MNase-seq
MNase-seq是micrococcalnucleasedigestion with deepsequencing（微球菌核酸抹消化結合深度的測序）的縮寫，是二空空八年以來用佇檢測人類基因組上核小體占用狀況的分子生物學技術。佇二空空九年才定名「MNase-seq」。 我簡來講，這个技術依賴賴來自金黃色葡萄球菌的非特異性核酸內外切抹著微球菌核酸響，用其結合和切割染色質上無結合卵白質的 DNA 區域。佮這个同時，這款被無消化佮組卵白猶是其他染色質結合卵白（譬如講轉錄因為）結合的 DNA。然後對卵白質中純化未切割的 DNA，並通過一種抑是濟種無仝的後一代測序方法進行測序。
MNase-seq 是用佇通過分析染色質會當佮性來評估表觀基因組狀態的四類方法之一。其他三種技術是 DNase-seq、FAIRE-seq 和 ATAC-seq。MNase-seq 主要是用對對這組卵白或者是其他染色質結合卵白結合的 DNA 區域來進行測序，其他三種測序的對象通常分別是：脫氧核糖核酸酸 I 過敏位點（DHS）、 測袂結合的 DNA 的染色質卵白質佮鬆散的染色質區域（通過標記物的轉座測定）。
歷史
一九五六年，頭擺佇金黃色葡萄球菌內底發現微球菌核酸響，卵白質佇一九六六年結晶檢測，特徵佇一九六七年予闡明。染色質的 MNase 消化消化是染色質結構早期研究的關鍵；用佇確定染色質的每一个核小體單位由大約兩百 bp 的 DNA 組成。這佮奧林斯佮奧林斯的「珠仔捾」模型做伙，證實了科恩伯格關於基本染色質結構的設想。進一步研究發現，MNase 無法度降解短於～一百四十 bp 的佮組卵白結合的 DNA，而且 DNase I 和 II 會當共結合的 DNA 降解至低到十 bp。這最終闡明矣核小體核心由～一百四十六 bp 的 DNA 包裹著，~ 五十 bp 的接頭 DNA 連接逐个核小體，而且十个連紲的 DNA 鹼基對隔地佮核小體的核心敆牢牢。
微球菌核酸抹消化自特徵佇一九六七年予闡明以來，除了用研究染色質結構外，一直以置於些核酸測序實驗。因為 MNase 優先消化腺允允佮胸腺石頭風的區域，此技術用來分析無染色質序列，譬如講酵母（激酒酵母）粒線體 DNA 佮食菌體 DNA。佇一九八空年代初期，MNase 消化予人確定熟似 SV 四十、果蠅（烏腹肚）、 酵母、佮猴山仔等染色體的核小體定相佮相關 DNA。一九八五年，頭一擺使用這款消化來研究染色質會當佮人類的基因表達的相關性。佇這項研究中，核酸抹佇檢測某寡致癌序列佮染色質佮核卵白的關聯。佇咧無測序抑是講陣列信息的情形下，利用 MNase 消化來確定核小體定位的研究一直繼續到二空空空年代初期。
隨著一九九空年代末和二空空年代初全基因組測序的出現，將純化的 DNA 序列佮激酒酵母、秀麗隱杆線蟲、烏腹肚、擬南芥、小鼠和人類的真核基因組進行較成做可能。MNase 消化首先予人應用激酒酵母佮秀麗隱杆線蟲的全基因組核小體佔研究。MNase 消化處理了後，通過微陣列進行分析，確定佗一寡 DNA 區域富含 MNase 抗性核小體。是因為 MNase 的微陣列分析通常用佇酵母的全基因組範圍和人類的有限基因組區域以確定核小體定位，這會當用做轉錄失活的推斷。
二空空八年開發出下一代測序的時，MNase 消化佮懸通量測序（即 Solexa / Illumina 測序）相結合以研究人類全基因組範圍內的核小體定位。一年後，術語「MNase-Seq」和「MNase-ChIP」終其尾予人創造出來，用佇咧染色質免疫沉底的微球菌核酸拍消化。自二空空八年頭一改應用以來，MNase-seq 已經予人用於對佮核小體占占跨真核生物表觀基因組學相關的 DNA 進行深度的測序。截到二空二空年二月，MNase-seq 猶是用測定染色質會當佮性。
Description
Chromatin is dynamic and the positioning of nucleosomes on DNA changes through the activity of various transcription factors and remodeling complexes , approximately reflecting transcriptional activity at these sites . DNA wrapped around nucleosomes are generally inaccessible to transcription factors . Hence , MNase-seq can be used to indirectly determine which regions of DNA are transcriptionally inaccessible by directly determining which regions are bound to nucleosomes .
