Amplicon-seq of WT DH10B E. coli expressing hokB from a plasmid
RNA was purified by GeneJET RNA Purification kit (Thermo Fisher Scientific) after 2h:15min and 3h:30min of growth. Libraries to examine RNA editing levels of hokB versions expressed from plasmids were constructed as follows: first, we used primer 23 (ACGACGCTCTTCCGATCTTCAGGTTCCGGCTCTAAG) and primer 24 (AGACGTGTGCTCTTCCGATCGAGTTTTTGTTCGGGCCCAAGC) to target the hokB transcript and add sequence tails that match Illumina's adapters. Notably, primer 23 is localized to the linker sequence between mcherry and hokB (only 3 bases overlap the sequence of hokB), while primer 24 is localized to the 3-prime untranslated region of the transcript with a sequence specific to the plasmid backbone. Thus, the primers can only amplify the cDNA of the hokB transcript that is transcribed from the plasmid. PCR conditions were as follows: initial denaturation for 3 minutes at 95C, followed by 10 cycles of:  1) denaturation for 20 seconds at 98C, 2) annealing for 15 seconds at 55C, 3) elongation for 10 seconds at 72C; at the end of the cycles a final elongation step for 60 seconds at 72C was added. Then, a second PCR  was carried out with primers 25 (AATGATACGGCGACCACCGAGATCTACACNNNNNNNNACACTCTTTCCCTACACGACGCTCTTCCGATCT) and 26 (CAAGCAGAAGACGGCATACGAGATNNNNNNNNGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT) using the first PCR product as a template. This PCR was used to add (oligos/adapters for the Illumina run with dual index). PCR conditions were as follows: Initial denaturation for 3 minutes at 95C, followed by 30 cycles of: 1) denaturation for 20 seconds at 98C, 2) annealing for 15 seconds at 60C, 3) elongation for 10 seconds at 72C; at the end of the cycles a final elongation step for 60 seconds at 72C was added. We used KAPA HiFi HotStart ReadyMix (Roche #07958935001) for the PCR reactions. Libraries were sequenced using 150-nucleotides singlepaired-end reads on the NovaSeq 6000 X platform (Illumina).