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Af-CUT&Tag: A Sensitive and Antibody-Free Chromatin Profiling Method Using Genetically Encoded Tags and High-Affinity Binders II

ABSTRACT: Transcription factors and chromatin features play critical roles in regulating gene expression. Experimental methods used for highly sensitive chromatin-binding protein profiling, such as CUT&Tag, are limited due to their high cost and technical restrictions, thereby impeding their widespread application. Therefore, we pioneered an antibody-free and sensitive tool for epigenetic analysis, Af-CUT&Tag. Application of the method requires prior generation of cell expressing a protein of interest (POI) tagged C-terminal short peptides. By leveraging the picomolar affinity between peptide and its binder, Tn5 fused with binder can specifically and highly sensitively recognize the peptide tagging protein. We show that the Af-CUT&Tag method is straightforward and can be applied to different proteins across cell lines and tissues, and extendable to single-cell analysis. With only minimal amounts of cellular or tissue material, it provides high sensitivity and yields abundant chromatin fragments per cell and offers unique insights into the regulation of early liver regeneration by the Hippo signaling pathway at the single-cell level. Together, we demonstrate that Af-CUT&Tag is a universal and comprehensive epigenetic analysis tool that can be extended to peptide and peptide-binding nanobody-Tn5 for epigenetic analysis.

ORGANISM(S): Mus musculus Homo sapiens

PROVIDER: GSE283705 | GEO | 2024/12/15

REPOSITORIES: GEO

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Af-CUT&Tag: A Sensitive and Antibody-Free Chromatin Profiling Method Using Genetically Encoded Tags and High-Affinity Binders III

Project description:Transcription factors and chromatin features play critical roles in regulating gene expression. Experimental methods used for highly sensitive chromatin-binding protein profiling, such as CUT&Tag, are limited due to their high cost and technical restrictions, thereby impeding their widespread application. Therefore, we pioneered an antibody-free and sensitive tool for epigenetic analysis, Af-CUT&Tag. Application of the method requires prior generation of cell expressing a protein of interest (POI) tagged C-terminal short peptides. By leveraging the picomolar affinity between peptide and its binder, Tn5 fused with binder can specifically and highly sensitively recognize the peptide tagging protein. We show that the Af-CUT&Tag method is straightforward and can be applied to different proteins across cell lines and tissues, and extendable to single-cell analysis. With only minimal amounts of cellular or tissue material, it provides high sensitivity and yields abundant chromatin fragments per cell and offers unique insights into the regulation of early liver regeneration by the Hippo signaling pathway at the single-cell level. Together, we demonstrate that Af-CUT&Tag is a universal and comprehensive epigenetic analysis tool that can be extended to peptide and peptide-binding nanobody-Tn5 for epigenetic analysis.

2024-12-15 | GSE283706 | GEO

Af-CUT&Tag: A Sensitive and Antibody-Free Chromatin Profiling Method Using Genetically Encoded Tags and High-Affinity Binders I

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scNanoSeq-CUT&Tag: a long-read single-cell CUT&Tag sequencing method for efficient chromatin modification profiling within individual cells

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Efficient transcription-coupled chromatin accessibility mapping in situ

Project description:Chromatin accessibility mapping is a powerful approach to identify potential regulatory elements. In the popular ATAC-seq method, Tn5 transposase inserts sequencing adapters into accessible DNA (‘tagmentation’). CUT&Tag is a tagmentation-based epigenomic profiling method in which antibody tethering of Tn5 to a chromatin epitope of interest profiles specific chromatin features in small samples and single cells. Here we show that by simply modifying the tagmentation conditions for histone H3K4me2/3 CUT&Tag, antibody-tethered tagmentation of accessible DNA sites is redirected to produce accessible DNA maps that are indistinguishable from the best ATAC-seq maps. Thus, DNA accessibility maps can be produced in parallel with CUT&Tag maps of other epitopes with all steps from nuclei to amplified sequencing-ready libraries performed in single PCR tubes in the laboratory or on a home workbench. As H3K4 methylation is produced by transcription at promoters and enhancers, our method identifies transcription-coupled accessible regulatory sites.

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Project description:We recently introduced CUT&Tag, an epigenomic profiling strategy in which antibodies are bound to chromatin proteins in situ in permeabilized nuclei, and then used to tether the cut-and-paste transposase Tn5. Activation of the transposase simultaneously cleaves DNA and adds DNA sequencing adapters (“tagmentation”) for paired-end DNA sequencing. Here, we introduce a streamlined CUT&Tag protocol that suppresses exposure artifacts to ensure high-fidelity mapping of the antibody-targeted protein and improves signal-to-noise over current chromatin profiling methods. Streamlined CUT&Tag can be performed in a single PCR tube from cells to amplified libraries, providing low-cost high-resolution genome-wide chromatin maps. By simplifying library preparation, CUT&Tag requires less than a day at the bench from live cells to sequencing-ready barcoded libraries. Because of low background levels, barcoded and pooled CUT&Tag libraries can be sequenced for ~$25 per sample, enabling routine genome-wide profiling of chromatin proteins and modifications that requires no special skills or equipment.

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