Distinct 5-methylcytosine profiles in poly(A)RNA from mouse bone marrow derived mast cells

Purpose: To transcriptome widely reveal 5-mC regulation by Tet2, we performed bisulfite-sequencing of mRNAs from Tet2-deficient BMMCs and the control cells.Methods: Mouse BMMCs were generated from bone marrow cells in RPMI-1640 medium with recombinant mouse IL-3 and SCF. BMMCs were stained to confirm the surface expression of Fc?RI and c-Kit. Cells with purity >97.5% were used for RNA isolation. Results: We carried out an unbiased global analysis of m5C in poly(A)RNA of mouse bone marrow derived mast cells (BMMCs).We show that there were indeed much more mCs in Tet2-deficient group compared with the control, more than half of which located in genic region. Furthermore, we observed that although much less mCs distributed in the three elements of exon regions, more mCs located in 3`-UTR for Tet2-deficient group compared with the control. Overall design: RNA was isolated from Tet2 deficient bone marrow derived mast cells (BMMC) and control cells and RNA-Bisulfite sequencing was performed. We performed two biological replicates and each contain two technical replicates

研究目的：为从转录组层面全面揭示Tet2对5-甲基胞嘧啶（5-mC）的调控作用，我们对Tet2缺陷型骨髓来源肥大细胞（Bone Marrow Derived Mast Cells, BMMCs，以下简称BMMCs）及对照细胞的mRNA进行了亚硫酸氢盐测序。
实验方法：BMMCs由小鼠骨髓细胞在添加重组小鼠IL-3与SCF的RPMI-1640培养基中诱导分化获得。通过染色验证细胞表面FcεRI与c-Kit的表达水平，选取纯度高于97.5%的细胞进行RNA提取。
实验结果：本研究对BMMCs的聚腺苷酸化RNA（poly(A) RNA）开展了无偏倚的全局m⁵C修饰分析。结果显示，Tet2缺陷组的mC修饰位点数量显著多于对照组，其中超过一半的修饰位点位于基因区域。进一步观察发现，尽管外显子区域的三个元件中mC修饰分布较少，但Tet2缺陷组中位于3'-UTR区域的mC修饰位点数量较对照组显著增多。
整体实验设计：分别从Tet2缺陷型BMMCs及对照细胞中提取RNA，进行RNA亚硫酸氢盐测序。本实验设置2次生物学重复，每一次生物学重复包含2次技术重复。