Omni ATAC-seq

We employed the omni-ATAC method of Corces. 80,000 viable naïve T-cells were pelleted and lysed in lysis buffer containing 10 mM Tris–HCl, 10 mM NACl, 3 mM MgCL₂, 0.1% NP40, 0.1% Tween20 and 0.01% Digitonin for 3 min on ice. Cells were washed with 1 mL of cold wash buffer (lysis buffer without NP40 or Digitonin) and nuclei were pelleted in a centrifuge at 800 RCF for 10 min at 4 degrees. Pelleted nuclei were transposed with Tn5 transposase (Illumina) in TD buffer (Illumina) supplemented with Digitonin (0.1%) and Tween20 (0.01%) for 30 min at 37. Transposed DNA was purified using Zymo DNA Clean and Concentrator-5 Kit (Zymo research) according to manufacturer’s instruction. DNA recoveries were measured on the Qubit fluorometer (Invitrogen). Library amplification was performed using Nextera DNA library prep kit with Nextera Index Kit (Illumina) as per manufacturers instruction. The number of PCR amplification cycles was determined by qRT-PCR using Quanitfast SYBR Green PCR mastermix (Qiagen) and Nextera Primer I5 and I7 Indexes for 5 cycles. The number of additional cycles was determined by a second round of qPCR performed on partially amplified libraries based on the CT value reading taken at 1/3 the fluorescence curve. Two step size selection was performed using AMPure XP beads (Beckman Coulter). Libraries were run on the LabChip GXII fragment analyser and quantitated on the Qubit fluorometer. Libraries were shipped on ice to Novogene (China) for pooling and sequencing on 2 lanes of the Illumina HiSeq at 2 × 150 paired end reads to generate 50 million reads per sample.