Next Generation Sequencing Facilitates Quantitative Analysis of Wild Type and Dicer LKO Liver mRNA Transcriptomes
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE189229 | GEO | 2021/11/22
REPOSITORIES: GEO
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Project description:Next Generation Sequencing Facilitates Quantitative Analysis of Wild Type and Dicer LKO Liver mRNA Transcriptomes
| PRJNA782011 | ENA
Project description:To investigate which mRNA and miRNA are involved in Dicer KO mouse tendon hypoplasticity, we performed RNA-seq and small RNA-seq using RNA from Achilles tendon of Cont (Dicer f/f), Scx HT (ScxCre/+ Knock In:Dicer +/+) and Dicer KO (ScxCre/+ Knock In:Dicer f/f) mice at 4 weeks of age. Achilles tendon from 4-week-old female mice was harvested, RNA was isolated using an RNA extraction kit. RNA libraries were generated and sequenced by K. K. DNAFORM (Tokyo, Japan). The libraries were sequenced by Illumina HISEQ4000 using Illumina provided protocol. Differential gene expression was analyzed with R Bioconductor DESeq2. We found that a large portion of tendon-fibroblast characteristic genes was downregulated in Dicer KO mice Achilles tendon compared to Cont and Scx HT.
2022-05-31 | GSE186353 | GEO
Project description:Dicer is an essential ribonuclease involved in the biogenesis of miRNA. Previous studies have reported that Dicer is dysregulated in multiple types of cancers. To investigate the downstream phosphoproteome of Dicer, we carried out phosphotyrosine profiling of the liver of Dicer knockout of mice. We employed antibody-based enrichment of phosphotyrosine containing peptides coupled with spike-in SILAC-based quantitation. Over 400 phosphoisites were identified in each condition while 290 phosphosites were quantitated in common. Amongst these, several key signaling molecules like receptor tyrosine kinase MET, MET, PDGFR, EPHA2, EPHB4 and IGF1R/INSR were hyperphosphorylated. Also, non-receptor tyrosine kinases of Src family and their downstream effector signaling partners including IRS2 and STAT3were found to be hyperphosphorylated. Our study indicates that there is significant alteration in the signaling pathways upon ablation of Dicer.
2016-07-08 | PXD000927 | Pride
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:In other to assess functional involvement of Klf5 in DR regulation, we made liver-specific Klf5 knockout mice. Ductular reaction upon cholestatic liver injury is severely suppressed in these mice. We conducted RNA-seq analysis on the BECs from control mice and Klf5 LKO mice upon DDC injury to further elucidate the Klf5 functions.
2018-03-16 | GSE97167 | GEO
Project description:mRNA profiles of healthy vs HUWE1 mutant cells were generated by deep sequencing, in triplicate, using Illumina HiSeq 4000 (single read, 75bp).
2021-03-06 | GSE130551 | GEO
Project description:mRNA profiles of HEK293T wild type (WT), AAG-/-, and ELP1-/- cells were generated by deep sequencing, in triplicate, using Illumina HiSeq 2500.
2020-02-26 | GSE129009 | GEO
Project description:We have shown that Chrnb4-cre; Dicerflox/flox retinas (Dicer CKO) display a cone dystrophy by postnatal day P21, independent of rod degeneration. To elucidate which miRNAs were involved in such phenotype, miRNA sequencing was performed in whole retinas of P21 and 3.5 month old control and Dicer CKO mice. This experiment aims to identify which miRNAs were differentially expressed in Dicer conditional knockout mice when compared to control mice.
2019-01-14 | E-MTAB-6132 | biostudies-arrayexpress
Project description:Purpose: The current study tests the hypothesis that peroxisome proliferator-activated receptor β (PPARβ) has a role in liver regeneration due to its effect in regulating energy homeostasis and cell proliferation. Methods: Wild-type (WT) and PPARβ-null mice (KO) male mice (3-5 month, C57BL/6) were kept in steel microisolator cages at 22°C with a 14-hr/10-hr light/dark cycle. Food and water were provided ad libitum throughout the entire feeding period. Standard PH was performed. All the animal experiments were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals under protocols approved by the University of California Davis Animal Care and Use Committee.Mouse liver RNA was prepared using TRIzol (Invitrogen, Carlsbad, CA). RNA concentration and integrity were determined by the Agilent 2100 Bioanalyzer using a RNA Nano Bioanalysis Chip. RNA-sequencing library preparation and sequencing was carried out by the Genome Sequencing Facility at University of Kansas Medical Center (Kansas City, KS). cDNA libraries were prepared with 2 µg of total RNA using the TruSeq RNA Sample Preparation Kit (Illumina). The libraries were clustered and sequenced on an Illumina HiSeq 2000 instrument with 100 bp paired end reads. Results: Differential gene expression profiling revealed the inhibition of expression in genes and pathways that are involved in metabolism and proliferation in regenerating KO livers. Specifically, PPARβ deficiency affected the activation of Akt and the expression of E2fs. Pathways that control glycolysis, FA synthesis as well as cell proliferation were de-regulated in regenerating PPARβ KO livers. Conclusions: The data suggest a role for PPARβ in regulating liver regeneration that is mediated at least in part through Akt and E2f-regulated pathways. mRNA profiles of 3-month old wild type (WT) and PPARbeta mice were generated by deep sequencing, one simple, using Illumina HiSeq 2000 instrument with 100 bp paired end reads
2013-05-18 | E-GEOD-47062 | 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 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