title: archaeal and bacterial 16rRNA gene : soil metagenome Irish grassland
design: Sequencing libraries were prepared for the V4 region of archaeal and bacterial 16S rRNA gene using the 515F and 926R primers (Parada et al., 2016). The first PCR was performed with the above-mentioned specific primers flanking Illumina overhang adapters sequences in a 25L PCR reaction containing 2x KAPA HiFi HotStart ReadyMix (Kapa Biosystems Inc, USA), 1M of each primer and 2.5L of DNA. For the 16S rRNA gene, the following conditions were applied: initial denaturation at 94C for 3 min, followed by 25 cycles of 94C for 45 s, 50C for 45 s and 72C for 1 min, and a final extension step at 72C for 5 min. The second PCR was conducted with the Nextera XT index kit (Illumina, San Diego, CA) following manufacturer's instructions. Amplicons were verified on 1% agarose gel and purified using AMPure XP magnetic beads (Beckman Coulter, USA). Concentrated samples and negative controls were pooled in equimolar ratios, diluted to 4 nM and sequenced with a 2 x 250 run on an Illumina MiSeq platform at Teagasc Next Generation DNA sequencing facility (Ireland).