Toti-N-glycan Recognition Enables Universal Multiplexed Single-Nucleus RNA Sequencing
Sample barcoding-based multiplex single-cell and single-nucleus sequencing (sc/sn-seq) offers substantial advantages by reducing costs, minimizing batch effects, and identifying artifacts, thereby advancing biological and biomedical research. Despite these benefits, universal sample barcoding has be...
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| Language: | English |
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American Association for the Advancement of Science (AAAS)
2025-01-01
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| Series: | Research |
| Online Access: | https://spj.science.org/doi/10.34133/research.0678 |
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| author | Yiran Guo Liang Zhang Xing Zhao Chang Xu Yiyang Li Zhaolong Gao Gaozhi Ou Peng Chen Wenshan Zheng Hao Pei Xin Liu Bi-Feng Liu Yiwei Li |
| author_facet | Yiran Guo Liang Zhang Xing Zhao Chang Xu Yiyang Li Zhaolong Gao Gaozhi Ou Peng Chen Wenshan Zheng Hao Pei Xin Liu Bi-Feng Liu Yiwei Li |
| author_sort | Yiran Guo |
| collection | DOAJ |
| description | Sample barcoding-based multiplex single-cell and single-nucleus sequencing (sc/sn-seq) offers substantial advantages by reducing costs, minimizing batch effects, and identifying artifacts, thereby advancing biological and biomedical research. Despite these benefits, universal sample barcoding has been hindered by challenges such as inhomogeneous expression of tagged biomolecules, limited tagging affinity, and insufficient genetic insertion. To overcome these limitations, we developed Toti-N-Seq, a universal sample multiplex method, by tagging Toti-N-glycan on cell surfaces or nuclear membranes via our engineered streptavidin–Fbs1 GYR variant fusion protein, which could be used not only for sc-seq but also for sn-seq. Instead of targeting lipids or proteins, we focused on targeting the ubiquitous N-glycans found on any species with accessible membranes, which minimizes the exchange between barcoded samples and avoids biased barcoding. Our technology can be broadly applied to multiple species and nearly all eukaryotic cell types, with an overall classification accuracy of 0.969 for sc-seq and of 0.987 for sn-seq. As a demonstration with clinical human peripheral blood mononuclear cells, our Toti-N-Seq achieved rapid one-step sample preparation (<3 min) for easily scaling up while keeping high fidelity of sample ratios, removing artifacts, and detecting rare cell populations (~0.5%). Consequently, we offer a versatile platform suitable for various cell types and applications. |
| format | Article |
| id | doaj-art-e254b75cc1ba42f1ac21a1c0d693b8b6 |
| institution | Kabale University |
| issn | 2639-5274 |
| language | English |
| publishDate | 2025-01-01 |
| publisher | American Association for the Advancement of Science (AAAS) |
| record_format | Article |
| series | Research |
| spelling | doaj-art-e254b75cc1ba42f1ac21a1c0d693b8b62025-08-20T03:36:31ZengAmerican Association for the Advancement of Science (AAAS)Research2639-52742025-01-01810.34133/research.0678Toti-N-glycan Recognition Enables Universal Multiplexed Single-Nucleus RNA SequencingYiran Guo0Liang Zhang1Xing Zhao2Chang Xu3Yiyang Li4Zhaolong Gao5Gaozhi Ou6Peng Chen7Wenshan Zheng8Hao Pei9Xin Liu10Bi-Feng Liu11Yiwei Li12Key Laboratory of Molecular Biophysics of MOE and Hubei Bioinformatics & Molecular Imaging Key Laboratory, Department of Biomedical Engineering, College of Life Science and Technology - The Key Laboratory for Biomedical Photonics of MOE at Wuhan National Laboratory for Optoelectronics, Huazhong University of Science and Technology, Wuhan 430074, China.College of Sports Medicine, Wuhan Sports University, Wuhan 430079, China.John A. Paulson School of Engineering and Applied Sciences, Harvard University, Cambridge 021238, MA, USA.MobiDrop (Zhejiang), Tongxiang, Zhejiang 314500, China.MobiDrop (Zhejiang), Tongxiang, Zhejiang 314500, China.Key Laboratory of Molecular Biophysics of MOE and Hubei Bioinformatics & Molecular Imaging Key Laboratory, Department of Biomedical Engineering, College of Life Science and Technology - The Key Laboratory for Biomedical Photonics of MOE at