Chromatin immunoprecipitation and high-throughput sequencing from paraffin-embedded pathology tissue
Chromatin immunoprecipitation and high-throughput sequencing from paraffin-embedded pathology tissue
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DOI：
10.1038/nprot.2011.406
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发表时间：
2011-12-01
期刊：
影响因子：
14.8
通讯作者：
Minucci, Saverio
中科院分区：
文献类型：
--
作者：
Fanelli, Mirco;Amatori, Stefano;Minucci, Saverio
福尔马林固定、石蜡包埋 (FFPE) 样品代表了病理样品储存的黄金标准。在这里，我们描述了病理组织染色质免疫沉淀 (PAT-ChIP)，这是一种通过 ChIP-seq 等技术对 FFPE 样品中的染色质进行提取和高通量分析的技术。从技术上讲，PAT-ChIP 的主要挑战是从 FFPE 样品中制备优质染色质。在这里，我们详细解释了建立稳健的染色质制备程序所需的方法、试剂的选择和故障排除步骤。其他步骤也根据现有技术进行了改编，以优化其在 PAT-ChIP-seq 中的使用。该协议从 PAT-ChIP 程序开始到结束需要 4 天。 PAT-ChIP 首次提供了使用患者来源的 FFPE 样本在全基因组范围内对组蛋白修饰和转录因子结合进行分析的机会。因此，该技术允许立即使用病理档案（甚至是几年前的档案）进行表观遗传分析和候选表观遗传生物标志物或靶标的识别。
Formalin-fixed, paraffin-embedded (FFPE) samples represent the gold standard for storage of pathology samples. Here we describe pathology tissue chromatin immunoprecipitation (PAT-ChIP), a technique for extraction and high-throughput analysis, by techniques such as ChIP-seq, of chromatin derived from FFPE samples. Technically, the main challenge of PAT-ChIP is the preparation of good-quality chromatin from FFPE samples. Here we provide a detailed explanation of the methodology used, the choice of reagents and the troubleshooting steps required to establish a robust chromatin preparation procedure. Other steps have also been adapted from existing techniques to optimize their use for PAT-ChIP-seq. The protocol requires 4 d from the start to the end of the PAT-ChIP procedure. PAT-ChIP provides, for the first time, the chance to perform analyses of histone modifications and transcription factor binding on a genome-wide scale using patient-derived FFPE samples. This technique therefore allows the immediate use of pathology archives (even those that are several years old) for epigenetic analyses and the identification of candidate epigenetic biomarkers or targets.