ChIP-seq –结合DNA测序分析蛋白与DNA的相互作用，确定结合位点。

ChIP：染色质免疫共沉淀技术（Chromatin Immunoprecipitation，ChIP）也称结合位点分析法。

ChIP测序也称为ChIP-seq，是一种用于分析蛋白质与DNA相互作用的方法。 ChIP-seq将染色质免疫沉淀（ChIP）与大规模平行DNA测序相结合，以确定DNA相关蛋白的结合位点。它可以用来地定位任何目标蛋白质的结合位点。

ChIP-seq测序特点：

很多实验涉及活检样品和稀有细胞群（干细胞，原代细胞等）

主要的挑战是以微量DNA样品构建NGS文库

ChIP-seq的ThruPLEX产品

ThruPLEX® DNA-seq Kit

Rubicon的ThruPLEX 技术，可以低至50 pg的DNA起始构建DNA文库，在提高灵敏度的同时可以构建多达96种illumina index引物文库。特别适用于ChIP-seq文库制备。

ThruPLEX用于ChIP-Seq的优势

产品特点

高灵敏度、高性能：以低至50 pg至50 ngDNA为起始材料

完整的文库构建试剂：优化的illumina index引物和接头试剂、酶、缓冲液及无核酸酶水

快速简便的工作流程：单管或单孔3步操作，2小时，无需转移操作

使用ThruPLEX进行ChIP-seq的引用文献

2016至今有11篇ThruPLEX用于ChIP-seq 的高影响因子文章发表。

• 应用领域

– 发育和干细胞生物学

– 肿瘤研究

SMARTer NGS products

Recent ChIP-seq Publications using ThruPLEX Technology

Rubicon’s collaborators and customers are constantly making scientific breakthroughs. Here are the latest published results obtained using ThruPLEX DNA-seq for chromatin mmunoprecipitation sequencing (ChIP-seq).

1.Baejen, C. et al. Genome-wide analysis of RNA Polymerase II termination at protein-coding. Genes. Mol. Cell 66, 1–12 (2017).

This paper used ChIP-seq, ChIP-qPCR, and other functional genomic methods to understand how RNA Pol II termination occurs in yeast. ThruPLEX DNA-seq kit was used to generate libraries for ChIP-seq. The author showed that the 3′-transition in budding yeast requires the Pol II elongation factor Spt5, and that polymerase II release from DNA requires the Rat1 exonuclease.

2.Maatouk, D.M. et al. Genome-wide identification of regulatory elements in Sertoli cells. Development 144, 720–30 (2017).

This study used ChIP-seq, DNaseI-seq, and RNA-seq to identify regulatory elements in mouse Sertoli cells during sex determination. ThruPLEX DNA-seq kit was used to prepare ChIP-seq libraries from FACS-sorted mouse Sertoli cells. By overlapping DNaseI-seq peaks with the chromatin landscape for H3K27ac, the authors were able to identify enhancers active only in Sertoli cells during the early stages of sex determination.

3.Liu, Y. et al. Transcriptional landscape of the human cell cycle. PNAS 114, 3473–78 (2017).

This paper investigated the transcriptional landscape across the cell cycles using a combination of ChIP-seq, DNase-seq, RNA-seq, and GRO-seq. ThruPLEX DNA-seq kit was used to prepare libraries for ChIP-seq and DNase-seq. Using the MCF-7 breast cancer cell line as a model, the authors revealed lag between transcription and steady-state RNA expression at the cell-cycle level. Other findings highlighted the importance of transcriptional and epigenetic dynamics during cell-cycle progression.

4.Warrick, J.I. et al. FOXA1, GATA3 and PPARγ cooperate to drive luminal subtype in bladder cancer: A molecular analysis of established human cell lines. Sci. Reps. 6, 38531 (2016).

This paper used ChIP-seq and RNA-seq to identify a set of human cell lines suitable for the study of molecular subtypes in bladder cancer. ChIP-seq libraries were prepared with ThruPLEX DNA-seq kit. The study shows that the combined overexpression of PPARγ, GATA3 and FOXA1 contributes to the transdifferentiation of bladder cancer cells from a more aggressive basal phenotype to a less invasive luminal phenotype.

5.Roy, N. et al. PDX1 dynamically regulates pancreatic ductal adenocarcinoma initiation and maintenance. Genes Dev. 30, 2669–83 (2016).

This study used ChIP-seq, RNA-seq, and BrU-seq to uncover the diverse functions of PDX1 at different stages of pancreatic ductal adenocarcinoma (PDA). ThruPLEX DNA-seq kit was used to prepare ChIP-seq libraries from PDX1-immunoprecipitated DNA. The authors reported distinct roles of PDX1 at different stages of PDA, suggesting that therapeutic approaches against this potential target need to account for its changing functions at different stages of carcinogenesis.

