Pore size syringe filter

For gene transduction, we employed a modified version of the retroviral infection method previously described [15 (link), 16 (link)]. We used the polycistronic retroviral vector designed to encode OCT3/4, KLF4, and SOX2 in the pMX retroviral vector (pMX-OKS) (Supplemental Fig. S1A). Concomitantly, pMX with GFP (pMX-GFP) was used as a control vector. To produce retroviral particles, Plat-A cells (in DMEM with 10% FBS without antibiotics) were transfected with the retroviral vector (pMX-OKS or pMX-GFP) using the FuGene HD transfection reagent (Promega, Madison, WI, USA) following the manufacturer’s instructions. The medium was replaced at 24 h after transduction, and the retrovirus-containing supernatant was harvested at 48 h after transduction. The supernatant was filtered through a 0.45-μm pore-size syringe filter (Sartorius Stedim Biotech, Goettingen, Germany). Infection of both cell lines with the retrovirus was conducted in the presence of 4 μg/mL polybrene (Nacalai Tesque, Kyoto, Japan) for 24 h. Non-transfected cells (MG-parental and NOS-parental), as well as cells transfected with GFP (MG-GFP and NOS-GFP), were used as control. All gene transduction procedures were performed in accordance with the National Institutes of Health Guidelines, and the study protocol was approved by the Kobe University Institutional Committee (Permission no. 30-18).

9 ml EDTA blood was collected in S-Monovettes^® (Sarstedt, Germany), stored at 4 °C, and centrifuged within 4 h after withdrawal at 1 841 × g for 8 min. Plasma was stored at − 80 °C. Thawed plasma was centrifuged at 16,000×g for 10 min at 4 °C and passed through a 0.8 µm pore size syringe filter (Sartorius, Germany). cfDNA was isolated from 1.8 to 5.4 ml (preferably 4 ml) plasma by affinity-based binding to magnetic beads according to the manufacturer’s instructions (QIAamp MinElute ccfDNA Kit, QIAGEN, Germany) and was eluted in 22 µl ultraclean water.