Profiling of DNA Methylation during RANKL Induced Osteoclastogenesis using Whole-Genome Bisulfite Sequencing

Purpose: The goals of this study are to describe the DNA methylation profile during RANKL induced osteoclastogenesis. Methods: Primary bone marrow cells were isolated and treated with M-CSF (30ng/mL) to generate bone marrow macrophages (BMMs). BMMs were then used to generate preosteoclast (pOC) stimulated by M-CSF (30ng/mL) together with RANKL (100ng/mL) for 24 h and mature osteoclast (mOC) stimulated by M-CSF (30ng/mL) together with RANKL (100ng/mL) for 72 h or more. DNA methylation profiles of BMM, pOC and mOC were generated by deep sequencing, using Illumina NovaSeq 6000. Results: After acquiring of clean reads, we used Hisat2 for spliced mapping. We mapped about 40 million sequence reads per sample of the WGBS results. Saturation rate analysis was then performed evaluating the gene coverage rate along with read depth increase. The transcripts mapping to difference functional region of the genome was then analyzed. Circos software was used to map the consistency of samples with mouse genome on a chromosomal level. The quantification of transcript intensity was analyzed using RPKM (Reads Per Kb per Million reads). Conclusions: Our study represents the first high-throughput WGBS sequencing data of DNA methylation during osteoclastogenesis. Overall design: DNA methylation of ostoeclast at distinct differentiation stages were determined using WGBS.