Simplified Epigenome Profiling Using Antibody-tethered Tagmentation.
Simplified Epigenome Profiling Using Antibody-tethered Tagmentation.
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DOI：
10.21769/bioprotoc.4043
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发表时间：
2021-06-05
期刊：
影响因子：
0.8
通讯作者：
Ahmad K
中科院分区：
文献类型：
--
作者：
Henikoff S;Henikoff JG;Ahmad K
我们之前介绍了靶下切割和标签化（CUT&Tag），这是一种表观基因组分析方法，其中Tn 5转座酶与感兴趣的染色质表位的抗体连接映射了小样品和单细胞中的特定染色质特征。使用CUT&Tag，将完整细胞或细胞核透化，然后连续添加一抗、二抗和结合抗体的嵌合蛋白A-转座酶融合蛋白。Mg++的添加激活转座酶并将测序衔接子原位插入相邻DNA中。我们已经调整了CUT&Tag，当使用组蛋白H3 K4 me 2，H3 K4 me 3或丝氨酸-5-磷酸化RNA聚合酶II抗体时，通过简单地修改转座酶激活条件，也可以绘制染色质可及性。使用这些抗体，我们重定向可访问的DNA位点的标签化，以产生具有非常高的信噪比和分辨率的染色质可及性图。从细胞核到扩增测序就绪文库的所有步骤都在单个PCR管中使用无毒试剂和廉价设备进行，使我们的简化策略适用于实验室，家庭工作台或教室。
We previously introduced Cleavage Under Targets & Tagmentation (CUT&Tag), an epigenomic profiling method in which antibody tethering of the Tn5 transposase to a chromatin epitope of interest maps specific chromatin features in small samples and single cells. With CUT&Tag, intact cells or nuclei are permeabilized, followed by successive addition of a primary antibody, a secondary antibody, and a chimeric Protein A-Transposase fusion protein that binds to the antibody. Addition of Mg++ activates the transposase and inserts sequencing adapters into adjacent DNA in situ. We have since adapted CUT&Tag to also map chromatin accessibility by simply modifying the transposase activation conditions when using histone H3K4me2, H3K4me3, or Serine-5-phosphorylated RNA Polymerase II antibodies. Using these antibodies, we redirect the tagmentation of accessible DNA sites to produce chromatin accessibility maps with exceptionally high signal-to-noise and resolution. All steps from nuclei to amplified sequencing-ready libraries are performed in single PCR tubes using non-toxic reagents and inexpensive equipment, making our simplified strategy for simultaneous chromatin profiling and accessibility mapping suitable for the lab, home workbench, or classroom.