·产品描述

       蛋白质-DNA相互作用在细胞功能方面起到了重要作用，比如信号传导、基因转录和染色质降解，DNA复制和重组，表观沉默等.辨别DNA结合蛋白的靶标以及了解蛋白质-DNA相互作用对于研究细胞各项过程有着重要的作用.染色质免疫共沉淀及后续的测序提供了一个研究蛋白质-DNA相互作用的强有力的工具.它可以检测活细胞内结合在特定序列上的蛋白.特别是，针对整个基因组上不同转录因子的ChIP抗体需求量很大，它们可以帮助研究基因组范围内的转录因子结合位点.ChIP分析要求参与实验的DNA含有的背景很低，这样才能识别真正的转录因子富集区域.现有的ChIP-Seq方法在研究蛋白-DNA互作过程中起到了重要的作用，但也有一些缺点：1.需要大量的细胞或组织产生足够的DNA文库，这些方法不能应用于含有有限DNA量的样本，如组织活检，胚胎组织等2.背景很高3.耗时（大于3d），不方便.为了解决这些问题，epigentek研发了EpiNext高灵敏染色质免疫共沉淀测序（Illumina）试剂盒.

       EpiNext高灵敏染色质免疫共沉淀测序（Illumina）试剂盒是一组完整的、被优化的试剂组合，它可以用来进行哺乳动物细胞或组织的染色质免疫共沉淀测序.这个试剂盒可以选择性的富集来源与不同物种，特别是哺乳动物的含有特异DNA序列的染色质片断，构建ChIP-Seq.文库一共Illumina平台的下一代测序，如Illumina Genome Analyzer II， HiSeq 和 MiSeq .优化的方案和组分可以用亚纳克级别的起始DNA进行非条形码序列（单个）和条形码序列（多重）DNA文库构建。

·产品特点

1.优化的缓冲液和方案使得ChIP背景极低，提高了反应的灵敏度和特异性 

2.抗体效率高，采用了最大化结合位点数量的融合型抗体 

3.高效率 阳性DNA和阴性对照产物之比大于500 

4.高特异性和灵活性可以用亚纳克级别的起始DNA进行非条形码序列（单个）和条形码序列（多重）DNA文库构建，每次反应用50000个细胞即可，范围为50，000-1，000，000个细胞，选择范围大 

5.快速的实验方案，从细胞、组织到DNA文库只要不到7h 

6.非常方便，此试剂盒含有ChIP-Seq的全部必要组分 

7. 超级HiFi扩增使得实验者可以在最小误差的情况下得到最大的DNA文库产率。

·产品说明书

The EpiNext™ ChIP-Seq High-Sensitivity Kit (Illumina) is a complete set of reagents required for carrying out a successful ChIP-Seq starting from mammalian cells or tissues. The kit is designed to ively enrich a chromatin fraction containing specific DNA sequences from various species, particularly mammals, and to prepare a ChIP-Seq library for next generation sequencing using Illumina platforms such as Illumina Genome Analyzer II, HiSeq and MiSeq systems. The optimized protocol and components of the kit allow capture of low abundance protein/DNA complexes with minimized non-specific background levels and the ability to construct both non-barcoded (singleplexed) and barcoded (multiplexed) ChIP-Seq libraries quickly with reduced bias. The kit has the following advantages and features:

• Optimized buffers and protocol allow minimal ChIP background by overcoming the weaknesses that cause non-specific enrichment, thereby increasing sensitivity and specificity of the ChIP reaction.
• Increased antibody ivity and capture efficiency through the use of unique chimeric proteins containing the maximum number of IgG binding domains coated on the strip-wells. This allows strong binding of any IgG subtype antibodies within a wide pH range regardless if they are in monoclonal or polyclonal form. 
• Highly efficient enrichment of targeted DNA. Enrichment ratio of positive to negative control > 500.
• High sensitivity and flexibility: Can be used for both non-barcoded (singleplexed) and barcoded (multiplexed) DNA library preparation. The input cell number can be as few as 50,000 cells with a range from 50,000 to 1,000,000 cells. A broad range of cell/tissue samples can be used, including samples with limited or tiny amounts of available chromatin.
• Fast and streamlined procedure:  the procedure from cell/tissues to library DNA is less than 7 hours. No clean-up is required between each step from ChIPed DNA to size ion, and all reactions take place in the same tube, thereby saving time and preventing handling errors, or loss of valuable samples. Gel-free size ion further reduces the preparation time.
• Highly convenient: the kit contains all required components for each step of ChIP-Seq, which are sufficient for both ChIP and ChIPed DNA library preparation, thereby allowing the ChIP-Seq to be the most convenient with reliable and consistent results. 
• Minimized bias: Ultra HiFi amplification and optional PCR-free step allow achievement of reproducibly high yields of DNA library with minimal sequence bias and low error rates.

Background Information 
Protein-DNA interaction plays a critical role for cellular functions such as signal transduction, gene transcription, chromosome segregation, DNA replication and recombination, and epigenetic silencing. Identifying the genetic targets of DNA binding proteins and knowing the mechanisms of protein-DNA interaction on a genome-wide scale is important for understanding cellular processes. Chromatin immunoprecipitation (ChIP) followed by next generation sequencing (ChIP-Seq) offers an advantageous tool for studying genome-wide protein-DNA interactions. It allows for detection that a specific protein binds to specific sequences in living cells. In particular, ChIP antibodies targeted against various transcriptional factors (TF) for genome-wide transcription factor binding site analysis by Chip-Seq is in high demand. Such analysis requires that ChIPed DNA contain minimal background for reliably identifying true TF-enriched regions. Currently used ChIP-Seq methods play an important role in identifying genome-wide protein-DNA interaction. However, these methods still have several drawbacks: (1) large amounts of cell/tissues are needed for obtaining a sufficient yield of library DNA, therefore these methods cannot be used for biological samples such as tumor biopsy and embryonic tissues whose amounts are limited; (2) the background levels of ChIPed DNA are high; and (3) the procedures are time consuming (>3 days) and inconvenient. To address this issue, Epigentek developed the EpiNext ChIP-Seq High Sensitivity Kit (Illumina) by combining its microplate-based ultra ChIP and high sensitive DNA library construction technologies. 

Principle & Procedure
The EpiNext ChIP-Seq High-Sensitivity Kit (Illumina) contains all necessary reagents required for carrying out a successful ChIP-Seq starting from mammalian cells or tissues. In the ChIP reaction, chromatin is isolated from cell/tissues and the target protein-DNA complex is immunoprecipitated using the antibody of interest. Immunoprecipitated DNA is then cleaned, released, and eluted. A positive control antibody (RNA polymerase II), a negative control non-immune IgG, and GAPDH primers are included in the kit, which can be used as a positive control to demonstrate the efficacy of the kit reagents and protocol. RNA polymerase II is considered to be enriched in the GAPDH gene promoter that is expected to be undergoing transcription in most growing mammalian cells and can be immunoprecipitated by RNA polymerase II antibody but not by non-immune IgG. In the library preparation, ChIPed DNA fragments are end repaired and dA tailed (end polishing) simultaneously.  Adaptors are then ligated to both ends of the polished DNA fragments for amplification and sequencing. Ligated fragments are size ed and purified using MQ binding beads, which allows quick and precise size ion of DNA. Size-ed DNA fragments are amplified with a high-fidelity PCR mix which ensures maximum yields from minimum amounts of starting material and provides highly accurate amplification of library DNA with low error rates and minimum bias. 

Starting Materials
Starting materials can include various tissue or cell samples such as culture cells from a flask or plate, fresh and frozen tissues, etc.

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