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SRP055996
Danio rerio
1138 Downloadable Samples
Illumina HiSeq 2500
Description
Spatial localization is a key determinant of cellular fate and behavior, but spatial RNA assays traditionally rely on staining for a limited number of RNA species. In contrast, single-cell RNA-seq allows for deep profiling of cellular gene expression, but established methods separate cells from their native spatial context. Here we present Seurat, a computational strategy to infer cellular localization by integrating single-cell RNA-seq data with in situ RNA patterns. We applied Seurat to spatially map 851 single cells from dissociated zebrafish (Danio rerio) embryos, inferring a transcriptome-wide map of spatial patterning. We confirmed Seurat’s accuracy using several experimental approaches, and used it to identify a set of archetypal expression patterns and spatial markers. Additionally, Seurat correctly localizes rare subpopulations, accurately mapping both spatially restricted and scattered groups. Seurat will be applicable to mapping cellular localization within complex patterned tissues in diverse systems. Overall design: We generated single-cell RNA-seq profiles from dissociated cells from developing zebrafish embryos (late blastula stage - 50% epiboly)
Publication Title
Spatial reconstruction of single-cell gene expression data.
Sample Metadata Fields
Subject
View Samples- Homo sapiens
- 13 Downloadable Samples
- Illumina HiSeq 2500
Description
Computational single-cell RNA-seq (scRNA-seq) methods have been successfully applied to experiments representing a single condition, technology, or species to discover and define cellular phenotypes. However, identifying subpopulations of cells that are present across multiple datasets remains challenging. Here, we introduce an analytical strategy for integrating scRNA-seq datasets based on common sources of variation, enabling the identification of shared populations across datasets and downstream comparative analysis. Implemented in our R toolkit Seurat (http://satijalab.org/seurat/), we use our approach to align scRNA-seq datasets of peripheral blood monocytes (PBMCs) under resting and stimulated conditions, hematopoietic progenitors sequenced using two profiling technologies, and pancreatic cell 'atlases' generated from human and mouse islets. In each case, we learn distinct or transitional cell states jointly across datasets, while boosting statistical power through integrated analysis. Our approach facilitates general comparisons of scRNA-seq datasets, potentially deepening our understanding of how distinct cell states respond to perturbation, disease, and evolution. Overall design: Human PBMCs were profiled using ddSeq and bulk RNA-seq. The ddSeq experiment was performed on unperturbed PBMCs. The bulk RNA-seq experiments were performed on both unperturbed and IFN-beta stimulated PBMC-derived populations (cDCs and pDCs) with three technical replicates.
Publication Title
Integrating single-cell transcriptomic data across different conditions, technologies, and species.
Sample Metadata Fields
Specimen part, Subject
View Samples- Homo sapiens
- 19 Downloadable Samples
Illumina HumanHT-12 V4.0 expression beadchip
Description
Breast cancer is a heterogeneous disease for which prognosis and treatment strategies are largely governed by the receptor status (estrogen, progesterone and Her2-neu) of the tumor cells. Gene expression profiling of whole breast tumors further stratifies breast cancer into several molecular subtypes which also co-segregate with the receptor status of the tumor cells. We postulated that cancer associated fibroblasts (CAFs) within the tumor stroma may exhibit subtype specific gene expression profiles and thus contribute to the biology of the disease in a subtype specific manner. Several studies have reported gene expression profile differences between CAFs and normal breast fibroblasts but in none of these studies were the results stratified based on tumor subtypes. To address whether gene expression in breast cancer associated fibroblasts varies between breast cancer subtypes, we compared the gene expression profiles of early passage primary CAFs isolated from twenty human breast cancer samples representing three main subtypes; seven ER+, seven triple negative (TNBC) and six Her2+. We observed significant expression differences between CAFs derived from Her2+ breast cancer and CAFs from TNBC and ER+ cancers, particularly in pathways associated with cytoskeleton and integrin signaling. In the case of Her2+ breast cancer, the signaling pathways found to be selectively up regulated in CAFs may contribute to the more invasive properties and unfavorable prognosis of Her2+ breast cancer. These data demonstrate that in addition to the distinct molecular profiles that characterize the neoplastic cells, CAF gene expression is also differentially regulated in distinct subtypes of breast cancer.
