Processing of human surgical samples for single-cell sequencing v1

Human clinical samples pose a challenge for single-cell RNA-seq (scRNA-seq) and single-nucleus ATAC-seq (snATAC-seq) analysis, due to their heterogeneity in cell type composition and degree of degradation or necrosis. Moreover, although some tissues can be sampled by surgical resection, most biospecimens are derived from core needle biopsies or fine needle aspirates, and must be allocated to pressing needs such as pathological analysis, thereby severely limiting the number of cells available for analysis. We have developed a protocol for obtaining robust, high-quality single-cell sequencing data from human biospecimens from a variety of biopsy types, enabling greater access to clinical cohorts. A critical factor is to limit ischemic time and transport to the lab to under an hour, and to restrict subsequent processing time to under 3 hours. This protocol uses a commercial tissue disruptor for dissociation. It was optimized on diverse human lung tumor samples, including pleural effusions, but is widely applicable to normal and diseased human clinical tissues from different organ sites.