Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Next-generation sequencing facilitates quantitative analysis of diaphysis and metaphysis from femur of 5-week-old Dlx3Oc-cKO and wild type (WT=Dlx3+/+) mice transcriptomes

ABSTRACT: Purpose: Next-generation sequencing (NGS) has revolutionized systems-based analysis of cellular pathways. The goals of this study are to compare NGS-derived femoral diaphysis and metaphysis transcriptome profiling (RNA-seq) to determine pathways and networks dependent on Dlx3 during bone development and homeostasis. Methods: mRNA profiles of diaphysis and metaphysis isolated from the femur of 5-week-old wild-type (WT) and Dlx3Oc-cKO (OC-cre;Dlx3f/-) conditional knockout mice were generated by deep sequencing, in triplicate, using Illumina HiSeq 2000. The sequence reads that passed quality filters were analyzed at the transcript isoform level by ANOVA (ANOVA) and TopHat. qRT-PCR validation was performed using SYBR Green assay. Results: RNA-Seq data were generated with Illumina's HiSeq 2000 system. Raw sequencing data were processed with CASAVA 1.8.2 to generate fastq files. Reads of 50 bases were mapped to the mouse transcriptome and genome mm9 using TopHat 1.3.2. Gene expression values (RPKM) were calculated with Partek Genomics Suite 6.6, which was also used for the ANOVA analysis to determine significantly differentially expressed genes. Conclusions: Our study represents the first detailed analysis of Dlx3Oc-cKO diaphysis and metaphysis from femurs, with biologic triplicates, generated by RNA-seq technology. The optimized data analysis workflows reported here should provide a framework for comparative investigations of expression profiles. We conclude that RNA-seq based transcriptome characterization would expedite genetic network analyses and permit the dissection of complex biologic functions. Diaphysis and metaphysis mRNA profiles of metaphysis and diaphysis from femurs of 5-wk-old (WT) and Dlx3Oc-cKO male mice were generated by deep sequencing, in triplicate, using Illumina HiSeq 2000.

ORGANISM(S): Mus musculus

SUBMITTER: Hong-wei Sun

PROVIDER: E-GEOD-53105 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

DLX3 regulates bone mass by targeting genes supporting osteoblast differentiation and mineral homeostasis in vivo.

Isaac J J Erthal J J Gordon J J Duverger O O Sun H-W HW Lichtler A C AC Stein G S GS Lian J B JB Morasso M I MI

Cell death and differentiation 20140620 9

Human mutations and in vitro studies indicate that DLX3 has a crucial function in bone development, however, the in vivo role of DLX3 in endochondral ossification has not been established. Here, we identify DLX3 as a central attenuator of adult bone mass in the appendicular skeleton. Dynamic bone formation, histologic and micro-computed tomography analyses demonstrate that in vivo DLX3 conditional loss of function in mesenchymal cells (Prx1-Cre) and osteoblasts (OCN-Cre) results in increased bon ...[more]

PMID: 24948010

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Project description:Purpose: Next-generation sequencing (NGS) has revolutionized systems-based analysis of cellular pathways. The goals of this study are to compare small non-coding RNA profiling (snRNA-seq) in WT oocyte, sperm and 2PN stage embryos to those sperm and 2PN stage embryos derived from WT, Dicer cKO and Drosha cKO. We further study the roles of sperm-borne small RNA on fertilization and pre-implantation embryonic development. Methods: Small RNA profiles of adult wild-type (WT) oocytes, adult WT sperm, 2PN stage embryos, adult Dicer cKO/Drosha cKO sperm, 2PN stage embryos were generated by deep sequencing in duplicate, using Ion Torrent Proton. The sequence reads that passed quality filters were analyzed at the small RNA level with two methods: Burrows–Wheeler Aligner (BWA) followed by ANOVA (ANOVA) and TopHat followed by Cufflinks. Results: Using an optimized data analysis workflow, we mapped about 30 million sequence reads per sample to the mouse genome (build mm9) and identified 16,014 small RNA (miRNA and endo-siRNA) in the oocyte, sperm and 2PN stage of WT and Dicer cKO/Drosha cKO mice with BWA workflow and 34,115 transcripts with TopHat workflow. Approximately 47% of the miRNAs showed differential expression between the WT and Dicer cKO sperm, ~52% of miRNAs were shown dysregulated in Drosha cKO sperm compared to those in WT sperm with a fold change ≥2.0 and p value <0.05. Data analysis with BWA and TopHat workflows revealed a significant overlap yet provided complementary insights in transcriptome profiling. Conclusions: Our study represents the first detailed analysis of small non-coding RNAs (miRNAs) in sperm and demonstrated that sperm-borne small RNAs are important for fertilization and early embrynic develoment, with biologic duplicates, generated by RNA-seq technology. The optimized data analysis workflows reported here should provide a framework for comparative investigations of small RNAs profiles in mouse sperm, oocytes and 2PN stage of embryos. Our results show that NGS offers a comprehensive and more accurate quantitative and qualitative evaluation of small RNA contents within sperm or oocytes/embryos. We conclude that RNA-seq based small RNAs characterization in gametes would expedite genetic network analyses and permit the dissection of complex biologic functions during fertilization and embryonic development.

Dataset Information

Next-generation sequencing facilitates quantitative analysis of diaphysis and metaphysis from femur of 5-week-old Dlx3Oc-cKO and wild type (WT=Dlx3+/+) mice transcriptomes

Publications

DLX3 regulates bone mass by targeting genes supporting osteoblast differentiation and mineral homeostasis in vivo.

Similar Datasets

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