GSM4500810: 24hpf_EM-seq_rep2; 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. EM-seq library construction was performed using the NEBNext Enzymatic Methyl-seq Kit (New England BioLabs, Ipswich, MA, USA) according to manufacturer instructions with modifications. 0.02ng of unmethylated lambda phage DNA (Promega, Madison, WI, USA) and 0.0001ng of pUC19 plasmid methylated at 100% of CpG sites (New England BioLabs, Ipswich, MA, USA) were used as spike-in controls to determine the efficiency of APOBEC deamination and TET2 oxidation, respectively. Briefly, 200ng of zebrafish gDNA was sonicated to an average insert size of 300bp. Input DNA concentration was selected according to the optimal input amount as recommended by the manufacturer. Sonicated DNA was end repaired followed by ligation of adapters to DNA overnight using NEXTFLEX Bisulfite-Seq Barcodes (PerkinElmer, Waltham, MA, USA). DNA was treated with TET2 for one hour. Following TET2 oxidation DNA was denatured with 0.1M NaOH then treated with APOBEC for three hours. DNA was 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. 300pmol of the combined libraries with 15% PhiX spike-in was sequenced on the Illumina HiSeq 10X platform using 150bp paired-end sequencing (high output mode) EM-seq (enzymatic methylation sequencing)