|
| Status |
Public on Dec 11, 2013 |
| Title |
Wnt3-/-_BMP4_12h |
| Sample type |
RNA |
| |
|
| Source name |
Wnt3-/- Epiblast-like cells (12h)
|
| Organism |
Mus musculus |
| Characteristics |
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: BMP4 for 12h
|
| 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)
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| |
|
| 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 |
cytokine-stimulation by BMP4 (500ng/ml)
EA1107_07_12h_KO_BMP4
|
| 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.
|
| |
|
| Submission date |
Aug 08, 2013 |
| Last update date |
Dec 11, 2013 |
| Contact name |
Kazuki Kurimoto |
| E-mail(s) |
[email protected]
|
| Organization name |
Nara Medical University
|
| Department |
School of medicine
|
| Lab |
Department of Embryology
|
| Street address |
840 Shijo-Cho, Kashihara
|
| City |
Nara |
| ZIP/Postal code |
634-8521 |
| Country |
Japan |
| |
|
| Platform ID |
GPL1261 |
| Series (1) |
| GSE49689 |
A Mesodermal Factor, T (BRACHYURY), specifies mouse germ cell fate by directly activating germline determinants. |
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