title: 18S Ecopile 2 Nov 20 Replicate 1
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). Fungal samples were sent to Novogene to sequence the v4-v5 18S rRNA region using as primers 528F (5-GCG GTA ATT CCA GCT CCA A-3) and 706R (5-AAT CCR AGA ATT TCA CCT CT-3). Libraries were constructed by Novogene specifications and sequenced by Illumina Novaseq 6000 system to get 2 x 250 bp reads.