Nuclei Isolation From Dissected Tissue

In this study, we specifically focused on the dissected AC, V1, SC, and MC from rats, as shown in Figure 1A. Nuclei were isolated from the frozen cortex according to an established protocol with minor modifications (Bakken et al., 2018; Thrupp et al., 2020). The whole operation was carried out on ice. Briefly, frozen tissue was cut into pieces and transferred to a 2 mL KIMBLE Dounce tissue grinder (Sigma #D8938-1SET) with 2 mL of ice-cold homogenization buffer [20 mM Tris pH 8.0 (Thermo Fisher Scientific)], 500 mM sucrose (Sigma), 50 mM KCl (Thermo Fisher Scientific), 10 mM MgCl₂ (Thermo Fisher Scientific), 0.1% NP-40 (Roche), 1× protease inhibitor cocktail (Roche), and 1% nuclease-free BSA, and 0.1 mM DTT). Tissues were homogenized by 15 strokes of the loose dounce pestle, and the homogenate was filtered through a 70 μM cell strainer (Falcon). Next, the filtered homogenate was homogenized by five strokes of the tight pestle to release nuclei, and it was again filtered through a 30 μM cell strainer (Sysmex) into a 15 mL centrifuge tube. Nuclei pellets were then obtained by centrifuging at 500 g for 5 min at 4°C. Nuclei were then washed twice with 1 ml of ice-cold blocking buffer (1× PBS supplemented with 1% BSA) followed by another step of centrifugation at 500 g for 5 min at 4°C. Finally, we resuspended the nuclei in 50 μL of 1× PBS containing 1% BSA and counted them with DAPI.

Schematic diagram for experimental and data analysis. (A) A total of four different cortex regions from adult rat brains were collected for single-nucleus assay for transposase-accessible chromatin using sequencing (snATAC-seq) profiling. (B) The analysis workflow for snATAC-seq profiles.