16S rRNA-seq of soil microbiota: rhizosphere soil
DNA was extracted from 25 mg fresh (frozen soil) with the DNeasy PowerSoil Pro Kit (Qiagen, Germany). Prokaryote community composition was determined by sequencing the V4-V5 region of the 16S rRNA gene with primer pair 515F and 926R (Walters et al., 2016). Sequencing library preparation involved two PCR steps. The first PCR reactions contained 2.5 L template DNA (5 ng L-1), 12.5 L KAPA HiFi HotStart ReadyMix (Roche, Ireland), and 0.5 L 1M forward and reverse adapted primers (Eurofins, UK). Samples were sequenced on a Illumina MiSeq Standard Flow Cell.