Methylome data derived from maternal-zygotic DNA methyltransferase 3aa−/− zebrafish

Genomic DNA methylation is an epigenetic marker mediated by DNA methyltransferases (Dnmts); in vertebrates, it comprises of a maintenance DNA methyltransferase, Dnmt1, and two de novo DNA methyltransferases (Dnmt3a and Dnmt3b). In zebrafish, there are two homologs of the mammalian Dnmt3a: Dnmt3aa and Dnmt3ab. A knockout (KO) mutant of zebrafish dnmt3aa was generated using the CRISPR/Cas9 genome-editing system as a new model for DNA methylation research. Since zebrafish dnmt3aa KO mutants were viable and fertile, a maternal-zygotic dnmt3aa deficient mutant (MZdnmt3aa) was generated. We performed whole-genome bisulfite sequencing (WGBS) to reveal the DNA methylation profile using this mutant and identified genomic regions with altered CpG methylation as differentially methylated regions (DMRs) in this mutant compared to those in the wild-type fish. We provided novel raw and processed datasets using the MZdnmt3aa KO mutant, and the raw data of WGBS are available through the Gene Expression Omnibus (GEO), accession number GSE178690.


a b s t r a c t
Genomic DNA methylation is an epigenetic marker mediated by DNA methyltransferases (Dnmts); in vertebrates, it comprises of a maintenance DNA methyltransferase, Dnmt1, and two de novo DNA methyltransferases (Dnmt3a and Dnmt3b). In zebrafish, there are two homologs of the mammalian Dnmt3a: Dnmt3aa and Dnmt3ab. A knockout (KO) mutant of zebrafish dnmt3aa was generated using the CRISPR/Cas9 genome-editing system as a new model for DNA methylation research. Since zebrafish dnmt3aa KO mutants were viable and fertile, a maternal-zygotic dnmt3aa deficient mutant (MZ dnmt3aa ) was generated. We performed whole-genome bisulfite sequencing (WGBS) to reveal the DNA methylation profile using this mutant and identified genomic regions with altered CpG methylation as differentially methylated regions (DMRs) in this mutant compared to those in the wild-type fish. We provided novel raw and processed datasets using the MZ dnmt3aa KO mutant, and the raw data of WGBS are available through the Gene Expression Omnibus (GEO), accession number GSE178690. ©

Value of the Data
• A new epigenomic dataset is provided for the MZ dnmt3aa −/ − mutant to study DNA methylation in zebrafish. • Since there are two dnmt3a genes in zebrafish, dnmt3aa and dnmt3ab , the WGBS data for the zebrafish MZ dnmt3aa −/ − mutant is useful for the detailed analysis of target genomic loci by Dnmt3a in vertebrates. • Enables genome-wide assessment in zebrafish regarding Dnmt3aa-mediated changes in DNA methylation. • Our dataset is useful for studies examining the effects of DNA methylation on transcription because hypomethylated differentially methylated regions (HypoDMRs) were able to be detected that overlap not only the transcription start sites and gene bodies, but also the transcription termination sites.

Data Description
Here, we present WGBS data for WT and MZ dnmt3aa −/ − zebrafish. A deletion of dnmt3aa gene was generated using CRISPR/Cas9. The dnmt3aa genomic DNA sequence and To investigate the DNA methylation profile changed by dnmt3aa knockout, we performed WGBS and identified hypoDMRs. The data is as follows: Supplementary

Zebrafish experiments and generation of dnmt3aa −/ − mutant fish using CRISPR/Cas9 system
Adult zebrafish and zebrafish larvae were maintained as described previously [2] . Genome editing using CRISPR/Cas9 system was performed as previously described [3] . Briefly, the pDR274 vector (Addgene Plasmid 42250) was digested with Bsa I, and annealed oligonucleotides for sgR-NAs were cloned into the pDR274 vector. The sequences of oligonucleotides for sgRNAs are listed in Table 1 . The sgRNA expression vectors were digested with Dra I, and the sgRNAs were synthesized using the MAXIscript TM T7 Kit (Invitrogen, Thermo Fisher Scientific). The synthesized sgRNAs were purified using the mirVana TM miRNA Isolation Kit (Invitrogen, Thermo Fisher Scientific). Moreover, the Cas9 expression vector (Addgene Plasmid 51815) was linearized using Not I, and Cas9 mRNA was synthesized using the mMESSAGE mMACHINE TM SP6 Kit (Invitrogen, Thermo Fisher Scientific). A mixture of three types of sgRNAs for the zebrafish dnmt3aa gene and Cas9 mRNA at appropriate concentrations was injected at the one-cell stage.
To identify the mutations, the target site of the CRISPR/Cas9 system on dnmt3aa was amplified using TaKaRa Ex Taq® (Takara Bio) with the primers listed in Table 2 . The amplicons were purified using the QIAquick PCR Purification Kit (QIAGEN) and then sequenced using the BigDye TM Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Thermo Fisher Scientific) and the primers listed in Table 2 .

