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USPPAR is a cost-effective, scalable, and highly sensitive single-cell RNA sequencing workflow compatible with
Ya-Wen Hsueh1,2, Po-Min Chiang1,2
1Institute of Clinical Medicine, College of Medicine, National Cheng Kung University, Tainan, Taiwan, R.O.C.
Plos Biology
|December 15, 2025
Summary
A new method called USPPAR enhances single-cell RNA sequencing (scRNA-seq) sensitivity and throughput for diverse samples. This cost-effective technique improves gene detection efficiency and enables high-resolution profiling across various tissues.
Area of Science:
- Molecular Biology
- Genomics
- Biotechnology
Background:
- Single-cell RNA sequencing (scRNA-seq) is crucial for biological research, but current high-capacity methods often lack the sensitivity of low-capacity methods.
- Existing protocols for cell or nuclei collection can be tissue-specific, hindering unbiased cross-sample comparisons.
Purpose of the Study:
- To develop a unified, sensitive, and high-capacity method for scRNA profiling applicable to diverse biological specimens.
- To overcome the limitations of existing scRNA-seq technologies in terms of sensitivity, throughput, and sample compatibility.
Main Methods:
- Developed the Unified framework by Split-Pool barcoding with optimal-efficiency PolydeoxyAdenylation for scRNA detection (USPPAR) using specific dsDNA substrates and controlled metal ion concentrations.
- Optimized terminal deoxynucleotide transferase activity for efficient polydeoxyadenylation of DNA ends.
- Incorporated lysine conjugation as an RNase inactivator and utilized partially chelated Cu²⁺ as a broad-spectrum nuclease inhibitor.
Main Results:
- USPPAR demonstrated gene detection sensitivity comparable to high-sensitivity methods and superior to existing high-cell-capacity platforms in HEK293 cells.
- Achieved high-sensitivity, high-resolution scRNA-seq in primary PBMCs and enabled one-pot extraction of RNA-stable nuclei from nuclease-rich tissues like the pancreas using the developed inhibitor.
- USPPAR matched cell-type coverage and achieved higher gene-detection efficiency compared to the 10× platform in mouse spleen and maize tissues.
Conclusions:
- USPPAR offers a unified, cost-effective, and sensitive solution for high-cell-capacity scRNA profiling across diverse specimens.
- The method overcomes key limitations in sensitivity and sample compatibility, providing a valuable tool for biological research.
- The developed nuclease inhibition strategy facilitates robust single-nucleus RNA sequencing from challenging tissues.
