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

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).

本数据集通过对经Hoxa11谱系示踪的小鼠在严重烧伤/肌腱切断术损伤后，采集其肌腱损伤部位的细胞开展单细胞RNA测序（scRNA）与单细胞核转座酶可及性测序（snATAC），实现对严重异位骨化诱导损伤后位于肢中间节段（zeugopod）的间充质干细胞（MSCs）的分化谱系示踪。本研究同时通过肢体制动处理，探究肢体制动在异常伤口愈合过程中对间充质干细胞的影响。实验设计概述：参照已发表研究（PMID 26274052）的方法，对Hoxa11CreERt2 +/- TdTom/tom小鼠实施烧伤及肌腱切断术。将术后小鼠分为两组，一组可正常活动，另一组则对受伤后肢进行制动固定。分别于第0天（未损伤基线）、第7天正常活动组、第7天制动组及第42天正常活动组采集肌腱损伤部位组织样本。将组织样本置于含750U/mlⅠ型胶原酶与7U/ml DispaseⅡ（Gibco）的罗威尔帕克纪念研究所培养基（RPMI）中，于37℃、160rpm持续振荡条件下消化20分钟。随后采用含2%胎牛血清（FBS）的磷酸盐缓冲液（PBS）终止消化，并通过40μm无菌细胞筛过滤。将细胞用含2% FBS的PBS洗涤后进行计数，以1000~1200个细胞/微升的浓度重悬。采用台盼蓝排斥法在Countess II自动细胞计数仪（赛默飞世尔科技）上评估细胞活力，仅选取活力＞85%的样本用于单细胞RNA测序。按照10x Genomics的实验指南，取足量试剂对5000个细胞的转录组进行测序。剩余的100,000个细胞则参照10x Genomics官方网站发布的单细胞ATAC测序细胞核分离实验方案完成细胞核分离操作。