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<p>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.</p>

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

  • 期刊论文

  • 出版模式：

    开放获取

  • 发表日期：

    2025年5月2日

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  • Yunxia Guo,
  • Junjie Ma,
  • Ruicheng Qi,
  • Rongrong Ma,
  • Xiaoying Ma,
  • Jitao Xu,
  • Kaiqiang Ye,
  • Yan Huang,
  • Xi Yang,
  • Jianyou Zhang,
  • Guangzhong Wang,
  • Xiangwei Zhao

摘要

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.