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Nano-CUT&Tag for mu...
Nano-CUT&Tag for multimodal chromatin profiling at single-cell resolution
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- Bárcenas-Walls, José Ramón (author)
- Stockholms universitet,Institutionen för biokemi och biofysik
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Ansaloni, Federico (author)
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Herve, Bastien (author)
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Strandback, Emilia (author)
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Nyman, Tomas (author)
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Castelo-Branco, Goncalo (author)
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- Bartošovič, Marek (author)
- Stockholms universitet,Institutionen för biokemi och biofysik
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(creator_code:org_t)
- 2024
- 2024
- English.
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In: Nature Protocols. - 1754-2189 .- 1750-2799. ; 19:3, s. 791-830
- Related links:
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https://urn.kb.se/re...
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https://doi.org/10.1...
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Abstract
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- The ability to comprehensively analyze the chromatin state with single-cell resolution is crucial for understanding gene regulatory principles in heterogenous tissues or during development. Recently, we developed a nanobody-based single-cell CUT&Tag (nano-CT) protocol to simultaneously profile three epigenetic modalities-two histone marks and open chromatin state-from the same single cell. Nano-CT implements a new set of secondary nanobody-Tn5 fusion proteins to direct barcoded tagmentation by Tn5 transposase to genomic targets labeled by primary antibodies raised in different species. Such nanobody-Tn5 fusion proteins are currently not commercially available, and their in-house production and purification can be completed in 3-4 d by following our detailed protocol. The single-cell indexing in nano-CT is performed on a commercially available platform, making it widely accessible to the community. In comparison to other multimodal methods, nano-CT stands out in data complexity, low sample requirements and the flexibility to choose two of the three modalities. In addition, nano-CT works efficiently with fresh brain samples, generating multimodal epigenomic profiles for thousands of brain cells at single-cell resolution. The nano-CT protocol can be completed in just 3 d by users with basic skills in standard molecular biology and bioinformatics, although previous experience with single-cell assay for transposase-accessible chromatin using sequencing (scATAC-seq) is beneficial for more in-depth data analysis. As a multimodal assay, nano-CT holds immense potential to reveal interactions of various chromatin modalities, to explore epigenetic heterogeneity and to increase our understanding of the role and interplay that chromatin dynamics has in cellular development. This protocol involves profiling gene regulatory dynamics in complex tissues at the single-cell level. This is achieved by generating two nanobody-Tn5 fusion proteins to recognize primary antibodies against widespread histone modifications.The nanobody-Tn5 fusions are combined with ATAC-seq to simultaneously profile three epigenetic modalities from the same single cell, thousands of cells at the same time. A bioinformatic pipeline, Nanoscope, for seamless analysis of nano-CT datasets is also described. Mapping of chromatin states at single-cell resolution is still challenging. This protocol describes nano-CT, a novel method to simultaneously characterize up to three epigenetic modalities at single-cell resolution.
Subject headings
- NATURVETENSKAP -- Biologi -- Biokemi och molekylärbiologi (hsv//swe)
- NATURAL SCIENCES -- Biological Sciences -- Biochemistry and Molecular Biology (hsv//eng)
Publication and Content Type
- ref (subject category)
- art (subject category)
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