SNARE-seq2 v2

2021 ◽  
Author(s):  
Nongluk Plongthongkum ◽  
Dinh H Diep ◽  
Song Chen ◽  
Blue Lake ◽  
Kun Zhang
Keyword(s):  
Gene Expression ◽  
High Throughput ◽  
Chromatin Accessibility ◽  
Single Experiment ◽  
Data Set ◽  
Whole Cells ◽  
High Throughput Method ◽  
Single Nucleus ◽  
Accessible Chromatin ◽  
Entire Procedure

To study the heterogeneity of complex tissues by joint profiling of gene expression and its regulation, we require an accurate and high-throughput method. Here we described improved high-throughput combinatorial indexing-based single-nucleus chromatin accessibility and mRNA expression sequencing 2 (SNARE-Seq2) co-assay. This protocol involves fixing and permeabilizing the nucleus followed by tagmentation, chromatin barcode ligation, reverse transcription, pooling and splitting for the next rounds of cell barcode ligation into cDNA and accessible chromatin (AC) on the same nucleus. The captured cDNA and AC are co-amplified before splitting and enrichment into single-nucleus RNA and single-nucleus AC sequencing libraries. The protocol can also be applied to both nuclei and whole cells to capture mRNA in the cytoplasm. This improvement allows us to generate hundreds of thousands of data set of each assay and can be scaled up to half a million cells from a single experiment. The entire procedure can be complete in 3.5 d for generating joint single-nucleus RNA and single-nucleus ATAC sequencing libraries.

SNARE-seq2 v2

2021 ◽  
Author(s):  
Nongluk Plongthongkum ◽  
Dinh H Diep ◽  
Song Chen ◽  
Blue Lake ◽  
Kun Zhang
Keyword(s):  
Gene Expression ◽  
High Throughput ◽  
Chromatin Accessibility ◽  
Single Experiment ◽  
Data Set ◽  
Whole Cells ◽  
High Throughput Method ◽  
Single Nucleus ◽  
Accessible Chromatin ◽  
Entire Procedure

To study the heterogeneity of complex tissues by joint profiling of gene expression and its regulation, we require an accurate and high-throughput method. Here we described improved high-throughput combinatorial indexing-based single-nucleus chromatin accessibility and mRNA expression sequencing 2 (SNARE-Seq2) co-assay. This protocol involves fixing and permeabilizing the nucleus followed by tagmentation, chromatin barcode ligation, reverse transcription, pooling and splitting for the next rounds of cell barcode ligation into cDNA and accessible chromatin (AC) on the same nucleus. The captured cDNA and AC are co-amplified before splitting and enrichment into single-nucleus RNA and single-nucleus AC sequencing libraries. The protocol can also be applied to both nuclei and whole cells to capture mRNA in the cytoplasm. This improvement allows us to generate hundreds of thousands of data set of each assay and can be scaled up to half a million cells from a single experiment. The entire procedure can be complete in 3.5 d for generating joint single-nucleus RNA and single-nucleus ATAC sequencing libraries.

SNARE-seq2 v2

2021 ◽  
Author(s):  
Nongluk Plongthongkum ◽  
Dinh H Diep ◽  
Song Chen ◽  
Blue Lake ◽  
Kun Zhang
Keyword(s):  
Gene Expression ◽  
High Throughput ◽  
Chromatin Accessibility ◽  
Single Experiment ◽  
Data Set ◽  
Whole Cells ◽  
High Throughput Method ◽  
Single Nucleus ◽  
Accessible Chromatin ◽  
Entire Procedure

To study the heterogeneity of complex tissues by joint profiling of gene expression and its regulation, we require an accurate and high-throughput method. Here we described improved high-throughput combinatorial indexing-based single-nucleus chromatin accessibility and mRNA expression sequencing 2 (SNARE-Seq2) co-assay. This protocol involves fixing and permeabilizing the nucleus followed by tagmentation, chromatin barcode ligation, reverse transcription, pooling and splitting for the next rounds of cell barcode ligation into cDNA and accessible chromatin (AC) on the same nucleus. The captured cDNA and AC are co-amplified before splitting and enrichment into single-nucleus RNA and single-nucleus AC sequencing libraries. The protocol can also be applied to both nuclei and whole cells to capture mRNA in the cytoplasm. This improvement allows us to generate hundreds of thousands of data set of each assay and can be scaled up to half a million cells from a single experiment. The entire procedure can be complete in 3.5 d for generating joint single-nucleus RNA and single-nucleus ATAC sequencing libraries.

