title: Illumina MiSeq paired end sequencing; Suppressor mutant screening lacking growth-essential tRNA modification in bacteria
design: Suppressor mutant screening lacking growth-essential tRNA modification in bacteria
library_construction_protocol: E. coli trmD-knockout (KO) strains were made for MG1655, BW25113, and XAC background, maintained by expressing TrmD from pBAD promoter on a temperature-sensitive plasmid. The cells were depleted of the plasmid, pre-existing TrmD protein, and pre-existing m1G37-tRNA by growing in liquid LB medium without the inducer arabinose, at non-permissive temperature for multiple cycles. The resultant depleted cell cultures were streaked out and grown on an LB plate without arabinose at non-permissive temperature 43C, and suppressor colonies were isolated. Distinct colonies were streak-purified, and genomic DNA was prepared by phenol extraction from an overnight cell culture. Sequencing library was prepared using Nextera XT DNA Library Prep Kit (Illumina).