|
| Status |
Public on Jun 24, 2013 |
| Title |
DKOG4_ES_DexPlus_3h_rep2 |
| Sample type |
RNA |
| |
|
| Source name |
mouse Dnmt3a-/-Dnmt3b-/- Gata4GR (DKOG4) ES cells, +Dex, 3hr, replicate 2
|
| Organism |
Mus musculus |
| Characteristics |
cell line: Line 16G4.3
genotype/variation: Dnmt3a-/-Dnmt3b-/-, stably expressing Gata4-glucocorticoid receptor ligand binding domain fusion gene
cell type: embryonic stem (ES) cells differentiating to primitive endoderm cells
dexamethasone: yes
time: 3h
|
| Treatment protocol |
100 nM dexamethasone (Dex) was added to ES cells stably expressing Gata4GR. The cells were recovered by trypsinization after 3 hours after addition of Dex, and used for RNA preparation.
|
| Growth protocol |
ES cells were cultured in Glasgow Minimum Essential Medium (GMEM, Sigma) supplemented with 10% fetal calf serum (FCS), 0.1 mM nonessential amino acids (Invitrogen), 1 mM sodium pyruvate, 0.1 mM 2-mercaptoethanol, and 2000 U/ml leukemia inhibitory factor (LIF) on gelatinized culture dishes without feeder cells.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated by using the QIAshredder and the RNeasy Plus Micro Kit with gDNA Eliminator column (Qiagen) according to the manufacturer's protocols. RNA was quantified and quality controlled using the NanoDrop ND-1000 Spectrophotometer (Thermo) and the 2100 Bioanalyzer (Agilent).
|
| Label |
Cy3
|
| Label protocol |
Amplification and Cy3 labeling of RNA was prepared from 50 ng of RNA using the Low Input Quick Amp Labeling kit (Agilent) according to the manufacturer's instructions (G4140-90040 One-Color Microarray-Based Gene Expression Analysis Protocol Version 6.5). Purification of Cy3-labeled cRNA was performed using the RNeasy Mini Kit (Qiagen). Quantity, quality and dye incorporation of Cy3-labeled cRNA were checked using the NanoDrop ND-1000 Spectrophotometer (Thermo) and the 2100 Bioanalyzer (Agilent).
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| |
|
| Hybridization protocol |
600 ng of Cy3-labeled cRNA was hybridized in Agilent hybridization chambers for 17 hours at 65 degrees C and 10 rpm according to the manufacturer's instructions (G4140-90040 One-Color Microarray-Based Gene Expression Analysis Protocol Version 6.5).
|
| Scan protocol |
Slide scanning was performed using the Agilent DNA Microarray Scanner G2505C with 3 um resolution and 20-bit dynamic range.
|
| Description |
Gene expression data from Dnmt3a-/-Dnmt3b-/- Gata4GR ES cells diffentiating to primitive endoderm cells with 3hr-culture in the presence of dexamethasone to activate Gata4GR transgene
|
| Data processing |
The scanned images were processed with Feature Extraction Software 10.7.3.1 (Agilent) using default parameters (protocol GE1_107_Sep09 and Grid 028005_D_F_20110406) to generate background subtracted and spatially detrended green Processed Signal intensities. Normalize signal intensities were obtained by a) removal of data of control probes, b) log2 transformation, and c) quantile normalization, which was performed using the normalizeQuantiles function of the limma package of the R.
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| |
|
| Submission date |
Mar 06, 2012 |
| Last update date |
Jun 24, 2013 |
| Contact name |
Yuichi Kumaki |
| E-mail(s) |
kuma@cdb.riken.jp
|
| Organization name |
RIKEN
|
| Department |
Center for Developmental Biology
|
| Street address |
2-2-3 Minatojima-minamimachi, Chuo-ku
|
| City |
Kobe |
| ZIP/Postal code |
650-0047 |
| Country |
Japan |
| |
|
| Platform ID |
GPL13912 |
| Series (1) |
| GSE36313 |
DNA Methylation Restricts Lineage-Specific Functions of Transcription Factor Gata4 during Embryonic Stem Cell Differentiation |
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