Single-cell RNA sequencing (scRNASeq) has advanced our understanding of lung biology, but its utility is limited by the need for fresh samples, loss of cell types by death or inadequate dissociation, and transcriptional stress responses induced during tissue digestion. Single-nucleus RNA sequencing (snRNASeq) has addressed these deficiencies in other tissues, but no protocol exists for lung tissue. We present a snRNASeq protocol and compare its results with those of scRNASeq. Two nuclear suspensions were prepared in lysis buffer on ice while one cell suspension was generated using enzymatic and mechanical dissociation. Cells and nuclei were processed using the 10× Genomics platform, and sequencing data were analyzed by Seurat. A total of 16,110 single-nucleus and 11,934 single-cell transcriptomes were generated. Gene detection rates were equivalent in snRNASeq and scRNASeq (∼1,700 genes and 3,000 unique molecular identifiers per cell) when mapping intronic and exonic reads. In the combined data, 89% of epithelial cells were identified by snRNASeq versus 22.2% of immune cells. snRNASeq transcriptomes are enriched for transcription factors and signaling proteins, with reduction in mitochondrial and stress-response genes. Both techniques improved mesenchymal cell detection over previous studies. Homeostatic signaling relationships among alveolar cell types were defined by receptor–ligand mapping using snRNASeq data, revealing interplay among epithelial, mesenchymal, and capillary endothelial cells. snRNASeq can be applied to archival murine lung samples, improves dissociation bias, eliminates artifactual gene expression, and provides similar gene detection compared with scRNASeq.

单细胞 RNA 测序 (scRNASeq) 提高了我们对肺生物学的理解，但其效用受到对新鲜样本的需求、死亡或解离不充分导致细胞类型丢失以及组织消化过程中诱导的转录应激反应的限制。单核 RNA 测序 (snRNASeq) 已经解决了其他组织中的这些缺陷，但没有针对肺组织的方案。我们提出了一个 snRNASeq 协议，并将其结果与 scRNASeq 的结果进行比较。在冰上裂解缓冲液中制备两种核悬浮液，同时使用酶促和机械解离产生一种细胞悬浮液。使用10X Genomics平台处理细胞和细胞核，Seurat分析测序数据。总共生成了 16,110 个单核和 11,934 个单细胞转录组。在映射内含子和外显子读数时，snRNASeq 和 scRNASeq 的基因检测率相等（每个细胞约 1,700 个基因和 3,000 个独特的分子标识符）。在综合数据中，89% 的上皮细胞被 snRNASeq 识别，而免疫细胞为 22.2%。snRNASeq 转录组富含转录因子和信号蛋白，减少了线粒体和应激反应基因。与之前的研究相比，这两种技术都改进了间充质细胞检测。肺泡细胞类型之间的稳态信号关系通过使用 snRNASeq 数据的受体-配体作图来定义，揭示上皮细胞、间充质细胞和毛细血管内皮细胞之间的相互作用。snRNASeq 可应用于存档的鼠肺样本，改善解离偏差，消除人为基因表达，