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

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.

doi:10.64898/2026.03.05.709668

bioRxiv · 10.64898/2026.03.05.709668

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

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.

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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.

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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.. 2026-03-07. Fixative eXchange (FX)-seq: Scalable Single-nucleus RNA Sequencing Analysis of PFA-fixed or FFPE Tissue. https://doi.org/10.64898/2026.03.05.709668

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