|
| Status |
Public on Feb 13, 2013 |
| Title |
Oct4_shutdown_Day4 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Oct4_shutdown_Day4
|
| Organism |
Mus musculus |
| Characteristics |
cell type: V6.5 embryonic stem cells
background strain: C57BL/6-129
treatment: shRNA knockdown of Oct4
|
| Growth protocol |
V6.5 (C57BL/6-129) murine ES cells were grown under typical ES conditions on irradiated mouse embryonic fibroblasts (MEFs). For location analysis, cells were grown for two passages off of MEFs, on gelatinized tissue-culture plates.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA extracted using Trizol following manufacturer's instructions
|
| Label |
Cy5
|
| Label protocol |
Cy-dye labeled cRNA samples were prepared using Agilent’s QuickAmp sample labeling kit. Input was 1 µg total RNA. Briefly, first and second strand cDNA are generated using MMLV-RT enzyme and an oligo-dT based primer. In vitro transcription is performed using T7 RNA polymerase and either cyanine 3-CTP or cyanine 5-CTP, creating a direct incorporation of dye into the cRNA.
|
| |
|
| Channel 2 |
| Source name |
Embyonic stem cells
|
| Organism |
Mus musculus |
| Characteristics |
cell type: V6.5 embryonic stem cells
background strain: C57BL/6-129
treatment: none
|
| Growth protocol |
V6.5 (C57BL/6-129) murine ES cells were grown under typical ES conditions on irradiated mouse embryonic fibroblasts (MEFs). For location analysis, cells were grown for two passages off of MEFs, on gelatinized tissue-culture plates.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA extracted using Trizol following manufacturer's instructions
|
| Label |
Cy3
|
| Label protocol |
Cy-dye labeled cRNA samples were prepared using Agilent’s QuickAmp sample labeling kit. Input was 1 µg total RNA. Briefly, first and second strand cDNA are generated using MMLV-RT enzyme and an oligo-dT based primer. In vitro transcription is performed using T7 RNA polymerase and either cyanine 3-CTP or cyanine 5-CTP, creating a direct incorporation of dye into the cRNA.
|
| |
|
| |
| Hybridization protocol |
Agilent (mouse 4x44k) expression arrays were hybridized according to our lab’s method, which differs slightly from the Agilent standard hybridization protocol. The hybridization cocktail consisted of 825 ng / 1.65 ug cy-dye labeled cRNA for each sample, Agilent hybridization blocking components, and fragmentation buffer. The hybridization cocktails were fragmented at 60°C for 30 minutes, and then Agilent 2X hybridization buffer was added to the cocktail prior to application to the array. The arrays were then hybridized for 16 hours at 60°C in an Agilent rotor oven set to maximum speed.
|
| Scan protocol |
Arrays were scanned using an Agilent scanner G2565BA and the data was extracted using Agilent’s Feature Extraction software v10.1.1.1.
|
| Data processing |
Data was extracted using Agilent’s Feature Extraction software v10.1.1.1 set to the default two-color gene expression protocol.
|
| |
|
| Submission date |
Feb 13, 2013 |
| Last update date |
Feb 13, 2013 |
| Contact name |
Richard A Young |
| E-mail(s) |
young_computation@wi.mit.edu
|
| Phone |
617-258-5219
|
| Organization name |
Whitehead Institute for Biomedical Research
|
| Lab |
Young Lab
|
| Street address |
9 Cambridge Center
|
| City |
Cambridge |
| State/province |
MA |
| ZIP/Postal code |
02142 |
| Country |
USA |
| |
|
| Platform ID |
GPL4134 |
| Series (2) |
| GSE44287 |
Master Transcription Factors and Mediator Establish Super-Enhancers at Key Cell Identity Genes [gene expression] |
| GSE44288 |
Master Transcription Factors and Mediator Establish Super-Enhancers at Key Cell Identity Genes |
|
