title: Metabarcoding microbiota sea lamprey: Sham control  at 12C 4weeks
design: At 5 or 8 weeks post-surgery, six lamprey larvae per group (n = 42 in total) were euthanized and dissected to collect individual intestinal content samples, which were immediately stored at 20 C until further processing. Total DNA was extracted using the E.Z.N.A Tissue DNA kit (Omega Biotek) following the manufacturers protocol. The V3V4 hypervariable region of the 16S rRNA gene was amplified with primers Bakt_341F (5-CCTACGGGNGGCWGCAG-3) and Bakt_805R (5-GACTACHVGGGTATCTAATCC-3), as described by Herlemann et al. (2011). PCR products were verified by agarose gel electrophoresis, purified with Mag-Bind RXNPure Plus magnetic beads (Omega Bio-tek), and indexed using Nextera XT adapters (Illumina). Sequencing was performed on an Illumina MiSeq platform with 2300 bp paired-end reads. Amplicons were randomly grouped into 3 replicates (R1, R2 and R3), each containing 2 larval samples of the same experimental group, to assess reproducibility.