RNA-mediated activity-based protein profiling reveals the substrates of a human dihydrouridine synthase

We adapted the iCLIP method to map DUS3L-dependent DHU sites in a transcriptome-wide manner by treating the cells with 5-FUrd. Flp-In T-Rex 293 cells expressing 3xFlag-DUS3L were treated with 5-FUrd, DUS3L-RNA were extracted and subject for Iclip library preparation.我们通过将细胞用5-氟尿嘧啶核苷（5-Fluorouridine，5-FUrd）处理，优化适配了单核苷酸分辨率紫外交联免疫沉淀测序（iCLIP）方法，以全转录组水平定位依赖于DUS3L的二氢尿嘧啶（DHU）位点。将表达3xFlag-DUS3L的Flp-In T-Rex 293细胞经5-FUrd处理后，提取其DUS3L结合RNA并进行iCLIP文库构建。