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

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.

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

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.

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 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 Methylome-wide analysis reveals epigenetic marks associated with resistance to tuberculosis in HIV-infected individuals from East Africa

2020 ◽  
Author(s):  
Catherine Stein ◽  
Penelope Bencheck ◽  
Jacquelaine Bartlett ◽  
Robert P Igo ◽  
Rafal S Sobota ◽  
...  
Keyword(s):  
Genome Wide Association Study ◽  
Sub Saharan Africa ◽  
Chromosome 1 ◽  
Chromosome 2 ◽  
Chromosome 5 ◽  
Epigenetic Marks ◽  
Genome Wide ◽  
A Genome ◽  
Immunodeficiency Virus ◽  
Sub Saharan

Background: Tuberculosis (TB) is the most deadly infectious disease globally and highly prevalent in the developing world, especially sub-Saharan Africa. Even though a third of humans are exposed to Myocbacterium tuberculosis (Mtb), most infected immunocompetent individuals do not develop active TB. In contrast, for individuals infected with both TB and the human immunodeficiency virus (HIV), the risk of active disease is 10% or more per year. Previously, we identified in a genome-wide association study a region on chromosome 5 that was associated with resistance to TB. This region included epigenetic marks that could influence gene regulation so we hypothesized that HIV-infected individuals exposed to Mtb, who remain disease free, carry epigenetic changes that strongly protect them from active TB. To test this hypothesis, we conducted a methylome-wide study in HIV-infected, TB-exposed cohorts from Uganda and Tanzania. Results: In 221 HIV-infected adults from Uganda and Tanzania, we identified 3 regions of interest that included markers that were differentially methylated between TB cases and LTBI controls, that also included methylation QTLs and associated SNPs: chromosome 1 (RNF220, p=4x10-5), chromosome 2 (between COPS8 and COL6A3 genes, p=2.7x10-5), and chromosome 5 (CEP72, p=1.3x10-5). These methylation results colocalized with associated SNPs, methylation QTLs, and methylation x SNP interaction effects. These markers were in regions with regulatory markers for cells involved in TB immunity and/or lung. Conclusion: Epigenetic regulation is a potential biologic factor underlying resistance to TB in immunocompromised individuals that can act in conjunction with genetic variants.

scholarly journals Nascent chromatin occupancy profiling reveals locus and factor specific chromatin maturation dynamics behind the DNA replication fork

2018 ◽  
Author(s):  
Mónica P. Gutiérrez ◽  
Heather K. MacAlpine ◽  
David M. MacAlpine
Keyword(s):  
Dna Replication ◽  
Replication Fork ◽  
Histone Variants ◽  
Replication Forks ◽  
Nucleosome Assembly ◽  
Genome Wide ◽  
Dna Binding Factors ◽  
Nucleotide Resolution ◽  
Kinetics Of ◽  
Regulatory Landscape

AbstractProper regulation and maintenance of the epigenome is necessary to preserve genome function. However, in every cell division, the epigenetic state is disassembled and then re-assembled in the wake of the DNA replication fork. Chromatin restoration on nascent DNA is a complex and regulated process that includes nucleosome assembly and remodeling, deposition of histone variants, and the re-establishment of transcription factor binding. To study the genome-wide dynamics of chromatin restoration behind the DNA replication fork, we developed Nascent Chromatin Occupancy Profiles (NCOPs) to comprehensively profile nascent and mature chromatin at nucleotide resolution. While nascent chromatin is inherently less organized than mature chromatin, we identified locus specific differences in the kinetics of chromatin maturation that were predicted by the epigenetic landscape, including the histone variant H2A.Z which marked loci with rapid maturation kinetics. The chromatin maturation at origins of DNA replication was dependent on whether the origin underwent initiation or was passively replicated from distal-originating replication forks suggesting distinct chromatin assembly mechanisms between activated and disassembled pre-replicative complexes. Finally, we identified sites that were only occupied transiently by DNA-binding factors following passage of the replication fork which may provide a mechanism for perturbations of the DNA replication program to shape the regulatory landscape of the genome.

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