|
Status |
Public on Feb 17, 2021 |
Title |
Dnmts TKO |
Sample type |
RNA |
|
|
Source name |
mouse Dnmt1-/-Dnmt3a-/-Dnmt3b-/- ES cells
|
Organism |
Mus musculus |
Characteristics |
cell line: Line 19-1 genotype: Dnmt1-/-Dnmt3a-/-Dnmt3b-/- cell type: embryonic stem cells (ESCs)
|
Growth protocol |
ES cells were cultured in feeder-free conditions in Glasgow Minimum Essential Medium (GMEM) (WAKO) containing 10% FCS (Invitrogen), 1 mM glutamine (WAKO), nonessential amino acid (WAKO) and 0.1 mM mercaptoethanol (WAKO) and supplemented with LIF (WAKO).
|
Extracted molecule |
total RNA |
Extraction protocol |
Total RNA was isolated by TRIZOL Reagent (Invitrogen) and purified using 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).
|
|
|
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 Dnmt1-/-Dnmt3a-/-Dnmt3b-/- ES cells
|
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.
|
|
|
Submission date |
Mar 08, 2012 |
Last update date |
Feb 17, 2021 |
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) |
GSE36371 |
Expression data from mouse wild type, PRDM14-overexpressing and Dnmts KO embryonic stem cells (ESCs). |
|