16S amplicon of zebrafish larvae exposed to loperamide
16S rRNA gene amplicons of the V6 region for the 45 conventional zebrafish samples, 2 mock community samples (Zymo Research DNA standard I D6305), 2 negative DNA extraction samples, and blank PCR control were prepared using 967F/1064R primers. A two-step PCR reaction using 200 ng of zebrafish DNA was performed in duplicate 50 L reactions as previously described (Eren et al., 2013; Huse et al., 2014). Each first step reaction included 25 L 2X Phusion Mastermix (Thermo Scientific F531S), 1.5 L of 10 M F/R primer mix, 13 - 20 L template DNA (200 ng), and 3.5  10.5 L nuclease-free water (up to 50 L). PCR amplification (step 1) conditions were denaturing at 98C for 3 min followed by 30 cycles of denaturation at 98C for 10 s, primer annealing at 56C for 30 s, and extension at 72C for 20 s, then a final extension at 72C for 20 s. Negative controls for the PCR reagents were prepared alongside zebrafish DNA samples, but with additional nuclease-free water input. PCR products were assed for concentration (Qubit DNA HS reagents) and expected size using agarose gel electrophoresis. A second PCR step was performed to attach sequencing barcodes and adaptors according to Illumina protocols. The PCR products were analyzed with 250 bp paired-end sequencing to obtain overlapping reads on an Illumina MiSeq at the Institut Pasteur Biomics platform.