Publication Title
Human breast cancer associated fibroblasts exhibit subtype specific gene expression profiles.
Sample Metadata Fields
Specimen part, Subject
View Samples- Mus musculus
- 14 Downloadable Samples
- Illumina HiSeq 2500
Description
Adult hematopoiesis has been studied in terms of progenitor differentiation potentials, whereas its kinetics in vivo is poorly understood. We combined inducible lineage tracing of endogenous adult hematopoietic stem cells (HSC) with flow cytometry and single-cell RNA sequencing to characterize early steps of hematopoietic differentiation in the steady state. Labeled cells, comprising primarily long-term HSC and some short-term HSC, produced megakaryocytic lineage progeny within one week, in a process that required only 2-3 cell divisions. Erythroid and myeloid progeny emerged simultaneously by 2 weeks, and included a progenitor population with expression features of both lineages. Myeloid progenitors at this stage showed diversification into granulocytic, monocytic and dendritic cell types, and rare intermediate cell states could be detected. In contrast, lymphoid differentiation was virtually absent within the first 3 weeks of tracing. These results show that continuous differentiation of HSC rapidly produces major hematopoietic lineages and cell types, and reveal fundamental kinetic differences between megakaryocytic, erythroid, myeloid and lymphoid differentiation. Overall design: We combined inducible lineage tracing of endogenous adult hematopoietic stem cells (HSC) with flow cytometry and single-cell RNA sequencing to characterize early steps of hematopoietic differentiation in the steady state.
Publication Title
Kinetics of adult hematopoietic stem cell differentiation in vivo.
Sample Metadata Fields
Specimen part, Subject
View Samples- Mus musculus
- 154 Downloadable Samples
- Illumina HiSeq 2500
Description
We present a detailed single cell time course of the macrophage response to Salmonella infection. By combining phenotypic fluorescent labels with single cell expression analysis we are able to identify gene modules associated with bacterial exposure and bacterial infection. We also identify other genetic clusters that are expressed heterogenously, ananlyzing both their regulation and their impact on infection Overall design: Analysis of 192 single cells across 4 time points after Salmonella exposure (MOI 1:1) with one of three different fluorescent labels indicating whether a given cell contained no intracellular bacteria (non-fluorescent), contained dead intracellular bacteria (only pHrodo positive), or contained live intracellular bacteria (pHrodo and GFP positive)
Publication Title
Pathogen Cell-to-Cell Variability Drives Heterogeneity in Host Immune Responses.
Sample Metadata Fields
No sample metadata fields
View Samples- Mus musculus
- 96 Downloadable Samples
- Illumina HiSeq 2500
Description
We present a detailed single cell analysis of the macrophage response to LPS from Salmonella enterica. By combining single cell transcriptional analysis, fluorescently labeled, LPS-coated beads, and cytometry we are able to distinguish the responses of macrophages that have internalized LPS-coated beads and those that have not. Overall design: Analysis of 96 single macrophages that were either: left untreated, were exposed to but did not internalize uncoated beads, were exposed to and internalized uncoated beads, were exposed to but did not internalize LPS-coated beads, or were exposed to and did internalize LPS-coated beads.
Publication Title
Pathogen Cell-to-Cell Variability Drives Heterogeneity in Host Immune Responses.
Sample Metadata Fields
No sample metadata fields
View Samples- Mus musculus
- 20 Downloadable Samples
- Illumina HiSeq 2000
Description
A time course of the macrophage response to Salmonella exposure analyzing the effects of input cell number as a control for single cell studies Overall design: Mouse macrophages were exposed to Salmonella enterica for different lengths of time. Libraries were constructed using either approximately 500,00 macrophages lysed directly on a tissue culture dish (bulk) or using only 150 cells isolated using FACS (sorted). All libraries were constructed in duplicate (bulk) or triplicate (sorted). All replicates are biological replicates
Publication Title
Pathogen Cell-to-Cell Variability Drives Heterogeneity in Host Immune Responses.
