A cost-effective and flexible workflow for high-resolution spatial transcriptomics in fixed tissue

The spatial heterogeneity of gene expression has driven the development of diverse spatial transcriptomics technologies. Here, we introduce photocleavage and ligation sequencing (PCL-seq), a spatial indexing method based on a light-controlled DNA labeling strategy applied to tissue sections. PCL-seq employs photocleavable oligonucleotides and sequence adapters to construct transcriptional profiles of specific regions of interest (ROIs), designated via microscopically controlled photo-illumination. In frozen mouse embryos, PCL-seq generates spatially aligned gene expression matrices and achieves high-quality data outputs, detecting approximately 170,000 unique molecular identifiers (UMIs) and 8,600 genes (irradiation diameter=100 µm). Moreover, PCL-seq is compatible with formalin-fixed and paraffin-embedded (FFPE) tissues, successfully identifying thousands of differentially enriched transcripts in the digits and vertebrae of FFPE mouse embryo sections. Additionally, PCL-seq achieves subcellular resolution, as demonstrated by differential expression profiling between nuclear and cytoplasmic compartments. These features establish PCL-seq as an accessible and versatile workflow for spatial transcriptomic analyses in both frozen and FFPE tissues at subcellular resolution. We designed a series of experiments to evaluate the performance of our newly developed sequencing method, PCL-seq. This method utilizes a photocleavable (PC) linker as a photosensitive compound, enabling precise photolytic cleavage and ligation of target regions within tissue sections. To verify the ability of PCL-seq to specifically amplify cDNA molecules from illuminated regions, we performed an experiment using co-cultured cells. In this setup, one cell type served as a non-illuminated background, while a small portion of the other cell type was selectively illuminated at varying ratios. The specificity of PCL-seq was assessed by comparing the differential genic reads between the two cell types through sequencing.Furthermore, we evaluated the data quality of PCL-seq on both cryo-preserved and FFPE (formalin-fixed paraffin-embedded) tissue sections, examining its ability to maintain spatial localization of gene expression. To test whether PCL-seq can achieve subcellular resolution, we irradiated the nuclear and cytoplasmic regions of HeLa cells separately and analyzed the resulting data. These experiments allowed us to systematically characterize the precision, spatial resolution, and versatility of PCL-seq under diverse experimental conditions.