|
Status |
Public on Feb 13, 2013 |
Title |
Med12_KD_Day3 |
Sample type |
RNA |
|
|
Channel 1 |
Source name |
Med12_KD_Day3
|
Organism |
Mus musculus |
Characteristics |
cell type: V6.5 embryonic stem cells background strain: C57BL/6-129 treatment: shRNA knockdown of Med12
|
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 |
|