Sequencing Full-Length Oyster Nuclear Receptors

Oligonucleotide primers for sequencing the full length of the oyster NR sequences CgRXR, CgRAR and CgPPAR (S1 Table) were designed with Primer-Blast at NCBI [46 (link)] based on C. gigas genomic DNA data [47 (link)] for each gene (GenBank: CGI_10004075 (CgRXR); CGI_10028545 (CgRAR); CGI_10011509 (CgPPAR)). Total RNA was extracted from frozen whole adults and mixed embryo oyster individuals (embryo toxicity test). Extraction, DNA digestion, reverse transcription, and amplicon visualization and purification were performed as described previously [24 (link)]. Amplicons were obtained by RT-PCR under the following conditions: 95°C for 2 min, thirty cycles of 95°C for 30 s, 60°C for 30 s, 72°C for 2 min, and a final extension at 72°C for 5 min, and cloned into a pGEM-T Easy vector (Promega). Vectors were purified using the PureLink Plasmid miniprep kit (Invitrogen), and were subsequently sequenced by Eurofins MWG Operon (Cologne, Germany). Each identified receptor sequence was confirmed by three independent successful cloning attempts.
The obtained coding DNA sequences (CDS) for all receptors (GenBank accession numbers: CgRXR1: KX590999; CgRXR2: KX591000; CgRAR: KX591001; CgPPAR: KX591002) were aligned to their associated genomic DNA sequence to identify isoforms and their intron/exon structure.