GSM9143333: ATAC_ANT_Training_m4; Mus musculus; ATAC-seq
Nuclear extraction. Mice were sacrificed within 40-60 minutes from behavioral testing. The anterior thalamus and anterior cingulate of C57Bl6/J mice were dissected, and nuclei were extracted using the percoll gradient method. The dissected tissue was homogenized with 54% percoll (Cytiva) in 1× buffer (50 mM Tris-HCl, pH 7.4; 25 mM KCl; 5 mM MgCl2; 250 mM sucrose) on ice, using a 23 G syringe and a 27 G syringe, 10 strokes each. After homogenization, 10% NP-40 substitute (Roche, cat. no. 11332473001) and 10% Tween-20 were added and incubated for 15 minutes on ice (0.1% final concentration for both NP-40 and Tween-20). Following the incubation, an equal volume of 1× buffer was added and mixed by pipetting. A percoll gradient was prepared by layering 31% and 35% percoll at the bottom of the tube. Nuclei were pelleted in the bottom layer by centrifugation in a swinging bucket (14,000 g, 10 minutes at 4°C). The nuclear pellet was carefully resuspended in blocking buffer (5% BSA/PBS) and incubated for 10 minutes on ice. After blocking, nuclei were labeled by incubation with Alexa Fluor 488-conjugated anti-NeuN antibody (1/2000) for 20 minutes on ice. Nuclei were then washed once in PBS and labeled with 7-AAD (Sigma, cat. no. SML1633). The 7-AAD singlet and NeuN-positive population was sorted by FACS, and the sorted nuclei were used for ATAC-seq. Libraries were prepared for sequencing using standard Illumina protocols