2 wk post wet-up soil eDNA replicate C
Environmental DNA was extracted from 5 g of soil thawed at 4 C for 1 hour. Soil was washed twice with 10 mM Tris-HCl (pH 8.0) by shaking horizontally for 15 minutes at 400 RPM at room temperature, followed by three cycles of alternating vortexing and hand mixing for 30 s each. Samples were then centrifuged at 4 C at 4,198  g for 20 min. Approximately 23 mL of wash fluid was collected and passed through a 0.22 m Steriflip-GP filter to remove cellular debris, followed by filtration through a 0.02 m filter to remove viral particles. The resulting filtrate was concentrated by isopropanol precipitation. Briefly, approximately 2025 mL of filtrate was amended with 23 L GlycoBlue, 2.3 mL of 3 M sodium acetate, and 23 mL of isopropanol, mixed thoroughly, and incubated at 20 C for at least 1 hour. Samples were centrifuged at 4,198  g for 20 minutes at 4 C, and the resulting DNA pellet was washed with freshly prepared cold 70% ethanol. After a final centrifugation (5 min at 4,198  g, 4 C) and removal of residual ethanol, pellets were air-dried for approximately 30 min and resuspended in 100 L of 10 mM Tris-HCl (pH 8.0). DNA libraries were prepared using the NEBNext Ultra DNA Library Preparation Kit following the manufacturers recommendations. Briefly, eDNA was fragmented by acoustic shearing using a Covaris S220 instrument. Fragmented DNA was end-repaired and adenylated, followed by ligation of sequencing adapters. Adapter-ligated DNA was indexed and enriched by limited-cycle PCR (817 cycles).