16S V3-V4 seq of 10 salinity intestinal microbiota of Urechis unicinctus
PCR amplification of the bacterial 16S rRNA genes V3V4 region was performed using the forward primer 338F (5-ACTCCTACGGGAGGCAGCA-3) and the reverse primer 806R (5-GGACTACHVGGGTWTCTAAT-3). The PCR components contained 5 l of Q5 reaction buffer (5), 5 l of Q5High-Fidelity GC buffer (5), 2 l (2.5 mM) of dNTPs, 1 l (10 uM) of each Forward and Reverse primer, 0.25 l of Q5 High-Fidelity DNA Polymerase (5U/l), 2 l of DNA Template, and 8.75 l of ddH2O. PCR amplicons were purified by Agencourt AMPure Beads (Beckman Coulter, Indianapolis, IN) and quantified using the PicoGreen dsDNA Assay Kit (Invitrogen, Carlsbad, CA, USA). After the individual quantification step, amplicons were pooled in equal amounts, and pair-end 2300 bp sequencing was performed using the Illlumina NovaSeq 6000 platform at Shanghai Personal Biotechnology Co., Ltd (Shanghai, China).