title: BS-seq of juvenile Panopea generosa
design: DNA was extracted using the Qiagen DNeasy according to manufacturer's instructions with slight modifications. Briefly, ~25mg samples were incubated with lysis buffer (ATL) and proteinase K at 56C for 1hr, mixing several times throughout. Samples were incubated an additional 10 minutes following addition of buffer AL. Prior to addition to the columns, 200l of 100% ethanol was added to each sample and instructions were followed to complete the extraction. Samples were eluted in 125-200l of buffer AE.  Samples were quantified using Qubit BR dsDNA kit and quality checked using 1.5% TAE gel. Reduced representation bisulfite sequencing (RRBS) libraries were generated. DNA samples were spiked with 0.5% (w/w) unmethylated lambda DNA (Promega cat: D1501) incubated with MSPI (20U l-1 NEB cat: R0106L) and 10x NEBuffer2 (NEB cat: B7002S) for digestion at the cut site of CC in the sequence CCGG.  Digested samples were processed with the EZ DNA Methylation-Gold Kit (Zymo Research Catalog Nos. D5005) for bisulfite conversion according to manufacturers instructions with a 30l input sample and a 12l elution. Sample quality and fragment size was assessed using the Agilent 2011 Bioanalyzer RNA pico chip. Samples passing quality control were then used for Illumina library preparation Illumina TurSeq DNA Methylation library prep kit according to manufacturers instructions. Samples were prepared for multiplex sequencing with 12 samples on a single lane using the Illumina DNA Methylation Kit Barcodes (Illumina Cat #: EGIDX81312).  The resulting libraries were quality controlled using the Qubit dsDNA high sensitivity kit for quantification and the Agilent Bioanalyzer DNA High Sensitivity Kit.  Libraries that passed QC were pooled in equal molar concentrations and submitted to Genewiz for sequencing on the Illumina HiSeq 2500 (2x100bp).