A universal cost-efficient sample labeling approach for multiplexed single cell RNA-seq based on recombinant HUH-endonuclease-agglutinin tagging

Quanyong Zhang; Maorong Li; Luemou Shen; Lujia Chen; Zhiheng Yuan; Xu Zhang; Hao Wang; Yang Yu; Fucheng Luo; Guangdun Peng; Jingjie Hu; Zhenmin Bao; Ning Song; Kai Chen

doi:10.1016/j.jgg.2025.10.004

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A universal cost-efficient sample labeling approach for multiplexed single cell RNA-seq based on recombinant HUH-endonuclease-agglutinin tagging

doi: 10.1016/j.jgg.2025.10.004

a. State Key Laboratory of Primate Biomedical Research and Institute of Primate Translational Medicine, Kunming University of Science and Technology, Kunming, Yunnan 650500, China;
b. Southern Marine Science and Engineering Guangdong Laboratory (Guangzhou), Guangzhou, Guangdong 511458, China;
c. Guangzhou Women and Children’s Medical Center, GMU-GIBH Joint School of Life Sciences, The Guangdong-Hong Kong-Macao Joint Laboratory for Cell Fate Regulation and Diseases, Guangzhou Medical University, Guangzhou, Guangdong 510623, China;
d. Shandong Provincial Key Laboratory of Animal Cell and Developmental Biology, School of Life Sciences, Shandong University, Qingdao, Shandong 266237, China;
e. Institute of Oceanology, Chinese Academy of Science, Qingdao, Shandong 266000, China;
f. Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, Guangdong 510530, China;
g. Key Laboratory of Tropical Aquatic Germplasm of Hainan Province & MOE Key Laboratory of Marine Genetics and Breeding, Ocean University of China, Sanya/Qingdao, Shandong 266100, China;
h. Archaeal Biology Center, Synthetic Biology Research Center, Shenzhen Key Laboratory of Marine Microbiome Engineering, Key Laboratory of Marine Microbiome Engineering of Guangdong Higher Education Institutes, Institute for Advanced Study, Shenzhen University, Shenzhen 518060, China

Funds:

the Principal Investigator Project of the Southern Marine Science and Engineering Guangdong Laboratory (YQ2024004)

National Thousand Talents Program and Yunnan Talents Program (XDYC-PYJL-2022-0020)

Guangzhou National Laboratory (GZNL2023A03005), and the Natural Science Foundation of Yunnan Province (202102AA100053).

This work was supported by the National Key Research and Development Program of China (2023YFC3402700)

Received Date: 2025-06-03
Accepted Date: 2025-10-22
Rev Recd Date: 2025-10-22

Available Online: 2025-10-29

Abstract

Abstract

Recent advances in single-cell transcriptomics have revolutionized our understanding of cellular diversity and tissue heterogeneity, providing unprecedented insights into biological and medical research. However, the high per-assay cost limits broader applications of this technology. Although sample-labeling strategies enabling multiplexing have emerged, current methods suffer from either impractical complexity for barcoding or high cost for preparing the index labeling reagents. To address these challenges, here we present HEATag, a universal cell membrane labeling approach that combines Duck circovirus HUH endonuclease (DCV) with wheat germ agglutinin (WGA) to efficiently tag cell membranes with indexed single-stranded DNA (indexed ssDNA). The DCV domain enables rapid, sequence-specific conjugation of indexed ssDNA, while the WGA domain ensures robust labeling of fresh or fixed cells across diverse species. This method is compatible with both commercial platforms and custom systems, readily adaptable to various single cell omics workflows. Therefore, HUH-endonuclease-agglutinin tagging (HEATag) provides a universal, cost-effective and scalable solution for high-throughput single-cell studies, enhancing library preparation efficiency and minimizing batch effects for single-cell researchers.
- Single-cell RNA sequencing,
- Sample labeling,
- HUH-endonuclease,
- Agglutinin,
- Glycan

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