Smart-RRBS for single-cell methylome and transcriptome analysis

Nat Protoc. 2021 Aug;16(8):4004-4030. doi: 10.1038/s41596-021-00571-9. Epub 2021 Jul 9.


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

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Review

MeSH terms

  • Amino Acid Sequence
  • Anti-Bacterial Agents / pharmacology
  • DNA (Cytosine-5-)-Methyltransferases / genetics
  • DNA (Cytosine-5-)-Methyltransferases / metabolism
  • DNA / metabolism*
  • Doxycycline / pharmacology
  • Epigenome
  • Gene Expression Regulation / drug effects
  • Gene Expression Regulation / physiology
  • Humans
  • Intracellular Signaling Peptides and Proteins
  • RNA, Messenger / genetics
  • RNA, Messenger / metabolism
  • Single-Cell Analysis*
  • Transcriptome


  • Anti-Bacterial Agents
  • FILIP1L protein, human
  • Intracellular Signaling Peptides and Proteins
  • RNA, Messenger
  • DNA
  • DNA (Cytosine-5-)-Methyltransferases
  • Doxycycline