Spatial Total RNA Sequencing of Formalin-Fixed Paraffin Embedded Tissues by spRandom-seq

Current sequencing-based spatial transcriptomic methods have been widely used to resolve gene expression; however, these methods are limited to fresh or fresh-frozen samples due to the low efficiency of oligo(dT) primers in capturing RNA transcripts in degraded samples. Here, we have developed a random primer-based spatial total RNA sequencing (spRandom-seq) technology for capturing full-length total RNAs in partially degraded (low RIN values) formalin-fixed paraffin-embedded (FFPE) tissues. Our spRandom-seq approach exhibited no discernible biases at the 3'-or 5'-ends within gene bodies and could be integrated with the commercially available 10X Visium and other well-established sequencing-based spatial transcriptomic platforms. Our spRandom-seq approach outperformed oligo(dT)-based 10X Visium with an 8-fold higher capturing rate for long-non-coding RNA (lncRNA) and other non-polyadenylated RNA biotypes, including miRNA, snRNA, and miscRNA. The unsupervised clustering with solely non-coding RNAs (ncRNAs) resulted in region-specific clusters aligned perfectly with mouse brain anatomic regions. Furthermore, in the clinical FFPE sections of breast cancer, our study revealed distinct expression patterns of MUCL1 and JCHAIN within the tumor region, highlighting the inherent heterogeneity. spRandom-seq provided a potential spatial method for clinical FFPE specimens with well-established and commercially available spatial platforms, ensuring ease of operation and commercial scalability for large-scale applications.