V1-V3 16S amplicon sequencing of coral from natural pH gradient at Maug caldera
Coral fragments were subsampled in a cryo-cart under liquid nitrogen and then homogenized (minimum of 2 min at 25 reps per second) under liquid nitrogen using a Retsch Cryomill. An aliquot of each coral homogenate was preserved in Trizol reagent. DNA was separated from RNA using organic solvent phase separation with chloroform, and subsequent DNA extraction was conducted using a back extraction buffer (BEB) (4 M guanidine thiocyanate; 50 mM sodium citrate; 1 M Tris, pH 8.0). After removal of aqueous phase containing RNA, BEB was added to the organic phase and interphase and then incubated at room temperature for 10 min. Samples were then centrifuged at 13,200 rpm for 15 min at 4C. The upper, aqueous phase (now containing DNA) was removed. DNA was precipitated using 100% isopropanol. DNA was pelleted using centrifugation, DNA pellets were washed twice with 70% ethanol, and then DNA pllets were resuspended in Nanopure water. Extracted DNA was submitted to BioAnalytical Services Lab at the Institute of Marine and Environmental Technology.