In a typical MNase-seq experiment , eukaryotic cell nuclei are first isolated from a tissue of interest . Then , MNase-seq uses the endo-exonuclease micrococcal nuclease to bind and cleave protein-unbound regions of DNA of eukaryotic chromatin , first cleaving and resecting one strand , then cleaving the antiparallel strand as well . The chromatin can be optionally crosslinked with formaldehyde . MNase requires Ca 二 + as a cofactor , typically with a final concentration of 一 mM . If a region of DNA is bound by the nucleosome core ( i . e . histones ) or other chromatin-bound proteins ( e . g . transcription factors ) , then MNase is unable to bind and cleave the DNA . Nucleosomes or the DNA-protein complexes can be purified from the sample and the bound DNA can be subsequently purified via gel electrophoresis and extraction . The purified DNA is typically ~ 百五 bp , if purified from nucleosomes , or shorter , if from another protein ( e . g . transcription factors ) . 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 . 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 . The positioning of nucleosomes elucidated , through MNase-seq , can then be used to predict genomic expression and regulation at the time of digestion .
Extended Techniques
MNase-ChIP / CUT & RUN sequencing
Recently , MNase-seq has also been implemented in determining where transcription factors bind on the DNA . Classical ChIP-seq displays issues with resolution quality , stringency in experimental protocol , and DNA fragmentation . Classical ChIP-seq typically uses sonication to fragment chromatin , which biases heterochromatic regions due to the condensed and tight binding of chromatin regions to each other . Unlike histones , transcription factors only transiently bind DNA . Other methods , such as sonication in ChIP-seq , requiring the use of increased temperatures and detergents , can lead to the loss of the factor . CUT & RUN sequencing is a novel form of an MNase-based immunoprecipitation . Briefly , it uses an MNase tagged with an antibody to specifically bind DNA-bound proteins that present the epitope recognized by that antibody . 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 . The use of this technique does not require high temperatures or high concentrations of detergent . Furthermore , MNase improves chromatin digestion due to its exonuclease and endonuclease activity . Cells are lysed in an SDS / Triton X 建一百 solution . Then , the MNase-antibody complex is added . And finally , the protein-DNA complex can be isolated , with the DNA being subsequently purified and sequenced . The resulting soluble extract contains a 二十五-fold enrichment in fragments under 五十 bp . This increased enrichment results in cost-effective high-resolution data .
Single-cell MNase-seq
Single-cell micrococcal nuclease sequencing ( scMNase-seq ) is a novel technique that is used to analyze nucleosome positioning and to infer chromatin accessibility with the use of only a single-cell input . First , cells are sorted into single aliquots using fluorescence-activated cell sorting ( FACS ) . The cells are then lysed and digested with micrococcal nuclease . The isolated DNA is subjected to PCR amplification and then the desired sequence is isolated and analyzed . The use of MNase in single-cell assays results in increased detection of regions such as DNase I hypersensitive sites as well as transcription factor binding sites .
