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

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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.