title: Illumina NovaSeq 6000 paired end sequencing; Whole genome sequencing analysis of evolved E.coli
design: Whole genome sequencing analysis of evolved E.coli
library_construction_protocol: 1 ml of evolved cells in the last cycle (cycle120) genomic DNA were extracted with genomic DNA prep kit (BIONEER). Three biological replicates from E.coli W3110 △ung strains harboring a mutator plasmid were grown overnight in LB medium with 35 μg/ml chloramphenicol antibiotic. On the next day, 3.5 μL of the overnight culture was passed into a new fresh LB medium 350 μL with 0.2% arabinose and 35 μg/ml chloramphenicol in 96-deep well plate (Bioneer) at 37oC with shaking. Bacterial cells were diluted every 4 hours and growth cycle was repeated up to 120 cycles. To create a continuous directed evolution system, E coli RNA polymerase is fused to the cytidine deaminase PmCDA1 linked with XTEN linker. E coli RNA polymerase allows targeting to E. coli genes. Cytidine deaminase introduces the C to T mutation on the target strand or the G to A mutation on the non-target strand. Genomic DNA was isolated using the BIONEER kit using the protocol for gram-negative bacteria.  The sequencing libraries were prepared according to the manufacturer's instructions of TruSeq DNA Nano Sample Preparation Kit(Illumina, Inc., San Diego, CA, USA). Briefly, Fragmentation of 100ng of genomic DNA was performed using adaptive focused acoustic technology(AFA; Covaris) and the fragmented DNA is end-repaired to create 5'-phosphorylated, blunt-ended dsDNA molecules. Following end-repair, DNA was size selected with bead-based method. These DNA fragments go through the addition of a single 'A' base, and ligation of the truseq indexing adapters.