RNA-seq analysis of gene expression profile in Setd2 WT and KO germinal center B cells and Phf19 WT and KO germinal center B cells

The goals of this study are to compare gene expression profiles of Setd2 WT and KO germinal center B cells and reveal the underlying mechanisms by which Setd2 regulates germinal center B cell responses. Further our study aims to compare gene expression profiles of Phf19 WT and KO germinal center B cells reveal the underlying mechanisms by which Phf19 regulates germinal center B cells responses. Finally, we combine the two profiles and investigate the relation shape betweeen Setd2 dependent regulation and Phf19 dependent regulation. Overall design: The germinal center B cell specific Setd2-deficient mice and WT control mice were immunized and 10e6 germinal center B cells were FACS sorted for RNA extraction, and the germinal center B cell specific Phf19-deficient mice and WT control mice were immunized and 10e6 germinal center B cells were FACS sorted for RNA extraction. Library were then generated with illumina protocol and subject to NGS and bioinformatic analysis.

本研究的核心目标如下：其一，比较Setd2野生型（WT）与敲除型（KO）生发中心B细胞的基因表达谱，揭示Setd2调控生发中心B细胞应答的潜在分子机制；其二，比较Phf19野生型与敲除型生发中心B细胞的基因表达谱，阐明Phf19调控生发中心B细胞应答的潜在分子机制；最后，本研究将整合上述两组表达谱数据，探究Setd2依赖型调控与Phf19依赖型调控之间的关联模式。总体实验设计：构建生发中心B细胞特异性Setd2敲除小鼠及野生型对照小鼠，经免疫接种后，通过荧光激活细胞分选（FACS）分选10^6个生发中心B细胞并提取总RNA；同理，构建生发中心B细胞特异性Phf19敲除小鼠及野生型对照小鼠，经免疫接种后分选10^6个生发中心B细胞并提取总RNA。随后采用Illumina建库流程构建测序文库，进行下一代测序（NGS）及生物信息学分析。