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T-RHEX-RNAseq – a tagmentation-based, rRNA blocked, random hexamer primed RNAseq method for generating stranded RNAseq libraries from very low numbers of lysed cells

ABSTRACT: T-RHEX-RNAseq – a tagmentation-based, rRNA blocked, random hexamer primed RNAseq method for generating stranded RNAseq libraries from very low numbers of lysed cells

PROVIDER: PRJEB56375 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	ERR10297679_1.fastq.gz	Fastqsanger.gz
	ERR10297679_2.fastq.gz	Fastqsanger.gz
	ERR10297680_1.fastq.gz	Fastqsanger.gz
	ERR10297680_2.fastq.gz	Fastqsanger.gz
	ERR10297681_1.fastq.gz	Fastqsanger.gz

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T-RHEX-RNAseq - a tagmentation-based, rRNA blocked, random hexamer primed RNAseq method for generating stranded RNAseq libraries directly from very low numbers of lysed cells.

Project description:BackgroundRNA sequencing has become the mainstay for studies of gene expression. Still, analysis of rare cells with random hexamer priming - to allow analysis of a broader range of transcripts - remains challenging.ResultsWe here describe a tagmentation-based, rRNA blocked, random hexamer primed RNAseq approach (T-RHEX-RNAseq) for generating stranded RNAseq libraries from very low numbers of FACS sorted cells without RNA purification steps.ConclusionT-RHEX-RNAseq provides an easy-to-use, time efficient and automation compatible method for generating stranded RNAseq libraries from rare cells.

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Improved tagmentation-based whole genome bisulfite sequencing for input DNA from less than 100 cells

Project description:We improved the tagmentation-based whole genome bisulfite sequencing technology, enabling stable library construction with complexity from minimally 0.5ng of initial genomic DNA, which equals less than 100 cells. This new approach is highly attractive for the complete methylome analysis of very limited cell numbers, e.g., pre-implantation embryos, or tiny biopsy specimens. Applying this newly modified T-WGBS, we sequenced the methylome of a rice strain using libraries constructed from 10 ng, 1 ng and 0.5 ng of input genomic DNA. Two libraries were independently constructed and sequenced for each aliquot of input genomic DNA

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Transposase assisted tagmentation of RNA/DNA hybrid duplexes

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: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:Generation of random numbers is a central problem for many applications in the field of information processing, including, e.g., cryptography, in classical and quantum regime, but also mathematical modeling, Monte Carlo methods, gambling and many others. Both, the quality of the randomness and efficiency of the random numbers generation process are crucial for the most of these applications. Software produced pseudorandom bit sequences, though sufficiently quick, do not fulfill required randomness quality demands. Hence, the physical hardware methods are intensively developed to generate truly random number sequences for information processing and electronic security application. In the present paper we discuss the idea of the quantum random number generators. We also present a variety of tests utilized to assess the quality of randomness of generated bit sequences. In the experimental part we apply such tests to assess and compare two quantum random number generators, PQ4000KSI (of company ComScire US) and JUR01 (constructed in Wroclaw University of Science and Technology upon the project of The National Center for Research and Development) as well as a pseudorandom generator from the Mathematica Wolfram package. Finally, we present our new prototype of fully operative miniaturized quantum random generator JUR02 producing a random bit sequence with velocity of 1 Mb/s, which successfully passed standard tests of randomness quality (like NIST and Dieharder tests). We also shortly discuss our former concept of an entanglement-based quantum random number generator protocol with unconditionally secure public randomness verification.

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