TELP Library Preparation with AI-labeled ssDNA

The AI-labeled ssDNA was used directly for library preparation with the TELP protocol except PCR amplification^{41 (link)}. Frist, mixing 28 μL of AI-labeled ssDNA, 1 μL of 10× EX buffer (Takara), 1 μL of 1 mM dCTP (NEB) and 1 μL of terminal deoxynucleotidyl transferase (TDT; NEB) for 37 °C for 35 min, 75 °C for 20 min. Second, the following extension mix to the above-mentioned TDT reaction: 6.2 μL of H₂O; 0.8 μL of KAPA2G Robust HS (KAPA); 12 μL of 5× KAPA buffer A (KAPA); 4.8 μL of 2.5 mM dNTP (Takara) and 6 μL extension primer (Ex primer, Supplementary Table 3). The extension program was as follows: (i) 95 °C for 3 min; (ii) 47 °C for 1 min, 68 °C for 2 min, 16 cycles and (iii) 72 °C for 10 min. Moreover, using exonuclease I (Exo I) (NEB) digest excess primers at 37 °C for 1 h. Then, the extended DNA was purified by MinElute PCR Purification Kit. Thirdly, mixing 8.4 μL DNA, 0.6 μL adapter containing unique molecular identifier (UMI), 10 μL 2x quick ligation buffer (NEB) and 1 μL Quick T4 ligase (NEB) for 20 °C 1 h. Then the reaction was purified with MinElute PCR Purification Kit. Then, the ligated DNA was added to DBCO-S-S-PEG3-Biotin (Click Chemistry Tools, Cat. No. A112-10) with the final concentration of 400 mM, and incubated in the thermomixer for 1 h at 37 °C (800 rpm), and purified by DNA Clean & Concentrator 5 (Vistech).