Heid Johanna, Cutler Ronald, Lee Moonsook, Vijg Jan, Maslov Alexander Y
Albert Einstein College of Medicine.
Res Sq. 2023 Aug 3:rs.3.rs-3186428. doi: 10.21203/rs.3.rs-3186428/v1.
Thus far, multiple techniques for single cell analysis have been developed, yet we lack a relatively simple tool to assess DNA and RNA from the same cell at whole-transcriptome and whole-genome depths. Here we present an updated method for physical separation of cytoplasmic RNA from the nuclei, which allows for simultaneous studies of DNA and RNA from the same single cell. The method consists of three steps - 1) immobilization of a single cell on solid substrate, 2) hypotonic lysis of immobilized single cell, and 3) separation of cytosol containing aqueous phase and immobilized nucleus. We found that DNA and RNA extracted from single cell using our approach is suitable for downstream sequencing-based applications. We demonstrated that the coverage of transcriptome and genome sequencing data obtained after DNA/RNA separation is similar to that observed without separation. We also showed that the separation procedure does not create any noticeable bias in observed mutational load or mutation spectra. Thus, our method can serve as a tool for simultaneous complex analysis of the genome and transcriptome, providing necessary information on the relationship between somatic mutations and the regulation of gene expression.
到目前为止，已经开发了多种单细胞分析技术，但我们仍然缺乏一种相对简单的工具，能够在全转录组和全基因组深度上对同一细胞的DNA和RNA进行评估。在此，我们提出一种从细胞核中物理分离细胞质RNA的更新方法，该方法能够同时对同一单细胞的DNA和RNA进行研究。该方法包括三个步骤：1）将单个细胞固定在固体基质上；2）对固定的单个细胞进行低渗裂解；3）分离含有水相的细胞质和固定的细胞核。我们发现，使用我们的方法从单细胞中提取的DNA和RNA适用于基于测序的下游应用。我们证明，DNA/RNA分离后获得的转录组和基因组测序数据的覆盖范围与未分离时观察到的相似。我们还表明，分离过程不会在观察到的突变负荷或突变谱中产生任何明显的偏差。因此，我们的方法可以作为一种同时对基因组和转录组进行复杂分析的工具，提供有关体细胞突变与基因表达调控之间关系的必要信息。
