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.
Label
biotin
Label protocol
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 protocol
Hybridization was performed according to manufacturer's (Affymetrix) instructions
Scan protocol
Scanning was performed according to manufacturer's (Affymetrix) instructions
Description
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 four samples using W692A retrovirus. Retroviral (W692A) 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).