Wuhan National Laboratory for Optoelectronics, Huazhong University of Science and Technology, Wuhan 430074, China.School of Sports, China University of Geosciences, Wuhan 430074, China.Key Laboratory of Molecular Biophysics of MOE and Hubei Bioinformatics & Molecular Imaging Key Laboratory, Department of Biomedical Engineering, College of Life Science and Technology - The Key Laboratory for Biomedical Photonics of MOE at Wuhan National Laboratory for Optoelectronics, Huazhong University of Science and Technology, Wuhan 430074, China.MobiDrop (Zhejiang), Tongxiang, Zhejiang 314500, China.MobiDrop (Zhejiang), Tongxiang, Zhejiang 314500, China.Key Laboratory of Molecular Biophysics of MOE and Hubei Bioinformatics & Molecular Imaging Key Laboratory, Department of Biomedical Engineering, College of Life Science and Technology - The Key Laboratory for Biomedical Photonics of MOE at Wuhan National Laboratory for Optoelectronics, Huazhong University of Science and Technology, Wuhan 430074, China.Key Laboratory of Molecular Biophysics of MOE and Hubei Bioinformatics & Molecular Imaging Key Laboratory, Department of Biomedical Engineering, College of Life Science and Technology - The Key Laboratory for Biomedical Photonics of MOE at Wuhan National Laboratory for Optoelectronics, Huazhong University of Science and Technology, Wuhan 430074, China.Key Laboratory of Molecular Biophysics of MOE and Hubei Bioinformatics & Molecular Imaging Key Laboratory, Department of Biomedical Engineering, College of Life Science and Technology - The Key Laboratory for Biomedical Photonics of MOE at Wuhan National Laboratory for Optoelectronics, Huazhong University of Science and Technology, Wuhan 430074, China.Sample barcoding-based multiplex single-cell and single-nucleus sequencing (sc/sn-seq) offers substantial advantages by reducing costs, minimizing batch effects, and identifying artifacts, thereby advancing biological and biomedical research. Despite these benefits, universal sample barcoding has been hindered by challenges such as inhomogeneous expression of tagged biomolecules, limited tagging affinity, and insufficient genetic insertion. To overcome these limitations, we developed Toti-N-Seq, a universal sample multiplex method, by tagging Toti-N-glycan on cell surfaces or nuclear membranes via our engineered streptavidin–Fbs1 GYR variant fusion protein, which could be used not only for sc-seq but also for sn-seq. Instead of targeting lipids or proteins, we focused on targeting the ubiquitous N-glycans found on any species with accessible membranes, which minimizes the exchange between barcoded samples and avoids biased barcoding. Our technology can be broadly applied to multiple species and nearly all eukaryotic cell types, with an overall classification accuracy of 0.969 for sc-seq and of 0.987 for sn-seq. As a demonstration with clinical human peripheral blood mononuclear cells, our Toti-N-Seq achieved rapid one-step sample preparation (<3 min) for easily scaling up while keeping high fidelity of sample ratios, removing artifacts, and detecting rare cell populations (~0.5%). Consequently, we offer a versatile platform suitable for various cell types and applications.https://spj.science.org/doi/10.34133/research.0678 |
| spellingShingle | Yiran Guo Liang Zhang Xing Zhao Chang Xu Yiyang Li Zhaolong Gao Gaozhi Ou Peng Chen Wenshan Zheng Hao Pei Xin Liu Bi-Feng Liu Yiwei Li Toti-N-glycan Recognition Enables Universal Multiplexed Single-Nucleus RNA Sequencing Research |
| title | Toti-N-glycan Recognition Enables Universal Multiplexed Single-Nucleus RNA Sequencing |
| title_full | Toti-N-glycan Recognition Enables Universal Multiplexed Single-Nucleus RNA Sequencing |
| title_fullStr | Toti-N-glycan Recognition Enables Universal Multiplexed Single-Nucleus RNA Sequencing |
| title_full_unstemmed | Toti-N-glycan Recognition Enables Universal Multiplexed Single-Nucleus RNA Sequencing |
| title_short | Toti-N-glycan Recognition Enables Universal Multiplexed Single-Nucleus RNA Sequencing |
| title_sort | toti n glycan recognition enables universal multiplexed single nucleus rna sequencing |
| url | https://spj.science.org/doi/10.34133/research.0678 |
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