KRBP72 eCLIP and partially edited A6 in T. brucei brucei KRBP72 RNAi cells

PCF T. brucei KRBP72-PTP tagged KRBP72 RNAi cell lines were grown in the presence or absence of 4 ug/mL doxycycline for 3 days. RNA was isolated using Trizol followed by phenol/chloroform extraction and ethanol precipitation. RNA samples were treated with DNase and phenol/chloroform extracted and ethanol precipitated. Two biological replicate experiments were performed, and western blot was used to validate the level of KRBP72 knockdown. cDNA was generated from DNase-treated RNA using gene-specific primers.

将带有KRBP72-PTP标签的布氏锥虫（Trypanosoma brucei）环期（procyclic form, PCF）KRBP72 RNA干扰（RNA interference, RNAi）细胞系，在含有或不含4 μg/mL多西环素的培养基中培养3天。采用Trizol试剂提取总RNA，随后经酚-氯仿抽提与乙醇沉淀完成纯化。所得RNA样品经脱氧核糖核酸酶（Deoxyribonuclease, DNase）处理后，再次进行酚-氯仿抽提与乙醇沉淀。本实验设置两次生物学重复，并通过蛋白质印迹（Western blot）验证KRBP72的敲低水平。以基因特异性引物，将经DNase处理的RNA反转录为互补脱氧核糖核酸（complementary DNA, cDNA）。