Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA 16802, USA.
Mayo Clinic, Department of Gastroenterology and Hepatology, Rochester, MN 55905, USA.
G3 (Bethesda). 2021 Jun 17;11(6). doi: 10.1093/g3journal/jkab101.
Chromatin immunoprecipitation followed by massively parallel, high throughput sequencing (ChIP-seq) is the method of choice for genome-wide identification of DNA segments bound by specific transcription factors or in chromatin with particular histone modifications. However, the quality of ChIP-seq datasets varies widely, with a substantial fraction being of intermediate to poor quality. Thus, it is important to discern and control the factors that contribute to variation in ChIP-seq. In this study, we focused on sonication, a user-controlled variable, to produce sheared chromatin. We systematically varied the amount of shearing of fixed chromatin from a mouse erythroid cell line, carefully measuring the distribution of resultant fragment lengths prior to ChIP-seq. This systematic study was complemented with a retrospective analysis of additional experiments. We found that the level of sonication had a pronounced impact on the quality of ChIP-seq signals. Over-sonication consistently reduced quality, while the impact of under-sonication differed among transcription factors, with no impact on sites bound by CTCF but frequently leading to the loss of sites occupied by TAL1 or bound by POL2. The bound sites not observed in low-quality datasets were inferred to be a mix of both direct and indirect binding. We leveraged these findings to produce a set of CTCF ChIP-seq datasets in rare, primary hematopoietic progenitor cells. Our observation that the amount of chromatin sonication is a key variable in success of ChIP-seq experiments indicates that monitoring the level of sonication can improve ChIP-seq quality and reproducibility and facilitate ChIP-seq in rare cell types.
染色质免疫沉淀结合大规模平行、高通量测序（ChIP-seq）是用于鉴定特定转录因子结合的 DNA 片段或具有特定组蛋白修饰的染色质的全基因组方法。然而，ChIP-seq 数据集的质量差异很大，相当一部分数据集的质量中等或较差。因此，辨别和控制导致 ChIP-seq 变化的因素非常重要。在这项研究中，我们专注于超声处理，这是一种用户可控的变量，用于产生剪切染色质。我们系统地改变从小鼠红细胞系固定染色质的剪切量，在进行 ChIP-seq 之前仔细测量所得片段长度的分布。这项系统研究补充了对其他实验的回顾性分析。我们发现超声处理的水平对 ChIP-seq 信号的质量有显著影响。过度超声处理会持续降低质量，而欠处理的影响因转录因子而异，对 CTCF 结合的位点没有影响，但经常导致 TAL1 结合或 POL2 占据的位点丢失。在低质量数据集未观察到的结合位点被推断为直接和间接结合的混合体。我们利用这些发现，在罕见的造血祖细胞中产生了一组 CTCF ChIP-seq 数据集。我们观察到染色质超声处理的量是 ChIP-seq 实验成功的关键变量，这表明监测超声处理的水平可以提高 ChIP-seq 的质量和可重复性，并促进在稀有细胞类型中进行 ChIP-seq。
