Cells, the basic units of biological structure and function, vary broadly in type and state. Single-cell genomics can characterize cell identity and function, but limitations of ease and scale have ...prevented its broad application. Here we describe Drop-seq, a strategy for quickly profiling thousands of individual cells by separating them into nanoliter-sized aqueous droplets, associating a different barcode with each cell’s RNAs, and sequencing them all together. Drop-seq analyzes mRNA transcripts from thousands of individual cells simultaneously while remembering transcripts’ cell of origin. We analyzed transcriptomes from 44,808 mouse retinal cells and identified 39 transcriptionally distinct cell populations, creating a molecular atlas of gene expression for known retinal cell classes and novel candidate cell subtypes. Drop-seq will accelerate biological discovery by enabling routine transcriptional profiling at single-cell resolution.
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•Drop-seq enables highly parallel analysis of individual cells by RNA-seq•Drop-seq encapsulates cells in nanoliter droplets together with DNA-barcoded beads•Systematic evaluation of Drop-seq library quality using species mixing experiments•Drop-seq analysis of 44,808 cells identifies 39 cell populations in the retina
Capturing single cells along with sets of uniquely barcoded primer beads together in tiny droplets enables large-scale, highly parallel single-cell transcriptomics. Applying this analysis to cells in mouse retinal tissue revealed transcriptionally distinct cell populations along with molecular markers of each type.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Differentiation of human embryonic stem cells (hESCs) provides a unique opportunity to study the regulatory mechanisms that facilitate cellular transitions in a human context. To that end, we ...performed comprehensive transcriptional and epigenetic profiling of populations derived through directed differentiation of hESCs representing each of the three embryonic germ layers. Integration of whole-genome bisulfite sequencing, chromatin immunoprecipitation sequencing, and RNA sequencing reveals unique events associated with specification toward each lineage. Lineage-specific dynamic alterations in DNA methylation and H3K4me1 are evident at putative distal regulatory elements that are frequently bound by pluripotency factors in the undifferentiated hESCs. In addition, we identified germ-layer-specific H3K27me3 enrichment at sites exhibiting high DNA methylation in the undifferentiated state. A better understanding of these initial specification events will facilitate identification of deficiencies in current approaches, leading to more faithful differentiation strategies as well as providing insights into the rewiring of human regulatory programs during cellular transitions.
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•Epigenetic and transcriptional dynamics in hESCs and hESC-derived populations•Lineage-specific remodeling at regions bound by OCT4, SOX2, and NANOG in hESCs•Germ-layer-specific switch to H3K4me1 or H3K27me3 at sites of high DNA methylation•Epigenetic dynamics frequently precede transcriptional activation
The epigenetic and transcriptional landscapes of three cell types representing each embryonic lineage derived from human embryonic stem cells are profiled, revealing distinct histone modification and DNA methylation dynamics that accompany lineage specification.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Single-cell transcriptomics reveals gene expression heterogeneity but suffers from stochastic dropout and characteristic bimodal expression distributions in which expression is either strongly ...non-zero or non-detectable. We propose a two-part, generalized linear model for such bimodal data that parameterizes both of these features. We argue that the cellular detection rate, the fraction of genes expressed in a cell, should be adjusted for as a source of nuisance variation. Our model provides gene set enrichment analysis tailored to single-cell data. It provides insights into how networks of co-expressed genes evolve across an experimental treatment. MAST is available at https://github.com/RGLab/MAST .
Single-cell RNA-seq can precisely resolve cellular states, but applying this method to low-input samples is challenging. Here, we present Seq-Well, a portable, low-cost platform for massively ...parallel single-cell RNA-seq. Barcoded mRNA capture beads and single cells are sealed in an array of subnanoliter wells using a semipermeable membrane, enabling efficient cell lysis and transcript capture. We use Seq-Well to profile thousands of primary human macrophages exposed to Mycobacterium tuberculosis.
