P6 ID4-EGFP+ undifferentiated spermatogonia, including those stained robustly (high) or weakly (low) for TSPAN8 were isolated by FACS. Overall design: Three replicate preparations of each population were used for independent RNA-seq using SMART-seq v4, Nextera XT libraries, Hiseq2500 sequencing, and TopHat/Bowtie/Cufflinks analyses.
TSPAN8 Expression Distinguishes Spermatogonial Stem Cells in the Prepubertal Mouse Testis.
Cell line, Subject
View SamplesLiver plays a profound role in the acute phase response (APR) observed in the early phase of acute bovine mastitis caused by Escherichia coli (E. coli). To gain an insight into the genes and pathways involved in hepatic APR of dairy cows we performed a global gene expression analysis of liver tissue sampled at different time points before and after intra-mammary (IM) exposure to E. coli lipopolysaccharide (LPS) treatment.
Gene expression profiling of liver from dairy cows treated intra-mammary with lipopolysaccharide.
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View SamplesTRF2 is a paralogue of TATA-box binding protein (TBP) with highest expression in testis. Although Trf2 inactivation in mice leads to arrested spermatogenesis, there is no direct evidence that Trf2 is recruited to chromatin to directly regulate gene expression. We used genetically modified mice where endogenous Trf2 has been modified to carry a TAP-TAG to perform ChIP-reChIP followed by deep sequencing. We found that Trf2 is recruited to all active promoters as a subunit of TFIIA/ALF complex together with TBP. To assess the effect of Trf2 inactivation on gene expression we performed RNA-seq on WT and Trf2-/- testes at 21 days of age when haploid cell gene expression is activated. Overall design: The testes from three 21 day old WT and three Trf2-/- males were taken to prepare total RNAs for deep sequencing.
TRF2 is recruited to the pre-initiation complex as a testis-specific subunit of TFIIA/ALF to promote haploid cell gene expression.
Specimen part, Subject
View SamplesThe platelet-derived growth factor receptor alpha (PDGFR) exhibits divergent effects in skeletal muscle. At physiological levels, signaling through this receptor promotes muscle development in growing embryos and proper angiogenesis in regenerating adult muscle. However, either increased PDGF ligands or enhanced PDGFR pathway activity causes pathological fibrosis. This excessive collagen deposition, which is seen in aged and diseased muscle, interferes with proper muscle function and limits the effectiveness of gene- and cell-based therapies for muscle disorders. Although compelling evidence exists for the role of PDGFR in fibrosis, little is known about the cells through which this pathway acts. Here we show that PDGFR signaling regulates a population of muscle-resident fibro/adipogenic progenitors (FAPs) that play a supportive role in muscle regeneration but may also cause fibrosis when aberrantly regulated. We found that FAPs produce multiple transcriptional variants of PDGFR with different polyadenylation sites, including an intronic variant that codes for a protein isoform containing a truncated kinase domain. This variant, upregulated during regeneration, acts as a decoy to inhibit PDGF signaling and to prevent FAP over-activation. Moreover, increasing expression of this isoform limits fibrosis in vivo, suggesting both biological relevance and therapeutic potential of modulating polyadenylation patterns in stem cell populations.
Intronic polyadenylation of PDGFRα in resident stem cells attenuates muscle fibrosis.
Sex, Specimen part, Treatment
View SamplesThe granulosa cells in the mammalian ovarian follicle respond to gonadotropin signalling and are involved in the processes of folliculogenesis and oocyte maturation. Studies on gene expression and regulation in human granulosa cells are of interest due to their potential for estimating the oocyte viability and IVF success. However, the post-transcriptional gene expression studies on miRNA level in the human ovary have been scarce. The current study determined the miRNA profile by deep sequencing of the two intrafollicular somatic cell types: mural and cumulus granulosa cells isolated from women undergoing controlled ovarian stimulation and in vitro fertilization. Overall design: Paired cumulus and mural granulosa samples were analysed from 3 women participating in IVF procedure. Libraries of all 6 samples were sequenced twice, generating 2 technical replicates for each sample. Differential gene expression study was performed on the pooled results of technical replicates.
Research resource: small RNA-seq of human granulosa cells reveals miRNAs in FSHR and aromatase genes.
