title: GSM7744982: ATAC-Seq - Vero-MERS-2; 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') in a final volume of 15 μL, and quantification was assessed using the following 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. Optimal PCR cycle number was determined as the qPCR cycle yielding fluorescence between 1/4 and 1/3 of the maximum fluorescence. The remaining DNA library was then amplified accordingly by PCR using previously reported barcoded primers for library multiplexing, purified with DNA Clean and Concentrator-5 (Zymo), and eluted into 20 μL final volume with water. Libraries were then subject to TapeStation 2200 High Sensitivity D1000 fragment size analysis (Agilent), MiSeq Nano v2 sequencing for library normalization, and NovaSeq 6000 paired-end sequencing for deep sequencing (Illumina).