Mapping a mammalian adult adrenal gland hierarchy across species by microwell-seq

Recently,single-cell RNA-seq technologies have been rapidly updated,leading to a revolution in biology.We previously developed Microwell-seq,a cost-effective and high-throughput single cell RNA sequencing(scRNA-seq)method with a very simple device.Most cDNA libraries are sequenced using an expensive Illumina platform.Here,we present the first report showing combined Microwell-seq and BGI MGISEQ2000,a less expensive sequencing platform,to profile the whole transcriptome of 11,883 individual mouse adult adrenal gland cells and identify 18 transcriptionally distinct clusters.Moreover,we performed a single-cell comparative analysis of human and mouse adult adrenal glands to reveal the conserved genetic networks in these mammalian systems.These results provide new insights into the sophisticated adrenal gland hierarchy and provide a benchmark,low-cost strategy for highthroughput single-cell RNA study.

scRNA-seq profiling of neonatal and adult thymus-derived CD4+ T cells by a T cell origin-time tracing model

It is well documented that the neonatal thymus-derived (neonatal-TD) regulatory T cells (Treg) are essential to prevent lethal autoimmune diseases and allergies, and neonatal and adult thymus possesses distinct output potentials for naïve T cells, including Treg. However, the molecular features and detailed functional differences between neonatal-TD and adult thymus-derived (adult-TD) T cells in terms of their ability to maintain immune homeostasis during long-term environmental influences are still largely unknown, partially due to the lack of appropriate animal models to precisely trace these cells at specific time points. In this study, neonatal-TD and adult-TD CD4+ T cells from the spleen and Peyer's patches were traced for 9 weeks by a T cell origin-time tracing mouse model and analysed by single-cell RNA sequencing. More Treg but fewer naïve T cells were found in neonatal-TD CD4+ T cells from both tissues than those from adult-TD counterparts. Interestingly, the neonatal-TD Treg in both the spleen and Peyer's patches exhibited augmented expression of Foxp3, Gata3, Ctla4, Icos, Il2ra, Tgfb1, and Nrp1, as well as enriched Gene Ontology terms like T cell activation and tolerance induction, indicating an enhanced immunosuppressive function. These results were further confirmed by flow cytometry analysis and in vitro immune suppression assays. Flow cytometry also revealed a significantly higher proportion of neonatal-TD Treg in total Treg than that of adult-TD counterparts, suggesting the longer lifespan of neonatal-TD Treg. To investigate the intrinsic features of neonatal-TD and adult-TD CD4+ T cells, a shortened tracing time was performed. Surprisingly, the neonatal-TD and adult-TD CD4+ T cells had similar proportions of Treg and did not exhibit significant differences in Foxp3, Gata3, Ctla4, Icos, Il2ra, and Tgfb1 expression levels after tracing for 12 days. On the other hand, neonatal-TD Treg present an increased Nrp1 expression level compared with adult-TD counterparts, indicating the enhanced stability. Together, our work reveals that the neonatal-TD Treg are more immunosuppressive, which is likely shaped primarily by environmental factors.