Explore new genomic applications with multiomic CUT&Tag

Background: Multiomics in chromatin profiling

In the field of epigenetics, “multiomics” typically refers to RNA and DNA sequencing from the same cell or sample type. Simultaneous profiling of the transcriptome and regulatory chromatin features, such as histone post-translational modifications (PTMs), has been a considerable challenge in epigenomics. Historically, researchers had to integrate RNA-seq and chromatin profiling data generated from separate cell samples, which can obscure direct links between gene expression, chromatin state, and cell identity. Chromatin immunoprecipitation sequencing (ChIP-seq), the leading chromatin mapping assay, was also notoriously unreliable and required high cell numbers, further impeding multiomic analysis.

Fortunately, advances in next-generation sequencing technology were soon met with an equally sensitive chromatin mapping strategy: ATAC-seq, assay for transposase-accessible chromatin using sequencing⁵. ATAC-seq uses a hyperactive Tn5 transposase to identify accessible regions of chromatin, thus providing a general overview of chromatin structure and identifying potential regulatory factors⁶. The use of Tn5 bypasses some of the most difficult and time-consuming aspects of ChIP-seq, including cross-linking, chromatin fragmentation, and library prep, all of which enables the use of extremely low cell numbers. ATAC-seq opened the door to analysis of rare cell types and precious patient samples, making it possible to leverage epigenomics for clinical research.

The development of single-cell ATAC-seq also drove major transformations in multiomics⁷. In the past decade, scientists have leveraged droplet-based and combinatorial indexing strategies to merge single-cell RNA-seq and ATAC-seq analysis, allowing researchers to examine multiple modalities from individual cells⁸. ATAC-seq has also been combined with DNA methylation analysis⁹, high-resolution microscopy¹⁰, and spatial profiling¹¹.

Single-cell and/or multiomic ATAC-seq assays have helped define new cell types, such as exhausted T cells¹², and understand the molecular complexity underlying complex metabolic diseases, including Type 2 Diabetes^13-15. However, the ability to pair these analyses with deeper profiling of histone PTMs and relevant chromatin-associated proteins was still reliant on ChIP-seq. Clearly, newer chromatin mapping strategies were needed!

Development of CUT&Tag and integration with RNA-seq

This is where CUT&Tag (Cleavage Under Targets and Tagmentation) comes into the story. In CUT&Tag, the Tn5 enzyme from ATAC-seq is fused to protein A/G (pAG-Tn5), resulting in selective integration of sequencing adapters at antibody-bound chromatin in intact nuclei^16,17. Similar to ATAC-seq, CUT&Tag bypasses the most challenging aspects of ChIP-seq, as there is no cross-linking, cell lysis, or chromatin fragmentation. In EpiCypher’s one-tube CUT&Tag protocol, tagmented fragments are directly PCR-amplified from the reaction mixture, further streamlining the workflow. Background is greatly reduced compared to ChIP-seq, despite using 100-fold fewer cells and 10-fold lower sequencing depths.

CUT&Tag, like ATAC-seq, is ideal for single-cell profiling¹⁷. Indeed, because CUT&Tag uses the same Tn5 technology as ATAC-seq, many of the single-cell ATAC-seq platforms – including multiomic strategies – can be leveraged for CUT&Tag.

To this point, Zhu et al. utilized CUT&Tag, RNA-seq, and combinatorial indexing to create Paired-Tag, which can profile histone PTMs and RNA at single-cell resolution. This innovative approach offers a comprehensive view of the regulatory landscape by combining transcriptomic and epigenomic data, enabling deeper insights into gene regulation and cellular heterogeneity.

Zhu et al. Joint profiling of histone modifications and transcriptome in single cells from mouse brain. Nature Methods 2021.

• Application: Development and proof-of-concept for Paired-Tag; distinguish cell types across multiple mouse brain tissues.
• Cell types: Adult mouse brain tissue (snap-frozen frontal cortex and hippocampus), human HeLa cells.
• Targets: H3K4me1, H3K4me3, H3K9me3, H3K27ac, and H3K27me3 (in tandem with RNA-seq).
• Significance of Study: Showcases the versatility of Tn5 and CUT&Tag for the joint study of chromatin structure and transcription – a key focus area in multiomics! Expect similar applications in the future.

