Systematic alteration of ATAC-seq for profiling open chromatin in cryopreserved nuclei preparations from livestock tissues
Systematic alteration of ATAC-seq for profiling open chromatin in cryopreserved nuclei preparations from livestock tissues
复制标题
DOI：
10.1038/s41598-020-61678-9
复制
发表时间：
2020-03-23
影响因子：
4.6
通讯作者：
Ross, P. J.
中科院分区：
文献类型：
--
作者：
Halstead, M. M.;Kern, C.;Ross, P. J.
转座酶可及染色质测定法(ATAC-SEQ)在过去几年中应用于描述染色质可及性已有很大发展，但其在组织中的适用性一直非常有限。为了在长期保存过程中保持核的结构，采用鸡肺组织制备的超低温保存的细胞核对ATAC-seq进行了优化。将测序数据与现有的DNase-SEQ、CHIP-SEQ和RNA-SEQ数据进行比较，以评估文库质量，最终得到一种改进的ATAC-SEQ方法，该方法能够从低温保存的细胞核制备中产生高质量的染色质可及性数据。利用这种方法，在鸡肺中发现的核小体无性区(NFR)重叠了一半的DNase-I超敏部位，与活跃的组蛋白修饰相一致，并特异性地标记了活跃表达的基因。值得注意的是，仅对亚核小体部分进行测序显著改善了信号，而测序后分离亚核小体读数并没有改善信号或峰叫声。这一改进的ATAC-SEQ技术的更广泛的适用性被测试使用冷冻保存的猪组织的核准备，导致在生物复制之间的NFR是高度一致的。此外，组织特异性NFR被富含与组织特异性功能相关的转录因子的结合基序，并标记了组织特异性过程中功能丰富的基因。总体而言，这些结果为优化ATAC-SEQ和分析动物组织中开放染色质的平台提供了见解。
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. The broader applicability of this modified ATAC-seq technique was tested using cryopreserved nuclei preparations from pig tissues, resulting in NFR that were highly consistent among biological replicates. Furthermore, tissue-specific NFR were enriched for binding motifs of transcription factors related to tissue-specific functions, and marked genes functionally enriched for tissue-specific processes. Overall, these results provide insights into the optimization of ATAC-seq and a platform for profiling open chromatin in animal tissues.