How to get quality DNA for ChIP Sequencing
Chromatin immunoprecipitation followed by sequencing, also known as ChIP-Seq, is a widely accepted epigenetic approach used to investigate protein-DNA interactions on a genome-wide scale. Through ChIP protocols, a DNA-bound protein is immunoprecipitated using specific antibodies, de-crosslinked, and the ChIP DNA is purified, which can then be amplified to get enough material for NGS (Next Generation Sequencing). This process is often used in academic settings to provide insight into gene regulation and its role in various diseases and biological pathways. While ChIP-Seq is becoming more widely used for drug screening and basic research, there are difficulties that must be overcome within the complicated ChIP protocols. One of these is obtaining clean and quality immunoprecipitated-DNA in order to be successful in downstream processes such as PCR amplification or NGS library generation.
Advantages to using ChIP-Seq
The biggest advantage that ChIP-Seq data provides is an unbiased approach to studying epigenetics where no prior knowledge is required. It is a precise investigation of epigenetic patterns, such as histone modifications or chromatin regulatory proteins, which are druggable targets through genome-wide profiling across multiple samples. But the ability to obtain such valuable data is dependent on recovering high-quality and concentrated ChIP DNA.
Why you need clean and concentrated ChIP DNA
The most successful sequencing outcomes from ChIP-Seq requires the recovery of high-quality ChIP DNA. Traditional (home brew) DNA purification methods for ChIP DNA samples can be complicated because the use of reagents such as phenol/chloroform and ethanol precipitation can lead to organic carry-over and co-precipitation. This residual matter can be inhibitory during downstream enzymatic steps and result in NGS library bias. Some commercial clean up methods for DNA purification have difficulties recovering the low amounts of DNA from de-crosslinked samples. Typical ChIP experiments are designed to generate immunoprecipitated DNA in the nanogram range, but it is often difficult to obtain purified DNA quantities over 1 ng. It is important that the small amount of purified ChIP DNA material is quality as the recommended ChIP DNA input range is 1-10 ng for the library prep step.
Top Tips for Quality DNA for ChIP-Seq
Two major factors in obtaining quality pulldown DNA for ChIP-Seq are use of an effective DNA clean up method and the ability to concentrate ChIP DNA in small volumes. As seen in an experiment by Zhong et al., a crucial step for quality DNA is purification of the sample which should occur after decrosslinking. There are many purification reagents to choose from on the market but many of them drastically decrease the overall DNA yield. It is important to find a purification kit that can also maintain the already small yield of ChIP assays. In addition to maintaining sample yield, this purification step needs to ensure there is no reagent interference in downstream applications, such as ligation or amplification during library prep. The other crucial step to ensure successful library amplification is concentrating purified DNA. After decrosslinking the sample, the starting material for library generation will become diluted and some samples may even have too large a volume to be amplified in one experiment.
Get clean and concentrated samples by using a ChIP DNA clean up kit that is optimized for recovering low amounts of DNA from chromatin and does not leave residue that will affect library prep. One of the most efficient kits that meets these criteria is the ChIP DNA Clean & Concentrator from Zymo Research. It is specifically formulated with a ChIP DNA binding buffer that promotes DNA absorption to the column in the presence of detergents, antibodies, and proteinases often used for ChIP. Unlike traditional phenol/chloroform and ethanol precipitation methods, this column-based kit comes with ready to- use buffers and allows for an easier and more efficient workflow. Because of its ease of use, this kit is perfect if it is your first-time doing ChIP-Seq and is also trusted by experienced researchers.
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- Jian Zhong, Zhenqing Ye, Samuel W. Lenz, Chad R. Clark, Adil Bharucha, Gianrico Farrugia, Keith D. Robertson, Zhiguo Zhang, Tamas Ordog and Jeong-Heon Lee. Purification of nanogram-range immunoprecipitated DNA in ChIP-Seq Application. BMC Genomics. 2017; 18:985
- Kidder BL, Hu G, Zhao K. ChIP-Seq: technical considerations for obtaining high-quality data. Nat Immunol. 2011; 12(10):918-22. doi: 10.1038/ni.2117.
- Ryuichiro Nakato, Toyonori Sakata. Methods for ChIP-seq analysis: A practical workflow and advanced applications. Methods. 2021; 187: 44-53
- Dickson BM, Tiedemann RL, Chomiak AA, Cornett EM, Vaughan RM, Rothbart SB. A physical basis for quantitative ChIP-sequencing. J Biol Chem. 2020; 295(47):15826-15837. doi: 10.1074/jbc.RA120.015353.