Mammary glands were collected from 8 pubescent (4.7-4.9 week-old) female mice and 8 adult (10 week old) female mice. Freshly sorted epithelial cells were submitted to a Fluidigm C1 System machine for single cell capture and cDNA synthesis. Cells were visualized under the microscope to ensure integrity of the captured single cells prior to cDNA preparation. Libraries were prepared using the Nextera XT kit and sequencing was carried out on an Illumina NextSeq 500 to achieve paired-end 75 bp reads. Overall design: RNA-seq profiling was completed for 221 cells from pubescent mice and 223 cells from adult mice.
Construction of developmental lineage relationships in the mouse mammary gland by single-cell RNA profiling.
Cell line, Subject
View SamplesMammary glands were collected from pre-pubescent (2 weeks old), pubescent (4.7- 4.9 weeks old) and adult (10 week-old) female mice. Freshly sorted epithelial cells were submitted to a Fluidigm C1 System machine for single cell capture and cDNA synthesis. Cells were visualized under a microscope to ensure integrity of the captured single cells prior to cDNA preparation. Libraries were prepared using the Nextera XT kit and sequencing was carried out on an Illumina Hiseq 2000 to achieve 100 bp paired-end reads. Overall design: RNA-seq profiling was completed for 144 cells from 8 pre-puberty (2 week old) mice, 136 cells from 8 pubescent (4.7-4.9 week old) mice and 66 cells from 8 adult (10 week old) mice.
Construction of developmental lineage relationships in the mouse mammary gland by single-cell RNA profiling.
Cell line, Subject
View SamplesMammary glands were collected from 8 pregnant (12.5 day) mice and 8 adult (10 week old) female mice. Basal epithelial cells were FACS sorted. Freshly sorted cells were submitted to a Fluidigm C1 System machine for single cell capture and cDNA synthesis. Cells were visualized under the microscope to ensure integrity of the captured single cells prior to cDNA preparation. Libraries were prepared using the Nextera XT kit and sequencing was carried out on an Illumina NextSeq 500 to achieve paired-end 75 bp reads. Overall design: RNA-seq profiling was completed for 75 cells from pregnant mice and 237 cells from adult mice.
Construction of developmental lineage relationships in the mouse mammary gland by single-cell RNA profiling.
Cell line, Subject
View SamplesMammary glands were collected from 8 pregnant (12.5 day) mice and 8 non-pregnant adult (10 week old) female mice. Epithelial cells were FACS sorted from the pregnant mice. Cells from the adult mice were FACS sorted as basal or luminal. Freshly sorted cells were submitted to a Fluidigm C1 System machine for single cell capture and cDNA synthesis. Cells were visualized under the microscope to ensure integrity of the captured single cells prior to cDNA preparation. Libraries were prepared using the Nextera XT kit and sequencing was carried out on an Illumina NextSeq 500 to achieve 75 bp paired-end reads. Overall design: 112 basal cells and 43 luminal cells were profiled from the adult mice. 123 total epithelial cells were profiled from the pregnant mice.
Construction of developmental lineage relationships in the mouse mammary gland by single-cell RNA profiling.
Cell line, Subject
View SamplesMammary glands of 8 adult (10 week-old) female mice were collected. Freshly sorted basal and luminal epithelial cells were submitted to a Fluidigm C1 System machine for single cell capture and cDNA synthesis. Cells were visualized under the microscope to ensure integrity of the captured single cells prior to cDNA preparation. Libraries were prepared using the Nextera XT kit and sequencing was carried out on an Illumina Hiseq 2000 to achieve 100bp paired-end reads. Overall design: 96 basal and 90 luminal cells were profiled from 8 mice.
Construction of developmental lineage relationships in the mouse mammary gland by single-cell RNA profiling.
Cell line, Subject
View SamplesEpithelial cells were isolated by FACS from the mammary glands of adult (10 week old) female mice. A basal subpopulation of the epithelial cells was also isolated. Freshly sorted cells were submitted to a 10X Genomics Chromium System for single cell capture. cDNA synthesis and library preparation was done according to the protocol supplied by the manufacturer. Sequencing was carried out on an Illumina NextSeq500 sequencer to achieve 75 bp paired-end reads. Overall design: Transcriptional profiling was completed for 4771 basal cells and 3302 total epithelial cells from 8 mice.
Construction of developmental lineage relationships in the mouse mammary gland by single-cell RNA profiling.
