S63-16S-Leaf-Endo
A two-step PCR approach was used with barcode tagged-templates and primers targeting the V4 region of the 16S rRNA gene for Archaea & Bacteria and the ITS2 region for Fungi using pooled primer sets to increase coverage of Archaeal, Bacterial, and Fungal taxa (Cregger et al., 2018  see Table S3). The first step of PCR included 2.5 M of peptide nucleotide acid (PNA) blockers for 16S rRNA amplifications (GGCAAGTCTTCTTCGGA and GGCTCAACCCTGGACAG) and 2.5 M of PNA targeting plant nuclear rRNA genes for ITS2 region (CGAGGGCACGTCTGCCTGG) were used to reduce amplification of plant material. Each reaction contained 2l of template DNA, 0.25 M of primer pair, 1X of KAPA HiFi HotStart ReadyMix, and molecular grade water for a total reaction volume of 25 l. PCR amplifications were performed with the conditions 95 C for 3 min, 25 cycles (30 cycles for endosphere) of 95 C for 30s, 78 C for 30S, 55 C for 30s and 72 C for 30s and a final extension of 72 C for 5 min.  The second step of PCRs were amplified following the Illumina 16S Metagenomic Sequencing Library Preparation note with the conditions 95 C for 3 min, 8 cycles of 95 C for 30s, 55 C for 30s and 72 C for 30s and a final extension of 72 C for 5 min.