Smart-RRBS for single-cell methylome and transcriptome analysis

Area of Science:

• Genomics
• Epigenetics
• Single-cell biology

Background:

• Investigating cellular heterogeneity requires methods that capture multiple molecular layers within individual cells.
• Understanding the interplay between DNA methylation and gene expression is crucial for deciphering epigenetic regulation.
• Previous methods have limitations in simultaneously profiling both epigenetic modifications and transcriptional states at a single-cell level.

Purpose of the Study:

• To present a detailed protocol for Smart-RRBS, a method for dual DNA methylation and transcriptome profiling in single cells.
• To guide researchers on the utility of Smart-RRBS for cancer research and the study of cellular heterogeneity.
• To enable in-depth analysis of rare cell populations and the regulatory consequences of epigenetic modifications.

Main Methods:

• The Smart-RRBS protocol combines Smart-seq2 for RNA profiling and reduced representation bisulfite sequencing (RRBS) for DNA methylation analysis.
• It involves the physical separation of messenger RNA (mRNA) from genomic DNA within individual cells.
• The method is applicable to both isolated single cells and micro-dissected tissue samples.

Main Results:

• Smart-RRBS generates paired epigenetic promoter and RNA-expression measurements for approximately 24% of protein-coding genes per cell.
• The protocol is effective for analyzing samples ranging from single cells to hundreds of cells from micro-dissected tissues.
• Manual processing of up to 192 samples takes approximately 3 days, excluding cell sorting and sequencing.

Conclusions:

• Smart-RRBS provides a robust method for paired epigenetic and transcriptomic profiling at the single-cell level.
• This technique facilitates the study of cellular heterogeneity and the functional impact of DNA methylation.
• The protocol is accessible with basic molecular biology expertise and standard laboratory equipment.