Kapa2g robust hs

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).

Leptin Receptor^db mice lack the functional, full-length Ob-Rb leptin receptor. Two microliters of HotSHOT DNA was combined with 23 mL of the PCR mixture. A total of 500 nM of each primer (forward: 5′-AGAACGGAC ACTCTTTGAAGTCTC-3′; reverse: 5′-CATTCAAACCATA GTTTAGGTTTGTGT-3′) was combined with PCR buffer, 2 mM MgCl₂, 0.2 mM dATP, 0.2 mM dCTP, 0.2 mM dGTP, and 0.2 mM dTTP (KAPA2G Robust HS; Kapa Biosystems, Cape Town, South Africa) to obtain a volume of 25 μL. Amplification was performed with a T100^TM Thermal Cycler system (Bio-Rad, Singapore). The PCR product obtained with the 25-μL mixture was digested by the direct addition of 25 μL of 1 × digestion cocktail containing 19 μL of water, 5 μL of 10 × CutSmart Buffer (New England Biolabs, Ipswich, MA, United States), and 1 μL of RsaI restriction enzyme (New England Biolabs) and overnight incubation of the mixture at 37°C. The digested products (50 μL) were analyzed in 4% agarose (Takara Biomedicals, Osaka, Japan) with 1 × TAE buffer containing 0.05% (v/v) GoldView^TM. Digestion with RsaI yielded 135-bp fragments in + / + mice and 135-, 108- and 27-bp fragments in heterozygotic db/ + mice.