title: 16S rRNA amplicon sequencing of pre-alpine grassland: Fendt soil at 0-5cm
design: 16S library preparation using the primers S-D-Bact-0008-a-S-16 and S-D-Bact-0343-a-A-15 (Klindworth et al., 2013). Amplification: 98C for 5 min; 98C for 10 s / 60C for 30 s / 72C for 30 s (25 cycles); 72C for 5 min. PCR reaction mixture (total 25 ul); 12.5 ul of NEB Next High Fidelity Master Mix, 0.5 ul of each primer(10 pmol), 1.5 ul of 3% BSA, 1 g of template DNA, an adequate amont of DPEC-treated water. Sample purification: Agencourt AMPure XP extraction kit (Beckman Coulter Company), the ratio of AMPure XP beads to PCR products is adapted to 0.8. Indexing PCR: Nextera XT Index Kit v2 set B, Amplification: 98C for 5 min; 98C for 10 s / 55C for 30 s / 72C for 30 s (8 cycles); 72C for 5 min. Sample purification: mentioned above. Products are pooled equimolar to a final concentration of 4 nM and sequenced using the MiSeq Reagent kit v3 (600 cycles) for paired end sequencing. Raw sequences were separated from adapters using AdapterRemoval v2.1.7, and reads were trimmed with a minimum read length of 50 bp and a Phred score of 15 (Schubert et al., 2016).