Paired-Tag: The Droplet Frontier

Improving Joint Single-cell Analysis with Droplet Paired-Tag

April 28, 2024 · By Stuart P. Atkinson

IGV tracks showing single-cell histone profiling — Single-cell histone modification tracks produced by Droplet Paired-Tag

1. Improving Joint Single-cell Analysis with Droplet Paired-Tag

Applying “Paired-Tag” (parallel analysis of individual cells for RNA expression and DNA from targeted tagmentation by sequencing) for joint epigenetic and gene expression profiling at single-cell resolution can provide an improved understanding of the epigenetic underpinnings of normal and disease-affected tissues. Research has linked dysregulated gene expression to disease development, and evidence now suggests that epigenetic factors play a significant potentially targetable role in developing and maintaining disease-associated transcriptomic profiles.

A series of introductory articles have described the development and application of Paired-Tag, first described in a Nature Methods article by Bing Ren at the University of California San Diego (now at the New York Genome Center). This third article focuses on a Nature Structural & Molecular Biology research article from the same team that reported “Droplet Paired-Tag” as a faster and more accessible methodology for joint epigenetic and gene expression profiling at single-cell resolution.

Improvements to the methodology include the implementation of a commercially available microfluidic platform (10x Chromium Single Cell Multiome) to introduce cellular barcodes and the simplification of the sequencing library preparation protocol, which brings the experimental timespan down to just 1.5 days and improves performance when identifying regulatory elements and correlating chromatin states of regulatory elements to putative target gene expression levels.

Stay tuned for the next article in this series that describes another recent application of Droplet Paired-Tag to support single-cell epigenomic and transcriptomic profiling in the Alzheimer’s disease-affected human brain, which affords insight into disease-associated molecular mechanisms.

Epigenome Technologies provides optimized Droplet Paired-Tag kits and services to researchers in the epigenetics field under an exclusive license from the Ludwig Institute for Cancer Research. Can Droplet Paired-Tag accelerate the field toward defining the dynamic, cell-type-specific epigenomic landscapes of complex tissues and disease and identify potential exciting therapeutic targets?

2. Introducing Droplet Paired-Tag

After nuclei permeabilization, targeted tagmentation employs primary antibodies specific to a histone modification precoupled with protein A–Tn5 transposase fusion proteins
Nuclei and barcoded beads undergo coencapsulation within droplets in a widely available microfluidic device
Beads contain two barcoded oligonucleotide types: a barcoded poly(dT) oligonucleotide to label cDNA (for reverse transcription) and a capturing oligonucleotide to label DNA fragments from tagmentation
Ligation next attaches the same barcoded capturing oligonucleotides to tagmented chromatin fragments and reverse transcription products (cDNA) from the same cells
After dissolving droplets, cDNA/chromatin fragments undergo purification and amplification before being split for library construction
Overall, Droplet Paired-Tag represents a quick and accessible means for joint epigenetic and gene expression profiling at single-cell resolution

Histone PTMs and Gene Expression from the Same Cell

Proof of concept for Droplet Paired-Tag analyzed H3K27ac and H3K27me3 and transcriptomes of mouse embryonic stem cells (mESCs)
The complexity of histone modification libraries generated with Droplet Paired-Tag compared favorably to Paired-Tag

Aggregated single-cell signals for H3K27ac and H3K27me3 resembled profiles from bulk CUT&Tag and ChIP-seq experiments and possessed high enrichment over peaks identified from ChIP-seq datasets
The number of H3K27ac peaks detected reached saturation after about 2,500 nuclei in Droplet Paired-Tag; meanwhile, Paired-Tag required 60% more nuclei to reach saturation by comparison

Transcriptomic libraries generated with Droplet Paired-Tag possessed comparable complexities to other single-cell RNA-seq tech and bulk mESC RNA-seq data

Gene expression levels positively correlated with H3K27ac at transcription start sites and inversely correlated with H3K27me3 deposition across gene bodies

These results indicate that Droplet Paired-Tag reliably captures high-quality joint epigenetic and transcriptomic profiles from the same cell in a more straightforward manner than Paired-Tag, which may accelerate future applications in understanding complex, disease-affected tissues

Droplet-Paired-Tag Integrates Histone Modification and Gene Expression Profiles at Gene Promoters in Mouse Brain Cells

Proof of concept for Droplet Paired-Tag in primary tissues focused on cells of the adult mouse frontal cortex

Clustering nuclei by their transcriptomic profiles identified twenty major types of neuronal/non-neuronal cells, which were independently recovered via histone modification profile analysis
Cell clusters agreed with those of Paired-Tag and single-nucleus RNA-seq datasets generated from cells of the mouse brain

Droplet Paired-Tag outperformed single-cell CUT&Tag and Droplet Paired-Tag with regards to histone modification profiling, with improvements to signal sensitivity and specificity contributing to the high-resolution separation of cell types

Droplet Paired-Tag yielded the lowest level of H3K27me3 within open chromatin regions, indicating minimal off-target Tn5 transposase activity, but a higher level of H3K27ac overlapping open chromatin regions

Multi-panel figure illustrating embeddings and statistics of RNA and DNA libraries — (A) UMAP embedding of Droplet Paired-Tag data from the frontal cortex, with nuclei clustered and annotated by both transcriptomic (gene expression) and epigenomic (H3K27ac and H3K27me3) profiles. (B) Venn diagram showing the overlap between cell-type annotations derived from transcriptome and epigenome clustering. (C) Genome browser views of gene expression and histone modification distributions over cell-type-specific marker genes. (D) Comparison of the number of unique transcripts and genes detected per cell between Droplet Paired-Tag and conventional Paired-Tag. (E, F) Box plots comparing the unique fragment counts and FRiP scores for H3K27ac (E) and H3K27me3 (F) between Droplet Paired-Tag and alternative single-cell epigenomic methods.

3. Droplet Paired-Tag Analysis Explores the World of Candidate Cis-Regulatory Elements in Mouse Brain Cells

Droplet Paired-Tag then characterized candidate cis-regulatory elements (cCRE) activity states via H3K27ac or H3K27me3 status in mouse brain cells

H3K27ac signals in promoter regions possessed a strong positive correlation with transcription at proximal cCREs, while the H3K27me3 signal possessed an overall inverse correlation
Transcription factor binding motif analysis of cCREs revealed the transcription factors involved in each case

Droplet Paired-Tag data predicted putative target genes of distal cCREs through joint epigenetic and gene expression profiling in the same cells

Overall, Droplet Paired-Tag data captured stronger cCRE-gene connections over background than Paired-Tag

Droplet Paired-Tag provides superior performance when evaluating cell-type-specific gene regulatory programs in complex tissues; therefore, future applications could identify disease-associated epigenetically dysregulated cCREs and directly link these cCREs to driver genes, thereby providing potential targets for gene-editing or epigenetic therapies

Bring The Power of Paired-Tag to Your Research

Droplet Paired-Tag represents an exciting commercially available platform for joint epigenetic and gene expression profiling at single-cell resolution. Overall, the speed and accessibility of Droplet Paired-Tag may enable even more significant leaps forward in our understanding of development and improve disease management. Here, the analysis of combined histone modification and transcriptomic maps in single mouse brain cells underscored the ability of Paired-Tag technology to identify genes subject to divergent epigenetic regulatory mechanisms in mouse brain cells, which has the potential to decipher disease-associated mechanisms in the future.