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

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.

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

Nature Communications ◽

10.1038/s41467-019-09982-5 ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 120

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.

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High-throughput single-cell epigenomic profiling by targeted insertion of promoters (TIP-seq)

The Journal of Cell Biology ◽

10.1083/jcb.202103078 ◽

2021 ◽

Vol 220 (12) ◽

Author(s):

Daniel A. Bartlett ◽

Vishnu Dileep ◽

Tetsuya Handa ◽

Yasuyuki Ohkawa ◽

Hiroshi Kimura ◽

...

Keyword(s):

Rna Polymerase ◽

Single Cell ◽

Rna Polymerase Ii ◽

High Throughput ◽

Single Cells ◽

Mapping Method ◽

Ctcf Binding ◽

Linear Amplification ◽

Site Mapping ◽

Chromatin Profiling

Chromatin profiling in single cells has been extremely challenging and almost exclusively limited to histone proteins. In cases where single-cell methods have shown promise, many require highly specialized equipment or cell type–specific protocols and are relatively low throughput. Here, we combine the advantages of tagmentation, linear amplification, and combinatorial indexing to produce a high-throughput single-cell DNA binding site mapping method that is simple, inexpensive, and capable of multiplexing several independent samples per experiment. Targeted insertion of promoters sequencing (TIP-seq) uses Tn5 fused to proteinA to insert a T7 RNA polymerase promoter adjacent to a chromatin protein of interest. Linear amplification of flanking DNA with T7 polymerase before sequencing library preparation provides ∼10-fold higher unique reads per single cell compared with other methods. We applied TIP-seq to map histone modifications, RNA polymerase II (RNAPII), and transcription factor CTCF binding sites in single human and mouse cells.

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

10.1101/568915 ◽

2019 ◽

Cited By ~ 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

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.

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Efficient chromatin accessibility mapping in situ by nucleosome-tethered tagmentation

eLife ◽

10.7554/elife.63274 ◽

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.

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Efficient chromatin accessibility mapping in situ by nucleosome-tethered tagmentation

10.1101/2020.04.15.043083 ◽

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.

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High-Resolution Protein−DNA Contacts for the Yeast RNA Polymerase II General Transcription Machinery†

Biochemistry ◽

10.1021/bi048993r ◽

2004 ◽

Vol 43 (40) ◽

pp. 12741-12749 ◽

Cited By ~ 23

Author(s):

Bo-Shiun Chen ◽

Subhrangsu S. Mandal ◽

Michael Hampsey

Keyword(s):

High Resolution ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Transcription Machinery ◽

General Transcription Machinery

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High-throughput, deterministic single cell trapping and long-term clonal cell culture in microfluidic devices

Lab on a Chip ◽

10.1039/c4lc01176g ◽

2015 ◽

Vol 15 (4) ◽

pp. 1072-1083 ◽

Cited By ~ 51

Author(s):

Huaying Chen ◽

Jane Sun ◽

Ernst Wolvetang ◽

Justin Cooper-White

Keyword(s):

High Throughput ◽

Clonal Growth ◽

Single Cells ◽

Microfluidic Devices ◽

Design Development ◽

Cell Trapping ◽

Single Cell Trapping ◽

Development And Validation

In this paper, the design, development and validation of a novel high throughput microfluidic device enabling both the robust and rapid trapping of 100's to 1000's of single cells and their in situ clonal growth is described.

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