Spatially exploring RNA biology in archival formalin-fixed paraffin-embedded tissues

The capability to spatially explore RNA biology in formalin-fixed paraffin-embedded (FFPE) tissues holds transformative potential for histopathology research. Here, we present pathology-compatible deterministic barcoding in tissue (Patho-DBiT) by combining in situ polyadenylation and computational innovation for spatial whole transcriptome sequencing, tailored to probe the diverse RNA species in clinically archived FFPE samples. It permits spatial co-profiling of gene expression and RNA processing, unveiling region-specific splicing isoforms, and high-sensitivity transcriptomic mapping of clinical tumor FFPE tissues stored for five years. Furthermore, genome-wide single nucleotide RNA variants can be captured to distinguish malignant subclones from non-malignant cells in human lymphomas. Patho-DBiT also maps microRNA regulatory networks and RNA splicing dynamics, decoding their roles in spatial tumorigenesis. Cellular-level Patho-DBiT dissects the spatiotemporal cellular dynamics driving tumor clonal architecture and progression. Patho-DBiT stands poised as a valuable platform to unravel rich RNA biology in FFPE tissues to aid in clinical pathology evaluation. Overall design: Patho-DBiT initiates with tissue section deparaffinization and heat-induced crosslink reversal. After tissue permeabilization, enzymatic in situ polyadenylation enables detection of various RNAs, followed by complementary DNA (cDNA) strand synthesis. Spatial barcoding is then achieved using microfluidic chips featuring 50 microchannels. These channels sequentially deliver horizontal and perpendicular barcodes, creating a unique barcode array. Post-imaging, the tissue undergoes digestion to extract barcoded cDNA to perform template switch, PCR amplification, rRNA removal, library preparation, and sequencing.