Callinectes sapidus anterior gill at 35ppt sample #1
Adult, intermolt Callinectes sapidus were held in the laboratory as described previously (Mitchell and Henry, 2014). Briefly, animals obtained from commercial fishers in East Point, FL, USA were shipped to Auburn University, AL, USA and held in 570 L recirculating tanks of full-strength seawater (35‰) at 24 °C for three weeks, with salinity being monitored daily. Following this acclimation period, one anterior (G3) and one posterior (G7) gill was removed from each of three individuals. Another set of anterior and posterior gills was subsequently sampled from individuals transferred to 10‰ for 1 week after being maintained at 35‰ for three weeks. In both cases, gills were transferred immediately to 3 mL of RNAgents Denaturing Solution (Promega, Madison, WI, USA) and kept on ice until homogenization. Total RNA was extracted from the 12 C. sapidus gills (n = 3 for four treatments: anterior and posterior gills at 35‰ and 10‰) using the RNAgents Total RNA Isolation System (Promega) as described previously (Havird et al., 2014; Mitchell and Henry, 2014). Protocols largely followed those used for previously published crustacean transcriptomes generated in our laboratories (Havird et al., 2014). Briefly, the SMART cDNA construction kit (ClonTech Laboratories Inc., Mountain View, CA, USA) was used as described in (Havird et al., 2014), with cDNA libraries being sent to the HudsonAlpha Institute for Biotechnology’s Genomic Services Lab (Huntsville, AL, USA). From these cDNA libraries, 100 bp paired-end reads were generated on an Illumina HiSeq 2000 (Illumina, San Diego, CA, USA), with each sample being sequenced on 1/6th of a lane.