Identify Flag-bound transcripts via RIP-seq

To identify the directly bound transcripts of Flag antibody (the backgroud control for our METTL16 RIP-seq), RNA immunoprecipitation sequencing (RIP-seq) was conducted HEK293T. Briefly, HEK293T cells were infected with pmiRNA1-empty vector. Only the GFP-positive cells were used for study and expanded in DMEM medium. The GFP-positive HEK293T cells were expanded in 150mm dish and collected at 70-80% confluency. The were cross-linked by 0.75 % formaldehyde (F8775, Sigma-Aldrich) with gentle rotation at room temperature for 10 min and the reaction was quenched by 125 nM glycine (final concentration) with shaking at room temperature for 5 min. Then cells were collected, lysed, and subjected to RIP with Flag antibody. Two biological replicates were included to confirm the data quality.为鉴定Flag抗体（本研究中METTL16 RIP-seq的阴性对照）的直接结合转录本，我们在HEK293T细胞中开展了RNA免疫共沉淀测序（RNA immunoprecipitation sequencing, RIP-seq）实验。简言之，我们以pmiRNA1空载体感染HEK293T细胞，仅选取GFP阳性细胞开展后续研究，并将其于DMEM培养基中扩增培养。将GFP阳性HEK293T细胞接种于150mm培养皿中扩增，待细胞汇合度达70%-80%时收集细胞。随后用0.75%甲醛（F8775，Sigma-Aldrich）室温轻柔旋转孵育10分钟对细胞进行交联，再以终浓度为125nM的甘氨酸室温振摇5分钟终止交联反应。收集细胞并裂解后，使用Flag抗体进行RNA免疫共沉淀实验。本实验设置2次生物学重复以验证数据质量。