USPPAR: un flujo de trabajo de secuenciación de ARN de célula única rentable, escalable y de alta sensibilidad...

Single-cell RNA sequencing (scRNA-seq) requires high sensitivity, throughput, and broad compatibility across specimens. Current high-capacity methods lack sensitivity compared to low-capacity counterparts. Moreover, tissue-specific methods for collecting cells/nuclei limit unbiased comparisons across samples. Here, we propose a Unified framework by Split-Pool barcoding with optimal-efficiency PolydeoxyAdenylation for scRNA detection (USPPAR). Using short and long dsDNA substrates, low Co²⁺ concentration, while eliminating all other metal-ion components, enabled terminal deoxynucleotide transferase to efficiently polydeoxyadenylate intractable blunt and 3' recessed dsDNA ends, which was unattainable with other systems. By benchmarking against six state-of-the-art technologies using HEK293, the efficient addition of PCR handles for cDNA amplification made USPPAR's gene detection sensitivity comparable to high-sensitivity methods and significantly higher than existing high-cell-capacity platforms. In primary PBMCs, USPPAR enabled high-sensitivity, high-resolution scRNA-seq, and lysine conjugation improved sensitivity as an RNase inactivator. Based on nuclease reporter and mRNA protection assays, partially chelated Cu²⁺ served as a potent, non-precipitating, broad-spectrum nuclease inhibitor across various pH levels. Beyond demonstrating high sensitivity in liver tissue, an organ with low nuclease activity, single-nucleus RNA sequencing (snRNA-seq) with this inhibitor enabled one-pot extraction of RNA-stable nuclei from nuclease-rich tissues, such as the pancreas. Finally, comparisons with reference datasets from the 10× platform using mouse spleen and maize tissues showed that USPPAR matched cell-type coverage while achieving higher gene-detection efficiency. With five key enzymes available and quality-controlled, USPPAR provides a unified, cost-effective, sensitive method for high-cell-capacity scRNA profiling of diverse specimens without special equipment.