Single-cell multi-omics ATAC+ gene expression is a single-cell multi-omics solution launched by 10x Genomics. It can obtain gene expression and chromatin accessibility maps of the same cell at the same time, thereby better distinguishing the final Terminal differentiated cells and cell types along developmental trajectories reveal complex interactions between regulatory elements, open chromatin, and gene expression.
1. Principle of 10x Genomics single-cell multi-omics ATAC+ gene expression technology
In order to obtain the transcriptome and epigenome of the same cell at the same time, 10x Genomics single-cell multi-omics ATAC+ gene Expression technology first uses Tn5 transposase to transpose nuclear DNA in a cell nuclear suspension. Tn5 transposase preferentially cuts nuclear DNA in open chromatin regions. The Chromium platform is then used for single-cell gene expression (GEX) and ATAC library preparation. This process wraps a single cell nucleus, reaction reagents and a single gel bead (Gel Bead) into a droplet (GEM). The gel beads also include a poly(dT) sequence with a specific barcode (10x Barcode), a unique molecular identifier (UMI), and a Spacer sequence with 10x Barcode. The poly(dT) sequence can capture poly(dA) tails. The mRNA is used to generate gene expression (GEX) libraries, and the Spacer sequence can add barcodes to the transposed DNA fragments to generate ATAC libraries. Sequence the two libraries obtained and match the sequencing data from the two libraries of the same cell through 10x Barcode to achieve simultaneous correlation of the transcriptome and epigenome of the same cell.
2. Advantages of 10x Genomics single-cell multi-omics ATAC+ gene expression technology
1. One cell, two interpretations
10x Genomics single-cell multi-omics ATAC+ gene expression technology can make full use of samples and obtain both transcriptome and epigenome information from a cell, maximizing the acquisition of multi-layer insights from limited samples.
Stanford University School of Medicine in the United States used 10x Genomics single-cell multi-omics ATAC+ gene expression technology to obtain colorectal cancer cell lines COLO320-DM (oncogene MYC amplified on ecDNA) and COLO320- from the same patient. Paired transcriptome and chromatin accessibility profiles were obtained in a total of 72,049 cells of HSR (oncogene MYC amplified on tandem chromosomal amplicons (HSRs)). Cellular clustering of single-cell ATAC-seq and single-cell RNA-seq data shows independent clustering of COLO320-DM and COLO320-HSR cell lines. Relative to chromosomal HSR? MYC-amplified COLO320-HSR, RNA expression as well as MYC accessibility scores were highly heterogeneous in ecDNA? MYC-amplified COLO320-DM, suggesting that variable activity of regulatory elements may explain cellular differences in oncogene expression.
2. Combine the two clustering results to identify cell types more accurately
10x Genomics single-cell multi-omics ATAC+ gene expression technology can simultaneously utilize two sets of transcriptome and epigenome data. Perform cell clustering on data to better characterize the cellular heterogeneity of complex cell populations and discover hidden insights. Additionally, epigenetic signatures can be more easily interpreted using gene expression markers.
Researchers at the Broad Institute of MIT and Harvard University used single-cell multi-omics ATAC+ gene expression technology to obtain high-quality epigenomes and transcripts from 34,774 cells in adult mouse skin. Group atlas, based on the analysis of these two data, found that not only cell types of different lineages can be distinguished, but also closely related types of cells, such as αhigh CD 34+ versus αlow CD 34+. RNA-based cellular clusters can also be distinguished by chromatin accessibility features, further confirming their identity, for example annotation of clusters based on the activity of lineage determinants revealed the global transcriptional activators Dlx3 and Sox9 and the repressor Zeb1 and Sox5 et al.
Some cellular states can be identified at higher resolution through chromatin or gene expression signatures, e.g. grouping of cell clusters based on clustering signatures revealed more pronounced chromatin accessibility differences between permanent and regenerative parts of hair follicles; conversely Cells corresponding to the granular layer are more easily distinguished as a distinct cluster at the gene expression level.
3. Transcriptome and epigenome correlation to discover new gene regulatory effects
10x Genomics single-cell multi-omics ATAC+ gene expression technology can combine regulatory elements with gene expression. Explore gene regulatory interactions that drive cell differentiation, development and disease.
