Shortcut barcoding and early pooling for scalable multiplex single-cell reduced-representation CpG methylation...

Area of Science:

• Epigenetics
• Genomics
• Molecular Biology

Background:

• DNA methylation is crucial for biological processes but challenging to analyze at the single-cell level.
• Existing technologies lack efficiency and robustness for routine single-cell DNA methylation studies.

Purpose of the Study:

• To develop a highly efficient and scalable single-cell technology for DNA methylation analysis.
• To enable robust characterization of methylation patterns and cellular heterogeneity.

Main Methods:

• Developed multiplex scalable single-cell reduced representation bisulfite sequencing (msRRBS).
• msRRBS involves pooling cell-specific barcoded DNA fragments before bisulfite conversion.
• The method avoids enzymatic modification or physical capture of DNA ends.

Main Results:

• Achieved high read mapping rates (62.5 ± 3.9%) and coverage of CpG islands (60.0 ± 1.4%) and promoters (71.6 ± 1.6%).
• Demonstrated high reproducibility in bulk cell duplicates (R = 0.97-0.99).
• Provided consistent coverage with low clean reads (1 Mb), outperforming conventional methods in cost and efficiency.

Conclusions:

• msRRBS is a unique, highly efficient approach for dissecting methylation heterogeneity.
• The method requires only 4 hours of hands-on time.
• Applicable to diverse multicellular systems, including cell lines and cancer models.