Single Cell Multi-Omics Library Construction via Nuclear Segmentation
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Solution Overview
Problem
Current single-cell multi-omics sequencing technologies face challenges in capturing comprehensive omics information due to low amounts of target substances, leading to library construction failures, low detection sensitivity, high noise, and poor repeatability, particularly in simultaneous sequencing of single-cell RNA and chromatin accessibility.
Innovation Solution
A method for constructing single-cell sequencing libraries that involves segmenting the nucleus and cytoplasm of a single cell to efficiently construct chromatin accessibility and transcriptome libraries in a microliter-scale reaction system, using Tn5 transposase for chromatin DNA library construction and reverse transcription for RNA libraries, with two amplification steps to enhance DNA yield and library quality.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Volume of moving object
If single-cell chromatin accessibility technology is carried out in a nanoliter-scale reaction system in a microfluidic chip, then the reaction system is miniaturized, but it is not conducive to the efficient separation of chromatin and RNA
Solution Approach 1:
The invention divides the single cell into nucleus and cytoplasm segments, allowing separate processing of chromatin (from nucleus) and RNA (from cytoplasm). This segmentation enables efficient separation and independent library construction for both omics types, resolving the contradiction between miniaturization and separation efficiency.
2Measurement precision
If the total amount of target substances in a single cell is very small, then the sensitivity of detection is improved, but it is very easy to cause losses of the substances during the operations
Solution Approach 1:
The invention performs preliminary enrichment and protection of target substances (chromatin and RNA) before library construction. By using specific buffers and conditions to preserve these scarce molecules throughout the workflow, minimal loss occurs while maintaining high detection sensitivity.
3Adaptability or versatility
If integrated sequencing between single-cell DNA or DNA methylation and single-cell RNA is performed, then multi-omics information is captured, but there is no related report on the integration among other different omics of a single cell, such as a simultaneous sequencing of single-cell RNA and chromatin accessibility
Solution Approach 1:
The invention segments the cell into nucleus and cytoplasm, enabling simultaneous but separate processing of chromatin and RNA. This segmentation strategy allows integration of multiple omics types (chromatin accessibility and transcriptome) while managing complexity through modular, parallel workflows.
Solution Approach 2:
The invention creates a universal platform that can simultaneously handle multiple omics types (chromatin accessibility and RNA sequencing) from a single cell. The methodology is adaptable to different omics combinations, providing multi-functionality without proportionally increasing complexity.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This approach enables simultaneous and efficient library construction for chromatin accessibility and RNA, reducing omics information loss and facilitating the integration and analysis of epigenome and transcriptome data, improving the study of single-cell gene expression regulation and disease surveillance.
Implementation Method 1
insertion of sequencing adaptors in the open chromatin regions by lysing the single cell and treating the single cell chromatin with Tn5 transposase
Implementation Method 2
reverse transcription and PCR amplification of the RNA containing a polyadenosine tail
Implementation Method 3
PCR amplification of the RNA containing a polyadenosine tail
Data Source
AI summary
Provided in the present invention is a method for constructing single cell sequencing libraries, comprising the following steps: a) lysing a single cell to obtain a single cell lysate; b) separating the nucleus and the cytoplasm in the single cell lysate obtained in step a) to obtain a nuclear solution and a total RNA solution; and c) constructing a chromatin DNA library with the nuclear solution obtained in step b) to obtain a chromatin-accessibility sequencing library of the single cell; and constructing a transcriptome library with the total RNA solution obtained in step b) to obtain a transcriptome sequencing library of the single cell.