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Highlights d The identity of old dermal fibroblasts becomes undefined and noisy d Old dermal fibroblasts acquire adipogenic traits d CR and HFD prevent and potentiate fibroblast aging, respectively d Loss of cell identity is a possible mechanism underlying aging

Single-cell RNA sequencing is at the forefront of high-resolution phenotyping experiments for complex samples. Although this methodology requires specialized equipment and expertise, it is now broadly applied in research. However, it is challenging to create broadly applicable experimental designs because each experiment requires the user to make informed decisions about sample preparation, RNA sequencing and data analysis. To facilitate this decision-making processes, in this Tutorial we summarize current methodological and analytical options, and discuss their suitability for a range of research scenarios. Specifically, we provide information about best practices to separate individual cells and provide an overview of current single-cell capture methods at different cellular resolutions and scales. RNA sequencing library preparation methods vary profoundly across applications and we discuss features important for an informed selection process. An erroneous or biased analysis can lead to misinterpretations or obscure biologically important information. We provide a guide to the major data processing steps and options for meaningful data interpretation. These guidelines will serve as a reference to support users in building a single-cell experimental framework from sample preparation to data interpretation, tailored to the underlying research context.

AbstractSingle-cell RNA sequencing (scRNA-seq) is the leading technique for charting the molecular properties of individual cells. The latest methods are scalable to thousands of cells, enabling in-depth characterization of sample composition without prior knowledge. However, there are important differences between scRNA-seq techniques, and it remains unclear which are the most suitable protocols for drawing cell atlases of tissues, organs and organisms. We have generated benchmark datasets to systematically evaluate techniques in terms of their power to comprehensively describe cell types and states. We performed a multi-center study comparing 13 commonly used single-cell and single-nucleus RNA-seq protocols using a highly heterogeneous reference sample resource. Comparative and integrative analysis at cell type and state level revealed marked differences in protocol performance, highlighting a series of key features for cell atlas projects. These should be considered when defining guidelines and standards for international consortia, such as the Human Cell Atlas project.

In the past decade, single-cell transcriptomics has helped to uncover new cell types and states and led to the construction of a cellular compendium of health and disease. Despite this progress, some difficult-to-sequence cells remain absent from tissue atlases. Eosinophils—elusive granulocytes that are implicated in a plethora of human pathologies1–5—are among these uncharted cell types. The heterogeneity of eosinophils and the gene programs that underpin their pleiotropic functions remain poorly understood. Here we provide a comprehensive single-cell transcriptomic profiling of mouse eosinophils. We identify an active and a basal population of intestinal eosinophils, which differ in their transcriptome, surface proteome and spatial localization. By means of a genome-wide CRISPR inhibition screen and functional assays, we reveal a mechanism by which interleukin-33 (IL-33) and interferon-γ (IFNγ) induce the accumulation of active eosinophils in the inflamed colon. Active eosinophils are endowed with bactericidal and T cell regulatory activity, and express the co-stimulatory molecules CD80 and PD-L1. Notably, active eosinophils are enriched in the lamina propria of a small cohort of patients with inflammatory bowel disease, and are closely associated with CD4+ T cells. Our findings provide insights into the biology of eosinophils and highlight the crucial contribution of this cell type to intestinal homeostasis, immune regulation and host defence. Furthermore, we lay a framework for the characterization of eosinophils in human gastrointestinal diseases.