Single-cell profiling of ectopic velvet grafts transplanted onto non-regenerative backskin to assess whether regenerative propensity is intrinsic to reindeer velvet

We have previously described the reindeer antler velvet as a highly unique mammalian model of adult skin regeneration as wounds on backskin form a raised, contractile scar devoid of appendages or pigment, whereas identical wounds in antler velvet exhibit scar-less regeneration. To ask whether regenerative capacity is inherent to cells within the velvet (and not due to factors derived from the antler environment), we transplanted full thickness velvet skin grafts onto dorsal backskin. This scRNA-Seq sample profiles cells within the ectopic velvet graft to assess their molecular resemblance to regenerative velvet or non-regenerative dorsal backskin. Overall design: To examine if the ability to regenerate skin is intrinsic to antler velvet, we performed autologous grafting to relocate a full thickness antler velvet graft to a non-regenerative location on the animal's back. As is expected following full-thickness skin grafting, grafts were observed to slough the epidermis and reveal the unpigmented dermal tissue to be well-integrated into the graft bed. Four weeks after grafting, the animals were again anesthetized and staples/sutures removed. The graft was then harvested with an 8mm biopsy punch to remove a full thickness piece of skin. To investigate whether ectopic velvet grafts retained regenerative function, we isolated single-cells from the 8mm biopsy punch and processed according to 10X Genomics Chromium Single Cell 3' Reagent Guidelines v3 Chemistry as per the manufacturer's protocol. In brief, single cells were sorted based on forward versus side scatter gating into 0.1% BSA–PBS and partitioned into Gel Bead-In-EMulsions (GEMs) using 10x GemCodeTM Technology. Next-Generation Sequencing was performed using the Illumina NovaSeq S2 Flow cells. All raw FASTQs were aligned to the bovine (Bos taurus) reference genome generated using cellranger mkref pipeline. The resulting gene barcode matrix was processed using Seurat (v3.1.1) R toolkit.