16S DNA-Seq of Populus x jackii twig endobiome: OKA5
Approximately 12 sterilised twigs were cut into small pieces (1-2 mm). 150 mg of tissues was bead beaten for 90 seconds and Genomic DNA was extracted with a DNeasy Plant Mini Kit (Qiagen). The V5-V6 region of the 16S rDNA gene using the chloroplast discriminating primers 799F/1115R. A two-step PCR approach was used to amplification. 25 L mixture containing 3 L of DNA extract, 0.2 M of both forward and reverse primer, 0.5 U of Phusion Hot Start II High-Fidelity DNA Polymerase (ThermoFisher), 1x of Phusion HF Buffer, 0.2 M of dNTPs, and 3% DMSO. PCR cycles were performed for 30 s at 98C for initial denaturation, followed by 15 s at 98C, 36 cycles of 30s at 64C, 30 s at 72C, and 10 m and 72C for final extension. Samples and purified with AMPure XP beads at a ratio of 0.75:1 (bead to DNA ratio; Beckman Coulter). A PhiX control library (Illumina) was spiked into the amplicon pool and Illumina MiSeq sequencing was performed using a MiSeq reagent kit V3 (paired end 300bp; Illumina).