Nuclei Isolation from Frozen Tissues

Single nuclei were obtained from flash-frozen tissues using sectioning and mechanical homogenization as previously described^{2 (link),54} . Slices of 5–10 mm thickness from frozen tissue were first sectioned with a cryostat in a 50-μm thickness section. All sections from each sample were homogenized using a 7 ml glass Dounce tissue grinder set (Merck) with 8–10 strokes of a loose pestle (A) and 8–10 strokes of a tight pestle (B) in homogenization buffer (250 mM sucrose, 25 mM KCl, 5 mM MgCl₂, 10 mM Tris-HCl, 1 mM dithiothreitol (DTT), 1× protease inhibitor, 0.4 U μl⁻¹ RNaseIn, 0.2 U μl⁻¹ SUPERaseIn and 0.1% Triton X-100 in nuclease-free water). Homogenate was filtered through a 40 μm cell strainer (Corning). After centrifugation (500g, 5 min, 4 °C), the supernatant was removed and the pellet was resuspended in storage buffer (1× PBS, 4% BSA and 0.2 U μl⁻¹ Protector RNaseIn). Nuclei were stained with 7-AAD viability staining solution (BioLegend), and positive single nuclei were purified by FACS using a MA900 Multi-Application Cell Sorter (Sony) and its proprietary software (Cell Sorter v.3.1.1) (Supplementary Fig. 7). Nuclei purification and integrity were verified by microscopy, and nuclei were further processed for multiome paired RNA and ATAC-seq using Chromium Controller (10x Genomics) according to the manufacturer’s protocol.