Single-Cell Joint RNA and DNA Library Generation via Tagmentation
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Solution Overview
Problem
Current methods face challenges in integrating datasets of different histone marks from various experiments and assessing transcriptional profiles along with chromatin states from the same cells, making it difficult to understand gene regulatory mechanisms effectively.
Innovation Solution
A method involving permeabilization of nuclei, tagging with antibodies and transposases, followed by tagmentation and reverse transcription, and subsequent ligation-based combinatorial barcoding to generate separate DNA and RNA libraries for sequencing, allowing for the joint analysis of gene expression and chromatin states in single cells.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If separate experiments are conducted for different histone marks, then each histone mark can be analyzed individually, but integration of datasets from different experiments becomes challenging
Solution Approach 1:
The patent combines multiple histone mark analyses into a single experiment by performing simultaneous ChIP and RNA sequencing from the same cell population. This merging approach allows direct comparison of different histone marks without the complexity of integrating data from separate experiments, while maintaining measurement precision for each mark.
Solution Approach 2:
The methodology creates a universal platform that can analyze multiple chromatin states and gene expression profiles in a single assay. This multi-functional approach enables the same experimental system to detect various histone modifications, DNA accessibility, and transcriptional activity simultaneously, eliminating the need for separate specialized experiments.
2Quantity of substance
If traditional bulk methods are used, then sufficient material is available for analysis, but cellular heterogeneity cannot be resolved
Solution Approach 1:
The patent segments the bulk sample analysis into single-cell resolution by performing chromatin accessibility and gene expression assays on individual cells. This segmentation allows the detection of cellular heterogeneity while maintaining sufficient material through the accumulation of data from many single cells, effectively resolving the contradiction between sample quantity and measurement precision.
3Quantity of substance
If chromatin state and gene expression are assessed from different cells, then sufficient material is available for each assay, but joint analysis from the same cells becomes necessary
Solution Approach 1:
The patent merges chromatin state analysis and gene expression profiling into a single joint assay performed on the same cell population. This combination preserves the correlation between chromatin accessibility and transcriptional activity by measuring both parameters from identical cells, eliminating the information loss that would occur if separate cell populations were analyzed.
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 comprehensive profiling of gene regulatory programs across different cell lineages and pathological conditions, providing insights into gene regulatory mechanisms and potential diagnostic markers.
Implementation Method 1
contacting the one or more nuclei with (i) an antibody that binds to a chromatin-associated protein or chromatin modification and (ii) a first transposase; wherein the first transposase is loaded with a nucleic acid comprising a first tag
Implementation Method 2
contacting the one or more nuclei with a ligase and a third tag comprising a second barcode selected from a second set of barcodes
Implementation Method 3
reverse transcribing the RNA in the one or more nuclei using primers comprising a second tag
Data Source
AI summary
The present invention relates to methods for the joint analysis of regulation of gene expression and gene expression in single cells. Provided are methods for obtaining gene expression information for a single nucleus, the methods comprising deriving a DNA library from the genomic DNA in one or more nuclei and deriving an RNA library from the RNA in one or more nuclei, sequencing the molecules in the RNA library and the DNA library, and correlating the RNA library and the DNA library for each of the one or more nuclei.