Stem cell differentiation trajectories in Hydra resolved at single cell resolution
ABSTRACT: The adult Hydra polyp continuously renews all of its cells using three separate stem cell populations but the genetic pathways enabling these dynamic tissue properties are not well understood. We used Drop-seq to sequence 25,052 Hydra cells and identified the molecular signatures of a broad spectrum of cell states, from stem cells to terminally differentiated cells. We constructed differentiation trajectories for each cell lineage and identified the transcription factors expressed along these trajectories, thus creating a comprehensive map of all developmental lineages in the adult animal. We unexpectedly found that neuron and gland cell differentiation transits through a common progenitor state, suggesting a shared evolutionary history for these secretory cell types. Finally, we have built the first gene expression map of the Hydra nervous system. By producing a comprehensive molecular description of the adult Hydra polyp, we have generated a resource for addressing fundamental questions regarding the evolution of developmental processes and nervous system function. Overall design: Drop-seq analysis of homeostatic Hydra vulgaris AEP (25,025 cells, 15 Drop-seq libraries total, 13 libraries for whole animal dissociations, 2 libraries for suspensions enriched for neuronal cells using FACS), ATAC-seq analysis of whole homeostatic Hydra magnipapillata 105 (3 replicated ATAC-seq libraries), Tag-seq analysis of epithelium specific gene expression of homeostatic Hydra vulgaris AEP (3 replicates for body column endoderm, 3 replicates for body column ectoderm).
Project description:Vsx2-GFP mouse retinas were dissected, FACS sorted for GFP+ cells and single-cell mRNAseq libraries generated with Drop-Seq Drop-Seq was performed on two different batches. Bipolar1-Bipolar4 are replicates from batch 1 and Bipolar5-6 are replicates from Batch 2
Project description:Hematopoietic stem cells give rise to diverse cell types in the blood system, yet our molecular understanding of the earliest fate decisions that generate this enormous diversity in humans remains incomplete. Here we leverage Drop-seq to individually profile more than 20,000 progenitors cells from human cord blood, without prior enrichment or depletion for individual lineages based on surface markers. Our data reveal a transcriptional 'atlas' of progenitor states in human cord blood, which we leverage to reconstruct differentiation trajectories from HSC to four downstream lineages. And we also demonstrate that Drop-seq data can be utilized to identify new heterogeneous markers of cell state. Overall design: CD34+ cells were enriched from human cord blood mononuclear cells, and mRNA profiles were generated by paired-end sequencing in Illumina HiSeq, with cDNA being amplified using the Drop-seq protocol, or a modified SMART-Seq2 protocol. Generally, 2-3 replicates were performed per biological sample.
Project description:Cell suspensions were prepared from mouse lungs dissociated at baseline or 14 days after intratracheal bleomycin injury, and single cell mRNA-seq libraries were generated with Drop-seq.
Project description:Single-cell RNA sequencing of mouse amygdala using Drop-Seq Overall design: Drop-Seq was performed for amygdala cells dissociated from male C57BL/6J mice in different experimental groups using different single-cell dissociation methods. Single-cell suspensions were generated using the Act-Seq protocol or conventional dissociation protocol, and processed through Drop-Seq to produce single-cell cDNA libraries attached to microbeads. cDNAs were amplified by PCR and prepared for sequencing through tagmentation.
Project description:A cell suspension was prepared from wild-type P14 mouse retinas, and single-cell mRNAseq libraries were generated with Drop-Seq. Drop-Seq was performed on four separate days using the same age (P14) and strain (C57BL/6). On day 1, replicate 1 was obtained. On day 2, replicates 2 and 3 were obtained. On day 3, replicates 4-6 were obtained. On day 4, replicate 7 was obtained.
Project description:During embryogenesis, cells acquire distinct fates by transitioning through transcriptional states. To uncover these transcriptional trajectories during zebrafish embryogenesis, we sequenced 38,731 cells and developed URD, a simulated diffusion-based computational reconstruction method. URD identified the trajectories of 25 cell types through early somitogenesis, gene expression along them, and their spatial origin in the blastula. Analysis of Nodal signaling mutants revealed that their transcriptomes were canalized into a subset of wild-type transcriptional trajectories. Some wild-type developmental branchpoints contained cells expressing genes characteristic of multiple fates. These cells appeared to trans-specify from one fate to another. These findings reconstruct the transcriptional trajectories of a vertebrate embryo, highlight the concurrent canalization and plasticity of embryonic specification, and provide a framework to reconstruct complex developmental trees from single-cell transcriptomes. This SuperSeries is composed of the SubSeries listed below. Overall design: Drop-seq analysis of wild-type embryos (38,731 cells, 12 timepoints, 28 samples total, 20-40 embryos per sample), SMART-seq2 analysis of wild-type and MZoep mutant embryos (52 wild-type and 364 MZoep cells from 50% epiboly stage), and 10X single-cell sequencing analysis of WT and MZoep mutant embryos (3,000 WT and 2,200 MZoep cells from 6-somites stage).
Project description:Wandering third instar larvae were dissected where the eye disc was separated from the antenna and brain. The tissues were dissociated into single cells and captured using Drop-seq. Single cell libraries were then generated from each cell and finally sequenced.
Project description:We generated 77-bp Illumina reads from single messenger RNA libraries from four diverse developmental stages of the two-spotted spider mite to maximally capture the complement of transcribed sequences across development. Adult, nymphal, larvae and embryonic stages were separated using sieves of various pore sizes, and mites of various developmental stages were carefully selected for transcriptome library preparation. Samples were a mix of males and females to capture male and female patterns of transcription, and were reared on beans (Phaseolus vulgaris cv California Red Kidney). The RNA-Seq data was used for validation of gene models predicted by EuGene, and to study patterns of gene expression across development. Gene expression for spider mites from adult, nymph, larvae and embryonic developmental stages was examined (technical replicates were generated).