design: The 10x Visium platform utilizes probe-based gene panels targeting approximately 18,000 genes in humans and 20,000 genes in mice. For each gene, a pair of probes is pre-designed to target specific RNA sequences. The left-hand probe (LHS) contains a Read 2 sequence for subsequent PCR amplification, while the right-hand probe (RHS) incorporates a poly(A) tail to facilitate capture by spatially barcoded oligonucleotides on the slide surface. Following hybridization with tissue RNA, the probe pairs are ligated to form a complete DNA strand, effectively transferring the RNA information onto the probe. The hybridized RNA template is then degraded, and the ligated probes are released through permeabilization (for FFPE samples, permeabilization conditions are standardized and do not require sample-specific optimization). The captured probes undergo on-slide extension to generate cDNA, which is subsequently recovered as single-stranded cDNA for library construction and sequencing, thereby enabling the acquisition of gene expression profiles. Each capture area on the slide features an oligo array, where each oligonucleotide comprises a capture probe complementary to the ligated product, along with a spatial barcode sequence that encodes positional information.