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Af-CUT&Tag: A Sensitive and Antibody-Free Chromatin Profiling Method Using Genetically Encoded Tags and High-Affinity Binders Fused to Tn5

ABSTRACT: Conventional chromatin profiling techniques are often limited by antibody availability and performance. Here, we introduce Af-CUT&Tag, a target antibody-free method that overcomes these limitations by using CRISPR-integrated peptide tags (HiBiT/ALFA-tag) recognized by engineered binders (LgBiT/NbALFA) fused to a Tn5 transposase. Af-CUT&Tag eliminates dependence on traditional target antibodies, achieving robust specificity and sensitivity with as few as 500 cells. It provides high-quality chromatin profiles, with improved signal-to-noise ratios and library quality compared with conventional antibody-based counterparts, while also enabling single-cell resolution (scAf-CUT&Tag). Applying Af-CUT&Tag to Hippo effectors (YAP1/TAZ) during liver regeneration revealed dynamic chromatin remodeling, including YAP1/TAZ-mediated control of lipid metabolism (e.g., Lpin1, Fasn) and heme clearance (Hpx, Trf). We further identify miR-122 as a critical regulator of these processes, impacting liver regeneration. The versatility of Af-CUT&Tag in cell lines, bulk tissues, and single nuclei establishes it as a powerful tool for studying gene regulation in development, disease, and regeneration. Keywords: Antibody-Free CUT&Tag; Chromatin Binding; Epigenetic Profiling; Peptide-binder; Single-cell analysis

ORGANISM(S): Mus musculus Homo sapiens

PROVIDER: GSE283706 | 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 Fused to Tn5

Project description:Conventional chromatin profiling techniques are often limited by antibody availability and performance. Here, we introduce Af-CUT&Tag, a target antibody-free method that overcomes these limitations by using CRISPR-integrated peptide tags (HiBiT/ALFA-tag) recognized by engineered binders (LgBiT/NbALFA) fused to a Tn5 transposase. Af-CUT&Tag eliminates dependence on traditional target antibodies, achieving robust specificity and sensitivity with as few as 500 cells. It provides high-quality chromatin profiles, with improved signal-to-noise ratios and library quality compared with conventional antibody-based counterparts, while also enabling single-cell resolution (scAf-CUT&Tag). Applying Af-CUT&Tag to Hippo effectors (YAP1/TAZ) during liver regeneration revealed dynamic chromatin remodeling, including YAP1/TAZ-mediated control of lipid metabolism (e.g., Lpin1, Fasn) and heme clearance (Hpx, Trf). We further identify miR-122 as a critical regulator of these processes, impacting liver regeneration. The versatility of Af-CUT&Tag in cell lines, bulk tissues, and single nuclei establishes it as a powerful tool for studying gene regulation in development, disease, and regeneration. Keywords: Antibody-Free CUT&Tag; Chromatin Binding; Epigenetic Profiling; Peptide-binder; Single-cell analysis

2024-12-15 | GSE283705 | GEO

Af-CUT&Tag: A Sensitive and Antibody-Free Chromatin Profiling Method Using Genetically Encoded Tags and High-Affinity Binders Fused to Tn5

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The two histone H2AZ variant, H2AZ.1 and H2AZ.2, differentially regulate PRC1-mediated histone H2A ubiquitinatione

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Transcriptional profiles of Tead1/4 Knockout and Unedited CT26 Cells

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CUT&Tag2for1: a modified method for simultaneous profiling of the accessible and silenced regulome in single cells

Project description:Cleavage Under Targets & Tagmentation (CUT&Tag) is an antibody-directed transposase-tethering chromatin profiling strategy for small samples and single cells. Previously, we showed that activation of tethered Tn5 transposase under low-salt conditions using antibodies that target promoters and enhancers produces high-resolution genome-wide chromatin accessibility maps. Here we show that low-salt CUT&Tag using a mixture of an antibody to the initiation form of RNA Polymerase II (Pol2 Serine-5 phosphate) and an antibody to repressive Polycomb domains (H3K27me3) followed by computational signal deconvolution produces efficient high-resolution maps of both the active and repressive regulomes. We have extended this CUT&Tag2for1 method to single cells using a novel deconvolution approach, thus producing high-quality multifactorial single-cell chromatin maps with a workflow that is identical to that for standard single-cell CUT&Tag. The ability to seamlessly map both the active and repressive regulatory elements in single cells provides a complete regulome profiling strategy suitable for high-throughput single-cell platforms.

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2020-07-15 | GSE145187 | GEO

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Project description:Methods derived from CUT&RUN and CUT&Tag enable genome-wide mapping of the localization of proteins on chromatin from as few as one cell. These and other mapping approaches focus on one protein at a time, preventing direct measurements of co-localization of different chromatin proteins in the same cells and requiring prioritization of targets where samples are limiting. Here we describe multi-CUT&Tag, an adaptation of CUT&Tag that overcomes these hurdles by using antibody-specific barcodes to simultaneously map multiple proteins in the same cells. Highly specific multi-CUT&Tag maps of histone marks and RNA Polymerase II uncovered sites of co-localization in the same cells, active and repressed genes, and candidate cis-regulatory elements. Single-cell multi-CUT&Tag profiling facilitated identification of distinct cell types from a mixed population and inference of cell type-specific gene expression. In sum, multi-CUT&Tag increases the “per cell” information content of epigenomic maps, facilitating direct analysis of the interplay of different proteins on chromatin.

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