Small RNA-Seq in epididymis of wild type and Alkbh3-/- mice. Small RNA-Seq in epididymis of wild type and Alkbh3-/- mice

Total RNA (100 μg) were denatured at 70 °C for 5 min and separated by a 15% TBE-Urea gel with 20/100 or 10/60 oligo length standard ladder (Integrated DNA Technologies, Coralville, IA). After visualized by SYBR Gold (Ref. S11494 from Life Technologies), tRFs in gels were cut according to sizes and recovered by small RNA PAGE recovery kit (Zymo Research, Irvine, CA, USA). The recovered tRFs was used to establish the library by NEB small RNA library preparation kit (E7330S). Overall design: Examination of small RNA difference in epididymis between wild type and Alkbh3-/- mice

取总RNA 100 μg，于70 ℃变性5 min，随后采用15% TBE-尿素变性凝胶进行电泳分离，电泳时加入20/100或10/60寡核苷酸长度标准分子量梯（购自Integrated DNA Technologies公司，位于美国爱荷华州科勒尔维尔）。经SYBR Gold染料（Life Technologies公司，货号S11494）染色显影后，根据片段大小切取凝胶中的tRNA衍生片段（tRFs），并使用小RNA PAGE回收试剂盒（Zymo Research公司，美国加利福尼亚州欧文市）完成样品回收。回收得到的tRFs通过NEB小RNA文库制备试剂盒（货号E7330S）构建测序文库。实验整体设计：探究野生型与Alkbh3基因纯合敲除（Alkbh3-/-）小鼠附睾组织中小RNA的表达差异。