title: FAB1: Soil microbial community structure predicts fungal necromass decomposition rates across diverse temperate forest types
design: We used high throughput sequencing (HTS) of bacterial (16S) and fungal (ITS) taxonomic markers to characterize microbial community richness and structure. In total, 36 soil samples were analyzed (three per tree monospecific plots). Total genomic DNA was extracted with PowerSoil Pro kits (MoBio, Carlsbad, CA, USA) from 0.25 g of freeze-dried soil following the manufacturer's instructions. DNA extraction (lysis tubes with no substrate added) and PCR blanks (molecular-grade water as a replacement for template DNA) were also included. For bacteria, the 515F-806R primer pair was chosen to target the V4 region of the 16S rRNA gene (Caporaso et al., 2012). For fungi, the 5.8S-Fun and ITS4-Fun primer pair (Taylor et al., 2017) was used to target the ITS2 region of the fungal rRNA operon. Samples were first amplified in individual 20 l reactions containing 10 l of Phusion Hot Start II High-Fidelity PCR Master Mix (Thermo Scientific, Waltham, MA, USA), 0.5 l of each 20 mM primer, 1 l of DNA template and 8 l of PCR-grade water. Thermocycling conditions were as follows: 1) 98C for 30 seconds, 2) 98C for 30 seconds, 3) 55C for30 seconds, 4) 72C for 30 seconds, repeat steps 2-4 34 times, 5) 72C for 10 minutes and 6) infinite hold at 4C. If initial PCRs were not successful, template DNA dilutions were performed. For all samples with amplicons, a second PCR was run under thermocycling conditions to add unique Golay barcodes and sequencing adaptors. PCR products were then cleaned using the Charm Just-a-Plate Purification and Normalization Kit (Charm Biotech, San Diego, CA, USA). Each sample was then pooled at equimolar concentration and sequenced with the MiSeq technology (2 x 250 bp V2 Illumina chemistry) at the University of Minnesota Genomics Center.