title: Young Eucalyptus replicate 1
design: DNA was extracted from approximately 500 mg of each soil sample using the Fast DNA Spin Kit For Soil (MP Biomedicals). The extracted DNA was dark in color due to a high level of humic material, so it was then purified using the last steps of the NucleoSpin Soil kit (Macherey-Nagel) protocol. DNA concentration and quality were measured using a NanoDrop. Soil bacterial diversity were assessed by next generation sequencing of the V4 variable region of the 16S rRNA gene using the primers 515FB (GTGYCAGCMGCCGCGGTAA) and 806RB (GGACTACNVGGGTWTCTAAT). PCR reactions with a barcode on the forward primer were used in a 28-cycle PCR using the HotStarTaq Plus Master Mix Kit (Qiagen, USA) under the following conditions: 94 C 3 min; 28 cycles of (94 C 30 s, 53 C 40 s, 72 C 1 min); 72 C 5 min. Following PCR amplification, PCR products were checked in 2% agarose gels to determine the success of PCR and the relative intensity of resulting bands. Multiple samples were pooled in equal proportions based on their molecular weight and DNA concentrations. Pooled samples were purified using calibrated Ampure XP beads. The pooled and purified PCR products were then used to prepare an Illumina DNA library. Sequencing was performed by MR DNA (www.mrdnalab.com, Shallowater, TX, USA) on a MiSeq paired-end 2x250 sequencing system, following the manufacturer's guidelines. Raw data were downloaded from Basespace and analysed using Mothur v1.39 software23.