GSM7914469: U2OS, 2, iMab, DIP; Homo sapiens; OTHER
Genomic DNA from cells was harvested using an AllPrep DNA/RNA/miRNA Universal Kit (QIAGEN, Cat No: 80224). Purified DNA was concentrated using Amicon Ultra-0.5 Centrifugal Filter Unit (Millipore, UFC501096) and concentration adjusted. DNA was fragmented using AFA Fiber Pre-Slit Snap-Cap microtubes (Covaris, 520045) and a Covaris instrument (M220 Focused-ultrasonicator) at Peak power: 50, Duty factor: 20, Cycles/Burst: 200, Time: 700 s, Setpoint: 20 °C producing 100–200 bp fragments. DNA fragments were stored at -80 °C until a pull-down experiment. A 96-well MaxiSorp plate (Thermo Scientific, 442404) was coated by adding 60 µL per well of 50 µg mL-1 streptavidin diluted in PBS and incubated overnight at 4 °C. Fragmented DNA was thawed and diluted in DPBS (ThermoFisher, 14190144), pH adjusted to 7.4. For each experiment 140 µg DNA were diluted in 525 µL DPBS and heated at 90 °C for 10 min followed by cooling down at the rate of 1 °C per min to 21 °C and kept on ice until the pull-down experiment. A streptavidin-coated plate was washed once with PBS and blocked with 200 µL per well of SuperBlock (ThermoFisher, 37515) for 2 h at room temperature Biotinylated iMab-MycAviTag was diluted to 49 µg in 525 µL of DPBS pH 7.4 (7 wells, 75 µL each) for each experiment. Blocked wells were washed once with PBS and Biotinylated iMab-MycAviTag solution was added. After incubating for 1 h at room temperature while shaking at 200 rpm, wells were washed twice with DPBS pH 7.4. Then, 75 µL of DNA solution was added to each well and incubated 16–20 h at 4 °C, and additionally, in the case of the MCF7 cell line, two independent control experiments were also carried out at an incubation temperature of 16 ℃. The supernatant was aspirated, each well washed and tap-dried seven times with 200 µL of DPBS pH 7.4 supplemented with 0.1% v/v Tween 20 and twice with DPBS only at room temperature of 24 ℃. Elution was performed by adding 100 µL of 100 mM Tris/acetate pH 10 supplemented with 1% w/v SDS to each well and incubating for 1 h at 40 °C while the plate was shaking at 200 rpm. Eluted DNA was collected, combined, and concentrated using ssDNA/RNA Clean & Concentrator kit (Zymo, Cat No: D7011). Libraries were constructed using a no-shearing IDT xGEN cfDNA & FFPE DNA library preparation kit following the manufacturer protocol