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Abstract：
Single cell sequencing of human heart tissue is technically challenging and methods to cryopreserve heart tissue for obtaining single cell information have not been standardized. Studies published to date have used varying methods to preserve and process human heart tissue, and have generated interesting datasets, but development of a biobanking standard has not yet been achieved. Heart transcription patterns are known to be regionally diverse, and there are few single cell datasets for normal human heart tissue.
Using pig tissue, we developed a rigorous and reproducible method for tissue mincing and cryopreservation that allowed recovery of high quality single nuclei RNA. We subsequently tested this protocol on normal human heart tissue obtained from organ donors and were able to recover high quality nuclei for generation of single nuclei RNA-seq datasets, using a commercially available platform from 10× Genomics. We analyzed these datasets using standard software packages such as CellRanger and Seurat.
Human heart tissue preserved with our method consistently yielded nuclear RNA with RNA Integrity Numbers of greater than 8.5. We demonstrate the utility of this method for single nuclei RNA-sequencing of the normal human interventricular septum and delineating its cellular diversity. The human IVS showed unexpected diversity with detection of 23 distinct cell clusters that were subsequently categorized into different cell types. Cardiomyocytes and fibroblasts were the most commonly identified cell types and could be further subdivided into 5 different cardiomyocyte subtypes and 6 different fibroblast subtypes that differed by gene expression patterns. Ingenuity Pathway analysis of these gene expression patterns suggested functional diversity in these cell subtypes.
Here we report a simple technical method for cryopreservation and subsequent nuclear isolation of human interventricular septum tissue that can be done with common laboratory equipment. We show how this method can be used to generate single nuclei transcriptomic datasets that rival those already published by larger groups in terms of cell diversity and complexity and suggest that this simple method can provide guidance for biobanking of human myocardial tissue for complex genomic analysis.
Using pig tissue, we developed a rigorous and reproducible method for tissue mincing and cryopreservation that allowed recovery of high quality single nuclei RNA. We subsequently tested this protocol on normal human heart tissue obtained from organ donors and were able to recover high quality nuclei for generation of single nuclei RNA-seq datasets, using a commercially available platform from 10× Genomics. We analyzed these datasets using standard software packages such as CellRanger and Seurat.
Human heart tissue preserved with our method consistently yielded nuclear RNA with RNA Integrity Numbers of greater than 8.5. We demonstrate the utility of this method for single nuclei RNA-sequencing of the normal human interventricular septum and delineating its cellular diversity. The human IVS showed unexpected diversity with detection of 23 distinct cell clusters that were subsequently categorized into different cell types. Cardiomyocytes and fibroblasts were the most commonly identified cell types and could be further subdivided into 5 different cardiomyocyte subtypes and 6 different fibroblast subtypes that differed by gene expression patterns. Ingenuity Pathway analysis of these gene expression patterns suggested functional diversity in these cell subtypes.
Here we report a simple technical method for cryopreservation and subsequent nuclear isolation of human interventricular septum tissue that can be done with common laboratory equipment. We show how this method can be used to generate single nuclei transcriptomic datasets that rival those already published by larger groups in terms of cell diversity and complexity and suggest that this simple method can provide guidance for biobanking of human myocardial tissue for complex genomic analysis.
摘要：
人类心脏组织的单细胞测序在技术上具有挑战性,并且冷冻保存心脏组织以获得单细胞信息的方法尚未标准化。迄今为止发表的研究使用了不同的方法来保存和处理人类心脏组织，并生成了有趣的数据集，但是生物标记标准的开发尚未实现。众所周知，心脏转录模式具有区域多样性，而且正常人心脏组织的单细胞数据集很少。
使用猪组织，我们开发了一种严格且可重复的组织切碎和冷冻保存方法，该方法可以回收高质量的单核RNA。我们随后在从器官供体获得的正常人心脏组织上测试了这个协议，并且能够恢复高质量的细胞核以生成单核RNA-seq数据集，使用10×基因组学的商业平台。我们使用CellRanger和Seurat等标准软件包分析了这些数据集。
用我们的方法保存的人心脏组织始终产生RNA完整性数大于8.5的核RNA。我们证明了该方法可用于正常人室间隔的单核RNA测序并描绘其细胞多样性。人IVS显示出乎意料的多样性,检测到23个不同的细胞簇,随后将其分类为不同的细胞类型。心肌细胞和成纤维细胞是最常见的鉴定细胞类型，可以进一步细分为5种不同的心肌细胞亚型和6种不同的成纤维细胞亚型，它们的基因表达模式不同。这些基因表达模式的独创性途径分析表明这些细胞亚型的功能多样性。
在这里，我们报告了一种简单的技术方法，该方法可以用普通的实验室设备进行冷冻保存和随后的人室间隔组织的核分离。我们展示了如何使用这种方法来生成单核转录组数据集，这些数据集可以在细胞多样性和复杂性方面与大型群体已经发表的数据集相媲美，并建议这种简单的方法可以为复杂的基因组分析提供指导。
使用猪组织，我们开发了一种严格且可重复的组织切碎和冷冻保存方法，该方法可以回收高质量的单核RNA。我们随后在从器官供体获得的正常人心脏组织上测试了这个协议，并且能够恢复高质量的细胞核以生成单核RNA-seq数据集，使用10×基因组学的商业平台。我们使用CellRanger和Seurat等标准软件包分析了这些数据集。
用我们的方法保存的人心脏组织始终产生RNA完整性数大于8.5的核RNA。我们证明了该方法可用于正常人室间隔的单核RNA测序并描绘其细胞多样性。人IVS显示出乎意料的多样性,检测到23个不同的细胞簇,随后将其分类为不同的细胞类型。心肌细胞和成纤维细胞是最常见的鉴定细胞类型，可以进一步细分为5种不同的心肌细胞亚型和6种不同的成纤维细胞亚型，它们的基因表达模式不同。这些基因表达模式的独创性途径分析表明这些细胞亚型的功能多样性。
在这里，我们报告了一种简单的技术方法，该方法可以用普通的实验室设备进行冷冻保存和随后的人室间隔组织的核分离。我们展示了如何使用这种方法来生成单核转录组数据集，这些数据集可以在细胞多样性和复杂性方面与大型群体已经发表的数据集相媲美，并建议这种简单的方法可以为复杂的基因组分析提供指导。
