|
| Status |
Public on Mar 30, 2012 |
| Title |
TCF7 shRNA Rep1 |
| Sample type |
RNA |
| |
|
| Source name |
murine EML Lin-SCA+CD34+ cells multipotential hematopoietic precursor cells treated with TCF7 shRNA
|
| Organism |
Mus musculus |
| Characteristics |
shRNA: TCF7
cell line: EML
cell type: murine EML Lin-SCA+CD34+ cells multipotential hematopoietic precursor cell
|
| Treatment protocol |
Total EML cells were washed twice in FACS buffer (0.5 % BSA, 1 mM EDTA, 1X PBS) and resuspended in 100 µl FACS buffer per 1x106 cells. CD34-FITC (1 µg per 1 x 106 cells; Ebiosciences) was added to the cells and incubated for 1 hour at 4 °C. Sca1-PE (0.06 µg per 1 x 106 cells; Ebiosciences) and Lineage Cocktail APC (5 µl per 1 x 106 cells; Miltenyi Biotec) were added to the cells and incubated for an additional 30 minutes. Lin-SCA+CD34+ and Lin-SCA-CD34- cells were collected using FACS Aria (Beckman). Lin-SCA+CD34+ cells (1x105) cells were transduced at an MOI=1 with a Tcf7 targeting shRNA construct TRCN0000012679 or a shRNA negative control. After a 24-hour incubation with the shRNA-containing virus, the cells were grown in EML GM for 24 hours, then cells were selected for 24 hours in puromycin (1.2 μg/ml). Cells were harvested (a total of four days after initial sort) and total RNA extracted.
|
| Growth protocol |
EML cell culture was maintained in expansion medium (IMDB, 20% heat inactivated horse serum, 100 ng/ml SCF [PeproTech])
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA from Tcf7 shRNA knockdown cells and control cells (transfected with scrambled shRNA) was purified using the RNeasy Plus kit from Qiagen.
|
| Label |
Biotin
|
| Label protocol |
Samples were labelled at the Stanford Functional Genomics Facility using standard Illumina protocols
|
| |
|
| Hybridization protocol |
Arrays were hybridized at the Stanford Functional Genomics Facility using standard Illumina protocols
|
| Scan protocol |
Arrays were scanned at the Stanford Functional Genomics Facility using standard Illumina protocols
|
| Description |
biological replicate 1
|
| Data processing |
Unbackground corrected data was exported from Beadstudio. The data was processed using the Bioconductor R lumi package. The data was subjected to background correction to make the data positive, a variance stabilizing transformation and quantile normalization.
|
| |
|
| Submission date |
Jun 17, 2011 |
| Last update date |
Mar 30, 2012 |
| Contact name |
Vince Schulz |
| E-mail(s) |
vincent.schulz@yale.edu
|
| Organization name |
Yale University
|
| Department |
Department of Pediatrics
|
| Lab |
Gallagher
|
| Street address |
333 Cedar St. LMP 4085
|
| City |
New Haven |
| State/province |
CT |
| ZIP/Postal code |
06519 |
| Country |
USA |
| |
|
| Platform ID |
GPL6887 |
| Series (1) |
| GSE30068 |
TCF7 is a key regulator of the switch of self-renewal and differentiation in a multipotential hematopoietic cell line |
|
