Single-Cell RNA Sequencing Defines Developmental Progression and Reproductive Transitions in Pneumocystis carinii

This study presents the first single-cell RNA sequencing (scRNA-seq) atlas of Pneumocystis carinii, generated from isolated organisms recovered from the bronchoalveolar lavage fluid of infected rats to map the life cycle of P. carinii. scRNA-seq profiling of wildtype P. carinii, The filtrates were centrifuged at 2500 x g to pellet fungal cells, which were resuspended in 0.85% ammonium chloride solution at 37°C. for 10 minutes to lyse red blood cells. After centrifugation, the resulting pellets were maintained in RPMI 1640 medium (Fisher Scientific, Pittsburgh, PA) supplemented with 20% fetal bovine serum (Cytiva, Sweden AB), MEM Non-Essential Amino Acids (Gibco, Pittsburgh, PA), MEM Vitamin Solution (Gibco, Pittsburgh, PA), penicillin-streptomycin (10,000 µg/mL,10,000 µg/mL; Gibco, Pittsburgh, PA), and vancomycin (5 mg/mL; Fisher Scientific, Pittsburgh, PA). Following extractions, incubation at 37°C for 30 minutes in T25 tissue culture flasks facilitated host cell adherence and enabled the enzymatic degradation of extracellular DNA using DNase I (Thermo Fisher Scientific, Waltham, MA). DNase I was applied at a concentration of 100 U/mL for effective DNA removal in cell culture systems. P. carinii organisms were collected from the supernatant, followed by centrifugation at 2500 x g to pellet and resuspended in 1 mL of 2% Ficoll-Hypaque before placement on a Ficoll gradient (38). We used a Ficoll-Hypaque gradient (Ficoll: Millipore, Billerica, MD; Hypaque: Sigma, St. Louis, MO). The gradient was prepared with Ficoll concentrations diluted in 16% sodium diatrizoate, ranging from 2% to 12% increments. Following centrifugation, each layer was collected, and the cells were rinsed with cold Dulbecco’s Phosphate-Buffered Saline (DPBS; Fisher Scientific, Pittsburgh, PA) and pelleted by centrifugation at 2500 × g. To minimize cell clumping, pellets were gently resuspended using a 20-gauge ball-tip gavage needle (Becton Dickinson & Co., Franklin Lakes, NJ) before final centrifugation at 2500 x g. The resulting samples were reconstituted in Mg²⁺ and Ca²-free RPMI 1640 medium (Fisher Scientific, Pittsburgh, PA) supplemented as described above.