Smart-RRBS for single-cell methylome and transcriptome analysis

The integration of DNA methylation and transcriptional state within single cells is of broad interest. Several single-cell dual- and multi-omits 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 similar to 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 similar to 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.

Automated summary

The integration of DNA methylation and transcriptional state within single cells is of broad interest for studying cellular heterogeneity and rare cell populations. The Smart-RRBS protocol combines Smart-seq2 and RRBS to generate paired epigenetic promoter and RNA-expression measurements for approximately 24% of protein-coding genes in typical single cells, and can also be applied to tissue samples comprising hundreds of cells. The protocol, excluding flow sorting of cells and sequencing, takes around 3 days to process up to 192 samples manually and requires basic molecular biology expertise and laboratory equipment.