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Single-Cell Methylation Sequencing

Epigenetic markings, such as DNA methylation, can be used to identify cell types and the regulatory elements underlying cell-specific function. In tissues containing a mixture of cell types, isolation and analysis of single cells is critical for eliminating noise from multiple epigenetic signatures. Single-cell methylation analysis also overcomes the lack of cell type-specific markers that would be needed to sort the diversity of cell types from complex tissues. Although RNA signatures can be analyzed in single cells to indicate cellular function, RNA expression does not elucidate regulatory elements that drive cellular diversity. Therefore, DNA methylation can be used as a stable indicator of cell type and epigenetic regulation.

As described in the paper by Luo et al.,Single Cell Methylomes Identify Neuronal Subtypes and Regulatory Elements in Mammalian Cortex”, Science (2017), the single nucleus methylcytosine sequencing (snmC-seq) method is a high-throughput, whole genome bisulfite sequencing approach to identify differentially methylated regions across thousands of cells to elucidate cellular diversity of complex tissues. It combines FACS-based isolation, bisulfite conversion, and Swift’s Accel-NGS® Adaptase™ Module with other commercially-available oligonucleotide primers, enzymes, and buffers (ordered separately) to construct next-generation sequencing (NGS) libraries from single neurons.

The snmC-seq method achieved a greater than two-fold increase in read mapping rate to improve data output compared to other published methods.  Additionally, it incorporates a three-dimensional indexing strategy for single-cell multiplexing during library preparation to enable 384-plex single-cell sequencing from 96 indexed libraries.

This high-throughput single-cell sequencing approach can be used for many applications, such as assessing normal tissue for regulation of cell differentiation, disease state to gain insight on epigenomic alterations, and cross-species to identify evolutionary conservation of epigenomic regulation.


Accel-NGS Adaptase Module for Single-Cell Methyl-Seq Library Preparation

The Swift advantage:
  • Provides an efficient workflow to construct NGS libraries from bisulfite-converted ssDNA from single cells.
  • Offers greater library complexity (or diversity) to retain a greater representation of the methylome, providing a two-fold increase in mapping rate over published protocols.
  • Generates library inserts greater than two-fold larger than existing post-bisulfite preparation methods to increase data output.
  • Utilizes dual indexing to enable 96-plex library sequencing of up to 384 cells.