GSM9712830: DRT3_MUT_Glu_rep1; Escherichia coli; Tn-Seq
Mutant library plates were grown at 37 °C for 18 hours, after which all colonies were scraped from the plates. Genomic DNA was extracted from these cells using the Wizard Genomic DNA Purification kit (Promega). Extracted gDNA was digested with MmeI (2 U MmeI per 1 µg gDNA) at 37 °C for 1 hour, followed by enzyme inactivation at 65 °C for 20 min. Digested DNA was purified using QIAquick PCR Purification columns (Qiagen). Sequencing adaptors were ligated onto digested mini-transposons with T4 DNA ligase at 16 °C for 14 hours, followed by another QIAquick PCR purification cleanup. PCR amplification and Illumina barcoding were performed for 25 cycles with Q5 Hot Start HF DNA polymerase (NEB), using an annealing temperature of 65 °C and an extension time of 20 sec. The 173 bp product was gel-extracted using the QIAquick Gel Extraction kit (Qiagen) and sequenced on the Element AVITI system in single-end mode, 150 reads per end.