Highlights • Diversity in the immune system is encoded by extensive cellular variation. • Heterogeneity exists at multiple molecular levels. • New single cell methods enable systematic studies of ...heterogeneity. • A fundamental challenge is to connect molecular variability with phenotypic diversity.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Extensive cellular heterogeneity exists within specific immune-cell subtypes classified as a single lineage, but its molecular underpinnings are rarely characterized at a genomic scale. Here, we use ...single-cell RNA-seq to investigate the molecular mechanisms governing heterogeneity and pathogenicity of Th17 cells isolated from the central nervous system (CNS) and lymph nodes (LN) at the peak of autoimmune encephalomyelitis (EAE) or differentiated in vitro under either pathogenic or non-pathogenic polarization conditions. Computational analysis relates a spectrum of cellular states in vivo to in-vitro-differentiated Th17 cells and unveils genes governing pathogenicity and disease susceptibility. Using knockout mice, we validate four new genes: Gpr65, Plzp, Toso, and Cd5l (in a companion paper). Cellular heterogeneity thus informs Th17 function in autoimmunity and can identify targets for selective suppression of pathogenic Th17 cells while potentially sparing non-pathogenic tissue-protective ones.
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•Atlas of Th17 single-cell RNA-seq profiles reveals extensive heterogeneity•Annotation approach relates single-cell profiles to legacy genomic signatures•Pathogenicity regulators co-vary with pro-inflammatory and regulatory modules•Functional validation of Th17 pathogenicity regulators: GRP65, TOSO, and PLZP
Single-cell RNA-seq coupled to a new functional annotation approach identifies distinct functional states of Th17 cells and the underlying molecular mechanisms that regulate their pathogenicity.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Encounters between immune cells and invading bacteria ultimately determine the course of infection. These interactions are usually measured in populations of cells, masking cell-to-cell variation ...that may be important for infection outcome. To characterize the gene expression variation that underlies distinct infection outcomes and monitor infection phenotypes, we developed an experimental system that combines single-cell RNA-seq with fluorescent markers. Probing the responses of individual macrophages to invading Salmonella, we find that variation between individual infected host cells is determined by the heterogeneous activity of bacterial factors in individual infecting bacteria. We illustrate how variable PhoPQ activity in the population of invading bacteria drives variable host type I IFN responses by modifying LPS in a subset of bacteria. This work demonstrates a causative link between host and bacterial variability, with cell-to-cell variation between different bacteria being sufficient to drive radically different host immune responses. This co-variation has implications for host-pathogen dynamics in vivo.
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•Single-cell RNA-seq reveals heterogeneity of host-pathogen encounters•PhoP-mediated LPS modifications occur in only a subset of bacteria during infection•Intracellular recognition of modified LPS induces heterogeneous type I IFN response•Heterogeneity is a key feature of pathogen population during infection
Functional heterogeneity in the response of host cells to infection is driven by cell-to-cell transcriptional variations in the population of infecting pathogens.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Immune checkpoint blockers (ICBs) have failed in all phase III glioblastoma (GBM) trials. Here, we show that regulatory T (Treg) cells play a key role in GBM resistance to ICBs in experimental ...gliomas. Targeting glucocorticoid-induced TNFR-related receptor (GITR) in Treg cells using an agonistic antibody (αGITR) promotes CD4 Treg cell differentiation into CD4 effector T cells, alleviates Treg cell-mediated suppression of anti-tumor immune response, and induces potent anti-tumor effector cells in GBM. The reprogrammed GBM-infiltrating Treg cells express genes associated with a Th1 response signature, produce IFNγ, and acquire cytotoxic activity against GBM tumor cells while losing their suppressive function. αGITR and αPD1 antibodies increase survival benefit in three experimental GBM models, with a fraction of cohorts exhibiting complete tumor eradication and immune memory upon tumor re-challenge. Moreover, αGITR and αPD1 synergize with the standard of care treatment for newly-diagnosed GBM, enhancing the cure rates in these GBM models.
Recent advances in cellular profiling have demonstrated substantial heterogeneity in the behaviour of cells once deemed 'identical', challenging fundamental notions of cell 'type' and 'state'. Not ...surprisingly, these findings have elicited substantial interest in deeply characterizing the diversity, interrelationships and plasticity among cellular phenotypes. To explore these questions, experimental platforms are needed that can extensively and controllably profile many individual cells. Here, microfluidic structures - whether valve-, droplet- or nanowell-based - have an important role because they can facilitate easy capture and processing of single cells and their components, reducing labour and costs relative to conventional plate-based methods while also improving consistency. In this article, we review the current state-of-the-art methodologies with respect to microfluidics for mammalian single-cell 'omics' and discuss challenges and future opportunities.
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IJS, NUK, SBMB, UL, UM, UPUK