Kod hot start

All cloning was performed using the Infusion system (Takara, 638909) and Stellar competent cells (Takara, 636763) as described by the manufacturer. PCR inserts were generated using GXL polymerase (Takara, R051A) or KOD HotStart (Millipore, 71086) and extracted using a gel extraction kit (Zymogen, D4002). Minipreps were performed using the Monarch system (NEB, T1010L) with vector backbones generated by EcoRI restriction enzyme digestion (ThermoFisher, FD0274, as per manufacturer’s instructions) or PCR with GXL polymerase/HiFi polymerase (Takara, R051A/639298). For the Pbp1-PS/Pub1-PS insertions into Ede1, a BshTI site was generated within the Ede1 construct. The PS domains were inserted into BshTI (ThermoFisher, FD1464) digested vector, removing the remnants of the restriction site. All obtained vectors were sequenced to confirm accuracy.

Point mutations (D137N, Q548A, D311A, T592A, T592E) in SAMHD1-coding plasmid pcDNA3.1-N-FLAG-SAMHD113 (link) were introduced by site-directed mutagenesis using complementary primer containing respective nucleotide exchanges. Mutagenesis PCR was performed with PfuUltra (Agilent Technologies) or KOD Hot Start (Merck Milipore) DNA Polymerase. Non-mutated template DNA was subsequently removed by DpnI (New England Biolabs) treatment of the PCR product. Mutations were verified by DNA sequencing.
The pJO19 HBV coding plasmid was linearized and used as a standard in quantification of HBV DNA copies in quantitative PCR (qPCR).

Genomic DNA was isolated from potential GLUL-KO cell lines using DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany). PCR using KOD Hot-start (Merck-Novagen, Darmstadt, Germany) was then performed to amplify GLUL with primers F: 5′-CTCCAGAACACCTTCCACCA-3′ and R: 5′-ACATTGCTGTCTCACCTTCC-3′. Cycling conditions were programmed as follows: 95 °C for 2 min, followed by 35 cycles of 95 °C for 20 s, 55 °C for 10 s, and 70 °C for 15 s. Further extension was carried out with Taq polymerase (Thermo Fisher Scientific) to add 3′ adenine overhangs for cloning into a T-vector containing complementary thymidine residues. PCR products were run on a gel where single bands obtained were extracted using Gel Extraction Kit (Machery-Nagel, Düren, Germany). Cleaned PCR products were then transformed in One Shot® TOP10 Chemically Competent E. coli (Thermo Fisher Scientific) using a TOPO TA cloning kit (Thermo Fisher Scientific, Invitrogen) before plating on LB agar plates (2.5% LB broth miller, Merck-Novagen and 1.5% BD Bacto™ Agar, Becton, Dickinson and Company, NJ, USA). Colonies were grown in 3 mL LB broth culture (2.5% LB broth miller, Merck-Novagen) before plasmid extraction (QIAprep Spin Miniprep Kit, Qiagen). Purified plasmids were subsequently sequenced via the BigDye® Terminator v3.1 cycle sequencing kit carried out by 1^st Base DNA Sequencing service (Singapore).