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主要针对极低起始量细胞进行实验,因此对核心酶原料有极高要求。CUT&Tag技术的核心原料酶是Hyperactive pA/G-Tn5 Transposase,它具有高活性,对微量DNA有高灵敏度和高亲和力,能有效抓取数十个细胞中的有限结合位点。正是由于Tn5转座酶对DNA有极高的亲和力,导致了纯化过程中易产生非特异核酸残留。诺唯赞生物针对非特异性核酸残留这一点进行攻克,定向改造进化Tn5酶,其灵敏度是野生型Tn5酶的1000倍,具有极低的核酸残留,大大减少了低起始量建库中非特异核酸占比。
wikipedia(要翻墙): 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.
发生在helix上的点突变可以有效提高Tn5活性,阻断了其内部的自抑制作用。Mg离子对于Tn5催化活性是必须的(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
该方法很大的特点就是,加入刀豆蛋白Abeads以后所有的建库步骤都在同一个tube中进行(single reaction tube)
schematic.jpg一抗二抗孵育,添加pA-Tn5,因为转座体本身对于暴露的DNA具有一定亲和力,所以对于没有正确结合到感兴趣位点的pA-Tn5一定要及时用较严格的条件洗掉
不过考虑到实际操作中可能洗着洗着就看不到细胞沉淀了(QAQ)
Efficient profiling of nucleosomes and RNAPII with CUT&-Tag
衡量ChIP-seq、CUT-RUN以及CUT-Tag的性能,在每个数据集都取8M reads的测序深度的前提下,发现尽管pattern相似,但是CUT-Tag具有最低的background:
methods_compare.jpg所以在测序数据量有限(稀有样本)的情况下,ChIP就无能为力了
可以看到,以K4me1为例,在相同测序深度条件下CUT-Tag的信号值是略高于CUT-RUN的
signal.jpg虽然基因的转录状态可以从组蛋白修饰简介推断出,但更直接的方法是检测RNA pol II,而且是engaged pol II(S2/S5-
phosphorylation (S2/5p),分子生物学背景知识),并把得到的peaks和GEO上已有的PRO-seq数据集进行了比较
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具有最高的检测染色质谱的效率,高sensitivity,下图示不同reads采样量条件下落在peaks内的reads占比
reads_fraction.jpgCUT&Tag simultaneously maps factor binding and accessible DNA
一个问题是pA-Tn5理论上也可以切割开放染色质,怎么把这些和特定TF的binding区分开?
we tested if pA-Tn5 tethered at transcription factors can be distinguished from accessible DNA sites in the genome
作者选择了一种核内因子NPAT做测试,因为它只会结合到约80个开放染色质区而且位置是已知的(chr1和chr6的组蛋白基因),方便区分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.
实验证明,主要reads还是富集在指定TF site的:
NPAT.jpgIn NPAT CUT&Tag profiles, ~99% of read counts accumulate at the promoters of the histone genes
但是在K562细胞中与ATAC-seq accessible profile作比较时,发现有约10%的reads还是分布在ATAC track里非histone region),这个就是untethered Tn5造成的了,如果我们只希望获得目标TF的binding site,就还是要尽可能减少那些不必要的其他区域的reads
ATAC_compare.jpgAdjusting the threshold and stringency of NPAT peak calling may improve detection
当然不只是NPAT这种结合位点少的,作者也对binding site丰富的factor做了测试,比如最经典的CTCF
CUT-Tag的精度足以提供某些感兴趣位点的superposition,整合多种染色质feature:
superposition.jpgCUT&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
作者甚至进一步提出了他们的scCUT&Tag,以轻柔的离心取代了beads,Tn5整合adaptor后把单个细胞分配到well中,对每个well的单细胞进行含index的PCR,最后把这些文库混合进行测序。这些单细胞集合成的profile与完全的bulk数据非常一致,而且大多数单细胞的track都落在bulk数据的peaks内,可见CUT-Tag方法在捕获单细胞feature上的可靠性
scCUT-Tag.jpg且CUT-Tag可以鉴定出不同细胞类型的tracks进而区分细胞群体
Discussion
chromatin profiling与RNA-seq相比的一大优势就是它不是单纯地测定表达量,而是chromatin states确实可以反映一种基因的表达状态,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就可以反映抑制状态,甚至有的文献报道的poised enhancer同时具有抑制性和激活性染色质状态(往往代表基因表达的蓄势中间态),有的经典promoter或enhancer会同时具有H3K4me3和H3K4me1两种修饰(同时具有promoter和enhancer的标志),这类调控元件有很大的发掘价值
与ATAC-seq map相比,ATAC-seq是间接地从motif数据或者chromatin profiling data推断TF binding,没有CUT-Tag可以直接验证TF实际结合到开放染色质区的数据更reliable
与ChIP相比的优势当然就是其作为unfixed in situ method的优势了
不过limitation还是有的,怎么减少batch效应,增强可重复性,怎么应用到快速的临床检测中:
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.
以及之前发现的low-level untargeted
accessible DNA sites和high-level CTCF-bound sites,当时是结合ATAC-seq数据发现的,如果有更好的算法可以对这两种无关factor进行建模,就完全可以不需要ATAC-seq或者其他先验数据。
不过还是要基于研究的目的,CUT-Tag可以同时profile TF binding 和其他accessible DNA也是一个很大的特点(统计学分析完全可以区分这两种feature),堪称“multi-OMIC” CUT&Tag
最后,一次CUT-Tag可不可以同时检测多种TF并且直接把不同TF的binding区分开?(或许可以用不同adaptor组装的Tn5去结合不同的抗体)
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.
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