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Single Cell Reduced Representation Bisulfite Sequencing Service

Creative Biolabs, as single-cell sequencing experts, can assist you with every step of single-cell reduced representation bisulfite sequencing. We can assist you whether you wish to start with a small test project or require routine high-throughput services.

Single Cell Reduced Representation Bisulfite Sequencing

By employing both dozens-of-cells (DC) and single-cell (SC) samples as starting materials, reduced representation bisulfite sequencing (RRBS), a highly precise method of DNA methylome profiling, was refined for examining the methylome landscape during embryonic development. There are only two copies of DNA molecules in a typical mammalian cell, and each comes from one of the two parents. When profiling a single cell's DNA methylome, it's critical to avoid DNA loss as much as possible, because all DNA pieces contain irreplaceable information.

A schematic of the single-cell RRBS (scRRBS) technique.Fig.1. A schematic of the single-cell RRBS (scRRBS) technique. (Guo, 2013)

Single Cell Reduced Representation Bisulfite Sequencing Workflow

The conventional RRBS process has five of them: (i) genomic DNA purification; (ii) restriction enzyme digestion; (iii) end repair and dA tailing; (iv) adapter ligation; and (v) bisulfite conversion. Cells are picked and lysed in 5 µl of lysis solution containing protease to release genomic DNA in the scRRBS method. The MspI digestion, end repair/dA tailing, and adapter ligation stages are completed by adding the respective reaction components progressively, with the enzymes in the processes inactivated by heating; Tango buffer is used for all of the reactions. Overnight incubation with premethylated sequencing adapters and highly concentrated T4 DNA ligase is used to ligate the DNA. The ligated DNA fragments are processed directly until bisulfite conversion, and the DNA is purified in the presence of carrier tRNA only after this step. The DNA is then PCR-amplified, with fragments between 200 and 700 bp being gel-selected and purified as the final library for sequencing.

Flowchart of the experimental procedures of the scRRBS technique.Fig.2. Flowchart of the experimental procedures of the scRRBS technique. (Guo, 2015)

Advantage of Single Cell Reduced Representation Bisulfite Sequencing

  • Applicability to subnanogram levels of DNA as starting material, down to a single cell.
  • The heterogeneity of DNA methylomes among individual cells to be studied.
  • Application of the technique to embryonic cells.

Published Data

Paper Title Smart-RRBS for single-cell methylome and transcriptome analysis
Journal Nature protocols
IF 13.491
Published 2021
Abstract They describe the protocol in depth and offer advice on how to use it. Their Smart-RRBS (reduced representation bisulfite sequencing) approach combines Smart-seq2 with RRBS, isolating mRNA from genomic DNA physically. In a typical single cell, it yields coupled epigenetic promoter and RNA-expression data for about 24% of protein-coding genes. It also works for tissue samples with hundreds of cells that have been microdissected. The methodology takes 3 days to process up to 192 samples manually, excluding flow sorting and sequencing. Basic molecular biology knowledge and laboratory equipment are required, including a UV sterilized PCR workstation, a DNA fluorometer, and a microfluidic electrophoresis system.
Methods Reduced representation bisulfite sequencing
Result The quality criteria on both axes are indicated by dashed lines on scatter plots of the two principal filters for RRBS and Smart-seq2 for 96 single cells. In the upper right quadrant, there were 84 (RRBS) and 91 passing cells (RNA-seq). The intersection of 80 cells that passed all quality checks for both assays is depicted in the Venn diagram at the bottom. The Venn diagram does not include one cell that failed both the RRBS and RNA-seq QCs.

Quality filters for Smart-RRBS data.Fig.3. Quality filters for Smart-RRBS data. (Gu, 2021)

Paper Title Identification of Smoking-Associated Differentially Methylated Regions Using Reduced Representation Bisulfite Sequencing and Cell type–Specific Enhancer Activation and Gene Expression
Journal Environmental health perspectives
IF 9.031
Published 2018
Abstract Cigarette smoke has been linked to cancer and cardiovascular disease. In illness investigations, smoking-associated differentially methylated regions (SMDMRs) have been discovered, but the causal link between changed DNA methylation and transcriptional change is unknown. Their goals were to finely resolve SM-DMRs and investigate the mechanism linking SM-DMRs to altered enhancer noncoding RNA (eRNA) and mRNA transcription in human circulating monocytes.
Methods Reduced representation bisulfite sequencing
Result Monocyte SM-DMRs were commonly found in putative gene regulation areas, according to RRBS. The most substantial monocyte DMR was found in both granulocytes and saliva DNA at a poised enhancer in the aryl-hydrocarbon receptor repressor gene (AHRR).

Genome distribution of CpGs captured by RRBS.Fig.4. Genome distribution of CpGs captured by RRBS. (Kim, 2018)

To learn more about the single-cell reduced representation bisulfite sequencing service, please feel free to contact us to confirm how we can be involved in your project.


  1. Guo, H., et al. Single-cell methylome landscapes of mouse embryonic stem cells and early embryos analyzed using reduced representation bisulfite sequencing. Genome research. 2013; 23(12): 2126-2135.
  2. Guo, H., et al. Profiling DNA methylome landscapes of mammalian cells with single-cell reduced-representation bisulfite sequencing. Nature protocols. 2015; 10(5): 645-659.
  3. Gu, H., et al. Smart-RRBS for single-cell methylome and transcriptome analysis. Nature protocols. 2021; 16(8): 4004-4030.
  4. Wan, M., et al. Identification of smoking-associated differentially methylated regions using reduced representation bisulfite sequencing and cell type–specific enhancer activation and gene expression. Environmental health perspectives. 2018; 126(4): 047015.
! ! For Research Use Only. Not for diagnostic or therapeutic purposes.