Determination of RNA decay rate in mammalian cells. Homo sapiens

In this work, we determined RNA degradation rate of whole transcriptome in HEK293 cells, by employing BRIC-seq (5'-bromo-uridine (BrU) immunoprecipitation chase-deep sequencing analysis) method. The cells were cultured under with 150 micro molar BrU for 24h. Then medium was replaced with normal growth medium, DMEM containing 10 FBS and antibiotics, to wash out BrU. After changing medium, cells were collected and total RNAs labeled with BrU were isolated by using RNAiso Plus reagent. BrU labeled RNAs were isolated by specific mouse monoclonal antibody against BrU, clone 2B1. Finally, isolated BrU labeled RNAs were analyzed by illumina platform.本研究采用BRIC-seq（5'-溴尿嘧啶免疫沉淀追踪深度测序分析法，即5'-bromo-uridine (BrU) immunoprecipitation chase-deep sequencing analysis）方法，测定了人胚肾293细胞（HEK293）的全转录组RNA降解速率。实验中，将细胞置于含150微摩尔BrU的培养基中培养24小时；随后更换为添加10%胎牛血清（FBS）及抗生素的DMEM正常生长培养基以洗脱BrU。换液后收集细胞，利用RNAiso Plus试剂提取总RNA，再通过针对BrU的特异性小鼠单克隆抗体（克隆号2B1）分离其中被BrU标记的RNA组分。最终，将纯化得到的BrU标记RNA通过Illumina测序平台完成测序分析。