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Mapping multiple chromatin targets with CUT&Tag

Profiling multiple chromatin targets from a single cell or small cell sample can unlock a wealth of information about transcriptional regulation and crosstalk between different genomic features. This type of multiomic analysis is impossible with ChIP-seq, due to the inherent noise and high cell input requirements. Incubation with multiple target-specific antibodies in ChIP-seq also poses a challenge: how can users know which reads are assigned to a given target?

This is where the versatility of pAG-Tn5 shines. Two groups have devised CUT&Tag-based methods that simultaneously map multiple histone PTMs within the same reaction, providing a comprehensive view of the chromatin landscape. In both strategies (Multi-CUT&Tag and MulTI-Tag), primary antibodies are associated with a distinct pAG-Tn5-adapter complex, allowing users to map two or more targets in a single sample. Sequencing reads are assigned to an antibody target via the appended adapter barcode, allowing for true multiomic profiling. Combined, these approaches enhance our understanding of gene regulation and shed light on the dynamic interplay between different epigenetic modifications.

Gopalan et al. Simultaneous profiling of multiple chromatin proteins in the same cells. Molecular Cell 2021.

• Application: Mouse embryonic stem cells (single cell and bulk), mouse trophoblast stem cells.
• Cell types: Mouse embryos (embryonic day 11), mouse brains (postnatal day 21).
• Targets: H3K27ac, H3K27me3, RNA Pol II phospho Ser2.
• Significance of study: The integration of histone PTM mapping with RNA Pol II profiling allows scientists to probe discrete mechanisms underlying transcriptional regulation, examine cell-type specific cis-regulatory elements, and define cell fate. This type of work is impossible with ChIP-seq and other next-generation mapping technologies! Expect applications across developmental biology, immunology, neuroscience, cancer research, and beyond.

Meers et al. Multifactorial profiling of epigenetic landscapes at single-cell resolution using MulTI-Tag. Nature Biotechnology 2022.

• Application: Development and proof-of-concept for MulTI-Tag; examined developmental trajectories during human embryonic stem cell differentiation in vitro.
• Cell types: Human K562 cells, H1 human embryonic stem cells, differentiated H1 hESCs (ectoderm, endoderm, mesoderm), mouse NIH3T3 cells.
• Targets: H3K4me2, H3K27me3, H3K36me3, RNA Pol II phospho Ser 5.
• Significance of study: Combined with work from Gopalan, this paper underscores the true flexibility of CUT&Tag and pAG-Tn5 for innovative platform development. Scientists now have multiple options for multiomic chromatin profiling analysis, which will open more doors to biomarker and therapeutic discovery.

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Summary and future multiomic CUT&Tag applications

CUT&Tag, with its ability to profile multiple histone modifications and other chromatin features alongside transcriptome profiling, has become a key tool in multiomics. Its adaptability when combined with single-cell technologies offers unprecedented insights into the epigenetic landscape and the dynamic interplay between chromatin states and gene expression.

Future integrations of CUT&Tag will provide deeper insights into regulatory mechanisms governing cellular functions and disease development. For example, the development of CUT&Tag-based chromatin accessibility assays (CUTAC) has allowed researchers to probe histone modifications and active regulatory regions in FFPE-embedded tissues^18,19, a feat that has been incredibly difficult to accomplish even with highly sensitive ATAC-seq approaches. CUT&Tag has also been leveraged in several studies for spatial profiling, which preserves tissue architecture in addition to providing high-resolution, single-cell chromatin profiling – see this blog to learn more.

Of course, multiomics encompasses dozens of other strategies, and many have been paired with ATAC-seq or Tn5 in previous papers. Drawing on these reports, other CUT&Tag multiomic applications include:

• DNA methylation (e.g. ATAC-Me¹⁹)
• Spatial profiling (e.g. Spatial CUT&Tag²⁰)
• Protein levels (e.g. PHAGE-ATAC²¹)
• Lineage tracing²²

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Tools to get started with CUT&Tag assay development

At EpiCypher, we are committed to advancing CUT&Tag technology and making innovative pAG-Tn5 enzymes accessible to researchers. Our IDEA Toolbox includes a variety of high-quality pAG-Tn5 enzymes, including uncharged His-pAG-Tn5 needed for Multi-CUT&Tag experiments. We also offer custom-loading Tn5 services, which are ideal for combinatorial indexing experiments. Together, these tools provide a robust foundation for CUT&Tag application development.

