Tip 100 plasmid midi kit

Three μg DNA was purified with a Qiagen-tip 100 Plasmid Midi Kit (#12145) and sequenced as previously described [30 (link)]. Base-calling was carried out using with Albacore v2.0.2. For long-read sequencing, plasmid DNA from strain C671-15 was isolated using a Qiagen-tip 100 Plasmid Midi Kit (#12145). Three μg of plasmid DNA was fragmented by ten passes through a size G23 needle. The library was constructed using the 1D Ligation Barcoding Kit (SQK-LSK108 and EXP-NBD103, Oxford Nanopore Technologies, Oxford, UK) with 'End-prep' incubations increased to 2x30 minutes and sequenced in an R9.4 flow cell (FLO-MIN106) with a MinION Mk1B (ONT). ONT’s Albacore v2.0.2 was used for base calling. ONT sequencing adaptors were removed with Porechop v0.2.0 [31 (link)]. Illumina reads were end-trimmed to q20 using Trimmomatic v0.36 [32 (link)]. The Hybrid assembly of Illumina and Nanopore reads, generated on NextSeq sequencing machines and ONT, were performed with Unicycler (v0.4.0) [33 (link)]. Using CLC Genomics Workbench (v10.1.1), inspection of BLAST-mapped Nanopore with reads longer than 10,000 bp confirmed the structure of the assembled plasmids. Likewise, CLC mappings of Illumina short-reads were inspected to correct eventual small-scale assembly errors (variants, homopolymers and INDELs).

The plasmid DNA used for transfection was purified with the Qiagen-tip 100 Plasmid Midi Kit (Qiagen, Germantown, MD, USA). Transfection was performed using Metafectene Pro (Biontex, San Diego, CA, USA) according to the manufacturer’s instructions. Namely, 5 μL of Metafectene Pro was mixed with 45 μL of 1× phosphate-buffered saline (PBS), and the necessary amount of plasmid DNA was adjusted with 1× PBS to a final volume of 50 μL in a separate tube. The two solutions were combined and incubated at room temperature for 15 min. After incubation, the mixture was added to the cells, and cells were incubated under normal growth conditions for 24 h. Then, the transfection mixture was replaced with fresh medium and cells were left to grow. After 24, 48, and 72 h of growth, cover clips with attached cells were taken from the Petri dishes and washed twice from the growth medium with 1× PBS before proceeding to fixing and immunostaining.