Researchers at the University of Washington applied single-cell multi-omics ATAC+ gene expression technology to the kidney tissue of 8-week-old male mice and obtained transcriptome and chromatin accessibility maps of 11,296 cells. And found associations between 1,260 remote sites and 321 genes. 44% of the sites were associated with the nearest TSS, and 21% were associated with the second closest TSS. The highest correlation is between the distal tubule cell marker gene Slc12a3 and a site 36 kb downstream of its TSS, overlapping its last exon. The accessibility of this site is slightly specific to distal tubule cells. high. Links between distal cis-regulatory elements and their target genes are useful for explaining differential expression in different cell types. For example, the cell type-specific expression of Slc6a 18 (a marker gene for type 2 proximal tubule S3 cells) is not reflected by cell type-specific promoter accessibility and its TSS is associated with a site 16 kb away, Accessibility of this site correlates with Slc6a18 expression.
3. Application fields of 10x Genomics single-cell multi-omics ATAC+ gene expression
Due to the limitations of single-omics, shortly after the emergence of single-cell ATAC-seq sequencing technology, single-cell ATAC A strategy of simultaneous application of -seq and single-cell RNA-seq technologies was adopted. At present, this strategy has been widely used in different fields such as organ development, disease and cancer mechanism research, and a total of nearly 50 articles have been published. The method of directly using single-cell multi-omics ATAC+ gene expression technology to analyze the transcriptome and epigenome of the same cell has also been applied to neonatal and adult mouse cerebral cortex, dexamethasone-treated lung cancer cells, mouse kidney, and mouse embryos. Tissues such as the forebrain and mouse skin during developmental stages. In December 2020, the first study using 10x Genomics single-cell ATAC+ gene expression technology to study the ecDNA hub driving intermolecular cooperative oncogene expression was also reported.
1. Case: Single-cell multi-omics ATAC+ gene expression analysis reveals the lineage determination mechanism in mouse skin
Published: November 2020
Published by : Broad Institute of MIT and Harvard University, etc.
Journal published: Cell
Impact factor: 38.637
1) Research background
Cell differentiation and function are regulated at multiple levels of gene regulation, including regulation of gene expression through changes in chromatin accessibility. However, differentiation is an asynchronous process, hampering current understanding of the regulatory events leading to cell fate decisions.
2) Materials and methods
Single-cell multi-omics ATAC+ gene expression technology (SHARE-seq), mouse skin, lung and brain tissue.
3) Research results
a. Multi-omics analysis can not only distinguish cell types of different lineages, but also distinguish closely related types of cells; RNA-based cell clusters can also be distinguished by Chromatin accessibility signatures further confirm identity; some cellular states can be identified at higher resolution through chromatin or gene expression signatures.
b. 63,110 peak-gene associations were identified in adult mouse skin, with a few individual peaks associated with 4 or more genes. High peak-gene-associated regulatory chromatin regions (DORCs) overlap with super-enhancers.
c. DORC is strongly enriched with known key regulatory genes for cross-lineage determination. DORCs differ significantly even between closely related cell populations, indicating that DORCs are highly cell type specific.
d. DORCs often become accessible before expression of their associated genes begins, consistent with lineage initiation. It has been long hypothesized that enhancer activation predicts target gene expression and implicates chromatin accessibility as a hallmark of lineage initiation.
e. Genome-wide changes in chromatin accessibility reflect the cellular state of lineage initiation, and chromatin potential may predict future cellular states on larger timescales than RNA velocity, particularly during differentiation period.
References:
1. King L. Hung, Kathryn E. Yost,Liangqi Xie, et al. EcDNA hubs drive cooperative intermolecular oncogene expression.[J].bioRxiv, 2020.11 .19.390278.
2. Ma Sai, Zhang Bing, LaFaveLindsay M et al. Chromatin potential identified by shared single-cell profiling of RNA and chromatin.[J] .Cell, 2020, 183: 1103-1116 .e20.
3. Cao Junyue, Cusanovich DarrenA, Ramani Vijay et al. Joint profiling of chromatin accessibility and gene expression in thousands of single cells.[J] .Science, 2018, 361: 1380-1385 .