scRNA sequencing of cells harvested from the tendon injury site after a severe burn/tenotomy injury with and without sciatic neurectomy

scRNA sequencing was performed on cells harvested from the injury site of mice that underwent a burn/tenotomy injury with sciatic neurectomy or sham neurectomy (skin incision) to study the role of nerves in the formation of heterotopic ossification and aberrant stem cell differentiation following traumatic injury. Overall design: C57B6 mice received pre-operative treatment with 0.06 mg/kg buprenorphine subcutaneously and were anesthetized with 2% isofluorane inhalation. A 10 mm longitudinal skin incision was made along the medial aspect of the left Achilles tendon. The Achilles tendon was separated from surrounding tissue and dissected at it's midsection. For the sciatic neurectomy, blunt dissection was performed through the biceps femoris to expose the sciatic nerve. The sciatic nerve was transected with reflection and suturing of the proximal nerve stump to the nearby semitendinosus muscle to prevent re-innervation. As a control, sham nerve injuries consisted of surgical exposure of the sciatic nerve without transection. Incisions were sutured closed with 5-0 Vicryl suture. Tenotomy injury site, consisting of tissue from the Achilles' tendon insertion at the gastrocnemius to the distal tendon enthesis at the calcaneus and surrounding tissue, excluding bone and skin, was harvested 7 days after injury. 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).