Functionalized Hydrogel Beads for Single-Cell Epigenomic Sequencing
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Solution Overview
Problem
Current single-cell sequencing technologies are limited in their ability to perform high-throughput epigenomic sequencing, lacking the capability to efficiently capture and analyze epigenetic modifications at a single-cell level.
Innovation Solution
The method involves preparing functionalized hydrogel beads that encapsulate single cells, allowing for cell lysis and capture of nucleic acids. The hydrogel beads are chemically modified to bind capture reagents, which specifically target epigenetic markers, enabling the formation of captured nucleic acids that can be barcoded and sequenced.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If traditional sequencing methods are used, then bulk population sequencing is achieved, but single-cell resolution and cellular heterogeneity cannot be resolved
Solution Approach 1:
The invention partitions bulk cell populations into individual single cells using microfluidic droplet generation, where each droplet contains at most one cell. This segmentation enables single-cell resolution while maintaining high throughput by processing millions of droplets in parallel through the microfluidic system.
Solution Approach 2:
The invention transitions from bulk population analysis to single-cell analysis by adding the dimension of spatial compartmentalization through microfluidic droplets. Each droplet serves as an isolated reaction chamber, enabling simultaneous processing of numerous single cells across parallel spatial dimensions.
2Measurement precision
If microfluidics-based single-cell sequencing methods are used, then single-cell resolution is achieved, but technical complexity increases making them challenging for biologists to perform
Solution Approach 1:
The invention uses universal adaptor ligation that can be applied across different sequencing platforms and epigenomic applications. The standardized adaptor design and modular workflow enable the same microfluidic system to perform multiple functions including chromatin accessibility sequencing, DNA methylation sequencing, and other epigenomic assays.
Solution Approach 2:
The invention employs disposable microfluidic chips and single-use reagent cartridges that eliminate the need for complex cleaning and sterilization procedures. Each chip is designed for single-use, reducing cross-contamination risks and simplifying the workflow for biologists who may not have extensive technical training.
3Ease of operation
If well plate-based methods are used, then ease of operation is maintained, but throughput is insufficient for high-throughput sequencing
Solution Approach 1:
The invention uses microfluidic pressure-driven flow systems to automatically transport droplets through various processing stages including lysis, capture reagent incubation, adaptor ligation, and sequencing library preparation. This hydraulic automation replaces manual well plate handling while maintaining operational simplicity through integrated fluid control.
Solution Approach 2:
The invention scales from low-throughput well plate formats to high-throughput microfluidic systems by changing key parameters including droplet generation rate, flow velocity, and reaction chamber dimensions. These parameter adjustments enable throughput increases of thousands-fold while maintaining the same basic operational workflow.
4Adaptability or versatility
If targeted transcriptome sequencing methods are used, then transcriptome analysis is achieved, but single-cell genome sequencing and other multiomic technologies are not well established
Solution Approach 1:
The invention employs universal adaptors that can ligate to various nucleic acid types including genomic DNA, RNA, and epigenetically modified DNA. The same microfluidic platform and capture reagent system can be applied across different omics disciplines, enabling high-throughput multiomic sequencing without requiring separate specialized systems for each application.
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 high-throughput, single-cell epigenomic sequencing, allowing for the interrogation of various epigenetic modifications at a scale surpassing existing technologies, with minimal instrumentation and reduced technical expertise.
Implementation Method 1
the functionalized hydrogel is chemically modified with a functional group capable of binding to a capture reagent
Implementation Method 2
binding of the capture reagent to (i) the nucleic acid, and (ii) the functional group of the functionalized hydrogel
Implementation Method 3
encapsulating single cells into particles comprising a cell lysis buffer and the functionalized hydrogel under conditions that allow cell lysis
Implementation Method 4
the capture reagent is also capable of binding to a nucleic acid
Implementation Method 5
the capture reagent is selected from the group consisting of an antibody, streptavidin, avidin, and aptamers
Data Source
AI summary
The present disclosure provides materials and methods for partitioning cells and high throughput, single-cell epigenetic sequencing. Methods for using chemically-modified hydrogels are also provided herein. Wherein determining the epigenomic state of a single-cell comprises preparing a functionalized hydrogel, wherein said functionalized hydrogel is chemically modified with a functional group capable of binding to a capture reagent.