title: 16S (V3V4) amplicons seq from R. nomadica jellyfish microbiome
design: The V3V4 hypervariable region of the bacterial 16S rRNA gene was amplified using a two-stage polymerase chain reaction (PCR) protocol. The primers used for the first round of amplification were 341F (CCTACGGGNGGCWGCAG) and 806R (GGACTACNVGGGTWTCTAAT). The first stage PCR amplification was carried out with DreamTaq Green PCR master mix (Thermo Scientific, USA), with each 50l reaction containing 25l of the master mix, 1l of each primer at 10 M concentration, 2 l of template DNA, and 21 l of nuclease-free water. Thermal cycling began with an initial denaturation at 95C for 5 minutes, followed by 32 cycles of denaturation at 95C for 30 seconds, annealing at 52C for 45 seconds, extension at 72C for 1 minute, and a final extension step at 72C for 7 minutes. The resulting PCR products were confirmed via electrophoresis on a 1% TAE agarose gel. The second stage of PCR was performed at the sequencing facility using 1l of the initial PCR product as a template, with the following conditions: an initial denaturation at 98C for 2 minutes, followed by 8 cycles of 98C for 10 seconds, 60C for 1 minute, and 68C for 1 minute. The final PCR libraries were pooled and sequenced using a 15% phiX spike-in on an Illumina MiSeq sequencer with V3 chemistry for 2x300 base paired-end reads. This sequencing was performed at the Genomics and Microbiome Core Facility (GMCF; Rush University, IL, USA).