Bacterial 16S rRNA gene amplicons from Haliclona oculata sponge: P_S5B
The DNAeasy PowerLyser Powersoil Kit was used to extract DNA from 0.2 g of the sponge specimen, with the final DNA elution in water and bead beating at 4000 rpm for 45 s. Bacterial-specific primers S-D-0564-a-S-15 (AYTGGGYDTAAAGNG) and S-D-Bact-1061-a-A-17 (CRRCACGAGCTGACGAC) were used to amplify a 500 bp region of the 16S rRNA gene from sponge-associated bacteria using HotStarTaq DNA Polymerase with the following reaction conditions: initial activation at 95 C (15 min), 28 cycles of dentaturation at 94 C (60 s), a step-down to 70 C (1 s), ramping rate of 0.4 C/s to 50 C for annealing (60 s), and a ramping rate of 0.8 C/s to 72 C for extension (60 s),  final extension at 72 C (10 min). Adaptor sequences from the TruSeq DNA LT Sample Prep Kit (Illumina) were used to preprare amplicon libraries for sequencing as outlined in the manufacturers protocol. PCR products were purified using magnetic Agencourt AMPure XP beads.