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 Preparation of optimized concanavalin A-conjugated Dynabeads® magnetic beads for CUT&Tag

PLoS ONE ◽  
2021 ◽  
Vol 16 (11) ◽  
pp. e0259846
Author(s):  
Yasuhiro Fujiwara ◽  
Yuji Tanno ◽  
Hiroki Sugishita ◽  
Yusuke Kishi ◽  
Yoshinori Makino ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Concanavalin A ◽  
Magnetic Particles ◽  
Magnetic Beads ◽  
Protein A ◽  
Con A ◽  
Established Method ◽  
Genomic Location ◽  
Tn5 Transposase ◽  
Selection Of

Epigenome research has employed various methods to identify the genomic location of proteins of interest, such as transcription factors and histone modifications. A recently established method called CUT&Tag uses a Protein-A Tn5 transposase fusion protein, which cuts the genome and inserts adapter sequences nearby the target protein. Throughout most of the CUT&Tag procedure, cells are held on concanavalin A (con A)-conjugated magnetic beads. Proper holding of cells would be decisive for the accessibility of Tn5 to the chromatin, and efficacy of the procedure of washing cells. However, BioMag®Plus ConA magnetic beads, used in the original CUT&Tag protocol, often exhibit poor suspendability and severe aggregation. Here, we compared the BioMag beads and Dynabeads® magnetic particles of which conjugation of con A was done by our hands, and examined the performance of these magnetic beads in CUT&Tag. Among tested, one of the Dynabeads, MyOne-T1, kept excessive suspendability in a buffer even after overnight incubation. Furthermore, the MyOne-T1 beads notably improved the sensitivity in CUT&Tag assay for H3K4me3. In conclusion, the arrangement and the selection of MyOne-T1 refine the suspendability of beads, which improves the association of chromatin with Tn5, which enhances the sensitivity in CUT&Tag assay.

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 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 Preparation of "stress-free" concanavalin A-conjugated Dynabeads magnetic beads for CUT&Tag

2021 ◽  
Author(s):  
Yasuhiro Fujiwara ◽  
Yuji Tanno ◽  
Hiroki Sugishita ◽  
Yusuke Kishi ◽  
Yoshinori Makino ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Concanavalin A ◽  
Magnetic Particles ◽  
Magnetic Beads ◽  
Protein A ◽  
Con A ◽  
Established Method ◽  
Genomic Location ◽  
Tn5 Transposase ◽  
Selection Of

Epigenome research has employed various methods to identify genomic location of proteins of interest, such as transcription factors and histone modifications. A recently established method called CUT&Tag uses a Protein-A Tn5 transposase fusion protein, which cuts the genome and inserts adapter sequences nearby the target protein. Throughout most of the CUT&Tag procedure, cells are held on concanavalin A (con A)-conjugated magnetic beads. Proper holding of cells would be decisive for the accessibility of Tn5 to the chromatin, and efficacy of the procedure of washing cells. However, BioMagPlus ConA magnetic beads, used in the original CUT&Tag protocol, often exhibit poor suspendability and severe aggregation. Here, we compared the BioMag beads and Dynabeads magnetic particles of which conjugation of con A was done by our hands, and examined the performance of these magnetic beads in CUT&Tag. Among tested, one of the Dynabeads, MyOne-T1, kept excessive suspendability in a buffer even after overnight incubation. Furthermore, the MyOne-T1 beads notably improved the sensitivity in CUT&Tag assay for H3K4me3. In conclusion, the arrangement and the selection of MyOne-T1 refine the suspendability of beads, which improves the association of chromatin with Tn5, which enhances the sensitivity in CUT&Tag assay.

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

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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