Divisions of Oncogenomics, Oncode Institute, Netherlands Cancer Institute, Amsterdam, the Netherlands.
Molecular Carcinogenesis, Oncode Institute, Netherlands Cancer Institute, Amsterdam, the Netherlands.
Life Sci Alliance. 2018 Dec 28;2(1):e201800115. doi: 10.26508/lsa.201800115. eCollection 2019 Feb.
Chromatin immunoprecipitation (ChIP)-seq analyses of transcription factors in clinical specimens are challenging due to the technical limitations and low quantities of starting material, often resulting in low enrichments and poor signal-to-noise ratio. Here, we present an optimized protocol for transcription factor ChIP-seq analyses in human tissue, yielding an ∼100% success rate for all transcription factors analyzed. As proof of concept and to illustrate general applicability of the approach, human tissue from the breast, prostate, and endometrial cancers were analyzed. In addition to standard formaldehyde fixation, disuccinimidyl glutarate was included in the procedure, greatly increasing data quality. To illustrate the sensitivity of the optimized protocol, we provide high-quality ChIP-seq data for three independent factors (AR, FOXA1, and H3K27ac) from a single core needle prostate cancer biopsy specimen. In summary, double-cross-linking strongly improved transcription factor ChIP-seq quality on human tumor samples, further facilitating and enhancing translational research on limited amounts of tissue.
由于技术限制和起始材料数量少，临床标本中的染色质免疫沉淀（ChIP）-seq 分析转录因子具有挑战性，通常导致低富集度和低信噪比。在这里，我们提出了一种优化的人类组织转录因子 ChIP-seq 分析方案，所有分析的转录因子的成功率约为 100%。作为概念验证，并说明该方法的一般适用性，分析了来自乳腺癌、前列腺癌和子宫内膜癌的人类组织。除了标准的甲醛固定外，双琥珀酰亚胺戊二酸酯也包含在该程序中，极大地提高了数据质量。为了说明优化方案的灵敏度，我们提供了来自单个核心针前列腺癌活检标本的三个独立因子（AR、FOXA1 和 H3K27ac）的高质量 ChIP-seq 数据。总之，双链交联强烈改善了人类肿瘤样本中转录因子 ChIP-seq 的质量，进一步促进和增强了对有限数量组织的转化研究。
