Revolutionizing epigenomics with single-cell CUT&Tag

Ellen N. Weinzapfel, PhD

Revolutionizing epigenomics with single-cell CUT&Tag

Single-cell epigenomics has transformed our understanding of cellular diversity and gene regulation. By analyzing individual cells, researchers can uncover the intricate mechanisms driving cellular states and how these processes go awry in disease. Immunotethering technologies, including pAG-Tn5 and CUT&Tag (Cleavage Under Targets & Tagmentation), have emerged as powerful tools in this research, offering precise chromatin profiling at unprecedented resolution and scale. EpiCypher is enabling exploratory method development with our IDEA Toolbox, which includes various pAG-Tn5 enzymes and custom-loading services. In this blog, we will explore three innovative applications of single-cell CUT&Tag: droplet-based strategies, spatial applications, and combinatorial indexing.

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Table of Contents

Single-Cell CUT&Tag background

CUT&Tag was specifically developed by Dr. Steven Henikoff for ultra-low input chromatin profiling, including single cells1,2. The improved sensitivity of CUT&Tag is directly enabled by pAG-Tn5: by selectively cleaving and inserting sequencing adapters at target-enriched chromatin, CUT&Tag effectively eliminates off-target signal, streamlines the workflow, and reduces sample loss. With EpiCypher’s direct-to-PCR strategy, tagmented fragments are amplified directly from the reaction mixture, and libraries can be quickly purified for next-generation sequencing.

Single-cell CUT&Tag is at the forefront of CUT&Tag literature, providing insights into cell-to-cell variability and rare cell populations. Multiple assay options are available, from droplet-based/microfluidics to combinatorial indexing strategies. Below we outline a few key methods and papers and how EpiCypher CUTANA™ CUT&Tag enzymes can help get you started.

Droplet-based single-cell CUT&Tag

Droplet-based strategies use microfluidics to encapsulate individual cells in droplets, allowing for the generation of uniquely barcoded sequencing libraries for single-cell profiling. The past decade has seen remarkable advances in droplet-based technologies, which can now process thousands of individual cells in parallel, providing a high-throughput strategy that is revolutionizing single-cell genomics3-6. Many of these platforms originally focused on ATAC-seq assays, which use Tn5 to cleave and ligate sequencing adapters at open chromatin. ATAC-seq has low cell input requirements, a rapid protocol, and high sensitivity, making it ideal for single-cell analysis. Importantly, the same hyperactive Tn5 is used for CUT&Tag assays, paving the way for innovative single-cell CUT&Tag applications.

In April 2021, two single-cell CUT&Tag papers that leverage droplet-based strategies were published in Nature Biotechnology7,8. These papers are outlined in detail below. Note that in each paper, CUT&Tag was first performed on bulk nuclei to selectively tagment antibody-bound chromatin, followed by single-cell library preparation on the 10x Genomics Chromium system. These studies demonstrate the potential of combining CUT&Tag with droplet-based technologies to achieve high-resolution chromatin profiling at the single-cell level.

Bartosovic et al. Single-cell CUT&Tag profiles histone modifications and transcription factors in complex tissues. Nature Biotechnology 2021.

  • Application: Deconvolute distinct cell types in mouse brain; integrate single-cell CUT&Tag with single-cell RNA-seq profiles.
  • Cell types: FACS-sorted oligodendrocyte precursors from mouse brain tissue, mouse ES cells, mouse fibroblasts (NIH-3T3), and mouse oligodendrocyte progenitor cell line.
  • Targets: H3K27me3, H3K4me3, H3K27ac, H3K36me3, OLIG2, and RAD21. Authors noted that the number of reads generated for transcription factors OLIG2 and RAD21 were lower than that for PTMs, but data were still useful.

Wu et al. Single-cell CUT&Tag analysis of chromatin modifications in differentiation and tumor progression. Nature Biotechnology 2021.

