(K-425) Drop-BS for high-throughput profiling of single-cell DNA methylomes

Genome-wide DNA methylation profile, or DNA methylome, is a critical component of the overall epigenomic landscape that modulates gene activities and cell fate. Single-cell DNA methylomic studies offer unprecedented resolution for detecting and profiling cell subsets based on methylomic features. Despite this, current single-cell methylomic technologies rely on tube or well plate-based methods, which pose challenges in scalability for processing a substantial number of single cells. Here we demonstrate a droplet-based microfluidic technology, Drop-BS, to construct single-cell bisulfite sequencing libraries for DNA methylome profiling. With Drop-BS, we open up new possibilities for comprehensive and high-throughput DNA methylome profiling at the single-cell level.

Drop-BS takes advantage of versatility offered by droplet microfluidics for pairing single cells and barcode beads, mixing various reagents, and conducting multi-step reactions. Drop-BS involves 5 major steps and 3 microfluidic devices to generate Illumina-compatible bisulfite sequencing library from single nuclei. 1) scDNA encapsulation and fragmentation: Single nuclei and lysis buffer containing micrococcal nuclease (MNase)were encapsulated into droplets with a microfluidic device for nuclei lysis and DNA fragmentation. 2) scDNA and single barcode bead droplets pairing and fusion: We applied alternating current (AC) voltage via an adjacent salt channel to produce dielectrophoresis (DEP)-based fusion between the scDNA and single-barcode bead droplets. 3) scDNA ligation and barcoding: The fused droplets were collected into a tube and exposed to UV. The barcoded oligonucleotides were released from barcode beads due to breakage of the photocleavable linker and appended to the fragmented gDNA via ligation reaction in droplets, completing scDNA barcoding. 4) Droplet-based bisulfite conversion: We used the bisulfite droplet device to generate droplets containing barcoded DNA and bisulfite. The droplets were incubated for bisulfite conversion to achieve a conversion rate of 99.0%. The droplets were then broken to pool the DNA. 5) Random priming and indexing PCR: We conducted random priming and indexing-PCR-based amplification to generate the library for sequencing.

After characterizing the Drop-BS platform with cell lines, we applied our technology to study prefrontal cortex (PFC) samples from mice and humans. We generated 1123 single-cell mouse PFC methylome with average 23,932 unique reads per cell and an average mapping efficiency of ~58%. Average 13,492 CpGs were covered per cell and a total of 11,421,613 CpGs were covered by the merged 1123 single-cell data. We also profiled two human brain samples with averagely 35,000- 41,000 unique reads per single cell. Our Drop-BS data revealed an average global mCG/CG of 71.73% and 76.12%, mCH/CH of 1.85% and 2.71%, for mouse and human PFCs respectively. The high mCH levels in the brain samples agree with literature. We also observed evident hypomethylation regions around TSS and higher methylation level throughout the gene body than that of adjacent intergenic regions. We performed Louvain clustering study to identify different brain cell types for both mouse and human PFCs tissue.

The challenges for bisulfite sequencing library preparation in droplets lie in genomic DNA fragmentation and bisulfite conversion. In Drop-BS, we utilized MNase (Micrococcal nuclease) to randomly cut genomic DNA into fragments within a specific size range by adjusting the treatment condition. We discovered that such optimization was important for increasing DNA recovery. We exploited a strategy of bisulfite conversion in droplets that substantially improved DNA recovery. By conducting pre-bisulfite adapter/barcode tagging, we avoided the complexity of conducting post-bisulfite adapter/barcode tagging in droplets. To summarize, Drop-BS takes advantage of the rapid speed of droplet microfluidics. The technology allows producing up to 10,000 single-cell bisulfite sequencing libraries in 2 d and the droplet-based process offers a bisulfite conversion rate of 99.0%. This innovative approach optimizes the process of single-cell DNA methylome profiling, showcasing the potential of droplet microfluidics in advancing epigenomic research.