MNase-seq
[3] Briefly, this technique relies on the use of the non-specific endo-exonuclease micrococcal nuclease, an enzyme derived from the bacteria Staphylococcus aureus, to bind and cleave protein-unbound regions of DNA on chromatin.
[17] In addition to being used to study chromatin structure, micrococcal nuclease digestion had been used in oligonucleotide sequencing experiments since its characterization in 1967.
[24] In the early 1980s, MNase digestion was used to determine the nucleosomal phasing and associated DNA for chromosomes from mature SV40,[25] fruit flies (Drosophila melanogaster),[26] yeast,[27] and monkeys,[28] among others.
[29] Studies utilizing MNase digestion to determine nucleosome positioning without sequencing or array information continued into the early 2000s.
MNase-based microarray analyses were often utilized at genome-wide scales for yeast[38][39] and in limited genomic regions in humans[40][41] to determine nucleosome positioning, which could be used as an inference for transcriptional inactivation.
[2] A year later, the terms “MNase-Seq” and “MNase-ChIP”, for micrococcal nuclease digestion with chromatin immunoprecipitation, were finally coined.
[3] Since its initial application in 2006,[1] MNase-seq has been utilized to deep sequence DNA associated with nucleosome occupancy and epigenomics across eukaryotes.
[45] This makes short-read, high-throughput sequencing ideal for MNase-seq as reads for these technologies are highly accurate but can only cover a couple hundred continuous base-pairs in length.
[46] Once sequenced, the reads can be aligned to a reference genome to determine which DNA regions are bound by nucleosomes or proteins of interest, with tools such as Bowtie.
[4] The positioning of nucleosomes elucidated, through MNase-seq, can then be used to predict genomic expression[47] and regulation[48] at the time of digestion.
[49][50] Classical ChIP-seq displays issues with resolution quality, stringency in experimental protocol, and DNA fragmentation.
Other methods, such as sonication in ChIP-seq, requiring the use of increased temperatures and detergents, can lead to the loss of the factor.
Digestion then specifically occurs at regions surrounding that transcription factor, allowing for this complex to diffuse out of the nucleus and be obtained without having to worry about significant background nor the complications of sonication.
[52] All four techniques are contrasted with ChIP-seq, which relies on the inference that certain marks on histone tails are indicative of gene activation or repression,[53] not directly assessing nucleosome positioning, but instead being valuable for the assessment of histone modifier enzymatic function.
[4] As with MNase-seq,[2] DNase-seq was developed by combining an existing DNA endonuclease[6] with Next-Generation sequencing technology to assay chromatin accessibility.
[54] Both techniques have been used across several eukaryotes to ascertain information on nucleosome positioning in the respective organisms[4] and both rely on the same principle of digesting open DNA to isolate ~140bp bands of DNA from nucleosomes[2][55] or shorter bands if ascertaining transcription factor information.
[5] Two major disadvantages of FAIRE-seq, relative to the other three classes, are the minimum required input of 100,000 cells and the reliance on crosslinking.
[8] ATAC-seq uses a hyperactive transposase to insert transposable markers with specific adapters, capable of binding primers for sequencing, into open regions of chromatin.