  • Application: Classify differential chromatin states during human embryonic stem cell differentiation, blood samples, and brain tumors.
  • Cell types: Human embryonic stem cells differentiated to endoderm; human primary glioblastoma; human PBMCs.
  • Targets: H3K27me3.

Spatial CUT&Tag

Spatial profiling refers to the analysis of chromatin states or gene expression in individual cells within the context of tissue architecture and cellular localization9. This approach is particularly useful for studying tissues with intricate organization, such as the brain, where spatial information is crucial for understanding cellular interactions and function. It can also be extremely useful when studying heterogeneous tumors and/or complex tumor microenvironments.

By definition, spatial profiling requires single-cell resolution and ultra-sensitive mapping technologies. Prior to the development of CUT&Tag, mapping specific chromatin features in spatial assays was incredibly challenging, and most assays focused on Tn5-based ATAC-seq. In 2022, the first spatial CUT&Tag paper was published, which used a combination of in situ CUT&Tag and droplet-based single-cell systems designed to record cellular localization10. The technology has continued to progress, and EpiCypher is excited to be partnering with AtlasXomics to advance this research.

Deng et al. Spatial-CUT&Tag: Spatially resolved chromatin modification profiling at the cellular level.. Science 2022.

  • Application: Proof-of-concept for spatial CUT&Tag; used to define unique mouse tissues and cell types during development; benchmarked by performing spatial CUT&Tag on mouse brain and compared with published single-cell CUT&Tag and RNA-seq data.
  • Cell types: Mouse embryos (embryonic day 11), mouse brains (postnatal day 21).
  • Targets: H3K27me3, H3K4me3, H3K27ac.

Combinatorial indexing single-cell CUT&Tag

Combinatorial indexing involves sequential tagging of cells with unique barcodes, enabling single-cell analysis without the need for physical separation. This method is efficient and scalable, allowing for comprehensive epigenomic profiling of complex tissues, including heterogeneous tumors and brain tissue11. Initially developed for whole genome sequencing12 and single-cell ATAC-seq13, combinatorial indexing has since been utilized for single-cell RNA-seq14, DNA methylation sequencing15, ChIP-seq16, and even chromatin conformation analysis17.

The pAG-Tn5 enzyme used in CUT&Tag is well-suited for combinatorial indexing applications, as demonstrated by Janssens et al. in their 2023 paper18. In this strategy, they used the Takara ICELL8 system to separate cells into nanowell plates containing a library of pAG-Tn5 enzymes, each loaded with distinct barcoded sequencing adapters. Following tagmentation the cells were removed from plates, pooled, and then split into a second plate for indexing PCR, thus adding a second round of barcodes. This split-pooling strategy was highly effective compared to other single-cell CUT&Tag approaches and was also used to develop the multiomic MulTI-Tag strategy19.

Janssens et al. Scalable single-cell profiling of chromatin modifications with sciCUT&Tag.. Nature Protocols 2023.

  • Application: Proof-of-concept, protocol paper; used to detect specific cell types in human blood.
  • Cell types: Human peripheral blood mononuclear cells (PBMCs).
  • Targets: H3K27me3, H3K4me1, me2, me3. Authors noted that most abundant marks should be prioritized to maximize yields and/or reads from single cells.

Summary

The versatility of single-cell CUT&Tag underscores its transformative impact on epigenomics research. From single-cell resolution to spatial profiling, pAG-Tn5 continues to expand the frontiers of what is possible in chromatin mapping. As these technologies evolve, they hold the promise of deeper insights into gene regulation and the epigenetic underpinnings of health and disease.

Stay tuned for our next blog, where we will discuss exciting multiomic CUT&Tag applications. Reach out to services@epicypher.com to learn about our custom-loading services that are ideal for single-cell and spatial CUT&Tag.

Explore our pAG-Tn5 enzymes for CUT&Tag assays

Tools to get started with CUT&Tag assay development

At EpiCypher, our scientists deliver cutting-edge CUT&Tag reagents with uncompromising quality. Uncharged, or unloaded, pAG-Tn5 enzymes are ideal for single-cell combinatorial indexing and other advanced CUT&Tag applications, which we will discuss in future blogs.

