v4-16S rRNA gene sequences from Cymbastela concentrica replicates 1 to 3, DNA extraction from PMA treated microbial cell enriched pellet (viable fraction)
A microbial enriched cell suspension was obtained from around 2 g of sponge sample, according to the method described by Thomas et al, The ISME Journal (2010) 4, 1557–1567. A 1 mL aliquot was treated with propidium monoazide dye. After photoactivation of the dye using a 750 W halogen lamp, cells were pelleted and stored at -80ºC for subsequent DNA extraction. Total community DNA from the PMA treated cell suspension was extracted using the PowerSoil®-htp 96 well DNA isolation kit. The conserved regions flanking the v4 region of the 16S rRNA gene found in bacteria and archaea were targeted using the primers 515F and 806R. PCR amplification of 16S rRNA genes and Illumina MiSeq amplicon sequencing were performed at the Ramaciotti Centre for Genomics (University of New South Wales, Sydney, Australia), according to the methodology described by Caporaso et al. (2012).
The University of New South Wales
Cymb4-6_PMA