GSM8776455: 42F_3; Homo sapiens; ATAC-seq
Nuclei Isolation and ATACseq: Cultured cells were freshly dissociated with TrypLE at 37℃ and washed with ice-cold PBS. Nuclei isolation and transposition was performed using the OMNI-ATACseq protocol (1) with some few adjustments 7. Briefly, 200, 000 cells were pelleted at 500 rcf for 5 minutes in a pre-chilled (4°C) fixed-angle centrifuge. Supernatant was carefully discarded in a 2-step method using a P1000 pipette followed by a P200 pipette. Pelleted cells were then resuspend-ed in 100 µL ice-cold lysis buffer comprising ATACseq resuspension buffer (A-RSB; 10 mM Tris-HCl, 10 mM NaCl, 3 mM MgCl2 in nuclease free H2O) supplemented with NP-40, tween-20 and digitonin at final concentrations of 0.1%, 0.1% and 0.01% respectively. After 3 minutes of lysis re-action on ice, 1ml of ice-cold wash buffer (A-RSB supplemented with tween-20; 0.1% final concen-tration) was added and the tubes inverted to mix. The nuclei were then pelleted by centrifugation, and suspension carefully discarded (as previously described). For transposition, nuclei were resus-pended in a 50 µL transposition mix (2x TD buffer, PBS, 0.01% digitonin, 0.1 % tween 20, nucle-ase-free H20 and TDE1Tn5 transposase enzyme). Transposition was performed at 37°C for 30 minutes in a thermomixer with shaking at 850 rpm. Transposed DNA was cleaned up using the MinElute® PCR purification kit. Libraries were generated as previously described (2), double-sized selected using AMPure XP beads and eluted in EB buffer. For sequencing, 16 libraries were pooled together and sequenced pair-end in a NextSeq P3.