Ribotag analysis of proprioceptive dorsal root ganglion neurons in cell culture. Ribotag analysis of proprioceptive dorsal root ganglion neurons in cell culture

We analyzed ribosome-associated RNA (raRNA) in dorsal root ganglion neurons in TrkC+ proprioceptive dorsal root ganglion neurons in cell culture. Overall design: All DRG neurons from young adult (3-6 mo) male mice expressing Rpl22HA/HA Runx3-Cre+ and age/sex matched Cre negative littermates (Rpl22HA/HA Runx3-Cre-) were harvested using density purification on Percoll and grown for 24h in serum-supplemented F12 medium. Ribosome-associated RNA was isolated using immunoprecipitation targeting the HA epitope. 10% of pre-immunoprecipitation lysates were used for input samples reflecting total RNA profile of the tissue. Cre negative samples were sequenced to control for background. N=3 for all four groups (Pulldown/Cre+, Pulldown/Cre-, Input/Cre+, Input/Cre-).

本研究在细胞培养体系中，针对TrkC阳性（TrkC+）本体感觉背根神经节（dorsal root ganglion, DRG）神经元内的核糖体结合RNA（ribosome-associated RNA, raRNA）开展分析。实验整体设计如下：选取3~6月龄年轻成年雄性小鼠，分别获取表达Rpl22HA/HA Runx3-Cre+的背根神经节神经元，以及年龄、性别匹配的Cre阴性同窝仔鼠（Rpl22HA/HA Runx3-Cre-）的背根神经节神经元；所有样本均通过Percoll密度纯化法分离获取，随后在添加血清的F12培养基中培养24小时。通过靶向HA表位的免疫沉淀技术分离核糖体结合RNA；取10%免疫沉淀前的裂解液作为输入样本，用以反映组织的总RNA表达谱。设置Cre阴性样本进行测序以扣除背景信号。四个实验组（下拉/Cre+、下拉/Cre-、输入/Cre+、输入/Cre-）每组的生物学重复数均为3（N=3）。