The molecular logic of Nanog-induced self-renewal in mouse embryonic stem cells. The molecular logic of Nanog-induced self-renewal in mouse embryonic stem cells

Identification of Nanog targets by transcriptomic changes of Nanog induction and depletion, identification of Nanog co-binding logic by ChIP-seq of Nanog co-binding factors with and without Nanog depletion. H3K27me3 with Nanog induction in +/- Lif. Overall design: RNA-seq performed on 44iN +/- Dox, SunTag Nanog & Nanog/Otx2 in +/- Dox +/- Lif. ChIP for Brg1, Esrrb, Sox2, Oct4 in 44iN +/- Dox. ATAC-seq in 44iN +/- Dox. H3K27me3 ChIP-seq in Suntag Nanog +/- Dox +/- Lif.

通过Nanog诱导与敲低后的转录组变化鉴定Nanog的靶基因，并通过在存在与不存在Nanog敲低的条件下对Nanog共结合因子开展染色质免疫沉淀测序（ChIP-seq）解析其共结合调控逻辑，同时分析伴随Nanog诱导、在添加/不添加白血病抑制因子（Leukemia Inhibitory Factor, LIF）条件下的H3K27me3修饰特征。
总体实验设计：对44iN细胞系在添加/不添加多西环素（Doxycycline, Dox）的条件下，以及携带SunTag标记Nanog、Nanog/Otx2的细胞系在添加/不添加Dox、添加/不添加LIF的条件下进行RNA测序（RNA-seq）；对44iN细胞系在±Dox条件下开展Brg1、Esrrb、Sox2、Oct4的染色质免疫沉淀（ChIP）实验；对44iN细胞系在±Dox条件下进行转座酶可及性测序（ATAC-seq）；对携带SunTag标记Nanog的细胞系在±Dox、±LIF条件下进行H3K27me3的ChIP-seq实验。