18S-28S 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. The analysis of fungal community composition was performed using the primer pair SR1R-Fw and LR12-R with Nanopore Universal Tag sequences were appended to both original primers. Amplification was conducted in two stages. In the first stage, PCR was performed with 25 L of repliQa HiFi ToughMix 2X (Quantabio, Beverly, Massachusetts, USA), 10 L of each tagged primer (0.5 M), and 10 L of genomic DNA (10 ng), adjusted to a final volume of 50 L with ultrapure demineralized water. In the second stage, 48 L of the first-round PCR product (~100 fmol) was combined with 2 L of barcoding primer (PCR Barcoding Expansion 1-12, EXP-PBC001, Oxford Nanopore Technologies) and 50 L of repliQa HiFi ToughMix 2X. 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.