For rodent samples, animals were selected between 7 and 9 weeks old, anesthetized with ketamine (75 mg/kg) and xylazine (5 mg/kg), and injected intraperitoneally with sodium heparin (400 U/kg). The abdomen was opened in sterile fashion, and the xiphoid process was lifted to reveal the underside of the diaphragm. The diaphragm was punctured and retracted to reveal the chest cavity. The rib cage was lifted, and an incision was made axially to reveal the trachea. The trachea was separated from the esophagus, opened using fine scissors, cannulated with a barbed fitting, and secured with 4-0 silk. The right ventricle was opened with scissors, and the pulmonary artery (PA) was cannulated with an 18-gauge feeding needle and secured with 4-0 monofilament polypropylene. An incision was made in the left ventricle, and the lungs were perfused via the PA with 100 ml of phosphate-buffered saline (PBS) containing sodium heparin (100 U/ml) and sodium nitroprusside (0.01 mg/ml) with concurrent airway ventilation. The heart-lung block was then removed from the chest to be perfused with enzyme. Pig samples were harvested from heparinized farm-raised pigs undergoing nonpulmonary terminal procedures. Samples were cut from distal regions of the lung and placed in ice-cold saline. A single lobule weighing approximately 2.5 g with associated large airway was dissected out and cannulated via a conducting airway for enzyme perfusion. Human samples were acquired from healthy tissues excised from the distal margins of lobectomies and/or tissues rejected from transplant. The human tissue pieces received were approximately 1 cm3 and did not contain airways or vessels of sufficient size to cannulate.

Dulbecco’s modified Eagle’s medium (DMEM) containing collagenase/dispase (1 mg/ml; Roche), elastase (3 U/ml; Worthington), and deoxyribonuclease (DNase) (20 U/ml; Worthington) preheated to 37°C was used to dissociate all tissues. For the rodent tissues, enzyme was instilled into the trachea 5× to 10× and also perfused through the vasculature (as the PA was still accessible). For pig tissue, enzyme was only instilled into the trachea 5× to 10×, and for human tissues, the enzyme mixture was delivered to the lung parenchyma repeatedly via a 25-gauge needle. In all cases, care was made to fully recruit and inflate the alveolar regions of the lung and to have the tissue fully saturated with enzyme. Following inflation and/or perfusion with enzyme, nonlung tissue was removed, including the heart and tracheas in the rodents. The remaining tissue and effluent were placed on a rocker at 37°C and 60 rpm until the tissue was soft enough to be dissociated, 20 min for the rodent tissues and 40 min for the large mammals. At this point, the lungs were pushed through a wire sieve using a weighing spatula until only dense connective tissue remained. The strainer was then rinsed with 20 ml of ice-cold DMEM containing 10% fetal bovine serum (FBS), 1% penicillin/streptomycin, 1% amphotericin, and 0.1% gentamicin. The tissue solution was spun down for 5 min at 300g, the supernatant was discarded, and the pellet was resuspended in red cell lysis buffer at a 1:1 ratio with pellet volume. After 60 s at room temperature, the buffer was diluted with 0.01% bovine serum albumin (BSA) in PBS (0.1 mg/ml) and spun down a second time. If necessary, this step was performed a second time. The pellet was resuspended again in 0.01% BSA in PBS and filtered through a 70-μm filter. The cells were then spun for 3 min at 300g, resuspended in 0.01% BSA in PBS, and passed twice through a 40-μm filter. The resulting cell suspension was counted, assessed for viability, and serially diluted to the desired concentration for single-cell library preparation.

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