title: GSM3764658: 21_DG; Rattus norvegicus; ATAC-seq
library_construction_protocol: The brains were removed, dissected out into discrete brain areas (striatum, dentate gyrus, Ammon's horn, prefrontal cortex, and cerebellum) and stored at − 80 °C until assayed.  The dentate gyrus was dissected out according to a previously described protocol76 modified for the rat brain. Briefly, the brain was cut sagittally to divide the hemispheres, which were then placed medial side up after removal of the regions posterior to lambda. The dentate gyrus and Ammon's horn (CA1 and CA3) (which were visible upon removal of thalamus and hypothalamus) were dissected out using fine tip surgical instruments. Liver and muscle tissues were also collected and stored at –80°C until analysis. The Omni-ATAC protocol on Nuclei Isolated from frozen tissue was slightly modified. A frozen tissue fragment approximately 5-10 mg was placed into a pre-chilled 2 ml Dounce homogenizer containing 0.5 ml of cold 1x homogenization buffer (320 mM sucrose, 0.1mM EDTA, 0.1% NP40, 5mM CaCl2, 3mM Mg(Ac)2, 10 mM Tris pH 7.8, 1x protease inhibitors(Roche, cOmplete), 167 uM β-mercaptoethanol, in water). Tissue was homogenized with approximately 10 strokes with the loose “A” pestle, followed by 20 strokes with the tight “B” pestle. Residual debris were removed by centrifugation for 1 min at 100 RCF. Avoiding pelleted debris, 400 ul was transferred to a pre-chilled 2 ml round bottom Lo-Bind Eppendorf tube. An equal volume (400 ul) of a 50% iodixanol solution (50% iodixanol in 1x homogenization buffer) was added and mixed by pipetting to make a final concentration of 25% iodixanol. 600 ul of a 29% iodixanol solution (29% iodixanol in 1x homogenization buffer containing 480 mM sucrose) was layered underneath the 25% iodixanol mixture. A clearly defined interface should be visible. In a similar fashion, 600ul of a 35% iodixanol solution (35% iodixanol in 1x homogenization containing 480 mM surcose) was layered underneath the 29% iodixanol solution. Again, a clearly defined interface should be visible between all three layers. In a swinging bucket centrifuge, nuclei were centrifuged for 20minute at 4800 RCF. After centrifugation, nuclei are present at the interface of the 29% and 35% iodixanol solutions. This nuclei band was collected in a 300 ul volume and transferred to a prechilled tube containing 1 ml of ATAC-seq RSB (10mM Tris pH 7.4, 10mM NaCl, 3mM MaCl2) with 0.1% Tween-20. Nuclei were pelleted by centrifugation at 500 RCF for 10 minutes in a pre-chilled 4°C fixed-angle centrifuge. Supernatant was removed using two pipetting steps, in which the supernatant was aspirated with p1000 pipette first, the remaining 100ul was carefully aspirated with a p200 pipette. 20 ul 2x TD buffer (20mM Tris pH 7.6, 10mM MgCl2, 20% Dimethyl Formamide) was added to the nuclear pellet and mixed by pipetting up and down 6 times. Nuclei were counted after addition of trypan blue which stains all nuclei. 50,000 counted nuclei were then transferred to a tube with 2x TD buffer being filled in up to 25ul. 25ul of transposition mix (2.5 ul transposase (100nM final), 16.5 ul PBS, 0.5 ul 1% digitonin, 0.5 ul 10% Tween-20, 5 ul H2O) was then added to the nuclei in the 25ul 2x TD buffer. Transposition reactions were mixed and incubated at 37°C for 30 minutes with simply tapping every 10 minutes. Reactions were cleaned up with Zymo DNA Clean and Concentrator 5 columns.