Npfu forte polymerase

Sodium citrate dihydrate (Dae-Jung, Korea) and citric acid (Duk-San Pure Chemical Co., Korea) were used to prepare a 1 M citrate solution. citric acid, itaconic acid (Sigma-Aldrich, USA) and cis-aconitic acid (Alfa Aesar, USA) were used for in the preparation of a standard curve. PCR was performed with nPfu-Forte polymerase (Enzynomics, Daejeon, Korea). Amplified DNA and plasmid were purified with MG Plasmid SV Miniprep Kit (Doctor Protein, Seoul, Korea). All endonucleases (NdeI, EcoRV, BamHI, SacI, XhoI, and NotI) were purchased from New England Biolabs (USA).

A first-strand cDNA was synthesized from SARS-CoV-2 RNA (NCCP43326) provided by the NCCP (National Culture Collection of Pathogen) using a SuperScript III first-strand synthesis system for RT-PCR (Invitrogen, Waltham, MA) according to the manufacturer’s instructions. Eight target regions were amplified with specific primers (see Electronic Supplementary Material Table S1) and nPfu Forte polymerase (Enzynomics, Daejeon, Korea) or Herculase II Fusion Enzyme with dNTPs Combo (Agilent Technologies, Santa Clara, CA). Through multiple overlap extension PCR [23 (link)], insert A was obtained from six PCR products and insert B was obtained from two PCR products. Primers were designed to include sequences with overlapping fragments for PCR stitching as well as extra base pairs including restriction enzyme sites or stop codons. SnaB1, BamH1, and Sal1 restriction enzymes (NEB, Ipswich, MA) were used to insert the final PCR products into the lentiviral vectors (Lugen, Seoul, Korea). Completed pCDH-A and pCDH-B vectors were verified through Sanger sequencing (Cosmogenetech, Seoul, Korea). Each lentivirus sample (1 × 10⁸ IFU/mL) was obtained through polyethylene glycol (PEG) precipitation using PEG-it Virus precipitation solution (SBI, Palo Alto, CA).