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High-resolution spatial transcriptomics in fixed tissue using a cost-effective PCL-seq workflow
Xue Dong1, Mengzhu Hu1, Xiaonan Cui2
1Single Cell Systems Biology Laboratory, College of Marine Life Sciences, Ocean University of China, Qingdao, Shandong 266101, China.
Genome Research
|August 5, 2025
Summary
We developed photocleavage and ligation sequencing (PCL-seq), a novel spatial transcriptomics method. This technique maps gene expression in tissues with subcellular resolution, applicable to both frozen and FFPE samples.
Area of Science:
- Molecular Biology
- Genomics
- Biotechnology
Background:
- Spatial heterogeneity in gene expression necessitates advanced spatial transcriptomics technologies.
- Existing methods face limitations in tissue compatibility and resolution.
Purpose of the Study:
- To introduce photocleavage and ligation sequencing (PCL-seq), a novel spatial transcriptomics method.
- To demonstrate PCL-seq's utility in frozen and FFPE tissues with subcellular resolution.
Main Methods:
- PCL-seq utilizes a light-controlled DNA labeling strategy with photocleavable oligonucleotides and ligation adapters.
- Regions of interest (ROIs) are designated via microscopically controlled photo-illumination for targeted transcriptional profiling.
- The method was applied to frozen mouse embryos and formalin-fixed paraffin-embedded (FFPE) mouse embryo sections.
Main Results:
- PCL-seq generated spatially aligned gene expression matrices from frozen mouse embryos, detecting ~170,000 UMIs and 8600 genes (100 µm illumination).
- The method successfully identified thousands of differentially enriched transcripts in FFPE mouse embryo sections (digits and vertebrae).
- Subcellular resolution was achieved, enabling differential expression profiling between nuclear and cytoplasmic compartments.
Conclusions:
- PCL-seq is an accessible and versatile workflow for spatial transcriptomic analysis.
- The technology is compatible with both frozen and FFPE tissues.
- PCL-seq provides high-quality data with subcellular resolution for detailed spatial gene expression studies.
