The Environmental Sample Processor (ESP) filtered seawater sequentially through 0.2 um pore size polyethersulfone filters. Seawater was evacuated from filters and followed twice with a 2 minute incubation with 1 ml of RNAlater™. RNAlater was evacuated, and filters were stored in the ESP until they were transferred to -80 C upon instrument recovery.
Filters were processed for DNA using the phenol-chloroform extraction method of Crump et al. (2003) after placing the filters into 1 ml of DNA extraction buffer. Extracted DNA was sheared ultrasonically to ~350 bp fragments, and library preparation was performed at the Georgia Genomics and Bioinformatics Core (GGBC) facility. Single-end 250 bp sequencing was performed using an Illumina HiSeq Rapid Run at Hudson Alpha Genomic Services Laboratory (Huntsville, AL, USA).
Single-cell sequencing: Seawater was transferred directly from the Niskin bottle to a 50 ml Falcon tube and placed on ice until brought back to lab. Each sampling day, 3 x 1 ml of seawater was preserved in cryovials using 100 ul of glyTe (5 ml glycerol, 3 ml Milli-Q H2O, 1 ml 100 x TE pH 8.0, 0.2 um filter sterilized after mixing the above, and stored in -20 C freezer). Preserved samples were then placed in a -80 C freezer. Samples were processed and sequenced at Bigelow Single Cell Genomics Center (Stepanauskas and Sieracki, 2007).
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