Dataset: DICE_sequences

See Howard et al. (2010), Rinta-Kanto et al. (2011), and Rinta-Kanto et al. (2012) for detailed methods, which are paraphrased below:

"In October 2006, seawater was collected from near-surface waters in the Gulf of Mexico off the coast of Dauphin Island (temp = 27 degrees C, salinity = 34) by lowering a clean bucket ~10 cm below the surface. Water was filtered through a 200 micrometer mesh into six 20-liter polyethylene Cubitainers (Fold-A-Carrier; Reliance Products, Ltd.). Three experimental Cubitainer microcosms were amended with sodium nitrate (10 uM) and potassium phosphate (0.6 uM) to induce bloom conditions; three control microcosms received no nutrient amendments. The microcosms were incubated at 27 degrees C with a 12-h-on/12-h-off light cycle (200 umol quanta per square meter per second) for the duration of the experiment. At the initial time point (T = 0), the full contents of one control and one experimental microcosm (20 liters each) were sacrificed for detailed chemical and biological measurements."

Howard et al. (2010):
"For DNA samples, water from each time point was sequentially filtered through 8-um and 3-um prefilters, and  particles were collected on 0.2-um pore-size polycarbonate filters. DNA was extracted using the PowerMax soil DNA isolation kit (MoBio Laboratories, Inc.). Concentrations were estimated by absorbance on a NanoDrop spectrophotometer (Thermo Scientific). DNA extracted from duplicate control and experimental samples was used as template for PCRs, followed by pyrosequencing of amplicons. Universal dmdA primers were modified from the work of Varaljay et al (2010). PCR products were run on 1% (dmdA amplicons) or 2% (bacterial and archaeal 16S rRNA amplicons) agarose gels, followed by purification using the Qiagen QIAquick gel extraction kit. Products were cleaned using the Ampure system (Agencourt Bioscience Corporation). Two separate pools were assembled, and these were sequenced on separate halves of the PicoTiterPlate (454 Life Sciences). 1,300 ng DNA was used in Roche GS FLX LR70 pyrosequencing at the University of South Carolina Environmental Genomics Facility (Columbia, SC).

For reads of high quality, adaptor and key sequences were removed and sequences were clustered using the CD-HIT program. For analysis of dmdA amplicons, reference sequences (the longest read in the cluster) were analyzed via  BLASTX against an in-house DmdA database. rRNA sequences were deposited in the Community Cyberinfrastructure for Advanced Marine Microbial Ecology Research and Analysis (CAMERA) database with project ID CAM-PROJ_DICE (sample CAM_S_A001= experimental microcosms; sample CAM_S_A002 = control microcosms). dmdA amplicon sequences were deposited in the NCBI Short Read Archive with project ID 49967."

Rinta-Kanto et al. (2011):
"The microarray was designed based on R. pomeroyi DSS-3 genes and their orthologs in 12 other Roseobacter  genomes (http://www.roseobase.org). The gene encoding DMSP demethylase (dmdA) was included on the array. Genes involved in DMSP cleavage to DMS (discovered in Roseobacter genomes subsequent to microarray final design) were not included on the array. Once a list of R. pomeroyi DSS-3 genes was compiled, orthologs for these genes in the other 12 Roseobacter genomes were identified using reciprocal BLASTs. Probes were designed from the orthologs using the Hierarchical Probe Design algorithm. The best probes selected by the program were queried against NCBI’s nr database using BLASTN, and only those probes with a best hit to a Roseobacter gene were retained. The final array consisted of 1578 probes targeting 431 genes. The complete list of probes is deposited in NCBI’s Gene Expression Omnibus database under accession number GPL10323. (The identifier codes for each probe begin with the R. pomeroyi DSS-3 locus tag followed by a number generated by the probe design algorithm describing the placement of the probe in the  tree generated during design.)"

Rinta-Kanto et al. (2012):
"RNA samples were collected from the microcosms on Day 5 by prefiltering ~1 L subsamples through 8.0 and 3.0 um poresize polycarbonate filters and collecting bacterioplankton on 0.2-um polycarbonate filters. Filters were placed in sterile 15 ml polypropylene tubes with lysis/binding buffer from the Ambion RNAqueous-kit. Cells were lysed using the Ambion RNAqueous Midi-kit (Applied Biosystems, Austin, TX). RNA was extracted from the lysate using Qiagen RNeasy kit. RNA was quantified after the extraction using a Nanodrop spectrometer.

Samples were processed as described in Poretsky et al. (2009) with some modifications (see Rinta-Kanto et al. 2012). Resulting amplified antisense RNAs were converted to doublestranded cDNA using Universal RiboClone cDNA Synthesis System (Promega). Concentrations of mRNA, amplified RNA and cDNA were measured using a Nanodrop spectrometer, and quality of the mRNA and cDNA was assessed using an Experion Automated Electrophoresis Station (Bio-Rad). Five ug of cDNA from each sample was used for pyrosequencing with the GS FLX sequencing system (Roche). Transcript libraries were deposited in the CAMERA database with a project ID CAM_PROJ_DICE."

References:
Poretsky, R.S., Hewson, I., Sun, S.L., Allen, A.E., Zehr, J.P., and Moran, M.A. (2009) Comparative day/night metatranscriptomic analysis of microbial communities in the North Pacific subtropical gyre. Environ Microbiol 11: 1358–1375. DOI: 10.1002/9781118010518.ch63
Varaljay, V. A., E. C. Howard, S. Sun, M. A. Moran (2009). Deep Sequencing of a DMSP-Degrading Gene (dmdA) Using PCR Primer Pairs Designed from Marine Metagenomic Data. Applied and Environmental Microbiology, vol. 76, p. 609. DOI: 10.1128/AEM.01258-09