scholarly journals CUT&Tag for efficient epigenomic profiling of small samples and single cells

2019 ◽  
Author(s):  
Hatice S. Kaya-Okur ◽  
Steven J. Wu ◽  
Christine A. Codomo ◽  
Erica S. Pledger ◽  
Terri D. Bryson ◽  
...  
Keyword(s):  
High Resolution ◽  
Rna Polymerase Ii ◽  
Single Cells ◽  
Protein A ◽  
Small Samples ◽  
Live Cells ◽  
Protein Activation ◽  
Tn5 Transposase ◽  
Fragment Libraries

AbstractMany chromatin features play critical roles in regulating gene expression. A complete understanding of gene regulation will require the mapping of specific chromatin features in small samples of cells at high resolution. Here we describe Cleavage Under Targets and Tagmentation (CUT&Tag), an enzyme-tethering strategy that provides efficient high-resolution sequencing libraries for profiling diverse chromatin components. In CUT&Tag, a chromatin protein is boundin situby a specific antibody, which then tethers a protein A-Tn5 transposase fusion protein. Activation of the transposase efficiently generates fragment libraries with high resolution and exceptionally low background. All steps from live cells to sequencing-ready libraries can be performed in a single tube on the benchtop or a microwell in a high-throughput pipeline, and the entire procedure can be performed in one day. We demonstrate the utility of CUT&Tag by profiling histone modifications, RNA Polymerase II and transcription factors on low cell numbers and single cells.

scholarly journals CUT&Tag for efficient epigenomic profiling of small samples and single cells

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Hatice S. Kaya-Okur ◽  
Steven J. Wu ◽  
Christine A. Codomo ◽  
Erica S. Pledger ◽  
Terri D. Bryson ◽  
...  
Keyword(s):  
High Resolution ◽  
Rna Polymerase Ii ◽  
Single Cells ◽  
Protein A ◽  
Small Samples ◽  
Live Cells ◽  
Protein Activation ◽  
Tn5 Transposase ◽  
Fragment Libraries

Abstract Many chromatin features play critical roles in regulating gene expression. A complete understanding of gene regulation will require the mapping of specific chromatin features in small samples of cells at high resolution. Here we describe Cleavage Under Targets and Tagmentation (CUT&Tag), an enzyme-tethering strategy that provides efficient high-resolution sequencing libraries for profiling diverse chromatin components. In CUT&Tag, a chromatin protein is bound in situ by a specific antibody, which then tethers a protein A-Tn5 transposase fusion protein. Activation of the transposase efficiently generates fragment libraries with high resolution and exceptionally low background. All steps from live cells to sequencing-ready libraries can be performed in a single tube on the benchtop or a microwell in a high-throughput pipeline, and the entire procedure can be performed in one day. We demonstrate the utility of CUT&Tag by profiling histone modifications, RNA Polymerase II and transcription factors on low cell numbers and single cells.

scholarly journals Simultaneous CUT&Tag profiling of the accessible and silenced regulome in single cells

2021 ◽  
Author(s):  
Derek H Janssens ◽  
Dominik J. Otto ◽  
Manu Setty ◽  
Kami Ahmad ◽  
Steven Henikoff
Keyword(s):  
High Resolution ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
High Throughput ◽  
Single Cells ◽  
Regulatory Elements ◽  
Small Samples ◽  
Signal Deconvolution ◽  
Chromatin Profiling

Cleavage Under Targets & Tagmentation (CUT&Tag) is an antibody-directed transposase tethering strategy for in situ chromatin profiling in small samples and single cells. We describe a modified CUT&Tag protocol 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 to produce high-resolution maps of both the active and repressive regulomes in single cells. The ability to seamlessly map active promoters, enhancers and repressive regulatory elements using a single workflow provides a complete regulome profiling strategy suitable for high-throughput single-cell platforms.

scholarly journals Efficient chromatin accessibility mapping in situ by nucleosome-tethered tagmentation

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Steven Henikoff ◽  
Jorja G Henikoff ◽  
Hatice S Kaya-Okur ◽  
Kami Ahmad
Keyword(s):  
Single Cells ◽  
Regulatory Elements ◽  
Chromatin Accessibility ◽  
Small Samples ◽  
H3k4 Methylation ◽  
Powerful Approach ◽  
Tn5 Transposase ◽  
Regulatory Sites

Chromatin accessibility mapping is a powerful approach to identify potential regulatory elements. A popular example is ATAC-seq, whereby 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 or H3K4me3 CUT&Tag, antibody-tethered tagmentation of accessible DNA sites is redirected to produce chromatin accessibility maps that are indistinguishable from the best ATAC-seq maps. Thus, chromatin 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.

scholarly journals Efficient chromatin accessibility mapping in situ by nucleosome-tethered tagmentation

