Samsung Genome Institute, Samsung Medical Center, Seoul 06351, South Korea.
Department of Health Sciences and Technology, SAIHST, Sungkyunkwan University, Seoul 06351, South Korea.
Genome Res. 2018 Jan;28(1):75-87. doi: 10.1101/gr.223263.117. Epub 2017 Dec 5.
Simultaneous sequencing of the genome and transcriptome at the single-cell level is a powerful tool for characterizing genomic and transcriptomic variation and revealing correlative relationships. However, it remains technically challenging to analyze both the genome and transcriptome in the same cell. Here, we report a novel method for simultaneous isolation of genomic DNA and total RNA (SIDR) from single cells, achieving high recovery rates with minimal cross-contamination, as is crucial for accurate description and integration of the single-cell genome and transcriptome. For reliable and efficient separation of genomic DNA and total RNA from single cells, the method uses hypotonic lysis to preserve nuclear lamina integrity and subsequently captures the cell lysate using antibody-conjugated magnetic microbeads. Evaluating the performance of this method using real-time PCR demonstrated that it efficiently recovered genomic DNA and total RNA. Thorough data quality assessments showed that DNA and RNA simultaneously fractionated by the SIDR method were suitable for genome and transcriptome sequencing analysis at the single-cell level. The integration of single-cell genome and transcriptome sequencing by SIDR (SIDR-seq) showed that genetic alterations, such as copy-number and single-nucleotide variations, were more accurately captured by single-cell SIDR-seq compared with conventional single-cell RNA-seq, although copy-number variations positively correlated with the corresponding gene expression levels. These results suggest that SIDR-seq is potentially a powerful tool to reveal genetic heterogeneity and phenotypic information inferred from gene expression patterns at the single-cell level.
在单细胞水平上同时进行基因组和转录组测序是一种强大的工具，可用于描述基因组和转录组的变异，并揭示相关关系。然而，在同一细胞中同时分析基因组和转录组在技术上仍然具有挑战性。在这里，我们报告了一种从单个细胞中同时分离基因组 DNA 和总 RNA（SIDR）的新方法，该方法实现了高回收率，同时最小化了交叉污染，这对于准确描述和整合单细胞基因组和转录组至关重要。为了可靠有效地从单个细胞中分离基因组 DNA 和总 RNA，该方法使用低渗裂解来保持核层粘连蛋白的完整性，然后使用抗体偶联的磁性微珠捕获细胞裂解物。使用实时 PCR 评估该方法的性能表明，它可以有效地回收基因组 DNA 和总 RNA。彻底的数据分析质量评估表明，SIDR 方法同时分离的 DNA 和 RNA 适用于单细胞水平的基因组和转录组测序分析。通过 SIDR（SIDR-seq）整合单细胞基因组和转录组测序表明，与传统的单细胞 RNA-seq 相比，遗传改变（如拷贝数和单核苷酸变异）通过单细胞 SIDR-seq 更准确地捕获，尽管拷贝数变异与相应基因的表达水平呈正相关。这些结果表明，SIDR-seq 有可能成为一种强大的工具，用于揭示单细胞水平上从基因表达模式推断出的遗传异质性和表型信息。
