ATAC-seq Protocol with Modifications

ATAC-seq analysis was performed as recently described with some modifications^{53 (link),54 (link)}. Briefly, 5 × 10⁴ cells were pretreated with 200 U ml⁻¹ DNase (Worthington, LS002006) in the culture medium for 30 min at 37 °C, then washed with PBS twice. Cell pellets were resuspended in 50 μl freezing media (10% DMSO, 50% FBS and 40% complete media) and transferred in an isopropyl alcohol chamber at −80 °C overnight. The next day, the frozen cell pellets were thawed and first incubated in L1 buffer (10 mM Tris–HCl pH 7.4, 10 mM NaCl, 3 mM MgCl₂, 0.1% Digitonin, 0.1% Tween-20 and 0.1% NP-40 supplemented with 1× Protease/Phosphatase inhibitors (Pierce)) for 3 min then resuspended in L2 buffer (10 mM Tris–HCl pH 7.4, 10 mM NaCl, 3 mM MgCl₂ and 0.1% Tween-20 supplemented with 1× Protease/Phosphatase inhibitors (Thermo Fisher Scientific, 78444)), centrifugated and resuspended in tagmentation buffer (25 μl 2× TD buffer (Illumina, 15027865), 2.5 μl Tn5 transposase (Illumina, 15027866), 16.5 μl PBS, 0.5 μl 1% digitonin, 0.5 μl 10% Tween-20, and 5 μl water) for additional 30 min at 37 °C, following manufacturer recommendations (Nextera DNA Sample Prep Kit, Illumina, 20015882). After DNA purification, adapter sequences were added to the fragmented DNA by PCR. Purified PCR products were sequenced using the Nextseq 500 Illumina genome analyzer.