The Power of Paired-Tag: Part 1

Single-Cell Joint Profiling of Histone Modifications and Gene Expression

February 26, 2024 · By Stuart P. Atkinson

Single-Cell UMAP embeddings of various histone marks and RNA — Single-Cell Histone modification UMAP embeddings (with and without RNA) showing the major class distinctions resolved by different histone modifications through Paired-Tag

1. The Importance of Joint Single-Cell Analysis

The identification of gene regulatory elements through the combination of epigenetic and transcriptomic profiling in single cells will improve our understanding of environmental contributions to disease and aging, identify new therapeutic targets for cancer Alzheimer’s disease, or metabolic disease and novel prognostic/diagnostic biomarkers, and clarify the genomic patterning underlying typical organism development. Next-generation sequencing-based techniques explore said profiles separately in bulk tissues; however, this lack of single-cell resolution may represent a barrier to collecting data from the small numbers of target cells present with the surrounding “uninvolved” cell types that comprise the majority of tissues.

Can the power of Paired-Tag (parallel analysis of individual cells for RNA expression and DNA from targeted tagmentation by sequencing) provide a solution to this problem?

2. Taking Epigenetic Profiling to Single-Cell Resolution

A team led by Bing Ren at the Ludwig Institute for Cancer Research/University of California San Diego reported that the Paired-Tag assay could generate cell-type-resolved epigenetic and transcriptomic maps in complex tissues by supporting the joint profiling of histone modifications and gene expression in single cells. We at Epigenome Technologies, under an exclusive license, now provide optimized Paired-Tag kits and services to researchers in the epigenetics field. Stay tuned for an upcoming article in this series that describes the recent application of Paired-Tag to support the single-cell epigenomic and transcriptomic analysis of the Alzheimer’s disease-affected human brain, which provides detailed insights into disease-associated molecular mechanisms.

Through a series of introductory articles, we now report on the vast potential of Epigenome Technologies’ single-cell profiling technology by discussing the Nature Methods and Nature Structural & Molecular Biology articles that helped to bring the only commercially available technology for joint profiling of histone modifications and gene expression in single cells to the market and then describing the most recent exciting applications.

How Paired-Tag Works

The Paired-Tag protocol has evolved significantly since the original publication of the article, and Single-Cell CUT&Tag versions (without RNA) are available as well. You can view our current protocols here.

Schematic of Paired-Tag Workflow — Paired-Tag in a Nutshell: antibody-Tn5 binds chromatin states → barcoding → sequencing.

Bringing CUT&Tag to Single-Cells

Isolated nuclei are incubated with validated antibodies and highly-active targeted-tagmentation enzyme

We maintain a curated list of single-cell capable antibodies (not all are!)
We partner with EpiCypher to incorporate highly-active enzymes into our custom reagents

The tagmentation reaction is performed in optimized conditions to minimize inserts at open chromatin
Nuclei are isolated into micro-partitions (plates, microwells, or droplets) - compatible with leading platforms
RT reactions are performed, and partition-specific barcodes appended to both scRNA and scCUT&Tag libraries

The partitioning can be repeated (Split&Pool or OAK)

Figure showing RNA/DNA UMI and minature IGV tracks — The distribution of unique DNA loci (epigenetic reads) and RNA UMIs across individual nuclei from deeply sequenced Paired-Tag and Paired-seq datasets. The plots span from the first quartile (Q1) to the third quartile (Q3) with the median marked by a central line; whiskers extend to a maximum of 1.5 times the IQR, and outliers are shown as individual dots.

Paired-Tag Provides High Epigenetic Coverage of Mouse Brain Nuclei

Accurate: Paired-Tag applied to adult mouse frontal cortex/hippocampus generated comparable results to bulk profiles.
High-Yield: Paired-Tag generated ~20,000 unique loci mapped per nucleus for DNA profiles and ~15,000 unique molecular identifiers per nucleus for RNA profiles (85% assigned to genic regions)
Best-in-Class Histone modification profiles display similar or higher numbers of unique loci captured per nucleus when compared to other single-cell histone modification profiling methods
Good Recovery: The capture efficiency of nuclear RNA remained similar to other commonly implemented scRNA-seq assays

Cataloging the epigenetic diversity of brain cell populations

The subsequent sequencing of ~70,000 mouse brain nuclei recovered 64,849 with matched DNA/RNA profiles

RNA datasets possessed reads mapping to genic regions and captured more intronic reads than comparable techniques, with transcriptomic profiles clustering nuclei into groups indicative of 22 cell types

Histone modification profiles also supported cell-type clustering, underscoring the encoding of gene expression in the overall epigenetic state as profiled by Paired-Tag

As per the original observation from ENCODE, broad regions of repressive modifications can separate major classes of cells, while permissive modifications distinguish cell types and can “fine-tune” cell states

Applying transcriptomic profiles supported the construction of genome-wide maps of five histone modification profiles in 22 mouse brain cell types

Bring The Power of Paired-Tag to Your Research

Paired-Tag represents an exciting commercially available means to jointly profile histone modifications and gene expression at the single-cell resolution and detect histone modifications and RNA transcripts in individual nuclei with an efficiency comparable to single-nucleus RNA-seq/ChIP–seq assays. Applying Paired-Tag technology may enable quantum leaps forward in our understanding of development and significantly improve disease management (and more!).