GSM8219596: L1626-GM12878_KAS; Homo sapiens; OTHER
500-mM N3-kethoxal solution was brought to 37C, then added to 1 mL of culture at a final concentration of 5 uM. Cells were then incubated for 5 minutes at 37c in a ThermoMixer at 1000 RPM. Cells were then pelleted by centrifugation, resuspended in 100 uL 1x PBS buffer, and DNA was immediately isolated using the Monarch Genomic DNA Purification Kit (NEB, Cat # T3010S), with the modification that elution was carried out with 87.5 uL 25 mM K3BO3 solution (pH 7.0). Biotin was clicked onto kethoxal-modified guanines by mixing 87.5 uL DNA, 2.5 uL 20 mM DBCO-PEG4-biotin (Sigma, Cat # 760749; DMSO solution), and 10 uL 10x PBS, and incubating at 37C for 90 minutes. DNA was isolated using AMPure XP beads and eluted in 130 uL 25 mM K3BO3 (pH 7.0), then sheared on a Covaris E220 for 120 seconds down to ~150-200 bp. Libraries were built on beads using the NEBNext Ultra II DNA Library Prep kit (NEB, Cat # E7645L). Biotin pull down was initiated py pipetting 20 uL Dynabeads MyOne Streptavidin T1 beads (ThermoFisher Scientific, Cat # 65306) into DNA lo-bind tubes. Beads were separated on magnet, resuspended in 200 uL of 1x TWB buffer (Tween Washing Buffer; 5 mM Tris-HCl pH 7.5; 0.5 mM EDTA; 1 M NaCl; 0.05% Tween 20), then separated on magnet again and  resuspended in 300 uL of 2x BB (Binding Buffer; 10 mM Tris-HCl pH 7.5, 1 mM EDTA; 2 M NaCl). The DNA (130 uL) was added together with 170 uL 0.1x TE buffer, and incubated at RT on rotator for ~15 minutes. Beads were separated on magnet, resuspended in 200 uL of 1x TWB, and incubated at 55C in a Termomixer for 2 minutes with shaking at 1000 rpm. Beads were again separated on magnet and the 200-uL 55C TWB wash step was repeated. Beads were separated on magnet and resuspended in 50 uL 0.1x TE. End repair was carried out by adding 7 uL NEB End Repair Buffer and 3 uL NEB End Repair Enzyme, incubating at 20C for 30 minutes, then at 65C for 30 minutes. End repair was followed by adaptor ligation by adding 2.5 uL NEB Adaptor, 1 uL NEB Ligation Enhancer and 30 uL NEB Ligation Mix, incubating at 20C for 20 minutes, then adding 3 uL USER Enzyme and incubating at 37C for 15 minutes. Beads were separated on magnet, resuspended in 200 uL of 1X TWB, then incubated at 55C in a Thermomixer for 2 minutes with shaking at 1000 rpm. Subsequently beads were separated on magnet and resuspended in 100 uL of 0.1X TE, separated on magnet again, resuspended in 15 uL of 0.1X TE Buffer, and transfered to PCR tubes. Beads were then incubated at 98C for 10 minutes, and libraries were amplified by adding 5 uL of i5 primer, 5 uL of i7 primer and 25 uL of 2X Q5 Hot Start Polymerase Mix, using the following PCR program: 30 seconds at 98C; 15 cycles of 98C for 10 seconds, 65C for 30 seconds, and 72C for 30 seconds; and a final extension at 72C for 5 minutes. Beads were separated on magnet and the final libraries were purified from the supernatant using 50 uL AMPure XP beads, eluting in 0.1X TE buffer.