Effects of sheared chromatin length on ChIP-seq quality and sensitivity.
Effects of sheared chromatin length on ChIP-seq quality and sensitivity.
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剪切染色质长度对ChIP-seq质量和灵敏度的影响。
DOI：
10.1093/g3journal/jkab101
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发表时间：
2021-06-17
期刊：
影响因子：
--
通讯作者：
Hardison RC
中科院分区：
文献类型：
--
作者：
Keller CA;Wixom AQ;Heuston EF;Giardine B;Hsiung CC;Long MR;Miller A;Anderson SM;Cockburn A;Blobel GA;Bodine DM;Hardison RC
染色质免疫沉淀后进行大规模平行高通量测序（ChIP-seq）是全基因组鉴定特定转录因子结合的DNA片段或染色质中具有特定组蛋白修饰的DNA片段的首选方法。然而，ChIP-seq数据集的质量差异很大，其中相当一部分是中等质量到较差质量。因此，重要的是要识别和控制导致ChIP-seq变异的因素。在这项研究中，我们专注于超声，用户控制的变量，以产生剪切染色质。我们系统地改变了小鼠红系细胞系固定染色质的剪切量，在ChIP-seq之前仔细测量所得片段长度的分布。这项系统性研究还通过对其他实验的回顾性分析进行了补充。我们发现，超声处理的水平对ChIP-seq信号的质量有显著影响。超声波处理一致地降低质量，而超声波处理不足的影响在转录因子之间不同，对CTCF结合的位点没有影响，但经常导致TAL 1占据的位点或POL 2结合的位点的丢失。在低质量数据集中未观察到的结合位点被推断为直接和间接结合的混合。我们利用这些发现在罕见的原代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的质量和再现性，并促进ChIP-seq在罕见细胞类型中的应用。
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.
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影响因子：
14.9
作者：
Sloan CA;Chan ET;Davidson JM;Malladi VS;Strattan JS;Hitz BC;Gabdank I;Narayanan AK;Ho M;Lee BT;Rowe LD;Dreszer TR;Roe G;Podduturi NR;Tanaka F;Hong EL;Cherry JM
通讯作者：
Cherry JM
影响因子：
3.9
作者：
Dogan N;Wu W;Morrissey CS;Chen KB;Stonestrom A;Long M;Keller CA;Cheng Y;Jain D;Visel A;Pennacchio LA;Weiss MJ;Blobel GA;Hardison RC
通讯作者：
Hardison RC
影响因子：
4.5
作者：
Zhou，Jian;Schor，Ignacio E.;Troyanskaya，Olga G.
通讯作者：
Troyanskaya，Olga G.
DOI：
10.1126/science.1256271
发表时间：
2014-08-22
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Lara-Astiaso D;Weiner A;Lorenzo-Vivas E;Zaretsky I;Jaitin DA;David E;Keren-Shaul H;Mildner A;Winter D;Jung S;Friedman N;Amit I
通讯作者：
Amit I
影响因子：
7
作者：
Xiang, Guanjue;Keller, Cheryl A.;Hardison, Ross C.
通讯作者：
Hardison, Ross C.