Cell Biology Center, Institute of Innovative Research, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama, Japan.
Division of Transcriptomics, Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, Japan.
Nat Protoc. 2020 Oct;15(10):3334-3360. doi: 10.1038/s41596-020-0375-8. Epub 2020 Aug 17.
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.
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来选择性激活或沉默特定基因来确定的。染色质免疫沉淀（ChIP）一直是绘制转录因子结合和组蛋白修饰位点的标准技术。最近，已经开发出替代 ChIP 的方法来满足对低投入表观基因组分析日益增长的需求。染色质整合标记（ChIL）随后进行测序（ChIL-seq）已被证明对于低投入样本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之前扩增了目标基因组序列。在用寡核苷酸偶联抗体（ChIL 探针）原位标记目标蛋白或修饰后，Tn5 转座酶介导的转位随后 T7 RNA 聚合酶介导的转录扩增附近的基因组序列。ChIL-seq 可在荧光显微镜下和基因组水平上检测抗体靶标定位。在这里，我们描述了 ChIL-seq 的详细方案，并评估了关键步骤的方法，包括 ChIL 探针反应、转位、原位转录和测序文库制备。该方案通常需要 3 天时间来准备测序文库，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储存数月，其制备和评估方案也有详细记录。还描述了一种可选的多目标分析（多目标 ChIL-seq）。我们预计，这里提出的方案将使 ChIL 技术更广泛地应用于分析珍贵样本，并促进进一步的应用。
