|
| Status |
Public on Jun 24, 2013 |
| Title |
DKO_Flk1_short_DexPlus_2h_rep2 |
| Sample type |
RNA |
| |
|
| Source name |
Flk1+/Ecad- mesoderm cells derived from mouse Dnmt3a-/-Dnmt3b-/- (DKO) ES cells, +Dex, 2hr, replicate 2
|
| Organism |
Mus musculus |
| Characteristics |
cell line: Line 16aabb
genotype/variation: Dnmt3a-/-Dnmt3b-/-
cell type: Flk1+/Ecad- mesoderm cells derived from embryonic stem (ES) cells
dexamethasone: yes
time: 2h
|
| Treatment protocol |
2 hours before cell collection, 100 nM dexamethasone was added to the cells. The cells were collected using 0.25% trypsin-EDTA, and single cell suspensions were stained using APC-conjugated anti-Flk1, biotinylated anti-E-cadherin (Eccd2, Takara Bio or DECMA-1, eBioscience) antibodies, and PE-conjugated streptavidin. Flk1 positive/E-cadherin negative cells were sorted by FACS-Aria (BD Biosciences).
|
| Growth protocol |
ES cells were plated onto a 10-cm dish with confluent OP9 stromal cells in the differentiation medium (alpha-MEM supplemented with 10% FCS and 50 uM 2-mercaptoethanol) for 4 days.
|
| Extracted molecule |
cytoplasmic RNA |
| Extraction protocol |
Cytoplasmic RNA was isolated by using the RNeasy Mini Kit (Qiagen) according to the manufacture's cytoplasmic RNA protocol with DNase digestion option. 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 Flk1+/Ecad- mesoderm cells derived from Dnmt3a-/-Dnmt3b-/- ES cells, with 2hr-culture in the presence of dexamethasone before sorting
'short' sample represents short time course experiment (0-3h). In this experiment, dexamethasone was added before FACS sorting.
'long' sample represents long time course experiment (0-72h). In this experiment, dexamethasone was added after FACS sorting.
|
| 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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