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Profiling DNA methylome landscapes of mammalian cells with single-cell reduced-representation bisulfite sequencing


The heterogeneity of DNA methylation within a population of cells necessitates DNA methylome profiling at single-cell resolution. Recently, we developed a single-cell reduced-representation bisulfite sequencing (scRRBS) technique in which we modified the original RRBS method by integrating all the experimental steps before PCR amplification into a single-tube reaction. These modifications enable scRRBS to provide digitized methylation information on 1 million CpG sites within an individual diploid mouse or human cell at single-base resolution. Compared with the single-cell bisulfite sequencing (scBS) technique, scRRBS covers fewer CpG sites, but it provides better coverage for CpG islands (CGIs), which are likely to be the most informative elements for DNA methylation. The entire procedure takes 3 weeks, and it requires strong molecular biology skills.

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Figure 1: Flowchart of the experimental procedures of the scRRBS technique.
Figure 2: The size distribution of the typical scRRBS libraries.
Figure 3: Schematic of the bioinformatic analysis procedures of the scRRBS data.
Figure 4: Box plots of the mapping efficiencies and the total unique CpG sites covered in our scRRBS data.
Figure 5: The methylation status of a representative locus of sperm-specific differentially methylated regions (DMRs).
Figure 6: The average DNA methylation levels across gene bodies and the flanking intergenic regions.


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We thank J. Qiao and L. Yan for their great help. The project was supported by the National Science Foundation of China (31322037 and 31271543) and the National Basic Research Program of China (2012CB966704 and 2011CB966303). This work is supported by a collaborative grant from the Center for Molecular and Translational Medicine.

Author information




L.W. and F.T. conceived the experiments and supervised the project. H.G., F.G., X.L., X.W. and X.F. carried out all of the experiments. P.Z. conducted the bioinformatic analyses. H.G., P.Z., L.W. and F.T. wrote the manuscript with contributions from all of the authors.

Corresponding authors

Correspondence to Lu Wen or Fuchou Tang.

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Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 The number of intersecting CpG sites and the union CpG sites of all possible combinations of all eight individual mESC scRRBS samples.

The blue fitting curve in panel a represents the average number of intersecting CpG sites. The red fitting curve in panel b represents the average number of union CpG sites. Only the CpG sites with more than 3-fold coverage were taken into consideration. The union CpG sites are the combined CpG sites, only if they are covered by at least one sample. The right y-axis indicates the proportion of all CpG sites in the mouse genome.

Supplementary Figure 2 Histogram of the distribution of the CpG sites with different methylation levels showing the digitized characteristics of DNA methylation measurements in our scRRBS dataset.

(a) Histograms of the distribution of CpG sites with different methylation levels in bulk hESC and human sperm cells. (b-c) Histograms of the distribution of CpG sites with different methylation levels in single human metaphase II oocytes (panel b) and single female pronuclei (panel c).

Supplementary Figure 3 Histogram of the distribution of CpG sites with different methylation levels showing the digitized characteristics of DNA methylation measurements in our scRRBS dataset.

(a) human sperm cells; (b) human male pronuclei.

Supplementary Figure 4 A heatmap view of a section of two chromosomes indicating the demethylation process of the parental genome after fertilization.

The color key from light blue to dark blue indicates the DNA methylation level from low to high, respectively. The white regions in the left panels indicate a lack of DNA methylation information. The red (panel a) or green (panel b) bars in the right panel represent the average DNA methylation level of the corresponding genomic region. The DNA methylation levels were calculated and presented based on 30 kb windows, only if these windows have more than 5 CpG sites covered.

Supplementary Figure 5 DNA methylation dynamics after fertilization.

The average DNA methylation levels of male and female pronuclei at different time points after intra-cytoplasmic sperm injection (ICSI), which indicates the demethylation pattern in paternal and maternal genomes after fertilization, respectively. The diamond and triangle in blue represent single sperm cells and single male pronuclei at different time points after ICSI, whereas ellipses and filled circles in red represent single metaphase II oocytes and single female pronuclei at different time points after ICSI, respectively. The gray triangle represents an outlier of the male pronuclei.

Supplementary information

Supplementary Data

Archive containing custom scripts used in this protocol. The overview of custom scripts, including the script names, functions and the corresponding protocol steps in which they should be used, is listed in Table 2. (ZIP 2 kb)

Supplementary Text and Figures

Supplementary Figures 1–5 (PDF 598 kb)

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Guo, H., Zhu, P., Guo, F. et al. Profiling DNA methylome landscapes of mammalian cells with single-cell reduced-representation bisulfite sequencing. Nat Protoc 10, 645–659 (2015).

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