Capturing Chromosome Conformation Across Length Scales.
Capturing Chromosome Conformation Across Length Scales.
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DOI：
10.3791/64001
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发表时间：
2023-01-20
期刊：
影响因子：
--
通讯作者：
Gibcus JH
中科院分区：
文献类型：
--
作者：
Yang L;Akgol Oksuz B;Dekker J;Gibcus JH
染色体构象捕获（3C）用于检测三维染色质相互作用。通常，与甲醛（FA）的化学交联用于固定染色质相互作用。然后，用限制性内切酶进行染色质消化，并随后对片段末端进行修饰，将三维（3D）接近转化为独特的连接产物。最后，在逆转交联、去除蛋白质和分离DNA后，将DNA剪切并准备进行高通量测序。基因座对的邻近结扎频率是它们在细胞群体三维空间共定位频率的量度。测序的Hi-C文库提供了所有位点对之间相互作用频率的全基因组信息。Hi-C的分辨率和精度依赖于有效的交联，维持染色质接触和染色质频繁而均匀的断裂。本文介绍了一种改进的原位Hi-C方案Hi-C 3.0，该方案通过结合两种交联剂（甲醛[FA]和二氨基酰戊二酸[DSG]）来提高交联效率，然后使用两种限制性内切酶（DpnII和DdeI）进行更精细的消化。Hi-C 3.0是一个单一的协议，用于精确定量基因组折叠特征在较小的尺度，如环和拓扑相关结构域（TADs），以及在较大的细胞核范围内的特征，如室。Hi-C 3.0是一种改进的Hi-C方案，它将甲醛和戊二酸二氨基酰交联剂与DpnII和DdeI限制性内切酶的混合物结合在一起，以提高染色质相互作用检测的信噪比和分辨率。
Chromosome conformation capture (3C) is used to detect three-dimensional chromatin interactions. Typically, chemical crosslinking with formaldehyde (FA) is used to fix chromatin interactions. Then, chromatin digestion with a restriction enzyme and subsequent religation of fragment ends converts three-dimensional (3D) proximity into unique ligation products. Finally, after reversal of crosslinks, protein removal, and DNA isolation, DNA is sheared and prepared for high-throughput sequencing. The frequency of proximity ligation of pairs of loci is a measure of the frequency of their colocalization in three-dimensional space in a cell population. A sequenced Hi-C library provides genome-wide information on interaction frequencies between all pairs of loci. The resolution and precision of Hi-C relies on efficient crosslinking that maintains chromatin contacts and frequent and uniform fragmentation of the chromatin. This paper describes an improved in situ Hi-C protocol, Hi-C 3.0, that increases the efficiency of crosslinking by combining two crosslinkers (formaldehyde [FA] and disuccinimidyl glutarate [DSG]), followed by finer digestion using two restriction enzymes (DpnII and DdeI). Hi-C 3.0 is a single protocol for the accurate quantification of genome folding features at smaller scales such as loops and topologically associating domains (TADs), as well as features at larger nucleus-wide scales such as compartments. Hi-C 3.0 is an improved Hi-C protocol that combines formaldehyde and disuccinimidyl glutarate crosslinkers with a cocktail of DpnII and DdeI restriction enzymes to increase the signal-to-noise ratio and the resolution of chromatin interaction detection.
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