Department of Surgery, University of Maryland School of Medicine, 670 W Baltimore Street, Baltimore, MD, 21201, USA.
Institute for Genome Sciences, University of Maryland School of Medicine, Baltimore, MD, USA.
Sci Rep. 2022 Jun 14;12(1):9851. doi: 10.1038/s41598-022-14099-9.
Single nuclei RNA sequencing (snRNA-seq) has evolved as a powerful tool to study complex human diseases. Single cell resolution enables the study of novel cell types, biological processes, cell trajectories, and cell-cell signaling pathways. snRNA-seq largely relies on the dissociation of intact nuclei from human tissues. However, the study of complex tissues using small core biopsies presents many technical challenges. Here, an optimized protocol for single nuclei isolation is presented for frozen and RNAlater preserved human kidney biopsies. The described protocol is fast, low cost, and time effective due to the elimination of cell sorting and ultra-centrifugation. Samples can be processed in 90 min or less. This method is effective for obtaining normal nuclei morphology without signs of structural damage. Using snRNA-seq, 16 distinct kidney cell clusters were recovered from normal and peri-transplant acute kidney injury allograft samples, including immune cell clusters. Quality control measurements demonstrated that these optimizations eliminated cellular debris and allowed for a high yield of high-quality nuclei and RNA for library preparation and sequencing. Cellular disassociation did not induce cellular stress responses, which recapitulated transcriptional patterns associated with standardized methods of nuclei isolation. Future applications of this protocol will allow for thorough investigations of small biobank biopsies, identifying cell-specific injury pathways and driving the discovery of novel diagnostics and therapeutic targets.
单细胞 RNA 测序 (snRNA-seq) 已发展成为研究复杂人类疾病的强大工具。单细胞分辨率使研究新的细胞类型、生物过程、细胞轨迹和细胞间信号通路成为可能。snRNA-seq 在很大程度上依赖于从人体组织中分离完整的细胞核。然而，使用小核心活检研究复杂组织存在许多技术挑战。这里提出了一种优化的用于冷冻和 RNAlater 保存的人肾活检样本的单细胞分离方案。由于消除了细胞分选和超速离心，该描述的方案快速、低成本且省时。样品可以在 90 分钟或更短的时间内处理。该方法可有效获得无结构损伤迹象的正常细胞核形态。使用 snRNA-seq，从正常和移植前急性肾损伤同种异体移植物样本中回收了 16 个不同的肾脏细胞簇，包括免疫细胞簇。质量控制测量表明，这些优化消除了细胞碎片，并允许获得高产量的高质量细胞核和 RNA 用于文库制备和测序。细胞分离不会诱导细胞应激反应，这再现了与标准化细胞核分离方法相关的转录模式。该方案的未来应用将允许对小生物库活检进行全面研究，确定细胞特异性损伤途径，并推动新的诊断和治疗靶点的发现。
