GSM9078811: P. fluorescens 55 transposon mutant library attached by S. marcescens Db10 D9+ssp4 strain, rep 2; Pseudomonas simiae; Tn-Seq
All cells were recovered from each plate in 1 ml PBS and genomic DNA was extracted using the DNeasy Blood & Tissue Kit (Qiagen). Genomic DNA (~10 µg) was sheared into 250 bp fragments by sonication using the QSonica Q800R sonicator (Amplitude 25%: 20x cycles of 15s on/off) and end repaired using the NEBNext End Repair Module (New England Biolabs). A polyC tail was added to 1 µg of end repaired DNA using Terminal Deoxynucleotidyl Transferase (NEB) with a mixture of 95% dCTP and 5% ddCTP as a substrate. Residual C-tailing reagents were removed using NucleoSeq gel filtration columns (Macherey-Nagel) and DNA fragments containing transposon insertion sites were amplified by PCR using Q5 Hot Start High-Fidelity DNA Polymerase (New England Biolabs) and oligonucleotide primers directed against the transposon sequence and the polyC tail. Size selection was performed using AMPure XP beads (Beckman) to enrich for an average fragment size of approximately 250 bp. Sequencing and index primers (NEB) were added by an additional round of PCR.