Fig. 2: Workflow for single-cell and single-nucleus analysis on human AT.

AT samples are collected from participants with informed consent, and metadata, including demographic and clinical data, are recorded. For snRNA-seq, AT can be snap frozen, allowing nuclei to be extracted from frozen samples. For scRNA-seq, fresh tissue is preferred, but fresh-frozen tissue can also be used if dissociated into single-cell suspensions before freezing. The samples are then processed to isolate cells or nuclei through enzymatic digestion or mechanical dissociation. Sample barcoding for multiplexing can be used to pool multiple samples in a single sequencing run, reducing costs and increasing throughput. Flow cytometry can be used to further sort and enrich specific cell populations before sequencing, or to remove low-quality nuclei or cells. Finally, the prepared samples can be loaded onto, for example, the 10x Chromium Controller, which uses advanced microfluidics to partition individual cells or nuclei into droplets, each containing a unique barcode for downstream sequencing and analysis. SVF, stromal vascular fraction; UMAP, uniform manifold approximation and projection.