GSM7956927: COLO320, DM, TPL, input, rep1; Homo sapiens; OTHER
Genomic DNA was then extracted using the Monarch gDNA Purification Kit (NEB T3010S) following the standard protocol but with elution using 50 µL 25 mM K3BO3 at pH 7.0. Click reaction was carried out by mixing 87.5 µL purified DNA, 2.5 µL 20 mM DBCO-PEG4-biotin (DMSO solution, Sigma 760749), and 10 µL 10× PBS in a final volume of 100 µL. The reaction was then incubated at 37◦C for 90 minutes. DNA was purified using AMPure XP beads by adding 50 µL beads 100 µL reaction, washing beads on a magnetic stand twice with 80% EtOH, and eluting in 130 µL 25mM K3BO3. Purified DNA was then sheared using a Covaris E220 instrument down to ∼200-400 bp size. Pulldown of biotin-labeled DNA was initiated by separating 10 µL of 10 mg/mL Dynabeads MyOne Streptavidin T1 beads (Life Technologies, 65602) on a magnetic stand, then washing with 180 µL of 1× TWB (Tween Washing Buffer; 5 mM Tris-HCl pH 7.5; 0.5 mM EDTA; 1 M NaCl; 0.05% Tween 20). Beads were then resuspended in 300 µL of 2× Binding Buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA; 2 M NaCl), sonicated DNA was added (diluted to a final volume of 300 µL if necessary), and beads were incubated for ≥15 minutes at room temperature on a rotator. Beads were separated on a magnetic stand and washed with 300 µL of 1× TWB and heated at 55◦C in a Thermomixer with shaking at 1,000 rpm for 2 minutes. The supernatant was removed on a magnetic stand, and the TWB wash and 55◦C incubation were repeated.   Libraries were prepared on beads using the NEBNext Ultra II DNA Library Prep Kit (NEB, #E7645).