RNA-seq of dinoflagellate
Three replicate samples were incubated with or without ZnO NPs for 1, 5 or 24 h, and total RNA was extracted using an RNeasy Plant Mini Kit (Qiagen, Hulsterweg, Germany). Three replicate samples pooled immediately after treatment with IMK medium were used for library preparation for the "0 h" group. Chilled cells in a microtube were treated with 100 L of acid-washed glass beads (7101,180 m; SigmaAldrich, MO, USA) and 450 L of Buffer RLT containing 10% (v/v) b-mercaptoethanol and vortexed for 1 min. Total RNA was extracted with the kit according to the manufacturers instructions. The quality of the RNA was verified using an Agient TapeStation (Agilent Technologies, CA, USA), and the quantity of RNA was determined by using a NanoDrop spectrophotometer (Thermo Fisher Scientific, MA, USA).A total of 4 g of total RNA from each sample was used for library preparation using a TruSeq stranded mRNA Sample Preparation Kit (Illumina, CA, USA) according to the manufacturers protocol. Three replicates from the same conditions were tagged with the same index sequence. After qualification and quantification of the cDNA libraries, the libraries were pooled at the same concentration.