Comparison of Droplet- and Microwell-based Methods to Analyze Cryopreserved Human Bronchoalveolar Lavage Cells by scRNA-Sequencing.

Single-cell and single-nuclei RNA sequencing (scRNA-seq) has revolutionized the exploration of tissue biology and cellular heterogeneity by delivering transcriptomic data at the individual cell level. Yet, the logistical challenge of utilizing fresh material has hindered investigations, particularly on human samples. Here, we aimed to address this limitation by implementing and comparing two cryopreservation and scRNA-seq methods for human bronchoalveolar lavage fluid (BALF) cells based on droplet and microwell entrapment. Four BALF samples were collected from routine diagnostic procedures and each sample was divided and processed using both techniques. Although the droplet-based method initially required a greater number of cells for fixation and cryopreservation, cells recovered post-sequencing and quality filtering displayed significantly higher counts of transcripts and genes per cell. This was particularly evident for alveolar macrophages, epithelial cells, mast cells and T cells, while both methodologies were similarly able to capture transcripts from neutrophils. Of note, the microwell-based approach uniquely identified fragile eosinophils. We performed single cell regulatory network inference and clustering (SCENIC) analyses and found that the ability to predict the activities of key transcription factors implicated in the differentiation and identity of BALF immune cells populations correlated with the amounts of transcripts and genes per cell. Our results can serve as a resource for the design of large-scale translational and clinical projects involving scRNA-seq analyses.