Cell line, Subject
View SamplesLong-lived quiescent mammary stem cells (MaSCs) are presumed to coordinate the dramatic expansion of ductal epithelium that occurs through the different phases of postnatal development, but little is known about the molecular regulators that underpin the activation of MaSCs. Here we show that ablation of the transcription factor Foxp1 in the mammary gland profoundly impairs ductal morphogenesis, resulting in a rudimentary tree throughout adult life. Foxp1-deficient glands were highly enriched for quiescent Tspan8hi MaSCs, which failed to become activated, even in competitive transplantation assays, and therefore harbor a cell-intrinsic defect. Luminal cells aberrantly expressed basal genes, suggesting that Foxp1 may also contribute to cell-fate decisions. Notably, Foxp1 was uncovered as a direct repressor of the Tspan8 gene in basal cells and deletion of Tspan8 could rescue the profound defects in ductal morphogenesis elicited by Foxp1 loss. Thus, a single transcriptional regulator, Foxp1, can control the exit of MaSCs from dormancy to orchestrate differentiation and development. Overall design: Basal and luminal epithelial cells were extracted from the mammary glands of floxed Foxp1 control and Foxp1 mammary gland conditional knockout mice. mRNA from three biological replicates of each cell population was profiled by RNA sequencing. All mice were female.
Foxp1 Is Indispensable for Ductal Morphogenesis and Controls the Exit of Mammary Stem Cells from Quiescence.
Specimen part, Cell line, Subject
View SamplesBreast tumors are characterized by inherent heterogeneity but the evolving cellular organization of breast tumors through progression remains poorly understood. Individual clones were tracked by combining mouse models of breast cancer with Confetti reporter strains. Expression profiling of individual clones sorted from tumors arising in K5- and Elf5-driven Pten/p53-deficient mice revealed distinct molecular signatures. Overall design: K5-rtTA-IRES-GFP and ElF5-rtTA-IRES-GFP transgenic mice were crossed with TetO-cre (JAX) and R26R-Confetti reporter strains to generate triple genetically modified mice. The mice were treated with medroxyprogesterone acetate (MPA) and dimethylbenz(a)anthracene (DMBA) to induce carcinogenesis. Three K5-driven and five Elf5-driven mammary tumors were selected. Individual live cells from each tumor were FACS sorted by the four Confetti fluorescent markers (to select individual clones) and by CD24 expression (high or low). Cell subsets for the eight tumors, four fluorescent markers and positive or negative CD24 status were profiled by RNA-seq (38 samples in all). Expression was quantified by counting RNA-seq reads at the gene level and (separately) at the exon level.
Intraclonal Plasticity in Mammary Tumors Revealed through Large-Scale Single-Cell Resolution 3D Imaging.
Specimen part, Cell line, Subject
View SamplesThe sexually dimorphic expression of genes across 26 somatic rat tissues was using Affymetrix RAE-230 genechips. We considered probesets to be sexually dimorphically expressed (SDE) if they were measurably expressed above background in at least one sex, there was at least a two-fold difference in expression (dimorphism) between the sexes, and the differences were statistically significant after correcting for false discovery. 14.5% of expressed probesets were SDE in at least one tissue, with higher expression nearly twice as prevalent in males compared to females. Most were SDE in a single tissue. Surprisingly, nearly half of the probesets that were (SDE) in multiple tissues were oppositely sex biased in different tissues, and most SDE probesets were also expressed without sex bias in other tissues. Two genes were widely SDE: Xist (female-only) and Eif2s3y (male-only). The frequency of SDE probesets varied widely between tissues, and was highest in the duodenum (6.2%), whilst less than 0.05% in over half of the surveyed tissues. The occurrence of SDE probesets was not strongly correlated between tissues. Within individual tissues, however, relational networks of SDE genes were identified. In the liver, networks relating to differential metabolism between the sexes were seen. The estrogen receptor was implicated in differential gene expression in the duodenum. To conclude, sexually dimorphic gene expression is common, but highly tissue-dependent. Sexually dimorphic gene expression may provide insights into mechanisms underlying phenotypic sex differences.
The incidence of sexually dimorphic gene expression varies greatly between tissues in the rat.
Sex, Specimen part
View SamplesMany heavy metals, including nickel (Ni), cadmium (Cd), and chromium (Cr) are toxic industrial chemicals with an exposure risk in both occupational and environmental settings that may cause harmful outcomes. While these substances are known to produce adverse health effects leading to disease or health problems, the detailed mechanisms remain unclear. To elucidate the processes involved in the of toxicity of nickel, cadmium, and chromium at the molecular level and to perform a comparative analysis, H4-II-E-C3 rat liver-derived cell lines were treated with soluble salts of each metal using concentrations derived from viability assays, and gene expression patterns were determined with DNA microarrays.
Exposure to nickel, chromium, or cadmium causes distinct changes in the gene expression patterns of a rat liver derived cell line.
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