Advantages of Single-Nucleus over Single-Cell RNA Sequencing of Adult Kidney: Rare Cell Types and Novel Cell States Revealed in Fibrosis
Advantages of Single-Nucleus over Single-Cell RNA Sequencing of Adult Kidney: Rare Cell Types and Novel Cell States Revealed in Fibrosis
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DOI：
10.1681/asn.2018090912
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发表时间：
2019-01-01
影响因子：
13.6
通讯作者：
Humphreys, Benjamin D.
中科院分区：
文献类型：
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作者：
Wu, Haojia;Kirita, Yuhei;Humphreys, Benjamin D.
背景肾脏和其他实体组织单细胞基因组研究的一个挑战是产生高质量的单细胞悬液，该悬液包含稀有或难以分离的细胞类型，并且没有RNA降解和人为转录应激反应。在成年小鼠肾脏上使用sNuc-DropSeq、DroNcseq和10 X Chromium平台进行单核RNA测序（snRNA-seq）。我们在单侧输尿管梗阻（UUO）手术后14天的小鼠纤维化肾脏中验证了snRNA-seq。我们在scRNA-seq数据集中识别了十个簇，但不存在肾小球细胞类型，其中一个簇主要由人为解离诱导的应激反应基因组成。相比之下，来自所有三个平台的snRNA-seq捕获了scRNA-seq数据集中未呈现的多种肾细胞类型，包括肾小球足细胞、系膜细胞和内皮细胞。未检测到应激反应基因。与已发表的scRNA-seq数据集相比，我们的snRNA-seq方案产生了20倍多的足细胞（分别为2.4%和0.12%）。出乎意料的是，单细胞和单核平台具有相同的基因检测灵敏度。结论snRNA-seq在成人肾脏中实现了与scRNA-seq相当的基因检测，并且具有显著的优势，包括降低解离偏倚，与冷冻样品相容，消除解离诱导的转录应激反应，并在发炎的纤维化肾脏上成功表现。
Background A challenge for single-cell genomic studies in kidney and other solid tissues is generating a high-quality single-cell suspension that contains rare or difficult-to-dissociate cell types and is free of both RNA degradation and artifactual transcriptional stress responses.Methods We compared single-cell RNA sequencing (scRNA-seq) using the DropSeq platform with single-nucleus RNA sequencing (snRNA-seq) using sNuc-DropSeq, DroNcseq, and 10X Chromium platforms on adult mouse kidney. We validated snRNA-seq on fibrotic kidney from mice 14 days after unilateral ureteral obstruction (UUO) surgery.Results A total of 11,391 transcriptomes were generated in the comparison phase. We identified ten clusters in the scRNA-seq dataset, but glomerular cell types were absent, and one cluster consisted primarily of artifactual dissociation-induced stress response genes. By contrast, snRNA-seq from all three platforms captured a diversity of kidney cell types that were not represented in the scRNA-seq dataset, including glomerular podocytes, mesangial cells, and endothelial cells. No stress response genes were detected. Our snRNA-seq protocol yielded 20-fold more podocytes compared with published scRNA-seq datasets (2.4% versus 0.12%, respectively). Unexpectedly, single-cell and single-nucleus platforms had equivalent gene detection sensitivity. For validation, analysis of frozen day 14 UUO kidney revealed rare juxtaglomerular cells, novel activated proximal tubule and fibroblast cell states, and previously unidentified tubulointerstitial signaling pathways.Conclusions snRNA-seq achieves comparable gene detection to scRNA-seq in adult kidney, and it also has substantial advantages, including reduced dissociation bias, compatibility with frozen samples, elimination of dissociation-induced transcriptional stress responses, and successful performance on inflamed fibrotic kidney.