16S sequencing of Tegula intertidal snails: whole body control vs. heat stress samples
A 350 bp region of the bacterial V4 region of the 16S rRNA gene was amplified using indexed forward (5-GTGCCAGCMGCCGCGGTAA-3) and reverse (5-GGACTACHVGGGTWTCTAAT-3) primers (Kozich et al., 2013) using the following protocol: 94 C for 3 min, 35 cycles of 94 C for 45 sec, 50 C for 60 sec, 72 C for 90 sec, and finally 72 C for 10 min. PCR reactions were performed in 25 uL volumes and contained 12.5 uL 2x Phusion High-Fidelity DNA Polymerase Master Mix (NEB), 5 uL ddH2O, 1.25 uL of each primer (10 uM), and 5 uL of template DNA. Amplifications were verified on a 1% agarose gel with GelRed and pooled. A double-sided bead cleanup using Cytiva Sera-Mag SpeedBeads Carboxyl Magnetic Beads (Fisher Scientific) was carried out to remove primer-dimers and a low amount of off-target larger PCR products. Quality and concentration of the pooled library was checked using a Bioanalyzer (Agilent, Santa Clara, CA, USA) and NEBs Library Quant Kit for Illumina following the manufacturers standard protocol. The library representing 18 total samples was then sequenced on an Illumina MiniSeq, yielding 300bp PE reads, using a mid-output reagent cartridge at the James Madison University Center for Genome and Metagenome Studies (CGEMS). Before loading, the library was combined with Illuminas PhiX control (30:70 16S:PhiX) to ensure a high-quality run despite the low diversity of the 16S library.