GSM6935220: Library prepared from human serum cfDNA extracted with TACS-TOPO scheme; Homo sapiens; OTHER
To a 1.5 mL microcentrifuge tube (Eppendorf), 50 µL of human serum, 100 µL of 10 mM Tris-HCl, pH 8.0, 2.5 µL of 500 mM EDTA, 0.75 µL of 5 M NaCl, 5 µL of 10% (w/v) SDS, and 5 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 25 µL of 3 M sodium acetate, pH 5.4, and 550 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 µL of 10 mM Tris-HCl, pH 8.0. [TACS ligation] A solution containing the target ssDNA was mixed with 2.5 µL of 10x TACS buffer [500 mM HEPES-KOH, pH 7.5, 50 mM MgCl2, 5% (v/v) Triton X-100] and 1 µL of rSAP (New England Biolabs, Ipswich, MA); the total volume was then adjusted to 11 µL with ddH2O. The reaction mixture was incubated at 37 °C for 15 min and 95 °C for 5 min. Next, 10 µL of 50% (w/v) PEG6000, 1 µL each of 10 mM ATP, 25 µM adaptor ([PHO]AGA[U]CGGAAGAGCGT*C*G*T*G[NH2]), 1 µL of 15 U/μL TdT (Takara Bio Inc, Shiga Japan), and 1 µL of 2 mg/ml TS2126 RNA ligase were added. TS2126 RNA ligase was prepared as described previously (Hisano O. et al., 2021). Note that TS2126 RNA ligase can be substituted with CircLigase II ssDNA ligase from Lucigen (Middleton, WI). The reaction was incubated at 37 °C for 15 min, 65 °C for 30 min, and 95 °C for 5 min.   [Replication of adaptor-tagged ssDNA] The adaptor-tagged ssDNA solution was mixed with 14 µL of ddH2O, 5 µL of 10x ExTaq buffer (Takara Bio), 4 µL of 2.5 mM dNTPs, 1 µL of 50 µM replicating primer ([PHO]ACACTCTTTCCCTACACGACGCTCTTCCGATCTTT), and 1 µL of 5 unit/µL hot start GeneTaq (Nippon Gene) followed by incubation at 95 °C for 3 min, 45 °C for 5 min, 55 °C for 5 min, and 65°C for 5 min.  [VOC-mediated second adaptor tagging] After complementary strand synthesis, 5 µL of 500 mM EDTA (Nacalai Tesque, Kyoto, Japan) and 1 µL of 150 mg/ml VOC (TA type) were added to the reaction mixture and incubated at 25 °C for 15 min.   [Purification of the library] After the second adaptor tagging, the reaction mixture was supplemented with 35 µL of Buffer B2 (3 M guanidine hydrochloride, 20% Tween 20) and 5 µL of Proteinase K (Qiagen) and incubated at 50 °C for 15 min. Then, 2 µL of Sera-Mag Carboxylate Magnetic Beads (Cytiva, Marlborough, MA) and 100 µL of isopropanol were mixed. After incubation at room temperature for 5 min, the magnetic particles were collected on a magnetic stand, and the supernatant was removed. The beads were washed twice with 200 µL of a solution containing 32% (v/v) PEG400, 1 M NaCl, and 50 mM Tris-HCl, pH 8.0. Finally, the beads were rinsed with 70% (v/v) ethanol, and the purified DNA was eluted with 25 µL of 10 mM Tris-Acetate, pH 8.0.   [UDG and APE 1 treatment, nick filling, and PCR amplification] To the purified library, 5 µL of 10×ExTaq Buffer, 4 µL of 2.5 mM dNTPs, 1 µL of 20 µM Amplification Univ (AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT), 1 µL of 20 µM Amplification Index-1 (CAAGCAGAAGACGGCATACGAGATCGTGATGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC), 1 µL of UDG (New England Biolabs), and 1 µL of APE 1 (New England Biolabs) were added. After adjusting the reaction volume to 50 µL with water, the reaction was incubated at 37 °C for 15 min, 72 °C for 5 min, and 95 °C for 1 min, followed by two cycles of 3-step incubations at 95 °C for 15 s, 55 °C for 30 s, and 72 °C for 1 min. The reaction mixture was purified with 90 µL of AxyPrep Mag PCR clean according to the manufacturer's instructions. The purified DNA was eluted in 21 µL of 10 mM Tris-Acetate, pH 8.0.