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| Status |
Public on Feb 04, 2015 |
| Title |
Quantitative Analysis of Wild Type and Dicer1-ifKO Hippocampal Transcriptomes (mRNA and small RNA) Through Next Generation Sequencing (mRNA-Seq). |
| Organism |
Mus musculus |
| Experiment type |
Expression profiling by high throughput sequencing
|
| Summary |
Methods: CaMKIIa-creERT2 (Erdmann et al., 2007) and Dicer1f/f (Harfe et al., 2005) were crossed to produce inducible forebrain-restricted Dicer1 knockout mice (Dicer-ifKO) mice. Hippocampal mRNA profiles of 3-month-old wild-type (WT) and (Dicer-ifKO) mice were generated by deep sequencing, in triplicate, using Illumina HiSeq 2500. Each sample included total RNA isolated from the hippocampus of 3 mice. In total, 12 mice per genotype were used. The sequence reads that passed quality filters were mapped to reference genome (GRCm38/mm10) using Bowtie 2 (2.0.5) and TopHat (2.0.6). SAM/BAM files were further processed with Samtools (0.1.18). Read count quantitations were obtained using Seqmonk (0.26.0). Normalization of read counts and differential expression analysis between genotypes was carried out using DESeq2 R package from Bioconductor (Release 2.13). qRT–PCR validation was performed using SYBR Green assays.
Results: We mapped about 13-14 million sequence reads per sample to the mouse genome (build GRCm38/mm10) and quantified 76,938 annotated transcripts. DESeq2 R package was used to normalize the counts and perform the differential expression. Differential analysis output was filtered by FDR threshold (padj < 0.1). This approach led us to identify 641 gene isoforms, corresponding to 314 genes that were differentially regulated in the mouse hippocampus upon Dicer ablation.
Conclusions: We extend here the characterization of inducible forebrain-restricted Dicer1 mutants confirming the initial memory improvement. Moreover, we describe several novel phenotypes associated with early Dicer loss in the mature brain including an exacerbated response to seizures, increased CA1 neuron excitability, a pronounced weight gain and enhanced induction of immediate early genes (IEGs) in relevant neuronal nuclei. To identify candidate genes that could explain these phenotypes, we conducted two complementary genomic screens for the miRNAs primarily affected and their targets. Overall, our results explain both the initial and late consequences of Dicer loss in excitatory neurons and indicate that Dicer and the miRNA system play a critical role regulating neuronal homeostasis and responsiveness.
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| Overall design |
Hippocampal mRNA profiles of 3-month-old wild-type (WT) and Dicer-ifKO (3 weeks upon tamoxifen administration) male mice were generated by deep sequencing, in triplicate, using Illumina HiSeq 2500. Each sample included total RNA isolated from the hippocampus of 3 mice. In total, 12 mice per genotype were used.
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| Contributor(s) |
LOPEZ-ATALAYA JP, BARCO A |
| Citation(s) |
25595182 |
| |
| Submission date |
Aug 08, 2014 |
| Last update date |
Sep 16, 2019 |
| Contact name |
Angel Barco |
| Organization name |
Instituto de Neurociencias (UMH-CSIC)
|
| Street address |
Av. Santiago Ramón y Cajal
|
| City |
Sant Joan d'Alacant |
| State/province |
Alicante |
| ZIP/Postal code |
03550 |
| Country |
Spain |
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| Platforms (1) |
| GPL17021 |
Illumina HiSeq 2500 (Mus musculus) |
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| Samples (6)
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| This SubSeries is part of SuperSeries: |
| GSE60263 |
Quantitative Analysis of Wild Type and Dicer1-ifKO Hippocampal Transcriptomes (mRNA and small RNA) Through Next Generation Sequencing. |
|
| Relations |
| BioProject |
PRJNA257833 |
| SRA |
SRP045378 |
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