title: 16S rRNA amplicon sequencing of the soil samples: Sterile soil on week 6 replicate 1
design: Genomic DNA was extracted from 0.30 g of soil using a NucleoSpin®Soil Kit (Macherey-Nagel, Düren, Germany). For cell lysis, buffer SL2 was used. Sequencing was performed on the Illumina MiSeq platform using the primers 27F and 357R. PCR conditions: 98 C for 5 min; 98 C for 10 s / 60 C for 30 s / 72 C for 30 s (30 cycles); 72 C for 5 min. PCR reaction (total 25 ul); 12.5 ul NEB Next High Fidelity Master Mix, 0.5 ul of each primer, 1.5 ul of 3% BSA, 1 g of template DNA. Sample purification: Agencourt AMPure XP extraction kit (Beckman Coulter, Krefeld, Germany), ratio of AMPure XP to PCR reaction is adapted to 0.8 to 1. Indexing PCR: Nextera® XT Index Kit v2 set A (Illumina, San Diego, CA, USA), 98 C for 5 min; 98 C for 10 s / 55 C for 30 s / 72 C for 30 s (8 cycles); 72 C for 5 min. Sample purification: Agencourt AMPure XP extraction kit. Products are pooled equimolar to a final concentration of 4 nM and sequenced using the MiSeq Reagent kit v3 (600 cycles; Illumina,San Diego, CA, USA) for paired end sequencing.