GSM7915897: Non-COVID_PAD; Homo sapiens; RNA-Seq
Anterior tibial arteries from long COVID patients were carefully dissected to remove the existing plaques and were cut into small pieces (1-3 mm3). All pieces were then incubated at 37°C with freshly prepared Liberase solution (5 mg/ml) (R&D) for 30 min. Following the incubation, the samples were vortexed, and the dissected tissues were incubated again with fresh Liberase solution for another 30 mins. The stop the incubation, endothelium media and 5% FBS was added to the digested tissues. Following digestion of human anterior tibial arteries (described above), prepared cells were resuspended in MAC sorting buffer containing 0.5% BSA as 1000 cells per μl. Following this, an estimated 10,000 cells per sample were captured as per instructions from the ChromiumTM single-cell 3' (10x Genomics) reagent kit user guide. Single cells were partitioned into microfluidic chambers using a 10x Genomics Chromium controller and then encapsulated in a gel bead containing sequencing adapters and primers, molecular barcodes, a unique molecular identifier (UMI), and oligo-dT to prime polyadenylated RNA transcripts to generate single-cell barcoded droplets (GEMs). After formation of GEMs, mRNA was reverse transcribed to barcoded cDNA libraries and resulting libraries were sequenced on an Illumina NovaSeq 6000 (Illumina).