github link
Accession IconSRP043666

RNA Sequencing Quantitative Analysis and identification of RNA editing sites of Wild Type and ADAR1 editing deficient (ADAR1E861A) murine fetal liver RNA

Organism Icon Mus musculus
Sample Icon 6 Downloadable Samples
Technology Badge IconIllumina HiSeq 2000

Submitter Supplied Information

Description
Purpose: RNA editing by ADAR1 is essential for hematopoietic development. The goals of this study were firstly, to identify ADAR1-specific RNA-editing sites by indentifying A-to-I (G) mismatches in RNA-seq data compared to mm9 reference genome in wild type mice that were not edited or reduced in editing frequency in ADAR1E861A editing deficient mice. Secondly, to determine the transcriptional consequence of an absence of ADAR1-mediated A-to-I editing. Methods: Fetal liver mRNA profiles of embryonic day 12.5 wild-type (WT) and ADAR1 editing-deficient (ADAR1E861A) mice were generated by RNA sequencing, in triplicate (biological replicates), using Illumina HiSeq2000. The sequence reads that passed quality filters were analyzed at the transcript level with TopHat followed by Cufflinks. qRT–PCR validation was performed using SYBR Green assays. A-to-I (G) RNA editing sites were identified as previously described by Ramaswami G. et al., Nature Methods, 2012 using Burrows–Wheeler Aligner (BWA) followed by ANOVA (ANOVA). RNA editing sites were confirmed by Sanger sequencing. Results: Using an optimized data analysis workflow, we mapped about 30 million sequence reads per sample to the mouse genome (build mm9) and identified 14,484 transcripts in the fetal livers of WT and ADAR1E861A mice with BWA. RNA-seq data had a goodness of fit (R2) of >0.94 between biological triplicates per genotype. Approximately 4.4% of the transcripts showed differential expression between the WT and ADAR1E861A fetal liver, with a LogFC=1.5 and p value <0.05. A profound upregulation of interferon stimulated genes were found to be massively upregulated (up to 11 logFC) in ADAR1E861A fetal liver compared to WT. 6,012 A-to-I RNA editing sites were identified when assessing mismatches in RNA-seq data of WT and ADAR1E861A fetal liver. Conclusions: Our study represents the first detailed analysis of fetal liver transcriptomes and A-to-I RNA editing sites, with biologic replicates, generated by RNA-seq technology. A-to-I RNA editing is the essential function of ADAR1 and is required to suppress interferon signaling to endogenous RNA. Overall design: Fetal liver mRNA profiles of E12.5 wild type (WT) and ADAR E861A mutant mice were generated by deep sequencing, in triplicate, using Illumina HiSeq 200.
PubMed ID
Total Samples
6
Submitter’s Institution
No associated institution
Alternate Accession IDs

Samples

Show of 0 Total Samples
Filter
Add/Remove
Accession Code
Title
Processing Information
Additional Metadata
No rows found
Loading...