title: GSM7908978: human non-diabetic ulcer; Homo sapiens; RNA-Seq
library_construction_protocol: Debrided diabetic foot ulcer (DFU) tissue was collected from one patient and immediately placed into a solution of Dulbecco's Modified Eagle Medium (DMEM; Sigma-Aldrich, St. Louis, MO, USA) with 10% fetal bovine serum (FBS; ThermoFisher, Waltham, MA, USA) and stored on normal ice (to keep samples at 4 °C). Then, debrided non-diabetic foot ulcer (NFU) tissue was collected from a second patient in the same fashion 1.5 h later. Samples remained on ice at the wound clinic for 1.5 to 3 h following debridement before transfer to the laboratory for processing. At the laboratory, samples were rinsed three times with 1x phosphate buffered saline (PBS; Fisher Scientific, Waltham, MA, USA) to cleanse samples of media and FBS that would quench the enzymatic digest solutions added next. Samples were cut into ~2–3 mm3 pieces with a scalpel and finely minced with fine scissors until a sludge-like consistency (<~0.1 mm3 pieces). Although samples were already small prior to mincing (<100 mm3), effort was made to ensure that a fine a consistency was reached, as finer tissue pieces have superior cellular yields. The resulting tissue was placed into a 50 mL conical tube containing 20 mL Liberase (Sigma-Aldrich) in PBS at a concentration of 0.5 mg/mL for enzymatic digest. The cell-digest suspensions were constantly agitated (rotated) for a total of 1.5 h at 37 °C. Every half hour, the sample was subjected to maximum speed on a vortex mixer (VWR) for 30 s to physically disrupt any tissue that had clumped together and thus maximize the tissue surface area exposed to enzymatic digestion at all times. The cellular and enzymatic solution was then pipetted through a 100 µm Nylon cell filter (Fisher-Scientific) into a new conical tube, and 20 mL of 10% FBS DMEM was added through the filter to quench the enzymatic reaction and release any cells trapped within the filter, maximizing downstream cell yield. Solutions were then spun at 300× g for 8 min at 4 °C in a centrifuge to pellet the cells, resuspended in 20 mL 10% FBS DMEM, and passed through a 70 µm Nylon cell filter. A 20 mL solution of 10% FBS DMEM was added through the filter to collect the remaining cells, and the solution was re-spun to obtain a final cell pellet. This pellet was resuspended to a final cellular concentration of 1200 cells/µL in 0.04% Bovine Serum Albumin (BSA; Sigma-Aldrich) in PBS in accordance with the maximum capture, concentration short of overloading, per specifications from 10X Genomics (Pleasanton, CA, USA). Single-cell RNA-seq (scRNA-seq) was performed at the Stanford Functional Genomics Facility (SFGF) for droplet-based microfluidic single cell RNA sequencing (scRNA-seq) using the 10x Chromium Single Cell platform (Single Cell 3′ v3, 10X Genomics, Pleasanton, CA, USA). A droplet of the cell suspensions, reverse transcription master mix, and partitioning oil was loaded onto a single cell chip and processed on the Chromium Controller. Reverse Transcription was performed at 53 °C for 45 min. cDNA was amplified for 12 cycles total (BioRad C1000 Touch thermocycler) with cDNA size selected using SpriSelect beads (Beckman Coulter, Indianapolis, IN, USA) and a ratio of SpriSelect reagent volume to sample volume of 0.6. cDNA was analyzed on an Agilent Bioanalyzer High Sensitivity DNA chip for qualitative control purposes. cDNA was fragmented using the proprietary fragmentation enzyme blend for 5 min at 32 °C, followed by end repair and A-tailing at 65 °C for 30 min. cDNA were double-sided size selected using SpriSelect beats. Sequencing adaptors were ligated to the cDNA at 20 °C for 15 min. cDNA was amplified using a sample-specific index oligo as primer, followed by another round of double-sided size selection using SpriSelect beads. Final libraries were analyzed on an Agilent Bioanalyzer High Sensitivity DNA chip for qualitative control purposes. Libraries were sequenced on a HiSeq 4000 Illumina platform targeting 50,000 reads per cell.