Fixative eXchange (FX)-seq: Scalable Single-nucleus RNA Sequencing Analysis of PFA-fixed or FFPE Tissue

Authors

Park, H.-E., Lee, Y. T., Lee, J., Ji, H., Song, Y.-L., Lee, J. W., Kim, S.-Y., Hur, J. K., Kim, E., Lee, C. W., Han, Y. D., Kim, H., Sohn, C. H.

Abstract

Single-nucleus RNA sequencing (snRNA-seq) of clinical formalin-fixed, paraffin-embedded (FFPE) samples has long been a challenge due to low reverse transcription (RT) yields. Here, we present Fixative-eXchange (FX)-seq, a highly scalable snRNA-seq method for heavily paraformaldehyde (PFA)-fixed and/or FFPE samples. We employ an organocatalyst to facilitate the removal of PFA crosslinks to increase RT yield and additional regiospecific Pt(II)-based crosslinking of RNA molecules to prevent leakage. FX-seq reveals cellular heterogeneity across multiple fixed samples by analyzing 321,710 nuclei, including PFA-fixed tissue, FFPE blocks, thin FFPE and hematoxylin and eosin (H&E)-stained sections from mouse brain and human cancer specimens such as gastrointestinal stromal tumor and colorectal cancer. FX-seq enables integrated analysis with pathologist annotation to label tumor and non-tumor regions of H&E-stained sections. FX-seq can also be applied to PFA-perfusion-based animal studies, large human cohort studies, and personalized drug treatment through precision medicine.

Preprint server: bioRxiv
The authors list and abstract were imported from bioRxiv on 08 Mar 2026.

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