Illumina HiSeq 2500 paired end sequencing; Illumina sequencing analysis of mutagenicity of EvoluGene
Illumina sequencing analysis of mutagenicity of EvoluGene
The ∆ung cells (cHYO057) harbouring a mutator plasmid (pHyo094) and a target plasmid (pHyo182) were grown overnight in LB broth supplemented with 100 μg/mL ampicillin and 35 μg/mL chloramphenicol (cycle #0). The overnight cultures (3.5 μl) were mixed with 350 μl of LB broth supplemented with 100 μg/mL ampicillin, 35 μg/mL chloramphenicol, 0.2% arabinose, and 0.1mM IPTG in a 96-deep well plate (Bioneer, South Korea) and incubated at 37oC for 4 hours (cycle #1). The dilution-incubation cycle was repeated up to 27 times. Cells taken at cycle 27 were grown in 15 mL of LB broth without arabinose and IPTG, and the target plasmids were extracted with Plasmid Miniprep Kit. The 3289-bp DNA fragments containing the pheS_A924G gene were amplified using primers 512 and 513 covering a region ranging from 1016 bp upstream from T7 promoter to 1069 bp downstream including T7 terminator. Samples were then prepared with TruSeq Nano DNA Kit (TruSeq Nano DNA Sample Preparation Guide, Part # 15041110 Rev. D)