Lamins organize the global three-dimensional genome from the nuclear periphery
To visualize if HiLands-B LADs indeed move away from the NL upon lamin loss, we selected two regions consisting of mostly HiLands-B and exhibiting decreased emerin DamID values upon lamin loss for FISH analyses using Oligopaint. We used FISH to label the HiLands-B LADs while immunostained on Emerin to mark the nuclear periphery. Using Imaris, we quantified the distance between the FISH signal and the NL and found that the two HiLands-B regions in TKO mESCs were farther away from the NL than in the WT.
Steps to reproduce
This dataset contains confocal images (lif files) acquired using a Leica SP5 confocal microscope equipped with a 63x/1.4 objective and an electron multiplying charge-coupled device camera. For each set of experiments, images were acquired as confocal stacks at 126 nm per step in the z-axis using the same laser setting. The cells were stained with FISH probes for HiLands-B regions, with Emerin antibody for marking the nuclear periphery, and with DAPI for the DNA. The Imaris software (Bitplane) was used for all the quantifications. For double blind analyses, one person coded each set of 3D FISH image with a number corresponding to its true feature. These images were then randomized and coded with another set of numbers. These randomized images were given to another person to perform the quantification. For each FISH experiment, at least 50 nuclei were quantified.