Shortcut barcoding and early pooling for scalable multiplex single-cell reduced-representation CpG methylation sequencing at single nucleotide resolution.
Liyao MaiZebin WenYulong ZhangYu GaoGuanchuan LinZhiwei LianXiang YangJingjing ZhouXianwei LinChaochao LuoWanwan PengCaiming ChenJiajia PengDuolian LiuSadie L MarjaniQian TaoYongping CuiJunxiao ZhangXuedong WuSherman M WeissmanXinghua PanPublished in: Nucleic acids research (2023)
DNA methylation is essential for a wide variety of biological processes, yet the development of a highly efficient and robust technology remains a challenge for routine single-cell analysis. We developed a multiplex scalable single-cell reduced representation bisulfite sequencing (msRRBS) technology. It allows cell-specific barcoded DNA fragments of individual cells to be pooled before bisulfite conversion, free of enzymatic modification or physical capture of the DNA ends, and achieves read mapping rates of 62.5 ± 3.9%, covering 60.0 ± 1.4% of CpG islands and 71.6 ± 1.6% of promoters in K562 cells. Its reproducibility is shown in duplicates of bulk cells with close to perfect correlation (R = 0.97-0.99). At a low 1 Mb of clean reads, msRRBS provides highly consistent coverage of CpG islands and promoters, outperforming the conventional methods with orders of magnitude reduction in cost. Here, we use this method to characterize the distinct methylation patterns and cellular heterogeneity of six cell lines, plus leukemia and hepatocellular carcinoma models. Taking 4 h of hands-on time, msRRBS offers a unique, highly efficient approach for dissecting methylation heterogeneity in a variety of multicellular systems.
Keyphrases
- single cell
- dna methylation
- highly efficient
- rna seq
- high throughput
- induced apoptosis
- genome wide
- cell cycle arrest
- single molecule
- gene expression
- physical activity
- endoplasmic reticulum stress
- signaling pathway
- randomized controlled trial
- oxidative stress
- cell death
- mesenchymal stem cells
- hydrogen peroxide
- cell therapy
- pi k akt