CUT&Tag for efficient epigenomic profiling of small samples and single cells
原文：https://doi.org/10.1038/s41467-019-09982-5
由于CUT&Tag主要針對極低起始量細胞進行實驗减途，因此對核心酶原料有極高要求。CUT&Tag技術(shù)的核心原料酶是Hyperactive pA/G-Tn5 Transposase兄淫，它具有高活性，對微量DNA有高靈敏度和高親和力抒钱，能有效抓取數(shù)十個細胞中的有限結(jié)合位點了袁。正是由于Tn5轉(zhuǎn)座酶對DNA有極高的親和力坦仍，導(dǎo)致了純化過程中易產(chǎn)生非特異核酸殘留。諾唯贊生物針對非特異性核酸殘留這一點進行攻克得糜，定向改造進化Tn5酶敬扛，其靈敏度是野生型Tn5酶的1000倍，具有極低的核酸殘留朝抖，大大減少了低起始量建庫中非特異核酸占比啥箭。
wikipedia（要翻墻）: https://en.wikipedia.org/wiki/Transposase#Transposase_Tn5
野生型Tn5活性是很低很低的：
Tn5 and other transposases are notably inactive. Because DNA transposition events are inherently mutagenic, the low activity of transposases is necessary to reduce the risk of causing a fatal mutation in the host, and thus eliminating the transposable element. One of the reasons Tn5 is so unreactive is because the N- and C-termini are located in relatively close proximity to one another and tend to inhibit each other.
發(fā)生在helix上的點突變可以有效提高Tn5活性，阻斷了其內(nèi)部的自抑制作用治宣。Mg離子對于Tn5催化活性是必須的（而MNase需要Ca2+)
The transposition of a transposon often needs only three pieces: the transposon, the transposase enzyme, and the target DNA for the insertion of the transposon.This is the case with Tn5, which uses a cut-and-paste mechanism for moving around transposons
Results
該方法很大的特點就是急侥，加入刀豆蛋白Abeads以后所有的建庫步驟都在同一個tube中進行（single reaction tube）
一抗二抗孵育砌滞，添加pA-Tn5，因為轉(zhuǎn)座體本身對于暴露的DNA具有一定親和力坏怪，所以對于沒有正確結(jié)合到感興趣位點的pA-Tn5一定要及時用較嚴(yán)格的條件洗掉
不過考慮到實際操作中可能洗著洗著就看不到細胞沉淀了（QAQ）
Efficient profiling of nucleosomes and RNAPII with CUT&-Tag
衡量ChIP-seq贝润、CUT-RUN以及CUT-Tag的性能，在每個數(shù)據(jù)集都取8M reads的測序深度的前提下铝宵，發(fā)現(xiàn)盡管pattern相似打掘，但是CUT-Tag具有最低的background:
所以在測序數(shù)據(jù)量有限（稀有樣本）的情況下，ChIP就無能為力了
可以看到鹏秋，以K4me1為例胧卤，在相同測序深度條件下CUT-Tag的信號值是略高于CUT-RUN的
雖然基因的轉(zhuǎn)錄狀態(tài)可以從組蛋白修飾簡介推斷出，但更直接的方法是檢測RNA pol II拼岳，而且是engaged pol II(S2/S5-
phosphorylation (S2/5p)，分子生物學(xué)背景知識)况芒，并把得到的peaks和GEO上已有的PRO-seq數(shù)據(jù)集進行了比較
We used an antibody to the S2/S5phosphorylation (S2/5p) forms of RNAPII, which distinguish engaged polymerase
CUT&Tag sensitively maps active sites in chromatin
CUT-Tag具有最高的檢測染色質(zhì)譜的效率惜纸，高sensitivity，下圖示不同reads采樣量條件下落在peaks內(nèi)的reads占比
CUT&Tag simultaneously maps factor binding and accessible DNA
一個問題是pA-Tn5理論上也可以切割開放染色質(zhì)绝骚，怎么把這些和特定TF的binding區(qū)分開耐版？
we tested if pA-Tn5 tethered at transcription factors can be distinguished from accessible DNA sites in the genome
作者選擇了一種核內(nèi)因子NPAT做測試，因為它只會結(jié)合到約80個開放染色質(zhì)區(qū)而且位置是已知的（chr1和chr6的組蛋白基因)，方便區(qū)分TF site和其他可能的accessible site
We used an antibody to the NPAT nuclear factor, a transcriptional coactivator of the replication-dependent histone genes, in CUT&Tag reactions.
