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Chromatin integration labeling for mapping DNA-binding proteins and modifications with low input.
Nature Protocols ( IF 13.1 ) Pub Date : 2020-08-17 , DOI: 10.1038/s41596-020-0375-8
Tetsuya Handa 1 , Akihito Harada 2 , Kazumitsu Maehara 2 , Shoko Sato 3 , Masaru Nakao 4 , Naoki Goto 4 , Hitoshi Kurumizaka 3 , Yasuyuki Ohkawa 2 , Hiroshi Kimura 1, 4
Affiliation  

Cell identity is determined by the selective activation or silencing of specific genes via transcription factor binding and epigenetic modifications on the genome. Chromatin immunoprecipitation (ChIP) has been the standard technique for mapping the sites of transcription factor binding and histone modification. Recently, alternative methods to ChIP have been developed for addressing the increasing demands for low-input epigenomic profiling. Chromatin integration labeling (ChIL) followed by sequencing (ChIL-seq) has been demonstrated to be particularly useful for epigenomic profiling of low-input samples or even single cells because the technique amplifies the target genomic sequence before cell lysis. After labeling the target protein or modification in situ with an oligonucleotide-conjugated antibody (ChIL probe), the nearby genome sequence is amplified by Tn5 transposase-mediated transposition followed by T7 RNA polymerase-mediated transcription. ChIL-seq enables the detection of the antibody target localization under a fluorescence microscope and at the genomic level. Here we describe the detailed protocol of ChIL-seq with assessment methods for the key steps, including ChIL probe reaction, transposition, in situ transcription and sequencing library preparation. The protocol usually takes 3 d to prepare the sequencing library, including overnight incubations for the ChIL probe reaction and in situ transcription. The ChIL probe can be separately prepared and stored for several months, and its preparation and evaluation protocols are also documented in detail. An optional analysis for multiple targets (multitarget ChIL-seq) is also described. We anticipate that the protocol presented here will make the ChIL technique more widely accessible for analyzing precious samples and facilitate further applications.



中文翻译:

染色质整合标记用于以低输入绘制 DNA 结合蛋白和修饰图谱。

细胞身份是通过转录因子结合和基因组上的表观遗传修饰选择性激活或沉默特定基因来确定的。染色质免疫沉淀 (ChIP) 已成为绘制转录因子结合和组蛋白修饰位点图谱的标准技术。最近,人们开发出了 ChIP 的替代方法,以满足对低输入表观基因组分析日益增长的需求。染色质整合标记 (ChIL) 和测序 (ChIL-seq) 已被证明对于低输入样品甚至单细胞的表观基因组分析特别有用,因为该技术在细胞裂解前扩增目标基因组序列。用寡核苷酸偶联抗体(ChIL 探针)标记靶蛋白或进行原位修饰后,附近的基因组序列通过 Tn5 转座酶介导的转座进行扩增,然后通过 T7 RNA 聚合酶介导的转录进行扩增。ChIL-seq 能够在荧光显微镜下和基因组水平上检测抗体靶点定位。在这里,我们描述了 ChIL-seq 的详细方案以及关键步骤的评估方法,包括 ChIL 探针反应、转座、原位转录和测序文库制备。该方案通常需要 3 天来准备测序文库,包括 ChIL 探针反应和原位转录的过夜孵育。ChIL 探针可以单独制备并保存数月,其制备和评估方案也有详细记录。还描述了多目标的可选分析(多目标 ChIL-seq)。我们预计此处介绍的协议将使 ChIL 技术更广泛地用于分析珍贵样品并促进进一步的应用。

更新日期:2020-08-17
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