TBP in mitotic bookmarking and reactivation of transcription [ChIP-Seq]. TBP in mitotic bookmarking and reactivation of transcription [ChIP-Seq]

To test of TBP binds in the same genomic regions in mitosis compared to interphase cells, we performed Chromatin ImmunoPrecipitation coupled with sequencing (ChIP-seq). Overall design: We used asynchronous mouse embryonic stem (ES) cells, or Nocodazole-synchronized mitotic ES cells as starting material for ChIP-seq. The cells were crosslinked with 1% formaldehyde, digested with MNase, and lightly sonicated to solubilize digested chromatin. This served as input for the ChIP. After extracting DNA from the ChIP experiment, we used paired end sequencing and mapped the reads to the mm10 genome. We then performed in silico size selection for reads under 100 bp and analyzed for enrichment at the TSS

为检测TBP在有丝分裂与间期细胞中是否结合于相同的基因组区域，我们开展了结合测序的染色质免疫共沉淀（Chromatin ImmunoPrecipitation coupled with sequencing, ChIP-seq）实验。实验设计概述：本研究以异步培养的小鼠胚胎干（mouse embryonic stem, ES）细胞，以及经诺考达唑（Nocodazole）同步化的有丝分裂期ES细胞作为ChIP-seq实验的起始材料。首先用1%甲醛对细胞进行交联处理，随后采用微球菌核酸酶（MNase）进行消化，并辅以轻度超声破碎以溶解消化后的染色质，以此作为染色质免疫共沉淀的输入样本。完成染色质免疫共沉淀实验后提取DNA，我们采用双端测序技术，并将测序读段比对至mm10小鼠参考基因组。随后对长度小于100 bp的测序读段进行计算机模拟长度筛选，并分析其在转录起始位点（transcription start site, TSS）处的富集情况。