16S rRNA amplicon library of Dryas octopetala leaves endophytes: Tatra Mountains, Poland, location T2
The leaves were sterilized through a sequential rinsing process involving 70% ethanol for 30 seconds, followed by a solution of 0.5% sodium hypochlorite for 2 minutes, and subsequently rinsed five times with distilled water, each rinse lasting 30 seconds. The DNA isolation was performed using Wizard Genomic DNA Purification Kit (Promega, Madison, Wisconsin, United States), according to manufacturer instructions. Microbial identification of bacteria was conducted by analysing the full-length 16S rRNA gene using the 16S Barcoding Kit (SQK-16S114.24) (Oxford Nanopore Technologies). The reaction mixture consisted of 10 L of barcoded primers, 10 L of gDNA (10 ng), and 25 L of repliQa HiFi ToughMix 2X (Quantabio, Beverly, Massachusetts, USA). The total volume was adjusted to 50 L with ultrapure demineralized water. For library purification, magnetic beads (AMPure XP Beads AXP, Beckman Coulter, USA, CA) were applied, and standardisation was performed based on fluorometric measurements (Quantus, Promega, Germany). After pooling, barcoded samples for bacteria or fungi were subjected to sequencing using flongle flow cell R10.4.1.