Bronchopulmonary dysplasia (BPD) is the most common complication of preterm birth, affecting approximately 18,000 infants annually in the United States. BPD causes interrupted alveolarization and vascularization in the lung, resulting in life-long chronic respiratory morbidity. Currently, there is a limited understanding of the underlying molecular pathogenesis and a shortage of evidence-based treatments for BPD. One of the most relevant animal models of BPD was generated in preterm baboons through an NHLBI-funded U01 program. As one of the past investigators of this program, we have an archive of frozen and paraffin-embedded lung tissue specimens from this model. Recent advances in single-cell genomic approaches have transformed our ability to interrogate cell types and their functional states from complex tissues. We propose that optimization and application of single-nucleus RNA-sequencing (snRNA-Seq) to our rare biorepository of non-human primate (NHP) BPD lung specimens would provide a unique opportunity to gain novel insights into the molecular pathogenesis of BPD at a single-cell resolution. Towards this goal, we have established a collaboration with Dr. Alex K. Shalek (MIT, Ragon Institute, Broad Institute), an expert and innovator in single-cell genomics, and developed the current research proposal to leverage our banked NHP BPD lung tissue specimens. The specific aims of this proposal are to: 1) optimize and validate protocols for snRNA-seq of frozen NHP lung specimens, and 2) apply these protocols to generate single-nucleus transcriptomes of developing lungs from prematurely delivered NHP infants and lungs with evolving and established BPD. To accomplish these aims, our laboratories will optimize and validate a workflow for single-nucleus isolation from control and BPD lung specimens. Power analysis strategies will be applied to determine the number of nuclei to be sequenced from each sample to achieve statistical significance across different comparisons. We will apply vigorous quality controls (QCs) to ensure the utility of our single-nucleus transcriptional profiles. Validation studies will employ bulk RNA-Seq on frozen lung samples as well as RNA in situ hybridization (RNA-ISH), immunohistochemistry and immunofluorescence on FFPE lung specimens. Final protocols will be applied to a cohort of control and BPD lung tissues to generate transcriptome maps of the NHP lungs during normal development and in early- and late- stage BPD at 3 different time points, thus providing a longitudinal assessment of molecular alterations associated with BPD. Overall, these studies will allow us to leverage a unique and well-preserved archive of newborn NHP lung specimens and generate an essential resource to inform us on temporal and spatial gene regulation at a single cell resolution during both normal lung development and BPD progression.