GSM5354437: Nuclear HK2 knockdown 1 (ATAC-seq); Homo sapiens; ATAC-seq
60,000 viable cells per sample were pelleted at 500 RCF at 4°C for 5 min. Supernatant was removed and cells were re-suspended in 50ul of cold resuspension buffer (RSB) containing 0.1% NP40, 0.1% Tween-20 and 0.01% digitonin. Cell suspension was incubated on ice for 3 minutes before being washed out with 1 ml of cold RSB containing 0.1% Tween-20. Cells were mixed by inversion prior to pelleting at 500 RCF at 4°C for 5 min. Supernatant was removed, cells were re-suspended in 50µl of transposition mix and incubated at 37°C for 1 hour in a thermomixer (Eppendorf) set to 1000 RPM. Transposition mix was purified using the Qiagen MinElute Reaction Cleanup Kit (Qiagen) and eluted in 20µl of water. 1µl of sample was taken into a qPCR reaction to determine the optimal number of PCR cycles required for amplification without reaching saturation and based off the measured cycle number, the remaining 19ul of each sample was amplified. Libraries were purified using AMPure XP beads (Beckman Coulter) using a double-sided bead cleanup protocol set to 0.7x-1.0x. This cleanup was performed twice to remove the large molecular fragments.