Multiple distinct, scale-dependent links between fungi and decomposition
Sawdust collected from the wood blocks was lyophilized and DNA was extracted from 50 mg thereof after bead beating in microcentrifuge tubes using a Qiagen DNeasy PowerPlant kit (Qiagen, Hilden, Germany) following Maynard et al. (2018). The fungal internal transcribed spacer (ITS) region was amplified via polymerase chain reaction (PCR) consisting of 35 cycles of denaturation at 95 C, annealing at 50 C, and extension at 72 C using modified versions of the ITS1F-KYO1 and ITS2-KYO2 primers in 96-well plates (Toju et al. 2016). Molecular tags for high-throughput sequencing were attached in a second PCR reaction (Toju et al. 2016) consisting of 8 cycles using identical temperature settings. We used gel electrophoresis to verify PCR product, then cleaned it via a magnetic bead purification method using Sera-Mag SpeedBeads (MilliporeSigma, Munich, Germany; Talbot et al. 2014). Then, we measured the DNA concentration in our cleaned PCR product using a Qubit fluorometer (Thermo Fisher, Waltham, MA, USA) and pooled our samples into a single library at equimolar concentration. Finally, we submitted the library for 2 x 300 Illumina MiSeq sequencing at the Stanford Functional Genomics Facility.