Antibody-guided Chromatin Tagmentation (ACT-seq)

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.

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CoBATCH for high-throughput single-cell epigenomic profiling

10.1101/590661 ◽

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.

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

10.1101/571208 ◽

2019 ◽

Cited By ~ 1

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.

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Rapid and Low-Input Profiling of Histone Marks in Plants Using Nucleus CUT&Tag

Frontiers in Plant Science ◽

10.3389/fpls.2021.634679 ◽

2021 ◽

Vol 12 ◽

Author(s):

Weizhi Ouyang ◽

Xiwen Zhang ◽

Yong Peng ◽

Qing Zhang ◽

Zhilin Cao ◽

...

Keyword(s):

Histone Modifications ◽

Chromatin Immunoprecipitation ◽

Binding Sites ◽

Protein A ◽

Transcriptional Factor ◽

Experimental Procedure ◽

Histone Marks ◽

Genome Wide ◽

Efficient Alternative ◽

Tn5 Transposase

Characterizing genome-wide histone posttranscriptional modifications and transcriptional factor occupancy is crucial for deciphering their biological functions. Chromatin immunoprecipitation followed by sequencing (ChIP-seq) is a powerful method for genome-wide profiling of histone modifications and transcriptional factor-binding sites. However, the current ChIP-seq experimental procedure in plants requires significant material and several days for completion. CUT&Tag is an alternative method of ChIP-seq for low-sample and single-cell epigenomic profiling using protein A-Tn5 transposase fusion proteins (PAT). In this study, we developed a nucleus CUT&Tag (nCUT&Tag) protocol based on the live-cell CUT&Tag technology. Our results indicate that nCUT&Tag could be used for histone modifications profiling in both monocot rice and dicot rapeseed using crosslinked or fresh tissues. In addition, both active and repressive histone marks such as H3K4me3 and H3K9me2 can be identified using our nCUT&Tag. More importantly, all the steps in nCUT&Tag can be finished in only 1 day, and the assay can be performed with as little as 0.01 g of plant tissue as starting materials. Therefore, our results demonstrate that nCUT&Tag is an efficient alternative strategy for plant epigenomic studies.

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SHERRY2: A method for rapid and sensitive single cell RNA-seq

10.1101/2021.12.25.474161 ◽

2021 ◽

Author(s):

Lin Di ◽

Bo Liu ◽

Yuzhu Lyu ◽

Shihui Zhao ◽

Yuhong Pang ◽

...

Keyword(s):

Gene Expression ◽

Single Cell ◽

Dynamic Range ◽

Single Cells ◽

Rna Seq ◽

Wide Dynamic Range ◽

Uniform Coverage ◽

Optimized Protocol ◽

Tn5 Transposase ◽

Higher Sensitivity

Many single cell RNA-seq applications aim to probe a wide dynamic range of gene expression, but most of them are still challenging to accurately quantify low-aboundance transcripts. Based on our previous finding that Tn5 transposase can directly cut-and-tag DNA/RNA hetero-duplexes, we present SHERRY2, an optimized protocol for sequencing transcriptomes of single cells or single nuclei. SHERRY2 is robust and scalable, and it has higher sensitivity and more uniform coverage in comparison with prevalent scRNA-seq methods. With throughput of a few thousand cells per batch, SHERRY2 can reveal the subtle transcriptomic differences between cells and facilitate important biological discoveries.

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Antibody-guided Chromatin Tagmentation for Two or More Factors (ACT2-seq)

10.21203/rs.3.pex-1695/v1 ◽

2021 ◽

Author(s):

Benjamin Carter ◽

Wai Lim Ku ◽

Keji Zhao

Keyword(s):

Single Cell ◽

Magnetic Beads ◽

Protein A ◽

Epigenetic Marks ◽

Tn5 Transposase

Abstract This protocol details the reagents and steps required to perform antibody-guided chromatin tagmentation for two or more factors (ACT2-seq, ACT2). Like its predecessor ACT-seq, ACT2 uses a fusion of protein A and Tn5 transposase to bind and profile epigenetic marks across the genome. ACT2 builds on the capabilities of ACT-seq by directly and concurrently profiling co-occupancy of epigenetic marks, which previously required laborious, expensive, and technically challenging approaches involving fluorescence, magnetic beads, or single-cell methods. ACT2 requires only standard pipetting and centrifugation techniques and can be completed in less than a single day of bench work.

