Division of Genetics and Genomics, Department of Pediatrics, Boston Children's Hospital, Boston, MA, USA.
Flow Cytometry Core Facility, Boston Children's Hospital, Boston, MA, USA.
J Mol Cell Cardiol. 2023 Jun;179:7-17. doi: 10.1016/j.yjmcc.2023.03.010. Epub 2023 Mar 26.
Single-cell approaches have become an increasingly popular way of understanding the genetic factors behind disease. Isolation of DNA and RNA from human tissues is necessary to analyze multi-omic data sets, providing information on the single-cell genome, transcriptome, and epigenome. Here, we isolated high-quality single-nuclei from postmortem human heart tissues for DNA and RNA analysis. Postmortem human tissues were obtained from 106 individuals, 33 with a history of myocardial disease, diabetes, or smoking, and 73 controls without heart disease. We demonstrated that the Qiagen EZ1 instrument and kit consistently isolated genomic DNA of high yield, which can be used for checking DNA quality before conducting single-cell experiments. Here, we provide a method for single-nuclei isolation from cardiac tissue, otherwise known as the SoNIC method, which allows for the isolation of single cardiomyocyte nuclei from postmortem tissue by nuclear ploidy status. We also provide a detailed quality control measure for single-nuclei whole genome amplification and a pre-amplification method for confirming genomic integrity.
单细胞方法已成为了解疾病背后遗传因素的一种越来越受欢迎的方式。为了分析多组学数据集，需要从人体组织中分离 DNA 和 RNA，从而提供有关单细胞基因组、转录组和表观基因组的信息。在这里，我们从人死后的心脏组织中分离出高质量的单个核细胞，用于 DNA 和 RNA 分析。从 106 个人体组织中获得了死后的人体组织，其中 33 人患有心肌疾病、糖尿病或吸烟史，73 人没有心脏病。我们证明了 Qiagen EZ1 仪器和试剂盒能够一致地分离出高产量的基因组 DNA，可用于在进行单细胞实验之前检查 DNA 质量。在这里，我们提供了一种从心脏组织中分离单个核细胞的方法，即 SoNIC 方法，该方法允许根据核倍性状态从死后组织中分离单个心肌细胞核。我们还提供了一种用于单个核细胞全基因组扩增的详细质量控制措施，以及一种用于确认基因组完整性的预扩增方法。
