Nucleic acid collection

After two weeks, the top 1 cm of sediment was collected by gently pouring off overlying water until approximately 1–2 mm of muddy, overlying water remained. Many meiofauna were present in the residual overlying water, so this was collected with the top 1 cm of sediment. A clean, plastic core extruder was used to gently push the top 1.5 cm of sediment out of the PVC tube. Sediment was collected in sterile weigh boats and briefly and gently homogenized with a sterile metal spatula before being stored in two RNAse/DNase-free 15 mL conical tubes per mesocosm for technical replicates. Sediment was then immediately flash frozen and stored at − 80 °C.

RNA and DNA were co-isolated using the Qiagen RNeasy PowerSoil Total RNA Kit followed immediately by the Qiagen RNeasy PowerSoil DNA Elution Kit. Phenol/ chloroform/ isoamyl alcohol 25:24:1 at pH 6.6 and 200 proof Molecular Grade Ethanol used for nucleic acid extraction and PCR cleanup (“PCR cleanup and library preparation” Section) were from Thermo Fisher. Nucleic acids were extracted according to manufacturer instructions, and 2 g of sediment were used in each extraction. There were 7 mesocosm samples and 2 technical replicates extracted per mesocosm. DNA was subsequently cleaned of residual RNA using Qiagen’s RNase A protocol. Briefly, digestion buffer and 0.5 µL of RNase A (Qiagen) was added to each sample and incubated for 40 min at 37 °C. DNA was separated using 200 µL of phenol / chloroform / isoamyl alcohol by centrifugation at 10,000 g for 5 min. The aqueous layer was removed and treated with 5 M NaCl and 100% ethanol. DNA precipitation occurred after a 30 min incubation at − 20 °C follow by centrifuging at 10,000 g for 10 min. Both RNA and DNA purity and concentration were determined using a NanoDrop 8000 (Thermo Scientific). The 260/280 values were between 1.95–2.2 and 1.7–1.85 for RNA and DNA, respectively. Also, RNA integrity was confirmed with gel electrophoresis by the presence of distinct 18S and 28S bands. Agarose, 50X TAE Buffer, SYBR Safe DNA Gel Stain, and 6X DNA Loading Dye used for gel electrophoresis were from Thermo Fisher. Extracted RNA and DNA samples were stored at − 80 °C.