Single nucleus RNA-sequencing: how it's done, applications and limitations

2021 ◽  
Author(s):  
Juliane Fischer ◽  
Thomas Ayers
Keyword(s):  
Gene Expression ◽  
Rna Sequencing ◽  
High Throughput ◽  
Isolated Nuclei ◽  
Whole Cells ◽  
Single Nucleus

Single nuclei RNA-sequencing (sNuc-Seq) is a methodology which uses isolated nuclei instead of whole cells to profile gene expression. By using droplet microfluidic technologies, users are able to profile thousands of single transcriptomes at high throughput from their chosen tissue. This article aims to introduce sNuc-Seq as a method and its utility in multiple tissue types. Furthermore, we discuss the risks associated with the use of nuclei, which must be considered before committing to a methodology.

scholarly journals ATAC-seq with unique molecular identifiers improves quantification and footprinting

2020 ◽  
Vol 3 (1) ◽  
Author(s):  
Tao Zhu ◽  
Keyan Liao ◽  
Rongfang Zhou ◽  
Chunjiao Xia ◽  
Weibo Xie
Keyword(s):  
Transcription Factor ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Pcr Amplification ◽  
Chromatin Accessibility ◽  
Link Type ◽  
Pcr Duplicates ◽  
Identify Transcription Factor ◽  
Accessible Chromatin ◽  
Accurate Quantification

AbstractATAC-seq (Assay for Transposase-Accessible Chromatin with high-throughput sequencing) provides an efficient way to analyze nucleosome-free regions and has been applied widely to identify transcription factor footprints. Both applications rely on the accurate quantification of insertion events of the hyperactive transposase Tn5. However, due to the presence of the PCR amplification, it is impossible to accurately distinguish independently generated identical Tn5 insertion events from PCR duplicates using the standard ATAC-seq technique. Removing PCR duplicates based on mapping coordinates introduces increasing bias towards highly accessible chromatin regions. To overcome this limitation, we establish a UMI-ATAC-seq technique by incorporating unique molecular identifiers (UMIs) into standard ATAC-seq procedures. UMI-ATAC-seq can rescue about 20% of reads that are mistaken as PCR duplicates in standard ATAC-seq in our study. We demonstrate that UMI-ATAC-seq could more accurately quantify chromatin accessibility and significantly improve the sensitivity of identifying transcription factor footprints. An analytic pipeline is developed to facilitate the application of UMI-ATAC-seq, and it is available at https://github.com/tzhu-bio/UMI-ATAC-seq.

scholarly journals Low-input ATAC&mRNA-Seq: a simple and robust method for simultaneous dual-omics profiling with low cell number

2021 ◽  
Author(s):  
Ruifang Li ◽  
Sara A Grimm ◽  
Paul A Wade
Keyword(s):  
Gene Expression ◽  
Strong Association ◽  
Regulation Of Gene Expression ◽  
Cell Number ◽  
Chromatin Accessibility ◽  
Robust Method ◽  
Data Types ◽  
Single Experiment ◽  
Low Input ◽  
Input Material

AbstractDeciphering epigenetic regulation of gene expression requires measuring the epigenome and transcriptome jointly. Single-cell multi-omics technologies have been developed for concurrent profiling of chromatin accessibility and gene expression. However, multi-omics profiling of low-input bulk samples remains challenging. Therefore, we developed low-input ATAC&mRNA-seq, a simple and robust method for studying the role of chromatin structure in gene regulation in a single experiment with thousands of cells, to maximize insights from limited input material by obtaining ATAC-seq and mRNA-seq data simultaneously from the same cells with data quality comparable to conventional mono-omics assays. Integrative data analysis revealed similar strong association between promoter accessibility and gene expression using the data of low-input ATAC&mRNA-seq as using single-assay data, underscoring the accuracy and reliability of our dual-omics assay to generate both data types simultaneously with just thousands of cells. We envision our method to be widely applied in many biological disciplines with limited materials.

