scRNA and snATAC sequencing of cells harvested from the tendon injury site after a severe burn/tenotomy injury
scRNA and snATAC sequencing of cells harvested from the tendon injury site after a severe burn/tenotomy injury in Hoxa11 lineage traced mice allowed for differentation tracing of MSCs located in zeugopod after severe heterotopic ossification inducing injury. The use of immobilization also allowed us to determine the effects of limb immoblization on MSCs during aberrant wound healing. Overall design: Burn and tenotomy was performed as previously described (PMID 26274052) in Hoxa11CreERt2 +/- TdTom/tom mice. Mice were allowed to ambulate normally or had their injured hindlimb immobilized.Tenotomy injury site was harvested from Day 0 (no injury baseline), Day 7 mobile (normal ambulation), Day 7 immobile, and Day 42 mobile mice. Tissue samples were digested for 20 minutes in 750U/ml Type 1 Collagenase and 7U/ml Dispase II (Gibco) in Roswell Park Memorial Institute (RPMI) medium at 37°C under constant agitation of 160rpm. Digestions were subsequently quenched with 2% FBS in PBS and filtered through 40µm sterile strainers. Cells were then washed in 2% FBS in PBS, counted and resuspended at a concentration of 1000-1200 cells/ul. Cell viability was assessed with Trypan blue exclusion on a Countess II (Thermo Fisher Scientific) automated counter and only samples with >85% viability were used for scRNA sequencing. Enough reagent was taken to sequence the transcriptome of 5000 cells according to 10x guidelines. 100,000 remaining cells were used for nuclei isolation according to the 10X Nuclei Isolation for Single Cell ATAC Sequencing protocol (available on their website).