title: RNA seq of pelagic crustacean zooplankton communicty
design: Direct RNA sequencing was performed using the Direct RNA Sequencing protocol with the SQK-RNA001 kit (ONT, USA, 2018a), with around 700 ng of poly-A RNA used as input for library preparation (instead of the recommended 500 ng). The poly-A RNA was ligated to 1.0 L reverse transcription (RT) adapter using T4 DNA ligase (NEB 0202). A reverse transcription reaction was performed on the RT ligated RNA, using SuperScript III Reverse Transcriptase (Invitrogen), and left to incubate in a thermal cycler at 50C for 50 minutes and 70C for 10 minutes. The products were then purified using 72 L of CleanNGS beads (CleanNA), followed by a washing step in 70% ethanol. Sequencing adapters preloaded with motor proteins were then ligated onto the cDNA and the final library was cleaned up once more using 40 l of the CleanNGS beads.