GSM6031528: hsc_aged_4; Mus musculus; Bisulfite-Seq
To isolate purified LT-HSCs from the murine bone marrow, density gradient centrifugation was performed to enrich low-density mononuclear cell (LDMNCs), followed by lineage depletion of mature hematopoietic cell lineages using rat anti-mouse biotin-conjugated lineage antibodies and Biotin Binder Dynabeads (Life Technologies). LT-HSCs were then isolated from lineage-negative bone marrow cells by fluorescence activated cell sorting. DNA was isolated from snap-frozen cell pellets (15.000 - 20.000 sorted cells) using the QIAamp DNA Micro Kit (Qiagen) according to the manufacturer's instructions. Tagmentation-based whole-genome bisulfite sequencing (TWGBS) libraries were prepared as described previously (Wang et al., Nat. Prot., 2013). We used 10-30 ng genomic DNA as input.