ITS amplicon of leaf microbiome: endophytes and health
PCR reaction was done for each sample under the following conditions: 30 s at 98 C; 35 cycles of denaturation at 98 C for 10 s, annealing at 54 C for 30 s, and extension at 72 C for 45 s; final step 10 min at 72 C33. The 25 L PCR mixture contained 12.5 L of Phusion High-Fidelity PCR Master Mix (New England Biolabs Inc., Beverly, MA), 2.5 L of each primer (1 M) and 50 ng of template DNA. PCR products were collected and purified using Qiagen Gel Extraction Kit (Qiagen, Germany). Sequencing libraries were generated using TruSeq DNA PCR-Free Sample Preparation Kit (Illumina, USA) following manufacturer's recommendations and then sequencing was conducted using the Illumina Novaseq 6000 Sequencer (Illumina, Inc., CA, USA).