What is Single-Cell ATAC Sequencing?
InquiryWhat is Single-Cell ATAC-Seq?
The DNA housed within the nuclei of eukaryotic cells is intricately organized, forming densely structured chromatin. During processes like DNA replication or transcription, it becomes imperative to unwind this sophisticated DNA packaging, enabling the binding of transcription factors and other regulatory elements. This section of exposed chromatin is termed "open chromatin." In this state, transcription factors and regulatory elements find their binding sites.
Consequently, delving into the open state of chromatin offers a means to comprehensively explore the genome-wide transcriptional regulation of genes. ATAC-seq (Assay for Transposase accessible chromatin with high-throughput sequencing) emerges as a valuable technique for investigating chromosome accessibility. It leverages DNA transposases in conjunction with high-throughput sequencing technology to analyze binding sites of transcription factors and histone modification molecules on DNA at a large scale.
The integration of single-cell technology with ATAC-seq, known as scATAC-seq, introduces an efficient method for probing chromatin openness at the single-cell level. scATAC-seq facilitates batch detection of chromatin openness, allowing for a systematic exploration of transcriptional gene regulation at the DNA level. This technique, coupled with biosignature analysis for identifying transcription factor binding sites, finds extensive applications in the study of cell differentiation and the tumor microenvironment.
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Single-cell ATAC-Seq vs. Single-Cell RNA-Seq
Single-cell ATAC-seq and single-cell RNA-seq are two distinct single-cell technologies that primarily focus on chromatin accessibility and gene expression, respectively, providing in-depth insights into cell function and regulation.
Differences in Experimental Library Construction
- Single-cell ATAC: Utilizes ATAC-seq (Assay for Transposase-Accessible Chromatin with high-throughput sequencing) technology, employing a modified Tn5 transposase to study chromatin accessibility across the entire genome. In the experimental process, single-cell ATAC can obtain data from hundreds to thousands of cells through physical isolation or a two-step composite indexing strategy.
- Single-cell RNA: Involves using live cells and extracting RNA sequences from the cytoplasm. Compared to ATAC, it requires a higher cell state and analyzes the expression levels of genes within cells.
Research Focus and Information Output
- Single-cell ATAC: Mainly focuses on the open state of chromatin, revealing the regulatory mechanisms of gene expression. Analyzing single-cell ATAC data can identify cell subpopulations, discover potential enhancer-gene interactions, and construct more accurate molecular models of cellular processes.
- Single-cell RNA: Concentrates on revealing the expression levels of individual genes within a single cell and cellular heterogeneity. Through single-cell RNA-seq, it is possible to identify cell types, explore dynamic changes in gene expression, and construct cell developmental trajectories.
Technical Platforms and Application Fields
- Single-cell ATAC: Based on ATAC-seq technology, for example, the Single Cell ATAC product on the 10x Genomics ChromiumTM platform, using a GEM (Gel Bead-in-Emulsion) to capture single cells. In research, single-cell ATAC is widely applied in fields such as oncology, disease research, neuroscience, developmental biology, and immunology.
- Single-cell RNA: Uses RNA-seq technology, such as single-cell RNA-seq. This technology is widely applied to reveal gene expression patterns at the single-cell level, particularly playing a crucial role in understanding cellular heterogeneity and developmental processes.
Single-Cell ATAC-seq Developed by 10x Genomics
Droplet-based platforms for single-cell ATAC-seq, such as the one developed by 10x Genomics, enable the exploration of chromatin accessibility in individual cells across diverse sample types. This cutting-edge technology not only facilitates the clustering of cells within samples but also unveils key regulatory factors present in distinct subpopulations or engaged in different biological processes.
10X Genomics platforms (from 10X Genomics)
The methodology involves subjecting a nuclear suspension of cells to a Tn5 transposase reaction, effectively excising open chromatin regions. The process continues as cell nuclei, gel beads with barcode sequences, and oil droplets are introduced into microfluidic chips, forming water-in-oil structures. Following linear amplification, fragments originating from the same nucleus are linked with identical barcode sequences. Subsequent PCR amplification adds the upper sequencing junction, culminating in the construction of high-throughput sequencing libraries.
The droplet-based single-cell ATAC-seq technique proves adept at generating high-quality libraries, facilitating the analysis of chromatin accessibility at the single-cell level within complex tissues. Noteworthy advantages of this method include its ability to provide comprehensive coverage of cell types, even rare ones, within a sample. Furthermore, it allows for the analysis of regulatory sequences at both the single-gene and single-cell levels, offering an objective framework for constructing developmental trajectories.
Single-Cell ATAC and Single-Cell Gene Expression Analysis
Strategies employing single-cell omics, combining single-cell transcriptome sequencing (scRNA-seq) and chromatin accessibility sequencing (scATAC-seq), offer a comprehensive understanding of cellular processes. While scRNA-seq delineates transcriptional states and expressions, scATAC-seq unveils chromatin opening states and the regulatory landscape linked to gene expression. The simultaneous application of both technologies establishes a direct connection between gene expression and the accessibility of regulatory elements, allowing for a more accurate reconstruction of molecular processes governing cellular physiology.
The integration of single-cell ATAC-Seq and single-cell gene expression into a multi-omics research model has emerged as the optimal approach to decipher dynamic changes in gene expression patterns and their underlying regulatory mechanisms. This model finds widespread application across research domains, including oncology, disease, neuroscience, developmental biology, and immunity, shaping the future landscape of single-cell development.
In a recent study, researchers generated single-cell chromatin accessibility and RNA expression profiles from human fetal cerebral cortex samples at 8 weeks of mid-gestation. This approach unraveled the dynamic developmental profiles of chromatin and gene regulation in the human cerebral cortex. The identification of 64,878 CRE-Gene pairs highlighted potential enhancer-gene interactions. Additionally, 185 genes (GPCs) with predictable expression levels based on chromatin accessibility were identified. The study also unveiled and characterized two precursor cell populations of astrocytes.
A single-cell epigenomic atlas of the human cerebral cortex. (Trevino et al., 2021)
Utilizing a deep learning model based on gene expression and chromatin accessibility, the researchers established a link between DNA sequences and chromatin accessibility. This model identified a disruptive mutation in an intron of NFIA, a gene previously implicated in autism, emphasizing the power of integrated multi-omics approaches in uncovering genetic variations associated with diseases.
Reference
- Trevino, Alexandro E., et al. "Chromatin and gene-regulatory dynamics of the developing human cerebral cortex at single-cell resolution." Cell 184.19 (2021): 5053-5069.