實驗證明，主要reads還是富集在指定TF site的：
In NPAT CUT&Tag profiles, ~99% of read counts accumulate at the promoters of the histone genes
但是在K562細胞中與ATAC-seq accessible profile作比較時充石，發(fā)現(xiàn)有約10%的reads還是分布在ATAC track里非histone region)寡键，這個就是untethered Tn5造成的了，如果我們只希望獲得目標(biāo)TF的binding site红省，就還是要盡可能減少那些不必要的其他區(qū)域的reads
Adjusting the threshold and stringency of NPAT peak calling may improve detection
當(dāng)然不只是NPAT這種結(jié)合位點少的，作者也對binding site豐富的factor做了測試，比如最經(jīng)典的CTCF
CUT-Tag的精度足以提供某些感興趣位點的superposition亭引，整合多種染色質(zhì)feature:
CUT&Tag profiles low cell number samples and single cells
CUT&Tag has the advantage that the entire reaction from antibody binding to adapter integration occurs within intact cells. The transposase and chromatin fragments remain bound together
作者甚至進一步提出了他們的scCUT&Tag，以輕柔的離心取代了beads皮获，Tn5整合adaptor后把單個細胞分配到well中焙蚓，對每個well的單細胞進行含index的PCR，最后把這些文庫混合進行測序洒宝。這些單細胞集合成的profile與完全的bulk數(shù)據(jù)非常一致购公，而且大多數(shù)單細胞的track都落在bulk數(shù)據(jù)的peaks內(nèi)，可見CUT-Tag方法在捕獲單細胞feature上的可靠性
且CUT-Tag可以鑒定出不同細胞類型的tracks進而區(qū)分細胞群體
Discussion
chromatin profiling與RNA-seq相比的一大優(yōu)勢就是它不是單純地測定表達量雁歌，而是chromatin states確實可以反映一種基因的表達狀態(tài)宏浩，比如：
chromatin profiling has the unique advantage of identifying silenced regions, which is a key aspect of establishing cell fates in development
states比如H3K27me3就可以反映抑制狀態(tài)，甚至有的文獻報道的poised enhancer同時具有抑制性和激活性染色質(zhì)狀態(tài)（往往代表基因表達的蓄勢中間態(tài)）将宪，有的經(jīng)典promoter或enhancer會同時具有H3K4me3和H3K4me1兩種修飾（同時具有promoter和enhancer的標(biāo)志）绘闷，這類調(diào)控元件有很大的發(fā)掘價值
與ATAC-seq map相比橡庞，ATAC-seq是間接地從motif數(shù)據(jù)或者chromatin profiling data推斷TF binding，沒有CUT-Tag可以直接驗證TF實際結(jié)合到開放染色質(zhì)區(qū)的數(shù)據(jù)更reliable
與ChIP相比的優(yōu)勢當(dāng)然就是其作為unfixed in situ method的優(yōu)勢了
不過limitation還是有的印蔗，怎么減少batch效應(yīng)扒最，增強可重復(fù)性，怎么應(yīng)用到快速的臨床檢測中：
While the ease and low cost of this pipeline is appealing, the primary virtue of automated chromatin profiling is the minimization of batch and handling effects, and thus maximum reproducibility. Such aspects are critical for clinical assays and testing for chromatintargeting drugs.
以及之前發(fā)現(xiàn)的low-level untargeted
accessible DNA sites和high-level CTCF-bound sites华嘹，當(dāng)時是結(jié)合ATAC-seq數(shù)據(jù)發(fā)現(xiàn)的吧趣，如果有更好的算法可以對這兩種無關(guān)factor進行建模，就完全可以不需要ATAC-seq或者其他先驗數(shù)據(jù)耙厚。
不過還是要基于研究的目的强挫，CUT-Tag可以同時profile TF binding 和其他accessible DNA也是一個很大的特點（統(tǒng)計學(xué)分析完全可以區(qū)分這兩種feature)，堪稱“multi-OMIC” CUT&Tag
最后薛躬，一次CUT-Tag可不可以同時檢測多種TF并且直接把不同TF的binding區(qū)分開?（或許可以用不同adaptor組裝的Tn5去結(jié)合不同的抗體）
In the future, we expect that barcoding of adapters will allow for multiple epitopes to be simultaneously profiled in single cells in large numbers, maximizing the utility of single-cell epigenomic profiling for studies of development and disease.
