EG grown on gelatin coated, 6-well plates, 300000 cells/well (30000 cells/cm2), in complete ES medium: DMEM, 15% FBS;1mM sodium pyruvate; 0.1 mM NEAA, 2 mM glutamate, 0.1 mM beta-mercapto ethanol,penicillin/streptomycin. Cells were incubated at 37 0C; 5% CO2. Medium was changed daily. On the day 3 Trizol was added and RNA extracted.t 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.
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 25 ul of DEPC dH2O.
Label
Cy3
Label protocol
Total RNA was labeled using Agilents Low RNA Input Fluorescent Linear Amplification kit, according to manufacturers instructions.
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.
Extracted molecule
total RNA
Extraction 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
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)
Description
Embryonic germ cells
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.
Global gene expression profiling reveals similarities and differences among mouse pluripotent stem cells
Data table header descriptions
ID_REF
Feature Number (FeatureNum)
VALUE
The experiment was done on two array platforms: NIA Mouse 44K Microarray v2.1 (GPL2552) (36 arrays, batch M1) and NIA Mouse 44K Microarray v2.2 (GPL4358) (14 arrays. batch M2). We used batch normalization for 2 groups of arrays since these arrays differed in the set of oligos and in the location of spots. Batch normalization is valid because in the 2nd set we used replications for the same cell lines in the same conditions as in the 1st set (standard LIF+ conditions). 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#arrayjoin . Then we estimated the average logintensity for each gene in all arrays for LIF+ conditions in the 1st batch (M1), and in all arrays (they were all for LIF+ conditions) in the 2nd batch (M2). The difference (M1-M2) was used as an adjustment factor which was added to the logintensity of that particular gene in the second batch. 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.
