A twin UGUA motif directs the balance between gene isoforms through CFIm and the mTORC1 signaling pathway

To identify APA changes in Tsc1-cKO mice, cTag-PAPERCLIP was performed on pAAV-Camk2a-iCre injected Tsc1-WT and Tsc1-floxed cTag-PABP mice. To identify CPSF6-dependent PAS, PAPERCLIP was performed on shCPSF6-BE2C cells with/without doxycycline treatment. Overall design: cTag-PAPERCLIP and PAPERCLIP library construction was performed as previously described (Hwang et al., Neuron 2017; Kunisky et al., Cell Rep 2021) and the libraries were sequenced on Illumina NextSeq

为鉴定Tsc1条件性敲除（Tsc1-cKO）小鼠中可变多聚腺苷酸化（alternative polyadenylation, APA）的变化，本研究对注射了pAAV-Camk2a-iCre的Tsc1野生型（Tsc1-WT）及Tsc1-floxed cTag-PABP小鼠开展了cTag-PAPERCLIP实验。为鉴定CPSF6依赖型多聚腺苷酸信号（polyadenylation signal, PAS），本研究对经/未经多西环素（doxycycline）处理的shCPSF6-BE2C细胞实施了PAPERCLIP实验。实验整体设计：cTag-PAPERCLIP与PAPERCLIP文库的构建流程参照既往发表的研究方法（Hwang等, Neuron 2017; Kunisky等, Cell Rep 2021）完成，随后将构建好的文库在Illumina NextSeq测序平台上进行测序。