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This protocol describes sc‑rDSeq, a scalable, droplet‑based method for full‑length, strand‑specific total RNA sequencing at single‑cell resolution. The protocol uses a refined set of 220 ribosomal‑depleted sequences (rDS) primers that selectively exclude ribosomal RNA during initial reverse transcription, enabling capture of both polyadenylated and non‑polyadenylated RNAs such as histone RNAs, noncoding RNAs, and enhancer RNAs, without requiring costly post‑amplification depletion steps. This method is useful for researchers who would like to detect not only gene expression variations, but also alternative splicing events and single nucleotide variations in complex heterogenous cellular systems, providing a more complete view of cellular heterogeneity and regulatory programs that remain invisible to conventional polyadenylated‑only sequencing approaches. Compared with existing full‑length protocols, which are often limited by high reagent costs or reliance on complex multistep microfluidics, sc-rDSeq provides a simpler, single-step microfluidic workflow compatible with standard inDrops platforms, which may reduce experimental complexity and cost relative to existing full-length total-RNA methods. A key improvement is the 10-fold increase in unique molecular identifiers per cell relative to 3' end‑based methods, at a reported reagent cost of approximately $0.08 per cell, making deep total transcriptome analysis more accessible. The protocol includes three major parts: sc‑rDSeq barcode synthesis, single‑cell co‑encapsulation, and library construction.

There is increasing clinical evidence that pancreatic dysfunction in diabetes needs to be viewed in the context of crosstalk with the liver as well as other organs. Our goal for this study was to develop a pancreas-liver co-culture system suited for mechanistic and therapy testing studies in the context of multi-organ cross talk. To achieve this goal, we developed a co-axial flow-focusing microfluidic device to fabricate multi-compartment hydrogel microcapsules. Each microcapsule contained two aqueous compartments or cores surrounded by poly(ethylene glycol) (PEG) hydrogel. Each microcapsule had pancreatic β-cells loaded into one compartment and hepatic cells into another compartment. Individual encapsulated cells assembled into pancreatic and hepatic cell spheroids over time. Characterization of microcapsules revealed enhanced hepatic and pancreatic function in microcapsules containing pancreas-liver co-cultures compared to microcapsules with one cell type only. Multicompartment microcapsules represent a novel microphysiological system type and hold the promise of increasing experiment throughput for mechanism discovery and drug development studies.

ABSTRACTHuman transmembrane serine protease 2 (TMPRSS2) has garnered substantial interest due to its clinical significance in various pathologies, notably its pivotal role in viral entry into host cells. The development of effective strategies to target TMPRSS2 is a current area of intense research and necessitates a consistent source of active TMPRSS2 with sufficient stability. Here, we comprehensively characterised human seminal‐fluid extracellular vesicles (SF‐EVs, also referred to as prostasomes), bearing a native source of surface‐exposed, enzymatically active TMPRSS2 as demonstrated by high‐sensitivity flow cytometry and a fluorometric activity assay. Additionally, we recombinantly produced human TMPRSS2 ectodomain in mammalian cells adopting a directed activation strategy. We observed comparable catalytic parameters and inhibition characteristics for both native SF‐EV‐associated and recombinant TMPRSS2 when exposed to serine protease inhibitor Nafamostat mesylate. Leveraging these findings, we developed a robust in vitro biochemical assay based on these SF‐EVs for the screening of TMPRSS2‐targeting compounds. Our results will accelerate the discovery and advancement of efficacious therapeutic approaches targeting TMPRSS2 and propel further exploration into the biological role of SF‐EV‐associated active TMPRSS2.