利用ATAC-seq技术在人免疫细胞中检测染色质开放性的方法建立

Detecting chromatin accessibility of human immune cells with ATAC-seq

基于转座酶和高通量测序的染色质分析技术(assay for transposase-accessible chromatin using sequencing,ATAC-seq)是近年来较为热门的一项用于检测染色质开放性的表观遗传组学技术。为确定适用于人原代免疫细胞的ATAC-seq实验方法,本研究探寻细胞裂解条件,发现0.1%NP-40能温和且较为彻底地裂解细胞。具体方法为使用Tn5转座酶切割染色质并连接测序接头,利用PCR扩增建库;运用2100Bioanalyzer毛细管电泳和统计分析文库中的DNA片段,得到片段呈周期性排布的ATAC-seq文库分布模式。使用一系列生物信息学工具确定文库质量,发现样本总比对率大于94%,线粒体基因组百分率低于38%,所得ATAC-seq信号在转录起始位点附近有明显富集,技术重复间可重复率约为80%。以上从多个维度表明实验及文库制备的整个过程质量良好,是较为理想的用于处理人原代免疫细胞的ATAC-seq实验方法。

The assay for trans posase accessible chroma tin using sequencing is a new epigenomic tec hnique that detects the profi-ling of open chromatin landscape.To build a practicable method for human primary immune cells,conditions for cell lysis were tested.0.1%NP40 was the most appropriate condition that could lyse cells mildly but thoroughly.Tn5 transposases were used to cut off DNA and load sequencing adapters simultaneously.Following transposition step,library was prepared using PCR amplification.The distribution of insert fragment showed the interval of approximately 200 bp,which was analyzed with Agilent 2100 Bioanalyzer.Using bioinformatic tools to ensure the library quality,the total mapping rate was more than 94%,and mitochondrial DNA percentage was less than 38%,the signal of ATAC seq was enriched at transcriptional start sites and the overlap between technical repetitions was approximately 80%。In concusion,we made a high quality li brary evaluated rmul-tidimensionally and it is an ideal ATAC seq method for processing human primary immune cells.