of significance between r Mean signal and r background (selected by user). |
| gNumBGUsed |
Number of local background regions or features used to calculate the background subtraction on this feature g channel. |
| rNumBGUsed |
Number of local background regions or features used to calculate the background subtraction on this feature r channel. |
| gIsWellAboveBG |
Boolean flag indicating if a feature is WellAbove Background or not. |
| rIsWellAboveBG |
Boolean flag indicating if a feature is WellAbove Background or not. |
| IsUsedBGAdjust |
A boolean used to flag features used for computation of global BG offset. |
| gBGUsed |
Background value (after global background adjust if turned ON) subtracted from the raw mean signal to generate the BG subtracted signal; this value is computed per channel. If global BG subtraction is used the column is identical for every feature in a given channel. |
| rBGUsed |
Background value (after global background adjust if turned ON) subtracted from the raw mean signal to generate the BG subtracted signal; this value is computed per channel. If global BG subtraction is used the column is identical for every feature in a given channel. |
| gBGSDUsed |
Standard deviation of background used in g channel. |
| rBGSDUsed |
Standard deviation of background used in r channel. |
| IsNormalization |
A boolean flag which indicates if a feature is used to measure dye bias. |
| gDyeNormSignal |
The dye-normalized signal in the indicated channel. |
| rDyeNormSignal |
The dye-normalized signal in the indicated channel. |
| gDyeNormError |
The standard error associated with the dye normalized signal. |
| rDyeNormError |
The standard error associated with the dye normalized signal. |
| DyeNormCorrelation |
Dye-normalized red and green pixel correlation. |
| ErrorModel |
Indicates the error model that you chose for Feature Extraction or that the software uses if you have chosen the Most Conservative option. |
