cfDNA was isolated from plasma of a healthy male individual obtained from the Genome Sequencing and Analysis Facility at the University of Texas at Austin. To prepare plasma, fresh blood was collected in 10-ml K+/EDTA venous blood collection tubes, mixed with an equal volume of phosphate-buffered saline without calcium or magnesium (PBS −/−; Thermo Fisher Scientific), gently layered over 15-ml Ficoll-Paque PLUS (GE Healthcare) in a 50-ml conical tube, and centrifuged at 400 × g for 35 min at room temperature. After centrifugation, plasma (top layer) was transferred into a clean tube and divided into 1-ml portions, which were stored at −80 °C.
To extract DNA, 400 μl of plasma was incubated with RNase I (Qiagen; 800 μg) as suggested by the manufacturer’s protocol and then processed by using a Qiagen DSP DNA Blood Mini Kit (Qiagen). The products were mixed and concentrated with an Oligo Clean and Concentrator kit (Zymo) and eluted in 10 μl of water per 1 ml plasma. The extracted DNA was analyzed by using a Bioanalyzer High Sensitivity DNA Analysis Kits (Agilent) and consistently contained 2–3 ng of predominantly 150–170 bp fragments per 1-ml plasma (see Supplementary Fig.S9 ).
To extract DNA, 400 μl of plasma was incubated with RNase I (Qiagen; 800 μg) as suggested by the manufacturer’s protocol and then processed by using a Qiagen DSP DNA Blood Mini Kit (Qiagen). The products were mixed and concentrated with an Oligo Clean and Concentrator kit (Zymo) and eluted in 10 μl of water per 1 ml plasma. The extracted DNA was analyzed by using a Bioanalyzer High Sensitivity DNA Analysis Kits (Agilent) and consistently contained 2–3 ng of predominantly 150–170 bp fragments per 1-ml plasma (see Supplementary Fig.