16s rRNA gene sequences of Gorgonia ventalin gall tissue microbiome
DNA from the ethanol-preserved copepods, galls or a normal tissue of the corals was extracted simultaneously using standard silica-based DNA extraction kit (Diatom DNAprep 100, Isogene, Moscow, Russia). Community analysis by 16S-rDNA gene amplicon sequencing targeting the V4 variable region was modified from using universal primers 515F (5 -GTGCCAGCMGCCGCGGTAA-3 ) and 806R (5 -GACTACHVGGGTWTCTAAT-3 ). The 515F primer was labeled with sample-specific Multiplex Identification DNA-tags (MIDs). 25 l PCR reactions contained 12,5  l 2x Taqman Environmental Mastermix 2.0 (ThermoFisher), 10pMol/ l V4-F-MID primer and 10pMol/ l V4-R-trP1 primer (Sigma Aldrich), 1 l template DNA and PCR grade water. PCR was performed as follows: heated lid 110 C, 95 C   10mins, followed by 40 cycles of 95 C   15 s, 50 C   20 s, 60 C   30 s, followed by 60 C   4mins and held at 12 C. Negative controls with PCR grade water occurred without amplification. PCR products were quantified in the QIAxcel (QIAGEN). PCR products were pooled in an equimolar concentration. The pool was cleaned using AMPure magnetic bead-based purification system (Beckman Coulter). The clean pool was quantified using the Bioanalyser (Agilent). The amplicon library was sequenced using an Ion 314 Chip by an Ion Torrent Personal Genome Machine (Life Technologies) at the Naturalis Biodiversity Center following manufacturer protocol.