GSM8625610: ATAC_B6_Chow_2; Mus musculus; ATAC-seq
Nuclei were isolated using a Dounce homogenizer and 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 1 mM β-mercaptoethanol). After homogenization, the samples were filtered through a 100 µm nylon mesh filter, and nuclei were recovered by OptiPrep/iodixanol (Sigma-Aldrich) density gradient centrifugation. Libraries were generated following Omni-ATAC protocol (Corces MR. et al. Nat Methods. 2017;14(10):959-962) with minor adjustments. Nuclei were resuspended in transposition mix (25 µl 2× TD buffer, 2.5 µl transposase, 16.5 µl PBS, 0.5 µl 1% digitonin, 0.5 µl 10% Tween-20, 5 µl H2O). After the transposition reaction, DNA was purified with DNA Clean & Concentrator-5 Kit (Zymo Research). Ten microliters of purified transposed DNA were amplified for 9-11 cycles (predetermined) using 25µl of 2 × Ultra II Q5 Master Mix (New England BioLabs) and 1 µl of both amplification primers (Buenrostro JD et al. Nat Methods. 2013;10(12):1213-1218; 1.25 μM final concentration). For cleanup and to remove primer dimers and large (>1,000 bp) fragments from the libraries, SPRIselect beads (Beckman Coulter) were used.