Coral Microbiome 16S rRNA Profiling

DNA was extracted from coral tissues using the UltraClean Soil DNA Kit (Mo Bio; Carlsbad, CA, United States) following the manufacturer’s instructions for maximum yield. The extracted genomic DNA was used for PCR amplifications of V3–V4 region of the 16S rRNA gene by using the following universal primers: Pro341F (CCTACGGGNBGCASCAG) (Takahashi et al., 2014 (link)) and Bact805R (GACTACHVGGGTATCTAATCC) (Herlemann et al., 2011 (link)). Each PCR mixture contained 5 μl of 10x PCR reaction buffer (Invitrogen), 1.5 μl of 50 mM MgCl2, 1 μl 10 mM dNTP mixture, 1 μl of 100 μM of each primer, 1 units of Taq polymerase, 3 μl of BSA (New England BioLabs), sterile MilliQ water up to 50 μl and 10 ng of DNA. Negative controls (with no template DNA) were included to assess potential contamination of reagents. The amplification products were purified with the GeneJET PCR purification kit (Fermentas, EU), quantified using the Qubit Kit (Invitrogen), and the quality (integrity and presence of a unique band) was confirmed by 1% agarose gel electrophoresis.