Robust single-cell discovery of RNA targets of RNA binding proteins and ribosomes [tia1_eclip]. Robust single-cell discovery of RNA targets of RNA binding proteins and ribosomes [tia1_eclip]

RNA binding proteins (RBPs) are critical regulators of gene expression and RNA processing that are required for gene function. Yet, the dynamics of RBP regulation in single cells is unknown. To address this gap in understanding, we developed STAMP (Surveying Targets by APOBEC Mediated Profiling), which efficiently detects RBP RNA interactions. STAMP does not rely on UV crosslinking or immunoprecipitation and, when coupled with single cell capture, can identify RBP and cell type specific RNA protein interactions for multiple RBPs and cell types in single pooled experiments. Pairing STAMP with long read sequencing also yields RBP target sites for full length isoforms. Finally, conducting STAMP using small ribosomal subunits (RiboSTAMP) allows analysis of transcriptome wide ribosome association in single cells. STAMP enables the study of RBP RNA interactomes and translational landscapes with unprecedented cellular resolution. Overall design: Crosslinking and immunoprecipitation of TIA1 bound RNA and sequencing from 2 technical replicates.

RNA结合蛋白（RNA binding proteins, RBPs）是基因表达与RNA加工过程的关键调控因子，是基因功能维持所必需的。然而，单细胞水平下RBP调控的动态机制仍有待阐明。为填补这一认知空白，我们开发了STAMP（Surveying Targets by APOBEC Mediated Profiling，即APOBEC介导的靶向检测技术），可高效检测RBP与RNA的相互作用。STAMP无需依赖紫外交联或免疫沉淀技术，当结合单细胞捕获策略时，可在单次混合实验中同时鉴定多种RBP及多种细胞类型特异性的RNA-蛋白质相互作用。将STAMP与长读长测序技术联用，还可获取对应全长转录本亚型的RBP结合位点。此外，以小核糖体亚基为研究对象开展STAMP实验（RiboSTAMP），可实现单细胞水平全转录组范围内的核糖体结合分析。STAMP能够以前所未有的细胞分辨率解析RBP的RNA相互作用组与翻译调控全景。总体实验设计：针对TIA1结合的RNA进行交联与免疫沉淀实验，并设置2个技术重复进行测序。