title: RNA-Seq of A. murrayi (whole body)
design: The whole sea pen tissues (pinnule and peduncle) were dissected, frozen in liquid nitrogen, and stored at -80C. The total RNA from the sea pen was extracted using the RNeasy Plant Mini Kit (QIAGEN), following the extraction steps according to the manufacturer's instructions. The RNA samples were eluted in DEPC-treated water in two 16 L steps. The RNA samples were treated with DNaseI enzyme (Invitrogen) to remove potential genomic DNA contamination. Subsequently, to clean impurities and residual DNaseI reaction remnants, they underwent a column cleanup process, following the instructions of the RNeasy Plant Mini Kit (QIAGEN). The concentrations of the RNA samples were estimated by fluorescence using the Qubit fluorometer (Thermo Fisher Scientific). The integrity of the RNA samples was confirmed by agarose gel electrophoresis (1%) stained with SYBR Safe (Invitrogen), and the RNA content was dried at 30C for 1 hour in speed vac mode V-AQ. The quality of RNA extracts (RIN value, fragment length distribution, and 28S/18S ratio) and their concentration were assessed by RNA concentrations were assessed using an Agilent 2100 bioanalyzer. After the QC procedures, mRNA was enriched using oligo(dT) beads. First, the mRNA was fragmented randomly by adding a fragmentation buffer. Then, the cDNA was synthesized using an mRNA template and random hexamers primer, after which a custom second-strand synthesis buffer (Illumina), dNTPs, RNase H, and DNA polymerase I were added to initiate the second-strand synthesis. Second, after a series of terminal repairs, A ligation, and sequencing adaptor ligation, the double-stranded cDNA library was completed through size selection and PCR enrichment. The GenOne Biotechnologies company (Brazil) performed cDNA library preparation and sequencing (150 bp paired-end reads). To exclude low-quality sequences, the raw reads were filtered by removing (i) reads with more than 50% of the qualities of the base lower than 5, (ii) reads only containing the adaptor sequence, and (iii) reads containing more than 10% of unknown nucleotide N.