GSM5426095: Sample 2_Wild-type zebrafish embryos 24 hours post-fertilisation; Danio rerio; Bisulfite-Seq
24hpf embryos were snap frozen in liquid nitrogen and stored at -80°C prior to DNA extraction. Genomic DNA (gDNA) was extracted from 24hpf embryos using the QIAGEN DNeasy Blood & Tissue Kit (QIAGEN, Chadstone, VIC, Australia) according to manufacturer instructions in two biological replicates. 1000ng of zebrafish gDNA and 5ng of unmethylated lambda phage DNA spike-in (Promega, Madison, WI, USA) was sonicated to an average insert size of 300bp. DNA was end repaired using Lucigen End-It DNA End-Repair Kit (LGC Biosearch Technologies, Novato, CA, USA) according to manufacturer instructions. Adapters were ligated to DNA overnight using NEXTFLEX Bisulfite-Seq Barcodes (PerkinElmer, Waltham, MA, USA). DNA was bisulfite converted using EZ DNA Methylation Kit (Zymo Research, Irvine, CA, USA) according to manufacturer instructions and PCR amplified (8 cycles). Library concentration was quantified through qPCR using KAPA Library Quantification Kit (Sigma-Aldrich, St. Louis, MO, USA) according to manufacturer instructions. The library was sequenced with 15% PhiX spike-in on Illumina HiSeq 10X platform using 150bp paired-end sequencing (high output mode).