Single cell and single nucleus transcriptomics of precision-cut liver slices

We performed single cells and single nucleus RNA sequencing of an organotypic ex vivo model of ductular reaction (DR) based on precision-cut liver slices (PCLS) from mouse and human livers, which faithfully preserves the native tissue architecture and key hepatic cell types. This system enables ex vivo propagation of DR from previously injured livers, as well as de novo induction of reactive BECs in uninjured tissues, providing an experimental framework for mechanistic dissection in disease-relevant settings. Overall design: Mouse and human PCLS were embedded in basement membrane extract (BME) and cultured on transwell inserts in expansion medium. Mouse PCLS were harvested after 96 hours, while human PCLS were collected after 144 hours of culture. At the respective timepoints, samples were processed for transcriptomic analysis. For single-nucleus RNA sequencing (snRNA-seq), PCLS were snap-frozen in liquid nitrogen and stored at –80 °C prior to nuclei isolation and library preparation. In contrast, for single-cell RNA sequencing (scRNA-seq), freshly collected PCLS were immediately dissociated and processed for single-cell suspension preparation and downstream library construction.