title: RRBS of Gill
design: 500 ng of purified  genomic DNA was simultaneously double digested for all 80 samples in a 96  well plate using the two frequent cutter enzymes AseI and NsiI (NEB-R0127L  and NEB-R0526L; Van Gurp 2016). Digested DNA was subsequently ligated to  custom y-yoked methylated sequencing adapters (Glenn et al., 2016) using a T4  DNA ligase (B9000S; New England Biolabs) with additional rATP to ensure  ligation of custom adapters. Digested and methylated adapter-ligated DNA was  bisulfite converted in a 96 well plate using the Zymo Research EZ DNA  Methylation-Lightning kit (D5031; Zymo Research) with a 16 minute  L-desulphonation step. Bisulfite converted DNA was then tagged and amplified  with Illumina adapters using 16 cycles of PCR (Glenn et al., 2016). Amplified  libraries were size selected to 300-600 base-pairs (bp) using the Zymo  Research Select-A-Size DNA clean & Concentrator (D4080; Zymo  Research). Size selection was  confirmed using the Agilent Bioanalyzer DNA high sensitivity chip (5067-4626;  Agilent Technologies). Libraries were subsequently pooled and sequenced with  a 10% PhiX spike-in on a full flow cell of the Illumina NextSeq 500 with 75  bp PE reads. Sequencing was completed at the North Carolina State Universitys  Genomic Sciences Laboratory (Raleigh, NC).