Endothelial Phospholipase C gamma2 Improves Outcomes of Diabetic Ischemic Limb Rescue Following VEGF Therapy

The single cell transcriptional profile from three diabetic and five non-diabetic human skin samples was analyzed The spatial transcriptomics of 1 non-diabetic and 1 diabetic human skin were analyzed Single-cell suspensions were generated from the unwounded human skin. Briefly, the tissue was chopped into small pieces and subjected to enzymatic dissociation using whole skin dissociation kit (cat. no. 130-101-540) in MACS C Tubes (cat. no. 130-093-237) (Miltenyi Biotec Inc., San Diego, CA). The samples were incubated in a water bath at 37°C for approximately 3 hours. After incubation, the samples were diluted by adding 0.5 mL of cold cell culture medium in order to stop the enzymatic reaction. The C Tubes were then tightly closed and attached upside down onto the sleeve of the gentleMACSâ„¢ Octo dissociator and UW human skin dissociation program was performed. The cell suspension was subjected to a pre-Separation Filter, 70 µm, placed on a 15 mL tube in order to remove the debris. RBC were lysed using 10X RBC lysis buffer (cat. no. 420301, BioLegend, San Diego, CA). The resulting cell suspension was utilized for single cell RNA sequencing using the 10x Genomics platform using Chromium Next GEM Single Cell 3' GEM, Library & Gel Bead Kit v3.1(10x Genomics, Inc.) and sequenced on an Illumina NovaSeq 6000 (Illumina, Inc). 20µm thick section was taken on visium spatial gene expression slide followed by permeabilization for 12 mins. cDNA libraries were synthesized using Visium spatial gene expression reagent kit as per manufacture?s recommendations (PN-100186, 100190; 10x Genomics) and sequenced using NovaSeq 6000 (Illumina, Inc). Generated reads were pre-processed using the standard Space Ranger (https://support.10xgenomics.com/spatial-gene-expression/software) pipeline to align the raw sequencing reads onto hg38 genome and obtained the respective h5-format and spatial image files for UW human skin. Resulting pre-processed files were loaded in Seurat for further downstream data processing and analysis.