Q sepharose resin slurry

Urine cfDNA was prepared from 22 to 90 mL of urine with Q-sepharose resin slurry (GE Healthcare, Chicago, Illinois, USA) at a ratio of 10 μL slurry per mL of urine and mixed as previously described [28 (link),35 (link)]. After 30 minutes, the urine/resin mixture was centrifuged for 10 minutes at 1,800 g. The supernatant was discarded, and resin was washed twice with 0.3 M LiCl/10 mM sodium acetate (pH 5.5), applying 2 mL per 100-μL resin. The resin was then transferred to a Micro Bio-Spin column (Bio-Rad, Hercules, California, USA), and the bound material was eluted by adding 3 separate 670-μL aliquots of 2 M LiCl/10 mM sodium acetate (pH 5.5). Next, the eluates were combined in 70% ethanol and passed over a QIAquick column (Qiagen, Hilden, Germany). The column was washed with 5 mL of 2 M LiCl in 70% ethanol, followed by 5 mL of 75 mM potassium acetate (pH 5.5) in 80% ethanol. We removed residual liquid by centrifuging the columns at 20,000 g for 3 minutes. Finally, bound DNA was eluted into 50 μL of nuclease-free water or 10 mM Tris-Cl (pH 8.5). DNA concentrations were measured using a Qubit dsDNA HS Assay Kit and Qubit 4.0 Fluorometer (Thermo Fisher Scientific, Waltham, Massachusetts, USA).

Urine samples were collected in cups pre-filled with 1–2 mL of 0.5 M EDTA. Shortly following collection, cfDNA was extracted from 22 to 90 ml of urine with Q-sepharose resin slurry (GE Healthcare, Chicago, Illinois)^{3 (link)}. Briefly, Q-sepharose resin was added to urine at a ratio of 10 ul slurry per ml of urine and mixed for 30 min. After centrifuging the mixture at 1800 × g for 10 min, the supernatant was discarded. The resin was washed twice with 0.3 M LiCl/10 mM sodium acetate (pH 5.5), transferred to a Micro Bio-Spin column (Bio-Rad, Hercules, California, USA), and the bound DNA was eluted with 70% ethanol and passed over a QIAquick column (Qiagen, Hilden, Germany). Columns were then washed with 2 M LiCl in 70% ethanol, followed by 75 mM potassium acetate (pH 5.5) in 80% ethanol. Finally, DNA was eluted in nuclease-free water or 10 mM Tris-Cl (pH 8.5). Urine cfDNA was quantified using the Qubit dsDNA High Sensitivity Assay kit (Thermo Fisher Scientific, Waltham, Massachusetts). cfDNA quality was assessed on an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, California).