GSM6765016: Sg, 3; Streptococcus gordonii; Tn-Seq
DNA isolation was performed with the Wizard genomic DNA isolation kit (Promega). A total of 5 μg of DNA suspended in 130 μl of TE were sheared by sonication (Covaris) to an average size of 300 bp and then purified and concentrated using a PCR cleanup kit (NEB Monarch) according to the manufacturer's instructions. Poly-C tails were added to sheared DNA using 1 μg DNA, 15 units TdT enzyme, dCTP (475 μM final concentration), and ddCTP (25 μM final concentration) in a total volume of 40 μl for 1 h at 37°C followed by 75°C for 20 min. DNA was purified using the Monarch PCR cleanup kit according to the manufacturer's instructions. DNA adjacent to each transposon insertion site was amplified by PCR using one primer targeting the poly-C tail (poly G primer, Table S3), and one targeting the end of the transposon (Tn end primer, Table S3). For PCR, 2.5 units of AccuPrime Pfx DNA polymerase (ThermoFisher), 1.5 μl of each primer (20 µM), and 250 ng template were used in a 50 µl reaction. Cycling conditions were 95°C for 2 min followed by 24 cycles of 95°C for 15 sec, 55°C for 30 sec, and 68°C for 2 min. A final 2 min extension at 68°C was also performed. PCR reactions were run on a 1.5% agarose gel and a range from 250-1000 bp was excised and purified using a gel extraction kit (NEB). Barcodes and primer sites for next-generation sequencing (Illumina) were added according to the manufacturer's protocol. Single-end sequencing was performed on an Illumina Nextseq 500/550 75-cycle High Output Kit v2 (Illumina) at the University of Louisville Center for Genetics and Molecular Biology.