title: Metabarcoding of bacterial 16S: Grassland_Amtvik_high_catenary_position_plot9_soil_0-10cm
design: Total DNA was extracted from 100 mg (soil depth 0-30 cm) or 400 mg (soil depth 40-50 cm) freeze-dried, homogenized soil using Macherey-Nagel NucleoSpin Soil kit (Dren, Germany). Bacterial community amplicons were generated by PCR amplification of the 16S rRNA gene, using the forward primer Pro341F and the reverse primer Pro805R for the V3-V4 region and a two-step PCR protocol adding individual Nextera barcodes at the second step. PCRs were run in technical duplicates and template concentrations and number of PCR cycles were optimized for each sample. PCR products were purified using Sera-Magmagnetic beads (GEHealthcare, IL, USA), concentrations were measured fluorometrically (Qubit Fluorometer, Invitrogen, Carlsbad, California, USA) and products mixed in equal amounts into four sequencing pools. Sequencing was performed by SciLifeLab (National Genomics Infrastructure, Uppsala, Sweden) on the MiSeq platform using the 2  250 bp paired-end chemistry (Illumina, San Diego, CA, USA).