RNA editing is a mutational mechanism that specifically alters the nucleotide content in sets of transcripts while leaving their cognate genomic blueprint intact. Editing has been detected from bulk RNA-seq data in thousands of distinct transcripts, but apparent editing rates can vary widely (from under 1% to almost 100%). These observed editing rates could result from approximately equal rates of editing within each individual cell in the bulk sample, or alternatively, editing estimates from a population of cells could reflect an average of distinct, biologically significant editing signatures that vary substantially between individual cells in the population. To distinguish between these two possibilities we have constructed a hierarchical Bayesian model which quantifies the variance of editing rates at specific sites using RNA-seq data from both single cells and a cognate bulk sample consisting of ~ 106 cells. The model was applied to data from murine bone-marrow derived macrophages and dendritic cells, and predicted high variance for specific edited sites in both cell types tested. We then 1 validated these predictions using targeted amplification of specific editable transcripts from individual macrophages. Our data demonstrate substantial variance in editing signatures between single cells, supporting the notion that RNA editing generates diversity within cellular populations. Such editing-mediated RNA-level sequence diversity could contribute to the functional heterogeneity apparent in cells of the innate immune system. Overall design: 26 samples were subjected to RNA-seq: 24 single WT macrophages, and 2 bulk samples (Apobec1 WT and KO macrophages), consisting of 500,000-1 million cells each.
RNA editing generates cellular subsets with diverse sequence within populations.
Specimen part, Cell line, Subject
View SamplesThe transcription factor MIST1 is required for final maturation of secretory cells of diverse tissues, including gastric digestive-enzyme secreting zymogenic (chief) cells (ZCs). Here, we show that MIST1 directly activates RAB26, RAB3D and several other genes.
RAB26 and RAB3D are direct transcriptional targets of MIST1 that regulate exocrine granule maturation.
Specimen part, Cell line
View SamplesIn this experiment, mucous neck cells from the gastric epithelium of normal, adult C57/B6 mice were laser-capture microdissected to determine gene expression in neck cells relative to pit cells, parietal cells, and zymogenic cells, whose expression profiles were previously deposited in GEO.
Evolution of the human gastrokine locus and confounding factors regarding the pseudogenicity of GKN3.
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View SamplesSelf-renewal and differentiation of spermatogonial stem cells (SSCs) provides the foundation for testis homeostasis, yet mechanisms that control their functions in mammals are poorly defined. We used microarray transcript profiling to identify specific genes whose expression are augmented in the SSC-enriched Thy1+ germ cell fraction of mouse pup testes. Comparisons of gene expression in the Thy1+ germ cell fraction to the Thy1-depeleted testis cell population identified 202 genes that are expressed 10-fold or higher in Thy1+ cells. This database provided a mining tool to investigate specific characteristics of SSCs and identify novel mechanisms that potentially influence their functions.
Colony stimulating factor 1 is an extrinsic stimulator of mouse spermatogonial stem cell self-renewal.
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View SamplesSpermatogonia expressing the highest levels of ID4 (ID4-GFP Bright) represent a population highly enriched for spermatogonial stem cells (SSC) while those expressing lower levels (ID4-GFP Dim) are the putative immediate progenitors. Comparing the transcriptome of these populations can provide insight into the SSC to progenitor transition. Overall design: Comparison of transcriptomes of ID4-GFP Bright and ID4-GFP Dim spermatogonia from postnatal day 8 mouse pups
ID4 levels dictate the stem cell state in mouse spermatogonia.
Specimen part, Subject
View SamplesP6 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 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 SamplesPaternal imprinting initiates in primordial germ cells (PGCs), and is considered largely completed at birth. The resulting postnatal spermatogonial stem cells (SSCs) thenself-renew and proliferate to populate the testicular niche, with sexual maturation enabling productive gametogenesis. Overall design: mRNA profiles of neonatal wild type (WT) mice testis were generated by deep sequencing using Illumina HiSeq 2000 Examination of 2 different histone modifications in mouse spermatogonia Please note that ChIPSeq_Kitplus samples are samples isolated with MACS CD117 microbeads from Miltenyi and ChIPSeq_Kitminus are samples that were not positively selected for Kit.
Transcription and imprinting dynamics in developing postnatal male germline stem cells.
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