Deoxyribonuclease (DNase I) is a nucleic acid endonuclease, and different chromatin regions are differently sensitive to DNase I. When a gene is in a transcriptionally active state, its sensitivity to DNase I degradation is more than 100-fold higher than that of a non-transcriptionally active region called DNase I hypersensitive site (DH site), i.e., open chromatin region. The traditional method for identifying DHSs is mainly end-labeled Southern Blotting, which involves several time-consuming and laborious steps. Using deoxyribonuclease I hypersensitive site sequencing (DNase-seq), Lifeasible can efficiently and specifically identify DHSs on a genome-wide basis.

Figure 1. Chromatin accessibility analysis by DNase-seq assay.

We used DNase I to digest nuclear chromatin partially and sequenced the recovered DNA to analyze chromatin accessibility at the genome-wide level and to quantify the relative abundance of DNase-sensitive chromatin on a genome-wide scale.

• End-capture. We used the appropriate amount of DNase I to treat the nuclear chromatin to form a cleavage site in the open chromatin region. Then we captured the 20 bp sequence at the end of the cleavage site by the specific cleavage action of restriction endonuclease Mme I for chromatin accessibility analysis.
• Double-hit. We controlled the amount of DNase I to form a double-hit on both sides of the regulatory proteins to release the DNA into small fragments, which were then sequenced to assess the chromatin accessibility of the region.

In comparison, end-capture can obtain more open chromatin sites, while double-hit provides a relatively simple operation process and higher signal-to-noise ratio. Meanwhile, the length of DNA fragments sequenced by double-hit (50-150 bp) is much longer than that of end-capture (20 bp), which can effectively overcome the problem of low comparison rate of complex genomes.

Technology characteristics

• DNase-seq has been widely used in various species with well-proven reliability.
• Targeted cleavage of DHS sites in open regions without cleavage of protected regions allows DNase-seq to infer possible locations of nucleosomes and changes in chromatin openness.
• DNase-seq requires millions of cells as samples. Now that we have developed single-cell DNase-seq, it is impossible to differentiate between dense chromatin regions and what is lost during sequencing (the disadvantage of no duplicates).
• DNase I suffers from cut preference, which needs to be eliminated computationally.
• The sequence information obtained does not fully restore unprotected sequences in open regions.
• Transcription factor binding sites can be inferred.

DNase-seq is a reliable and robust technique for identifying regulatory elements active on the genome without needing a priori information for other epigenetic studies. Lifeasible is here to help you with chromatin studies using this technological tool. Please feel free to contact us for more details.

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For research use only, not intended for any clinical use.

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