ChIP-seq analysis of condensin complex in cultured mammalian cells
ChIP-seq analysis of condensin complex in cultured mammalian cells
复制标题
培养哺乳动物细胞中凝缩蛋白复合物的 ChIP-seq 分析
DOI：
10.1007/978-1-4939-6545-8_16
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发表时间：
2017
影响因子：
--
通讯作者：
T.
中科院分区：
文献类型：
--
作者：
Sakata;T.;Shirahige;K.;Sutani;T.
ChIP-seq，或染色质免疫沉淀结合大规模平行DNA测序，是一种强大的技术，以高分辨率在全基因组范围内研究体内蛋白质-DNA相互作用。在这里，我们描述了一个ChIP-seq协议优化的人有丝分裂染色体上的凝聚素I复合物的分析。该方案包括通过两种交联试剂进行密集的细胞固定和通过核酸酶和超声处理进行彻底的染色质剪切的程序，这两者都有助于提高缩合蛋白I ChIP-seq谱的信噪比。优化的方案也可能有助于通过ChIP-seq探索其他“很难看到”蛋白的染色体结合位点。
ChIP-seq, or chromatin immunoprecipitation combined with massively parallel DNA sequencing, is a powerful technique to investigate in vivo protein–DNA interactions on a genome-wide scale at high resolution. Here we describe a ChIP-seq protocol optimized for analysis of condensin I complex on human mitotic chromosomes. The protocol includes procedures of intensive cell fixation by two cross-linking reagents and thorough chromatin shearing by nuclease and sonication treatments, both of which contribute to improving the signal-to-noise ratio of condensin I ChIP-seq profiles. The optimized protocol may also be helpful to explore chromosomal binding sites of other “hard-to-see” proteins by ChIP-seq.