GSE132230
Profiling of mRNA, lncRNA and circRNA in Apoptotic Bodies during Osteoclastogenesis by RNA-seq
Purpose: The goals of this study are to describe the RNA contents in apoptotic bodies (ABs) derived from osteoclasts at different stages and to compare the differences among those ABs from difference cell stages. The biological function of these ABs are also predicted and evaluated. 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. RNA 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 to the mouse genome (GRCm38). 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 Fragments Per Kilobase of exon modelper Million mapped fragments (FPKM). Conclusions: Our study represents the first high-throughput deep RAN sequencing data of cell derived apoptotic bodies. The biological prediction and analysis also revealed that ABs derived from osteoclast of different stages have distinct biological role. Followed by low throughput validation and detailed experiments, we identified that pOC derived ABs are more pro-angiogenic while mOC derived ABs are more pro-osteogenic. Overall design: Apoptotic bodies derived from ostoeclast at distinct differentiation stages were isolated and undergo with deep RNA-seq to reveal their inner RNA content.