GSM7793714: QL5; Homo sapiens; ATAC-seq
Genomic DNA was extracted using the Quick-DNA Microprep Kit (D3020; Zymo Research). Briefly, 10^5cells were resuspended in 1 ml of cold ATAC-seq resuspension buffer (RSB; 10 mM Tris-HCl pH 7.4, 10 mM NaCl and 3 mM MgCl2in water). Cells were centrifuged at 500×gfor 5 min in a prechilled fixed-angle centrifuge and the supernatant was carefully aspirated. Cell pellets were then resuspended in 50 µl RSB containing 0.1% NP40, 0.1% Tween-20 and 0.01% digitonin y pipetting up and down three times. This cell lysis reaction was incubated on ice for 3 min. After lysis, 1 ml RSB containing 0.1% Tween-20 (without NP40 or digitonin) was added, and the tubes were inverted to mix. Nuclei were then centrifuged for 10 min at 500×gin a prechilled fixed-angle centrifuge. Supernatant was removed, and nuclei were resuspended in 50 µl of transposition mix (2.5 µl transposase (100 nM final), 16.5 µl PBS, 0.5 µl 1% digitonin, 0.5 µl 10% Tween-10 and 5 µl water) by pipetting up and down six times. Transposition reactions were incubated at 37 °C for 30 min in a shaking thermomixer. Reactions were cleaned up with QIAquick PCR spin columns. Library quantitation was used to determine the number of amplification cycles by plotting linear Rn vs. cycle and determining the cycle number corresponding to one-fourth of maximum fluorescent intensity