Genotyping of MZ dnmt3aa −/ − zebrafish
The amputated fins of MZ dnmt3aa −/ − mutants were digested overnight in lysis buffer (100 mM NaCl, 20 mM Tris-HCl pH 8, 50 mM EDTA pH 8, 0.5% SDS, and 36 ug/ml Proteinase K) at 55 °C. Proteinase K was inactivated by incubation at 98 °C for 10 min. A heteroduplex mobility assay (HMA) was performed to detect the dnmt3aa mutations. The target site of the CRISPR/Cas9 system on exon 1 of the dnmt3aa gene was amplified using BIOTAQ TM DNA Polymerase (Meridian Bioscience). The primers used are listed in Table 2 . The amplicons were analyzed using polyacrylamide gel electrophoresis.

Whole-genome bisulfite library preparation and sequencing of zebrafish sample
Two pools, WT and MZ dnmt3aa −/ − , were prepared, each consisting of 10 larvae at 2 dpf. Genomic DNA was then extracted by digesting each pool with proteinase K and sodium dodecyl sulfate. DNA samples were prepared by mixing zebrafish genomic DNA and unmethylated lambda DNA (Promega) as spike controls at a ratio of 10 0 0:1. DNA samples (10 0 ng) were subjected to bisulfite treatment using an EZ DNA Methylation-Gold Kit (Zymo Research). Bisulfitetreated DNA samples (50 ng) were subjected to ten cycles of PCR using random primers from the TruSeq DNA Methylation Kit (Illumina) to add adapters. Libraries with adapters were sequenced on a HiSeq X Five Sequencing System (Illumina) with 150 bp single-end reads. WGBS data were obtained from a single experiment. Bisulfite treatment, library preparation, and sequencing were performed by Takara Bio, Inc. (Shiga, Japan).

WGBS data processing of zebrafish sample
The read sequences in FASTQ format were trimmed by removing six bases from the random primers of the TruSeq DNA Methylation Kit (Illumina) and the last 50 bases of low-quality reads. After trimming, the reads were aligned to the zebrafish (danRer10) and lambda phage genomes using Bismark (version 0.10.1) [4] and Bowtie (version 1.0.0) [5] . Once aligned, reads corresponding to PCR duplicates were removed and methylation calls were performed. The bisulfite conversion rates based on spike control for all samples were over 99%, and the error rates were as follows: WT, 0.3% and MZ dnmt3aa −/ − mutant, 0.3%. Methylated cytosine sites were identified using binomial distribution with a false discovery rate ≤ 0.05, calculated using the Benjamini-Hochberg (BH) method and read depth ≥ 5. Data analysis was performed using Takara Bio, Inc. (Shiga, Japan), as previously described [6] .

Identification of HypoDMRs with TTSs, TSSs, gene bodies, and intergenic regions
The genomic positions of HypoDMRs overlapping with TTSs, TSSs, gene bodies, and intergenic regions were determined using bedtools intersect (version 2.28.0) [8] . HypoDMRs that overlapped with TTSs and the other three (TSSs, gene bodies, and intergenic regions) were included in the HypoDMRs that overlapped with TTSs. HypoDMRs that overlapped with TSSs and the other two (gene bodies and intergenic regions) were included in HypoDMRs that overlapped with TSSs. HypoDMRs that overlapped with both gene bodies and intergenic regions were included in the HypoDMRs that overlapped with gene bodies. HypoDMRs that did not overlap with TTSs, TSSs, or gene bodies were included in the HypoDMRs that overlapped with an intergenic region. In addition, genes with the HypoDMRs were extracted concurrently.

Ethics Statements
All zebrafish experiments were carried out with approval from the Hiroshima University Animal Research Committee (Permit Number: F18-2-7).

Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data Availability
Identification of transcription termination defects at DNA hypomethylated transcription termination sites in DNA methyltransferase 3a-deficient vertebrates [