Single-Nucleus Isolation from White Matter

All tissue was dissected into 30 mg sections from DLF white matter with a scalpel blade and stored at − 80° C. On the day of single-nucleus encapsulation, tissue was placed in a 7 mL dounce with ice cold buffer (0.5 M sucrose, 2 M KCl, 1 M MgCl2, 1 M Tricine-KOH pH 7.8, spermine, spermidine, DTT, RNasin, H2O) and dounced with a tight pestle ten times to mechanically dissociate and homogenize the tissue. To further dissociate the tissue, 5% IGEPAL-CA630 was added to the homogenate and dounced 5 times. The homogenate was then strained through a 40 μm cell strainer (VWR) on ice. 5 mL of 50% iodixanol was added to the homogenate and mixed. In a 13 mL ultraclear ultracentrifuge tube, 1 mL of 40% iodixanol was added with 1 mL of 30% iodixanol carefully layered on top, followed by 10 mL of the tissue homogenate. The tissue was then ultracentrifuged in a swing-bucket rotor at 10,000xg for 18 min. Following ultracentrifugation, the 35% iodixanol layer was collected and the nuclei were counted. Nuclei were diluted with 30% iodixanol to 90,000 nuclei/mL for inDrops chip loading. Nuclei were isolated for inDrops using a previously described method [24 (link)].