The integration of DNA methylation and transcriptional state within single cells is of broad interest. Several single-cell dual- and multi-omics approaches have been reported that enable further investigation into cellular heterogeneity, including the discovery and in-depth study of rare cell populations. Such analyses will continue to provide important mechanistic insights into the regulatory consequences of epigenetic modifications. We recently reported a new method for profiling the DNA methylome and transcriptome from the same single cells in a cancer research study. Here, we present details of the protocol and provide guidance on its utility. Our Smart-RRBS (reduced representation bisulfite sequencing) protocol combines Smart-seq2 and RRBS and entails physically separating mRNA from the genomic DNA. It generates paired epigenetic promoter and RNA-expression measurements for ~24% of protein-coding genes in a typical single cell. It also works for micro-dissected tissue samples comprising hundreds of cells. The protocol, excluding flow sorting of cells and sequencing, takes ~3 d to process up to 192 samples manually. It requires basic molecular biology expertise and laboratory equipment, including a PCR workstation with UV sterilization, a DNA fluorometer and a microfluidic electrophoresis system.

单细胞内 DNA 甲基化和转录状态的整合具有广泛的意义。已经报道了几种单细胞双组学和多组学方法，可以进一步研究细胞异质性，包括发现和深入研究稀有细胞群。此类分析将继续为表观遗传修饰的调控后果提供重要的机制见解。我们最近报道了一种在癌症研究中分析来自相同单细胞的 DNA 甲基化组和转录组的新方法。在这里，我们介绍协议的详细信息并提供有关其实用性的指导。我们的 Smart-RRBS（简化代表性亚硫酸氢盐测序）方案结合了 Smart-seq2 和 RRBS，需要将 mRNA 从基因组 DNA 中物理分离出来。它为典型单细胞中约 24% 的蛋白质编码基因生成配对的表观遗传启动子和 RNA 表达测量值。它还适用于包含数百个细胞的显微解剖组织样本。该协议不包括细胞流分选和测序，需要约 3 天才能手动处理多达 192 个样本。它需要基本的分子生物学专业知识和实验室设备，包括带紫外线消毒的 PCR 工作站、DNA 荧光计和微流体电泳系统。