PS 218
Total RNA was extracted from sponge tissue samples using TRIzol Reagent and the PureLink RNA Mini Kit (Invitrogen) with on-column PureLink DNase treatment, following the manufacturers protocol. Extracted total RNA was successively cleaned-up with the RNeasy MinElute cleanup kit (Qiagen), also following the manufacturers protocol. Final RNA concentration was checked with Qubit RNA BR Assay Kit and Qubit 2.0 Fluorometer (Invitrogen, CA, USA). RNA was stored at -80 C until further analysis. Quality of the extracted RNA was further assessed on a Qubit 3.0 Fluorometer (Invitrogen, CA, USA) with the RNA BR Assay Kit and the TapeStation RNA ScreenTape at Competence Centre for Genomic Analysis (CCGA) in Kiel where the library preparation and sequencing took place. Ribosomal RNA from prokaryotes and eukaryotes was removed using the Illumina Ribo-Zero Plus rRNA depletion kit. Eighteen cDNA libraries were then prepared with the TruSeq stranded total RNA kit according to the pre-release protocol and sequenced on one lane of the Illumina NovaSeq6000 S1 FlowCell, using a pair end (150bp length) sequencing strategy.