library_construction_protocol: Tumor samples from a vehicle control- or a regorafenib-treated mouse were embedded in Tissue-Tek optimal cutting temperature (OCT) compound immediately after surgical resection and frozen with isopentane in a liquid nitrogen bath then stored at – 80°C. OCT embedded blocks were cryosectioned at a thickness of 10 µm in a cryostat and tissue sections were placed within the frames on a Visium Spatial Transcriptomic Library Preparation Glass Slides. Visium spatial gene expression libraries were prepared for sequencing using 10x Genomics protocols. Enzymatic fragmentation and size selection are used to optimize the cDNA amplicon size. P5, P7, i7 and i5 sample indexes, and TruSeq Read 2 (read 2 primer sequence) are added via End Repair, A-tailing, Adaptor Ligation, and PCR. The final libraries contain the P5 and P7 primers used in illumina amplification.