• His-pAG-Tn5, uncharged (EpiCypher 15-1028)
• pAG-Tn5, uncharged (EpiCypher 15-1025)
• pAG-Tn5, loaded with standard sequencing adapters (EpiCypher 15-1017 and 15-1117)
• Fluorescent pAG-Tn5, loaded with Cy5-tagged sequencing adapters (EpiCypher 15-1026)
• Create a custom loaded pAG-Tn5 library – contact our genomics team at services@epicypher.com
• Click here for all other CUT&Tag reagents – including kits, enzymes, antibodies, and more!

Stay tuned for additional blogs about CUT&Tag applications, including CUTAC-FFPE profiling of biobanked patient samples. If you are interested in custom Tn5 loading services or need a product quote, fill out the form below.

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

• Zhang et al. Characterizing cellular heterogeneity in chromatin state with scCUT&Tag-pro. Nature Biotechnology 2022.
• Bartosovic & Castelo-Branco. Multimodal chromatin profiling using nanobody-based single-cell CUT&Tag. Nature Biotechnology 2022.
• Janssens et al. CUT&Tag2for1: a modified method for simultaneous profiling of the accessible and silenced regulome in single cells. Genome Biology 2022.
• Sati et al. HiCuT: An efficient and low input method to identify protein-directed chromatin interactions. PLoS Genetics 2022.
• Cameron et al. Mapping the genomic landscape of CRISPR–Cas9 cleavage. Nature Methods 2017.

References

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2. Vandereyken K et al. Methods and applications for single-cell and spatial multi-omics. Nat Rev Genet 24, 494-515 (2023). https://doi.org/10.1038/s41576-023-00580-2.
3. Kreitmaier P et al. Insights from multi-omics integration in complex disease primary tissues. Trends Genet 39, 46-58 (2023). https://doi.org/10.1016/j.tig.2022.08.005.
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6. Li Z et al. Identification of transcription factor binding sites using ATAC-seq. Genome Biol 20, 45 (2019). https://doi.org/10.1186/s13059-019-1642-2.
7. 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.
8. Zhu C et al. An ultra high-throughput method for single-cell joint analysis of open chromatin and transcriptome. Nat Struct Mol Biol 26, 1063-70 (2019). https://doi.org/10.1038/s41594-019-0323-x.
9. Barnett KR et al. ATAC-Me Captures Prolonged DNA Methylation of Dynamic Chromatin Accessibility Loci during Cell Fate Transitions. Mol Cell 77, 1350-64.e6 (2020). https://doi.org/10.1016/j.molcel.2020.01.004.
10. Chen X et al. ATAC-see reveals the accessible genome by transposase-mediated imaging and sequencing. Nat Methods 13, 1013-20 (2016). https://doi.org/10.1038/nmeth.4031.
11. 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.
12. Belk JA et al. Epigenetic regulation of T cell exhaustion. Nat Immunol 23, 848-60 (2022). https://doi.org/10.1038/s41590-022-01224-z.
13. Chiou J et al. Single-cell chromatin accessibility identifies pancreatic islet cell type- and state-specific regulatory programs of diabetes risk. Nat Genet 53, 455-66 (2021). https://doi.org/10.1038/s41588-021-00823-0.
14. Rai V et al. Single-cell ATAC-Seq in human pancreatic islets and deep learning upscaling of rare cells reveals cell-specific type 2 diabetes regulatory signatures. Mol Metab 32, 109-21 (2020). https://doi.org/10.1016/j.molmet.2019.12.006.
15. Ackermann AM et al. Integration of ATAC-seq and RNA-seq identifies human alpha cell and beta cell signature genes. Mol Metab 5, 233-44 (2016). https://doi.org/10.1016/j.molmet.2016.01.002.
16. 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.
17. 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.
18. Henikoff S et al. Epigenomic analysis of formalin-fixed paraffin-embedded samples by CUT&Tag. Nat Commun 14, 5930 (2023). https://doi.org/10.1038/s41467-023-41666-z.
19. Henikoff S et al. Direct measurement of RNA Polymerase II hypertranscription in cancer FFPE samples. bioRxiv 2024.02.28.582647 (2024). https://doi.org/10.1101/2024.02.28.582647.
20. 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.
21. Fiskin E et al. Single-cell profiling of proteins and chromatin accessibility using PHAGE-ATAC. Nat Biotechnol 40, 374-81 (2022). https://doi.org/10.1038/s41587-021-01065-5.
22. Lareau CA et al. Massively parallel single-cell mitochondrial DNA genotyping and chromatin profiling. Nat Biotechnol 39, 451-61 (2021). https://doi.org/10.1038/s41587-020-0645-6.