Protocol for genotyping cephalopod sex using a skin swab and quantitative PCR

Summary The coleoid cephalopods (octopus, cuttlefish, and squid) are emerging model organisms for neuroscience, development, and evolutionary biology. Determining their sex early in life is critical for population management and controlled experiments. Here, we present a protocol to non-invasively determine the sex of multiple cephalopod species as young as 3 hours post-hatching using a skin swab and quantitative PCR (qPCR). We describe steps for designing qPCR primers, swabbing live animals, extracting DNA, running the qPCR, and analyzing the results. For complete details on the use and execution of this protocol, please refer to Rubino et al.1 Highlights Swab live cephalopods as early as 3 hours post-hatching Extract DNA from cephalopod skin swabs Perform qPCR-based sex determination Design and validate qPCR primers for new species Competing Interest Statement The authors have declared no competing interest.