Soil microbiome of Quercus lobata seedlings
DNA was used in multilocus metabarcoding for two loci: 16S targeting bacteria and archaea and Fungal ITS targeting fungi. Primer sequences were as follows. 16S 515f: GTGYCAGCMGCCGCGGTAA, 806r: GGACTACNVGGGTWTCTAAT (78). Fungal ITS 5F: GGAAGTAAAAGTCGTAACAAGG, 5.8SR: CAAGAGATCCGTTGTTGAAAGTT (79). This first PCR was performed using a 15uL reaction mixture containing 7.5 uL QIAGEN Multiplex Plus Taq PCR 2x Master Mix, 3nM of each primer, and 2uL of DNA template. The first PCR was done in triplicate with the above primers containing Illumina (San Diego, CA) Nextera adaptor sequences in the 5 primer sequence, then products were confirmed by gel electrophoresis, cleaned using Sera-Mag Serapure beads (Sigma-Aldrich, Darmstadt, Germany), quantified using the Qubit DNA BR Assay (Thermo Fisher, Walthamm, MA) and pooled at equimolar levels by sample. Illumina Nextera indices were added through an additional indexing PCR done with Kapa HiFi HotStart Ready Mix (Roche, Indianapolis, IN, USA) according to manufacturers guidelines. Indexed samples were confirmed by gel electrophoresis, cleaned, quantified, and pooled as described in the first PCR, except samples were pooled together rather than primers.