ATAC-seq of Visceral Adipose Tissue

ATAC-seq was carried out using the Omni-ATAC protocol [12 (link)]. Visceral adipose tissue (1 g) was placed in cold 1 × homogenization buffer (320 mM sucrose, 0.1 mM EDTA, 0.1% NP40, 5 mM CaCl2, 3 mM Mg(Ac)2, 10 mM Tris pH 7.8, 1 × protease inhibitors (Roche, cOmplete), and 167 μM β-mercaptoethanol, in water), homogenized with Dounce homogenizers, and residual debris was precleared by using 80 um nylon mesh filter. Nuclei were then collected by layering with iodixanol mixture. 50,000 counted nuclei were transferred to a tube containing the transposition mix (25 μl 2 × TD buffer, 2.5 μl transposase (100 nM final), 16.5 μl PBS, 0.5 μl 1% digitonin, 0.5 μl 10% Tween-20, 5 μl water) and mixed by pipetting up and down six times. Transposition reactions were incubated at 37 °C for 30 min in a thermomixer with shaking at 1,000 r.p.m. Reactions were cleaned up with Zymo DNA Clean and Concentrator 5 columns. Library amplification was done with 2 × KAPA HiFi mix (Kapa Biosystems) and 1.25 µM indexed primers using the following PCR conditions: 72 °C for 5 min; 98 °C for 30 s; and 10–11 cycles at 98 °C for 10 s, 63 °C for 30 s, and 72 °C for 1 min.