Fresh colon tumor tissues were embedded in optimal cutting temperature (OCT) compound and cryosectioned onto gene expression slides. Tissue sections were fixed, stained, and imaged for morphological annotation. Permeabilization was performed to release mRNA, which was captured by spatially barcoded probes. cDNA was synthesized, amplified, and sequenced to generate spatial gene expression profiles. After mRNA capture on spatially barcoded slides, reverse transcription was performed to generate cDNA with positional barcodes. Second-strand synthesis was followed by denaturation and cDNA amplification. Amplified cDNA was enzymatically fragmented, end-repaired, and poly(A)-tailed. Adaptors were ligated, and the library was indexed by PCR. Final libraries were quantified and sequenced on an Illumina platform to obtain spatial gene expression data.