embryonic stem cell Strain: 129SvEv-Tac Gender: male Individual Identifier: 251279910099
Extracted molecule
total RNA
Extraction protocol
One ml of TrizolTM (Invitrogen) was added to 1 well. Total RNA was extracted using Phase lock gelTM (Eppendorf) columns according to manufacturer protocol. RNA was precipitated with isopropanol, washed with 70% ethanol and dissolved in 30 ul of DEPC dH2O.
Label
Cy3
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).
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
Label
Cy5
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).
Hybridization 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) http://www.chem.agilent.com/scripts/literaturePDF.asp?iWHID=33696
Description
This arrays was previously submitted as GSM137384 and was normalized with arrays in GSE5914. Since we are reusing it in this experiment we re-submit it as a new sample as the normalized values are different from the previous submitted values.
Data processing
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.
Identification of Pou5f1, Sox2, and Nanog downstream target genes with statistical confidence by applying a novel algor
Data table header descriptions
ID_REF
Feature Number (FeatureNum). Please check the platform file for the annotation.
VALUE
The normalized VALUE among all the arrays in the series. The experiment was done on three array platforms: We used batch normalization for 3 groups of arrays since these arrays differed in the set of oligos and in the location of spots. First we normalized each set of arrays based on UMR signal (Cy5) as follows: (1) Take values from the columns gDyeNormSignal,rDyeNormSignal in the raw files and log-transform them using: log10(max(x,1)). These values will be referenced below as Xgi and Xri where i is array number. (2) Take average of Xri for the oligo among all arrays within the same platforms: AverXr = average(Xri). (3) For each array estimate Yi = Xgi-Xri+AverXr (4) Round Yi to 4 decimal digits. This is standard protocols when one platform of arrays used in the experimenti, which can be found at http://lgsun.grc.nia.nih.gov/ANOVA/help.html. Algorithm for removing Cy5 bleaching effect: Because some arrays showed slight reduction of Cy5 signal (UMR) due to ozone bleaching we compensated for this effect as follows. First we selected 100 genes with the highest variances of Cy5 signal and with average log10-signal >2.5, and estimated the average Cy5 signal for these genes in each array which roughly represented the degree of bleaching effect. Then we used a linear regression to fit Cy5 log signal for each gene as a function of the bleaching effect (average log Cy5 signal of 100 genes described above), and the Cy3 signal in the same array. Then the effect of bleaching was subtracted. If after this correction,log Cy5 signal in array differed by > 3xSD from the mean log Cy5 signal it was replaced by the mean. Algorithm for combining with earlier data (time points > 24 hr):Data on earlier time points (3-24 hr) was combined with data on later time points (24-120 hr) that was adjusted as follows: xt = xt - x24 + y24, where xt and xt are log gene expression for time point >24hr before and after adjustment, respectively, x24 is log gene expression in the experiment, and y24 is log gene expression in the new experiment. For the majority of genes we used the same oligo sequence in both data sets that were combined. However, for some genes that had no common oligo we combined data from different oligos. The data listed contains non-redundant genes which differ either in gene symbol or in the U-cluster (NIA Mouse Gene Index, ver. mm7 http://lgsun.grc.nia.nih.gov/geneindex/mm7/). Please see the supplementary raw data files for the complete set of spots for the arrays.
