Project description:We have used RNA-seq to examine circular RNAs from RNase R treated poly(A)-/ribo- RNAs in human embryonic stem cells Examine circular RNAs in human embryonic stem cells
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
Project description:We have used RNA-seq to examine circular RNAs from poly(A)-/ribo- RNAs in human and mouse embryonic stem cells In order to identify novel circular RNAs from different species
Project description:Accumulating evidence suggests important roles of RNAs interacting with genomic regions in the regulation of genome functions including X chromosome inactivation and gene expression. However, no method to identify RNAs interacting with a given genomic region in a non-biased manner has been reported. Here, we used engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) combined with the RNA-Seq analysis (enChIP-RNA-Seq) to perform non-biased search of RNAs interacting with telomeres. In enChIP-RNA-Seq, the target genomic regions are captured with an engineered DNA-binding molecule such as a TAL protein. Subsequently, RNAs are purified and subjected to the RNA-Seq analysis. The detected RNAs contained known telomere-binding RNAs including telomerase RNA and Cajal body-specific RNAs. In addition, we detected many novel telomere-binding RNAs. We confirmed binding of candidate RNAs to telomeres by the enChIP-RT-PCR analysis. Identified novel telomere-binding RNAs may play important roles in telomere functions. In addition, our results suggest that enChIP-RNA-Seq analysis would be useful for identification of RNAs interacting with specific genomic regions. RNAs associated with telomeres were identified by using the enChIP technology combined with deep sequencing using Illumina Miseq. Briefly, to isolate telomeres, a TAL protein, Telomere-TAL (Tel-TAL), recognizing a 19-bp sequence containing an array of TTAGGG (telomere repeats) was fused with 3xFLAG tag and NLS (3xFN-Tel-TAL) and LexA protein (3xFNLDD)11 were expressed in a mouse hematopoietic cell line, Ba/F3, respectively. The cells were crosslinked with formaldehyde, and crosslinked chromatin was fragmented by sonication. Subsequently, chromatin complexes containing 3xFN-Tel-TAL or 3xFNLDD were immunoprepicitated with anti-FLAG M2 Ab. Supplementary URL: http://www.nature.com/srep/2013/131108/srep03171/full/srep03171.html
Project description:Proteomic analysis of Ago2-/- oocytes revealed that Ago2 interacted with endogenous small interfering RNAs (endo-siRNAs) to repress mRNA translation globally.
Project description:Accumulating evidence suggests important roles of RNAs interacting with genomic regions in the regulation of genome functions including X chromosome inactivation and gene expression. However, no method to identify RNAs interacting with a given genomic region in a non-biased manner has been reported. Here, we used engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) combined with the RNA-Seq analysis (enChIP-RNA-Seq) to perform non-biased search of RNAs interacting with telomeres. In enChIP-RNA-Seq, the target genomic regions are captured with an engineered DNA-binding molecule such as a TAL protein. Subsequently, RNAs are purified and subjected to the RNA-Seq analysis. The detected RNAs contained known telomere-binding RNAs including telomerase RNA and Cajal body-specific RNAs. In addition, we detected many novel telomere-binding RNAs. We confirmed binding of candidate RNAs to telomeres by the enChIP-RT-PCR analysis. Identified novel telomere-binding RNAs may play important roles in telomere functions. In addition, our results suggest that enChIP-RNA-Seq analysis would be useful for identification of RNAs interacting with specific genomic regions.