ChIP-seq文庫制備ThruPLEX DNA-seq Kit

ChIP-seq –結合DNA測序分析蛋白與DNA的相互作用，確定結合位點。

ChIP：染色質免疫共沉淀技術（Chromatin Immunoprecipitation，ChIP）也稱結合位點分析法。

ChIP測序也稱為ChIP-seq，是一種用于分析蛋白質與DNA相互作用的方法。 ChIP-seq將染色質免疫沉淀（ChIP）與大規模平行DNA測序相結合，以確定DNA相關蛋白的結合位點。它可以用來地定位任何目標蛋白質的結合位點。

ChIP-seq測序特點：

很多實驗涉及活檢樣品和稀有細胞群（干細胞，原代細胞等）

主要的挑戰是以微量DNA樣品構建NGS文庫

ChIP-seq的ThruPLEX產品

ThruPLEX® DNA-seq Kit

Rubicon的ThruPLEX 技術，可以低至50 pg的DNA起始構建DNA文庫，在提高靈敏度的同時可以構建多達96種illumina index引物文庫。特別適用于ChIP-seq文庫制備。

ThruPLEX用于ChIP-Seq的優勢

產品特點

高靈敏度、高性能：以低至50 pg至50 ngDNA為起始材料

完整的文庫構建試劑：優化的illumina index引物和接頭試劑、酶、緩沖液及無核酸酶水

快速簡便的工作流程：單管或單孔3步操作，2小時，無需轉移操作

使用ThruPLEX進行ChIP-seq的引用文獻

2016至今有11篇ThruPLEX用于ChIP-seq 的高影響因子文章發表。

• 應用領域

– 發育和干細胞生物學

– 腫瘤研究

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產品

ThruPLEX® DNA-seq Kit

Rubicon公司成立于2000年，總部位于美國密歇根州。產品都擁有技術，檢控嚴格，擁有cGMP級生產能力，是單細胞測序文庫構建的。2017年1月，Rubicon 成為Takara的一員，并且發展良好。

華雅旗下紅榮微再（上海）生物工程技術有限公司2018年，與TAKARA繼續合作，授權代理（上海）Rubicon和Cellartis 試劑。紅榮微再以“傳遞科學價值，服務科學研究”為宗旨，主營干細胞、醫療、細胞治療、器官再生四大板塊的產品。

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