16S rRNA (V3-V4) of Adult Aquatic  Hydroptilidae sp. - whole body microbiome
We extracted DNA from whole-body freshwater larval insects, adult aquatic insects, adult terrestrial insects, and riparian spiders. DNA was extracted using a protocol designed by the Surette Lab at McMaster University, KingFisher Protocol for Low Biomass Samples, following DNA isolation methods as described by Stearns et al. (2015) with modifications including using the entire supernatant and eluting with 50 uL of elution buffer. The extracted DNA was used to amplify the V3-V4 hypervariable region of the 16S rRNA gene using a nested PCR approach where the 8F (3  AGAGTTTGATCCTGGCTCAG  5) and 926R (5  CCGTCAATTCCTTTRAGTTT  3) region of the 16S gene was amplified at 94 C for 5 min, followed by 15 cycles at 94 C for 30 s. This reaction was used as the template for the second phase of PCR which used 341F (5  CCTACGGGAGGCAGCAG  3) and 806R (5  GGACTACNVGGGTWTCTAAT  3) Illumina adapted primers (IDT, Coralville, Iowa) which underwent 5 min at 94 C and 5 cycles at 94 C, 47 C, and 72 C for 30 s each, followed by an additional 25 cycles at 94 C, 50 C, and 72 C for 30 s each, with a final extension at 72 C for 10 min. PCR products were visualized on a 1.5% agarose gel and sequenced on the Illumina MiSeq platform (paired-end reads, 2 x 300 base pairs or bp) at the Farncombe Institute (McMaster University, Hamilton, Ontario).