Single cell ATAC sequencing in Skeletal cells

To determine lineage and differentiation relationship among skeletal cells in native environment, we performed tracking of clonal and sub-clonal mitochondrial mutations across population. Specifically, we utilized PolGD257A/D257A mice with a mitochondrial DNA “mutator” phenotype that provide a rich substrate of somatic mutations to facilitate clonal lineage tracing among skeletal cells. Here, CD24+CD29+SSCs, CD29+COPs, CD24+CAPs, CD24-CD29-cells, THY+ osteolineage cells, CD200-CD105+ adipolineage cells and CD45+ hematopoietic cells were isolated from the femurs of 3-month-old PolGD257A/D257A mice, co-labelled with both FACS and CITE-seq antibodies, and were subjected to a modified single cell ATAC-seq method for single cell sequencing of mtDNA variants. Our data establish that CD24+CD29+SSCs share a clonal lineage with adipolineage and osteolineage cells, providing the first evidence of clonal multipotency in skeletal cells and thus, establishes the direct in vivo evidence for our model of skeletal cell differentiation. Overall design: Here, CD24+CD29+SSCs, CD29+COPs, CD24+CAPs, CD24-CD29-cells, THY+ osteolineage cells, CD200-CD105+ adipolineage cells and CD45+ hematopoietic cells were isolated from the femurs of 3-month old PolGD257A/D257A mouse femurs co-labelled with both FACS and CITE-seq antibodies, and were subjected to a modified single cell ATAC-seq method for single cell sequencing of mtDNA variants.