RGED RGED / Next Generation Sequencing Facilitates Quantitative Analysis of ribo-tag pulled down Transcriptomes in renal proximal tubule epithelial cells from Plasmodium Chabaudi Chabaudi infected Egfp.l10Pepck and Egfp.l10PepckSlc40a1Pepckdelta/delta mice

Public on 2021-11-25

Description

Purpose: Next-generation sequencing (NGS) is emmerged as a highthrough-put technique to analyze cellular pathways. The goals of this study are to profile NGS-derived renal proximal tubule epithelial cells (RPTEC) transcriptome (RNA-seq), to identify transcription signature in RPTEC upon Plasmodium Chabaudi Chabaudi (Pcc) infection and steady state (NI). We also would like to understand how iron export affect the transcriptome in this cell compartment. Therefore, we will compare the transcriptome between Pcc infected (or steady state) Egfp.l10Pepck and Egfp.l10PepckSlc40a1Pepckdelta/delta mice.; Methods: TRAP (Translating Ribosome Affinity Purification) were performed in the kidneys of Pcc infected (or steady state) 10-16 weeks Egfp.l10Pepck and Egfp.l10PepckSlc40a1Pepckdelta/delta mice. RPTEC TRAP mRNA profiles were generated by next generation sequencing, in triplicate, using Illumina High output kit v2.5 and NextSeq500 sequencer.; Results: Using an optimized data analysis workflow, we mapped an average 32.54 million sequence reads per sample to the mouse genome (build mm39) and identified 19,052 transcripts in the TRAP mRNA of in the kidneys of Pcc infected or steady state 10-16 week old Egfp.l10Pepck and Egfp.l10PepckSlc40a1Pepckdelta/delta mice. Differential gene expression was performed using the DESeq2 R package (v.1.32) and gene expression was modeled by genotype and condition. Differentially expressed genes were considered for genes with an adjusted p-value<0.05 and an absolute log2 fold change>0.; 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.

Overall Design

TRAP (Translating Ribosome Affinity Purification) were performed in the kidneys of Pcc infected (or steady state) 10-16 weeks Egfp.l10Pepck and Egfp.l10PepckSlc40a1Pepckdelta/delta mice. RPTEC TRAP mRNA profiles were generated by next generation sequencing, in triplicate, using Illumina High output kit v2.5 and NextSeq500 sequencer.

Curator

yq_pan

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