scholarly journals Quantifying epigenetic modulation of nucleosome breathing by high-throughput AFM imaging

2021 ◽  
Author(s):  
Sebastian Ferdinand Konrad ◽  
Willem Vanderlinden ◽  
Jan Lipfert
Keyword(s):  
High Throughput ◽  
Single Molecule ◽  
Mode Of Action ◽  
Chromatin Accessibility ◽  
Wild Type ◽  
Analysis Pipeline ◽  
Data Set ◽  
Single Molecule Level ◽  
Afm Imaging ◽  
Conformational Landscape

Nucleosomes are the basic units of chromatin and critical to the storage and expression of eukaryotic genomes. Chromatin accessibility and gene readout are heavily regulated by epigenetic marks of which post-translational modifications of histones play a key role. However, the mode of action and the structural implications on the single-molecule level of nucleosomes is often still poorly understood. Here, we apply a high-throughput AFM imaging and analysis pipeline to investigate the conformational landscape of the nucleosome variants H3K36me3, H3S10phos and H4K5/8/12/16ac. Our data set of >25,000 nucleosomes reveals nucleosomal unwrapping steps corresponding to 5 bp DNA. We find that H3K36me3 nucleosomes unwrap significantly more than wild type nucleosomes and additionally unwrap stochastically from both sides similar to CENP-A nucleosomes and in contrast to the highly anti-cooperative unwrapping of wild type nucleosomes. Nucleosomes with H3S10phos or H4K5/8/12/16ac modifications show unwrapping populations similar to wild type nucleosomes and also retain the same level of anti-cooperativity. Our findings help putting the mode of action of these modifications into context: While H3K36me3 likely partially acts by directly affecting nucleosome structure on the single-molecule level, H3S10phos and H4K5/8/12/16ac must predominantly act through higher-order processes. Our analysis pipeline is readily applicable to other nucleosome variants and will facilitate future high-resolution studies of the conformational landscape of nucleoprotein complexes.

Genomic and epigenomic active vitamin Dresponses in human colonic organoids

2021 ◽  
Author(s):  
Jinchao Li ◽  
David Witonsky ◽  
Emily Sprague ◽  
Dereck Alleyne ◽  
Margaret C Bielski ◽  
...  
Keyword(s):  
Gene Expression ◽  
Vitamin D ◽  
Differentially Expressed Genes ◽  
Chromatin Accessibility ◽  
Differentially Expressed ◽  
Active Vitamin ◽  
Host Environment ◽  
Genome Wide ◽  
Ideal System ◽  
Accessible Chromatin

Background & Aims: Active vitamin D, 1α,25(OH)2D3, is a nuclear hormone with roles in colonic homeostasis and carcinogenesis; yet, mechanisms underlying these effects are incompletely understood. Organoids are an ideal system to study genomic and epigenomic host-environment interactions. We utilize colonic organoids to measure 1α,25(OH)2D3 responses on genome-wide gene expression and chromatin accessibility over time. Methods: Human colonic organoids were treated in triplicate with 100nM 1α,25(OH)2D3 or vehicle control for 4 and 18 hours (h) for chromatin accessibility, and 6 and 24h for gene expression. ATAC- and RNA-sequencing were performed. Differentially accessible peaks were analyzed using DiffBind and EdgeR; differentially expressed genes were analyzed using DESeq2. Motif enrichment was determined using HOMER. Results: At 6h and 24h, 2870 and 2721 differentially expressed genes, respectively (false discovery rate, FDR<5%) were identified with overall stronger responses with 1α,25(OH)2D3. Vitamin D treatment led to stronger chromatin accessibility especially at 4h. The vitamin D receptor (VDR) motif was strongly enriched among accessible chromatin peaks with 1α,25(OH)2D3 treatment accounting for 30.5% and 11% of target sequences at 4h and 18h, respectively (FDR<1%). Genes such as CYP24A1, FGF19, MYC, FOS and TGFBR2 showed significant transcriptional and chromatin accessibility responses to 1α,25(OH)2D3 treatment with accessible chromatin located distant from promoters for some gene regions. Conclusions: Assessment of chromatin accessibility and transcriptional responses to 1α,25(OH)2D3 yielded new observations about vitamin D genome-wide effects in the colon facilitated by application of human colonic organoids. This framework can be applied to study host-environment interactions between individuals and populations in future.

