TnSeq sample from Agrobacterium fabrum str. C58 - 2YT Ampicillin
Transposon insertional mutant libraries of A. tumefaciens and B. abortus representing approximately 200,000  1 million mutants were cultured in liquid or plate growth conditions and pooled. DNA was extracted from the pools using cholorform:phenol:isoamyl alcohol followed by ethanol precipitation according to the ThermoFisher Scientific protocol. Illumina libraries were prepared for each sample using an adapted protocol of the Illumina DNA Prep kit. DNA was quantified with a Thermo Scientific Nanodrop 2000c and diluted to pools that included 500ng in nuclease-free H2O. Libraries were tagmented with the bead-linked Tn5 transposase of the Illumina DNA Prep kit to fractionate the genomic DNA and ligate the Illumina adaptors. Tagmented libraries underwent three rounds of nested PCR to amplify the transposon-containing fragments, first using primers specific to the transposon and the unique sequence of adaptor 1, then the full-length adaptor 1 and adaptor 2 with the Tn-specific sequence, and finally with the Illumina indices i7 and i5 in unique dual index pairs per library sample. PCR reactions used either Illumina Enhanced PCR Master mix or KAPA HiFi HotStart ReadyMix and all DNA purification cleanups used a 1:1 ratio of Illumina Sample purification beads, washing twice with fresh 80% EtOH and eluting with Illumina resuspension buffer or 10 mM Tris HCl ph 8.6. The final libraries were quantified with a Qubit fluorometer (Invitrogen) using the Qubit HS dsDNA assay kit and integrity was checked with the Fragment Analyzer automated electrophoresis (Agilent Technologies). Each library sample was prepared and loaded onto the Illumina NovaSeq X Plus S4 Flow Cell  PE 150 to generate 150 bp paired-end reads. Raw reads were demultiplexed with bcl-convert (v3.8.2-12-g85770e0b). Reads were then trimmed with fastp version 0.23.1 using default settings. Transposon sequences were limited to R2 and further processed for analysis.