title: 16S Ecopile 5 Nov 20 Replicate 3
design: Two grams of sample were taken from each Ecopile and the bacteria within sample were resuspended in a saline solution (5 mL of sterile NaCl 0.85%) by vigorous shaking at 2000 rpm for 30 minutes in a Multi Reax agitator.  The liquid phase was centrifuged at 300 g for 30 seconds to pellet the soil particles and subsequently the supernatant was centrifuged at 8000 g for 10 minutes to pellet the microorganisms. This pellet was finally resuspended in 1 mL of PBS. Sixty L of lysozyme (1 mg/mL) were added to each sample, followed by an incubation at 37C for 1 h. Isolation of total DNA from each sample was carried out in triplicate using the FastDNA Spin Kit for Soil (MP Biomedicals, USA) according to manufacturer indications. The isolated DNA was quantified using Nanodrop spectrophotometer 2000c (Thermo Fisher Scientific) and Qubit 4 fluorometer (Invitrogen). For bacterial 16S profiling, the isolated DNA was PCR amplified to sequence the V3-V4 16S rRNA amplicons using the primers: 341F (5-CCT ACG GGN GGC WGC AG-3) and 805R (5-GAC TAC HVG GGT ATC TAA TCC-3). Libraries were prepared with Illumina MiSeq v3 reagent kit according to suppliers specifications and sequenced by Illumina MiSeq System to get 2 x 300 bp reads.