Approximately 1000ng of DNA was purified from zebrafish embryos and digested with MspI, then end repaired using Klenow -exo (New England Biolabs ID: R0106S and M0212S). Methylated barcodes (BioO Scientific ID: 511912) were ligated to each sample using T4 DNA ligase (New England Biolabs ID: M0202S), and bisulfite treated according to manufacturer’s protocol (Zymo Research ID: D5005). Following bisulfite treatment libraries were PCR amplified for 20 cycles using Platimun Taq (ThermoFisher ID: 10966018) and the standard primers and protocol from BioO Scientific methylated barcodes kit. High-throughput sequencing was by Illumina’s protocol for either 50bp single end or 125bp paired end runs on an Illumina HiSeq 2500.