10 beta competent e coli

The coding sequences for the ADAR1 p110 isoform and ADAR1 p150 isoform were amplified from the constructs previously generated in our lab (Bahn et al., 2015 (link)) and cloned into the pcDNA4-TO-FLAG-myc-His vector (Invitrogen) using restriction enzymes NotI-HF (NEB) and BstBI (NEB). ADAR2 mutant constructs (EAA, E396A, and E488Q) were generated by introducing the recoding mutations to the pcDNA4-ADAR2-WT construct previously generated in our lab (Tran et al., 2019 (link)). In general, the ADAR2 coding sequences were reamplified to introduce mutations using overlap extension PCR, followed by digestion and ligation into the pcDNA4-TO-FLAG-myc-His vector via the restriction enzymes NotI-HF (NEB) and XbaI (NEB). All PCR reactions were performed using the Q5® Hot Start High-Fidelity 2X Master Mix (NEB). The primers used for PCR reactions are listed in Table S1. NEB 10-beta Competent E.coli were used for plasmid construction.