Te saturated phenol

All human sera and plasmas used in the current study were purchased from commercial sources and are summarized in Supplementary Table S2. The procedure described below represents cfDNA preparation from 100 µL of human serum. However, the serum or plasma volume used differed depending on the experimental purpose (the starting serum/plasma volume is indicated in each data). In such cases, the mixing ratios of the reagents were maintained.
To a 1.5-mL microcentrifuge tube (Eppendorf), 100 µL of human serum, 400 µL of 10 mM Tris–HCl, pH 8.0, 5 µL of 500 mM ethylenediaminetetraacetic acid (EDTA), 1.5 µL of 5 M NaCl, 10 µL of 10% (w/v) SDS, and 10 µL of 20 mg/mL proteinase K (Qiagen) were added and incubated at 50 °C for 1 h. After the protease digestion, extractions were performed twice with TE Saturated Phenol (Nippon Gene) and once with phenol–chloroform-isoamyl alcohol (25:24:1) (Nacalai Tesque). Then, the aqueous phase was transferred to a new tube, supplemented with 50 µL of 3 M sodium acetate, pH 5.4, and 1100 µL of isopropanol. The mixture was centrifuged at 15,000 × g for 5 min, and the supernatant was discarded by decantation. Then, the DNA pellet was rinsed with 70% (v/v) ethanol and dissolved in 10 mM Tris–HCl, pH 8.0.