library_construction_protocol: Tissue block and library preparation for spatial transcriptomic (ST) analysis RNAlater-preserved tissue blocks were thawed at room temperature (RT), retrieved from the cryomold with a pair of RNase AWAY (surface decontaminant, 7000TS1, ThermoFisher) treated forceps, dabbed with clean Kimwipes to remove excess liquid, and incubated in a new cryomold filled with OCT at RT for 10 min to further remove RNAlater residual. At the end of incubation, tissue blocks were transferred to a new cryomold filled with OCT, positioned to desired orientation without creating bubbles, and left frozen at −20°C before trimming. Frozen OCT blocks were trimmed to desired size (~8 mm on each side) to reduce the chances of tissue folding during serial sectioning. To increase the precision of tissue capture, a cryo-resistant plastic grid with matching capture areas was attached to the back of each Visium Spatial Gene Expression Slide (2000233, 10x Genomics) and 12 microslides (Superfrost+ and ColorFrost+, EF15978Z, Daigger Scientific). Gird-attached slides and tissue blocks were left equilibrated inside the cryostat chamber for 30 min prior to a serial tissue scan, which was performed to ensure the capture of the desired ROI with matching morphology to the terminal MRI. Specifically, tissue blocks were sectioned at 10 µm, captured with microslides, and stained every 120 µm with 3% Sudan black (199664-25G, Sigma-Aldrich) in ethylene glycol (BDH1125-1LP, VWR) solution at 56˚C for 30 sec in a water bath to color myelin. After Sudan black (SB) stain, microslide was rinsed with running tap water for 1 min, transferred to hematoxylin (100% Surgipath SelecTech Hematoxylin 560MX, 3801575, Leica) at 56˚C for 30 sec to color gray matter and white matter lesions. After hematoxylin staining, the microslide was rinsed with running tap water again for 1 min, coverslipped (Premium Cover Glasses, EF15972L, Daigger Scientific) with UltraPureTM glycerol (15514-011, Invitrogen), and visualized with a brightfield microscope (Leitz Laborlux S Wild GMBH). When the morphology of the tissue section meets the desired target, the adjacent section is captured and mounted onto the Visium Spatial Gene Expression Slide without covering the fiducial frame to facilitate image alignment in the downstream data analysis. In parallel, pre- and post-Visium captured tissue sections were made and kept at −20°C for long-term storage. At the end of tissue scans, Visium Spatial Transcriptomics slides containing the tissue sections of interest were transported on dry ice and kept at −80°C for long-term storage or immediately proceeded to staining and library preparation. Slides were thawed on a pre-warmed Thermocycler Adapter (3000380, 10x Genomics) at 37˚C for 1 min to minimize tissue damage caused by condensation. To stain myelin, 1 mL of filtered 1% SB/ethylene glycol was pipetted directly onto the leveled tissue slide and incubated for 5 minutes at RT. After removing excess SB solution, stained slide was dipped in 50 mL of ddH2O (351-029-131CS, Quality Biological) in a Falcon tube 5 times, dipped in 800 mL of ddH2O 15 times, and then another 15 times in a separate glass beaker containing 800 mL of ddH2O. To get an optimal contrast between myelin and gray matter, tissue was dried for 1 min (the time was extended if the tissue section was not dry) before being stained with 1 mL Nuclear Fast Red (NFR, ab246831, Abcam) for 10 min at RT on a level surface and rinsed again as described for SB staining. To avoid bubbles, slides were first saturated with 5 mL of ddH2O on a leveled surface and gradually replaced with 80% glycerol solution containing 5% RNase inhibitor (AM2684, Thermo Fisher Scientific) through steady vacuuming off ddH2O from one end of the slide and adding glycerol with a pipette at the opposite end simultaneously. At the end of solution swapping, the homogenous mounting media was coverslipped and proceeded immediately to 4X tiling imaging with a Nikon Eclipse Ci microscope. After imaging, the coverslip was rinsed off by submerging the slide in 3X SSC buffer (46-020-CM, Corning) diluted in ddH2O, then rinsed briefly in 1X SSC buffer to remove excess mounting media. After imaging and coverslip removal, the slide was enzymatically permeabilized with Visium Spatial Gene Expression Reagent Kit (PN-1000184, Spatial 3' v1, 10x Genomics) at 37 ˚C for 20 min, which was determined by Tissue Optimization protocol (PN-1000193, 10x Genomics). The cDNA library was then prepared following the manufacturer's protocol (Visium Spatial Gene Expression Reagents Kits User Guide, Rev D). The cycle number for cDNA amplification for each library (TableS2) was determined using the Cq value obtained from the qPCR steps detailed in the manufacturer's protocol. All libraries were sequenced using the Illumina Novaseq S2 platform. Library quantity and quality were assessed using Qubit™ dsDNA HS Assay Kit (Q32854, Invitrogen™) with a Qubit™ 4 Fluorometer (Q33226, Thermo Fisher Scientific) and High Sensitivity DNA Kit (5067-4626, Agilent Technologies) with a 2100 Bioanalyzer instrument (G2939BA, Agilent Technologies), respectively.