2020 ◽  
Author(s):  
Steven Henikoff ◽  
Jorja G. Henikoff ◽  
Hatice S. Kaya-Okur ◽  
Kami Ahmad
Keyword(s):  
Single Cells ◽  
Regulatory Elements ◽  
Chromatin Accessibility ◽  
Small Samples ◽  
H3k4 Methylation ◽  
Powerful Approach ◽  
Tn5 Transposase ◽  
Regulatory Sites

AbstractChromatin accessibility mapping is a powerful approach to identify potential regulatory elements. A popular example is ATAC-seq, whereby 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 or H3K4me3 CUT&Tag, antibody-tethered tagmentation of accessible DNA sites is redirected to produce chromatin accessibility maps that are indistinguishable from the best ATAC-seq maps. Thus, chromatin 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.

scholarly journals Multiplexed dynamic imaging of genomic loci in single cells by combined CRISPR imaging and DNA sequential FISH

2017 ◽  
Author(s):  
Yodai Takei ◽  
Sheel Shah ◽  
Sho Harvey ◽  
Lei S. Qi ◽  
Long Cai
Keyword(s):  
Single Cells ◽  
Embryonic Stem ◽  
Dynamic Imaging ◽  
Live Cells ◽  
Chromosome Dynamics ◽  
Cellular Processes ◽  
Sequential Fish ◽  
Telomere Dynamics ◽  
Subtelomeric Regions

ABSTRACTVisualization of chromosome dynamics allows the investigation of spatiotemporal chromatin organization and its role in gene regulation and other cellular processes. However, current approaches to label multiple genomic loci in live cells have a fundamental limitation in the number of loci that can be labelled and uniquely identified. Here we describe an approach we call “track first and identify later” for multiplexed visualization of chromosome dynamics by combining two techniques: CRISPR labeling and DNA sequential fluorescence in situ hybridization (DNA seqFISH). Our approach first labels and tracks chromosomal loci in live cells with the CRISPR system, then barcodes those loci by DNA seqFISH in fixed cells and resolves their identities. We demonstrate our approach by tracking telomere dynamics, identifying 12 unique subtelomeric regions with variable detection efficiencies, and tracking back the telomere dynamics of respective chromosomes in mouse embryonic stem cells.

scholarly journals Antibody-guided Chromatin Tagmentation (ACT-seq)

2019 ◽  
Author(s):  
Benjamin Carter ◽  
Keji Zhao ◽  
Wai Lim Ku ◽  
Jee Youn Kang ◽  
Qingsong Tang
Keyword(s):  
Single Cell ◽  
Specific Antibody ◽  
Binding Proteins ◽  
Single Cells ◽  
Protein A ◽  
Histone Variants ◽  
Chromatin Binding ◽  
Histone Tail ◽  
Genome Wide ◽  
Tn5 Transposase

Abstract ACT-seq is a streamlined method for mapping genome-wide distributions of histone tail modifications, histone variants, and chromatin-binding proteins in a small number of or single cells. ACT-seq utilizes a fusion of Tn5 transposase to Protein A that is targeted to chromatin by a specific antibody, allowing chromatin fragmentation and sequence tag insertion specifically at genomic sites presenting the relevant antigen. The Tn5 transposase enables the use of an index multiplexing strategy (iACT-seq), which enables construction of thousands of single-cell libraries in one day by a single researcher without the need for drop-based fluidics or visual sorting. The protocol described here is intended for use with bulk-cell samples. The single-cell iACT-seq protocol is separate.

scholarly journals A Non-Destructive, Tuneable Method to Isolate Live Cells for High-Speed AFM Analysis

2021 ◽  
Vol 9 (4) ◽  
pp. 680
Author(s):  
Christopher T. Evans ◽  
Sara J. Baldock ◽  
John G. Hardy ◽  
Oliver Payton ◽  
Loren Picco ◽  
...  
Keyword(s):  
Atomic Force Microscopy ◽  
High Resolution ◽  
High Speed ◽  
Single Cells ◽  
Sample Surface ◽  
Live Cells ◽  
Contact Mode ◽  
Force Microscopy ◽  
Atomic Force ◽  
Afm Analysis

Suitable immobilisation of microorganisms and single cells is key for high-resolution topographical imaging and study of mechanical properties with atomic force microscopy (AFM) under physiologically relevant conditions. Sample preparation techniques must be able to withstand the forces exerted by the Z range-limited cantilever tip, and not negatively affect the sample surface for data acquisition. Here, we describe an inherently flexible methodology, utilising the high-resolution three-dimensional based printing technique of multiphoton polymerisation to rapidly generate bespoke arrays for cellular AFM analysis. As an example, we present data collected from live Emiliania huxleyi cells, unicellular microalgae, imaged by contact mode High-Speed Atomic Force Microscopy (HS-AFM), including one cell that was imaged continuously for over 90 min.

