GSM9047358: iTF-Microglia Day 18, untreated, rep2; Homo sapiens; ATAC-seq
Chromatin was extracted from 100,000 cells, which were harvested by mechanical dissociation, washed with 50 μL cold 1X PBS, and centrifuged at 300×g for 5 minutes. The pellet was resuspended in 50 μL cold lysis buffer (10 mM Tris-HCl pH 7.5, 10 mM NaCl, 3 mM MgCl2, 0.1% NP-40, 0.1% Tween-20, 0.01% Digitonin, and nuclease-free H2O) and incubated for 3 minutes on ice. After lysis, 1 mL of wash buffer (10 mM Tris-HCl pH 7.5, 10 mM NaCl, 3 mM MgCl2, 0.1% Tween-20, and nuclease-free H₂O) was added, mixed by gentle inversion, and centrifuged at 500×g for 10 minutes at 4°C. The nuclei pellet was resuspended in 50 μL transposition mix (Tagment DNA TDE1 Enzyme and Buffer, Illumina) and incubated at 37°C for 30 minutes with shaking (1000 rpm). DNA was purified using the Qiagen MinElute Reaction Cleanup Kit and eluted in 11 μL. Libraries were generated using Nextera XT Index primers (Illumina) and Q5 Hot Start Master Mix (NEB), followed by PCR amplification (30 sec at 98°C; [10 sec at 98°C, 30 sec at 63°C, 1 min at 72°C] ×10 cycles).