GSM6268817: K562 WT rep2 [ATAC-seq]; Homo sapiens; ATAC-seq
For each ATAC-seq assay, 50,000 cells were collected and washed with PBS. After centrifugation, resuspend cell pellet by gently pipetting up and down 10 times with 100 mL cold Lysis Buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% NP-40, 0.1% Tween-20, 0.01% Digitonin) and then keep on ice for 3 minutes. Add 1 mL Wash Buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% Tween-20) to the tube and mix gently. The nuclei were then pelleted by centrifuging at 1000 x g for 10 minutes at 4 °C. Resuspend nuclei pellet with 1 x Transposition Reaction Buffer (50 mM Tris-acetate, pH 7.5, 150 mM potassium acetate, 10mM magnesium acetate, 4 mM spermidine, 0.1% Tween-20, 0.01% Digitonin). Add 5 mL homemade ATAC-seq Tnp complex, then mix well gently and place in a 37 °C thermomixer with 1000 rpm shaking for 30 minutes. After the reaction, DNA was purified with MinElute Reaction Cleanup Kit (QIAGEN, Cat. 28206). Elute DNA with 15 mL elution buffer, then set up PCR reaction by mixing with 2.5 mL each indexed primer (25 mM) and 20 mL NEBNext High-Fidelity 2X PCR Master Mix (NEB, M0541S). Run the following PCR program: 72 °C for 5 minutes; 98 °C for 30 seconds; 11 cycles of 98 °C for 10 seconds, 63 °C for 30 seconds, and 72 °C for 1 minute; 72 °C for 5 minutes. Purify 200-650 bp DNA fragments for sequencing.