Instrument: Illumina HiSeq 2500
Strategy: ATAC-seq
Source: GENOMIC
Selection: other
Layout: PAIRED
Construction protocol: Adherent cell cultures were dissociated and 100 000 cells were washed twice in cold PBS at 4°C. Cells were resuspended in 100 uL Hypotonic Cell Lysis Buffer (0.1% Sodium Citrate Tribasic Dihydrate, 0.1% Triton X-100) and titurated until cells were dissolved. Samples were incubated on ice 30 minutes, centrifuged at 2000 g for 5 minutes at 4°C, and pellet resuspended in 100 uL Normal Cell Lysis Buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% IGEPAL CA-630), titurated, incubated on ice 30 minutes, centrifuged at 2000 g for 5 minutes at 4°C, and supernatant removed. The transposase reaction was carried out by titurating in 25 uL per sample in TD Buffer (10 mM Tris-HCl, pH 8.00, 5 mM Magnesium Chloride) with 5 uL Transposase (Illumina Nextera Kit), and incubated at 37°C for 30 minutes, and 8.5 uL of 100mM EDTA was added, samples transferred to ice, and DNA recovered using MinElute PCR Purification columns (Qiagen). Libraries were generated by PCR in 50 uL reaction (25 uL sample, 10 uL 5x Phusion HF buffer, 0.5 uL Phusion Polymerase, 1 uL 10mM dNTPs, 0.5 uL of each custom Illumina primers at 12.5 uM). The PCR reaction followed 98°C for 30 seconds, followed by 12 cycles of 98°C for 10s, 63°C for 30s, 72°C for 1 min, followed by 72°C for 5 min. DNA was recovered using GeneRead Purification columns (Qiagen). The libraries were sequenced to 50 million reads per sample on Illumina HiSeq 2500 using Nextera Sequencing Primers.