RNA-seq of Crassostrea virginica female gonad
Simultaneous DNA and RNA extraction was conducted for female gonad and sperm samples using the Zymo Quick-DNA/RNA Microprep Plus kit (Cat # D7005). A total of 10-15 mg of frozen gonad tissue and 50 L of sperm in seawater solution were used as inputs for females and males, respectively. Manufacturers instructions were used, with the following exceptions. After addition of 300 L DNA/RNA Shield (1X), 30 L PK Digestion Buffer, and 15 L Proteinase K, female samples were incubated at 55C overnight. Four volumes of DNA/RNA Lysis Buffer (200 L) were added to sperm samples. All centrifugation steps were conducted at 16,000 rcf. DNase I digestion was performed for RNA. Concentration of eluted DNA and RNA were verified with Qubit dsDNA High Sensitivity or RNA High Sensitivity assays (Thermo Fisher Scientific), respectively, and quality confirmed on a BioAnalyzer.