Sample Metadata Fields
No sample metadata fields
View Samples- Mus musculus
- 768 Downloadable Samples
- Illumina HiSeq 2500
Description
Direct programming via the overexpression of transcription factors (TFs) aims to control cell fate at a precision that will be instrumental for clinical applications. However, direct programming of terminal fates remains an obscure process. Taking advantage of the rapid and uniquely efficient programming of spinal motor neurons by overexpression of Ngn2, Isl1 and Lhx3, we have characterized gene expression, chromatin and transcription factor binding time-course dynamics during complete motor neuron programming. Our studies point to a surprisingly dynamic programming process. Promoter chromatin and expression analysis reveals at least three distinct phases of gene activation, while programming factor binding shifts from one set of targets to another, controlling regulatory region activity and gene expression. Furthermore, our evidence suggest that the enhancers and genes activated in the final stage of motor neuron processing are dependent on the combined activities of Isl1 and Lhx3 factors with Ebf and Onecut TFs that are themselves activated midway through the programming process. Our results suggest an unexpected multi-stage model of motor neuron programming in which the programming TFs require activation of a set of intermediate regulators before they complete the programming process. Overall design: Gene expression was characterized by single-cell RNA-seq during the direct programming of ES cells into motor neurons using over-expression of Ngn2-Isl1-Lhx3 programming factors.
Publication Title
A Multi-step Transcriptional and Chromatin State Cascade Underlies Motor Neuron Programming from Embryonic Stem Cells.
Sample Metadata Fields
Specimen part, Cell line, Treatment, Subject
View Samples- Mus musculus
- 10 Downloadable Samples
- Illumina HiSeq 2500
Description
Direct programming via the overexpression of transcription factors (TFs) aims to control cell fate at a precision that will be instrumental for clinical applications. However, direct programming of terminal fates remains an obscure process. Taking advantage of the rapid and uniquely efficient programming of spinal motor neurons by overexpression of Ngn2, Isl1 and Lhx3, we have characterized gene expression, chromatin and transcription factor binding time-course dynamics during complete motor neuron programming. Our studies point to a surprisingly dynamic programming process. Promoter chromatin and expression analysis reveals at least three distinct phases of gene activation, while programming factor binding shifts from one set of targets to another, controlling regulatory region activity and gene expression. Furthermore, our evidence suggest that the enhancers and genes activated in the final stage of motor neuron processing are dependent on the combined activities of Isl1 and Lhx3 factors with Ebf and Onecut TFs that are themselves activated midway through the programming process. Our results suggest an unexpected multi-stage model of motor neuron programming in which the programming TFs require activation of a set of intermediate regulators before they complete the programming process. Overall design: For bulk cell RNA-seq analysis, cells were collected at different time points after NIL induction and RNA isolated using TRIzol LS (Life Technologies) followed by purification using Qiagen RNAeasy kit
Publication Title
A Multi-step Transcriptional and Chromatin State Cascade Underlies Motor Neuron Programming from Embryonic Stem Cells.
Sample Metadata Fields
Specimen part, Cell line, Subject
View Samples- Homo sapiens
- 64 Downloadable Samples
- Illumina HiSeq 2500, Illumina HiSeq 2000
Description
RNA-Seq is an effective method to study the transcriptome, but can be difficult to apply to scarce or degraded RNA from fixed clinical samples, rare cell populations, or cadavers. Recent studies have proposed several methods for RNA-Seq of low quality and/or low quantity samples, but their relative merits have not been systematically analyzed. Here, we compare five such methods using a comprehensive set of metrics, relevant to applications such as transcriptome annotation, transcript discovery, and gene expression. Using a single human RNA sample, we constructed and deeply sequenced 10 libraries with these methods and two control libraries. We find that the RNase H method performed best for low quality RNA, and can even effectively replace oligo (dT) based methods for standard RNA-Seq. SMART and NuGEN had distinct strengths for low quantity RNA. Our analysis allows biologists to select the most suitable methods and provides a benchmark for future method development. Overall design: Examination of 9 different RNA-Seq libraries starting from total RNA from 5 distinct methods; also 3 control RNA-Seq libraries
Publication Title
Comparative analysis of RNA sequencing methods for degraded or low-input samples.
Sample Metadata Fields
Specimen part, Cell line, Subject
View Samples