title: Municiple Waste Struvite Plot 2  Unbalanced 2019   Nematodes 18S Library
design: Soil sampling was conducted 51 days after fertiliser application following cutting of grass silage (July 2019). Nine sample cores were taken to a depth of 10cm per plot and stored at -20C until ready for analysis. A total of 40 composite samples were obtained (8 treatments X 5 replicate plots). Defrosted samples were sieved through a 2mm mesh and homogenised. 25g sub-samples of soil were shaken in 25ml of deionised water for 10 minutes at 95rpm and centrifuged for 2 minutes at 1500rpm. The supernatant was discarded, and remaining samples were dried overnight at 28C in petri dishes before homogenising using a mortar and pestle. Finally, 0.25g sub-samples were obtained. Total DNA was extracted from 0.25g soil sub-samples using Qiagen DNeasy PowerSoil Pro kit, as per manufacturers instructions. Total DNA quality and quantity was assessed by NanoDrop instrument and agarose gel electrophoeresis using 1% agarose gels, before outsourcing to a sequencing company. Nematode 18S V4 region rRNA were sequenced  using MN18F and 22R primer pair on Illumina paired-end platform