title: GSM8630439: HepG2, 5.0%FA + flash freezing; Homo sapiens; ATAC-seq
library_construction_protocol: Cells were pelleted and resuspended in 1 ml of fixative buffer (0.1-5% Formaldehyde in DMEM) and incubated at room temperature for 5 minutes. Afterwards, cells were pelleted and washed in DMEM + 0.1% BSA, pelleted again, and either snap-frozen by submerging the tubes in liquid nitrogen or cryopreserved. Cells and nuclei were permeabilized in lysis buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% Igepal CA-630, 0.1% Tween-20, 0.01% Digitonin, 1% BSA) and incubated for 5 minutes on ice. Ice-cold wash buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% Tween-20, 1% BSA) was added and mixed by inversion. Nuclei were pelleted and resuspended in transposition mix (25 μl of Diagenode 2x Tn5 reaction buffer, 0.5 μl of 1% Digitonin, 0.5 μl of 10% Tween-20, 16.5 μl of PBS, 5 μl of water, and 2.5 μl of Custom Tn5 at a 1:4 dilution). The mixture was thoroughly mixed by pipetting and incubated for 30 minutes at 37°C. Transposition was quenched by adding 50 μl of 2x Tn5 stop solution (40 mM EDTA, 2 mM spermidine), followed by incubation at 37°C for 15 minutes. The reactions were then cooled and pooled on ice. The pooled cells were centrifuged and resuspended in buffer EB. Samples were de-crosslinked at 68°C for 30 minutes. DNA was purified using the MinElute PCR purification kit according to the manufacturer's instructions. For PCR amplification, 10 μl of each transposed DNA was mixed with 2.5 μl of 25 μM i5 primer, 2.5 μl of 25 μM i7 primer, and 25 μl of 2x NEBNext Ultra II Q5 Master Mix. The mixture was amplified (72°C for 5 min, 98°C for 30 s, followed by 10 cycles of 98°C for 10 s, 63°C for 30 s, 72°C for 1 min, hold at 4°C). Final libraries were cleaned using Ampure XP at a 0.8x ratio (40 μl of beads per 50 μl of reaction) and eluted in EB (Qiagen).