Analysis of cfDNA with denaturing polyacrylamide gel electrophoresis

The following were added to 4 μL of purified cfDNA: 4 μL of 2.5× TACS buffer (125 mM HEPES-KOH (pH 7.5), 12.5 mM MgCl₂, 1.25% (v/v) Triton-X100, and 50% (w/v) polyethylene glycol (PEG) 6000 [Nacalai Tesque, Kyoto, Japan]), 1 μL of 0.05 mM 7-propargylamino-7-deaza-ddATP-6-FAM (Jena Bioscience, Jena, Germany), 0.4 μL of internal standard solution (see Additional file 1: Supplementary Methods), and 0.5 μL of TdT (Takara Bio Inc.); the reaction volume was adjusted to 15 μL with water. After incubating the reaction mixture at 37 °C for 30 min, 7.5 μL of buffer B2 (3 M guanidine hydrochloride, 20% (v/v) Tween 20) and 1 μL of 20 mg/mL proteinase K were added, and the mixture was incubated at 55 °C for 15 min. Next, the DNA was recovered with solid-phase reversible immobilization (SPRI) [60] as follows. The proteinase K-treated reaction was combined with 1 μL of Sera-Mag carboxylate-modified magnetic particles (GE Healthcare, Chicago, IL, USA.) and 72 μL of binding buffer (300 mM NaCl, 3 mM Tris-HCl [pH 8.0], 0.3 mM EDTA, 0.015% (v/v) Tween 20, 70% (v/v) ethanol). After incubation at room temperature for 5 min, the beads were washed with 70% (v/v) ethanol. Purified DNA was eluted with 4 μL of 10 mM Tris-acetate (pH 8.0) and analyzed on a 10% Novex TBE-Urea gel (Thermo Fisher Scientific). Fluorescence images were obtained using Typhoon Trio+ and analyzed with ImageQuant software (GE Healthcare).