High sensitivity dna assay

For whole genome sequencing of AP3_16^T strain, DNA was isolated following a previously described protocol (Furmanczyk et al., 2017a (link)). The isolated genomic DNA was used to prepare two types of libraries: (1) paired-end library with average insert size 500 bp (using KAPA HTP Library Preparation Kit for Illumina platforms according to manufacturer’s protocol) (Kapa Biosystems, United States), (2) Nextera^® Mate Pair library with average insert size 8 kbp (using Illumina protocol) (Illumina, United States). The libraries were verified using a 2100 Bioanalyzer (Agilent, United States) High-Sensitivity DNA Assay and KAPA Library Quantification Kit for the Illumina (Kapa Biosystems, United States). Sequencing was performed using an Illumina MiSeq (MiSeq Reagent Kit v3, 600 cycles) (Illumina, United States) with read length of 300 bp.

After conidial germination in YEPD medium at 25°C for 24 h, the mycelia of different strains were collected by centrifugation for RNA extraction. Total RNA was extracted using TRIzol Reagent (Invitrogen). A NanoDrop spectrophotometer (Thermo Scientific) was employed to determine the RNA concentration, quality and integrity. Poly-T oligo-attached magnetic beads were purified to enrich mRNA. First-strand cDNA was obtained using random oligonucleotides and SuperScript II. Second-strand cDNA synthesis was subsequently performed using DNA polymerase I and RNase H. The resulting cDNA fragments were further adenylated at the 3’ ends and ligated with Illumina PE adaptor oligonucleotides. PCR was performed to enrich the DNA fragments with ligated adaptors on both ends. After purification using an AMPure XP system and quantification using an Agilent high-sensitivity DNA assay, the obtained sequencing library was sequenced on a NovaSeq 6000 platform (Illumina).