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Next Generation Sequencing Analysis of CircRNAs/linear RNA in Isolated Renal Proximal Tubule tissues from Acute kidney injury mice

ABSTRACT: The goal of this study was to find the differentially expressed circRNAs/linear RNA in AKI mice model compared to normal mice. We established cisplatin-induced AKI mice models and then extracted RNAs from isolated renal tubular tissues for Next Generation Sequencing(NGS) at different time points during early stage of AKI. CircRNA library was constructed by NEB Next®Ultra™ small RNA Sample Library Prep Kit for Illumina®. NGS was performed by using Illumina HiSeq 2500 Genome Sequencers.The original image data file was transformed into Raw Data by Base Calling. Clean Data was obtained by removing reads that containing joints and more than 5% N (undetermined base information). Mapped Reads were obtained by sequence alignment between Clean Reads and reference genome sequenced using BWA software package. CIRI software was used to predict circRNAs. Finally, we identified 2162 circRNAs and our study represents the first detailed analysis of AKI mice circRNA transcriptomes, which provide a framework for investigations of circRNAs expression profiles in AKI

ORGANISM(S): Mus musculus

PROVIDER: GSE130814 | GEO | 2020/02/01

REPOSITORIES: GEO

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Project description:Purpose: To profile the circular RNAs (circRNAs), microRNAs (miRNA), and mRNAs expression in BPA exposed GC-2 cells and normally cultured cells. Methods: For circRNA sequencing, total RNA was depleted of rRNA and then linear RNAs by RNase R digestion. cDNA library was constructed according to the Illumina TruSeq library preparation protocol. The Hiseq3000 sequencing platform (Illumina, San Diego, CA, USA) was used for circRNA sequencing. The sequence depth is 10G. Sequences were mapped using STAR software against GRCm38/mm10 mouse reference genome in UCSC Known Genes databases. CIRCexplorer software was applied to analyze the back-spliced junction for circular RNA prediction. Small RNA sequencing was performed on Hiseq2500in 10M sequence depth after regular small RNA library preparation. After adaptor trimming by FASTXToolkit (0.0.14), reads were aligned to the mouse genome (GRCm38/mm10) with Bowtie software (version 1.1.0). miRBase v21 and miRDeep2 were used to annotate or forecast new miRNAs respectively.Hiseq2500 platform was used to sequence mRNA in 6G depth after strand-specific library construction with TruSeq RNA LTPrep Kit (Illumina, San Diego, CA, USA). Clean reads were aligned to reference genome (mouse GRCm38/mm10) with STAR software. And StringTie and Cuffcompare were used for transcription assembly and novel transcription prediction. Results: A total of 4706 and 6486 circRNAs were identified in normally grow GC-2 cells and BPA treated cells respectively, with most not reported in circBase. According to the screening criterion of two folds, 4821 circRNAs were up-regulated and 2855 circRNAs were down-regulated compared to control group. The amounts and kinds of circRNAs were generally enhanced under BPA stress. Similar to circRNAs, miRNA sequencing also showed that more miRNAs were up-regulated than down-regulated under BPA stress (689 up and 98 down by ≥2 folds). In mRNA sequencing, 2978 genes were up-regulated and 2381 were down-regulated by at least 2 folds after BPA treatment. Conclusions: Our study was the first to delineate the full landscape of circRNAs/miRNAs/mRNAs in BPA induced reproductive toxicity. Many functional circRNAs and miRNAs were found to be activated under BPA exposure. They hold great promise to be developed as sensitive biomarkers for BPA exposure and targets for BPA toxity blocking.

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Next Generation Sequencing Analysis of CircRNAs/linear RNA in Isolated Renal Proximal Tubule tissues from Acute kidney injury mice

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