DNA-Seq of Coemansia sp. RSA 2523
Sporulating hyphae were scraped from the surface and placed in 2x CTAB (cetyltrimethylammonium bromide) buffer for DNA extraction. DNA extraction followed the CTAB protocol of Gardes and Bruns (1993), but with the following modifications: tissues were ground using a micropestle attached to a drill press and subjected to two rounds of freezing and thawing, the phenol:chloroform wash step was followed by an additional chloroform wash step, and samples were left at -20C overnight for the isopropanol precipitation step. After extraction, DNA was treated with RNase A and quantified by both Nanodrop 2000 spectrophotometer (ThermoFisher Scientific, Waltham, MA, USA) and Qubit 4.0 Fluorometer (Invitrogen, Waltham, MA, USA). Sequencing libraries were generated using NEBNext DNA Library Prep Kit (New England BioLabs, Ipswich, MA, USA) following manufacturer's recommendations using 1.0 g DNA per sample and indices were added to each sample. Genomic DNA was randomly fragmented to a size of 350 bp by shearing, then DNA fragments were end polished, A-tailed, and ligated with the NEBNext adapter for Illumina sequencing, and further PCR-enriched by P5 and indexed P7 oligos. The PCR products were purified (AMPure XP system) and resulting libraries were analyzed for size distribution by Agilent 2100 Bioanalyzer and quantified using real-time PCR. Sequencing was performed on the Illumina NovaSeq 6000 sequencing platform with NovaSeq XP v1 reagent kits, following 2x150 indexed chemistry.