Ammonium chloride lysis

All animal work in this study was carried out in accordance with regulations set by the United Kingdom Home Office and the Animal Scientific Procedures Act of 1986. Bone marrow was isolated from the spine, femora, tibiae, and ilia of 8 weeks and 72 weeks old C57BL/6J mice. Red blood cell depletion was performed with ammonium chloride lysis (STEMCELL Technologies), and lineage-negative cells were isolated using the EasySep Mouse Hematopoietic Progenitor Cell Isolation Kit (STEMCELL Technologies).
The lineage-depleted cells were stained with the following fluorophore-conjugated monoclonal antibodies: Cd105-PE, clone MJ7/18, Miltenyi; Cd4-Vioblue, clone REA604, Miltenyi; Cd11b-Vioblue, clone REA592, Miltenyi; Cd117-Pe Vio770, clone REA791, Miltenyi; Cd8a-Vioblue, clone 53-6.7, Miltenyi; Cd50-Vioblue, clone REA421, Miltenyi; Cd45R-Vioblue, clone REA755, Miltenyi; GR1-Vioblue, clone REA810, Miltenyi; Sca-APC, clone REA422, Miltenyi; Cd48-APC Cy7, clone HM48-1, Miltenyi; Cd150-BV510, clone TC15-12F12, Cd34-PeCy5, MEC147, Miltenyi. Approximately 10,000 LK (Lin−, Cd117+) cells per sample were sorted using the BD FACSMelody cell sorter (BD Biosciences, San Jose, California) into 1× PBS containing 4% BSA. For low-input and single-cell qPCR, a pool of 25 cells and single LSK Cd150+ Cd48− Cd34− HSCs respectively were sorted directly into Smart-seq2 lysis buffer^{51 (link)}.

The femurs, tibiae, iliac crest, humeri, and vertebrae of 6-16 weeks old mice were crushed, washed with 10 mL of PBS supplemented with 2% heat-inactivated FBS, and strained through 70-μm meshes. Cell suspension was depleted of red blood cells by ammonium chloride lysis (STEMCELL Technologies), and stained with the lineage depletion kit (19816A, STEMCELL Technologies) following the manufacturer’s instructions and passed through magnetic columns. Lineage-depleted cells were resuspended in 100 μl of PBS supplemented with 2% FCS containing the following antibodies against: c-Kit (APC-Cy7, clone 2B8, 105826, Biolegend), Sca-1 (BV421, clone D7, 108128, Biolegend), CD45 (FITC, clone 30-F11, 103108, Biolegend), Flt3 (PE, clone A2F10, 135306, Biolegend) and Il-7R⍺ (BV605, clone A7R34, 135041, Biolegend). Cells were incubated at 4°C for 30 minutes in the dark, washed, and resuspended in 100 μl of PBS supplemented with 2% FCS containing streptavidin (BV510, 405234, Biolegend). Cells were further incubated at 4°C for 15 minutes, washed and resuspended in 500 μl of PBS supplemented with 2% FCS containing 0.5 μl 7AAD (A1310, Life Technologies). Cells (lineage^- c-Kit⁺ population, and lineage^- c-Kit^lo Sca-1⁺ population) were bulk sorted using a Becton Dickinson Influx sorter.