RNAseq of Trichodesmium: environmental sample
Trichodesmium colonies were collected using plankton nets (200 m mesh, 50 cm diameter) deployed at 15 meters for 15 minutes. Net cod-ends were emptied into an acid-cleaned bucket and transferred to a laminar flow hood inside a trace-metal clean bubble laboratory. 50 colonies (in triplicate) were randomly handpicked using plastic Pasteur pipettes and individually washed through 3 steps before allocation into 150 mL bottles. Bottles were filtered onto 0.2 m Supor filters, flash frozen with liquid N2 and stored at -80 C. RNA was extracted used an All Prep DNA/RNA kit (Qiagen, Manchester, UK), DNase-treated with a Qiagen DNase kit, and cleaned with ZYMO RNA Clean and Concentrator kit. Depletion of rRNA from total RNA was performed using the Ribo-Zero Bacteria Magnetic Kit, and strand-specific sequencing libraries were prepared using the ScriptSeq v2 RNA-Seq Library Preparation Kit (Illumina, originally EpiCentre). Paired end sequencing was carried out using an Illumina MiSeq instrument.