18S of experimental pond community
Microbial communities were sampled from each experimental pond five times over the course of the experiment; 48 hours prior to plastics addition (March 7, 2023), on day 16 (March 23, 2023), day 36 (April 23, 2023), day 56 (May 3, 2023), and on day 94 (June 9, 2023). Water samples from each pond were collected using a vertical integrated water sampler, with water taken from both sides and the center of each pond, transferred and homogenized in sterile 5-gallon plastic buckets. Using a MasterFlex peristaltic pump (Masterflex L/S Portable Sampling Pump, Avantor, Radnor, Pennsylvania, USA), 2-3 liters were filtered through a 47mm, 0.22m Millipore Express PLUS membrane filter (Merck KGaA, Darmstadt, Germany). Filters were immediately frozen in liquid nitrogen prior to storage at -80C. For each sample, DNA was extracted from half of the filter following the QIAGEN DNeasy PowerSoil Pro Kit protocol (DNeasy PowerSoil Pro Kit Handbook, Qiagen, Hilden, Germany). Step 16 was modified by allowing 50L of the elution buffer to rest on the filter membrane before the final elution. For each sample, amplicon libraries targeting the V4-V5 region of the 16S rRNA gene and V9 region of the 18S rRNA gene were generated following the Earth Microbiome Project protocol at the Argonne National Labs Environmental Sample Preparation & Sequencing Facility. Library preparation was conducted using the 515-806R primer set for 16S and 1389F-1510R primer set for 18S, and amplicons were sequenced on a 2151bp paired-end NextSeq 2000.
SUB15192021
18S_JRD444