GSM7434181: Female_Saline_1; Rattus norvegicus; ATAC-seq
Flash-frozen NAc tissue was added to a tube containing 500µl lysis buffer (1mM Tris-HCl pH 7.4, 1mM NAc, 0.3 mM MgCl2, 0.01% Tween-20, 0.01% Igepal in nuclease free water, 0.001% Digitonin, 0.1% BSA), homogenized using a motorized homogenizer and RNase free pestle, and incubated on ice for 5 minutes. Samples were then pipette mixed 15x and incubated on ice for an additional 10 minutes. Following lysis, 4 samples from same sex and treatment were grouped into a single sample, and 2mL chilled wash buffer (10mM Tris-HCl pH 7.4, 10mM NACl, 3mM MgCl2, 1% BSA, 0.1% Tween-20) was added. Samples were then passed through 70µM and 40µM Scienceware Flowmi Cell Strainers. Samples were then centrifuged at 250 rcf for 5 min at 4°C. Supernatant was removed and the pellet was resuspended in 1mL of chilled wash buffer (10mM Tris-HCl pH 7.4, 10mM NACl, 3mM MgCl2, 1% BSA, 0.1% Tween-20). 100µL of sample was then stained with 7-aminoactinomycin D and used to set a representative gate for fluorescence activated cell sorting (FACs). This gate was then used to purify nuclei further. Following FACs, samples were spun at 250 rcf for 10 min at 4°C to remove any remaining debris. Samples were then loaded into individual wells of the Chromium NextGem Single Cell Chip using 4 of the 8 available wells. FACS-sorted nuclei were subject to 10X GEM generation, transposase barcoding, and library construction using a Chromium Next GEM Single Cell ATAC Kit v2. Libraries were quantified and underwent sequencing on an Illumina sequencing platform (NextSeq 500) using a dual-indexed, paired-end strategy (Read 1N, 50 cycles; i7 index, 8 cycles; i5 index, 16 cycles; Read 2N, 50 cycles).