Biochemical analysis of chromatin targeting by Tn5 transposase
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ABSTRACT:
PROVIDER: PRJNA535498 | ENA |
REPOSITORIES: ENA
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Project description:Tn5 transposase is used as a tool for detecting nucleosome-free regions of genomic DNA in eukaryotes, but its DNA target site in chromatin has not been understood. In the present study, the well-positioned dinucleosomes were reconstituted, and the Tn5 transposase target sites were mapped in the dinucleosomes in vitro. We found that Tn5 transposase preferentially targets near the entry-exit DNA region within the nucleosome, if the linker DNA exists between two nucleosomes. This specific DNA targeting by Tn5 did not depend on the linker DNA length and DNA sequence. Tn5 transposase becomes to target the middle of the linker DNA, in addition to the entry-exit site of the nucleosome, if the linker DNA length extends to 30 base pairs. These in vitro data provide direct evidence for the Tn5 target sites in the nucleosome, resulting important information for interpretation of the Tn5-transposase-based genomics methods, which have been interpreted as linker or nucleosome-free DNA regions in genomes.
2019-09-18 | GSE130322 | GEO
Project description:Deep sequencing of single cell-derived genomic DNA and/or cDNAs brings novel insights into oncogenesis and embryogenesis. However, traditional library preparation for RNA-Seq requires multiple steps, including shearing the target DNA/RNA and following sequential enzymatic reactions, which result in consequent sample loss and stochastic variation at each step. Such variation may significantly affect the output from sequencing. We have found that a new technique of library preparation using hyperactive Tn5 transposase for the next-generation sequencer of Illumina's platform provided high-quality libraries from 100ng of short-length (average 700~800 bp) single-cell level cDNA. This new method reduced the number of steps in the protocol, which resulted in improved reproducibility and reduced variation among the specimens. Two methods of library preparation (sonication, tagmentation with hyperactive Tn5 transposase) were compared in the case of RNA-Seq for single-cell level cDNA. Technical triplicates were used.
2012-05-25 | E-GEOD-38198 | biostudies-arrayexpress
Project description:Massively parallel DNA sequencing of thousands of samples in a single machine-run is now possible, but the preparation of the individual sequencing libraries is expensive and time-consuming. Tagmentation-based library construction, using the Tn5 transposase, is efficient for generating sequencing libraries but currently relies on undisclosed reagents, which severely limits development of novel applications and the execution of large scale projects. Here, we present simple and robust procedures for Tn5 transposase production and optimized reaction conditions for tagmentation-based sequencing library construction. We further show how molecular crowding agents both modulate library lengths and enable efficient tagmentation from sub-picogram amounts of cDNA. Comparison of single-cell RNA-sequencing libraries generated using produced and commercial Tn5 demonstrated equal performances in terms of gene detection and library characteristics. Finally, as naked Tn5 can be annealed to any oligonucleotide of choice, for example molecular barcodes in single-cell assays or methylated oligonucleotides for bisulfite sequencing, custom Tn5 production and tagmentation enables innovation in sequencing-based applications.
2014-07-14 | GSE58652 | GEO
Project description: Not available
| S-EPMC6731594 | biostudies-literature
Project description:The analysis of chromatin features in single cells centers around the use of Tn5 transposase and exploits its activity to simultaneously fragment target DNA and integrate adapter sequences of choice. This reaction provides a direct readout in the transposase-accessible chromatin in single cells (scATAC-seq) assay to map open chromatin regions. Furthermore, by targeting Tn5 to antibody-bound chromatin epitopes, features like histone modifications can be mapped in single cells. Thus, enhancing Tn5 activity to improve genomic coverage for scATAC-seq or facilitating multi-omics readout of chromatin features via Tn5 together with the transcriptome is of great interest. Here, we address these issues by optimizing scATAC-seq for an increased number of integrations per cell. In addition, we provide a protocol that combines mapping of histone modification with scRNA-seq from the same cell. Our experimental workflows improve the results obtained from the downstream data analysis and serve to better resolve epigenetic heterogeneity and transcription regulation in single cells.
