ATAC-seq Protocol for Profiling Chromatin Accessibility

ATAC-seq was performed as described^{58 (link)}. Fifty thousand cells were centrifuged at 500 × g for 5 min at 4 °C. Cell pellets were washed once with 1x PBS and cells were pelleted by centrifugation using the previous settings. Cell pellets were resuspended in 25 μl of lysis buffer (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl₂, 0.1% IGEPAL CA-630) and centrifuged immediately 500 × g for 10 min at 4 °C. The cell pellet was resuspended in the transposase reaction mix (25 μL 2 × TD buffer (Nextera DNA Sample Preparation Kit), 2.5 μL Illumina Tn5 transposase and 22.5 μL nuclease-free water). The transposition reaction was carried out for 30 min at 37 °C. Directly following transposition, the sample was purified using a QIAGEN MinElute Purification Kit. Then, we amplified library fragments using NEBNext 2x PCR master mix and 1.25 M of Nextera PCR primers, using the following PCR conditions: 72 °C for 5 min; 98 °C for 30 s; and thermocycling at 98 °C for 10 s, 63 °C for 30 s, and 72 °C for 1 min. The libraries were purified using a QIAGEN PCR purification kit yielding a final library concentration of ~30 nM in 20 μL. Libraries were amplified for a total of 10–13 cycles and were subjected to high-throughput sequencing using the Illumina HiSeq 2500 Sequencer.