ITS DNA-Seq of Populus deltoides twig endobiome: MSH1
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). We targeted the ITS1 rDNA gene with the universal fungal primers, ITS1F/ITS2. A two-step PCR approach was used to amplification. 25 L mixture containing 4 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. ITS1 samples were purified three times with AMPure XP beads at a ratio of 0.8:1 (bead to DNA ratio; Beckman Coulter) to eliminate primer dimers. 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).