NKX2-2 based nuclei sorting on human archival pancreas enables the enrichment of islet endocrine populations for single nucleus RNA sequencing. NKX2-2 based nuclei sorting on human archival pancreas enables the enrichment of islet endocrine populations for single nucleus RNA sequencing

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 dissociation might alter gene expressions. In this work, 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 human pancreata. We innovated fluorescence-activated nuclei sorting (FANS) based on the positive signal of NKX2-2 antibody to enrich for 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. We observed comparable endocrine cellular composition and cell type signature gene expression between our snRNA-seq libraries and conventional scRNA-seq libraries generated with live cells from freshly isolated human islets. Our work fills a technological gap and helps to unleash archival pancreatic tissue for molecular profiling targeting the endocrine population. We expect that our protocol can be used to enrich nuclei for transcriptomics study from various populations in the pancreas and in different organs/tissues. Overall design: Nuclei were isolated using citric acid method from archival frozen human pancreata. Islet endocrine population was enriched by FANS based on the positive signal of NKX2-2 labelling. Resulting nuclei were processed using 10x Genomics Chromium Next GEM Single Cell 3’ Reagent Kits v 3.1.

当前用于解析人类胰腺内分泌细胞单细胞转录组的研究方法，几乎完全依赖新鲜分离的胰岛。然而，人类胰岛的可获取性极为有限。
此外，胰岛分离及后续单细胞解离过程中的繁复操作步骤，可能会改变基因的表达模式。
本研究交叉对比了五种细胞核分离方案，最终选定柠檬酸法作为从人类胰腺中分离细胞核的最优策略，所获细胞核具备高RNA完整性与低细胞质污染水平。
我们基于NKX2-2抗体的阳性信号，对荧光激活细胞核分选（fluorescence-activated nuclei sorting, FANS）技术进行了创新改良，以从胰腺全细胞核池中富集内分泌细胞群体的细胞核。
本研究的样本制备流程可构建高质量的单细胞核基因表达文库，同时保留内分泌细胞群体的多样性。
我们观察到，本研究的单细胞核RNA测序（single-nucleus RNA sequencing, snRNA-seq）文库与采用新鲜分离人类胰岛活细胞构建的传统单细胞RNA测序（single-cell RNA sequencing, scRNA-seq）文库，在内分泌细胞组成及细胞类型标记基因表达上具有可比性。
本研究填补了一项技术空白，有助于解锁存档胰腺组织用于针对内分泌细胞群体的分子解析研究。
我们预期本研究的方案可用于富集胰腺及其他不同器官/组织中各类细胞群体的细胞核，以供转录组学研究使用。
实验整体设计：采用柠檬酸法从存档冷冻人类胰腺组织中分离细胞核；基于NKX2-2标记的阳性信号，通过FANS富集胰岛内分泌细胞群体的细胞核；所得细胞核使用10x Genomics Chromium Next GEM 单细胞3'试剂试剂盒v3.1进行后续处理。