核酸酶靶向切割和釋放 (CUT&RUN)技術(shù)是由Steven henikoff博士團(tuán)隊(duì)開發(fā)的一種染色質(zhì)圖譜分析方法，基于Ulrich Laemmli博士的染色質(zhì)免疫切割技術(shù) (ChIC)，融合蛋白A與微球菌核酸酶 (pA-MNase)，選擇性原位切割與抗體結(jié)合的染色質(zhì)。在CUT&RUN中，細(xì)胞或細(xì)胞核固定化在固相載體上，從溶液中分離出pAG-MNase裂解的DNA片段。該方法與二代測(cè)序(NGS)兼容，可提供高質(zhì)量的組蛋白翻譯后修飾(PTMs)和染色質(zhì)相關(guān)蛋白(如轉(zhuǎn)錄因子；Figure 1)。

FIGURE 1 Overview of the CUTANA™ CUT&RUN protocol.

ChIP-seq是組蛋白PTMs和染色質(zhì)相關(guān)蛋白全基因組定位的主要方法。在這種方法中，染色質(zhì)通過超聲或酶消化破碎，然后免疫沉淀目標(biāo)特異性片段。盡管進(jìn)行優(yōu)化，但ChIP-seq需要大量細(xì)胞(通常為105 - 106個(gè)細(xì)胞)而且需要深度測(cè)序input 染色質(zhì)與免疫沉淀物質(zhì)(通常為 >3000萬 reads/次)來從背景中解析信號(hào)。

ChIC和CUT&RUN通過將基因組片段靶向釋放到溶液中，徹-底改變了染色質(zhì)調(diào)控。通過這一創(chuàng)新，背景顯著減少，允許使用少量細(xì)胞且每個(gè)反應(yīng)僅需300 - 800萬reads/次對(duì)組蛋白PTMs和染色質(zhì)相關(guān)蛋白進(jìn)行高分辨率基因組圖譜的繪制(Figure 2)。簡(jiǎn)化的工作流程和節(jié)省的成本使ChIC/CUT&RUN適用于高通量研究表觀遺傳生物學(xué)。

FIGURE 2 Representative genome browser tracks show CUTANA™ CUT&RUN results using 500,000 K562   cells.  Clear peaks with the expected distribution profile are observed using 3-8 million sequencing  reads per reaction for a variety of epigenetic targets, including histone PTMs (H3K4me3, H3K27me3,  H3K27ac), transcription factors (CTCF), epigenetic reader proteins (BRD4), writer enzymes (MLL1),   and chromatin remodelers (SMARCA4).  Rabbit IgG antibody shown as a negative control (top track).

CUTANA™ChIC/CUT&RUN試劑盒包含48個(gè)反應(yīng)的材料，專為多通道移液而設(shè)計(jì)，以實(shí)現(xiàn)CUT&RUN的高通量?jī)?yōu)勢(shì)。該試劑盒包括陽(yáng)性(H3K4me3)和陰性(Rabbit IgG)對(duì)照抗體，以及SNAP-CUTANA™ K-MetStat Panel (16個(gè)DNA條形碼設(shè)計(jì)核小體攜帶廣泛研究的賴氨酸甲基化PTMs)的分裝分量。K-MetStat Panel加入到對(duì)照反應(yīng)中，直接監(jiān)測(cè)實(shí)驗(yàn)成功與否并幫助排除故障。此外，在pAG-MNase切割后，將剪切的E. coli DNA添加到所有反應(yīng)中，以控制文庫(kù)構(gòu)建并使NGS標(biāo)準(zhǔn)化。該試劑盒與細(xì)胞和細(xì)胞核兼容，包括凍存和交聯(lián)樣品(Figure 3)。

FIGURE 3 Heatmaps show CUT&RUN signal (red) and background (blue) of H3K4me3-enriched regions flanking annotated transcription start sites (TSS, +/- 2 kb). Gene rows are aligned across conditions, showing that genome-wide enrichment is preserved across sample types.

盡管建議從500,000個(gè)細(xì)胞開始，但只需使用5,000個(gè)細(xì)胞即可生成可比較的數(shù)據(jù)(Figure 4)。對(duì)照組的加入以及與不同靶類型、樣本和低細(xì)胞數(shù)的兼容性，使該試劑盒成為染色質(zhì)圖譜實(shí)驗(yàn)的首-選解決方案。                                 

FIGURE 4 Representative genome browser tracks for H3K4me3 (low abundance target) and H3K27me3  (high abundance target) CUT&RUN experiments using decreasing amounts of K562 cells. At 5,000   cells, data quality is largely indistinguishable from standard conditions (500,000 cells).

保存條件

OPEN KIT IMMEDIATELY and store components at room temperature, 4℃, and -20℃ as indicated (see User Manual corresponding to Kit Version 3). Stable for 6 months upon date of receipt.

數(shù)據(jù)示例

FIGURE 1： CUT&RUN DNA fragment size  distribution analysis.  CUT&RUN was performed  as described above.   Library DNA was analyzed  by Agilent Tapestation® .  This analysis confirmed  that mononucleosomes  were predominantly  enriched in CUT&RUN (~300 bp peaks represent 150 bp nucleosomes + sequencing  adapters).

FIGURE 2： SNAP-CUTANA™ K-MetStat Spikein Controls. DNA-barcoded designer  nucleosomes (dNucs)  representing 16 K-methyl PTMs: mono-, di-, and tri-methylation at H3K4, H3K9, H3K27, H3K36,  and H4K20, as well as  unmodified control, were spiked into CUT&RUN  reactions prior to the  addition of antibodies (IgG, H3K4me3). Spike-in barcodes were counted and  normalized from  raw fastq files using the shell  script and analysis sheet available at  epicypher/19-1002.  Barcodes for IgG (top;  normalized to total reads) and H3K4me3 (bottom; normalized to on-target)  antibodies are  shown. The spike-ins confirmed optimal  experimental conditions (H3K4me3  antibody  specifically recovered the target dNuc, while IgG  showed no preferential enrichment).

FIGURE 3： CUT&RUN genome-wide heatmaps. CUT&RUN was performed as described above. Peaks were  called with MACS2. Heatmaps show  two replicates (“Rep") of IgG (left) and H3K4me3 (right) kit  control antibodies in aligned rows  ranked by intensity (top to bottom) and colored  such that red  indicates high localized enrichment  and blue denotes background signal.

FIGURE 4： Representative gene browser tracks. CUT&RUN was performed as described above. A representative  168 kb window at the TRMT2A gene is shown for two replicates (“Rep") of IgG and H3K4me3 kit control   antibodies. Representative tracks are also  shown for antibodies to H3K27me3 and the  transcription  factor CTCF. The CUT&RUN kit  produced the expected genomic distribution for  each target. Images  were generated using the Integrative Genomics Viewer (IGV, Broad Institute).

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