Transposase assisted tagmentation of RNA/DNA hybrid duplexes
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA600433 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR12074059_1.fastq.gz | Fastqsanger.gz | |||
| SRR12074059_2.fastq.gz | Fastqsanger.gz | |||
| SRR12074060_1.fastq.gz | Fastqsanger.gz | |||
| SRR12074060_2.fastq.gz | Fastqsanger.gz | |||
| SRR12074061_1.fastq.gz | Fastqsanger.gz |
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Project description:Tn5-mediated transposition of double-strand DNA has been widely utilized in various high-throughput sequencing applications. Here, we report that the Tn5 transposase is also capable of direct tagmentation of RNA/DNA hybrids in vitro. As a proof-of-concept application, we utilized this activity to replace the traditional library construction procedure of RNA sequencing, which contains many laborious and time-consuming processes. Results of Transposase assisted RNA/DNA hybrids Co-tagmEntation (termed “TRACE-seq”) are comparable to traditional RNA-seq methods in terms of detected gene number, gene body coverage, gene expression measurement, library complexity, and differential expression analysis; at the meantime, TRACE-seq enables a one-tube library construction protocol and hence is more rapid (within 6h) and convenient. We expect this tagmentation activity on RNA/DNA hybrids to have broad potentials on RNA biology and chromatin research.
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Project description:Tn5-mediated transposition of double-strand DNA has been widely utilized in various high-throughput sequencing applications. Here, we report that the Tn5 transposase is also capable of direct tagmentation of RNA/DNA hybrids in vitro. As a proof-of-concept application, we utilized this activity to replace the traditional library construction procedure of RNA sequencing, which contains many laborious and time-consuming processes. Results of Transposase-assisted RNA/DNA hybrids Co-tagmEntation (termed 'TRACE-seq') are compared to traditional RNA-seq methods in terms of detected gene number, gene body coverage, gene expression measurement, library complexity, and differential expression analysis. At the meantime, TRACE-seq enables a cost-effective one-tube library construction protocol and hence is more rapid (within 6 hr) and convenient. We expect this tagmentation activity on RNA/DNA hybrids to have broad potentials on RNA biology and chromatin research.
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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.
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Project description:As the most abundant and best-characterized internal mRNA modification, N6-methyladenosine (m6A) emerges to play a critical regulatory role in wide range of physiological and pathological processes, including gametogenesis, neuronal development, obesity and tumorigenesis. Methylated RNA immunoprecipitation coupled with next-generation sequencing (MeRIP-seq) facilitates transcriptome-wide m6A profiling, also is the most widely used technique to understand the biological significance of m6A. However, it typically requires over 100 μg of total RNA or 107 cells as input materials, hampering its application in limited samples. Here, we develop tMeRIP-seq, a transposase assisted MeRIP-seq method to achieve m6A profiling using ultra-low amount of input RNA. By marrying Tn5 tagmentation to m6A-specific immunoprecipitation, tMeRIP-seq largely improves the efficiency of library construction and reduces the input materials to as little as 60 ng total RNA or 103 cells. We apply this method on a small droplet of human blood and recapitulate the m6A profile previously reported using conventional protocol. We find tMeRIP-seq is a convenient and powerful method to examine m6A in ultra-low input material, potentially providing m6A as a new layer of bio-marker for liquid biopsy.
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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.
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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.
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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