2023-11-08 | GSE235501 | GEO
Project description:The analysis of chromatin features in single cells centers around the use of Tn5 transposase and exploits its activity to simultaneously fragment target DNA and integrate adapter sequences of choice. This reaction provides a direct readout in the transposase-accessible chromatin in single cells (scATAC-seq) assay to map open chromatin regions. Furthermore, by targeting Tn5 to antibody-bound chromatin epitopes, features like histone modifications can be mapped in single cells. Thus, enhancing Tn5 activity to improve genomic coverage for scATAC-seq or facilitating multi-omics readout of chromatin features via Tn5 together with the transcriptome is of great interest. Here, we address these issues by optimizing scATAC-seq for an increased number of integrations per cell. In addition, we provide a protocol that combines mapping of histone modification with scRNA-seq from the same cell. Our experimental workflows improve the results obtained from the downstream data analysis and serve to better resolve epigenetic heterogeneity and transcription regulation in single cells.
2023-11-08 | GSE235499 | GEO
Project description:The analysis of chromatin features in single cells centers around the use of Tn5 transposase and exploits its activity to simultaneously fragment target DNA and integrate adapter sequences of choice. This reaction provides a direct readout in the transposase-accessible chromatin in single cells (scATAC-seq) assay to map open chromatin regions. Furthermore, by targeting Tn5 to antibody-bound chromatin epitopes, features like histone modifications can be mapped in single cells. Thus, enhancing Tn5 activity to improve genomic coverage for scATAC-seq or facilitating multi-omics readout of chromatin features via Tn5 together with the transcriptome is of great interest. Here, we address these issues by optimizing scATAC-seq for an increased number of integrations per cell. In addition, we provide a protocol that combines mapping of histone modification with scRNA-seq from the same cell. Our experimental workflows improve the results obtained from the downstream data analysis and serve to better resolve epigenetic heterogeneity and transcription regulation in single cells.
2023-11-08 | GSE235497 | GEO
Project description:The analysis of chromatin features in single cells centers around the use of Tn5 transposase and exploits its activity to simultaneously fragment target DNA and integrate adapter sequences of choice. This reaction provides a direct readout in the transposase-accessible chromatin in single cells (scATAC-seq) assay to map open chromatin regions. Furthermore, by targeting Tn5 to antibody-bound chromatin epitopes, features like histone modifications can be mapped in single cells. Thus, enhancing Tn5 activity to improve genomic coverage for scATAC-seq or facilitating multi-omics readout of chromatin features via Tn5 together with the transcriptome is of great interest. Here, we address these issues by optimizing scATAC-seq for an increased number of integrations per cell. In addition, we provide a protocol that combines mapping of histone modification with scRNA-seq from the same cell. Our experimental workflows improve the results obtained from the downstream data analysis and serve to better resolve epigenetic heterogeneity and transcription regulation in single cells.
2023-11-08 | GSE235504 | GEO
Project description:The analysis of chromatin features in single cells centers around the use of Tn5 transposase and exploits its activity to simultaneously fragment target DNA and integrate adapter sequences of choice. This reaction provides a direct readout in the transposase-accessible chromatin in single cells (scATAC-seq) assay to map open chromatin regions. Furthermore, by targeting Tn5 to antibody-bound chromatin epitopes, features like histone modifications can be mapped in single cells. Thus, enhancing Tn5 activity to improve genomic coverage for scATAC-seq or facilitating multi-omics readout of chromatin features via Tn5 together with the transcriptome is of great interest. Here, we address these issues by optimizing scATAC-seq for an increased number of integrations per cell. In addition, we provide a protocol that combines mapping of histone modification with scRNA-seq from the same cell. Our experimental workflows improve the results obtained from the downstream data analysis and serve to better resolve epigenetic heterogeneity and transcription regulation in single cells.
2023-11-08 | GSE235503 | GEO
Project description:The analysis of chromatin features in single cells centers around the use of Tn5 transposase and exploits its activity to simultaneously fragment target DNA and integrate adapter sequences of choice. This reaction provides a direct readout in the transposase-accessible chromatin in single cells (scATAC-seq) assay to map open chromatin regions. Furthermore, by targeting Tn5 to antibody-bound chromatin epitopes, features like histone modifications can be mapped in single cells. Thus, enhancing Tn5 activity to improve genomic coverage for scATAC-seq or facilitating multi-omics readout of chromatin features via Tn5 together with the transcriptome is of great interest. Here, we address these issues by optimizing scATAC-seq for an increased number of integrations per cell. In addition, we provide a protocol that combines mapping of histone modification with scRNA-seq from the same cell. Our experimental workflows improve the results obtained from the downstream data analysis and serve to better resolve epigenetic heterogeneity and transcription regulation in single cells.
2023-11-08 | GSE235502 | GEO