Optimization and validation of single-nucleus RNA sequencing for non-human primate BPD lungs

支气管肺发育不良(BPD)是早产最常见的并发症，影响大约 在美国，每年有18,000名婴儿。BPD导致肺泡化和血管化中断 肺部疾病，导致终生慢性呼吸道疾病。目前，人们对这一问题的了解有限 BPD的潜在分子发病机制和循证治疗的缺乏。其中最 相关的BPD动物模型是通过NHLBI资助的U01计划在早产狒狒身上建立的。AS 作为该项目过去的研究人员之一，我们有一份冰冻和石蜡包埋的肺组织档案 这个模型的标本。单细胞基因组方法的最新进展已经改变了我们的能力 从复杂的组织中询问细胞类型及其功能状态。我们建议优化和 单核RNA测序在我们稀有的非人灵长类生物库中的应用 (NHP)BPD肺标本将提供一个独特的机会，以获得对分子的新见解。 单细胞分辨率下BPD的发病机制。为了实现这一目标，我们与Dr。 亚历克斯·K·沙莱克(麻省理工学院、拉贡研究所、博德研究所)，单细胞基因组学专家和创新者，以及 开发了当前的研究方案，以利用我们储存的NHP BPD肺组织样本。具体的 该建议的目的是：1)优化和验证冰冻NHP肺标本的SnRNA-SEQ方法， 以及2)应用这些方案来产生早熟发育的肺的单核转录本 分娩的NHP婴儿和肺部有BPD的发展和确定。为了实现这些目标，我们的实验室 将优化和验证从对照和BPD肺样本中分离单核的工作流程。电源 将应用分析策略来确定每个样本中待测序的核的数量 在不同的比较中实现统计意义。我们将应用严格的质量控制(QC)来 确保我们的单核转录图谱的实用性。验证研究将使用批量RNA-Seq 冰冻肺标本以及RNA原位杂交(RNA-ISH)、免疫组织化学和 FFPE肺标本的免疫荧光。最终协议将应用于控制和BPD队列 肺组织在正常发育期间和早期和晚期生成NHP肺的转录组图谱 在3个不同的时间点进行BPD阶段，从而提供相关的分子变化的纵向评估 与BPD。总体而言，这些研究将使我们能够利用独特的和保存完好的新生儿NHP档案 肺标本，并产生一个重要的资源，以告知我们的时间和空间基因调控在一个 正常肺发育和BPD进展期间的单细胞分辨率。

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.