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DIRECT: Digital Microfluidics for Isolation-Free Shared Library Construction of Single-Cell DNA Methylome and Transcriptome

ABSTRACT: DIRECT: Digital Microfluidics for Isolation-Free Shared Library Construction of Single-Cell DNA Methylome and Transcriptome

PROVIDER: PRJNA1004262 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR25604920_1.fastq.gz	Fastqsanger.gz
	SRR25604920_2.fastq.gz	Fastqsanger.gz
	SRR25604921_1.fastq.gz	Fastqsanger.gz
	SRR25604921_2.fastq.gz	Fastqsanger.gz
	SRR25604922_1.fastq.gz	Fastqsanger.gz

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DIRECT: Digital Microfluidics for Isolation-Free Shared Library Construction of Single-Cell DNA Methylome and Transcriptome

Project description:Integrated single-cell transcriptome and DNA methylome profiling has provided insight into the complex regulatory networks of cells. Existing methods are based on picking a single-cell and performing library construction in a tube, which is costly and cumbersome. Here, we propose DIRECT, a digital microfluidics-based method for simultaneous analysis of the methylome and transcriptome in a single library construction. The accuracy of DIRECT is demonstrated in comparison with bulk and single-omics data, and the high CpG site coverage of DIRECT allows for precise analysis of copy number variation information, enabling expansion of single cell analysis from two- to three-omics. By applying DIRECT to monitor the dynamics of mouse embryonic stem cell differentiation, the relationship between DNA methylation and changes in gene expression during differentiation was revealed. DIRECT enables accurate, robust, and reproducible single-cell DNA methylation and gene expression co-analysis at a lower cost and with greater efficiency, broadening the application scenarios of single-cell multi-omics analysis and revealing a more comprehensive and fine-grained map of cellular regulatory landscapes.

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Project description:Single-cell DNA methylation sequencing is a powerful method for elucidating important physiological and pathological processes, identifying cell subpopulations, and constructing epigenetic regulatory networks. Existing methylome analyses typically require substantial starting materials, complex operations, and high cost and are susceptible to contamination. These problems have impeded the development of single-cell methylome technology for rare cell profiling. In this work, we report Digital Microfluidics-based single-cell Reduced Representation Bisulfite Sequencing (Digital-scRRBS), the first microfluidics-based single-cell methylome library construction platform, which is an automatic, efficient, reproducible, and reagent-economy approach to dissect the single-cell methylome. Taking advantage of our uniquely designed digital microfluidic chip, we realized efficient single-cell isolation in less than 15 seconds. Furthermore, with the advantages of a confined environment, superhydrophobic surface, and nano-scale reaction volume of our digital microfluidic chip, more amplifiable DNA is retained for library construction compared to other approaches. We have successfully constructed single-cell methylation sequencing libraries with a unique genome mapping rate of up to 53.6%, covering up to 2.26 million CpG sites. The application of Digital-scRRBS allows us to discriminate cell identity and dynamically monitor DNA methylation levels during drug administration. Digital-scRRBS provides the technology for widespread application of single-cell methylation methods as a versatile tool for epigenetic analysis in rare cells and highly heterogeneous populations.

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Project description:Single-cell joint analysis of methylome and transcriptome reveals how the methylation regulates the transcriptional activity. However, traditional bench-top protocols for single-cell DNA methylation and RNA transcription co-detection are labor-intensive, cost-ineffective and contaminant-prone. Herein we establish DMF-scMT-seq, a highly-efficient and cost-effective method to simultaneously analyze single-cell DNA methylation and transcriptional activity based on digital microfluidics. DMF-scMT-seq automates the workflow of single-cell isolation, cellular hypotonic lysis, nucleic acid separation and methylome/transcriptome library construction in a contactless and addressable way. The system ensures high accuracy (R>0.85), high gene detection ability (14697 genes per cell at 4 million sequencing depth), and high CpG coverage (677198 CpG sites per cell at 1 million sequencing depth). By using DMF-scMT-seq, the relationship of DNA methylation and RNA transcription under different genomic contexts is resolved. We further apply DMF-scMT-seq to study the dynamics of transcription regulation with methylation-inhibiting anti-tumor Decitabine, and identify the methylated promoter/gene body driven genes in response to Decitabine treatment for the first time. It is anticipated that DMF-scMT-seq provides the potential to characterize the epigenetic regulatory network in other biological single-cell systems.

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