library_construction_protocol: Frozen tissue slides were thawed at 37°C for 2 minutes, fixed with 1% formaldehyde in PBS with RNase inhibitor for 10 minutes, and quenched with glycine for 5 minutes. The tissue was washed with DPBS-RI and DI water, then permeabilized with Triton X-100 and RNase inhibitor for 30 minutes. After washing with DPBS-RI, the tissue was treated with HCl to disrupt protein structure and remove histones, then washed with a buffer containing RNase inhibitor. A transposition mixture was added and incubated at 37°C for 60 minutes twice. The reaction was stopped with EDTA and RNase inhibitor, and the tissue was washed with NEB3.1 buffer and DPBS-RI before proceeding to the in-situ reverse transcription reaction. mRNAs were then captured by the biotinylated RT-primer (poly-dT primer with UMIs and a universal linker sequence, followed by reverse transcription to synthesize the complementary DNA (cDNA). Both genomic fragments and cDNA in the tissue were then ligated to spatial barcodes A and B sequentially. Barcoded gDNA fragments and cDNA were then released after reverse crosslinking. Afterward, the biotin-labeled cDNA was enriched by the streptavidin bead and separated from the gDNA-contained supernatant. cDNA was followed by template switching, and the cDNA library construction, whereas, gDNA was followed by Enzymatic Methyl-seq conversion, splint ligation, and DNA library construction. In brief, 200 µl of supernatant was collected for methylated gDNA detection and library construction. The supernatant was purified and eluted in 84 µl of nuclease-free water. The Enzymatic Methylation-seq Conversion module was used to convert unmethylated cytosines to uracil. The DNA sample was divided into PCR tubes, and a TET2 reaction mixture was added. After incubation at 37°C, the sample was purified with SPRI beads and eluted. The separated beads containing cDNA were used to generate a cDNA library. Beads were washed, then incubated in a TSO solution with agitation at room temperature and 42°C. PCR was run, and the clear PCR solution was transferred to qPCR tubes with Evagreen dye. After qPCR, the amplified product was purified with SPRI beads and eluted in nuclease-free water. The Nextera XT DNA Library Prep Kit was used for cDNA library preparation. Library Strategy: Spatial-DMT-seq (Spatial DNA Methylation and RNA Transcriptomic Sequencing)