ChIP-sequencing, also called ChIP-seq, is a widely accepted epigenetic method used to investigate protein interactions with DNA on a genome-wide scale. ChIP-sequencing mixes chromatin immunoprecipitation (ChIP) with massively parallel DNA sequencing to find the binding sites of DNA-associated proteins.
A DNA-bound protein is immunoprecipitated through ChIP protocols using specific antibodies, de-crosslinked, and the ChIP DNA is purified. The ChIP DNA is then enhanced to obtain sufficient data for NGS (Next Generation Sequencing). This process gives an understanding of gene regulation, is often used in academic settings, and plays a vital role in numerous viruses and biological paths. ChIP-Seq is generally used for drug screening and basic research. Some hurdles need to be resolved within complex chip protocol. One of them is to obtain a clean and quality immunoprecipitated-DNA to be successful in downstream processes such as NGS library generation or PCR amplification.
Benefits Of Using ChIP-Sequencing
The most significant benefit that ChIP-Seq information gives is an unbiased approach to studying epigenetics where former knowledge is not needed. It is a thorough study of epigenetic patterns, such as chromatin regulatory proteins or histone modifications. They are druggable targets through genome-wide profiling across numerous samples. But attaining such valuable information relies on recovering superior quality and concentrated ChIP DNA.
Why Do You Require Clean and Concentrated ChIP DNA
The most effective sequencing result from ChIP-Seq requires the recovery of high-quality ChIP DNA. Traditional (homebrew) DNA purification methods use reagents such as phenol/chloroform and ethanol precipitation. These reagents can lead to organic carry-over and co-precipitation, making standard (homebrew) DNA purification methods for ChIP DNA samples complex.
This residual issue results in NGS library bias and is inhibitory during downstream enzymatic steps. Some commercial DNA purification clean-up techniques have trouble retrieving small amounts of DNA from de-crosslinked samples. While most ChIP investigations are intended to produce immunoprecipitated DNA in the nanogram range, it is sometimes challenging to get pure DNA levels greater than 1 ng. As the recommended ChIP DNA input range is 1–10 ng for the library preparation process, the small quantity of purified ChIP DNA material must be of superior quality.
Best Advice for Quality DNA for ChIP-Sequencing
Two significant factors in obtaining quality pulldown DNA for ChIP-Seq are as follows:
- The use of an effective DNA clean-up method.
- The ability to concentrate ChIP DNA in minor volumes.
As realized in testing, a crucial step for quality DNA is purifying the sample after decrosslinking. There are many purification reagents to choose from on the market, but they mainly drastically decline the overall DNA yield. Exploring a purification kit that can also uphold the already small yield of ChIP assays is essential.
This purification process must ensure no reagent interference in subsequent applications, including ligation or amplification during library preparation, and maintain sample yield. Concentrating pure DNA is the second critical step in ensuring successful library amplification. The beginning material for library generation will dilute after decrosslinking the sample, and some samples may even have too much volume to be amplified in a single experiment.
Use a ChIP DNA clean-up kit of clean and concentrated samples. These samples are enhanced for recovering a small amount of DNA from chromatin. Do not leave a residue that will harm library preparation. MBP INC provides one of the most efficient kits that meet these criteria: the DNA Clean & Concentrators, including Genomic DNA Clean & Concentrator-10, EZ DNA Methylation Kit, and many more.
These kits are specially designed with a DNA binding buffer that, in the presence of detergents, antibodies, and proteinases, encourages DNA absorption to the column. These kits are frequently used for ChIP. In contrast to traditional phenol/chloroform and ethanol precipitation procedures, this column-based kit includes ready-to-use buffers and enables a simpler, more effective workflow. This kit is ideal even if you are performing ChIP-Seq for the first time due to its simplicity of use.
Conclusion
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