TBC1D3 suppression of the Histone Lysine N-methyltransferase G9a promotes the generation of neural progenitors and cortical expansion [ChIP-seq]

we conducted chromotin Immunoprecipitation sequencing (ChIP-seq) using human cerebral organoids treated with TAT-Scrambled (Short for TAT-Scr) with the sequence of YGRKKRRQRRR-FRVRYWFQGCHSEDPWR and TAT-465-481, a special peoptide designed for inhibition of TBC1D3, with the sequence of YGRKKRRQRRR-EGPWFRHYDFRQSCWVR. Overall design: Briefly, tissues were fixed in 1% formaldehyde to cross-link histone and non-histone protein to DNA for 10 to 20 minutes at room temperature and the crosslink was stopped by the addition of glycine. Then cells were lysated to release nucleus and micrococcal nuclease was used to digest chromatin into DNA/protein fragments.A part of the diluted chromatin in each group was transferred into a new microfuge tube to be saved as the input sample and H3K9me2 (Cell Signaling Technology, 4658s) was used as the immunoprecipitating antibody to be added into diluted chromatin mixture. Incubated IP samples at 4°C for overnight with rotation. Added ChIP-Grade Protein G Magnetic Beads to each IP reaction and incubated for 2h at 4°C with rotation and then the chromatin from antibody/Protein G magnetic beads was eluted and reversed. DNA in the chromatin mixture was purified and sequenced.

我们针对经TAT-Scrambled（简称TAT-Scr，序列为YGRKKRRQRRR-FRVRYWFQGCHSEDPWR）以及靶向抑制TBC1D3的特异性肽段TAT-465-481（序列为YGRKKRRQRRR-EGPWFRHYDFRQSCWVR）处理的人类大脑类器官，开展了染色质免疫共沉淀测序（Chromatin Immunoprecipitation sequencing，ChIP-seq）实验。实验整体设计如下：简要而言，将组织置于1%甲醛中于室温下固定10至20分钟，使组蛋白与非组蛋白交联至DNA，随后加入甘氨酸终止交联反应。之后裂解细胞以释放细胞核，使用微球菌核酸酶将染色质消化为DNA-蛋白质复合物片段。将各组中稀释后的部分染色质转移至新的微量离心管中，作为输入样本留存；向剩余的染色质稀释混合物中加入组蛋白H3K9me2抗体（Cell Signaling Technology，货号4658s）作为免疫沉淀抗体。将免疫沉淀（IP）样品于4℃条件下旋转孵育过夜。向每一组IP反应体系中加入ChIP级蛋白G磁珠，于4℃条件下旋转孵育2小时，随后洗脱并逆转抗体与磁珠结合的染色质复合物。纯化染色质混合物中的DNA并进行测序。