title: RNA-seq of Karlodinium veneficum: control with f/2 medium
design: RNA was extracted from filtered cells with an RNeasy Mini Kit (Qiagen) following the manufacturers protocol. RNA concentration was measured using a NanoDrop spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA) and the integrity of RNA was evaluated by electrophoresis. Contaminating DNA was digested using a DNA-free DNA Removal Kit (Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturers protocol. RNA collected at T1hr was combined into a single composite sample per treatment for sequencing. The quality and integrity of the RNA in the composite samples were assessed using a fragment analyzer (Advanced Analytical Technologies, Inc., Ankeny, IA, USA). Library preparation and sequencing of composite RNA samples were conducted at Delaware Biotechnology Institute (DBI; Newark, DE, USA). NEXTflex Poly(A) Beads (Perkin-Elmer, Waltham, MA, USA) were used to isolate messenger RNA (mRNA) to limit both ribosomal RNA and prokaryotic mRNA contamination in the final sequence library. A pooled library was prepared using the NEXTflex Rapid Directional RNA-Seq Kit (Perkin-Elmer) following the manufacturers protocol. The library concentration was measured using a Qubit fluorometer (Thermo Fisher Scientific), and the library quality was assessed using a fragment analyzer (Advanced Analytical Technologies, Inc.). Sequencing of 101 base pair single-end reads from the pooled library was performed on a single lane on an Illumina HiSeq 2500 system (Illumina, San Diego, CA, USA).