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Single-cell ATAC-seq Signal Extraction and Enhancement with SCATE

10.1101/795609 ◽

2019 ◽

Cited By ~ 1

Author(s):

Zhicheng Ji ◽

Weiqiang Zhou ◽

Hongkai Ji

Keyword(s):

Single Cell ◽

State Of The Art ◽

Single Cells ◽

Regulatory Elements ◽

Single Cell Sequencing ◽

Statistical Framework ◽

Genome Wide ◽

Cell Subpopulations ◽

Accessible Chromatin ◽

Regulatory Landscape

AbstractSingle-cell sequencing assay for transposase-accessible chromatin (scATAC-seq) is the state-of-the-art technology for analyzing genome-wide regulatory landscape in single cells. Single-cell ATAC-seq data are sparse and noisy. Analyzing such data is challenging. Existing computational methods cannot accurately reconstruct activities of individual cis-regulatory elements (CREs) in individual cells or rare cell subpopulations. We present a new statistical framework, SCATE, that adaptively integrates information from co-activated CREs, similar cells, and publicly available regulome data to substantially increase the accuracy for estimating activities of individual CREs. We show that using SCATE, one can better reconstruct the regulatory landscape of a heterogeneous sample.

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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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Topologically Dependent Abundance of Spontaneous DNA Damage in Single Human Cells

10.1101/859686 ◽

2019 ◽

Author(s):

Qiangyuan Zhu ◽

Yichi Niu ◽

Michael Gundry ◽

Kuanwei Sheng ◽

Muchun Niu ◽

...

Keyword(s):

Dna Damage ◽

Single Cell ◽

Genome Stability ◽

De Novo ◽

Single Cells ◽

Human Cells ◽

Single Nucleotide Variants ◽

Genome Wide ◽

First Time

AbstractIn the studies of single-cell genomics, the large endeavor has been focused on the detection of the permanent changes in the genome. On the other hand, spontaneous DNA damage frequently occurs and results in transient single-stranded changes to the genome until they are repaired. So far, successful profiling of these dynamic changes has not been demonstrated by single-cell whole-genome amplification methods. Here we reported a novel single-cell WGA method: Linearly Produced Semiamplicon based Split Amplification Reaction (LPSSAR), which allows, for the first time, the genome-wide detection of the DNA damage associated single nucleotide variants (dSNVs) in single human cells. The sequence-based detection of dSNVs allows the direct characterization of the major damage signature that occurred in human cells. In the analysis of the abundance of dSNVs along the genome, we observed two modules of dSNV abundance, instead of a homogeneous abundance of dSNVs. Interestingly, we found that the two modules are associated with the A/B topological compartments of the genome. This result suggests that the genome topology directly influences genome stability. Furthermore, with the detection of a large number of dSNVs in single cells, we showed that only under a stringent filtering condition, can we distinguish the de novo mutations from the dSNVs and achieve a reliable estimation of the total level of de novo mutations in a single cell.

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Genomic sequences and RNA binding proteins predict RNA splicing kinetics in various single-cell contexts

10.1101/2021.05.02.442314 ◽

2021 ◽

Author(s):

Ruiyan Hou ◽

Yuanhua Huang

Keyword(s):

Single Cell ◽

Rna Splicing ◽

Binding Proteins ◽

Rna Binding ◽

Genomic Sequence ◽

Rna Binding Proteins ◽

Single Cells ◽

Regulatory Machinery ◽

Kinetic Rates ◽

Splicing Efficiency

RNA splicing is a key step of gene expression in higher organisms. Accurate quantification of the two-step splicing kinetics is of high interests not only for understanding the regulatory machinery, but also for estimating the RNA velocity in single cells. However, the kinetic rates remain poorly understood due to the intrinsic low content of unspliced RNAs and its stochasticity across contexts. Here, we estimated the relative splicing efficiency across a variety of single-cell RNA-Seq data with scVelo. We further extracted three large feature sets including 92 basic genomic sequence features, 65,536 octamers and 120 RNA binding proteins features and found they are highly predictive to RNA splicing efficiency across multiple tissues on human and mouse. A set of important features have been identified with strong regulatory potentials on splicing efficiency. This predictive power brings promise to reveal the complexity of RNA processing and to enhance the estimation of single-cell RNA velocity.

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