ATAC-seq Protocol for Chromatin Accessibility

ATAC-seq was performed from two biological replicates at four time points as described (Buenrostro et al., 2013 (link)). Briefly, 5 × 10⁴ cells at each time point were harvested and treated with Nextera Tn5 Transposase (Illumina, FC-121–1030) for 45 min at 37°C. Library fragments were amplified using 1× NEBNext High-Fidelity 2× PCR Master Mix (NEB, M0541S) and 1.25 μM of custom Nextera PCR primers 1 and 2. PCR amplification was done with 11 cycles, determined by KAPA Real-Time Library Amplication Kit (Peqlab, KK2701) to stop prior to saturation. Then, the samples were purified using Qiagen MinElute PCR Purification Kit (Qiagen, 28004) and with Agencourt AMPure XP beads (Beckman Coulter, A63881) in 3:1 ratio. The libraries were sequenced at 2 × 50 bp on HiSeq2000 (TAL, Göttingen). The average read numbers obtained were 3 × 10⁷.