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Single-cell RNA-Seq transcriptome analysis of circular RNAs in mouse embryos

ABSTRACT: Single-cell RNA-Seq transcriptome analysis of circular RNAs in mouse embryos

PROVIDER: PRJNA231896 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR1051260_1.fastq.gz	Fastqsanger.gz
	SRR1051260_2.fastq.gz	Fastqsanger.gz
	SRR1051270_1.fastq.gz	Fastqsanger.gz
	SRR1051270_2.fastq.gz	Fastqsanger.gz
	SRR1051280_1.fastq.gz	Fastqsanger.gz

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Single-cell RNA-Seq transcriptome analysis of circular RNAs in mouse embryos

Project description:We reported a new method of single-cell universal poly(A)-independent RNA sequencing (SUPeR-seq) to obtain single cell whole transcriptome analysis that covers both poly(A) plus and poly(A) minus RNA species. We built libraries of single mouse ESC , 10pg, 100pg and 1ng diluted mouse ES total RNA, single HEK293T cell and mouse early embryos before 4-cell stage. We analyzed these data using bioinformatics and find a good coverage of non-polyadenylated RNAs, circular RNA for example. to make a comparison,we also used mouse ES cells and HEK293T cells to build libraries using the protocol publish by F.Tang in 2009(sample name Tang2009_*). We also de novo assembled new genes in mouse ESCs and early embryo samples, further validated known and novel zygotic genes with a-Aminatine treated 2-cell embryos(sample name SUPeR-seq_a-AM_treated_2-cell*).

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Project description:Purpose: We are using the illumina sequencing to compare the false positive and true positive circular RNA findings to confine the method to detect the true circular RNAs Methods: The testis whole transcriptome profiling was generated from 4-week mouse testis using illumina Nextseq, duplicated. The sequence reads that passed quality filters were analyzed at the transcript isoform level with TopHat followed by Cufflinks. Results: our data suggest that circular RNAs identified based on junction sequences in the RNA-seq reads, especially those from Illumina Hiseq sequencing, mostly result from template-switching events during reverse transcription by MMLV-derived reverse transcriptases. It is critical to employ reverse transcriptases lacking terminal transferase activity (e.g., MonsterScript) to construct sequencing library or perform RT-PCR for identification and quantification of true circular RNAs. Conclusions: Our study represents the first detailed analysis of retinal transcriptomes, with biologic replicates, generated by RNA-seq technology. The optimized data analysis workflows reported here should provide a framework for comparative investigations of expression profiles. Our results show that NGS offers a comprehensive and more accurate quantitative and qualitative evaluation of mRNA content within a cell or tissue. We conclude that RNA-seq based transcriptome characterization would expedite genetic network analyses and permit the dissection of complex biologic functions. The wild type mouse testis RNAs were constructed NGS library by two different enzyme, then parallel sequenced in illumina Nextseq

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Project description:Recently, it has been demonstrated that genomes of many species express single stranded RNAs with covalently closed ends, named circular RNAs. Their regulatory potential and functional relevance are just starting to be revealed. Here we present a novel computational tool, seekCRIT (seek for differentially expressed Circular RNAs In Transcriptome), that identifies circular RNAs and detects their differential expression between two conditions. Using seekCRIT we identified the circular RNAs that are expressed in the neural retina and determined that the majority of them (74%) are expressed in both, ischemic and normal conditions. We identified over 40 circular RNAs that were differentially expressed between both conditions and validated these experimentally using qRT-PCR. The high validation rate of 90% with a false discovery rate (FDR) of < 5% demonstrates the accuracy and reliability of seekCRIT.

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