scholarly journals CoBATCH for high-throughput single-cell epigenomic profiling

2019 ◽  
Author(s):  
Qianhao Wang ◽  
Haiqing Xiong ◽  
Shanshan Ai ◽  
Xianhong Yu ◽  
Yaxi Liu ◽  
...  
Keyword(s):  
Single Cell ◽  
Binding Proteins ◽  
Single Cells ◽  
Protein A ◽  
High Signal ◽  
Chromatin Binding ◽  
Genome Wide ◽  
Single Cell Profiling ◽  
Genomic Regions ◽  
Tn5 Transposase

ABSTRACTAn efficient, generalizable method for genome-wide mapping of single-cell histone modifications or chromatin-binding proteins is so far lacking. Here we develop CoBATCH, combinatorial barcoding and targeted chromatin release, for single-cell profiling of genomic distribution of chromatin-binding proteins in cell culture and tissue. Protein A in fusion to Tn5 transposase is enriched through specific antibodies to genomic regions and Tn5 generates indexed chromatin fragments ready for the library preparation and sequencing. Importantly, through a combinatorial barcoding strategy, we are able to measure epigenomic features up to tens of thousands single cells per experiment. CoBATCH produces not only high signal-to-noise features, but also ~10,000 reads per cells, allowing for efficiently deciphering epigenetic heterogeneity of cell populations and subtypes and inferring developmental histories. Thus, obviating specialized device, CoBATCH is easily deployable for any laboratories in life science and medicine.

scholarly journals Mapping Histone Modifications in Low Cell Number and Single Cells Using Antibody-guided Chromatin Tagmentation (ACT-seq)

2019 ◽  
Author(s):  
Benjamin Carter ◽  
Wai Lim Ku ◽  
Qingsong Tang ◽  
Jee Youn Kang ◽  
Keji Zhao
Keyword(s):  
Single Cells ◽  
Protein A ◽  
Histone Variants ◽  
Cell Number ◽  
Attractive Alternative ◽  
Chromatin Binding ◽  
Epigenetic Marks ◽  
Genome Wide ◽  
Tn5 Transposase ◽  
Intensive Procedures

ABSTRACTModern next-generation sequencing-based methods have empowered researchers to assay the epigenetic states of individual cells. Existing techniques for profiling epigenetic marks in single cells often require the use and optimization of time-intensive procedures such as drop fluidics, chromatin fragmentation, and end repair. Here we describe ACT-seq, a novel and streamlined method for mapping genome-wide distributions of histone tail modifications, histone variants, and chromatin-binding proteins in a small number of or single cells. ACT-seq utilizes a fusion of Tn5 transposase to Protein A that is targeted to chromatin by a specific antibody, allowing chromatin fragmentation and sequence tag insertion specifically at genomic sites presenting the relevant antigen. The Tn5 transposase enables the use of an index multiplexing strategy (iACT-seq), which enables construction of thousands of single-cell libraries in one day by a single researcher without the need for drop-based fluidics or visual sorting. We conclude that ACT-seq present an attractive alternative to existing techniques for mapping epigenetic marks in single cells.

scholarly journals Defining proximity proteomics of post-translationally modified proteins by antibody-mediated protein A-APEX2 labeling

2021 ◽  
Author(s):  
Xinran Li ◽  
Jiaqi Zhou ◽  
Wenjuan Zhao ◽  
Qing Wen ◽  
Weijie Wang ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Protein A ◽  
Protein Protein Interactions ◽  
Efficient Tool ◽  
Post Translational Modifications ◽  
Protein Activation ◽  
Genome Wide ◽  
Powerful Approach ◽  
Modified Proteins

AbstractProximity labeling catalyzed by promiscuous enzymes, such as APEX2, has emerged as a powerful approach to characterize multiprotein complexes and protein-protein interactions. However, current methods depend on the expression of exogenous fusion proteins and cannot be applied to post-translational modifications. To address this limitation, we developed a new method to label proximal proteins of interest by antibody-mediated protein A-APEX2 labeling (AMAPEX). In this method, a modified protein is bound in situ by a specific antibody, which then tethers a protein A-APEX2 (pA-APEX2) fusion protein. Activation of APEX2 labels the nearby proteins with biotin; these proteins are then purified using streptavidin beads and are identified by mass spectrometry. We demonstrate the utility of this approach by profiling the binding proteins of histone modifications including H3K27me3, H3K9me3, H3K4me3, H4K5ac and H4K12ac, and we verified the genome-wide colocalization of these identified proteins with bait proteins by published ChIP-seq analysis. Overall, AMAPEX is an efficient tool to identify proteins that interact with modified proteins.

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