Single-cell technologies are becoming increasingly widespread and have been revolutionizing our understanding of cell identity, state, diversity and function. However, current platforms can be slow to apply to large-scale studies and resource-limited clinical arenas due to a variety of reasons including cost, infrastructure, sample quality and requirements. Here we report DNBelab C4 (C4), a negative pressure orchestrated, portable and cost-effective device that enables high-throughput single-cell transcriptional profiling. C4 system can efficiently allow discrimination of species-specific cells at high resolution and dissect tissue heterogeneity in different organs, such as murine lung and cerebral cortex. Finally, we show that the C4 system is comparable to existing platforms but has huge benefits in cost and portability and, as such, it will be of great interest for the wider scientific community.

Spatial transcriptomics analysis allows the examination of the biological characteristics and spatial distribution of individual lung cells at a single-cell resolution. However, due to the presence of cavities in the alveoli of the lungs, it is challenging to section them for spatial transcriptomics experiments. Here, we present a protocol for acquiring high-quality fresh mouse lung spatial transcriptomics data. We describe steps for lung perfusion, acquiring frozen slices, collecting cDNA from lung sections, and data analysis. For complete details on the use and execution of this protocol, please refer to Jiang et al.¹.

Axolotl (Ambystoma mexicanum) is an excellent model for investigating regeneration, the interaction between regenerative and developmental processes, comparative genomics, and evolution. The brain, which serves as the material basis of consciousness, learning, memory, and behavior, is the most complex and advanced organ in axolotl. The modulation of transcription factors is a crucial aspect in determining the function of diverse regions within the brain. There is, however, no comprehensive understanding of the gene regulatory network of axolotl brain regions. Here, we utilized single-cell ATAC sequencing to generate the chromatin accessibility landscapes of 81,199 cells from the olfactory bulb, telencephalon, diencephalon and mesencephalon, hypothalamus and pituitary, and the rhombencephalon. Based on these data, we identified key transcription factors specific to distinct cell types and compared cell type functions across brain regions. Our results provide a foundation for comprehensive analysis of gene regulatory programs, which are valuable for future studies of axolotl brain development, regeneration, and evolution, as well as on the mechanisms underlying cell-type diversity in vertebrate brains.

Thermal injuries represent one of the most severe forms of trauma to the human body, with a high annual incidence of burn victims globally. Skin regeneration and wound healing following thermal injury constitute a complex process involving various cell types and cytokine interactions. By Single-cell ATAC sequencing (scATAC-seq), we elucidated the molecular mechanisms underlying the dermal regeneration and healing processes following thermal injury in a rat model. Tissue samples were harvested for sequencing at predetermined intervals (0 h, 12 h, 24 h, 3 d, 7 d, 11 d, 15 d, and 19 d post-injury), yielding 28,179 high-quality single cells. Our comprehensive analysis revealed 28 distinct cell populations throughout the regenerative process, encompassing various subsets of keratinocytes, fibroblasts, and immune cells, exhibiting temporal heterogeneity across samples. Furthermore, we investigated the chromatin accessibility landscape of individual cell types and identified enriched transcription factor binding motifs, corroborating the robustness and validity of our data. Our dataset provides a valuable resource for further elucidation of burnt skin regeneration and healing processes.

Proper placentation is essential for embryonic growth and viability, yet the spatial organization and interactions of placental cell types remain incompletely understood. Here, we present a spatiotemporal transcriptomic atlas of the mouse placenta (STAMP) from embryonic days 9.5 to 18.5 at single-cell resolution. This atlas delineates major placental cell types and developmental trajectories. We observed labyrinth region expansion through branching morphogenesis, with trophoblast progenitor cells declining and terminally differentiated trophoblast cells increasing from E12.5 onward, and glycogen cells (GCs) transitioned from the junctional zone (JZ) to the maternal decidua. Furthermore, we found two novel GC subclusters with distinct spatial distributions and molecular features. Analysis of defective placentas revealed an increased number of GCs and altered macrophage distribution in the labyrinth layer. Transmission electron microscopy and glycogen content examination confirmed sluggish glycogen breakdown, while macrophage accumulation correlated with tissue remodeling and immune responses. Our spatial transcriptomic analysis elucidates mechanisms underlying placental abnormalities and embryonic lethality. This atlas enhances understanding of mouse placental development, aids in identifying developmental defects and pathogenic causes in dysfunctional placentas, and provides valuable insights for optimizing in vitro embryo culture conditions.

Spatial transcriptomics enables a single-cell resolution view of gene expression patterns in tissues, providing insight into their biological functions. However, applying this approach to the skin presents inherent challenges. Here, we present a protocol for preparing mammalian skin samples encompassing hair follicles for spatial transcriptomics. We describe steps for sample preparation, embedding, acquisition of frozen slices, RNA quality control, tissue mounting, fixation, staining, and imaging. We then detail procedures for permeabilization, reverse transcription, and cDNA collection. For complete details on the use and execution of this protocol, please refer to Chen et al.¹.