Single-Cell Mirror Bisulfite Sequencing – A Method for Single-Base Resolution of 5hmC at the Single-Cell Level
Principle Investigators: Xueguang Sun, PhD, & Ying Sun, PhD
The 5-hydroxymethylcytosine (5hmC) is an intermediate product of the DNA demethylation process. 5hmC has been identified as a new epigenetic player that is important in the regulation of nervous system development, as well as in cognition, memory, and other neurological functions. In various studies on neurodevelopment and neurodegenerative diseases, the levels of 5hmC could be differentially regulated during development and/or disease pathogenesis, suggesting the potentially critical roles of 5hmC in these neural developmental and disease processes.
The abundance of 5hmC is one to several orders lower than 5mC in the mammalian genome. Therefore, the detection of 5hmC is technically challenging. Although a number of methods have been developed to analyze 5hmC in bulk sample, they have never been applied to single-cell analysis due to their respective technical limits.
We are developing a novel bisulfite sequencing method to analyze 5hmC at single-cell level. This method uses single-strand extension coupled with two enzymatic treatments (β-glucosyltransferase and a CpG methylase), and bisulfite conversion to interrogate 5hmC sites, which could provide unique insights into the dynamics of DNA methylation turnover and the extent of cellular heterogeneity in this epigenetic mark.