Adult oysters n=3 (191.93 g+/- 56.45) (Mean +/- SD) were anesthetized by overnight holding in tanks (6L) with MgCl2 (50g/l). The following day, mantle tissue was taken and placed at -80C and oysters placed back into seawater. After 7 days, oysters were subjected to a 40C, 1 hour acute heat shock. Tissue samples were taken immediately following heat shock exposure and placed at -80C.
Genomic DNA was isolated using DNAzol (Molecular Research Center) from mantle tissue taken prior to and post heat shock (three oysters). Methylation enrichment was performed using the MethylMiner Kit (Invitrogen) following the manufacturer’s instructions. Specifically, DNA was sheared by sonication on a Covaris S2 (Covaris) (parameters: 10 cycles at 60 seconds each, duty cycle of 10%, intensity of 5, 100 cycles/burst). Sheared DNA was used as input DNA and incubated with MBD-Biotin Protein coupled to M-280 Streptavidin Dynabeads following the manufacturer’s instructions (MethylMiner (Invitrogen)). Enriched, methylated DNA was eluted from the bead complex with 1M NaCl and purified by ethanol precipitation. DNA was further purified using PCR purification columns (Qiagen) prior to labeling.
A custom DNA tiling array containing 697,753 probes covering 9158 full-length C. gigas genes including 2kb upstream of the start site was used. Probes were designed using an interval size of 100bp and a window size of 25bp.
Samples were labeled using the NimbleGen Dual-Color DNA Labeling Kit and the arrays were processed according to the manufacturer’s recommendations (Roche NimbleGen, Madison, Wisconsin) and imaged at 5um using a GenePix 4000B microarray scanner (Molecular Devices, Sunnyvale, CA).
Fluorescent intensities from the Cy3 (input) and Cy5 (IP) channels were extracted from each array using DEVA 1.2.1 (Roche NimbleGen; Madison, WI) and the output was processed using the Bioconductor package, Ringo (Toedling 2007). Control probes were discarded and the data from each array was initially normalized using the Tukey biweight mean method. Paired pre- and post-heat shock log2(IP/Input) values were adjusted using a linear regression fit (setting m = 1 and b = 0) to compensate for variation in data compression between arrays. To determine differential methylation, the input channels from the paired datasets were loess normalized and the results were used to calculate a threshold value, T, equivalent to 3 s.d from the mean. Applying this back to each pairwise comparison of pre- and post-heat shock log2(IP/Input) ratios, differential methylation was assigned to a given probe when the absolute value of the paired ratio exceeded T. For our studies, we concentrated on runs of at least 3 adjacent probes identified as differentially hyper- or hypo-methylated and localized to a gene body (plus 1000bp upstream). The R programing language  was used for data processing, including the generation of bedGraph and GFF tracks for visualization in IGV (Robinson 2011).