|
| Status |
Public on Jan 28, 2008 |
| Title |
Wildtype vs DNA Methylation Deficient mESC Gene Expression |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Wildtype Mouse ES Cell
|
| Organism |
Mus musculus |
| Characteristics |
Wildtype mouse ES Cell (J1) RNA
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Trizol Extracted and cleaned with Qiagen RNeasy kit
|
| Label |
Cy5 or Cy3
|
| Label protocol |
Cy5 or Cy3 Labelled with Agilent Low Input Linear Amplification Kit
|
| |
|
| Channel 2 |
| Source name |
TKO Mouse ES Cell
|
| Organism |
Mus musculus |
| Characteristics |
Dnmt3a/3b -/-; Dnmt1 KD mouse ES Cells (Meissner et al., 2005. Nucleic Acid Research. 33:5868-77)
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Trizol Extracted and cleaned with Qiagen RNeasy kit
|
| Label |
Cy3 or Cy5
|
| Label protocol |
Cy3 or Cy5 Labelled with Agilent Low Input Linear Amplification Kit
|
| |
|
| |
| Hybridization protocol |
750ng of RNA for each channel was labelled using Agilent Low Input Linear Amplification protocol. Labelled cRNA mixed with Agilent 10x Control targets and Agilent 25x Fragmentation Buffer. This was incubated at 37C for 30 min and the reaction was stopped by the addition of 2x Agilent hybridization buffer. This mix was hybridized to arrays for 17 hours at 65C at 4rpms. Arrays were washed as per Agilent protocol and scanned immediately
|
| Scan protocol |
The arrays were scanned with Agilent DNA microarray scanner (G2565BA), and probe features were extracted using Feature Extraction Software (version 8.5)
|
| Description |
All cell cultures were maintained at 37 °C with 5% CO2. The culture of mouse ESCs were maintained in DMEM supplemented with 15% heat-inactivated FBS , LIF and penicillin/streptomycin. mESCs were grown in feeder-free for 2 passages before harvesting of RNA. RNA was isolated using Trizol.
|
| Data processing |
Arrays were processed using Agilent Feature Extraction (Version 8.5). Log 10 ratio were calculated from processed Cy5/ processed Cy3 signals. Replicates 1 and 3 were labeled - J1 with Cy5 and TKO with Cy3. Replicate 2 was labeled J1 with Cy3 and TKO with Cy5.
|
| |
|
| Submission date |
Sep 26, 2007 |
| Last update date |
Aug 14, 2011 |
| Contact name |
Yin Shen |
| E-mail(s) |
yshen@mednet.ucla.edu
|
| Organization name |
UCLA
|
| Street address |
695 Charles E. Young Dr.
|
| City |
Los Angeles |
| State/province |
CA |
| ZIP/Postal code |
90095 |
| Country |
USA |
| |
|
| Platform ID |
GPL2872 |
| Series (1) |
| GSE9172 |
Mapping Promoter CpG Methylation in mESCs |
|
