Quantitative Analysis of Wild Type and Dicer1-ifKO Hippocampal Transcriptomes (mRNA and small RNA) Through Next Generation Sequencing.
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
SUBMITTER: jose lopez
PROVIDER: E-GEOD-60263 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
Publications
Blocking miRNA Biogenesis in Adult Forebrain Neurons Enhances Seizure Susceptibility, Fear Memory, and Food Intake by Increasing Neuronal Responsiveness.
Fiorenza Anna A Lopez-Atalaya Jose P JP Rovira Victor V Scandaglia Marilyn M Geijo-Barrientos Emilio E Barco Angel A
Cerebral cortex (New York, N.Y. : 1991) 20150116 4
The RNase Dicer is essential for the maturation of most microRNAs, a molecular system that plays an essential role in fine-tuning gene expression. To gain molecular insight into the role of Dicer and the microRNA system in brain function, we conducted 2 complementary RNA-seq screens in the hippocampus of inducible forebrain-restricted Dicer1 mutants aimed at identifying the microRNAs primarily affected by Dicer loss and their targets, respectively. Functional genomics analyses predicted the main ...[more]
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Project description: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 small RNA 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. After trimming of the adapter sequences and masking for low-complexity or low-quality sequence, the inserts were sorted in separated files according to their lengths. The reads in which no match of the adapter sequences is found were discarded. Inserts of length 18-50 were pooled together and mapped to reference genome (GRCm38/mm10) using Burrows-Wheeler Alignment Tool (BWA) v.0.5.9. We used BWA to map the inserts with a maximum of 2 mismatches. Inserts mapping to several positions on the reference sequences with the same mapping quality were attributed at random to one of the position with a mapping quality of 0. Files were further processed using Samtools v.0.1.18 to get binary files (BAM). Read count quantitations were obtained using Seqmonk (0.26.0). We counted reads that mapped to annotated features (miRNAs, 2035; miscRNA, 491; mRNA 76938; rRNA, 341; snoRNA, 1602; snRNA, 1431; tRNAs, 26248; tRNA, 22). The DESeq2 R package from Bioconductor was used to normalize the counts and perform the differential expression analysis. Results: We mapped about 17-22 million sequence reads per sample to the mouse genome (build GRCm38/mm10) and quantified 109,108 annotated features. 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 119 annotated features (including 96 miRNAs) 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. Hippocampal small RNA 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.
2015-02-04 | E-GEOD-60262 | biostudies-arrayexpress
Project description: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. 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.
2015-02-04 | E-GEOD-60261 | biostudies-arrayexpress
Project description:DNA replication requires the faithful propagation of both genetic and epigenetic information. There is evidence that DNA polymerases play a role in transcriptional silencing, but the extent of their contribution and how it relates to heterochromatin maintenance is unclear. To get a better understanding of the role of POL2A, the catalytic subunit of the DNA polymerase epsilon, into maintaining heterochromatin silencing, we performed mRNA-seq on Arabidopsis plants carrying a new hypomorphic pol2a mutation. We deepened our analysis by profiling transcriptomes of other silencing mutants or combinations of pol2a with other mutations.
2020-06-04 | E-MTAB-8939 | biostudies-arrayexpress
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2018-09-30 | E-MTAB-7203 | biostudies-arrayexpress
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2010-05-28 | E-GEOD-20870 | biostudies-arrayexpress
Project description:DNA replication requires the faithful propagation of both genetic and epigenetic information. There is evidence that DNA polymerases play a role in transcriptional silencing, but the extent of their contribution and how it relates to heterochromatin maintenance is unclear. Analyzing a new hypomorphic pol2a mutant allele, we find that POL2A, the catalytic subunit of the DNA polymerase epsilon, maintains heterochromatin silencing and nuclear organization. We also found that POL2 inhibits DNA methylation and histone H3 lysine 9 methylation, which both correlate with 24-nucleotide small RNA accumulation. In this experiment, we sequenced small-RNA libraries generated from immature inflorescences of pol2a-10 and pol2a-12 mutants and wild-type plants.
2020-06-04 | E-MTAB-8938 | biostudies-arrayexpress
Project description:Drought stress is a major problem around the world and although progress in understanding how vegetable crops and model plants adapt to drought have been made, there is still little information about how fruit crops deal with moderate drought stress. In this study, we investigated the response of two apple genotypes: a drought-sensitive genotype (M26) and a drought-tolerant genotype (MBB). Our results of the morphology, physiology and biochemistry under moderate drought stress, indicated that relative water content (RWC) and leaf area (LA) were not significant changes in two genotypes. However, it had larger leaf mass per area (LMA), and accumulated higher free proline (CFP), soluble sugars (CSS) and malonaldehyde (MDA) in the leaves. Thus, it appears that the MBB genotype could produce more osmosis-regulating substances. Phosphoproteomic was analyzed from leaves of both genotypes under moderate drought stress using the isobaric tags for relative and absolute quantification (iTRAQ) technology. A total of 595 unique phosphopeptides, 682 phosphorylated sites and 446 phosphoproteins were quantitatively analyzed in the two genotypes. Motif analyses of the phosphorylation sites showed that six motifs including [PxsP], [sP], [sD], [Rxxs], [sxP] and [sxs] were enriched. We identified 12 and 48 PLSC phosphoproteins in M26 and MBB, respectively. Among these, 9 PLSC phosphoproteins were common to both genotypes, perhaps indicating a partial overlaps of the mechanisms to moderate drought stress. Gene ontology analyses revealed that the PLSC phosphoproteins present a unique combination of metabolism, transcription, translation and protein processing, suggesting that the response in apple to moderate drought stress encompasses a new homeostasis of major cellular processes. The basic trend was an increase in protein abundance related to drought and organic substance upon moderate drought stress between two genotypes. These increases were higher in the drought-tolerant genotype (MBB) than in the drought-sensitive genotype (M26). The 23 differentially expressed mRNA encoding phosphoproteins were analysis by quantitative real-time PCR (qRT-PCR). Our study is the first to address the phosphoproteome of a major fruit crop, apple rootstocks, in response to moderate drought stress, and provide insights into the molecular regulation mechanisms of apple rootstock under moderate drought stress.
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2013-10-14 | E-GEOD-24696 | biostudies-arrayexpress
Project description:Objective: To determine the effects of age and topographic location on gene expression in human neural retina. Methods: Macular and peripheral neural retina RNA were isolated from human donor eyes for DNA microarray and quantitative RTPCR analyses. Results: Total RNA from human donor retina preserved integrity. Hierarchic clustering analysis demonstrates the gene expression profiles of young, old, macula and peripheral retina cluster into four distinct groups. Genes which are highly expressed in macular, peripheral, young or old retina are identified, including inhibitors of Wnt Signaling Pathway (DKK1, FZD10 and SFRP2) shown preferably expressed in the periphery. Conclusions: The transcriptome of the human retina is affected by age and topographic location. Wnt pathway inhibitors in the periphery may maintain peripheral retinal cells in an undifferentiated state. Understanding the effects of age and topographic location on gene expression may lead to the development of new therapeutic interventions for age-related eye diseases. Twleve youang and older retinal macular or prepheral RNA samples are used for DNA microarray study to compare the effects of aging and anatomic location on gene expression in human retina.
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