GSM8574626: ATAC-seq_PF-382_FTO_int8del; Homo sapiens; ATAC-seq
First a resuspension buffer was made of 10mM Tris-HCl, pH 7.5, 10mM NaCl, 3mM MgCl2. Briefly, 50,000 cells were washed in ice cold 1X PBS and pelleted by centrifugation. The pellet was lysed in 50 µL of lysis buffer comprised of 48.5 µL resuspension buffer, 0.5 µL 10% NP-40 (Sigma), 0.5 µL 10% Tween-20 (Sigma), 0.5 µL 1% Digitonin (Promega). Following incubation on ice for 3 minutes samples were washed with 1 mL wash buffer comprised of 990µL resuspension buffer, 10 µL 10% Tween-20. Nuclei were retained following centrifugation at 500g for 10 mins at 4oC. Transposition mixes were made with the following reagents 25µL 2X TD/Tagment DNA Buffer (Illumina), 16.5µL 1X PBS, 0.5µL 10% Tween-20 (Sigma), 0.5µL 1% Digitonin (Promega), 2.5µL Tn5 Transposase/Tagment DNA Enzyme 1 (Illumina) and 5µL nuclease-free H2O. Nuclei were gently resuspended in the transposition mix and incubated for 30 minutes at 37oC on a thermomixer at 1,000 rpm. After transposition DNA was purified with AMPure XP beads at a 1.8x ratio. Purified transposed DNA was amplified by PCR by using the NEBNext High-Fidelity 2X PCR Master Mix (NEB) with ATAC-seq primers. PCR cycle number was optimised by spiking in 100X SYBR Green I followed by qPCR to ensure amplification did not reach saturation. Amplified libraries were purified by AMPure XP beads at a 1.8x ratio. Libraries were sequenced at ~30 million paired reads per sample on an NextSeq (Illumina).