title: plankton above deep-water coral reef GRR10
design: Accession: SRX338073  Experimental design: Seawater was collected above deep-water coral reefs off Norway. One L samples were filtered (0.2 µm) and plankton nucleic acids were extracted. RNA of 5.0 and 0.5 ng was converted to single-stranded cDNA by reverse transcription with random hexamers (iScript kit, BioRad, Hercules, CA). Variable V1-V3 regions of SSU rRNA from DNA and cDNA was PCR amplified using domain specific forward primers of Bacteria 9bfm (5’-GAGTTTGATYHTGGCTCAG-3’; Muhling et al. 2008) and Eukarya Euk1Afm (5’-CTGGTKGATYCTGCCAG-3’; Diez et al. 2001), in combination with the “universal” reverse primer 518rm (5'-ATTACCGCGGSTGCTGG-3'; Muyzer et al. 1993). Taxon coverage was attempted optimized by a modification of primers Euk1Afm and 518rm (1-2 degenerate positions). Amplicons from 10-100 ng of nucleic acids were generated in triplicate using a two-step protocol (Berry et al. 2011). PCR was performed in 20 µl containing 0.5 µl template, 0.5 µM of each primer, 250 µM of each dNTP, 20 µg ml-1 bovine serum albumin and 0.4 U Phusion DNA polymerase in HF buffer (Finnzymes, Espoo, Finland); 98°C for 1 min, cycling at 98°C for 10 sec, 53°C for 30 sec, 72°C for 1 min, a final 72°C for 5 min. Following 20 cycles reactions were pooled, cleaned (ExoSAP-IT, GE Healthcare, UK) and 2 µl amplified for 10 further cycles including sequencing adapters. Amplicons of expected size and purity following agarose gel electrophoresis were cleaned (Agencourt AMPure beads, Beckman Coulter, Massachusetts, USA) and spectrophotometrically quantified (Qubit, Invitrogen, Carlsbad, USA). Emulsion PCR and pyrosequencing was performed at the Norwegian High-Throughput Sequence Centre (University of Oslo).