title: GSM7744992: ATAC-Seq - Vero-HKU5-SARS-4; Chlorocebus sabaeus; ATAC-seq
library_construction_protocol: ATAC-seq with iDAPT-MS profiling: VeroE6 cells were washed and suspended in 1xPBS. Cells were pelleted (500 x g, 5 min, 4 °C), incubated in 500 μL nuclei extraction buffer (20 mM HEPES-KOH pH 7.9, 10 mM KCl, 0.5 mM spermidine, 0.1% Triton X-100, 20% glycerol, and 1x EDTA-free protease/phosphatase inhibitors [Thermo] in ultrapure water) per ~5e6 cells for 10 min on ice, and 50 μL DMSO mixed into the nuclear suspension prior to freezing cells at -80 °C. We found these lysis conditions to inactivate live SARS-CoV-2 virus for downstream handling and be compatible with high-quality ATAC-seq/iDAPT-MS profiling. 1.25 nmol MEDS-A/B, corresponding recombinant transposase (TP [transposase-peroxidase] or T [Tn5 transposase]) enzyme, and 0.5 nmol hemin chloride per sample were incubated at room temperature for 1 hr. Nuclei were thawed on ice, pelleted (1000 x g, 10 min, 4 °C), and resuspended in tagmentation buffer (20% dimethylformamide, 10 mM MgCl2, 20 mM Tris-HCl pH 7.5, 33% 1xPBS, 1% BSA, 0.01% digitonin, 0.1% Tween-20, 500 μM biotin-phenol, and 1x protease/phosphatase inhibitor) without transposome. Nuclei were counted with trypan blue (Thermo) with a Countess 3 (Thermo), and 5e6 nuclei per condition was resuspended in tagmentation buffer and 1 nmol transposome equivalent in a total volume of 500 μL, followed by incubation at 37 °C for 30 min with agitation on a thermomixer (1,000 rpm). 10 μL of tagmentation mix was saved for quality assessment as described below for ATAC-seq sample preparation.  ATAC-seq only: The OmniATAC sample preparation protocol was used as previously described with modifications where indicated below. Normalized transposase enzyme (in 2xDB) was mixed with 12.5 pmol MEDS-A/B (1.25 μL in water) and incubated at room temperature for 1 hr. In the meantime, 100,000 cells were centrifuged at 500 x g for 5 min at 4°C. Cells were resuspended in 50 μL lysis buffer 1 (LB1: 10 mM Tris-HCl pH 7.5, 10 mM NaCl, 3 mM MgCl2, 0.01% digitonin, 0.1% Tween-20, and 0.1% NP-40) with trituration, incubated on ice for 3 min, and then further supplemented with 1 mL lysis buffer 2 (LB2: 10 mM Tris-HCl pH 7.5, 10 mM NaCl, 3 mM MgCl2, and 0.1% Tween-20). Nuclei were pelleted (500 x g, 10 min, 4 °C), resuspended with 50 μL tagmentation reaction mixture (20% dimethylformamide, 10 mM MgCl2, 20 mM Tris-HCl pH 7.5, 33% 1xPBS, 0.01% digitonin, 0.1% Tween-20, and transposome complex in 50 μL total volume), and incubated at 37 °C for 30 min with agitation on a thermomixer (1,000 rpm). After tagmentation, DNA libraries were extracted with DNA Clean and Concentrator-5 (Zymo) and eluted with 21 μL water. To determine optimal PCR cycle number for library amplification, quantitative PCR was performed similarly as previously reported on a StepOnePlus Real-Time PCR (Applied Biosystems) with the StepOne v2.3 software. 2 μL of each ATAC-seq library was added to 2x NEBNext Master Mix (NEB) and 0.4x SYBR Green (Thermo Fisher) with 1.25 μM of each primer (Primer 1.1: 5'-AATGATACGGCGACCACCGAGATCTACACTAGATCGCTCGTCGGCAGCGTCAGATGTGTAT-3'; Primer 2.1: 5'-CAAGCAGAAGACGGCATACGAGATTCGCCTTAGTCTCGTGGGCTCGGAGATGT-3'