Additional single-cell CUT&Tag and spatial CUT&Tag papers of interest

References

  1. Kaya-Okur HS et al. CUT&Tag for efficient epigenomic profiling of small samples and single cells. Nat Commun 10, 1930 (2019). https://doi.org/10.1038/s41467-019-09982-5.
  2. Kaya-Okur HS et al. Efficient low-cost chromatin profiling with CUT&Tag. Nat Protoc 15, 3264-83 (2020). https://doi.org/10.1038/s41596-020-0373-x.
  3. Zilionis R et al. Single-cell barcoding and sequencing using droplet microfluidics. Nat Protoc 12, 44-73 (2017). https://doi.org/10.1038/nprot.2016.154.
  4. Mazutis L et al. Single-cell analysis and sorting using droplet-based microfluidics. Nat Protoc 8, 870-91 (2013). https://doi.org/10.1038/nprot.2013.046.
  5. Buenrostro JD et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-90 (2015). https://doi.org/10.1038/nature14590.
  6. Heumos L et al. Best practices for single-cell analysis across modalities. Nat Rev Genet 24, 550-72 (2023). https://doi.org/10.1038/s41576-023-00586-w.
  7. Wu SJ et al. Single-cell CUT&Tag analysis of chromatin modifications in differentiation and tumor progression. Nat Biotechnol 39, 819-24 (2021). https://doi.org/10.1038/s41587-021-00865-z.
  8. Bartosovic M et al. Single-cell CUT&Tag profiles histone modifications and transcription factors in complex tissues. Nat Biotechnol 39, 825-35 (2021). https://doi.org/10.1038/s41587-021-00869-9.
  9. Moffitt JR et al. The emerging landscape of spatial profiling technologies. Nat Rev Genet 23, 741-59 (2022). https://doi.org/10.1038/s41576-022-00515-3.
  10. Deng Y et al. Spatial-CUT&Tag: Spatially resolved chromatin modification profiling at the cellular level. Science 375, 681-6 (2022). https://doi.org/10.1126/science.abg7216.
  11. Vitak SA et al. Sequencing thousands of single-cell genomes with combinatorial indexing. Nat Methods 14, 302-8 (2017). https://doi.org/10.1038/nmeth.4154.
  12. Amini S et al. Haplotype-resolved whole-genome sequencing by contiguity-preserving transposition and combinatorial indexing. Nat Genet 46, 1343-9 (2014). https://doi.org/10.1038/ng.3119.
  13. Cusanovich DA et al. Multiplex single cell profiling of chromatin accessibility by combinatorial cellular indexing. Science 348, 910-4 (2015). https://doi.org/10.1126/science.aab1601.
  14. Cao J et al. Comprehensive single-cell transcriptional profiling of a multicellular organism. Science 357, 661-7 (2017). https://doi.org/10.1126/science.aam8940.
  15. Mulqueen RM et al. Highly scalable generation of DNA methylation profiles in single cells. Nat Biotechnol 36, 428-31 (2018). https://doi.org/10.1038/nbt.4112.
  16. Ai S et al. Profiling chromatin states using single-cell itChIP-seq. Nat Cell Biol 21, 1164-72 (2019). https://doi.org/10.1038/s41556-019-0383-5.
  17. Mulqueen RM et al. High-content single-cell combinatorial indexing. Nat Biotechnol 39, 1574-80 (2021). https://doi.org/10.1038/s41587-021-00962-z.
  18. Janssens DH et al. Scalable single-cell profiling of chromatin modifications with sciCUT&Tag. Nat Protoc 19, 83-112 (2024). https://doi.org/10.1038/s41596-023-00905-9.
  19. Meers MP et al. Multifactorial profiling of epigenetic landscapes at single-cell resolution using MulTI-Tag. Nat Biotechnol 41, 708-16 (2023). https://doi.org/10.1038/s41587-022-01522-9.