Name: GSM5677916
Instrument: HiSeq X Ten
Strategy: OTHER
Source: GENOMIC
Selection: other
Layout: PAIRED
Construction protocol: ACE-seq library preparation for the DNA hydroxymethylation profiling was performed as previously described {Schutsky et al, 2018 PMID: 30295673;Wang et al, 2021 PMID: 32822044}. Briefly, 100 ng of genomic DNA extracted from sea urchin, lancelet and zebrafish embryos and adult tissues was spiked with 1ng of CpG methylated λ phage DNA (Wisegene) and 0.5 ng of all-C hydroxymethylated pUC19 plasmid DNA (Wisegene) and sonicated to ~300 bp fragments using a M220 focused ultrasonicator (Covaris) with the following parameters: peak incident power, 50W; duty factor, 20%; cycles per burst, 200; treatment time, 75 sec. Sonicated DNA (125 ul total volume) was concentrated to 16.6 ul using AMPure beads (1 volume DNA : 2 volumes AMPure beads). 5hmC protection reaction was performed using β-glucosetransferase (10U/ul, New England Biolabs) in Cutsmart Buffer according to manufacturer's instructions at 37°C for 1 hr, followed by 10 min at 65°C. The DNA was denatured in DMSO at 95°C for 10 min and immediately placed on dry ice for 2 mins allowing the samples to freeze. C and 5mC deamination reaction was performed using the APOBEC3A enzyme (NEBNext® Enzymatic Methyl-seq Kit, New England Biolabs) with the following ramping conditions: 4°C for 10 min, 4°C - 50°C 2:15 min per degree of the ramp, 50°C for 10 min. Deaminated DNA was then purified using AMPure beads (1 volume DNA : 1 volume AMPure beads) and subjected to low input library preparation using Accel-NGS Methyl-Seq DNA kit (Swift Biosciences). Briefly, DNA was denatured and subjected to an adaptase reaction, followed by primer extension, adapter ligation, and 15 cycles of indexing PCR. Library size and consistency was determined by the Agilent 4200 Tapestation system. The libraries were quantified using the KAPA library quantification kit (Roche).