Q5 high fidelity 2x master mix

The fungal ITS2 region was targeted for amplification and sequencing with primers that append barcodes and Illumina adapters, similar to those described by (Taylor et al., 2016 (link)). See Key Resource Table for full primer sequences. Notably, this results in the Illumina read-one from the reverse (LSU) side of the ITS reference sequence and the read-two from the 5.8S side. The ITS2 region was amplified and Illumina adapters appended by PCR in 25 μ 1 volume with Q5 High-Fidelity 2X master mix (NEB, # M0492L). PCR conditions were as follows: initial denaturation at 95°C for 2 minutes, followed by 32 cycles of 98°C for 15 seconds, 52°C for 20 seconds, 72°C for 45 seconds, and a final 2 minute extension at 72°C. We performed triplicate PCR reactions for each sample, then combined triplicates and cleaned the PCR products with Axygen AxyPrep Mag PCR cleanup beads (Corning, #MAG-PCR-CL-50) using 1.8X volumes of beads diluted to 62.5% in water (v/v) to remove large primer-dimers. Cleaned products were quantified then multiplexed at even abundances and sequenced on an Illumina MiSeq in paired-end mode with 300 cycles each and a 5% PhiX library spike-in to provide increased base diversity. All sequences have been deposited at NCBI under the BioProject PRJNA559026.

Total RNA was reverse transcribed using ProtoScript II Reverse
Transcriptase (New England Biolabs, MA, USA). Gene-specific reverse
transcription primers TV_004 and TV_007 were used for ORF10 and ORF61
transcripts, respectively. Traditional PCR was used to confirm the
existence of ORF10 and ORF61 transcripts using the following primers:
ORF10_c3244 forward TV_017, reverse TV_004; ORF10_c2410 forward TV_018,
reverse TV_004; ORF61_c8714 forward TV_025, reverse TV_010; ORF61_c20295
forward TV_026, reverse TV_010. Each PCR reaction mixture contained 12.5
μL Q5® High-Fidelity 2X Master Mix (NEB), 10 μM
each primer, 1 μL cDNA, and nuclease-free water to a final volume
of 25 μl. PCR was performed with pre-denaturation at 98°C
for 30 s, amplification with 35 cycles of denaturation at 98°C
for 10 s, annealing at 56°C for 30 s, and extension at
72°C for 15 s, followed by a final extension at 72°C for 2
min. PCR products were loaded onto 1.5% agarose gels and the expected
bands were excised and purified according to the NucleoSpin Gel and PCR
Clean-Up kit instructions (Clontech/Takara, Kyoto, Japan). The purified
PCR products were cloned into pCR-Blunt II-TOPO Vector
(Invitrogen/Thermofisher). Subsequently, DNA minipreps were prepared
from ten colonies per culture and Sanger sequenced by Genewiz (NJ,
USA).