Statde Key Laboratory of Genetic Engineering School of Life Science, Fudan University, Shanghai 200438, China.
Novoprotein Scientific Inc., Wujiang 215200, China.
Yi Chuan. 2021 Apr 20;43(4):362-374. doi: 10.16288/j.yczz.21-016.
The emerging cleavage under target and tagment (CUT&Tag) technology uses Tn5 transposase to cleavage near the DNA binding site of target protein and study the generated DNA fragments by the next-generation sequencing. It can quickly identify protein-DNA interactions, which greatly simplifies the experimental process of ChIP-Seq. After CUT&Tag tagment reaction, DNA recovery or other post-processing is required to perform library construction PCR. Different recovery methods have significant impact respectively. By establishing Streptavidin beads recovery CUT&Tag(srCUT&Tag), we can quickly and conveniently complete the product recovery of CUT&Tag. We carried out CUT&Tag assay of H3K4me3, RNA Polymerase II (RNA polymerase II, RNAPII), transcription factor CTCF and HMGA1 in K562 cells with different recovery methods, including ethanol precipitation, fragment separation magnetic beads (SPRI) Magnetic bead recovery, direct PCR method, as well as our srCUT&Tag recovery method. The results show that among the CUT&Tag results of four different targets, the SPRI magnetic bead recovery and our srCUT&Tag methods have higher recovery efficiency than the direct PCR method and ethanol precipitation method. All CUT&Tag results showed that the recovery of SPRI magnetic beads would lose most of the product fragments less than 150 bp. In the recovery of CTCF and HMGA1, direct PCR lost most of the fragments larger than 300 bp and has significant difference from result of other recovery method. This enables srCUT&Tag to provide more real and higher-resolution information than other recovery method. In summary, the newly established srCUT&Tag recovery method can improve the efficiency of CUT&Tag library construction and obtain better data quality compared with the existing CUT&Tag product recovery method, providing a better technical choice for epigenetics research.
新兴的靶标和片段（CUT&Tag）技术使用 Tn5 转座酶在靶蛋白的 DNA 结合位点附近进行切割，并通过下一代测序研究生成的 DNA 片段。它可以快速识别蛋白质-DNA 相互作用，极大地简化了 ChIP-Seq 的实验过程。在 CUT&Tag 片段化反应后，需要进行 DNA 回收或其他后处理才能进行文库构建 PCR。不同的回收方法有显著的影响。通过建立链霉亲和素珠回收 CUT&Tag（srCUT&Tag），我们可以快速方便地完成 CUT&Tag 的产物回收。我们使用不同的回收方法在 K562 细胞中进行了 H3K4me3、RNA 聚合酶 II（RNA polymerase II，RNAPII）、转录因子 CTCF 和 HMGA1 的 CUT&Tag 检测，包括乙醇沉淀、片段分离磁珠（SPRI）磁珠回收、直接 PCR 法以及我们的 srCUT&Tag 回收法。结果表明，在四种不同靶标 CUT&Tag 的结果中，SPRI 磁珠回收和我们的 srCUT&Tag 方法的回收效率均高于直接 PCR 法和乙醇沉淀法。所有 CUT&Tag 结果均表明，SPRI 磁珠回收会丢失大部分小于 150 bp 的产物片段。在 CTCF 和 HMGA1 的回收中，直接 PCR 法丢失了大部分大于 300 bp 的片段，与其他回收方法的结果有显著差异。这使得 srCUT&Tag 能够提供比其他回收方法更真实、更高分辨率的信息。综上所述，新建立的 srCUT&Tag 回收方法可以提高 CUT&Tag 文库构建的效率，并获得比现有 CUT&Tag 产物回收方法更好的数据质量，为表观遗传学研究提供了更好的技术选择。
