Liu Zhiheng, Zhang Lirong, Chen Yupeng
Tianjin Institute of Urology, The Second Hospital of Tianjin Medical University, Tianjin Medical University, Tianjin, 300211, China.
Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Tianjin Medical University, Tianjin, 300070, China.
J Biol Methods. 2024 Jul 10;11(2):e99010015. doi: 10.14440/jbm.2024.0009. eCollection 2024.
Spatiotemporal regulation of gene expression is essential for maintaining cellular homeostasis throughout kidney development and disease progression. Transcription factors (TFs) and epigenetic modifications play pivotal roles in controlling gene expression. Profiling chromatin modifications across the genome, along with the distribution and target regulation by TFs in specific kidney cell types, is crucial for understanding the dynamic changes in gene expression. Here, we presented a comprehensive workflow for epigenomic, cistromic, and transcriptomic analyses of primary kidney tubular cells. Specifically, our methodologies included the isolation of primary kidney tubular epithelial cells, RNA extraction, assay for transposase-accessible chromatin using sequencing, ultra-low-input micrococcal nuclease-based native chromatin immunoprecipitation, cleavage under targets and release using nuclease, and subsequent bioinformatic analysis. This protocol provides a methodological framework for investigating the roles of TFs and epigenetic modifications in kidney development and diseases.
基因表达的时空调控对于在整个肾脏发育和疾病进展过程中维持细胞内稳态至关重要。转录因子(TFs)和表观遗传修饰在控制基因表达中起关键作用。分析全基因组的染色质修饰,以及TFs在特定肾细胞类型中的分布和靶标调控,对于理解基因表达的动态变化至关重要。在这里,我们展示了一个用于原代肾小管细胞表观基因组、顺式作用元件组和转录组分析的综合工作流程。具体而言,我们的方法包括原代肾小管上皮细胞的分离、RNA提取、转座酶可及染色质测序分析、基于超低输入微球菌核酸酶的天然染色质免疫沉淀、靶标切割和核酸酶释放,以及后续的生物信息学分析。该方案为研究TFs和表观遗传修饰在肾脏发育和疾病中的作用提供了一个方法框架。