|
| Status |
Public on Jun 27, 2013 |
| Title |
Adipocyte_PU1_rep3 |
| Sample type |
RNA |
| |
|
| Source name |
3T3-L1 adipocyte, day 10, adeno-PU1 72hrs
|
| Organism |
Mus musculus |
| Characteristics |
cell line: 3T3-L1
cell type: adipocytes
treatment: adeno-PU1
|
| Treatment protocol |
For adipocyte infections, purified adenoviruses were incubated in low-serum media (maintenance media with 0.5% FBS) containing 0.5μg/mL poly-L-lysine for two hours at 25C. Adipocytes were washed once with PBS then incubated with adenovirus (5mL of diluted virus per 10cm dish). After four hours, additional low-serum media was added (5mL per 10cm dish). After 16 hours, low serum media was replaced with growth media and cells were incubated under standard conditions until harvest (24-72 hours post-infection).
|
| Growth protocol |
3T3-L1 pre-adipocytes were maintained in DMEM (Hi glucose) + 10% FBS + 1% PennStrep. For differentiation, cells were grown to confluence and media was changed (day -2). Two days later (day 0), a DMI cocktail was added consisting of 1uM dexamethasone, 0.5mM 3-isobutyl-1-methylxanthine and 10ug/mL insulin. On day 2, media was changed to maintenance media + 10ug/mL insulin. On day 4, media was changed to maintenance media. By day 7, differentiation to mature adipocytes was considered complete and cells were infected with adenovirus.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from adipocytes using TRIzol (Invitrogen) followed by purification with the RNeasy mini kit (Qiagen).
|
| Label |
Cy3
|
| Label protocol |
One-color RNA spike-in was diluted at 1:7500 in Dilution Buffer (Agilent, Cat # 5188-5282). T7 promoter primer from Low Input Quick Amp Labeling Kit one-color (Agilent 5190-2305) was diluted at 0.8 ul of T7 primer plus 1 ul of H2O. The reaction mixture was made by combining 2 ul of diluted spike-in, 1.5 ul of RNA (100 ng/ul) and 1.8 ul of diluted T7 primer. The rest of labeling and purification were done according to the manufacturer's protocol for Cy3 one-color labeling.
|
| |
|
| Hybridization protocol |
Cy3-labeled cRNA was fragmented at 60 oC for 30 minutes with the following combination 720 ng of Cy3 cRNA, 6 ul of 10X Blocking Agent, 1.2 ul of 25X fragmentation buffer and H2O to 30 ul. The hybridization cocktail was made by mixing 25 ul of fragmented Cy3 cRNA with 25 ul of 2x GEx hybridization buffer HI-RPM. Following the manufacturer's protocol, 45 ul of the cocktail was loaded onto each array of the 8X array/slide. The assembled array was incubated at 65 oC on a rotator at 10 rpm for 17 hours. Array slides were disassembled in GE Wash Buffer 1 at room temperature (RT). They were washed in GE Wash Buffer 1 at RT for 1 minute. They were washed again in GE Wash Buffer 2 at 37 oC for 1 minute. Slides were put in slide holder for scanning.
|
| Scan protocol |
Slides were scanned using Agilent SureScan at 3 um following manufacturer's instruction. Raw data were extracted using Agilent Feature Extraction software.
|
| Description |
PU1-Ad3
|
| Data processing |
Data was quantile normalized in R with the limma package's function normalizeBetweenArray. FDRs were calculated using the SAMR package.
|
| |
|
| Submission date |
Jun 26, 2013 |
| Last update date |
Jun 27, 2013 |
| Contact name |
Joanna R DiSpirito |
| E-mail(s) |
rdo2@mail.med.upenn.edu
|
| Organization name |
University of Pennsylvania
|
| Department |
IDOM
|
| Lab |
Mitchell A. Lazar
|
| Street address |
Smilow Center for Translational Research, 3400 Civic Center Boulevard
|
| City |
Philadelphia |
| State/province |
PA |
| ZIP/Postal code |
19104 |
| Country |
USA |
| |
|
| Platform ID |
GPL10787 |
| Series (2) |
| GSE48344 |
Pruning of the adipocyte cistrome by hematopoietic master regulator PU.1 (expression) |
| GSE48345 |
Pruning of the adipocyte cistrome by hematopoietic master regulator PU.1 |
|
