The goal of this study was to profile single cells from the lungs of four species of mammal in as comparable a fashion as possible. We chose mice, rats, pigs, and human donors to cover the full range of generally used translational animal models. The intent was to devise a single method of single-cell dissociation and sequencing, which could be applied to tissues from each species. The following protocol and enzymatic concentrations were optimized to provide the highest cell yield (approximately 5 to 10% of cells in the lung) without sacrificing cell viability (approximately 80 to 85% viable). This was largely achieved, although tissues from the larger mammals required slightly longer enzymatic digestion times. Total in-house preparation time from tissue acquisition to library capture was between 1.5 and 2.0 hours for all samples. To increase the statistical power of the human dataset, an additional 12 batches were sequenced from cryopreserved dissociations from 11 separate individuals. These samples were dissociated via a different method by a collaborating laboratory (see the next two sections); the CCA described here allows excellent batch alignment between these two distinct approaches to dissociation and sample handling.

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