Unraveling the whole genome DNA methylation profile of zebrafish kidney marrow through Oxford Nanopore sequencing

Zebrafish is a widely used model organism for investigating human diseases, including hematopoietic disorders. However, a comprehensive methylation baseline for zebrafish primary hematopoietic organ, the kidney marrow (KM), is still lacking. We employed Oxford Nanopore Technologies (ONT) sequencing to profile DNA methylation in zebrafish KM by generating four KM datasets, with two groups based on the presence or absence of red blood cells. Our findings revealed that blood contamination in the KM samples reduced read quality and altered methylation patterns. Compared with whole-genome bisulfite sequencing (WGBS), the ONT-based methylation profiling can cover more CpG sites (92.4% vs 70%-80%), and exhibit less GC bias with more even genomic coverage. And the ONT methylation calling results showed a high correlation with WGBS results when using shared sites. This study establishes a comprehensive methylation profile for zebrafish KM, paving the way for further investigations into epigenetic regulation and the development of targeted therapies for hematopoietic disorders. Wild-type Tubingen (TU) zebrafish lines were obtained from the Zebrafish International Resource Center (ZIRC, USA). The study received approval from the Committee on the Use of Laboratory and Research Animals (CULATR) at the University of Hong Kong (HKU). Kidney marrow from 8-month-old WT zebrafish was collected, dissociated in 0.9X PBS via pipetting, and filtered through a 40 µm nylon cell strainer (Corning, NY, USA). Some samples underwent treatment with eBioscience™ 1x RBC Lysis Buffer (Invitrogen, MA, USA) to lyse red blood cells, and these samples were designated as the KM group. Samples without red blood cell lysis were categorized as the KMB group. The resulting cells were centrifuged at 500g for 5 minutes, resuspended, filtered, and washed twice with 0.9X PBS for DNA extraction. Genomic DNA was extracted using QIAamp DNA Kits (Qiagen, Germany).