Comparison to other Chromatin Accessibility Assays
MNase-seq is one of four major methods ( DNase-seq , MNase-seq , FAIRE-seq , and ATAC-seq ) for more direct determination of chromatin accessibility and the subsequent consequences for gene expression . 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 , not directly assessing nucleosome positioning , but instead being valuable for the assessment of histone modifier enzymatic function .
DNase-seq
As with MNase-seq , DNase-seq was developed by combining an existing DNA endonuclease with Next-Generation sequencing technology to assay chromatin accessibility . Both techniques have been used across several eukaryotes to ascertain information on nucleosome positioning in the respective organisms and both rely on the same principle of digesting open DNA to isolate ~ 一百四十 bp bands of DNA from nucleosomes or shorter bands if ascertaining transcription factor information . Both techniques have recently been optimized for single-cell sequencing , which corrects for one of the major disadvantages of both techniques ; that being the requirement for high cell input .
At sufficient concentrations , DNase I is capable of digesting nucleosome-bound DNA to 十 bp , whereas micrococcal nuclease cannot . Additionally , DNase-seq is used to identify DHSs , which are regions of DNA that are hypersensitive to DNase treatment and are often indicative of regulatory regions ( e . g . promoters or enhancers ) . An equivalent effect is not found with MNase . As a result of this distinction , DNase-seq is primarily utilized to directly identify regulatory regions , whereas MNase-seq is used to identify transcription factor and nucleosomal occupancy to indirectly infer effects on gene expression .
FAIRE-seq
FAIRE-seq differs more from MNase-seq than does DNase-seq . FAIRE-seq was developed in 兩千空七 and combined with Next-Generation sequencing three years later to study DHSs . FAIRE-seq relies on the use of formaldehyde to crosslink target proteins with DNA and then subsequent sonication and phenol-chloroform extraction to separate non-crosslinked DNA and crosslinked DNA . The non-crosslinked DNA is sequenced and analyzed , allowing for direct observation of open chromatin .
MNase-seq does not measure chromatin accessibility as directly as FAIRE-seq . However , unlike FAIRE-seq , it does not necessarily require crosslinking , nor does it rely on sonication , but it may require phenol and chloroform extraction . Two major disadvantages of FAIRE-seq , relative to the other three classes , are the minimum required input of 一百 , 零 cells and the reliance on crosslinking . Crosslinking may bind other chromatin-bound proteins that transiently interact with DNA , hence limiting the amount of non-crosslinked DNA that can be recovered and assayed from the aqueous phase . Thus , the overall resolution obtained from FAIRE-seq can be relatively lower than that of DNase-seq or MNase-seq and with the 一百 , 零 cell requirement , the single-cell equivalents of DNase-seq or MNase-seq make them far more appealing alternatives .
ATAC-seq
ATAC-seq is the most recently developed class of chromatin accessibility assays . ATAC-seq uses a hyperactive transposase to insert transposable markers with specific adapters , capable of binding primers for sequencing , into open regions of chromatin . PCR can then be used to amplify sequences adjacent to the inserted transposons , allowing for determination of open chromatin sequences without causing a shift in chromatin structure . ATAC-seq has been proven effective in humans , amongst other eukaryotes , including in frozen samples . As with DNase-seq and MNase-seq , a successful single-cell version of ATAC-seq has also been developed .
ATAC-seq has several advantages over MNase-seq in assessing chromatin accessibility . ATAC-seq does not rely on the variable digestion of the micrococcal nuclease , nor crosslinking or phenol-chloroform extraction . It generally maintains chromatin structure , so results from ATAC-seq can be used to directly assess chromatin accessibility , rather than indirectly via MNase-seq . ATAC-seq can also be completed within a few hours , whereas the other three techniques typically require overnight incubation periods . The two major disadvantages to ATAC-seq , in comparison to MNase-seq , are the requirement for higher sequencing coverage and the prevalence of mitochondrial contamination due to non-specific insertion of DNA into both mitochondrial DNA and nuclear DNA . Despite these minor disadvantages , use of ATAC-seq over the alternatives is becoming more prevalent .