title: NextSeq of Escherichia coli B REL607: a single mutant with point mutation at position 170 of the gene lepA amino acid Q to E, used in the competition experiments against wild type and double mutant,isolate 11
design: Overnight grown cultures at 37 C were used to extract DNA of all the mutant strains using the described protocol of GenElute Bacterial Genomic DNA kit (Sigma, Catalog no. NA2110-1KT). The protocol of whole-genome sequencing used in this study is adapted from (Picelli et al., 2014). Tagmentation is carried out using the Illumina Nextera XT DNA sample preparation. 5 l of DNA sample with 5x TAPS-DMF-MgCl2 buffer was mixed with 10 l of Tn5-beads (in washing buffer: 30mM Tris pH=8, 50mM NaCl, 0.1% Triton X-100). After mixing, the plate was incubated at 55 C for 15 minutes. Tn5 transposase was stripped off by adding NT buffer to the tagmented DNA solution. The beads were washed using 0.6% SDS and incubated at 55 C for 5 minutes. The samples were ready for the final PCR enrichment, and the PCR reaction was performed using the kit protocol. The PCR profile was run for eight cycles, including the denaturation at 98 C, annealing at 65 C, and extension at 72 C. The PCR samples were analyzed on the BioAnalyzer (Agilent 2100) and processed for sequencing. The samples were proceeded for the Illumina sequencer and converted the raw data into fastq files using bcl2fastq.