16SrRNA_Initial_Terrestrial_2_F
For amplicon sequencing, 9 l of DNA extract was combined with 6 l of GoTaq Flexi Buffer (5x, Promega), 3 l of MgCl 2 (25 mM), 0.24 l of each primer, 0.6 l of dNTP (10 mM), 1.8. l of bovine serum albumin (10 mg ml-1), 8.82 l of MilliQ water, and 0.3 l of Hot start Taq (5U ml-1, Promega). PCR was performed at 95C for 2 minutes, followed by 36 cycles (bacteria) or 38 cycles (fungi) of 94C for 40 seconds, 58C for 40 seconds, and 72C for 1 minute. For protists, the PCR protocol involved 98C for 30 seconds, followed by 30 cycles of 98C for 10 seconds, 64C for 20 seconds, and 72C for 25 seconds, with a final cycle at 72C for 5 minutes. Up to 3 PCR products were pooled, and the amplicons were purified using the DNA Clean & Concentrator-5 kit (Zymo, Freiburg, Germany). The purified samples were sent to Eurofins Genomics Europe Shared Services GmbH (Konstanz, Germany) for quantification, barcoding, and paired-end sequencing using the Illumina MiSeq v3 platform (Illumina Inc., San Diego, Ca, USA).