Systematic alteration of ATAC-seq for profiling open chromatin in cryopreserved nuclei preparations from livestock tissues

The use of Assay for Transposase-Accessible Chromatin (ATAC-seq) to profile chromatin accessibility has surged over the past years, but its applicability to tissues has been very limited. With the intent of preserving nuclear architecture during long-term storage, cryopreserved nuclei preparations from chicken lung were used to optimize ATAC-seq. Sequencing data were compared with existing DNase-seq, ChIP-seq, and RNA-seq data to evaluate library quality, ultimately resulting in a modified ATAC-seq method capable of generating high quality chromatin accessibility data from cryopreserved nuclei preparations. Using this method, nucleosome-free regions (NFR) identified in chicken lung overlapped half of DNase-I hypersensitive sites, coincided with active histone modifications, and specifically marked actively expressed genes. Notably, sequencing only the subnucleosomal fraction dramatically improved signal, while separation of subnucleosomal reads post-sequencing did not improve signal or peak calling. Overall, these results provide insights into the optimization of ATAC-seq and a platform for profiling open chromatin in animal tissues.

过去数年间，利用转座酶可及性染色质测序（Assay for Transposase-Accessible Chromatin, ATAC-seq）分析染色质可及性的研究呈爆发式增长，但其在组织样本中的应用却受到极大限制。为实现长期储存过程中细胞核结构的完整保留，研究人员以鸡肺来源的低温保存细胞核制备物为实验材料，对ATAC-seq流程进行优化。将所得测序数据与已公开的脱氧核糖核酸酶I测序（DNase-seq）、染色质免疫共沉淀测序（ChIP-seq）及RNA测序（RNA-seq）数据进行比对以评估文库质量，最终开发出一种改良型ATAC-seq方法，可从低温保存的细胞核制备物中获取高质量的染色质可及性数据。运用该方法，在鸡肺中鉴定得到的核小体缺失区域（NFR）与半数脱氧核糖核酸酶I超敏感位点存在重叠，且与活性组蛋白修饰特征相符，可特异性标记活跃表达的基因。值得注意的是，仅对亚核小体组分进行测序即可显著提升信号强度，而测序后再分离亚核小体测序读段则无法改善信号或提升峰识别效果。综上，本研究既为ATAC-seq的优化提供了理论思路，也为动物组织的开放染色质分析搭建了可靠技术平台。