scholarly journals Chromatin Accessibility Is Associated with Artemisinin Biosynthesis Regulation in Artemisia annua

Molecules ◽  
2021 ◽  
Vol 26 (4) ◽  
pp. 1194
Author(s):  
Limeng Zhou ◽  
Yingzhang Huang ◽  
Qi Wang ◽  
Dianjing Guo
Keyword(s):  
Gene Expression ◽  
Artemisia Annua ◽  
Glandular Trichomes ◽  
Regulation Of Gene Expression ◽  
Glandular Trichome ◽  
Chromatin Accessibility ◽  
Biosynthetic Genes ◽  
Nuclear Molecules ◽  
Accessible Chromatin ◽  
Artemisinin Biosynthesis

Glandular trichome (GT) is the dominant site for artemisinin production in Artemisia annua. Several critical genes involved in artemisinin biosynthesis are specifically expressed in GT. However, the molecular mechanism of differential gene expression between GT and other tissue types remains elusive. Chromatin accessibility, defined as the degree to which nuclear molecules are able to interact with chromatin DNA, reflects gene expression capacity to a certain extent. Here, we investigated and compared the landscape of chromatin accessibility in Artemisia annua leaf and GT using the Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) technique. We identified 5413 GT high accessible and 4045 GT low accessible regions, and these GT high accessible regions may contribute to GT-specific biological functions. Several GT-specific artemisinin biosynthetic genes, such as DBR2 and CYP71AV1, showed higher accessible regions in GT compared to that in leaf, implying that they might be regulated by chromatin accessibility. In addition, transcription factor binding motifs for MYB, bZIP, C2H2, and AP2 were overrepresented in the highly accessible chromatin regions associated with artemisinin biosynthetic genes in glandular trichomes. Finally, we proposed a working model illustrating the chromatin accessibility dynamics in regulating artemisinin biosynthetic gene expression. This work provided new insights into epigenetic regulation of gene expression in GT.

Transposon Insertion Sequencing, a Global Measure of Gene Function

2020 ◽  
Vol 54 (1) ◽  
pp. 337-365
Author(s):  
Tim van Opijnen ◽  
Henry L. Levin
Keyword(s):  
Dna Sequencing ◽  
High Throughput ◽  
Bacterial Infections ◽  
Single Experiment ◽  
Transposon Insertion ◽  
High Throughput Method ◽  
High Throughput Dna Sequencing ◽  
Complete Set ◽  
Living Organisms ◽  
Transposon Insertion Sequencing

The goal of genomics and systems biology is to understand how complex systems of factors assemble into pathways and structures that combine to form living organisms. Great advances in understanding biological processes result from determining the function of individual genes, a process that has classically relied on characterizing single mutations. Advances in DNA sequencing has made available the complete set of genetic instructions for an astonishing and growing number of species. To understand the function of this ever-increasing number of genes, a high-throughput method was developed that in a single experiment can measure the function of genes across the genome of an organism. This occurred approximately 10 years ago, when high-throughput DNA sequencing was combined with advances in transposon-mediated mutagenesis in a method termed transposon insertion sequencing (TIS). In the subsequent years, TIS succeeded in addressing fundamental questions regarding the genes of bacteria, many of which have been shown to play central roles in bacterial infections that result in major human diseases. The field of TIS has matured and resulted in studies of hundreds of species that include significant innovations with a number of transposons. Here, we summarize a number of TIS experiments to provide an understanding of the method and explanation of approaches that are instructive when designing a study. Importantly, we emphasize critical aspects of a TIS experiment and highlight the extension and applicability of TIS into nonbacterial species such as yeast.

Sign in / Sign up

Export Citation Format

Share Document