strain background: C57BL/6 genotype/variation: Wnt3 Knockout; Wnt3 (-/-) gender: male cell type: Wnt3 (-/-) mESCs bearing the BV transgenes cell subtype: Epiblast-like cells (EpiLCs) stimulated with: none for 0h
Treatment protocol
EpiLCs derived from mESCs were cultured by plating 2 x 10^3 cells in a well of a lipidure U-bottom 96-well plate and induced into floating aggregates in GMEM supplemented with 15% Knockout Serum Replacement (KSR; Invitrogen), leukemia inhibitory factor (LIF; 1000 u/ml; Merk Millipore), 0.1 mM NEAA, 1 mM sodium pyruvate, 0.1 mM 2-mercaptoethanol, 100 U/ml penicillin, 0.1 mg/ml streptomycin, and 2 mM L-glutamine.
Growth protocol
mESCs maintained in N2B27medium supplemented with 2i (PD0325901, 0.4 uM: BioVision, Milpitas, CA; CHIR99021, 3 uM: BioVision) and LIF (1000 u/ml). They were induced into EpiLCs in a well of a 12-well plate coated with human plasma fibronectin (Merck Millipore) (16.7 ug/ml) in N2B27medium containing Activin A (20 ng/ml; Peprotech), bFGF (12 ng/ml; Invitrogen), and KSR (1%; Invitrogen). Medium was changed 24 hrs later, followed by another 24 hr-cultivation.
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated with Qianeg RNeasy Micro according to manufacture's instruction.
Label
biotin
Label protocol
cDNAs were amplified as described in Kurimot et al., 2006, Nucreic Acids Research. Biotinylated cRNA were prepared from amplified cDNA according to the standard Affymetrix protocol (Affymetrix GeneChip Expression Analysis Technical Manual, 2004, Affymetrix: Eukaryotic One-Cycle Target Labeling Assay)
Hybridization protocol
Hybridization was performed according to the standard Affymetrix protocol (Affymetrix GeneChip Expression Analysis Technical Manual, 2004, Affymetrix: Eukaryotic Target Hybridization)
Scan protocol
The microarray image data were processed with the GeneChip Scanner 3000 (Affymetrix)
Description
EA1107_02_KO-D2EpiLC
Data processing
CEL data were generated by GeneChip Operating Software and then subjected to the dCHIP software (Dec17, 2010 build). Data were normalized together with the default settings. The Model-Based Expression Indices (MBEI) were calculated using the PM/MM difference mode with log-2 transformation of signal intensity and truncation of low values to zero.
