snCED-seq: high-fidelity cryogenic enzymatic dissociation of nuclei for single-nucleus RNA-seq of FFPE tissues

Yunxia Guo, Junjie Ma, Ruicheng Qi, Rongrong Ma, Xiaoying Ma, Jitao Xu, Kaiqiang Ye, Yan Huang, Xi Yang, Jianyou Zhang (), Guangzhong Wang () and Xiangwei Zhao ()
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Yunxia Guo: Southeast University
Junjie Ma: Chinese Academy of Sciences
Ruicheng Qi: Chinese Academy of Sciences
Rongrong Ma: Yangzhou University
Xiaoying Ma: Southeast University
Jitao Xu: Southeast University
Kaiqiang Ye: Southeast University
Yan Huang: Southeast University
Xi Yang: Southeast University
Jianyou Zhang: Yangzhou University
Guangzhong Wang: Chinese Academy of Sciences
Xiangwei Zhao: Southeast University

Nature Communications, 2025, vol. 16, issue 1, 1-19

Abstract: Abstract Recent advances have shown that single-nucleus RNA sequencing (snRNA-seq) can be applied to formalin-fixed, paraffin-embedded (FFPE) tissues, opening avenues for transcriptomic analysis of archived specimens. Yet, isolating intact nuclei remains difficult due to RNA cross-linking. Here, we introduce a cryogenic enzymatic dissociation (CED) strategy for rapid, high-yield and fidelity nuclei extraction from FFPE samples and validate its utility with snRandom-seq (snCED-seq) using male C57/BL6 mice. Compared with conventional approaches, CED delivers a tenfold increase in nuclei yield with significantly reduced hands-on time, while minimizing secondary RNA degradation and preserving intranuclear transcripts. snCED-seq enhances gene detection sensitivity, lowers mitochondrial and ribosomal contamination, and increases overall gene expression quantification. In Alzheimer’s disease studies, it distinguished two astrocyte subpopulations, microglia, and oligodendrocytes, revealing cellular heterogeneity. Additionally, snCED-seq identify major cell types in a single 50 μm FFPE human lung section. Our results demonstrate that snCED-seq is robust for FFPE specimens and poised to enable multi-omics analyses of clinical samples.

Date: 2025
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DOI: 10.1038/s41467-025-59464-0

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