title: GSM5893293: tam1_ATAC-seq; Mus musculus; ATAC-seq
library_construction_protocol: Permeabilized nuclei were obtained by resuspending cells in 250 µL Nuclear Permeabilization Buffer (10 mM Tris-HCL [pH 7.5], 10 mM NaCl, 3mM MgCl2, 0.1% Tween-20 [Sigma], 0.1% IGEPAL-CA630 [Sigma] and 0.01% Digitonin [Promega] in water) and incubating for 10 minutes on a rotator at 4°C. Nuclei were then pelleted by centrifugation for 5 minutes at 500 x g at 4°C. The pellet was resuspended in 20 µL ice-cold Tagmentation Buffer (33 mM Tris-acetate [pH 7.8] [BP-152, Thermo Fisher Scientific], 66 mM K-acetate [P5708, Sigma], 11 mM Mg-acetate [M2545, Sigma], 16% DMF [DX1730, EMD Millipore] in Molecular biology water [46000-CM, Corning]). An aliquot was then counted to determine nuclei concentration. Approximately 50,000 nuclei were resuspended in 10 µL ice-cold Tagmentation Buffer and incubated with 1 µL Tagmentation enzyme (FC-121-1030; Illumina) at 37°C for 30 minutes while shaking at 500 rpm. The tagmentated DNA was purified using the MinElute PCR purification kit (28004, Qiagen). The libraries were amplified using NEBNext High-Fidelity 2X PCR Master Mix (M0541, NEB) with primer extension at 72°C for 5 minutes and denaturation at 98°C for 30 seconds, followed by 8 cycles of denaturation at 98°C for 10 seconds, annealing at 63°C for 30 seconds, and extension at 72°C for 60 seconds. Amplified libraries were then purified using the MinElute PCR purification kit (28004, Qiagen) and two size selection steps were performed using SPRIselect bead (B23317, Beckman Coulter) with 0.55X and 1.5X bead-to-sample volume ratios, respectively.