Specimen part, Subject
View SamplesThe platelet-derived growth factor receptor alpha (PDGFR) exhibits divergent effects in skeletal muscle. At physiological levels, signaling through this receptor promotes muscle development in growing embryos and proper angiogenesis in regenerating adult muscle. However, either increased PDGF ligands or enhanced PDGFR pathway activity causes pathological fibrosis. This excessive collagen deposition, which is seen in aged and diseased muscle, interferes with proper muscle function and limits the effectiveness of gene- and cell-based therapies for muscle disorders. Although compelling evidence exists for the role of PDGFR in fibrosis, little is known about the cells through which this pathway acts. Here we show that PDGFR signaling regulates a population of muscle-resident fibro/adipogenic progenitors (FAPs) that play a supportive role in muscle regeneration but may also cause fibrosis when aberrantly regulated. We found that FAPs produce multiple transcriptional variants of PDGFR with different polyadenylation sites, including an intronic variant that codes for a protein isoform containing a truncated kinase domain. This variant, upregulated during regeneration, acts as a decoy to inhibit PDGF signaling and to prevent FAP over-activation. Moreover, increasing expression of this isoform limits fibrosis in vivo, suggesting both biological relevance and therapeutic potential of modulating polyadenylation patterns in stem cell populations.
Intronic polyadenylation of PDGFRα in resident stem cells attenuates muscle fibrosis.
Sex, Specimen part, Treatment
View SamplesThe granulosa cells in the mammalian ovarian follicle respond to gonadotropin signalling and are involved in the processes of folliculogenesis and oocyte maturation. Studies on gene expression and regulation in human granulosa cells are of interest due to their potential for estimating the oocyte viability and IVF success. The current study determined the mRNA profile by deep sequencing of the two intrafollicular somatic cell types: mural and cumulus granulosa cells isolated from women undergoing controlled ovarian stimulation and in vitro fertilization. Overall design: Paired cumulus and mural granulosa samples were analysed from 3 women participating in IVF procedure. Differential gene expression study was performed. The identified gene expression profile was also used for predicting targets for miRNAs that were also identified from the same samples (GSE46489).
Research resource: small RNA-seq of human granulosa cells reveals miRNAs in FSHR and aromatase genes.
Specimen part, Subject
View SamplesTo reveal distinct transcriptomes associated with spermatogonial stem cell renewal vs. initiation of differentiation, single-cell transcriptomes from Adult Human spermatogonia were subdivided into subpopulations based on the levels of ID4 mRNA (determined in this experiment). This correlates with distinct fates of corresponding mouse spermatogonia when assayed by transplantation, with ID4-EGFPbright cells highly enriched for SSCs, and ID4-EGFPdim cells enriched for progenitors. We used the Fluidigm C1 instrument to capture individual spermatogonia for SMART-Seq2 single-cell RNA-seq. Overall design: Nine replicate preparations of Adult Human spermatogonia were used for this study. Data are from a total of 635 cells. Cells were binned into quartiles according to ID4 mRNA levels (Q1 = ID4-high, Q4=ID4-low, Q2 and Q3 have intermediate ID4 mRNA levels) to facilitate comparisons.
The Mammalian Spermatogenesis Single-Cell Transcriptome, from Spermatogonial Stem Cells to Spermatids.
Specimen part, Subject
View SamplesTo reveal distinct transcriptomes associated with spermatogonial stem cell renewal vs. initiation of differentiation, single-cell transcriptomes from Adult ID4-EGFP+ spermatogonia were subdivided into subpopulations that displayed distinct fates when assayed by transplantation, with ID4-EGFPbright cells highly enriched for SSCs, and ID4-EGFPdim cells enriched for progenitors. We used the Fluidigm C1 instrument to capture individual spermatogonia for SMART-Seq2 single-cell RNA-seq. Overall design: Four replicate preparations of Adult mouse ID4-EGFP+ spermatogonia were used for this study. Data are from a total of 300 cells. Cells were binned into quartiles according to EGFP epifluorescence intensity (Q1 = EGFP-bright, Q4=EGFP-dim, Q2 and Q3 have intermediate EGFP fluorescence) to facilitate comparisons.
The Mammalian Spermatogenesis Single-Cell Transcriptome, from Spermatogonial Stem Cells to Spermatids.
Specimen part, Subject
View SamplesTo reveal distinct transcriptomes associated with spermatogonial stem cell renewal vs. initiation of differentiation, single-cell transcriptomes from P6 ID4-EGFP+ spermatogonia were subdivided into subpopulations that displayed distinct fates when assayed by transplantation, with ID4-EGFPbright cells highly enriched for SSCs, and ID4-EGFPdim cells enriched for progenitors. We used the Fluidigm C1 instrument to capture individual spermatogonia for SMART-Seq2 single-cell RNA-seq. Overall design: Five replicate preparations of mouse P6 ID4-EGFP+ spermatogonia were used for this study. Data are from a total of 278 cells. Cells were binned into quartiles according to EGFP epifluorescence intensity (Q1 = EGFP-bright, Q4=EGFP-dim, Q2 and Q3 have intermediate EGFP fluorescence) to facilitate comparisons.
The Mammalian Spermatogenesis Single-Cell Transcriptome, from Spermatogonial Stem Cells to Spermatids.
Specimen part, Subject
View Samples