PDF(Pubmed)
Abstract：
BACKGROUND: Current approaches to profile the single-cell transcriptomics of human pancreatic endocrine cells almost exclusively rely on freshly isolated islets. However, human islets are limited in availability. Furthermore, the extensive processing steps during islet isolation and subsequent single cell dissolution might alter gene expressions. In this work, we report the development of a single-nucleus RNA sequencing (snRNA-seq) approach with targeted islet cell enrichment for endocrine-population focused transcriptomic profiling using frozen archival pancreatic tissues without islet isolation.
RESULTS: We cross-compared five nuclei isolation protocols and selected the citric acid method as the best strategy to isolate nuclei with high RNA integrity and low cytoplasmic contamination from frozen archival human pancreata. We innovated fluorescence-activated nuclei sorting based on the positive signal of NKX2-2 antibody to enrich nuclei of the endocrine population from the entire nuclei pool of the pancreas. Our sample preparation procedure generated high-quality single-nucleus gene-expression libraries while preserving the endocrine population diversity. In comparison with single-cell RNA sequencing (scRNA-seq) library generated with live cells from freshly isolated human islets, the snRNA-seq library displayed comparable endocrine cellular composition and cell type signature gene expression. However, between these two types of libraries, differential enrichments of transcripts belonging to different functional classes could be observed.
CONCLUSIONS: Our work fills a technological gap and helps to unleash frozen archival pancreatic tissues for molecular profiling targeting the endocrine population. This study opens doors to retrospective mappings of endocrine cell dynamics in pancreatic tissues of complex histopathology. We expect that our protocol is applicable to enrich nuclei for transcriptomics studies from various populations in different types of frozen archival tissues.
RESULTS: We cross-compared five nuclei isolation protocols and selected the citric acid method as the best strategy to isolate nuclei with high RNA integrity and low cytoplasmic contamination from frozen archival human pancreata. We innovated fluorescence-activated nuclei sorting based on the positive signal of NKX2-2 antibody to enrich nuclei of the endocrine population from the entire nuclei pool of the pancreas. Our sample preparation procedure generated high-quality single-nucleus gene-expression libraries while preserving the endocrine population diversity. In comparison with single-cell RNA sequencing (scRNA-seq) library generated with live cells from freshly isolated human islets, the snRNA-seq library displayed comparable endocrine cellular composition and cell type signature gene expression. However, between these two types of libraries, differential enrichments of transcripts belonging to different functional classes could be observed.
CONCLUSIONS: Our work fills a technological gap and helps to unleash frozen archival pancreatic tissues for molecular profiling targeting the endocrine population. This study opens doors to retrospective mappings of endocrine cell dynamics in pancreatic tissues of complex histopathology. We expect that our protocol is applicable to enrich nuclei for transcriptomics studies from various populations in different types of frozen archival tissues.
摘要：
背景：目前人类胰腺内分泌细胞的单细胞转录组学方法几乎完全依赖于新鲜分离的胰岛。然而,人类胰岛的可用性有限。此外，胰岛分离和随后的单细胞溶解过程中的大量处理步骤可能会改变基因表达。在这项工作中,我们报道了一种单核RNA测序(snRNA-seq)方法的发展,该方法利用不经胰岛分离的冷冻存档胰腺组织进行靶向胰岛细胞富集,用于内分泌群体集中的转录组学分析.
结果：我们交叉比较了五种细胞核分离方案，并选择了柠檬酸方法作为从冷冻存档人胰腺中分离具有高RNA完整性和低细胞质污染的细胞核的最佳策略。我们基于NKX2-2抗体的阳性信号创新了荧光激活的细胞核分选，以从胰腺的整个细胞核池中富集内分泌群体的细胞核。我们的样品制备程序生成了高质量的单核基因表达文库，同时保留了内分泌种群的多样性。与使用新鲜分离的人胰岛的活细胞生成的单细胞RNA测序（scRNA-seq）文库相比，snRNA-seq文库显示了相当的内分泌细胞组成和细胞类型特征基因表达.然而,在这两种类型的库之间，可以观察到属于不同功能类别的转录本的差异富集。
结论：我们的工作填补了技术空白，并有助于释放冷冻的胰腺组织档案，用于针对内分泌人群的分子谱分析。这项研究为复杂组织病理学的胰腺组织中内分泌细胞动力学的回顾性映射打开了大门。我们希望我们的方案适用于在不同类型的冷冻档案组织中从各种人群中富集转录组学研究的细胞核。
结果：我们交叉比较了五种细胞核分离方案，并选择了柠檬酸方法作为从冷冻存档人胰腺中分离具有高RNA完整性和低细胞质污染的细胞核的最佳策略。我们基于NKX2-2抗体的阳性信号创新了荧光激活的细胞核分选，以从胰腺的整个细胞核池中富集内分泌群体的细胞核。我们的样品制备程序生成了高质量的单核基因表达文库，同时保留了内分泌种群的多样性。与使用新鲜分离的人胰岛的活细胞生成的单细胞RNA测序（scRNA-seq）文库相比，snRNA-seq文库显示了相当的内分泌细胞组成和细胞类型特征基因表达.然而,在这两种类型的库之间，可以观察到属于不同功能类别的转录本的差异富集。
结论：我们的工作填补了技术空白，并有助于释放冷冻的胰腺组织档案，用于针对内分泌人群的分子谱分析。这项研究为复杂组织病理学的胰腺组织中内分泌细胞动力学的回顾性映射打开了大门。我们希望我们的方案适用于在不同类型的冷冻档案组织中从各种人群中富集转录组学研究的细胞核。
