Lin- Sca1+ cKit+ GFP+ (transduced) mouse bone marrow cells were sorted using a BD FACSAria directly into RLT RNA lysis buffer with 2-ME (Qiagen). Total RNA was Dnase1-treated on Rneasy mini-columns according to manufacturer's instructions (Qiagen Rneasy mini-kit). Total RNA quantity and integrity were verified using Agilent Technologies Bioanalyzer 2100.
50ng of total RNA from each sample was amplified using the RiboAmp RNA amplification kit (Arcturus). First-round aRNA was converted into double-strand cDNA again using the Arcturus RiboAmp kit. Second-round dscDNA was then biotinylated in a second amplification using the Enzo High Yield T7 transcription kit according to the manufacturer's instructions. Labeled cRNA was purified using Qiagen Rneasy mini-columns.
Hybridization was performed according to manufacturer's (Affymetrix) instructions
Scanning was performed according to manufacturer's (Affymetrix) instructions
Primary bone marrow mononuclear cells were harvested from 5-6 week old C57BL/6 mice (Jackson Labs, Bar Harbor, ME) and subjected to immunomagnetic negative selection using the Lineage cell depletion Kit (Miltenyi Biotech, Auburn, CA) to enrich for stem and progenitor cells (Lin- cells). To further enrich the stem cell compartment, Lin- cells underwent positive selection for the expression of Sca1 (Lin-Sca1+;LS) using the Anti-Sca-1 MicroBead Kit (Miltenyi Biotech). LS cells (6x105 in 3ml) were plated in ultra-low adhesion 6-well plates (Costar, Corning, NY) and incubated overnight at 37oC, 5% CO2 in transplant media [RPMI, 20% FCS, penicillin and streptomycin, 10 ng/ml IL-3, 20 ng/ml IL-6 (R&D Systems, Minneapolis, MN), 10 ng/ml SCF (Stem Cell technologies, Vancouver, Canada)]. Twelve hours later, 6x105 cells in 2ml of fresh transplant media were added to 6-well plates (Cellstar, GBO, NC) coated with 100 mg Retronectin (Takara, Madison WI). Independent retroviral transductions were performed on three samples using control (GFP/MigR1) retrovirus. Retroviral (GFP) supernatants (2 ml), 4 µl of 40 mg/ml polybrene and 40µl 1M Hepes were added to each well and the cells centrifuged for 90 minutes at 1400 g, 37oC. Cells were subjected to a second round of transduction 24 hours later as described above. 48 hours following the second transduction, cells were collected (kept as independent samples) and stained with PE conjugated anti-cKit (BD Bioscience), APC conjugated anti-Sca1 (Ebioscience), and biotin conjugated anti-lineage cocktail (Miltenyi Biotech). PerCP Cy5.5 conjugated Streptavidin (BD Bioscience) was added to identify differentiated cells marked with biotin conjugated antibodies. 7-AAD (Molecular Probes) was used to exclude dead cells from the sort. 25,000 transduced (GFP+), live, Lin- (7-AAD/PerCP Cy5.5-), Sca1+ cKit+ (APC-PE ++) cells were sorted directly into 600ul of RLT RNA lysis buffer (Qiagen) using a BD FACSAria cell sorter (BD Bioscience).