title: NextSeq 550 sequencing; Whole genome "TraDIS-Xpress" assay to determine gene roles during growth of E. coli in different concentrations of the antibiotics trimethoprim and sulfamethoxazole
design: Whole genome "TraDIS-Xpress" assay to determine gene roles during growth of E. coli in different concentrations of the antibiotics trimethoprim and sulfamethoxazole
library_construction_protocol: Large TraDIS-Xpress transposon mutant library described in Yasir et al. 2020 Genome Res. Feb;30(2):239-249. Made using a mini-Tn5 transposon conferring kanamycin resistance and incorporating an IPTG-inducible outward-transcribing tac promoter in kananmycinR-3' transposon end. Mutant library estimated to consist of at least 380,000 mutants. 10e7 cfu of mutant library was grown overnight at 37Â°C in 1 ml LB broth. LB broth was unsupplemented for controls (published elsewhere) and supplemented with the following: trimethoprim at 2 x MIC trimethoprim at 1 x MIC trimethoprim at 0.5 x MIC trimethoprim at 0.25 x MIC sulfamethoxazole at 2 x MIC sulfamethoxazole at 1 x MIC sulfamethoxazole at 0.5 x MIC sulfamethoxazole at 0.25 x MIC Each antibiotic growth condition was performed with 0.2 mM or 1 mM IPTG to induce expression of the outward-transcribing transposon tac promoter or without IPTG for no induction of transcription from this transposon promoter.  Each of these growth conditions was performed in duplicate to give a total of 48 samples.   Zymo research Quick-DNA (TM) Fungal/Bacterial 96 kit (2 x 96 preps) (Cat. no. D6006)  Illumina Nextera DNA Library Preparation Kit (96 samples) (Cat. No. FC-121-1031) with customised oligonucleotides to allow PCR from transposon ends and sequencing of the generated fragments using a standard Illumina NextSeq 500/550 High Output v2 kit.