Single-Cell Chromatin Sequencing With Targeted DNA Fragment Barcoding
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Solution Overview
Problem
Current technologies lack effective methods for labeling nuclear target-associated DNA in single cells, which is essential for comprehensive analysis of gene expression and chromatin structure.
Innovation Solution
A method involving permeabilization of cells to allow entry of DNA digestion enzymes and binding reagents, followed by barcoding of nuclear target-associated DNA fragments using oligonucleotide barcodes, enabling the generation of barcoded DNA fragments for sequencing and analysis.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If current massively parallel single cell gene expression technology is used, then cell throughput is improved (>10000 cells), but the ability to label and analyze nuclear target-associated DNA in single cells is insufficient
Solution Approach 1:
The method segments the single cell analysis process into distinct functional modules: (1) cell permeabilization to enable reagent entry, (2) binding reagent-specific oligonucleotide attachment to nuclear targets, (3) DNA digestion enzyme treatment to generate fragments, and (4) oligonucleotide barcode attachment for identification. This segmentation allows each module to be optimized independently while maintaining overall system versatility for different nuclear targets.
Solution Approach 2:
The invention creates a universal platform that can analyze multiple types of nuclear targets (DNA, chromatin, protein-associated DNA) using the same basic methodology. The binding reagents can be swapped to target different nuclear components, and the barcoding system universally identifies all fragments, enabling the system to handle both gene expression analysis and nuclear target-associated DNA analysis with the same cellular throughput capability.
2Difficulty of detecting and measuring
If cell permeabilization is performed to allow entry of binding reagents and DNA digestion enzymes, then nuclear target accessibility is improved, but cell integrity is compromised
Solution Approach 1:
The method performs preliminary cell permeabilization before introducing the binding reagents and DNA digestion enzymes. This preliminary action creates controlled access pathways that allow the necessary reagents to enter the nucleus while minimizing uncontrolled cellular damage. The permeabilization is optimized to be sufficient for reagent entry but not so extreme as to cause complete cell disintegration, thereby balancing accessibility with retained structural integrity for reliable analysis.
3Measurement precision
If oligonucleotide barcodes are attached to nuclear target-associated DNA fragments, then DNA identification precision is improved, but process complexity increases
Solution Approach 1:
The invention uses binding reagent-specific oligonucleotides as intermediaries that first attach to the nuclear targets and then serve as attachment points for the actual barcodes. This intermediary step simplifies the overall process by decoupling the target recognition function from the barcoding function, making each step more manageable and reducing the complexity of direct barcode attachment while maintaining high identification precision.
Solution Approach 2:
The barcoding process is designed to be self-assembling through complementary base pairing between the binding reagent-specific oligonucleotides and the barcode oligonucleotides. This self-service mechanism eliminates the need for complex enzymatic ligation or chemical coupling steps, reducing process complexity while ensuring high precision attachment of barcodes to the correct DNA fragments.
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
Enables detailed labeling and sequencing of nuclear target-associated DNA, allowing for accurate determination of genome and methylome information, as well as chromatin structure, in single cells.
Implementation Method 1
contacting the nuclear target with a digestion composition comprising a DNA digestion enzyme to generate a plurality of nuclear target-associated dsDNA fragments each comprising a single-stranded overhang
Implementation Method 2
a binding reagent capable of specifically binding to the nuclear target, wherein each binding reagent comprises a binding reagent specific oligonucleotide comprising a unique identifier sequence for the binding reagent
Implementation Method 3
each oligonucleotide barcode of the first plurality of oligonucleotide barcodes comprises a first target-binding region capable of hybridizing to the plurality of nuclear target-associated dsDNA fragments
Data Source
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AI summary
Disclosed herein include systems, methods, kits, and compositions for labeling nuclear target-associated DNA in a cell. Some embodiments provide digestion compositions comprising a DNA digestion enzyme and a binding reagent capable of specifically binding to the nuclear target. Some embodiments provide conjugates comprising a transposome and a binding reagent capable of specifically binding to a nuclear target. The transposome can comprise a transposase (e.g., Tn5 transposase), a first adaptor having a first 5' overhang, and a second adaptor having a second 5' overhang. The methods can comprise contacting a permeabilized cell comprising a nuclear target associated with dsDNA, such as genomic DNA (gDNA), with the compositions provided herein to generate a plurality of nuclear target-associated dsDNA fragments (e.g., nuclear target-associated gDNA fragments) each comprising the one or two single- stranded overhangs.