Lamins organize the global three-dimensional genome from the nuclear periphery

Published: 14-05-2018| Version 1 | DOI: 10.17632/436tkbfmx3.1
Contributors:
Jiabiao Hu,
Yixian Zheng,
Xiaobin Zheng

Description

To visualize whether HiLands-P chromatin is decondensed, we used Oligopaint method -a modified fluorescence in situ hybridisation (FISH) technique specifically for imaging large Chromatic loci-to quantify four selected HiLands-P regions from chromosomes 1, 4, 13, and 14. After the hybridisation, we used Imaris software to quantify both the volumes and surface areas of these FISH signal in WT and TKO mESCs. The comparison demonstrated that both the volumes and surface areas of these regions increased significantly in TKO mESCs.

Files

Steps to reproduce

This datasets contain 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-P regions, 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.