RNA-seq of Cassiopea: round 1 control
For RNAseq, 3/4 of the rhopalia from each animal (approximately 12 per animal) were pooled for each sequencing sample. For qPCR, 6 rhopalia were amputated, and 2 rhopalia were pooled per animal in 3 separate extraction. After amputation, specimens were transferred into Stellar Scientific Deadbolt 1.5 mL tubes, and were immediately flash frozen in liquid nitrogen and stored in a -80C freezer. For extraction, tubes were seated on a liquid-nitrogen-cooled mortar and were homogenized physically with a cooled pestle (Thomas Scientific). The Plant Spectrum Total RNA kit (Sigma) was used to isolate the RNA from the homogenized specimens. Ground tissues were immediately vortexed in 1 mL of Lysis buffer, double the amount specified by the kit. After material from the binding solution was applied to the columns, they were treated with a DNase digestion (Sigma). When needed, samples were additionally cleaned and concentrated using the Zymo Research RNA Clean and Concentration Kit and eluted into 13 uL. Paired-end 150 bp RNA sequencing was performed at the UC Berkeley sequencing core facility using the Illumina NovaSeq 6000.