#Materials and Methods ##Experimental Design Adult oysters from three founder populations (Dabob Bay, Fidalgo Bay, and Oyster Bay) grown for 19 months at Clam Bay, WA were used for this experiment. All oysters were held at 8°C for two weeks at the University of Washington prior to the experiment. Oyster from each population were subjected to acute temperature stress (38°C water for 1 hour; n=22), mechanical stress (1000 rpm x 5 min; n=22) or served as controls (mainted at 8°C ; n=22). All oysters were placed back in 8°C seawater and sampled at 1 hour post stress (n=8), 24 hours post stress (n=8), and six oysters were monitored daily for 14 days for mortality. Ctenidia and mantle tissue was dissected and stored in RNAzol RT (Molecular Research Center, Inc.) at -80°C for later analysis. ##RNA Isolation Ctenidia stored in 1mL RNAzol RT (Molecular Research Center, Inc.) at -80°C were thawed and homogenized with disposable plastic pestles (ThermoFisher). RNA was isolated according to the manufacturer's protocol for total RNA isolation. Briefly, 400μL of 0.1% DEPC-H2O was added to the homogenate, vortexed for 15 seconds, and incuabated at room temperature for 15 minutes. The samples were centrifuged for 15 minutes, 16,000g, at room temperature. After centrifugation, 750μL of the supernatent was transferred to a clean tube, added an equal volume of isopropanol (750μL), mixed, and incubated at room temperature for 15 minutes. The samples were centrifuged at 12,000g for 10 minutees at room temperature. The supernatatent was discarded and the pellets were washed with 500μL of 75% ethanol (made with 0.1% DEPC-H2O) and centrifuged at 4,000g for 3 minutes at room temperature. Repeated wash step one time. Ethanol was removed and pellets were resuspended in 100μL of 0.1% DEPC-H2O. Samples were quantified using a NanoDrop1000 (ThermoFisher) and stored at -80C. ##DNase Treatment and Reverse Transcription Prior to creating cDNA, total RNA was treated with DNase to remove reisdual genomic DNA (gDNA) using the Turbo DNA-free Kit (Ambion/Life Technologies). The manufacturer's rigorous protocol was followed. Briefly, 1.5μg of total RNA was treated in 0.5mL tubes in a reaction volume of 50μL. The samples were incubated with 1μL of DNase for 30 minutes at 37C. An additional 1μL of DNase was added to each sample and incubated at 37°C for an additional 30 minutes. The DNase was inactivated with 0.2 volumes of the inactivation reagent according to the manufacturer's protocol. Samples were quantified using a NanoDrop1000 (ThermoFisher) and verified to be free of gDNA via quantitative PCR (qPCR). Reverse transcription was performed using M-MLV Reverse Transcriptase (Promega) with oligo dT primers (Promega), using 500ng of DNased RNA. The RNA was combined with 0.25ug of primers in a volume of 74.75uL, incubated at 70°C for 5 minutes in a thermal cycler without a heated lid (PTC-200; MJ Research), and immediately placed on ice. Added 25.25uL to each RNA-primer mix of a master mix of 5x Reverse Transcriptase Buffer (Promega), 10mM each of dNTPs (Promega), and M-MMLV Reverse Transcriptase (50U/reactions). Samples were incubated at 42°C for 1hr and then at 95°C for 3 minutes in a thermal cycler without a heated lid (PTC-200; MJ Research) and then stored at -20°C. ## Quantitative PCR Quantitative PCR was completed using primers developed with the O. lurida Transcriptome V3 which can be found at XXXX. This transcriptome was annotated using verified SwissPro and GoSlim ID's. Following annotation, the transcriptome was filtered for stress responses and E values. Based on function, gene targets were visually selected from the remaining list. Sequence contigs were then pulled from the transcriptome using the seqinR package (XXXX,XXXX). These contigs were then run through NCBI primer blast to develop primers sequences for qPCR. Primers were then verified to work using standard qPCR method as described below. Once primers were verified to work, full sample sets from the treatments were run in replicates to determine differences in expression. Protocol for Quantitative PCR was as follows. A master mix for each target gene was created using Ssofast evagreen supermix (10ul per reaction) (BioRad, USA) with forward and reverse primers (0.5 ul each primer per per reaction) (Integrated DNA Technologies). Sample cDNA was diluted 1μL to 9μL so as to eliminate pipette error due to the small volume needed for analysis. In each well 11μL of master mix was added followed by 9μL sample. The samples were run on white qPCR plates (xxxx) with optically clear lids (XXXX) in a BioRad CFX Real Time Thermocycler (BioRad). The program run was for 40 cycles with reads occuring before and after the termination step (95°C for 10 min, 95°C for 30 sec, 60°C for 1 min, 72°C for 30 sec, repeat 40 times, termination 95°C for 1 minute). ##Statistical Analysis To generate data for use in statistical analysis, raw fluorescence data was exported from the qPCR runs on the BioRad CFX with no baseline subtraction. This data was then read as a CSV into R and formatted to run with the qPCR analysis package, qpcR (XXXX,2015). The qpcR package then fits the raw fluorescence data to the sigmoidal model (xxxx,xxxx) which generates efficiency and cycle thresholds for each sample. Taking these data, I then generated expression values using the equation >Expression=1/(1+Efficiency)^Cycle Threshold. This value was then normalized using expression values from a normalizing gene (XXXX). These values were then run through an ANOVA and a post-hoc Tukey's Honestly Significant Differences to determine significant differences between normalized expression values.