ATAC-seq Library Preparation from Hippocampus

ATAC-seq sample preparation was performed for eight biological replicates/conditions according to detailed protocols obtained from Hongjun Song’s lab at the University of Pennsylvania (Buenrostro et al., 2015 (link); Su et al., 2017 (link)). In brief, one hippocampus was homogenized with a Dounce tissue grinder (Wheaton 357544) in 2 mL HB buffer (1 mM DTT, 0.15 mM spermine, 0.5 mM spermidine, protease inhibitor (Sigma-Aldrich 04693159001), 0.3% IGEPAL-630, 0.25 M sucrose, 25 mM MgCl₂, 20 mM Tricine-KOH). The homogenate was filtered through a 40-μm strainer and centrifuged over one volume of cushion buffer (0.5 mM MgCl₂, 0.5 mM DTT, protease inhibitor, 0.88 M sucrose) at 2,800g for 10 min in a swinging bucket centrifuge at 4°C. The nuclei pellet was resuspended in 20 μL PBS. Then, 1 μL of the nuclei sample was stained with Hoechst 33342 to calculate nuclei concentration, and 50,000 nuclei per sample were used for downstream library preparation. Libraries were prepared using the Nextera DNA library prep kit (Illumina FC-121-1030), and quality was analyzed using the D1000 ScreenTape Assay (Agilent 5067-5582). Paired-end 50 bp sequencing to a depth of ~40 million reads per sample was performed using the Illumina HiSeq 2500 system at the UCLA Broad Stem Cell Research Center Sequencing Core.