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Smart-RRBS for single-cell methylome and transcriptome analysis
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Journal Article
Smart-RRBS for single-cell methylome and transcriptome analysis
2021
Overview
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.
This is a protocol for profiling the methylome and transcriptome of a single cell by using Smart-seq2 and reduced representation bisulfite sequencing.
Publisher
Nature Publishing Group UK,Nature Publishing Group
Subject
/ Anti-Bacterial Agents - pharmacology
/ Biomedical and Life Sciences
/ Computational Biology/Bioinformatics
/ DNA
/ DNA (Cytosine-5-)-Methyltransferases - genetics
/ DNA (Cytosine-5-)-Methyltransferases - metabolism
/ Gene Expression Regulation - drug effects
/ Gene Expression Regulation - physiology
/ Humans
/ Intracellular Signaling Peptides and Proteins
/ Methods
/ Protocol
