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Multiplex generation and single cell analysis of structural variants in mammalian genomes [IVT-seq]

ABSTRACT: Multiplex generation and single cell analysis of structural variants in mammalian genomes [IVT-seq]

PROVIDER: PRJNA1189512 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR31445343_1.fastq.gz	Fastqsanger.gz
	SRR31445343_2.fastq.gz	Fastqsanger.gz
	SRR31445344_1.fastq.gz	Fastqsanger.gz
	SRR31445344_2.fastq.gz	Fastqsanger.gz
	SRR31445345_1.fastq.gz	Fastqsanger.gz

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Multiplex generation and single cell analysis of structural variants in mammalian genomes [IVT-seq]

Project description:Studying the functional consequences of structural variants (SVs) in mammalian genomes is challenging because: 1) SVs arise much less commonly than single nucleotide variants or small indels; and 2) methods to generate, map and characterize SVs in model systems are underdeveloped. To address these challenges, we developed Genome-Shuffle-seq, a method that enables the multiplex generation and mapping of thousands of SVs (deletions, inversions, translocations, extrachromosomal circles) throughout mammalian genomes. We also demonstrate the co-capture of SV identity with single-cell transcriptomes, facilitating the measurement of SVs’ impact on gene expression. We anticipate Genome-Shuffle-seq will be broadly useful for the systematic exploration of the functional consequences of SVs on gene expression, chromatin landscape, and 3D nuclear architecture, while also initiating a path towards a minimal mammalian genome.

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Project description:In vitro transcription (IVT) is a technology of vital importance that facilitated the production of mRNA therapeutics and drove numerous breakthroughs in RNA biology. T7 polymerase-produced RNAs can begin with either 5′-triphosphate guanosine (5′-pppG) or 5′-triphosphate adenosine (5′-pppA), generating potential agonists for the RIG-I/type I interferon response. While it is established that IVT can yield highly immunogenic double-stranded RNA (dsRNA) via promoterless transcription, the specific contribution of initiating nucleosides to this process has not been previously reported. Our study shows that IVT-derived RNAs containing 5′-pppA are significantly more immunogenic compared with their 5′-pppG counterparts. We observed heightened levels of dsRNAs triggered by IVT with 5′-pppA RNA, activating the RIG-I signaling pathway in cultured cells, as well as in ex vivo and in vivo mouse models where the IFN-β gene was substituted with the mKate2+ reporter. Elevated levels of dsRNA were found in both short 5′-pppA RNAs and full-length mRNAs, including those of COVID-19 vaccines. These findings reveal the unexpected source of IVT RNA immunogenicity, offering valuable insights for both academic research and medical applications of this technology.

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