School of Life Sciences, Biomedical Pioneering Innovation Center, Peking University, Beijing 100871, People's Republic of China.
Changping Laboratory, Beijing 102206, People's Republic of China.
Proc Natl Acad Sci U S A. 2023 Dec 5;120(49):e2310367120. doi: 10.1073/pnas.2310367120. Epub 2023 Nov 27.
Existing single-cell bisulfite-based DNA methylation analysis is limited by low DNA recovery, and the measurement of 5hmC at single-base resolution remains challenging. Here, we present a bisulfite-free single-cell whole-genome 5mC and 5hmC profiling technique, named Cabernet, which can characterize 5mC and 5hmC at single-base resolution with high genomic coverage. Cabernet utilizes Tn5 transposome for DNA fragmentation, which enables the discrimination between different alleles for measuring hemi-methylation status. Using Cabernet, we revealed the 5mC, hemi-5mC and 5hmC dynamics during early mouse embryo development, uncovering genomic regions exclusively governed by active or passive demethylation. We show that hemi-methylation status can be used to distinguish between pre- and post-replication cells, enabling more efficient cell grouping when integrated with 5mC profiles. The property of Tn5 naturally enables Cabernet to achieve high-throughput single-cell methylome profiling, where we probed mouse cortical neurons and embryonic day 7.5 (E7.5) embryos, and constructed the library for thousands of single cells at high efficiency, demonstrating its potential for analyzing complex tissues at substantially low cost. Together, we present a way of high-throughput methylome and hydroxymethylome detection at single-cell resolution, enabling efficient analysis of the epigenetic status of biological systems with complicated nature such as neurons and cancer cells.
现有的基于单碱基亚硫酸氢盐的 DNA 甲基化分析方法受到 DNA 回收率低的限制,而单碱基分辨率下 5hmC 的测量仍然具有挑战性。在这里,我们提出了一种无亚硫酸盐的单细胞全基因组 5mC 和 5hmC 分析技术,命名为 Cabernet,它可以在高基因组覆盖度下实现单碱基分辨率的 5mC 和 5hmC 特征分析。Cabernet 利用 Tn5 转座酶进行 DNA 片段化,从而能够区分不同等位基因,以测量半甲基化状态。使用 Cabernet,我们揭示了早期小鼠胚胎发育过程中 5mC、半 5mC 和 5hmC 的动态变化,揭示了仅由主动或被动去甲基化控制的基因组区域。我们表明,半甲基化状态可用于区分复制前和复制后细胞,与 5mC 图谱结合使用时,可以更有效地对细胞进行分组。Tn5 的特性自然使 Cabernet 能够实现高通量单细胞甲基组谱分析,我们对小鼠皮质神经元和胚胎发育第 7.5 天 (E7.5) 胚胎进行了探测,并以高效率为数千个单细胞构建了文库,证明了其在以低得多的成本分析复杂组织方面的潜力。总之,我们提出了一种高通量单碱基分辨率甲基组和羟甲基组检测方法,能够有效地分析具有神经元和癌细胞等复杂性质的生物系统的表观遗传状态。
Proc Natl Acad Sci U S A. 2023-12-5
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