Pnp tr tips

For atomic force microscopy, isolated nuclei in PBS were spun (500g, 3 minutes) on to 12mm coverslips coated with Poly-D-Lysine (1 μg/μl was used to coat coverslips for 5 minutes, then rinsed with purified water and left to dry overnight). Coverslips were transferred to a glass slide, secured with vacuum grease, and covered in a PBS droplet for AFM. Indentation experiments were performed on an MFP-3D Bio Atomic Force Microscope (Oxford Instruments) mounted in a Ti-U fluorescent inverted scope (Nikon Instruments, Melville, NY) and used Asylum Research 13, Igor Pro 6.34A software. Nanoworld PNP-TR tips were calibrated for their spring constant using the thermal noise method and used for probing isolated nuclei. A trigger force of 2nN, an approach Velocity constant of at 2 μm/s and a force distance of 6 μm were used to generate a force map with 12 points across 2μm². Hand4.2-Gal4 was used to drive expression of GFP, and thus only GFP-positive nuclei were selected for indentation. The software was used to calculate the Young’s modulus using the Hertz equation⁸⁹ . Any poor fits to the indentation curve were excluded. Then, the average Young’s modulus was calculated from the force map.