Isolation of Mouse Hematopoietic Stem Cells

CD150⁺CD34⁻Kit⁺Sca1⁺Lineage⁻ HSCs were isolated from mouse BM by fluorescence-activated cell sorting (FACS)^{16 (link),17 (link)}. Briefly, WBMCs were stained with APC-cKit antibody and cKit-positive cells enriched using anti-APC magnetic beads and LS columns (Miltenyi Biotec). The cKit-enriched cells were then stained with a biotin lineage antibody cocktail (biotin-CD4, -CD8, -CD45R/B220, -TER119, -Gr1, and -CD127), before being stained with anti-CD34, anti-cKit, anti-Sca1, anti-CD150, and streptavidin-APC/eFluor780 for 90 min (see Supplementary Table 1 for antibody details). Cells were purified using a FACS AriaII (BD) and BD FACS Diva 8 software by direct sorting into wells containing HSC media using PI as a live/dead stain (see Supplementary Fig. 3A for representative FACS gating).