ISS1 transposon TnSeq library for S. anginosus 980/01 cultured on BHI - replicate B
The ISS1 transposon mutant library was constructed according to protocols.io [https://www.protocols.io/view/transposon-insertion-sequencing-tn-seq-library-pre-rm7vzn6d5vx1/v1] with minor modifications. DNA was mechanically sheared using Covaris nto 300-500 bp fragment sizes, followed by end repair and TA-adaptor ligation using NEB Ultra II End Repair and Ligation Modules. Ligation reaction was purified by Ampure XP magnetic beads and library restriction digest was perfomed using SmaI for 2 h at 25C to cleave the pGH9:ISS1 plasmid 33 bp upstream of the sequence encoding ISS1 to minimise the amount of TnSeq reads mapping to plasmid. The digested library was purified using Ampure XP beads, and the amount of DNA recovered was quantified using the Qubit dsDNA HS assay kit (Thermo Fisher Scientific) according to the manufacturers instructions. One hundred nanograms of library DNA was PCR amplified for 20 cycles according to the NEBNext Ultra II DNA library prep kit protocol. Library amplification utilized the specific ISS1 primer containing Nextera XT 5 overhang (P5) with barcode index sequence and a TAadaptor compatible unique indexing Nextera XT PCR (P7) primer per library.