title: Tn-Seq of S. meliloti: The deltapSymAB transposant library passed through rich media (replicate 2)
design: Transposon mutagenesis was accomplished in the wild-type and deltapSymAB strains in parallel. Equal volumes of transposon delivery donor and recipient were mixed as bi-parental matings and plated on rich medium and incubated at 30°C for 6 hr. Mating mixtures were collected in rich medium with 10% glycerol and aliquots were stored at -80°C. To accomplish equivalent coverage of each genome with transposon insertions, ~675,000 and 360,000 colonies were pooled for the wild-type and pSymAB strains, respectively. In duplicate, 1x109 cells from each of the two clone libraries were transferred into 500 ml of either rich or defined medium at 30°C. Cells were then pelleted and DNA was extracted and fragmented to an average molecular weight of 1000 bp. The resulting DNA fragments were appended with short 3 oligo-C homopolymer tails, and these samples were used as the template for a two-round PCR that gave rise to the final Illumina-ready libraries. In the first round, a transposon end-specific primer and an oligo-G primer were used. After clean-up, a portion of the first-round product was used as the template for the second-round reaction employing a nested transposon-specific primer and a reverse index-incorporating primer. A series of three primers were designed to incorporate base diversity in the opening cycles of Illumina sequencing, and a series of eight 2BAR primers were designed to uniquely identify each experimental condition in a single multiplexed sequencing sample. After PCR amplification of transposon-flanking sequences with concomitant incorporation of Illumina adapters and barcodes, the samples were size-selected for 200-600-bp fragments, and sequenced as 50-bp single-end reads. Raw reads were used as input into a custom-built Tn-seq analytical pipeline.