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The Power of Paired-Tag: Joint Profiling of Histone Modifications and Gene Expression in Single Cells (Part 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. 

Epigenome Technologies Takes 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 incubation of permeabilized nuclei with specific antibodies targets a protein A-fused Tn5 transposase to chromatin to simultaneously promote sequential tagmentation and reverse transcription

  • A ligation-based combinatorial barcoding strategy then adds DNA barcodes to both chromatin DNA fragments and complementary DNA from reverse transcription ("Split&Pool")

  •  Chromatin DNA and cDNA derived from barcoded nuclei next undergo purification and amplification before being split into separate sequencing libraries


Many RNA transcripts and DNA fragments per nucleus

  • Paired-Tag applied to adult mouse frontal cortex/hippocampus generated comparable results to bulk profiles

  • 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)

  • Histone modification profiles display similar or higher numbers of unique loci captured per nucleus when compared to other single-cell histone modification profiling methods

  • 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!).

The subsequent application of Paired-Tag to mouse brain cells delivered the first report of combined histone modification and transcriptomic maps in single cells, and an upcoming article describing the second half of this Nature Methods study will explain how the integrative analysis of maps created by implementing Paired-seq identified genes subject to divergent epigenetic regulatory mechanisms.

By Stuart P. Atkinson


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