Abstract:
This dataset focuses on protists in the phylum Ciliophora collected at stations spanning the continental shelf in the northeastern Gulf of Mexico aboard the R/V Bellows research cruise, BE-1301. The dataset contains raw sequences from cloned rRNA genes, 95 per sample, 25 samples for oceanographic stations covering the northeast Gulf of Mexico continental shelf and DeSoto Canyon in February and July of 2012. The raw sequences contain primer segments and about 5% non-ciliate sequences, mostly radiolarians and acantharians. Edited sequences were binned at 95% similarity for ecological analysis and sequences representative of bins have been uploaded to Genbank as numbers MT973819 to MT973954. This dataset supports the publication: Snyder, R. A., Moss, J. A., Santoferrara, L., Head, M., & Jeffrey, W. H. (2021). Ciliate microzooplankton from the Northeastern Gulf of Mexico. ICES Journal of Marine Science. doi:10.1093/icesjms/fsab002
Suggested Citation:
Snyder, Richard A., Joseph A. Moss, and Wade H. Jeffrey. 2021. Dataset for: Ciliate microzooplankton from the Northeastern Gulf of Mexico. Distributed by: GRIIDC, Harte Research Institute, Texas A&M University–Corpus Christi. doi:10.7266/WBVVRTXM
Data Parameters and Units:
The headers are-Station, Lat (latitude), Long (longitude), Sample Date, Station Depth, Sample Depth (m), Chl a1 mg L-1, Chl b1 mg L-1, Nitrate + Nitrite1, mg L-1, Ortho P1 mg L-1, Kd2, PAR, Samples for Microsc.3, Samples for Molec., sample # month, day, site, depth
Methods:
Ten liters for each sample were filtered onto 0.22µm Sterivex® filters using a multichannel peristaltic pump. Duplicate filters were obtained from each sample location, if possible, to provide a backup in case of sample processing difficulties and to provide archived samples. Filters were frozen at -20°C onboard ship. Between collections, bottles and tubing rigs were rinsed with 10% HCL, 95% ethanol, and 18 ohm purified water. Upon arrival to lab, all samples were kept at -80°C until processing or for archival purposes.
Filters were removed from Sterivex units with sterile hacksaw blades and forceps and inserted into separate 2ml bead-beading tubes (Powersoil; MoBio®). Lysis buffer was added and filters were subjected to 3 freeze-thaw (liquid nitrogen/75°C) cycles. Tubes were agitated using a PowerLyzer homogenizer (MoBio®) using 90 second bursts at setting S3500. DNA extraction was done according to the manufacturer’s instructions. DNA concentration was estimated using a Nanodrop® 1000 and stored at -20°C.
For each sample, 763 bp fragments of the 18S rRNA gene were amplified using the ciliate-specific primer set 384F (5’ YTB GAT GGT AGT GTA TTG GA 3’) and 1147R (5’ GAA CGA AAG WTA RGG GAT CA 3’) (Dopheide et al. 2008). PCR was performed in quadruplicate for each sample (2 to 5ng template DNA/reaction) with final reagent concentrations in 50μl volumes containing: 3.0 mM MgCl2 (Roche), PCR buffer (FastTaq 10x/Green), 0.50µM each primer, 0.04 U of FastStart Taq DNA polymerase (Roche), 0.2 mM PCR Nucleotide MixPlus (Roche). Reactions proceeded for 30 cycles of 94°C for 30 s, 60°C for 60 s, and 72°C for 1 min, with a final elongation for 10 min. PCR products were verified via 0.8% agarose gels, excised using sterile scalpels, and purified with a QIAGEN Gel Extraction Kit (QIAGEN, Valencia, CA, U.S.A).
Purified DNA was cloned into pCR 2.1 TOPO vectors (Life Technologies; Carlsbad, Calif.) following the manufacturer’s protocol and transformed into electrocompetent cells (MegaX DH10B T1; Life Technologies) using a BIO-RAD MicroPulser set to the manufacturer’s specifications. Transformed cells were incubated for 1 hour at 37° C and screened via selective growth on LB media (ampicillin 0.1%, kanamycin 0.1%, x-gal 0.08%) for 18-24 hours. For each sample, a library of 96 random clones were grown for 12-14 hours with selective LB broth and shipped in 10% glycerol stocks for Sanger Sequencing (Beckman Coulter Genomics, Danver, MA). Sequences were obtained for a total of 2194 clones.
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