Roseobacter denitrificans OCh114 GTA DNA
Two independent biological replicates of GTA particles from each of the three strains (R. mobilis 45A6, R. pomeroyi DSS-3, and R. denitrificans OCh 114) were concentrated and prepared for sequencing. The two replicates of concentrated particles for each strain were separately purified by CsCl step gradients according to standard protocols (Thurber et al 2009), the 1.3  1.5 density interface was collected, and presence of particles verified by SYBR Gold staining. Nucleic acids were extracted from the purified particles by formamide disruption of the protein capsids followed by a CTAB extraction and ethanol precipitation of the nucleic acids (Thurber et al 2009). After extraction, the DNA was prepared for amplification. The extracted nucleic acids were Covaris fragmented (Covaris, Inc., Woburn, Massachusetts, USA) to 300-400 bp fragments to unify the template size according to the manufacturers instructions. The dsDNA fragments were amplified using Accel-NGS 1S Plus DNA library and indexing kits (Swift Biosciences, Ann Arbor, MI, USA) according to manufacturers instructions. After amplification, three L of the extracted DNA were stained with ethidium bromide and visualized on a 2% agarose gel to verify amplification. Gel images were taken using an Alpha Imager EC (www.alphainnotech.com). GTA amplicons were comparable to the kit positive control and no amplification was observed in negative control reactions. Amplified DNA from the two replicate GTA preps from each of the strains were gel purified (0.8% agarose), extracted with sterile scalpels, and processed using the Zymo Large Fragment DNA recovery kit (Zymo Research, Irvine, CA). Paired-end (PE) libraries with 320bp insert size were constructed and 6 pmol of each GTA preparation was multiplexed and sequenced on a single lane of an Illumina MiSeq in the KAUST Bioscience Core Lab (Thuwal, Saudi Arabia).