Genome sequence of N2-fixer Stutzerimonas stutzeri strain MBI-RS3
Genomic DNA was extracted and prepared for sequencing using the Rapid Barcoding Kit 96 (SQK-RBK110.96, Oxford Nanopore Technologies), following the manufacturers protocol (Version: RBK_9126_v110_revO_24Mar2021). High molecular weight genomic DNA (50 ng per sample) was transferred to 1.5 mL Eppendorf DNA LoBind tubes and adjusted to 9 L with nuclease-free water. Each sample was barcoded using 1 L of Rapid Barcode (RB01-96), mixed thoroughly, and incubated at 30C for 2 minutes, followed by 80C for 2 minutes. The barcoded DNA samples were pooled and purified using AMPure XP beads (Beckman Coulter, A63881), washed twice with 80% ethanol, and eluted in 15 L of Elution Buffer (EB). DNA libraries were quantified using a Qubit fluorometer (Thermo Fisher Scientific), and 11 L of pooled DNA was ligated to 1 L of Rapid Adapter F (RAP-F) for 5 minutes at room temperature. The prepared library was kept on ice until loading onto an Oxford Nanopore MinION flow cell (FLO-MIN106D, R9.4.1) for sequencing.