|
| Status |
Public on Apr 12, 2013 |
| Title |
Pax5-2 |
| Sample type |
RNA |
| |
|
| Source name |
Pax5
|
| Organism |
Mus musculus |
| Characteristics |
cell type: Ebf1-/- pre-pro-B cell line
transduction: MigR-Pax5 virus
|
| Treatment protocol |
High titer virus was generated using a CaPO4 transfection protocol. Cultures were supplemented with fresh medium at 12h post-infection and harvested for downstream applications at indicated time points post-infection.
|
| Growth protocol |
Ebf1-/- progenitors were propagated and maintained on OP9 stromal cells in Opti-MEM, 10% FCS, primatone, L-glutamine, and Pen/Strep supplemented with 10 ng/ml each of IL-7, flt3-ligand (FL) and stem cell factor (SCF).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Target preparation and hybridization protocols were conducted as described in the Ovation Pico WTA System V2 User Guide (NuGEN) and the Affymetrix GeneChip Expression Analysis Technical Manual. Briefly, [50ng] of total RNA was converted to first-strand cDNA using reverse transcriptase primed by poly(T) and random oligomers that incorporated an RNA priming region. Second-strand cDNA synthesis was followed by ribo-SPIA linear amplification of each transcript using an isothermal reaction with RNase, RNA primer and DNA polymerase, and the resulting cDNA was fragmented, assessed by Bioanalyzer, and biotinylated by terminal transferase end labeling. cDNA yields ranged from 5-9ug
|
| Label |
biotin
|
| Label protocol |
Protocols were conducted as described in the Affymetrix GeneChip Expression Analysis Technical Manual. Quality and quantity of extracted RNA was tested on a bioanalyser by UPENN Microarray Core Facility. the resulting cDNA was fragmented, assessed by Bioanalyzer, and biotinylated by terminal transferase end labeling. cDNA yields ranged from 5-9ug
|
| |
|
| Hybridization protocol |
Biotinylated targets were heated at 99 °C for 5 min and hybridized for 16 h at 45 °C. The microarrays were then washed at low (6× SSPE) and high (100 mM MES, 0.1 M NaCl) stringency and stained with streptavidin-phycoerythrin. Fluorescence was amplified by adding biotinylated anti-streptavidin and an additional aliquot of streptavidin-phycoerythrin stain.
|
| Scan protocol |
GeneChips were scanned using the GeneArray Scanner 3000 7G.
|
| Data processing |
The data were analysed using Partek Genomics Suite, version 6.5 (Partek). Robust multichip average (RMA) with default settings was used to normalize data.
|
| |
|
| Submission date |
Apr 11, 2013 |
| Last update date |
Apr 12, 2013 |
| Contact name |
David Allman |
| E-mail(s) |
dallman@mail.med.upenn.edu
|
| Phone |
215 746-5547
|
| Organization name |
University of Pennsylvania
|
| Street address |
36th & Hamilton Walk, 230 John Morgan Building
|
| City |
Philadelphia |
| State/province |
PA |
| ZIP/Postal code |
19104-6160 |
| Country |
USA |
| |
|
| Platform ID |
GPL6246 |
| Series (1) |
| GSE46004 |
Expression data comparing EBF1 versus Pax5 induced genes |
|
