Histones, transcription factors, and other DNA-binding proteins help regulate the expression of our genes and are involved in many physiological and pathological processes. When studying protein/DNA interactions, the method of choice should ideally ensure that:
true target protein-enriched genome regions are reliably identified, particularly from limited biological samples;
enrichment of protein/DNA complexes is accomplished with minimized non-specific background levels; and
mapping of interaction sites is with minimal bias and at high resolution.
One of the most popular methods for analyzing such interactions is to perform a chromatin immunoprecipitation followed by sequencing (ChIP-seq).
The main limitation of ChIP-seq is that it requires a large amount of starting material to produce a strong enough signal over background noise. Also, the use of cross-linking during an initial fixation step can lead to the masking of epitopes recognized by ChIP antibodies. Advancements like ChIP-exo and ChIPmentation allow for reduced cell numbers or increased resolution. ChIP-exo provides high-resolution mapping, but is time-consuming and necessitates an ample supply of input cells. ChIPmentation uses transposase and sequencing-compatible adaptors to enable the integration of ligation during the ChIP process, but follows a traditionally slow (about 2 days) ChIP procedure and cannot achieve high-resolution mapping.
Cleavage under targets and release using nuclease (CUT&RUN-sequencing) and cleavage under target and tagmentation (CUT&Tag-sequencing) were recently developed for mapping protein-DNA interactions from low input material, and have significantly improved mapping resolution. However, both are offered at a high cost. CUT&RUN-sequencing employs expensive pA/MNase fusion protein that has significant A/T sequence bias, causing the target protein-bound DNA region profiles to be seriously affected by the level of MNase digestion. CUT&Tag-sequencing displays the same digestion bias and is less specific due to off-target accessibility of chromatin caused by the Tn5 transposase enzyme.
To address these issues, EpiGentek has developed two new techniques, CUT&RUN Fast and CUT&Tag In-Place-Sequencing, for rapidly enriching protein-bound DNA and mapping genome-wide protein/DNA interactions. These innovative approaches combine the advantages of ChIP-exo, ChiPmentation, and CUT&RUN-sequencing with the fastest procedures in convenient all-in-one kits to reliably identify true target protein-enriched regions and achieve high-resolution mapping.
Both techniques utilize a unique nucleic acid cleavage enzyme mix, which has low sequence bias, to simultaneously fragment chromatin and cleave/remove unbound DNA sequences in both ends of the target protein/DNA complex in situ. DNA occupied by the protein of interest is unaffected, thereby minimizing immunocapture/sequencing background. With these techniques, library DNA generation can be achieved in just a few hours starting from as little as 500 cells or 100 ng of isolated chromatin.
Due to the popularity of the CUT&RUN-sequencing and CUT&Tag-sequencing methods for ChIP-based procedures, as well as the concerns in regard to improving upon their cost, reliability, and convenience, the EpiNext™ CUT&RUN Fast Kit and the EpiNext™ cTIP (CUT&Tag In-Place)-Sequencing Kit will certainly meet the demands of investigators expressing such concerns. EpiGentek has also applied these novel methodologies to the study of other epigenetic factors, RNA methylation in particular.
The EpiQuik CUT&RUN m6A RNA Enrichment Kit and the EpiNext CUT&RUN RNA m6A-Seq Kit are designed to specifically capture and enrich RNA fragments containing the m6A modification from low input RNA specimens with minimal non-specific background levels. As the most common and abundant eukaryotic RNA modification, m6A accounts for over 80% of all RNA methylation and affects virtually every facet of ribonucleic acid biology. Interestingly, m6A exhibits both pro- and anti-viral activities, depending on the virus species and host cell type. Research tools for investigating this epigenetic mark may prove useful in the fight against the SARS-CoV-2 virus and COVID-19.
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