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<p>Bone marrow cells were flushed from the femurs and tibiae of three mice and pooled. After standard erythrocyte lysis nucleated cells were incubated with normal rat serum for 15 min at 4 degrees Celsius. Subsequently cells were stained with a panel of biotinylated lineage-specific antibodies (murine progenitor enrichment cocktail, containing anti-CD5, anti-CD45R, anti-CD11b, anti-TER119, anti-Gr-1, and anti-7-4, Stem Cell Technologies, Vancouver, Canada), FITC-anti-Sca-1 and APC-anti-c-kit (Pharmingen). Cells were washed twice, and incubated for 30 minutes with streptavidin-PerCP (Pharmingen). After two washes cells were resuspended in PBS with 1% BSA, and purified using a MoFlo flow cytometer. The lineage-depleted bone marrow cell population was defined as the 5% cells showing least PerCP-fluorescence intensity. Stem cell yield across all BXD samples varied from 16,000 to 118,000 Lin-Sca-1+ c-kit+ cells. A small aliquot of each sample of purified cells was functionally tested for stem cell activity by directly depositing single cells in a cobblestone area forming cell assay. The remainder of the cells was immediately collected in RNA lysis buffer. Total RNA was isolated using StrataPrep Total RNA Microprep kit (Stratagene) as described by the manufacturer. RNA pellets were resolved in 500 microliters absolute ethanol, and sent on dry ice by courrier to GNF, La Jolla, CA.</p>
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