Single HSCs were allowed to expand in vivo for four months to guarantee that the transplanted cell was a stem cell. Recipient mice that were positive for reconstitution were euthanized four months after transplantation, and the blood, bone marrow, spleen and thymus were collected. All tissues were prepared for flow cytometry as described above and stained with CD45.1 (FITC, clone A20, BioLegend) and CD45.2 (APC, clone 104, BioLegend) antibodies. CD45.2+ cells were sorted from each tissue on a Synergy cell sorter (Sony Biotechnology), spun down and frozen at −80 °C. Genomic DNA was then extracted from the CD45.2+ bone-marrow cells and from a tail biopsy that had been collected at 2 weeks of age from the same mouse that provided the single HSC. Genomic DNA was extracted with the Puregene Cell and Tissue kit (Qiagen) following the manufacturer’s instructions.
