16S rRNA gene amplicon seq, grass soil inoculated with sterile H2O
Amplification of 16S rRNA gene fragment variable V3-V4 region using primers 335F (CADACTCCTACGGGAGGC)/769R (GYGBGGGKAKCAAACAGGAT) (Dorn-In et al. 2015).  PCR using NEBNext high fidelity polymerase, total volume of 25 l (15 ng DNA template, 12.5 l polymerase, 5 pmol of each primer). PCR cond. 5 min 98 C; 30x 10 s 98 C, 30 s 60 C, 30 s 72 C; 5 min 72 C. Purification using AMPure XP beads, quantification via PicoGreen assay. Indexing PCR: Nextera XT Index Kit v2, total volume of 25 l (10 ng DNA template, 12.5 l NEBNext high fidelity polymerase, 2.5 l of each indexing primer) PCR cond.: 30s 98 C; 8x 10 s 98 C, 30s 55 C, 30s 72 C; 5min 72 C. Purification using AMPure XP beads, qualification+quantification via Fragment Analyzer instrument, pooling in equimolar ratio of 4nM.