Tn5 tagments and transports oligos to single-strand DNA for high throughput sequencing
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ABSTRACT:
PROVIDER: PRJNA859175 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR20231337_1.fastq.gz | Fastqsanger.gz | |||
| SRR20231337_2.fastq.gz | Fastqsanger.gz | |||
| SRR20231338_1.fastq.gz | Fastqsanger.gz | |||
| SRR20231338_2.fastq.gz | Fastqsanger.gz | |||
| SRR20231339_1.fastq.gz | Fastqsanger.gz |
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Project description:Tn5 transposon tagments double-strand DNA and RNA/DNA hybrid to generate nucleic acids ready to be amplified for high throughput sequencing. The nucleic acid substrates of Tn5 transposon need to be explored to increase the applications of Tn5. Here, we find that Tn5 transposon is able to transport oligos into the 5’ end of single-strand DNA, which are more than 140 base pairs of nucleic acids. Based on this property of Tn5, we develop a tagmentation-based and ligation-enabled single-strand DNA sequencing method, TABLE-seq. Through a series of reaction temperature, time, and enzyme concentration tests, we managed to apply this TABLE-seq to strand-specific RNA sequencing, starting with as low as 30 picograms of total RNA. Moreover, compared with traditional dUTP based strand-specific RNA sequencing, this method detects more genes, has higher strand-specificity, and shows more evenly distributed reads across genes. Together, our results provide insights into the properties of Tn5 transposons and expand the applications of Tn5 in cutting-edge sequencing techniques.
2023-01-30 | GSE208316 | GEO
Project description:SLC19A1 transports immunoreactive cyclic dinucleotides
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Project description:Background: Next-generation sequencing (NGS) is fundamental to the current biological and biomedical research. Construction of sequencing library is a key step of NGS. Therefore, various library construction methods have been explored. However, the current methods are still limited by some shortcomings. Results: This study developed a new NGS library construction method, Single strand Adaptor Library Preparation (SALP), by using a novel single strand adaptor (SSA). SSA is a double-stranded oligonucleotide with a 3ʹ overhang of 3 random nucleotides, which can be efficiently ligated to the 3′ end of single strand DNA by T4 DNA ligase. SALP can be started with any denatured DNA fragments such as those sheared by Tn5 tagmentation, enzyme digestion and sonication. When started with Tn5-tagmented chromatin, SALP can overcome a key limitation of ATAC-seq and become a high-throughput NGS library construction method, SALP-seq, which can be used to comparatively characterize the chromatin openness state of multiple cells unbiasly. In this way, this study successfully characterized the comparative chromatin openness states of four different cell lines, including GM12878, HepG2, HeLa and 293T, with SALP-seq. Similarly, this study also successfully characterized the chromatin openness states of HepG2 cells with SALP-seq by using 105 to 500 cells. Conclusions: This study developed a new NGS library construction method, SALP, by using a novel kind of single strand adaptor (SSA), which should has wide applications in the future due to its unique performance.
2018-01-31 | GSE104162 | GEO
Project description:We generated Tn5 segmentation data on naked genomic DNA to explore the intrinsic Tn5 insertion preference along the whole genome
2021-10-01 | GSE164997 | GEO
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:In recent years, tagmentation-based library preparation using a hyperactive version of the Tn5 transposase gained more and more popularity. The limited hands-on time, robustness and high efficiency of the method are essential for the processing of next-generation sequencing libraries form little input material like single cells or the processing of hundreds of samples simultaneously. The hyperactive Tn5 is commercially available (Nextera XT DNA library preparation kit), however, high-throughput experiments with hundreds of samples are costly. Here, we present a highly reproducible Tn5 transposase purification strategy via an N-terminal His6-Sumo3 tag and the workflow for the tagmentation-based NGS library preparation. We demonstrate that NGS libraries processed with the in-house produced Tn5 are of the same quality like those processed with the Nextera XT DNA library preparation kit and that the purification of the transposase is reproducible across institutes. Producing the Tn5 transposase in-house allows for customized experimental design and reduces costs of large-scale experiments dramatically. We describe a novel single cell polyadenylation site mapping protocol that benefits from the fact that the in-house produced Tn5 can be loaded with any desired linker oligonucleotide for tagmentation.
2017-11-13 | GSE101519 | GEO
Project description:In recent years, tagmentation-based library preparation using a hyperactive version of the Tn5 transposase gained more and more popularity. The limited hands-on time, robustness and high efficiency of the method are essential for the processing of next-generation sequencing libraries from little input material like single cells or the processing of hundreds of samples simultaneously. The hyperactive Tn5 is commercially available (Nextera XT DNA library preparation kit), however, high-throughput experiments with hundreds of samples are costly. Here, we present a highly reproducible Tn5 transposase purification strategy via an N-terminal His6-Sumo3 tag and the workflow for the tagmentation-based NGS library preparation. We demonstrate that NGS libraries processed with the in-house produced Tn5 are of the same quality like those processed with the Nextera XT DNA library preparation kit and that the purification of the transposase is reproducible across institutes. Producing the Tn5 transposase in-house allows for customized experimental design and reduces costs of large-scale experiments dramatically. We describe a novel single cell polyadenylation site mapping protocol that benefits from the fact that the in-house produced Tn5 can be loaded with any desired linker oligonucleotide for tagmentation.
2017-11-13 | GSE101518 | GEO
Project description:To be able to study where Tn5 inserts among repeat sequences of the genome, ATAC-seq was performed using a custom insert. The resulting DNA was then mechanically sheared and sequenced using PacBio
2024-07-19 | E-MTAB-14238 | 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:This experiment includes the sequencing files for different hp1a-tn5 hybrids, together with standard ATAC-seq and ChIP-seq data used to evaluate the best construct.
2021-08-17 | E-MTAB-9648 | biostudies-arrayexpress