Comparison of Droplet- and Microwell-based Methods to Analyze Cryopreserved Human BAL Cells by Single-Cell RNA Sequencing

Single-cell and single-nucleus RNA sequencing (scRNA-seq) has revolutionized the exploration of tissue biology and cellular heterogeneity by delivering transcriptomic data at the individual-cell level. However, 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 BAL fluid (BALF) cells on the basis of 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 postsequencing, 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, whereas 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 analyses and found that the ability to predict the activities of key transcription factors implicated in the differentiation and identity of BALF immune cell 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.