6.Luizon, M.R. et al. Genomic characterization of metformin hepatic response. PLOS Genet. 12, e1006449 (2016).

This study identified genes and regulatory elements involved in metformin hepatic response in human hepatocytes using ChIP-seq and RNA-seq. ChIP-seq libraries were prepared with ThruPLEX DNA-seq kit. The paper provided a comprehensive genome-wide understanding of metformin-dependent response in human hepatocytes and identified potential candidates for the treatment of type 2 diabetes.

7.Spangle, J.M. et al. PI3K/AKT signaling regulates H3K4 methylation in breast cancer. Cell Rep. 15, 1–13 (2016).

Using ChIP-seq and RNA-seq, this study investigated the role of PI3K/AKT pathway activation in preclincal models of breast cancer. ChIP-seq libraries were prepared using ThruPLEX DNA-seq kit to measure H3K4me3 and KDM subcellular localization. The authors demonstrated the importance of the PI3K/AKT signaling pathway in regulating the epigenetic landscape in breast malignancies, and more specifically that the expression of cell-cycle genes regulated by the AKT/KDM complex is associated with advanced-stage breast cancer.

8.Hojo, H. et al. Sp7/Osterix Is restricted to bone-forming vertebrates where it acts as a Dlx co-factor in osteoblast specification. Dev. Cell 37, 1–16 (2016).

This study used ChIP-seq and RNA-seq to analyze the role of the transcription factor Sp7/Osterix during bone formation in mice. The authors generated a transgenic mouse line in which a biotin motif and three FLAG epitopes were attached to the C-terminus of the Sp7 protein to enable the binding sites of Sp7 could be identified by ChIP. ThruPLEX DNA-seq kit was used to generate ChIP-seq libraries to identify osteoblast enhancers. The authors concluded that the appearance of Sp7 within the Sp family was likely to have played a key role in the emergence of bone-forming osteoblasts during vertebrate evolution.

9.Cejas, P. et al. Chromatin immunoprecipitation from fixed clinical tissues reveals tumor-specific enhancer profiles. Nat Med. 22, 685–691 (2016).

This paper describes a new method called fixed-tissue chromatin immunoprecipitation sequencing (FiT-seq) for reliable extraction of soluble chromatin from FFPE tissue samples for accurate detection of histone marks. ThruPLEX DNA-seq kit was used to generate FiT-seq libraries from FFPE specimens and ChIP-seq libraries fresh frozen samples, and data from the two sample types were demonstrated to be concordant with each other. The study shows that FiT-seq allows tumor-specific enhancers and super enhancers to be elucidated and correlated with known oncogenic drivers to enhance understanding in how chromatin states affect gene regulation.

10.Si, S. et al. Loss of Pcgf5 affects global H2A monoubiquitination but not the function of hematopoietic stem and progenitor cells. PLOS One 11, e0154561 (2016).

This paper analyzed the role of the Polycomb-group (PcG) RING finger protein Pcgf5 in hematopoietic stem and progenitor cells (HSPCs) using ChIP-seq and RNA-seq. Libraries for ChIP-seq were generated using ThruPLEX DNA-seq kit. ChIP-seq analysis confirmed the reduction in H2AK119ub1 levels observed in pcgf5-deficient HSPCs but revealed no significant association with gene expression levels. The authors concluded that Pcgf5 regulates histone H2AK119 monoubiquitination in vivo, but its role in hematopoiesis is marginal.

11.O'Brien, L.L. et al. Differential regulation of mouse and human nephron progenitors by the Six family of transcriptional regulators. Development 143, 595–608 (2016).

This paper compared the regulatory actions of the transcription factor Six2 in mouse and human kidney progenitor cells during nephrogenesis using ChIP-seq and RNA-seq. Sequencing libraries were constructed from mouse and human ChIP DNA using ThruPLEX DNA-seq kit. The study demonstrated the existence of a different regulation of Six-factors between human and mouse nephron progenitors, and provided a potential mechanistic link to the lengthened period of progenitor cell self-renewal and nephrogenesis of the human kidney.

ChIP-seq的ThruPLEX产品

ThruPLEX® DNA-seq Kit

Rubicon公司成立于2000年，总部位于美国密歇根州。产品都拥有技术，检控严格，拥有cGMP级生产能力，是单细胞测序文库构建的。2017年1月，Rubicon 成为Takara的一员，并且发展良好。

华雅旗下红荣微再（上海）生物工程技术有限公司2018年，与TAKARA继续合作，授权代理（上海）Rubicon和Cellartis 试剂。红荣微再以“传递科学价值，服务科学研究”为宗旨，主营干细胞、医疗、细胞治疗、器官再生四大板块的产品。

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