Transposase assisted tagmentation of RNA/DNA hybrid duplexes
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ABSTRACT:
PROVIDER: PRJNA600433 | ENA |
REPOSITORIES: ENA
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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.
2020-07-29 | GSE143422 | GEO
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.
2021-09-07 | GSE167307 | GEO
Project description:Establishment of Transposase-assisted Low-input m6A Sequencing Technique
| PRJNA704264 | ENA
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: Not available
| S-EPMC7402673 | biostudies-literature
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:Tn5 transposase and tagmentation procedures for massively scaled sequencing projects
| PRJNA253099 | ENA
Project description:R-Loops are unique RNA-containing chromatin structures, which participate in various key biological processes and associate with multiple human diseases. Accurately and comprehensively profiling R-Loops in the genome is crucial to study their functions under the physiological and pathological conditions. However, the existing methodologies have produced broad discrepancies in R-Loop profiling and an independent strategy is urgently needed. Here, we constructed an artificial DNA-RNA hybrid recognition sensor protein GST-His6-2XHBD with the hybrid-binding domain of RNase H1, and found GST-His6-2XHBD could specifically interact with DNA-RNA hybrids and behaves similarly to the anti-DNA-RNA hybrid S9.6 antibody in DRIPc-seq. Furthermore, we established a convenient method, R-loop CUT&Tag, by combination of GST-His6-2XHBD with a Tn5-based cleavage under targets and tagmentation approach. R-Loop CUT&Tag generates highly specific signals for native R-Loops, and can sensitively detect the R-Loop signals at the promoter, genebody and enhancer. R-Loop CUT&Tag also provides possibilities to genome-widely map the native R-Loops with limited materials, and may benefit the resolving of the broad discrepancies between multiple R-Loop mapping methods.
2021-02-22 | GSE156400 | GEO
Project description:Induction of somatic oncogenic mutations by the domesticated DNA transposase PGBD5 in cerebellar progenitor cells promotes medulloblastoma development.
2024-03-27 | GSE249785 | GEO
Project description:Genomic rearrangements are a hallmark of childhood cancers, but the mutational processes underlying most of these variants remain unknown. We identified piggyBac transposable element derived 5 (PGBD5) as a highly expressed, enzymatically active domesticated human DNA transposase in a large subset of pediatric solid tumors, including rhabdoid tumors. Ectopic expression of PGBD5 in primary human cells was sufficient to induce fully penetrant cell transformation both in vitro and in immunodeficient mice in vivo. This activity required specific catalytic aspartic acid residues in the PGBD5 transposase domain as well as cellular non-homologous end-joining DNA repair, and was associated with distinct structural rearrangements defined by specific DNA sequence motifs. Similar genomic alterations, some recurrent, were found in primary human rhabdoid tumors. Thus, PGBD5 represents a new class of developmental oncogenic mutators in childhood solid tumors.
2016-12-05 | GSE81160 | GEO