11-GBR ITS2
Small fragments (1 cm) for each of A. millepora and P. lutea (n = 4) were removed from independent colonies and immediately preserved in RNAlater (Ambion, Life Technologies; Vega Thurber et al. 2009) and stored at 20C until processing. Prior to DNA extraction, excess RNAlater solution was removed (Vega Thurber et al. 2009, Tout et al. 2015) before fragments were air-picked into sterile PBS-EDTA. DNA was extracted from the tissue slurry using the Qiagen DNeasy Plant Mini Kit according to the manufacturers instructions. To disrupt the Symbiodiniaceae cells, 200 l of 0.5 mm sterile glass beads (BioSpec) were bead-beaten at 30 Hz for 90 s with a Tissue Lyser II (Qiagen). Quantity and quality of DNA was checked using a NanoDrop 2000C spectrophotometer (Thermo Fisher Scientific). PCR amplifications were performed in triplicate reactions with a Qiagen Multiplex PCR Kit. PCR amplification of the internal transcribed spacer 2 (ITS2) region was done using the primers ITSintfor2 and ITS2-reverse (Illumina sequencing adapters underlined), and following the PCR conditions of Arif et al. (2014). For individual PCR reactions, DNA was aliquoted to 1250 ng l1, with 10 l Qiagen Mix, 0.5 l of each 10 M primer mix, 1 l of DNA template, and DNAsefree water to adjust the reaction volume to 20 l. A 5 l aliquot of each PCR product was run on an 1% agarose gel to visualise successful amplification. Samples were purified and indexed to add Nextera XT indexing and sequencing adapters (Illumina) ac - cording to the manufacturers protocol. Indexed amplicons were again purified, quantified on the QuBit (Quant-IT dsDNA Broad Range Assay Kit; Invitrogen) and pooled in equimolar ratios on the BioAnalyser (Agilent Technologies). The final pooled library was purified on a 2% agarose gel to remove excess primer-dimers. The library was sequenced at 8 pmol with 10% phiX on the Illumina Miseq, 2 300 bp end version 3 chemistry ac cording to the manufacturers specifications, at the Ramaciotti Centre for Genomics.