RNA-Seq

Single-cell RNA sequencing (scRNA-seq) has revolutionized the study of cellular heterogeneity, but the rapid expansion of analytical tools has proven to be both a blessing and a curse, presenting researchers with significant challenges. Here, we present SeuratExtend, a comprehensive R package built upon the widely adopted Seurat framework, which streamlines scRNA-seq data analysis by strategically integrating essential tools and databases. SeuratExtend offers a user-friendly and intuitive interface for performing a wide range of analyses, including functional enrichment, trajectory inference, gene regulatory network reconstruction, and denoising. The package integrates multiple databases, such as Gene Ontology and Reactome, and incorporates popular Python tools like scVelo, Palantir, and SCENIC through a unified R interface. We illustrate SeuratExtend's capabilities through case studies investigating tumor-associated high-endothelial venules and autoinflammatory diseases, as well as showcase its novel applications in pathway-level analysis and cluster annotation. SeuratExtend enhances data visualization with optimized plotting functions and carefully curated color schemes, ensuring both aesthetic appeal and scientific rigor. The package's effectiveness has been demonstrated through successful workshops and training programs, establishing its value in both research and educational contexts. SeuratExtend empowers researchers to harness the full potential of scRNA-seq data, making complex analyses accessible to a wider audience. The package, along with comprehensive documentation, tutorials, and educational resources, is freely available at GitHub, providing a valuable resource for the single-cell genomics community.

Cell-cell communication (CCC) is crucial for cellular function and tissue homeostasis. Existing methods for protein-oriented CCC detection often overlook metabolite-mediated CCC (mCCC), and adapting them to mCCC analysis is challenging due to fundamental differences in the underlying biological mechanisms. To fill this gap, we developed MEBOCOST, an algorithm built on scRNA-seq and metabolic flux balance analysis to detect mCCC among single cells. Comprehensive benchmarking analyses based on simulation, spatial, CRISPR screen, and clinical patient data demonstrated the robustness of MEBOCOST in detecting biologically significant mCCC events. We applied MEBOCOST to scRNA-seq datasets of human white adipose tissues and unraveled macrophages were the predominant source of mCCC reprogramming in obese patients. Moreover, analysis in mice brown adipose tissue successfully recapitulated known and further uncovered new mCCC events, including a glutamine-mediated endothelial-to-adipocyte communication, which was experimentally verified to regulate adipocyte differentiation. Therefore, MEBOCOST is a valuable tool for researchers investigating mCCC in diverse biological contexts and disease samples. MEBOCOST is freely available at https://github.com/kaifuchenlab/MEBOCOST.

The gene-regulatory dynamics governing drug responses in cancer are yet to be fully understood. Here, we report a pipeline capable of producing high-throughput pharmacotranscriptomic profiling through live-cell barcoding using antibody-oligonucleotide conjugates. This pipeline combines drug screening with 96-plex single-cell RNA sequencing. We show the potential of this approach by exploring the heterogeneous transcriptional landscape of primary high-grade serous ovarian cancer (HGSOC) cells after treatment with 45 drugs, with 13 distinct classes of mechanisms of action. A subset of phosphatidylinositol 3-OH kinase (PI3K), protein kinase B (AKT) and mammalian target of rapamycin (mTOR) inhibitors induced the activation of receptor tyrosine kinases, such as the epithelial growth factor receptor (EGFR), and this was mediated by the upregulation of caveolin 1 (CAV1). This drug resistance feedback loop could be mitigated by the synergistic action of agents targeting PI3K-AKT-mTOR and EGFR for HGSOC with CAV1 and EGFR expression. Using this workflow could enable the personalized testing of patient-derived tumor samples at single-cell resolution.

BACKGROUND: The enteric nervous system (ENS) is a complex network of interconnected ganglia within the gastrointestinal (GI) tract. Among its diverse functions, the ENS detects bowel luminal contents and coordinates the passing of stool. ENS defects predispose to GI motility disorders. Previously, distinct enteric neuron types were cataloged by dye-filling techniques, immunohistochemistry, retrograde labeling, and electrophysiology. Recent technical advances in single cell RNA-sequencing (scRNA-seq) have enabled transcriptional profiling of hundreds to millions of individual cells from the intestine. These data allow cell types to be resolved and compared using their transcriptional profiles (“clusters”) rather than relying on antibody labeling. As a result, greater diversity of enteric neuron types has been appreciated. Because each scRNA-seq study has relied on different methods for cell isolation and library generation, numbers of neuron clusters and cell types detected differ between analyses. Cell counts in each dataset are particularly important for characterization of rare cell types since small numbers of profiled cells may not sample rare cell types. Importantly, each dataset, depending on the isolation methods, may contain different proportions of cells that are not detected in other datasets. Aggregation of datasets can effectively increase the total number of cells being analyzed and can be helpful for confirming the presence of low-abundance neuron types that might be absent or observed infrequently in any single dataset. RESULTS: Here we briefly systematically review each Mus musculus single cell or single nucleus RNA-sequencing (snRNA-seq) ENS dataset. We then reprocess and computationally integrate these select independent scRNA-seq enteric neuron datasets from the myenteric plexus with the aim to identify new cell types, shared marker genes across juvenile to adult ages, dataset differences, and achieve some consensus on transcriptomic definitions of enteric neuronal subtypes. CONCLUSIONS: Data aggregation generates a consensus view of enteric neuron types and improves resolution of rare neuron classes. This meta-atlas offers a deeper understanding of enteric neuron diversity and may prove useful to investigators aiming to define alterations among enteric neurons in disease states. Future studies face the challenge of connecting these deep transcriptional profiles for enteric neurons with historical classification systems.