ATAC-seq Protocol for Chromatin Profiling

ATAC-seq was performed as previously described (25 (link)). Briefly, 50,000 cells were washed with 50 μl cold PBS and resuspended in 50 μl lysis buffer (10 mM Tris–HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.2% [vol/vol] IGEPAL CA-630). The suspension of nuclei was then centrifuged for 10 min at 500g at 4°C, followed by the addition of 50 μl transposition reaction mix (25 μl TD buffer, 2.5 μl Tn5 transposase, and 22.5 μl nuclease-free H₂O) from the Nextera DNA library Preparation Kit (96 samples) (FC-121-1031; Illumina). Samples were then PCR amplified and incubated at 37°C for 30 min. DNA was isolated using a MinElute Kit (QIAGEN). ATAC-seq libraries were first subjected to five cycles of pre-amplification. To determine the suitable number of cycles required for the next round of PCR, the libraries were assessed by quantitative PCR as described (26 (link)), and the libraries were then PCR amplified for the appropriate number of cycles according to the qRT-PCR results. Libraries were purified with a Qiaquick PCR (QIAGEN) column, and the library’s concentration was measured using a KAPA Library Quantification Kit (KK4824) according to the manufacturer’s instructions. Finally, the ATAC libraries were sequenced on the NextSeq 500 sequencing platform using a NextSeq 500 High Output Kit v2 (150 cycles) (FC-404-2002; Illumina) according to the manufacturer’s instructions.