GSM9789799: Before competition biol rep 3; Pseudomonas aeruginosa PAO1; OTHER
Bacterial cells were harvested and the plasmids were extracted using Maxi Plasmid DNA Kit (Geneaid). 10 ng of the extracted plasmid DNA was used as the template for a first-round PCR amplification (18 cycles) of the hcp1 gene region using target-specific primers with adapter overhangs (listed in Supplementary Table X). Amplicons of the expected size (~561 bp) were gel-purified using a QIAquick Gel Extraction Kit. To generate sequencing libraries, 10 ng of the purified first-round PCR products was subjected to a second-round PCR (8 cycles) using KAPA HiFi HotStart ReadyMix to incorporate Illumina i5 and i7 index sequences (Illumina DNA/RNA UD Indexes Set D, Tagmentation). The indexed amplicons were subsequently purified using SPRI beads (Beckman Coulter), quantified, and validated for the expected library fragment size. The individual libraries were then normalized to 2 nM, pooled, and quantified using the KAPA Library Quantification Kit for Illumina Sequencing Platforms. The final pooled libraries were further validated using an Agilent 5200 Fragment Analyzer.