title: RNA-seq of filtered seawater
design: Total nucleic acids were extracted using a Macherey-Nagel NucleoMag DNA/RNA Water Kit following manufacturers instructions for Sterivex filter units with 1 mL of Lysis Buffer MC1, addition of bead beating step by transferring lysate to a Macherey-Nagel Bead Tube Type A. Total RNA  (250 ng of Qubit quantified total RNA input) were prepared for metatranscriptomic sequencing at the University of Connecticut Center for Genome Innovation (https://cgi.uconn.edu/) using the Illumina Stranded Total RNA Prep, Ligation with Ribo-Zero Plus library preparation kit (Illumina, San Diego, CA) following the manufacturers protocol. Libraries were validated for length and adapter dimer removal using the Agilent TapeStation 4200 D1000 High Sensitivity assay (Agilent Technologies, Santa Clara, CA, USA), then quantified and normalized using the dsDNA High Sensitivity Assay for Qubit 3.0 (Life Technologies, Carlsbad, CA, USA). Sample libraries were then prepared for Illumina sequencing on the NovaSeq 6000 by denaturing and diluting the libraries per manufacturers protocol (Illumina, San Diego, CA, USA). All samples were combined into one sequencing pool, proportioned according to expected number of reads, and run as one sample pool using a S4 200 cycle v1.5 flow cell targeting 60M 100bp paired end reads per sample.