Integrated analysis of chromatin conformation by Hi-C and electron tomography

在这一年中，我们进行了进一步的ChromEMT实验，然后通过3D断层扫描进行电子显微镜（EM）成像。今年，我们与冲绳科学技术研究所合作，获得了高分辨率图像。这些图像证实了ChromEMT协议是正确的，尽管在图像中观察到的对比度仍然需要一些改进。我们的下一个目标是标记特定的RNA分子，以便在EM图像中进行识别。我们使用针对特定RNA的初级未修饰探针，具有附着的生物素部分和荧光标记的二级探针用于通过光学显微镜进行确认，由具有附着的纳米金珠的抗生物素抗体组成的三级探针，以及由具有附着的不同颜色的荧光团的抗体组成的四级探针。由于只有初级探针（相对便宜）对RNA具有特异性，因此这种设置使我们能够在靶向不同RNA时最大限度地降低成本。我们使用XIST作为协议开发的目标。在光学显微镜中，来自二级和四级探针的重叠信号证实了我们的探针正在工作。早期使用链霉亲和素的尝试失败了，因为它无法进入细胞核。相比之下，三级探针上的1.4nm纳米金珠可以进入细胞核。对于EM中的可视化，我们使用黄金增强来增加金珠的大小。虽然EM断层扫描证实探针能够进入细胞核，但我们目前仍然有很强的背景信号，其中第三探针在细胞核内虚假结合。

During this year, we have performed further ChromEMT experiments following by electron microscope (EM) imaging by 3D tomography. This year, in collaboration with Okinawa Institute of Science and Technology, we obtained high-resolution images. The images confirmed that the ChromEMT protocol is working correctly, though the contrast observed in the images still needs some improvement.Our next aim is to label specific RNA molecules for identification in the EM images. We use a primary unmodified probe against a specific RNA, a secondary probe with an attached biotin moiety and a fluorescent label for confirmation by light microscopy, a tertiary probe consisting of an anti-biotin antibody with a nanogold bead attached, and a quaternary probe consisting of an antibodywith an attached fluorophore at a different color. As only the primary probe (which is relatively cheap) is specific to the RNA, this setup allows us to minimize costs when targeting different RNAs. We are using XIST as a target for protocol development. Overlapping signals from the secondary and quaternary probe in the light microscope confirmed that our probes are working. An earlier attempt using streptavidin failed, as it was unable to enter the nucleus. In contrast, the 1.4 nm nanogold bead on the tertiary probe can enter the nucleus. For visualization in EM, we use gold enhancement to increase the size of the gold bead. While EM tomography confirmed that the probes are able to enter the nucleus, we currently still have a strong background signal with spurious binding of the tertiary probe inside the nucleus.