GSM6284911: TS169_08_THP1_CCS1477_48h_100nM; Homo sapiens; ATAC-seq
The Assay for Transposase Accessible Chromatin (ATAC-seq) was performed following the Omni- ATAC improved version of the conventional protocol (Corces et al., 2017) using 5x10^4 viable THP1 cells. Cell pellets were washed once in cold PBS, re-suspended in 50μl of cold lysis buffer (10mM Tris-HCl pH 7.5, 10mM NaCl, 3mM MgCl2, 0.1% v/v NP-40, 0.1% v/v Tween-20, 0.01% Digitonin) and further incubated on ice for 3min. Cells were then washed in 1ml of wash buffer (10mM Tris-HCl pH 7.5, 10mM NaCl, 3mM MgCl2, 0.1% v/v Tween-20) and nuclei were pelleted by centrifugation for 10min at 500xg at 4°C. Supernatant was discarded and the nuclei were re-suspended in 50μl of transposition reaction buffer (Nextera DNA Sample Preparation Kit, Illumina) containing 16.5μl PBS, 0.1% v/v Tween-20, 0.01% Digitonin, 5μl nuclease-free water, 25μl 2X TD Buffer and 2.5μl Tn5 Transposase. The reaction was incubated at 37°C for 30min at 1,000rpm and purified using the MinElute Reaction Cleanup Kit (QIAGEN). DNA was amplified using NEB Next High-Fidelity PCR Master Mix (New England Biolabs) and 1.25μM of custom PCR primers and conditions (Buenrostro et al., 2013). The PCR reaction was monitored to reduce GC and size bias by pre-amplifying the full libraries for five cycles and taking an aliquot to run for 25 cycles using the same PCR cocktail and SYBR Green. The remaining 45μl reaction was amplified for 6-7 additional cycles as determined by qPCR and DNA libraries were purified using the MinElute Reaction Cleanup Kit (QIAGEN). Fragments of 200– 1000bp were selected using AMPure beads (Beckman Coulter) and quantified by quantitative PCR with a KAPA Library Quantification Kit (Kapa Biosystems). Libraries were prepared for sequencing using a Microplex Library Preparation Kit (Diagenode). 200-800bp fragments were selected using AMPure beads (Beckman Coulter, Brea, CA) and quantified by qPCR with a KAPA Library Quantification Kit (Kapa Biosystems, Basel, Switzerland). Sequencing was performed using a Novaseq SP sequencer system (Illumina) generating around 80 million reads per sample.