|
| Status |
Public on Sep 25, 2010 |
| Title |
Induced G1E-ER-GATA-1_ETO2 knockdown_Rep1 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Control siRNA-treated G1E-ER-GATA-1_Cy5
|
| Organism |
Mus musculus |
| Characteristics |
cell line: G1E-ER-GATA-1
cell type: Control siRNA-treated/b-estradiol-treated murine ES cells
tissue: erythroid
strain: GATA-1-null
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using TRIZOL (Invitrogen).
RNA quality was checked by Nanodrop: A260/280>1.7 and 260/230>1.6
|
| Label |
Cy5
|
| Label protocol |
1.2 ug of total RNA in a total volume of 8.3 uL was incubated with T7 promoter primer and Cy5-CTP. After denaturation at 65℃, MMLV-RT, dNTP, RNaseOUT, DTT and 5x buffer were added and incubated at 40℃ for 2h. Transcription master mix, containing 4x buffer, DTT, NTPmix, PEG, RNaseOUT, pyrophosphatase, T7 RNA polymerase and Cy5-CTP, were added and incubated 40℃ for 2h.
|
| |
|
| Channel 2 |
| Source name |
ETO2 siRNA-treated G1E-ER-GATA-1_Cy3
|
| Organism |
Mus musculus |
| Characteristics |
cell line: G1E-ER-GATA-1
cell type: ETO2 siRNA-treated/b-estradiol-treated murine ES cells
tissue: erythroid
strain: GATA-1-null
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using TRIZOL (Invitrogen).
RNA quality was checked by Nanodrop: A260/280>1.7 and 260/230>1.6
|
| Label |
Cy3
|
| Label protocol |
1.2 ug of total RNA in a total volume of 8.3 uL was incubated with T7 promoter primer and Cy3-CTP. After denaturation at 65℃, MMLV-RT, dNTP, RNaseOUT, DTT and 5x buffer were added and incubated at 40℃ for 2h. Transcription master mix, containing 4x buffer, DTT, NTPmix, PEG, RNaseOUT, pyrophosphatase, T7 RNA polymerase and Cy3-CTP, were added and incubated 40℃ for 2h.
|
| |
|
| |
| Hybridization protocol |
Cy5- and Cy3-labeled cRNAs were combined with blocking agent and fragmentation buffer and incubated 60℃ 30 min to fragment RNA, and GEx Hybridization buffer was added to stop the reaction. The mixture was hybridized to the Agilent 4 x 44K whole mouse genome microarray (G4122F) at 65℃ 17h.
|
| Scan protocol |
After washing with GE wash buffer data were collected using Agilent microarray scanner.
The settings were as follows:
Dye channel: Red & Green, Scan resolution: 5um, Scanning Mode: single pass
|
| Data processing |
VALUE was determined as log10 ratio of normalized Cy5 (red) signal to normalized Cy3 (green) signal. Normalization was based on EDGE3 software Liner&LOWNESS (Locally Weighted Liner Regression).
|
| |
|
| Submission date |
Sep 23, 2010 |
| Last update date |
Sep 24, 2010 |
| Contact name |
Tohru Fujiwara |
| E-mail(s) |
fujiwara-to@apple.email.ne.jp
|
| Phone |
+81-22-717-7165
|
| Organization name |
Tohoku University Graduate School of Medicine
|
| Department |
Hematology and Rheumatology
|
| Street address |
2-1 Seiryo-cho, Aoba-ku
|
| City |
Sendai |
| ZIP/Postal code |
980-8575 |
| Country |
Japan |
| |
|
| Platform ID |
GPL4134 |
| Series (2) |
| GSE24333 |
Expression data from ETO2 knockdown G1E-ER-GATA-1 cells |
| GSE24359 |
Expression data from ETO2/LMO2 knockdown G1E-ER-GATA-1 cells |
|
